Biological magnetic microsphere and preparation method and application thereof

文档序号：527166 发布日期：2021-06-01 浏览：20次中文

阅读说明：本技术 一种生物磁性微球及其制备方法和应用 (Biological magnetic microsphere and preparation method and application thereof ) 是由郭敏吴亮徐丽琼于雪于 2020-11-27 设计创作，主要内容包括：本发明第一方面提供了一种生物磁性微球,包括磁性微球本体,所述磁性微球本体外表面具有至少一种带有线性主链和支链的聚合物,所述线性主链的一端固定于磁性微球本体外表面,聚合物的其他端游离于磁性微球本体外表面,所述生物磁性微球的聚合物的支链末端连接有生物素。本发明还提供了基于第一方面提供的生物磁性微球进行的改进,以及制备方法和应用。本发明的生物磁性微球操作使用便捷,可在溶液中的快速分散和快速沉聚,无需使用高速离心机等大型实验设备；用途广泛,还可通过生物素连接具有可选择性的纯化介质(如亲和素、亲和蛋白、多肽/蛋白标签等),可普遍、大规模应用于蛋白包括但不限于抗体类物质等目标物的分离纯化。(The invention provides a biomagnetic microsphere, which comprises a magnetic microsphere body, wherein the outer surface of the magnetic microsphere body is provided with at least one polymer with a linear main chain and a branched chain, one end of the linear main chain is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is dissociated from the outer surface of the magnetic microsphere body, and the tail end of the branched chain of the polymer of the biomagnetic microsphere is connected with biotin. The invention also provides improvement based on the biomagnetic microspheres provided by the first aspect, and a preparation method and application thereof. The biomagnetic microspheres are convenient to operate and use, can be rapidly dispersed and rapidly precipitated in a solution, and do not need large experimental equipment such as a high-speed centrifuge; the method has wide application, can be connected with a selective purification medium (such as avidin, affinity protein, polypeptide/protein tag and the like) through biotin, and can be widely and massively applied to the separation and purification of target substances such as proteins including but not limited to antibody substances.)

1. A biological magnetic microsphere comprises a magnetic microsphere body, and is characterized in that: the outer surface of the magnetic microsphere body is provided with at least one polymer with a linear main chain and a branched chain, one end of the linear main chain is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the tail end of the branched chain of the polymer of the biological magnetic microsphere is connected with biotin or biotin analogues.

2. The biomagnetic microsphere of claim 1, wherein: the branched ends of the polymer are linked to a purification medium by a linking element, and the linking element comprises the biotin or biotin analogue.

3. The biomagnetic microsphere of claim 2, wherein: the purification medium contains an avidin type tag, a polypeptide type tag, a protein type tag, an antibody type tag, an antigen type tag or a combination thereof;

preferably, the avidin-type tag is avidin, an avidin analog that binds biotin, an avidin analog that binds a biotin analog, or a combination thereof;

more preferably, the end of the branched chain of the polymer of the biomagnetic microsphere is connected with biotin; the purification medium is avidin, and forms the binding function of an affinity complex with the biotin;

more preferably, the avidin is streptavidin, modified streptavidin, a streptavidin analog, or a combination thereof;

preferably, the polypeptide-type tag is selected from any one of the following tags or variants thereof: a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag comprising a WRHPQFGG sequence, a tag comprising a variant sequence of WRHPQFGG, a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, and combinations thereof;

preferably, the protein-based tag is selected from any one of the following tags or variants thereof: an affinity protein, SUMO tag, GST tag, MBP tag, or a combination thereof; more preferably one, said affinity protein is selected from the group consisting of: protein a, protein G, protein L, modified protein a, modified protein G, modified protein L, and combinations thereof.

4. The biomagnetic microspheres according to any one of claims 2 or 3, wherein: the purification media is attached to the branched ends of the polymer by a linking element comprising an affinity complex;

preferably, the biotin or biotin analogue is linked to avidin or avidin analogue by affinity complex action, and the purification medium is linked directly or indirectly to the avidin or avidin analogue;

more preferably one, said purification medium is covalently linked to biotin or a biotin analogue at the end of the branch of said polymer by an avidin-type tag-purification medium, via a linking element forming an affinity complex between the avidin-type tag and the biotin or biotin analogue;

in a further preferred form, the purification medium is covalently linked to the complex by avidin-purification medium, the linking element of the affinity complex being formed with biotin or a biotin analogue at the end of the branch of the polymer.

5. The biomagnetic microspheres according to any one of claims 2-4, wherein: the purification medium is attached to the ends of the branches of the polymer in a manner that: covalent bonds, supramolecular interactions, linking elements, or combinations thereof.

Preferably, the covalent bond is a dynamic covalent bond; more preferably, the dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof;

preferably one, said supramolecular interaction is selected from: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, and combinations thereof;

more preferably, the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction.

6. The biomagnetic microsphere of claim 1, wherein: further comprising avidin bound to said biotin or biotin analogue; wherein the biotin or biotin analogue and the avidin form an affinity complex; preferably, the avidin is streptavidin, modified streptavidin, a streptavidin analog, or a combination thereof.

7. The biomagnetic microsphere of claim 6, wherein: also included are affinity proteins linked to the avidin or avidin analogs.

8. The biomagnetic microsphere according to any one of claims 1 to 7, wherein: the size of the magnetic microsphere body is selected from any one of the following particle size scales or a range between any two of the following particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 65 μm, 40 μm, 45 μm, 50 μm, 25 μm, 1 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm; the diameter sizes are averages;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.1-10 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.2-6 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.4-5 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.5-3 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.2-1 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.5-1 μm;

in one preferred mode, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm;

in a preferred embodiment, the magnetic microsphere body has an average diameter of 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, or 1000nm, with a deviation of ± 20%, more preferably ± 10%.

9. The biomagnetic microsphere according to any one of claims 1 to 8, wherein: the linear backbone of the polymer is a polyolefin backbone or an acrylic polymer backbone;

preferably, the linear backbone of the polymer is a polyolefin backbone and is provided by the backbone of an acrylic polymer;

more preferably, the monomer unit of the acrylic polymer is one of acrylic acid, acrylate, methacrylic acid, methacrylate or a combination thereof.

10. The biomagnetic microsphere of claim 1, wherein: the branches of the polymer are covalently bound to biotin or a biotin analogue through a covalent bond based on a functional group;

preferably, the covalent bond based on a functional group refers to a covalent bond formed by the functional group participating in covalent coupling, wherein the functional group is carboxyl, hydroxyl, amino, sulfhydryl, salt form of carboxyl, salt form of amino, formate, or a combination of the foregoing functional groups.

11. The biomagnetic microsphere according to any one of claims 1 to 10, wherein: the linear backbone of the polymer is covalently coupled to the outer surface of the magnetic microsphere body either directly or indirectly through a linking group.

12. According to claims 1 to 11Any one of the biomagnetic microspheres is characterized in that: the magnetic microsphere body is SiO₂A wrapped magnetic material;

preferably, the magnetic material is an iron oxide, an iron compound, an iron alloy, a cobalt compound, a cobalt alloy, a nickel compound, a nickel alloy, a manganese oxide, a manganese alloy, or a combination thereof;

more preferably, the magnetic material is Fe₃O₄、γ-Fe₂O₃Iron nitride, Mn₃O₄、FeCrMo、FeAlC、AlNiCo、FeCrCo、ReCo、ReFe、PtCo、MnAlC、CuNiFe、AlMnAg、MnBi、FeNiMo、FeSi、FeAl、FeSiAl、BaO·6Fe₂O₃、SrO·6Fe₂O₃、PbO·6Fe₂O₃GdO, or a combination thereof.

13. The method for preparing the biomagnetic microspheres according to claim 1, wherein the biomagnetic microspheres comprise: the method comprises the following steps:

(1) chemically modifying the magnetic microsphere body by using an amino silane coupling agent, and introducing amino to the outer surface of the magnetic microsphere body to form an amino modified magnetic microsphere A;

the magnetic microsphere body is SiO₂A wrapped magnetic material;

(2) covalently coupling acrylic acid molecules to the outer surface of the magnetic microsphere A by utilizing covalent reaction between carboxyl and amino, and introducing carbon-carbon double bonds to form a carbon-carbon double bond-containing magnetic microsphere B;

(3) under the condition of not adding a cross-linking agent, polymerizing acrylic monomer molecules by utilizing the polymerization reaction of carbon-carbon double bonds to obtain an acrylic polymer, wherein the acrylic polymer has a linear main chain and a branched chain containing functional groups, and is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain; forming acrylic polymer modified magnetic microspheres C;

(4) and covalently coupling biotin or biotin analogues to the tail end of the polymer branched chain through functional groups contained in the polymer branched chain to obtain the biomagnetic microspheres.

14. A method for preparing biomagnetic microspheres according to any one of claims 2-5, wherein: the method comprises the following steps:

the magnetic microsphere body is SiO₂A wrapped magnetic material;

(4) covalently coupling biotin or biotin analogue to the tail end of the polymer branched chain through a functional group contained in the polymer branched chain to obtain biotin or biotin analogue modified biomagnetic microspheres D;

(5) connecting a purification medium with biotin or biotin analogues at the tail end of the polymer branched chain in the biomagnetic microspheres D to obtain the biomicrosphere microspheres combined with the purification medium;

preferably, a covalent linking complex of avidin or an avidin analogue and a purification medium is bound to the end of the polymer branch chain, and the binding of the biotin or the biotin analogue and the avidin or the avidin analogue to form an affinity complex is performed to obtain the biomagnetic microspheres with the purification medium;

independently and optionally, comprises (6) magnet sedimentation of the biomagnetic microspheres, liquid phase removal and washing;

independently optionally, comprising replacement of the covalently linked complex of the avidin or avidin analog with the purification medium.

15. The method for preparing the biomagnetic microspheres according to claim 7, wherein the biomagnetic microspheres comprise: the method comprises the following steps:

preferably, the preparation method of the biomagnetic microspheres comprises the following steps:

(4) covalently coupling biotin to the tail end of the polymer branched chain through a functional group contained in the polymer branched chain to obtain biotin-modified biomagnetic microspheres D;

(5) binding the avidin-avidin covalent connection compound E to the tail end of the branched chain of the polymer, and forming the binding effect of an affinity compound between biotin and avidin to obtain the biomagnetic microspheres bound with the avidin;

independently optionally, comprising (6) magnet sedimentation of the biomagnetic microspheres, removal of the liquid phase, washing;

independently optionally, a replacement of the avidin-avidin covalent linkage complex E is included.

16. Use of the biomagnetic microspheres according to any one of claims 1-12 for separation and purification of proteinaceous substances;

in a preferred mode, the biological magnetic microsphere is applied to separation and purification of antibody substances;

the application may optionally further comprise the reuse of biomagnetic microspheres when the purification media is attached to the branched ends of the polymer via a linking element comprising an affinity complex.

17. The use of the biomagnetic microspheres according to any one of claims 2-5 or 7 for separating and purifying antibody substances, wherein: the purification medium is affinity protein;

preferably, the affinity protein is linked to the polymer branch in the form of a biotin-avidin-affinity protein;

preferably, the antibody species includes antibodies, antibody fragments, antibody fusion proteins, antibody fragment fusion proteins;

when the affinity protein is linked to the end of the branch of the polymer via a linking element comprising an affinity complex, the use may optionally further comprise the reuse of the biomagnetic microspheres, i.e. comprise the reuse after the exchange of the affinity protein.

18. A biological magnetic microsphere comprises a magnetic microsphere body, and is characterized in that: the outer surface of the magnetic microsphere body is provided with at least one polymer with a linear main chain and a branched chain, one end of the linear main chain is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, the tail end of the branched chain of the polymer of the magnetic microsphere is connected with biotin, and the biotin is further connected with avidin through the binding effect of an affinity compound.

19. A biological magnetic microsphere comprises a magnetic microsphere body, and is characterized in that: the outer surface of the magnetic microsphere body is provided with at least one polymer with a linear main chain and a branched chain, one end of the linear main chain is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, the tail end of the branched chain of the polymer of the magnetic microsphere is connected with a purification medium, and the purification medium is selected from: avidin-type tags, polypeptide-type tags, protein-type tags, antibody-type tags, antigenic-type tags, and combinations thereof;

preferably, the avidin-type tag is avidin, an avidin analog that binds biotin, an avidin analog that binds a biotin analog, or a combination thereof;

more preferably, the avidin is streptavidin, modified streptavidin, a streptavidin analog, or a combination thereof;

in a preferred embodiment, the protein-based tag is selected from any one of the following tags or variants thereof: an affinity protein, SUMO tag, GST tag, MBP tag, or a combination thereof; more preferably one, the affinity protein is selected from the group consisting of protein a, protein G, protein L, modified protein a, modified protein G, modified protein L, and combinations thereof;

in a more preferable mode, the outer surface of the magnetic microsphere body is provided with at least one polymer with a linear main chain and branched chains, one end of the linear main chain is covalently fixed on the outer surface of the magnetic microsphere body, and the other end of the polymer is free from the outer surface of the magnetic microsphere body; the branched chain end of the polymer of the biomagnetic microsphere is connected with affinity protein; further preferably, there is binding of the affinity complex in a branched backbone between the affinity protein and the linear backbone of the polymer; more preferably, the affinity protein is selected from the group consisting of protein a, protein G, protein L, modified protein a, modified protein G, modified protein L, and combinations thereof.

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to a biomagnetic microsphere and a preparation method and application thereof.

Background

The separation and purification of proteins, including but not limited to antibodies, antibody fragments or fusion proteins thereof, is an important downstream link in the production process of biological drugs, and the effect and efficiency of separation and purification directly affect the quality and production cost of protein drugs. For protein purification, agarose gel and other materials are commonly used as purification columns or purification microsphere carriers at present. In the prior art, for the separation and purification of antibody substances (such as antibody molecules, antibody fragments or fusion proteins thereof), production technicians mainly separate and purify antibody molecules in fermentation broth or reaction solution through Protein A (Protein A for short) affinity adsorption columns. In the protein A column, the specific binding is carried out between the protein A fixed on the carrier and the specific site at the Fc end of the antibody molecule, thereby realizing the specific and high-efficiency separation of the antibody from the solution. At present, the carrier in the commonly used protein A column mainly adopts materials such as agarose gel and the like.

The three-dimensional porous structure of the gel-like material is beneficial to increasing the specific surface area of the material, so that the sites capable of being combined with a purification medium (such as immobilized protein A) are increased, and the specific binding capacity to a target protein (including an antibody) is increased. Although the three-dimensional porous structure of the carrier material can greatly increase the number of binding sites of proteins (including antibodies), the porous structure in the carrier can also increase the retention time of the proteins during protein elution, and discontinuous spaces or dead spaces in the carrier can also prevent the proteins from being eluted from the material, so that the retention ratio is increased. If the binding sites with the protein are only fixed on the outer surface of the carrier, although the protein product can be prevented from entering the interior of the material, the retention time and the retention ratio of the protein during elution are greatly reduced; however, if only the outer surface of the carrier is used, the specific surface area of the carrier is greatly reduced, and the number of binding sites of the protein is greatly reduced, thereby reducing the purification efficiency.

The polymer is a high molecular compound and can be formed by polymerizing monomer molecules. The monomer molecules with active sites are adopted for polymerization, the polymerization product can be rich in a large number of active sites, the number of the active sites is greatly increased, and corresponding binding sites can be formed or introduced through the active sites. The polymer has various types and structures, molecular chains are mutually crosslinked to form a net structure, the linear structure of a single linear molecular chain is adopted, the branched structure with a plurality of branched chains (such as structures of branched structures, dendritic structures, comb-shaped structures, hyperbranched structures and the like) is also adopted, and the polymers with different structural types have wide application in different fields.

In the prior art, purification columns for protein separation and purification mainly use covalent coupling to fix the purification medium. Taking protein A as a purification medium and taking a protein A column for purifying antibody substances as an example, the protein A column for separating and purifying antibodies, antibody fragments or fusion proteins thereof and the like mainly adopts a covalent coupling mode to fix the protein A, and the protein A is covalently coupled to a carrier through cysteine at the C terminal. Although the covalent coupling mode can ensure that the purification medium (such as protein A) is firmly fixed on the carrier, after the purification column (such as protein A column) is used for many times, the binding performance of the purification medium (such as protein A) is reduced, and the purification effect is reduced. Therefore, in order to guarantee higher purification efficiency and quality, operating personnel need in time with the whole changes of filler in the affinity chromatography column, and this process not only consumptive material quantity is big, consumes a large amount of manual works and time moreover, leads to the purification with high costs.

Disclosure of Invention

The invention provides a biomagnetic microsphere which can be used for separating and purifying target objects, particularly protein substances (including but not limited to antibody proteins), can be combined with the target objects in a high-flux manner, can effectively reduce the retention ratio of the target objects during elution, can conveniently replace a purification medium (such as affinity protein), has the characteristics of rapidness, high flux, reusability and renewable use, and can greatly reduce the purification cost of the target objects. For example, when affinity protein is used as a purification medium, the purification cost of antibody protein can be greatly reduced.

1. The invention provides a biomagnetic microsphere, which comprises a magnetic microsphere body, wherein the outer surface of the magnetic microsphere body is provided with at least one polymer with a linear main chain and a branched chain, one end of the linear main chain is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the tail end of the branched chain of the polymer of the biomagnetic microsphere is connected with biotin or biotin analogues. The biotin or biotin analogue can be used as a purification medium, and can also be used as a connecting element for further connecting other types of purification media.

The biomagnetic microspheres are also called biotin magnetic microspheres or biotin magnetic beads.

The term "immobilized" refers to the linear backbone being "immobilized" on the outer surface of the magnetic microsphere body by covalent bonding.

Further, the linear backbone is covalently immobilized to the outer surface of the magnetic microsphere body in a direct manner or in an indirect manner via a linker (linking element).

Further, the number of the polymer branches is plural; preferably at least 3.

In one preferred embodiment, the size of the magnetic microsphere body is selected from any one of the following particle size scales or a range between any two of the following particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 65 μm, 40 μm, 45 μm, 50 μm, 25 μm, 1 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm; the diameter dimensions are averages.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.1 to 10 μm.

In one preferred mode, the diameter of the magnetic microsphere body is selected from 0.2 to 6 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.4 to 5 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.5 to 3 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.2 to 1 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.5 to 1 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm.

In a preferred embodiment, the magnetic microsphere body has an average diameter of 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, or 1000nm, with a deviation of ± 20%, more preferably ± 10%.

In one preferred embodiment, the polymer main chain is a polyolefin main chain or an acrylic polymer main chain. The acrylic polymer is defined in the noun and term section. In one preferred form, the polyolefin backbone is simultaneously an acrylic polymer backbone (i.e., the linear backbone of the polymer is the polyolefin backbone and is provided by the backbone of the acrylic polymer).

Preferably, the monomer unit of the acrylic polymer is selected from one or a combination of acrylic monomer molecules such as acrylic acid, acrylate, methacrylic acid, methacrylate and the like. The acrylic polymer may be obtained by polymerization of one of the above monomers or by copolymerization of a corresponding combination of the above monomers.

In one preferred embodiment, the polymer branches are covalently bonded to biotin or biotin analogue via a covalent bond based on functional groups, which covalently bonds biotin or biotin analogue to the polymer branch ends. Can be obtained by covalent reaction of functional groups contained in branched chains of polymer molecules on the outer surface of the biomagnetic microsphere and biotin or biotin analogues. Among the preferred embodiments of the functional group is a specific binding site (defined in detail in the "noun and term" section of the detailed description).

The covalent bond based on the functional group refers to a covalent bond formed by the functional group participating in covalent coupling. Preferably, the functional group is carboxyl, hydroxyl, amino, mercapto, a salt form of carboxyl, a salt form of amino, a formate group, or a combination of the foregoing functional groups. One of the preferred forms of the salt of the carboxyl group is the sodium salt form such as COONa; the salt form of the amino group may be preferably an inorganic salt form or an organic salt form, including, but not limited to, hydrochloride, hydrofluoride, and the like. The combination of the functional groups refers to all branched chains of all polymer molecules on the outer surface of one magnetic microsphere, and allows the participation in the formation of covalent bonds based on different functional groups; in the case of biotin, all biotin molecules on the outer surface of one biotin magnetic microsphere may be covalently linked to different functional groups, but one biotin molecule can be linked to only one functional group.

In one preferred embodiment, the linear backbone of the polymer is covalently coupled to the outer surface of the magnetic microsphere body directly or indirectly via a linking group.

In one preferred embodiment, the magnetic microsphere body is SiO₂A wrapped magnetic material. Alternatively, SiO₂Can be a silane coupling agent with an active site.

In a preferred mode, the magnetic material is selected from one or a combination of iron oxide, iron compound, iron alloy, cobalt compound, cobalt alloy, nickel compound, nickel alloy, manganese oxide and manganese alloy;

further, Fe is preferable₃O₄、γ-Fe₂O₃Iron nitride, Mn₃O₄、FeCrMo、FeAlC、AlNiCo、FeCrCo、ReCo、ReFe、PtCo、MnAlC、CuNiFe、AlMnAg、MnBi、FeNiMo)、FeSi、FeAl、FeSiAl、MO·6Fe₂O₃One or a combination of GdO; wherein Re is a rare earth element; m is Ba, Sr, Pb, i.e., MO.6Fe₂O₃Is BaO.6Fe₂O₃、SrO·6Fe₂O₃Or PbO.6Fe₂O₃。

2. The second aspect of the present invention provides a biomagnetic microsphere, and on the basis of the biomagnetic microsphere provided by the first aspect of the present invention, the biotin or biotin analogue is further connected with a purification medium as a connecting element. Namely: the branched ends of the polymer are linked to a purification medium by a linking element, and the linking element comprises the biotin or biotin analogue.

The purification medium may contain, but is not limited to, an avidin-type tag, a polypeptide-type tag, a protein-type tag, an antibody-type tag, an antigenic-type tag, or a combination thereof.

In one preferred embodiment, the avidin-type tag is avidin, an avidin analog that binds biotin, an avidin analog that binds a biotin analog, or a combination thereof.

In one preferred mode, the end of a branched chain of the polymer of the biomagnetic microsphere is connected with biotin; the purification medium is avidin, and forms affinity complex binding action with the biotin.

The avidin may be, but is not limited to, streptavidin, modified streptavidin, streptavidin analogs, or combinations thereof.

In a preferred embodiment, the polypeptide-type tag is selected from any one of the following tags or variants thereof: a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag comprising a WRHPQFGG sequence, a tag comprising a variant sequence of WRHPQFGG, a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, or a combination thereof. The Streg tag contains WSHPQFEK and variants thereof.

In a preferred embodiment, the protein tag is selected from any one of the following tags or variant proteins thereof: affinity proteins, SUMO tags, GST tags, MBP tags and combinations thereof.

In one preferred embodiment, the affinity protein is selected from the group consisting of protein a, protein G, protein L, modified protein a, modified protein G, modified protein L, and combinations thereof.

In a preferred embodiment, the antibody-type tag is any one of an antibody, a fragment of an antibody, a single chain fragment, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any one, or a variant of any one.

In a preferred embodiment, the antibody type tag is an anti-protein antibody.

In a preferred embodiment, the antibody-type tag is an antibody against a fluorescent protein.

In a preferred embodiment, the antibody-type tag is a nanobody;

in one preferred embodiment, the antibody-type tag is a nanobody against a protein.

In a preferred embodiment, the antibody-type tag is a nanobody against a fluorescent protein.

In one preferred embodiment, the antibody type tag is a nanobody against green fluorescent protein or a mutant thereof.

In a preferred embodiment, the antibody-type tag is an Fc fragment.

The attachment means of the purification medium to the biotin or the biotin analogue include, but are not limited to: covalent bonds, non-covalent bonds (e.g., supramolecular interactions), linking elements, or combinations thereof.

In a preferred embodiment, the covalent bond is a dynamic covalent bond; more preferably, the dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In a preferred embodiment, the supramolecular interaction is: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, or combinations thereof.

In one preferred embodiment of the biomagnetic microspheres, the purification medium is linked to the branched ends of the polymer by a linking member comprising an affinity complex.

In one of the preferred modes, the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction.

In one preferred form, the biotin or biotin analogue is linked to avidin or avidin analogue by affinity complex action, and the purification medium is linked directly or indirectly to the avidin or avidin analogue.

In one of the preferred modes, the purification medium is covalently linked to biotin or a biotin analogue at the end of the branch of the polymer by an avidin-type tag-purification medium, via a linking element forming an affinity complex between the avidin-type tag and the biotin or biotin analogue; in a further preferred form, the purification medium is covalently linked to the complex by avidin-purification medium, the linking element of the affinity complex being formed with biotin or a biotin analogue at the end of the branch of the polymer.

3. The third aspect of the present invention provides a biomagnetic microsphere, and on the basis of the biomagnetic microsphere provided by the first aspect of the present invention, further, the biotin or the biotin analogue is used as a connecting element, and further, avidin or the avidin analogue is connected through an affinity complex binding effect.

The biological magnetic microspheres also become avidin magnetic microspheres or avidin magnetic beads.

The avidin or avidin analogue can be used as a purification medium, and can also be used as a connecting element to be further connected with other types of purification media. Wherein said biotin or biotin analogue and said avidin or avidin analogue form an affinity complex binding therebetween.

In a preferred embodiment, the biomagnetic microspheres provided by the first aspect of the present invention further comprise avidin bound to the biotin. Wherein, biotin and avidin form affinity complex binding action. Namely: the tail end of a branched chain of the polymer of the biomagnetic microsphere is connected with biotin; the purification medium is avidin, and forms affinity complex binding action with the biotin.

In a preferred embodiment, the avidin is any one of streptavidin, modified streptavidin, streptavidin analogs, or a combination thereof.

4. The fourth aspect of the present invention provides a biomagnetic microsphere, which is based on the biomagnetic microsphere provided by the third aspect of the present invention, and further comprises an affinity protein linked to the avidin or avidin analogue. At the moment, biotin or biotin analogue, avidin or avidin analogue are used as connecting elements, and affinity complex binding effect is formed between the biotin or biotin analogue and avidin analogue; the affinity protein serves both as a purification medium and as a linking element, preferably as a purification medium.

The biomagnetic microspheres also become affinity protein magnetic microspheres or affinity protein magnetic beads.

In a preferred embodiment, on the basis of the biomagnetic microspheres provided by the second aspect of the present invention, the purification medium is an avidin, and the biomagnetic microspheres further include avidin linked to the avidin, and biotin bound to the avidin, the biomasses being linked to the branches of the polymer; wherein the purification medium is connected to the polymer branch chains through a connecting element, and an affinity complex formed by biotin and avidin is included in the connecting element.

In a preferred embodiment, the affinity protein is one of protein a, protein G, protein L, or a modified protein thereof.

5. The fifth aspect of the present invention provides a method for preparing the biomagnetic microspheres (refer to fig. 3) provided by the first aspect of the present invention, comprising the following steps:

(1) chemically modifying the magnetic microsphere body, and introducing amino to the outer surface of the magnetic microsphere body to form an amino modified magnetic microsphere A; when the magnetic microsphere body is SiO₂In the case of the coated magnetic material, the coupling agent is preferably an aminosilicone coupling agent.

In one preferred embodiment, the magnetic microsphere body is chemically modified by a coupling agent.

When the magnetic microsphere body is SiO₂When the magnetic material is wrapped, the magnetic microsphere body can be chemically modified by using a silane coupling agent. The silane coupling agent is preferably an amino silane coupling agent.

(2) Covalently coupling acrylic acid molecules to the outer surface of the magnetic microsphere A by utilizing covalent reaction between carboxyl and amino, and introducing carbon-carbon double bonds to form a carbon-carbon double bond-containing magnetic microsphere B.

(3) Under the condition of not adding a cross-linking agent, polymerizing acrylic monomer molecules (such as sodium acrylate) by utilizing the polymerization reaction of carbon-carbon double bonds to obtain an acrylic polymer, wherein the obtained acrylic polymer has a linear main chain and a branched chain containing functional groups, and the polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain to form the acrylic polymer modified magnetic microsphere C.

The definition of the functional groups of the acrylic monomer molecules and the polymer branches is shown in the noun and term part.

Preferably, the functional group is carboxyl, hydroxyl, amino, mercapto, formate, ammonium salt, salt form of carboxyl, salt form of amino, formate group, or a combination of the foregoing functional groups; the "combination of functional groups" refers to the functional groups contained in all the branched chains of all the polymers on the outer surface of one magnetic microsphere, and the types of the functional groups can be one or more. The meaning of "combination of functional groups" as defined in the first aspect is identical.

Further preferably, the functional group is a specific binding site.

(4) Biotin or biotin analogue is covalently coupled to the end of the branched chain of the polymer through a functional group contained in the branched chain of the polymer, so that the biomagnetic microsphere (a biotin magnetic microsphere) combined with the biotin or biotin analogue is obtained.

6. The sixth aspect of the present invention provides a method for preparing the biomagnetic microspheres provided by the second aspect of the present invention, comprising the following steps:

(i) providing the biomagnetic microspheres of claim 1; the production can be carried out by the steps (1) to (4) of the fifth aspect.

(ii) And connecting the purified medium with biotin or biotin analogue at the tail end of the polymer branched chain of the biomagnetic microsphere to obtain the biomagnetic microsphere combined with the purified medium.

7. The seventh aspect of the present invention provides a method for preparing the biomagnetic microspheres provided by the second aspect of the present invention, comprising the following steps:

(i) providing the biomagnetic microspheres of claim 1; the production can be carried out by the steps (1) to (4) of the fifth aspect.

(ii) A covalent connection complex of avidin or an avidin analogue and a purification medium (such as an avidin-purification medium covalent connection complex) is used as a raw material for providing the purification medium, the covalent connection complex is bonded to the tail end of a branched chain of a polymer, and the biotin or the biotin analogue and the avidin or the avidin analogue form the binding action of the affinity complex, so that the biomagnetic microspheres with the purification medium are obtained.

Independently and optionally, the method comprises (6) settling the biomagnetic microspheres by using a magnet, removing the liquid phase and washing.

Independently optionally, comprising replacement of the purification medium, may be achieved by replacing the covalently linked complex of the avidin or avidin analogue and the purification medium.

8. The eighth aspect of the present invention provides a method for preparing the biomagnetic microspheres provided by the fourth aspect of the present invention (for example, fig. 3), comprising the following steps:

(1) chemically modifying the magnetic microsphere body, and introducing amino to the outer surface of the magnetic microsphere body to form an amino modified magnetic microsphere A; when it is at homeThe magnetic microsphere body is SiO₂In the case of the coated magnetic material, the coupling agent is preferably an aminosilicone coupling agent.

In one preferred embodiment, the magnetic microsphere body is chemically modified by a coupling agent.

When the magnetic microsphere body is SiO₂When the magnetic material is wrapped, the magnetic microsphere body can be chemically modified by using a silane coupling agent. In this case, the coupling agent is preferably an aminosilicone coupling agent.

One of the preferred embodiments of the functional group is a specific binding site.

Other preferred modes of the functional group are in accordance with the above first aspect.

(4) Biotin is covalently coupled through functional groups contained in the branched chains of the polymer to obtain biotin-modified biomagnetic microspheres D (a biotin magnetic microsphere).

(5) The avidin-avidin covalent linking compound E is combined to the tail end of the branched chain of the polymer through the specific binding action between biotin and avidin, and the binding action of the affinity compound is formed between biotin and avidin to obtain the biomagnetic microsphere F (the biomagnetic microsphere F is combined with avidin and is an avidin magnetic microsphere).

Independently optionally comprises (6) magnet sedimentation of the biomagnetic microspheres F, removal of the liquid phase and washing.

Also independently optionally comprising step (7) replacing the avidin-avidin covalent linking complex E.

9. The ninth aspect of the invention provides the application of the biomagnetic microspheres of the first to fourth aspects of the invention in separation and purification of protein substances.

In a preferred mode, the biological magnetic microsphere is applied to separation and purification of antibody substances.

The antibody substance refers to a protein substance containing antibodies and antibody fragments, including but not limited to antibodies, antibody fragments, antibody fusion proteins and antibody fragment fusion proteins.

The use of the purification media when attached to the branched ends of the polymer via a linking element comprising an affinity complex may optionally further comprise the reuse of the biomagnetic microspheres, i.e. comprise the reuse after replacement of the purification media.

10. The tenth aspect of the present invention provides the use of the biomagnetic microspheres of the first aspect to the fourth aspect of the present invention in separation and purification of antibody substances, particularly in separation and purification of antibodies, antibody fragments, antibody fusion proteins, and antibody fragment fusion proteins.

The purification medium is an affinity protein.

In one preferred form, the affinity protein is linked to the polymer branch in the form of a biotin-avidin-affinity protein.

When the affinity protein is linked to the end of the branched chain of the polymer via a linking member comprising an affinity complex (e.g., the biomagnetic microspheres of the fourth aspect), the application may optionally further comprise recycling the biomagnetic microspheres, i.e., the affinity protein may be reused after replacement.

11. The eleventh aspect of the invention provides a biomagnetic microsphere. The outer surface of the magnetic microsphere body is provided with at least one polymer with a linear main chain and a branched chain, one end of the linear main chain is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the tail end of the branched chain of the polymer of the magnetic microsphere is connected with a purification medium; the purification medium is selected from an avidin-type tag, a polypeptide-type tag, a protein-type tag, an antibody-type tag, an antigen-type tag, or a combination thereof.

In one preferred form, the avidin-type tag is avidin, an avidin analog that binds biotin, an avidin analog that binds a biotin analog, or a combination thereof.

Preferably, the avidin is streptavidin, modified streptavidin, a streptavidin analog, or a combination thereof.

In a preferred embodiment, the polypeptide-type tag is selected from any one of the following tags or variants thereof: a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag comprising a WRHPQFGG sequence, a tag comprising a variant sequence of WRHPQFGG, a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, and combinations thereof; the Streg tag contains WSHPQFEK or a variant thereof.

In a preferred embodiment, the protein-based tag is selected from any one of the following tags or variants thereof: affinity proteins, SUMO tags, GST tags, MBP tags and combinations thereof.

In a preferred mode, the outer surface of the magnetic microsphere body is provided with at least one polymer with a linear main chain and branched chains, one end of the linear main chain is covalently fixed on the outer surface of the magnetic microsphere body, and the other end of the polymer is free from the outer surface of the magnetic microsphere body; the branched chain end of the polymer of the magnetic microsphere is connected with affinity protein.

Preferably, further, the affinity protein has a binding effect of an affinity complex to a branched backbone between the linear backbone of the polymer.

More preferably one, the affinity protein is selected from the group consisting of protein a, protein G, protein L, modified protein a, modified protein G, modified protein L or a combination thereof.

The main advantages and positive effects of the invention include:

one of the cores of the present invention is the biomagnetic microsphere structure, on the outer surface of the magnetic microsphere body, polymer molecules with linear main chains are covalently immobilized, and these polymer molecules also have a large number of functionalized branched chains, and the functionalized branched chains are connected with purification media (including but not limited to biotin, avidin, affinity protein, polypeptide tags, protein tags, etc.). Through the structure, a large amount of purification media suspended at the side ends of the linear main chain of the polymer are provided on the outer surface of the biomagnetic microsphere, so that a high retention ratio caused by a traditional net structure is avoided, and a large amount of target (such as antibody) binding sites can be provided by overcoming the limitation of the specific surface area. Wherein the kind of the purification medium can be selected according to the kind of the substance to be purified. When the purification medium is attached to the polymer arms in the form of an affinity complex, with a strong non-covalent interaction; further, the branched backbone between the purification medium (e.g., affinity protein) and the linear backbone of the polymer may also allow for binding of the affinity complex, allowing for easy replacement of the purification medium (e.g., affinity protein). When the target substance to be separated and purified is an antibody-like protein, the purification medium is usually an affinity protein. The preparation of the magnetic microspheres and the explanation of the principle part are combined for understanding.

The core of the invention is also the construction process (preparation method) of the biomagnetic microsphere structure: through chemical modification of the outer surface, a plurality of binding sites are provided on the outer surface of a magnetic bead (the outer surface of a biomagnetic microsphere body), then polymer molecules are covalently connected to the single binding sites on the outer surface of the magnetic bead, the polymer molecules are covalently connected to the single binding sites on the outer surface of the magnetic bead through one end of a linear main chain, a large number of side branched chains are distributed along the linear main chain, and the side branched chains carry nascent binding sites, so that the amplification of the binding sites is multiple times, dozens of times, hundreds of times and even thousands of times, and then a specific purification medium is connected to the nascent binding sites of the polymer branched chains according to specific purification requirements, so as to realize the capture of corresponding specific target molecules (particularly biochemical molecules, including but not limited to antibody-type protein molecules. In addition, the single binding site of the biomagnetic microsphere can be covalently linked to only one linear polymer backbone, or can be covalently linked to two or more linear backbones, so that the chain accumulation is not caused, and the retention ratio is preferably increased.

Preferably, one binding site leads out only one linear backbone, which in this case provides a larger space for the linear backbone to move.

Preferably, one binding site leads out only two linear backbones, providing as much space for movement of the linear backbones as possible.

The main advantages and positive effects of the invention also include:

(1) according to the structural design, the surface of the magnetic microsphere is coated by the polymer carrying a large number of branched chains with a special structure, the limitation of specific surface area is overcome, a large number of purified medium binding sites are provided, the number of purified media which can be bound on the surface of the magnetic microsphere is multiplied by multiple times, more than ten times, more than one hundred times and even more than one thousand times, and then a high-flux binding target object is realized, and preferably the target object is a protein substance; so that the biomagnetic microspheres can efficiently capture target substances (such as target proteins, including but not limited to antibodies, antibody fragments, or fusion proteins thereof) onto the magnetic microspheres from a mixed system, and high-throughput binding, i.e. high-throughput separation, is realized.

(2) The flexibility of the polymer chain can be utilized, the polymer chain can flexibly swing in a reaction and purification mixed system, the activity space of a purification medium is enlarged, the capture rate and the combination amount of protein are increased, the rapid and sufficient combination of a target object is promoted, and the high efficiency and the high flux are realized.

(3) The structure design of the invention enables the biomagnetic microspheres to realize high-efficiency elution of purified target (such as target protein, including but not limited to antibody, antibody fragment, or fusion protein thereof) during elution, effectively reduces the retention time and retention ratio of the target, and realizes high efficiency and high yield. The purification medium can be connected to the tail end of the branched chain of the polymer, on one hand, the structure of the polymer can not form a net structure, so that the branched chain is not accumulated, discontinuous space and dead angle can be avoided, and high detention time and high detention proportion caused by the traditional net structure are avoided; on the other hand, the branched chains of the polymer further play a space separation role, so that the purification medium can be fully distributed in the mixed system and is far away from the surfaces of the magnetic microspheres and the internal skeleton of the polymer, the efficiency of capturing the target object is increased, the retention time and the retention proportion of the target object can be effectively reduced in the subsequent elution step, and the separation with high flux, high efficiency and high proportion is realized. The structural design of the invention can utilize the high flexibility of the linear main chain and has the advantage of high magnification of the number of the branched chains, thereby better realizing the combination of high speed and high flux and the separation of high efficiency and high proportion (high yield).

(4) The purification medium (such as affinity protein) of the biomagnetic microsphere can be connected to the tail end of a polymer branched chain on the outer surface of a magnetic bead in a non-covalent strong binding force manner in an affinity compound manner; when the purification medium (such as affinity protein) needs to be updated and replaced, the purification medium can be conveniently and quickly eluted from the microspheres and recombined with a new purification medium, and the purification performance of the magnetic microspheres can be quickly recovered, so that the biological magnetic microspheres can be repeatedly regenerated and used, and the separation and purification cost is reduced.

(5) The biological magnetic microsphere is convenient to operate and use. When the magnetic microspheres combined with the target object are separated from the system, the operation is convenient, the aggregation state and the position of the magnetic microspheres can be efficiently controlled by only using a small magnet, the rapid dispersion or rapid sedimentation of the magnetic microspheres in the solution is realized, the separation and purification of the target object (such as an antibody) become simple and rapid, large-scale experimental equipment such as a high-speed centrifuge and the like are not needed, and the separation and purification cost is greatly reduced.

(6) The biological magnetic microsphere provided by the invention has wide application, and the purification medium is selective. According to the specific type of the purification substrate, a corresponding purification medium can be flexibly carried in the magnetic microsphere system, so that the capture of specific target molecules (particularly protein substances) is realized. For example, the affinity protein can be selected for targeting, and is generally applied to separation and purification of antibody substances including but not limited to antibodies, antibody fragments, or fusion proteins thereof on a large scale.

Drawings

FIG. 1 is a schematic structural diagram of a biomagnetic microsphere provided by a first aspect of the present invention. Wherein the magnetic microsphere body is made of SiO₂Encapsulated Fe₃O₄For example, and using biotin as the purification medium. In the figure, the number of polymer molecules (4) is only used for the sake of simplicity and illustration, and does not mean that the number of polymer molecules on the outer surface of the magnetic microsphere is limited to 4, but can be controlled and adjusted according to the content of each raw material in the preparation process. Similarly, the number of branches pendant from the side ends of the linear backbone is also illustrative and is not intended to limit the number of side branches of the polymer molecules of the present invention.

FIG. 2 is a schematic structural diagram of a biomagnetic microsphere provided by a fourth aspect of the present invention. Protein a is shown as a purification medium, and is bound to the ends of the branches of the brush structure by the "biotin-avidin-protein a" approach. Wherein the biomagnetic microsphere body is made of SiO₂Encapsulated Fe₃O₄For example. In the figure, the number of polymer molecules and the number of branches at the side end of the main chain are only illustrative, and the number of side chains of the polymer molecule of the present invention is not limited.

FIG. 3 is a flow chart of a method for preparing biomagnetic microspheres according to a fourth aspect of the invention. Wherein, the preparation process from the amino modified magnetic microsphere A to the biomagnetic microsphere D corresponds to the preparation method of the biomagnetic microsphere provided by the fifth aspect.

FIG. 4 shows the results of the experiment before and after the biomagnetic microsphere D (a biotin magnetic bead) is combined with the protein A-eGFP-avidin. RFU value test results, incubate 1 time. Protein A-eGFP-avidin (SPA-eGFP-avidin) is prepared through an in-vitro protein synthesis system, supernatant (abbreviated as IVTT supernatant) after IVTT reaction is obtained, and the change of solution RFU values before and after combination with the biomagnetic microspheres D is compared. Of the two proteins, protein A-eGFP-avidin, avidin with the number 1 was Streptavidin, and avidin with the number 2 was Tamvavidin 2. "Total" corresponds to the RFU value of IVTT Supernatant before binding (before biomagnetic microsphere treatment), and "Supernatant" corresponds to the RFU value of IVTT Supernatant after binding (after biomagnetic microsphere treatment).

FIG. 5 shows the experimental results of preparing biomagnetic microspheres F (a protein A magnetic bead): RFU value test results; protein a-eGFP-avidin saturation binding. And repeatedly reacting the biomagnetic microspheres D with the solution (abbreviated as IVTT supernatant) obtained after the IVTT expressing the protein A-eGFP-avidin to obtain the biomagnetic microspheres F which are saturated and combined by the protein A-eGFP-avidin. Wherein, the avidin corresponding to 1 is Streptavidin, the avidin corresponding to 2 is Tamvavidin2, and the super corresponds to IVTT supernatant which is not treated by the biomagnetic microspheres; flow-through 1(Flow through liquid 1), Flow-through 2(Flow through liquid 2), and Flow-through 3(Flow through liquid 3) are respectively corresponding to the magnetic microspheres to be incubated with avidin-protein A continuously (capture binding) and eluted (unbind release) for three times, and IVTT supernatant with the same source and the same dosage is used each time to obtain Flow through liquids 1,2 and 3 in sequence.

FIG. 6 shows the result of the experiment for separating and purifying antibody with biomagnetic microsphere F, in which avidin is Streptavidin. And (3) incubating the biomagnetic microspheres F with an antibody IgG solution, eluting, capturing, separating and eluting the antibody IgG from the antibody solution, releasing the elution into an eluent, and performing a corresponding test result of denaturing polyacrylamide gel electrophoresis (SDS-PAGE) on the eluent containing the purified antibody. Wherein, lanes 1,2,3,4 correspond to the antibody elution bands with the bed volumes of 2. mu.l, 6. mu.l, 18. mu.l, and 54. mu.l of SPA-magnetic microspheres, respectively, and lane 5 is the antibody elution band with the bed volume of 7.5. mu.l of protein A agarose column from Biotechnology engineering (Shanghai) GmbH (hereinafter, abbreviated as "Biotechnology Co.") of positive control commercial origin. Wherein M corresponds to Marker molecular weight.

FIG. 7 shows the result of repeated use of the biomagnetic microspheres F, wherein Streptavidin is contained in the biomagnetic microspheres F. And (3) incubating the biomagnetic microspheres F with an antibody solution (binding the antibody), washing, eluting (releasing the antibody), repeating for three times, and performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis test on the eluate. Wherein, lanes 1,2,3,4 correspond to the antibody elution bands with the bed volumes of 2. mu.l, 6. mu.l, 18. mu.l, and 54. mu.l of SPA-magnetic microspheres, respectively, and lane 5 is the antibody elution band with the bed volume of 7.5. mu.l of protein A agarose column from Biotechnology engineering (Shanghai) GmbH (hereinafter, abbreviated as "Biotechnology Co.") of positive control commercial origin. Wherein M corresponds to Marker molecular weight.

FIG. 8 shows the experimental results of the regeneration of the biomagnetic microspheres: the fluorescence relative value test result; protein A-eGFP-Tamvavidin2 was used. And repeatedly reacting the biomagnetic microspheres D with the IVTT solution of the protein A-eGFP-avidin to obtain biomagnetic microspheres F (1) which are saturated and combined by the protein A-eGFP-avidin. And then eluting the protein A-eGFP-avidin from the biological magnetic microspheres D, and combining the biological magnetic microspheres D with a solution obtained after the IVTT reaction of the fresh protein A-eGFP-avidin for the second time to obtain the biological magnetic microspheres F (2) which are saturated and combined by the protein A-eGFP-avidin for the second time. And repeating the steps to obtain the biomagnetic microspheres F (3) which are saturated and combined by the protein A-eGFP-avidin for the third time. Wherein, the supernatant corresponds to IVTT supernatant which is not treated by the biomagnetic microspheres; and (3) continuously incubating the Flow-through liquid 1(Flow-through1), the Flow-through liquid 2(Flow-through2) and the Flow-through liquid 3(Flow-through3) with avidin-protein A for three times respectively corresponding to the magnetic microspheres, and sequentially obtaining the Flow-through liquids 1,2 and 3 by using the same IVTT supernatant (new suction) each time.

FIG. 9 shows the regeneration of biomagnetic microspheres and the experimental results of the separation and purification of antibodies after the regeneration; protein A-eGFP-Tamvavidin2 was used. And eluting the biomagnetic microspheres F saturated and combined by the protein A-eGFP-avidin by using a denaturing buffer solution, and performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test on an eluted solution containing the protein A-eGFP-avidin. Lanes 1,2, and 3 represent the bands of protein a-eGFP-avidin-containing eluates eluted from the biomagnetic beads F after the first saturation binding, the second saturation binding, and the third saturation binding, respectively. Lane 4 shows the bands of purified antibody protein eluted with the elution buffer after the third time of incubation of the regenerated biomagnetic microspheres F with commercially available newborn bovine serum. Wherein M corresponds to Marker molecular weight.

FIG. 10 shows the results of the protein G loading test using the biomagnetic beads G (a ProteinG magnetic bead ): RFU value test results. And (3) incubating the biological magnetic microspheres D and the supernatant of the IVTT of the protein G-eGFP-avidin for three times to obtain the biological magnetic microspheres G which are saturated and combined by the protein G-eGFP-avidin. Wherein Supernatant corresponds to IVTT Supernatant which is not treated by the biomagnetic microspheres; three incubations gave the corresponding flow-through solutions: FT 1(flow-through liquid 1), FT 2(flow-through liquid 2), and FT 3(flow-through liquid 3).

FIG. 11 shows the results of the experiment for separating and purifying antibody using biomagnetic beads G (ProteinG magnetic beads): and (3) after the biomagnetic microspheres G are incubated with an antibody IgG solution, eluting, capturing, separating and eluting the antibody IgG from the antibody solution, releasing the antibody IgG into an eluent, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test on the corresponding eluent containing the purified antibody. Wherein M corresponds to Marker molecular weight.

FIG. 12 shows the RFU value test result of binding of biomagnetic microsphere H (a magnetic bead with anti EGFP nano-antibody) to eGFP protein. And (3) incubating the biomagnetic microspheres H with IVTT supernatant of the eGFP protein, and combining the eGFP protein. Wherein, Total corresponds to IVTT supernatant which is not processed by the biomagnetic microspheres; flow-through corresponds to Flow-through incubated once.

FIG. 13. result of experiment for separating and purifying eGFP protein by using biomagnetic microsphere H (an anti EGFP magnetic bead): and (3) carrying out elution after the biological magnetic microspheres H and the eGFP solution are incubated, capturing, separating and eluting the eGFP from the stock solution, releasing the eGFP into the eluent, and correspondingly carrying out SDS-PAGE test on the eluent containing the purified eGFP. Wherein M corresponds to Marker molecular weight Marker.

Nucleotide and/or amino acid sequence listing

SEQ ID No. 1, nucleotide sequence of protein A, length 873 bases.

SEQ ID No. 2, nucleotide sequence of tamavidin2, 423 bases in length.

3, nucleotide sequence of mEGFP, 714 bases in length.

SEQ ID No. 4, nucleotide sequence of protein G antibody binding region, 585 bases in length.

SEQ ID No. 5, amino acid sequence of the nano antibody anti-eGFP, length 117 amino acids.

SEQ ID No. 6, the nucleotide sequence of mScarlet, 693 bases in length.

Detailed Description

The invention will be further elucidated with reference to the embodiments and examples, to which reference is made. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, without specific conditions being noted in the following examples, are preferably carried out according to, with reference to, the conditions as indicated in the specific embodiments described above, and may then be carried out according to conventional conditions, for example "Sambrook et al, molecular cloning: a Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), "A Laboratory Manual for cell-free protein Synthesis" Experimental Manual for expressed by Alexander S.Spirin and James R.Swartz.cell-free protein synthesis: methods and protocols [ M ] 2008 ", etc., or according to the conditions recommended by the manufacturer.

Unless otherwise indicated, percentages and parts referred to in this invention are percentages and parts by weight.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

The temperature units in this application are, unless otherwise specified, degrees Celsius (. degree. C.).

Nouns and terms

The following is an explanation or description of the meanings of the partially related terms or terms used in the present invention in order to facilitate a better understanding of the present invention. The corresponding explanations or illustrations apply to the present invention in its entirety, both as follows and as described above. In the present invention, when a cited document is referred to, the definitions of the related terms, nouns and phrases in the cited document are also incorporated, but in case of conflict with the definitions in the present invention, the definitions in the present invention shall control. In the event that a definition in a cited reference conflicts with a definition in the present disclosure, the cited components, materials, compositions, materials, systems, formulations, species, methods, devices, etc. are not to be construed as limiting.

Magnetic beads: the ferromagnetic or magnetizable microspheres, which can also be described as magnetic beads, have a fine particle size, preferably in the range from 0.1 μm to 1000. mu.m in diameter. Examples of magnetic beads of the present invention include, but are not limited to: magnetic microsphere A, magnetic microsphere B, magnetic microsphere C, biomagnetic microsphere D (a biotin magnetic bead), biomagnetic microsphere F (a protein A magnetic bead), biomagnetic microsphere G (a protein G magnetic bead), biomagnetic microsphere H (a magnetic bead with anti EGFP nano antibody), and biomagnetic microsphere K (antibody magnetic bead).

A magnetic microsphere body: magnetic beads with modified sites (magnetic microspheres with bindable sites). For example silica coated magnetic material particles, more specifically as aminated silica coated magnetic material particles.

Magnetic microspheres A: amino-modified magnetic microspheres.

Magnetic microspheres B: magnetic microspheres containing carbon-carbon double bonds.

Magnetic microspheres C: the acrylic polymer modifies the magnetic microsphere.

Biotin magnetic beads: the magnetic beads to which biotin or a biotin analogue is bound are capable of specifically binding to a substance having an avidin-type label. The advantages include that the target protein can be expressed integrally in a fusion protein mode after being marked by the avidin or the avidin protein mutant, and the application mode is simple and convenient. Also known as biotin magnetic microspheres. The biotin or biotin analogue can be used as a purification medium and also as a linker element.

And (3) biological magnetic microspheres D: a magnetic microsphere combined with biotin or biotin analogues, a biotin magnetic bead. The biotin can be used as a purification medium and also as a connecting element.

Avidin magnetic beads: magnetic beads to which avidin or an avidin analog is bound. Capable of specifically binding to a substance bearing a biotin-type label. Also known as avidin magnetic microspheres.

The biomagnetic microsphere F: a magnetic microsphere combined with protein A, a protein A magnetic bead. Can be formed by combining biotin-modified biomagnetic microspheres D and avidin-protein A covalent connection complexes E.

Affinity protein magnetic beads: the magnetic microsphere combined with the affinity protein can be used for separating and purifying antibody substances. Also known as affinity protein magnetic microspheres.

The biomagnetic microsphere G: a magnetic microsphere combined with protein G, a protein G magnetic bead. For example, the biotin-modified biomagnetic microspheres D can be combined with avidin-protein G covalent connection complexes.

The biomagnetic microsphere K: magnetic beads bound to antibody type tags. Can be used for separating and purifying the target substance capable of being specifically combined with the target substance. Also known as antibody magnetic microspheres or antibody magnetic beads.

Nano antibody magnetic beads: magnetic beads bound with nanobodies. Can be used for separating and purifying the target substance capable of being specifically combined with the target substance. Also known as nanobody magnetic microspheres.

And (3) biological magnetic microspheres H: a magnetic bead of nano antibody, a magnetic microsphere (an anti EGFP magnetic bead) combined with nano antibody anti EGFP. Can be combined by an avidin-anti EGFP covalent connecting complex.

Polymers, broadly included in the present invention are oligomers and polymers having at least three structural units or a molecular weight of at least 500Da (which molecular weight may be characterized in a suitable manner, such as number average molecular weight, weight average molecular weight, viscosity average molecular weight, etc.).

Polyolefin chain: refers to a polymer chain free of heteroatoms covalently linked only by carbon atoms. The invention mainly relates to a polyolefin main chain in a comb structure; such as the linear backbone of an acrylic polymer.

Acrylic polymer: refers to a homopolymer or copolymer having a structure of-C (COO-) -C-unit, the copolymerization form of said copolymer is not particularly limited, preferably capable of providing a linear main chain and a metered amount of pendant group COO-; the acrylic polymer is allowed to contain a hetero atom in the linear main chain. Wherein the carbon-carbon double bond may also allow the presence of other substituents, as long as the progress of the polymerization reaction is not impaired, e.g. methyl substituents (corresponding to-CH)₃C (COO-) -C-). Wherein COO-can be present in the form of-COOH, in the form of a salt (e.g., sodium salt), or in the form of a formate (preferably an alkyl formate, such as methyl formate-COOCH)₃Ethyl formate-COOCH₂CH₃(ii) a May also be hydroxyethyl formate-COOCH₂CH₂OH), and the like. Specific structural forms of the-C (COO-) -C-unit structure include, but are not limited to, -CH (COOH) -CH₂-、-CH(COONa)-CH₂-、-MeC(COOH)-CH₂-、-MeC(COONa)-CH₂-、-CH(COOCH₃)-CH₂-、-CH(COOCH₂CH₂OH)-CH₂-、-MeC(COOCH₃)-CH₂-、-MeC(COOCH₂CH₂OH)-CH₂-any one of the like or any combination thereof. Wherein Me is methyl. The linear main chain of one polymer molecule may have only one kind of the above-mentioned unit structure (corresponding to a homopolymer), or may have two or more kinds of unit structures (corresponding to a copolymer).

Acrylic monomer molecule: the monomer molecule which can be used for synthesizing the acrylic polymer has a basic structure of C (COO ═ C, and examples thereof include CH (cooh) ═ CH₂、CH(COONa)＝CH₂、CH₃C(COOH)＝CH₂、CH₃C(COONa)＝CH₂、CH(COOCH₃)＝CH₂、CH(COOCH₂CH₂OH)＝CH₂、CH₃C(COOCH₃)＝CH₂、CH₃C(COOCH₂CH₂OH)＝CH₂And the like.

Branched chain: chains of the present invention are attached to the branch point and have independent ends. In the present invention, the branched chain and the side chain have the same meaning and may be used interchangeably. In the present invention, the branched chain means a side chain or a side group bonded to the linear main chain of the polymer, and may be a short branched chain such as a carboxyl group, a hydroxyl group, an amino group, or a long branched chain containing a large number of atoms, without any particular requirement for the length or size of the branched chain. The structure of the branched chain is not particularly limited, and the branched chain may be linear or branched with a branched structure. The branches may also contain additional side chains or side groups. The number, length, size, degree of re-branching, and other structural features of the branched chains are preferably such that the net structure is not formed as much as possible, and the branched chains are not accumulated to increase the retention ratio, and in this case, the flexible swing of the linear backbone can be smoothly exerted.

Branched chain skeleton: the branched chain skeleton is formed by connecting skeleton atoms in sequence in a covalent bond or non-covalent bond mode, and is connected to the main chain of the polymer in sequence from the tail end of the branched chain. The functional group at the end of the polymer is linked to the main chain of the polymer via a branched backbone. The cross point of the branched skeleton and the main chain is the branching point of the leading-out branched chain. For example, branching between the purification media and the linear backbone of the polymerThe scaffold, taking affinity protein as purification medium as example, the affinity protein at the end of the polymer branch chain can be sequentially passed through avidin, biotin, and propylenediamine residue (-NH-CH)₂CH₂CH₂-NH-), carbonyl (residue after amidation of the carboxyl group) to the polyolefin backbone of the polymer.

The end of the branch includes the end of all branches. In the case of a linear main chain, in addition to being fixed to one end of the magnetic microsphere body, the other end of the linear main chain must be connected to a branch point, and thus, is also broadly included in the scope of the "branched end" of the present invention. Therefore, the polymer attached to the outer surface of the magnetic microsphere body of the present invention has at least 1 branch point.

Functional groups of polymer branches: the functional group has reactivity, or has reactivity after being activated, and can directly carry out covalent reaction with reactive groups of other raw materials, or carry out covalent reaction with reactive groups of other raw materials after being activated, so as to generate covalent bond connection. One of the preferred ways to function as a functional group for the polymer branches is a specific binding site.

Direct linkage refers to linkage in which interaction occurs directly without the aid of spacer atoms. Forms of such interactions include, but are not limited to: covalent means, non-covalent means, or a combination thereof.

Indirect attachment means by means of at least one attachment element, in which case at least one spacer atom is involved. The connecting elements include, but are not limited to: linker peptides, affinity complex linkages, and the like.

Immobilization, immobilized, immobilization, and the like "immobilization" means a covalent bonding means.

The "linkage"/"binding" means such as carrying, linking, binding, capturing, etc. is not particularly limited and includes, but is not limited to, covalent means, non-covalent means, etc.

Covalent mode: directly bonded by covalent bonds. The covalent means includes, but is not limited to, dynamic covalent means, which means direct bonding by dynamic covalent bond.

Covalent bond: the method comprises common covalent bonds such as amide bonds and ester bonds, and also comprises dynamic covalent bonds with reversible properties. The covalent bond comprises a dynamic covalent bond. A dynamic covalent bond is a chemical bond with reversible properties including, but not limited to, imine bonds, acylhydrazone bonds, disulfide bonds, or combinations thereof. The meaning of which is understood by those skilled in the chemical arts.

Non-covalent means: including but not limited to, coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions and other supramolecular interaction modes.

Supramolecular interaction: including but not limited to coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, and combinations thereof.

Linking elements, also referred to as linking groups, refer to elements used to link two or more non-adjacent groups, including at least one atom. The linking means between the linking member and the adjacent group is not particularly limited, and includes, but is not limited to, covalent means, non-covalent means, and the like. The internal connection of the linking member is not particularly limited, and includes, but is not limited to, covalent and non-covalent.

A covalent linking element: the spacer atoms from one end of the linker to the other are all covalently linked.

Specific binding site: in the present invention, the specific binding site refers to a group or a structural site having a binding function on a polymer branch chain, the group or the structural site having a specific recognition and binding function for a specific target, and the specific binding can be achieved by a binding action such as coordination, complexation, electrostatic force, van der waals force, hydrogen bond, covalent bond, or other interaction.

Covalent attachment of the complex: compounds that are linked directly or indirectly by covalent means are also referred to as covalent linkers.

Avidin-purification medium covalent linking complex: the compound formed by covalent connection has one end of avidin and the other end of purification medium, and the two are directly connected through covalent bonds or indirectly connected through covalent connecting elements.

Avidin-avidin covalent linkage complex E: avidin-purification medium covalent linkage complexes with affinity proteins as purification media; or avidin-avidin complex E; the compound formed by covalent connection has one end of avidin and the other end of avidin, and the two are directly connected through covalent bonds or indirectly connected through covalent connecting groups. Such covalent attachment means include, but are not limited to, covalent bonds, linking peptides, and the like. Such as: Streptavidin-Protein a complex, Streptavidin-Protein a fusion Protein, Streptavidin-enhanced green fluorescent Protein-Protein a fusion Protein (Protein a-eGFP-Streptavidin), Protein a-eGFP-tamvadin 2, Protein g-eGFP-avidin fusion Protein, Protein g-eGFP-tamvadin 2 fusion Protein, and the like.

Affinity complex: non-covalently linked complexes formed by two or more molecules through specific binding interactions, relying on extremely strong affinity, such as: the complex formed by the interaction of biotin (or a biotin analogue) with avidin (or an avidin-like substance). The manner of binding of biotin to an affinity complex of avidin is well known to those skilled in the art.

Purifying the substrate, also called target, material to be separated from the mixed system. The purification substrate in the present invention is not particularly limited, but preferably the purification substrate is a protein-based substance (in this case, also referred to as a target protein).

A purification medium capable of specifically binding to the purification substrate to capture the purification substrate, thereby separating the purification substrate from the mixed system. The purification medium attached to the end of the branch of the polymer of the invention is a functional element having the function of binding a purification substrate. When the purification medium is covalently linked to an adjacent group, it behaves as a functional group with the function of binding the purification substrate.

Affinity protein: specifically binds to a target protein and has a high affinity binding force, such as protein A, protein G, protein L, modified protein A, modified protein G, modified protein L, and the like.

Protein A: protein A, a 42kDa surface Protein, was originally found in the cell wall of Staphylococcus aureus. It is encoded by the spa gene, the regulation of which is controlled by the DNA topology, cellular osmolarity and a two-component system known as ArlS-ArlR. Due to their ability to bind immunoglobulins, have been used in research related to the field of biochemistry. Can be specifically combined with the Fc of the antibody, is mainly used for purifying the antibody and can be selected from any commercial products. The terms "Protein A", "SPA", and "Protein A" are used interchangeably herein.

Protein G: protein G, an immunoglobulin binding Protein, is expressed in group C and group G streptococci, similar to Protein a, but with different binding specificities. It is a 65kDa (G148 protein G) and 58kDa (C40 protein G) cell surface protein, which is primarily used for antibody purification by specific binding to antibodies or certain functional proteins, and can be selected from any commercially available product.

Protein L: protein L, is limited to those antibodies that specifically bind kappa (. kappa.) light chains. In humans and mice, most antibody molecules contain a kappa light chain and the remaining lambda light chain. Is mainly used for antibody purification and can be selected from any commercial product.

Biotin: the biotin can be combined with avidin, and has strong binding force and good specificity.

Avidin: avidin, which can bind biotin with strong binding force and good specificity, is used as Streptavidin (Streptavidin, abbreviated as SA), analogues thereof (Tamvavidin 2, abbreviated as Tam2), modified products thereof, mutants thereof, and the like.

Biotin analogues, meaning non-biotin molecules capable of forming a specific binding with avidin similar to "avidin-biotin", preferably one of them being a polypeptide or protein, such as those developed by IBAPolypeptides comprising the WSHPQFEK sequence used in the series (e.g.II、Etc.), and similar polypeptides containing the WNHPQFEK sequence. WNHPQFEK can be regarded as WSMutated sequences of HPQFEK.

Avidin analogs, which refer to non-avidin molecules capable of forming specific binding with biotin similar to "avidin-biotin", preferably one of which is a polypeptide or protein. The avidin analogs include, but are not limited to, derivatives of avidin, homologous species of avidin (homologues), variants of avidin, and the like. Such avidin analogs are, for example, Tamavidin1, Tamavidin2, etc. (ref.: FEBS Journal,2009,276, 1383-.

Biotin-type label: the biotin type label comprises the following units: biotin, an avidin analog capable of binding avidin analogs, and combinations thereof. The biotin-type tag is capable of specifically binding avidin, an avidin analog, or a combination thereof. Therefore, the method can be used for separating and purifying protein substances including but not limited to protein substances marked by avidin type labels.

Avidin-type tag: the avidin type tag comprises the following units: avidin, avidin analogs that bind biotin analogs, and combinations thereof. The avidin-type tag is capable of specifically binding biotin, a biotin analog, or a combination thereof. Therefore, the method can be used for separating and purifying protein substances including but not limited to protein substances labeled by biotin type labels.

Polypeptide type tag: the polypeptide-type tag of the present invention refers to a tag comprising a polypeptide tag or a derivative of a polypeptide tag. The polypeptide tag refers to a tag of a polypeptide structure consisting of amino acid units, wherein the amino acid can be a natural amino acid or an unnatural amino acid.

Protein type tag: the polypeptide-type tag of the present invention includes a tag comprising a protein tag or a derivative of a protein tag. The protein tag refers to a tag of a protein structure consisting of amino acid units, wherein the amino acid can be natural amino acid or unnatural amino acid.

Antibody type tag: the antibody-type tag of the present invention refers to a tag containing an antibody-type substance, which is capable of specifically binding to a corresponding target, such as an antigen. Examples of the antibody type tag also include an anti eGFP nanobody that can specifically bind to eGFP protein.

Antigenic tag: the antigenic tag of the present invention refers to a tag containing an antigenic substance, which is capable of specifically binding to an antibody substance.

A peptide is a compound in which two or more amino acids are linked by peptide bonds. In the present invention, the peptide and the peptide fragment have the same meaning and may be used interchangeably.

Polypeptide, peptide composed of 10-50 amino acids.

Protein, peptide composed of more than 50 amino acids. The fusion protein is also a protein.

Derivatives of polypeptides, derivatives of proteins: any polypeptide or protein to which the present invention relates, unless otherwise specified (e.g., specifying a particular sequence), is understood to also include derivatives thereof. The derivatives of the polypeptide and the derivatives of the protein at least comprise C-terminal tags, N-terminal tags, C-terminal tags and N-terminal tags. Wherein, C terminal refers to COOH terminal, N terminal refers to NH₂The meaning of which is understood by those skilled in the art. The label can be a polypeptide label or a protein label. Some examples of tags include, but are not limited to, histidine tags (typically containing at least 5 histidine residues; such as the 6 XHis, HHHHHHHHHH; such as the 8 XHis tag), Glu-Glu, c-myc epitopes (EQKLISEEDL),A Tag (DYKDDDDK), a protein C (EDQVDPRLIDGK), Tag-100(EETARFQPGYRS), a V5 epitope Tag (V5 epitope, GKPIPNPLLGLDST), VSV-G (YTDIEMNRLGK), Xpress (DLYDDDDK), hemagglutinin (YPYDVPDYA), beta-galactosidase (beta-galactosidase), thioredoxin (thioredoxin), histidine-site thioredoxin (His-batch thioredoxin), IgG knotBinding domain (IgG-binding domain), intein-chitin binding domain (intein-chitin binding domain), T7 gene 10(T7 gene 10), glutathione S-transferase (GST), Green Fluorescent Protein (GFP), Maltose Binding Protein (MBP), and the like.

Protein-based substances, in the present invention, broadly refer to substances containing polypeptides or protein fragments. For example, polypeptide derivatives, protein derivatives, glycoproteins, and the like are also included in the category of protein substances.

Antibody, antigen: the present invention relates to antibodies, antigens, and, unless otherwise specified, domains, subunits, fragments, single chains, single chain fragments, variants thereof are also understood to be encompassed. For example, reference to an "antibody" includes, unless otherwise specified, fragments thereof, heavy chains lacking light chains (e.g., nanobodies), Complementarity Determining Regions (CDRs), and the like. For example, reference to "antigen" includes, unless otherwise specified, epitopes (epitopes), epitope peptides.

The antibody substance, including but not limited to antibodies, fragments of antibodies, single chains of antibodies, single chain fragments, antibody fusion proteins, fusion proteins of antibody fragments, and the like, and derivatives and variants thereof, of the present invention may be any substance that can produce antibody-antigen specific binding.

The antigenic substances, as used herein, include, but are not limited to, antigens known to those skilled in the art and substances capable of performing an antigenic function and specifically binding to the antibody substances.

Anti-protein antibodies: refers to an antibody that specifically binds to a protein.

Nanobody against fluorescent protein: refers to a nanobody capable of specific binding to a fluorescent protein.

Homology (homology), unless otherwise specified, means at least 50% homology; preferably at least 60% homology, more preferably at least 70% homology, more preferably at least 75% homology, more preferably at least 80% homology, more preferably at least 85% homology, more preferably at least 90% homology; also such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology. The description object is exemplified by homologous sequences such as the omega sequences mentioned in the present description. Homology here refers to similarity in sequence, and may be equal in numerical similarity (identity).

Homologs, which refer to substances having homologous sequences, may also be referred to as homologues.

"variant," or "variant," refers to a substance that has a different structure (including, but not limited to, minor variations) but retains or substantially retains its original function or property. Such variants include, but are not limited to, nucleic acid variants, polypeptide variants, protein variants. Means for obtaining related variants include, but are not limited to, recombination, deletion or deletion, insertion, displacement, substitution, etc. of the building blocks. Such variants include, but are not limited to, modified products, genetically engineered products, fusion products, and the like. To obtain the gene modification product, the gene modification can be performed by, but not limited to, gene recombination (corresponding to the gene recombination product), gene deletion or deletion, insertion, frame shift, base substitution, and the like. Gene mutation products, also called gene mutants, belong to one type of gene modification products. One of the preferred modes of such variants is a homologue.

Modified product: including but not limited to chemically modified products, amino acid modifications, polypeptide modifications, protein modifications, and the like. The chemical modification product refers to a product modified by chemical synthesis methods such as organic chemistry, inorganic chemistry, polymer chemistry and the like. Examples of the modification method include ionization, salinization, desalinization, complexation, decomplexing, chelation, decomplexing, addition reaction, substitution reaction, elimination reaction, insertion reaction, oxidation reaction, reduction reaction, and post-translational modification, and specific examples thereof include oxidation, reduction, methylation, demethylation, amination, carboxylation, and vulcanization.

"mutant", mutant, as used herein, unless otherwise specified, refers to a mutant product that retains or substantially retains its original function or property, and the number of mutation sites is not particularly limited. Such mutants include, but are not limited to, gene mutants, polypeptide mutants, and protein mutants. Mutants are one type of variant. Means for obtaining relevant mutants include, but are not limited to, recombination, deletion or deletion of structural units, insertion, displacement, substitution, and the like. The structural unit of the gene is basic group, and the structural units of the polypeptide and the protein are amino acid. Types of gene mutations include, but are not limited to, gene deletions or deletions, insertions, frameshifts, base substitutions, and the like.

"modified" products, including but not limited to derivatives, modified products, genetically engineered products, fusion products, etc., of the present invention, can retain their original function or property, and can optimize, alter their function or property.

Eluent (target protein for example): eluting the target protein; after elution, the target protein is present in the eluent.

Washing solution (taking target protein as an example): eluting impurities such as impure protein and the like; after elution, the impure protein is carried away by the washing liquid.

The binding force is as follows: binding capacity, e.g., binding capacity of magnetic microspheres to a protein.

Affinity force: substrate solutions of different concentration gradients were used, substrate concentration when magnetic microspheres bound only 50% substrate.

IVTT: in vitro transcription and translation, an In vitro transcription and translation system, is a cell-free protein synthesis system. The cell-free protein synthesis system takes exogenous target mRNA or DNA as a protein synthesis template, and can realize the synthesis of target protein by artificially controlling and supplementing substrates required by protein synthesis, substances such as transcription and translation related protein factors and the like. The cell-free protein synthesis system of the present invention is not particularly limited, and may be any one or any combination of cell-free protein synthesis systems based on yeast cell extracts, escherichia coli cell extracts, mammalian cell extracts, plant cell extracts, insect cell extracts.

In the present invention, the term "translation-related enzymes" (TRENs) refers to an enzyme substance required in the synthesis process from a nucleic acid template to a protein product, and is not limited to an enzyme required in the translation process. Nucleic acid template: also referred to as genetic template, refers to a nucleic acid sequence that serves as a template for protein synthesis, including DNA templates, mRNA templates, and combinations thereof.

Flow-through liquid: and (3) the clear liquid collected after the magnetic beads are incubated with the system containing the target protein, wherein the clear liquid contains residual target protein which is not captured by the magnetic beads. For example, in example 2, a solution containing an avidin-avidin complex is added to an affinity column and passed through the column to form a solution, such as: flow-through1, flow-through2 and flow-through3, respectively representing the first, second and third passes of the solution.

RFU, Relative Fluorescence Unit value (Relative Fluorescence Unit).

eGFP: enhanced green fluorescence protein (enhanced green fluorescence protein). In the present invention, the eGFP broadly includes wild-type and variants thereof, including but not limited to wild-type and mutants thereof.

mEGFP: a206K mutant of eGFP.

"optionally" means that there may or may not be any selection criterion that can implement the technical solution of the present invention. In the present invention, the term "optional" means that the present invention can be implemented as long as it is applied to the technical means of the present invention.

In the present invention, preferred embodiments such as "preferred" (e.g., preferred, preferable, preferably, preferred, etc.), "preferred", "more preferred", "better", "most preferred", etc. do not limit the scope and protection of the invention in any sense, do not limit the scope and embodiments of the invention, and are provided as examples only.

In the description of the invention, references to "one of the preferred", "in a preferred embodiment", "some preferred", "preferably", "preferred", "more preferred", "further preferred", "most preferred", etc. preferred, and references to "one of the embodiments", "one of the modes", "an example", "a specific example", "an example", "by way of example", "for example", "such as", "such", etc. do not constitute any limitation in any sense to the scope of coverage and protection of the invention, and the particular features described in each mode are included in at least one embodiment of the invention. The particular features described in connection with the various modes can be combined in any suitable manner in any one or more of the particular embodiments of the invention. In the present invention, the technical features or technical aspects corresponding to the respective preferred embodiments may be combined in any suitable manner.

In the present invention, "any combination thereof" means "more than 1" in number, and means a group consisting of the following cases in an inclusive range: "optionally one of them, or optionally a group of at least two of them".

In the present invention, the description of "one or more", etc. "has the same meaning as" at least one "," a combination thereof "," or a combination thereof "," and a combination thereof "," or any combination thereof "," any combination thereof ", etc., and may be used interchangeably to mean" 1 "or" greater than 1 "in number.

In the present invention, "and/or" means "either one of them or any combination thereof, and also means at least one of them.

The prior art means described in the modes of "usually", "conventionally", "generally", "often", etc. are also referred to as the content of the present invention, and if not specifically stated, they may be regarded as one of the preferred modes of the partial technical features of the present invention, and it should be noted that they do not constitute any limitation to the scope of the invention and the protection scope.

All documents cited herein, and documents cited directly or indirectly by such documents, are hereby incorporated by reference into this application as if each were individually incorporated by reference.

It is to be understood that within the scope of the present invention, each of the above-described technical features of the present invention and each of the technical features described in detail below (including but not limited to the examples) may be combined with each other to constitute a new or preferred technical solution as long as it can be used for implementing the present invention. Not described in detail.

1. The invention provides a biomagnetic microsphere, which comprises a magnetic microsphere body, wherein the outer surface of the magnetic microsphere body is provided with at least one polymer with a linear main chain and a branched chain, one end of the linear main chain is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the tail end of the branched chain of the polymer of the magnetic microsphere is connected with biotin or biotin analogues.

The biomagnetic microspheres of the first aspect of the invention are also referred to as biotin magnetic microspheres or biotin magnetic beads.

A typical structure of the biomagnetic microspheres is shown in FIG. 1.

The biotin or biotin analogue can be used as a purification medium, and can also be used as a connecting element for further connecting other types of purification media.

Compared with the gel porous materials commonly used at present, such as agaroses, most of the commercially available microspheres adopt the agaroses. The porous material possesses a rich pore structure, thereby providing a large specific surface area and a high binding capacity for a purified substrate, but accordingly, when proteins are adsorbed or eluted, protein molecules are required to additionally enter or escape from complex pore channels inside the porous material, which takes more time and is easier to retain. In contrast, the binding site for capturing the target protein provided by the invention only utilizes the outer surface space of the biomagnetic microspheres, and can be directly released into eluent without passing through a complex reticular channel during adsorption and elution, so that the elution time is greatly reduced, the elution efficiency is improved, the retention ratio is reduced, and the purification yield is improved.

1.1. Magnetic microsphere body

In the present invention, the volume of the magnetic microsphere body can be any feasible particle size.

The smaller particle size is beneficial to realizing that the magnetic microspheres are suspended in a mixing system and are more fully contacted with protein products, and the capture efficiency and the binding rate of the protein products are improved. In some preferred modes, the diameter size of the magnetic microsphere body is any one of the following particle size scales (deviation may be ± 25%, ± 20%, ± 15%, ± 10%) or a range between any two particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 65 μm, 40 μm, 45 μm, 50 μm, 25 μm, 1 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm. Unless otherwise specified, the diameter size refers to an average size.

The volume of the magnetic microsphere body can be any feasible particle size.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.1-10 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.2-6 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.4-5 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.5-3 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.2-1 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.5-1 μm.

In some preferred embodiments, the average diameter of the magnetic microsphere body is about 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, 1000nm, and the submultiples may be ± 25%, ± 20%, ± 15%, and ± 10%.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm.

In some preferred modes, the diameter of the magnetic microsphere body is 1 μm, 10 μm, 100 μm, 200 μm, 500 μm, 800 μm, 1000 μm, and the deviation range can be ± 25%, 20%, 15%, 10%.

Different magnetic materials can provide different types of activation sites, can create differences in the manner in which the purification media are bound, and can also differ in the ability to disperse and settle with a magnet, and can also create selectivity for the type of substrate being purified.

The magnetic microsphere body and the magnetic microsphere comprising the magnetic microsphere body can be quickly positioned, guided and separated under the action of an external magnetic field, and can be endowed with various active functional groups such as hydroxyl, carboxyl, aldehyde group, amino and the like on the surface of the magnetic microsphere by surface modification or chemical polymerization and other methods.

In some preferred modes, the magnetic microsphere body is SiO₂A wrapped magnetic material. Wherein, SiO₂The wrapping layer may include a silane coupling agent with its own active site.

In some preferred forms, the magnetic material is selected from: iron compounds (e.g., iron oxides), iron alloys, cobalt compounds, cobalt alloys, nickel compounds, nickel alloys, manganese oxides, manganese alloys, zinc oxides, gadolinium oxides, chromium oxides, and combinations thereof.

In some preferred embodiments, the iron oxide is, for example, magnetite (Fe)₃O₄) Maghemite (gamma-Fe)₂O₃) Or a combination of the two oxides, preferably ferroferric oxide.

In some preferred forms, the magnetic material is selected from: fe₃O₄、γ-Fe₂O₃Iron nitride, Mn₃O₄、AlNi(Co)、FeCrMo、FeAlC、AlNiCo、FeCrCo、ReCo、ReFe、PtCo、MnAlC、CuNiFe、AlMnAg、MnBi、FeNi(Mo)、FeSi、FeAl、FeNi(Mo)、FeSiAl、BaO·6Fe₂O₃、SrO·6Fe₂O₃、PbO·6Fe₂O₃GdO, and combinations thereof. Wherein, the Re is a rare earth element, rhenium.

1.2. Polymer structures providing a high number of branch ends

The outer surface of the magnetic microsphere body is provided with at least one polymer with a linear main chain and a branched chain, one end of the linear main chain is fixed on the outer surface of the magnetic microsphere body, and the other end of the polymer is free from the outer surface of the magnetic microsphere body.

The term "immobilized" refers to being "immobilized" on the outer surface of the magnetic microsphere body by covalent bonding.

In some preferred embodiments, the polymer is covalently coupled to the outer surface of the magnetic microsphere body directly or indirectly through a linking element.

The polymer has a linear main chain, and at the moment, the polymer has the advantages of high flexibility of the linear main chain and high magnification of the number of branched chains, and can better realize the combination of high speed and high flux, and the separation of high efficiency and high proportion (high yield).

For the magnetic microsphere, one end of the polymer is covalently coupled to the outer surface of the magnetic microsphere body, the rest ends including all branched chains and all functional groups are dissolved in the solution and distributed in the outer space of the magnetic microsphere body, and the molecular chain can be fully stretched and swung, so that the molecular chain can be fully contacted with other molecules in the solution, and the capture of the target protein can be further enhanced. When the target protein is eluted from the magnetic microspheres, the target protein can directly get rid of the constraint of the magnetic microspheres and directly enter the eluent. Compared with the polymer physically wound on the outer surface of the magnetic microsphere body or integrally formed with the magnetic microsphere body, the polymer covalently fixed through one end of the linear main chain (in some preferred modes, a single linear main chain of the polymer is covalently fixed, and in other preferred modes, 2 or 3 linear main chains are covalently led out from the fixed end of the main chain) can effectively reduce the stacking of the molecular chain, strengthen the stretching and swinging of the molecular chain in the solution, strengthen the capture of the target protein, and reduce the retention ratio and the retention time of the target protein during elution.

1.2.1. The polymer main chain of the biomagnetic microsphere provided by the invention

In some preferred embodiments, the linear backbone is a polyolefin backbone or an acrylic polymer backbone.

In other preferred embodiments, the linear backbone of the polymer is an acrylic polymer backbone. The polyolefin main chain may be a linear main chain containing only carbon atoms, or may contain hetero atoms (hetero atoms are non-carbon atoms) in the linear main chain.

In some preferred embodiments, the backbone of the polymer is a polyolefin backbone. The monomer unit of the acrylic polymer is acrylic acid, acrylate, methacrylic acid, methacrylate and other acrylic monomer molecules or a combination thereof. The acrylic polymer may be obtained by polymerization of one of the above monomers or by copolymerization of an appropriate combination of the above monomers.

In some preferred embodiments, the linear backbone of the polymer is a polyolefin backbone. Specifically, for example, the polyolefin backbone is a backbone provided by a polymerization product of one of acrylic acid, acrylate, methacrylic acid, methacrylate, or a combination thereof (a backbone provided by a copolymerization product thereof), or a backbone of a copolymerization product formed by polymerization of the above monomers. The polymerization product of the above monomer combination is exemplified by acrylic acid-acrylic ester copolymer, and also methyl methacrylate-hydroxyethyl methacrylate copolymer (MMA-HEMA copolymer), acrylic acid-hydroxypropyl acrylate copolymer. The above-mentioned monomers participate in the polymerization to form a copolymerization product, such as maleic anhydride-acrylic acid copolymer, for example.

In some preferred embodiments, the linear backbone is a polyolefin backbone and is provided by the backbone of an acrylic polymer.

In some preferred embodiments, the linear backbone is an acrylic polymer backbone.

In other preferred embodiments, the backbone of the polymer is an acrylic polymer backbone. May be a main chain of the polyolefin (main chain is only carbon atoms) or may contain a hetero atom (hetero atom: non-carbon atom) in the main chain.

In other preferred embodiments, the polymer backbone is a block copolymer backbone comprising polyolefin blocks, for example, a polyethylene glycol-b-polyacrylic acid copolymer (within the scope of acrylic copolymers). It is preferable that the flexible swing of the linear main chain is smoothly exerted, that the accumulation of the branched chain is not caused, and that the residence time or/and the ratio is not increased.

In other preferred embodiments, the backbone of the polymer is a condensation-polymerized backbone. The condensation polymerization type main chain refers to a linear main chain which can be formed by condensation polymerization between monomer molecules or oligomers; the polycondensation main chain may be of a homo-type or a co-type. Such as polypeptide chains, polyamino acid chains, and the like. Specifically, for example, an epsilon-polylysine chain, an alpha-polylysine chain, gamma-polyglutamic acid, polyaspartic acid chain, etc., an aspartic acid/glutamic acid copolymer, etc.

The number of linear backbones to which one binding site of the outer surface of the magnetic microsphere body may be covalently coupled may be 1 or more.

In some preferred modes, only one linear main chain is led out from one binding site on the outer surface of the magnetic microsphere body, so that a larger movement space can be provided for the linear main chain.

In other preferred modes, only two linear main chains are led out from one binding site on the outer surface of the magnetic microsphere body, and a larger movement space is provided for the linear main chains as much as possible.

One end of a main chain of the polymer is covalently coupled to the outer surface of the magnetic bead (the outer surface of the biomagnetic microsphere), the rest ends including all branched chains and all functional groups are dissolved in the solution and distributed in the outer space of the magnetic bead, and the molecular chain can be fully stretched and swung, so that the molecular chain can be fully contacted with other molecules in the solution, and the capture of the target protein can be further enhanced. When the target protein is eluted from the magnetic beads, the target protein can directly get rid of the constraint of the magnetic beads and directly enter the eluent; compared with the polymer physically wound on the outer surface of the magnetic bead or integrally formed with the magnetic bead, the polymer covalently fixed through one end of the linear main chain (most preferably, a single linear main chain of the polymer is covalently fixed, and in addition, the fixed end of the main chain is covalently led out 2 or 3 linear main chains) provided by the method can effectively reduce the stacking of the molecular chain, strengthen the stretching and swinging of the molecular chain in the solution, enhance the capture of the target protein, and reduce the retention ratio and the retention time of the target protein during elution.

1.2.2. The polymer branched chain of the biomagnetic microsphere provided by the invention

The number of the branched chains is related to factors such as the size of the magnetic microsphere body, the type of the skeleton structure of the polymer, the chain density (particularly the branched chain density) of the polymer on the outer surface of the magnetic microsphere body and the like.

The number of polymer branches is plural, at least 3. The number of side branches is related to the size of the magnetic microsphere, the length of the polymer main chain, the linear density of the side branches along the polymer main chain, the chain density of the polymer on the outer surface of the magnetic microsphere and other factors. The amount of polymer branches can be controlled by controlling the feed ratio of the raw materials.

The branched polymer has at least 3 branches.

Each branch end is independently bound or unbound to the purification medium.

When the branch termini are bound to the purification medium, each branch terminus is independently bound directly to the purification medium or is indirectly joined to the purification medium through a linking element.

When the purification medium is bound to the end of the branched chain, the number of the purification medium may be 1 or more.

In some preferred embodiments, at least 3 purification media are bound to one molecule of the branched polymer.

1.3. Binding mode of biotin or biotin analogue

The manner in which the biotin or biotin analogue is attached to the branched ends of the polymer is not particularly limited.

The means by which the biotin or biotin analogue is attached to the end of the branch of the polymer include, but are not limited to: covalent bonds, supramolecular interactions, or combinations thereof.

In some preferred forms, the covalent bond is a dynamic covalent bond; more preferably, the dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In some preferred forms, the supramolecular interaction is selected from the group consisting of: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, and combinations thereof.

In some preferred embodiments, the branches of the polymer are covalently bound to biotin or a biotin analogue via a functional group-based covalent bond, whereby the biotin or biotin analogue is covalently bound to the ends of the polymer branches. Can be obtained by covalent reaction of functional groups contained in branched chains of polymer molecules on the outer surface of the biomagnetic microsphere and biotin or biotin analogues. Among the preferred embodiments of the functional group is a specific binding site (defined in detail in the "noun and term" section of the detailed description).

Purification media (purification element)

The purification medium is a functional element for specifically capturing the target from the mixed system, i.e. the purification medium and the target molecule to be separated and purified can be specifically combined. The captured target molecules can be eluted and released under proper conditions, so that the purposes of separation and purification are achieved.

When the protein substance is taken as a target object, the purification medium and the target protein or a purification label carried in the target protein can mutually form specific binding action. Therefore, substances that can be used for the target protein purification tag can be used as an alternative to the purification medium; peptides or proteins used as purification media may also be used as an alternative to purification tags in the protein of interest.

2.1. Type of purification Medium

The purification medium may contain, but is not limited to, an avidin-type tag, a polypeptide-type tag, a protein-type tag, an antibody-type tag, an antigenic-type tag, or a combination thereof.

In one preferred embodiment, the avidin-type tag is avidin, an avidin analog that binds biotin, an avidin analog that binds a biotin analog, or a combination thereof.

In some preferred forms, the purification medium is: avidin, an avidin analog that can bind biotin or an analog thereof, biotin, a biotin analog that can bind avidin or an analog thereof, an affinity protein, an antibody, an antigen, DNA, or a combination thereof.

In some preferred modes, the branched chain end of the polymer of the biomagnetic microsphere is connected with biotin; the purification medium is avidin, and forms affinity complex binding action with the biotin.

In some preferred embodiments, the avidin is any one of streptavidin, modified streptavidin, streptavidin analogs, or a combination thereof.

Such avidin analogs, e.g., tamavidin1, tamavidin2, and the like. Tamavidin1 and Tamavidin2 are proteins found by Yamamoto et al in 2009 to have the ability to bind biotin (Takakura Y et al Tamavidins: Novel avidin-like biotin-binding proteins from the tamogitateke mushroom [ J ]. FEBS Journal,2009,276,1383-1397), which have a strong affinity for biotin similar to streptavidin. The thermal stability of Tamavidin2 is superior to that of streptavidin, and its amino acid sequence may be retrieved from relevant database, such as UniProt B9A0T7, or optimized with codon conversion and optimizing program to obtain DNA sequence.

Such as a WSHPQFEK sequence or a variant sequence thereof, a WRHPQFGG sequence or a variant sequence thereof, and the like.

In some preferred forms, the purification medium is: a polypeptide tag, a protein tag, or a combination thereof.

In some preferred forms, the purification medium is an affinity protein.

Examples of such affinity proteins include, but are not limited to, protein a, protein G, protein L, modified protein a, modified protein G, modified protein L, and the like.

The definition of antibody, antigen, refers to the term moiety, which is understood to also include, but is not limited to, domains, subunits, fragments, heavy chains, light chains, single chain fragments (e.g., nanobodies, heavy chains lacking light chains, heavy chain variable regions, complementarity determining regions, etc.), epitopes (epitopes), epitope peptides, variants of any of the foregoing, and the like.

In some preferred forms, the polypeptide tag is selected from any one of the following tags or variants thereof: a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a tag comprising a WSHPQFEK sequence, a tag comprising a variant sequence of WSHPQFEK, a tag comprising a WRHPQFGG sequence, a tag comprising a variant sequence of WRHPQFGG, a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, or a combination thereof.

In some preferred embodiments, the protein tag is selected from any one of the following tags or variants thereof: an affinity protein, SUMO tag, GST tag, MBP tag, or a combination thereof; more preferably one, the affinity protein is selected from the group consisting of protein a, protein G, protein L, modified protein a, modified protein G, modified protein L or a combination thereof.

In some preferred embodiments, the antibody-type tag is any one of an antibody, a fragment of an antibody, a single chain fragment, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any one, or a variant of any one.

In some preferred embodiments, the antibody-type tag is an anti-protein antibody.

In some preferred embodiments, the antibody-type tag is an anti-fluorescent protein antibody.

In some preferred embodiments, the antibody-type tag is a nanobody.

In some preferred embodiments, the antibody-type tag is a nanobody against a protein.

In some preferred embodiments, the antibody-type tag is a nanobody against a fluorescent protein.

In some preferred modes, the antibody type tag is a nanobody against green fluorescent protein or a mutant thereof.

In some preferred embodiments, the antibody-type tag is an Fc fragment.

2.2. Loading mode of purification medium

The manner in which the purification medium is attached to the biotin or biotin analogue is not particularly limited.

The attachment means of the purification medium to the biotin or biotin analogue include, but are not limited to: covalent bonds, non-covalent bonds (e.g., supramolecular interactions), linking elements, or combinations thereof.

In some preferred forms of the biomagnetic microspheres, the purification medium is attached to the branched ends of the polymer via a linking element comprising an affinity complex.

In some preferred forms, the biotin or biotin analogue binds to avidin or avidin analogue by affinity complex action, and the purification medium is directly or indirectly attached to the avidin or avidin analogue.

In some preferred forms, the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction.

In some preferred embodiments, the affinity complex selection criteria are: the magnetic microsphere has good specificity and strong affinity, and also provides a site for chemical bonding, so that the affinity compound can be covalently connected to the tail end of a branched chain of a polymer, or can be covalently connected to the outer surface of the magnetic microsphere body after chemical modification, such as a binding site of the outer surface, the tail end of a main chain of a linear polymer and the tail end of a branched chain type polymer. Such as a combination of: biotin or its analogs and avidin or its analogs, antigens and antibodies, and the like.

When the loading mode comprises dynamic covalent bonds and supermolecular interactions (especially affinity complex interactions), a reversible loading mode is formed, and the purification medium can be unloaded from the tail end of the branched chain under certain conditions, so as to be updated or replaced.

And (4) updating the purified medium, wherein the purified medium is the same in type before and after updating corresponding to the regeneration of the magnetic microspheres.

The purified medium is replaced, and the types of the purified medium before and after replacement are different corresponding to the change of the magnetic microspheres.

In some preferred embodiments, the purification medium is avidin, and further comprises biotin bound to the avidin, wherein biotin serves as a linking element; wherein, biotin and avidin form affinity complex binding action.

In some preferred modes, the purification medium is an affinity protein, and further comprises avidin linked to the affinity protein, and biotin bound to the avidin; wherein, the biotin and the avidin form affinity complex binding action, and the affinity complex is used as a connecting element.

In some preferred modes, the branched chain end of the polymer of the biomagnetic microsphere is connected with biotin, avidin and a purification medium in sequence. More preferably, the purification medium is an antibody or antigen. The means of linkage between the avidin and the purification medium include, but are not limited to: covalent bonds, non-covalent bonds, linking elements, or combinations thereof.

In some preferred embodiments, the purification medium is attached to the biomagnetic microspheres at the end of a polymer branch via the following attachment elements: including, but not limited to, nucleic acids, oligonucleotides, peptide nucleic acids, aptamers, deoxyribonucleic acids, ribonucleic acids, leucine zippers, helix-turn-helix motifs, zinc finger motifs, biotin, avidin, streptavidin, anti-hapten antibodies, and the like, combinations thereof. Of course, the linking element may also be a double stranded nucleic acid construct, a duplex, a homo-or hetero-hybrid (a homo-or hetero-hybrid selected from DNA-DNA, DNA-RNA, DNA-PNA, RNA-RNA, RNA-PNA or PNA-PNA), or a combination thereof.

2.3. Mechanism of action of the purification Medium

The force of the purification medium to capture the target molecule in the reactive purification mixed system may be selected from: including but not limited to covalent bonds, supramolecular interactions, combinations thereof.

In some preferred embodiments, the target is bound to the end of the polymer branch of the biomagnetic microspheres by the following force: biotin-avidin binding, Streg tag-avidin binding, avidin-avidin binding, histidine tag-metal ion affinity, antibody-antigen binding, or a combination thereof. The Streg tag, which mainly includes but is not limited to peptide tags developed by IBA corporation that can form specific binding with avidin or its analogs, usually contains the WSHPQFEK sequence or its variant sequences.

2.4. Regeneration and reuse of purification media

When the purification medium is connected to the tail end of the polymer branched chain of the biomagnetic microsphere in a reversible mode such as an affinity complex, a dynamic covalent bond and the like, the purification medium can be eluted from the tail end of the polymer branched chain under a proper condition, and then a new purification medium is recombined.

Take the example of affinity complex interaction as the affinity complex interaction force between biotin and streptavidin.

The extremely strong affinity between biotin and streptavidin is the binding effect of a typical affinity complex, which is stronger than the action of a common non-covalent bond and weaker than the action of a covalent bond, so that the purification medium can be firmly bound at the tail end of a polymer branched chain on the outer surface of a magnetic bead, and the streptavidin can be eluted from the specific binding position of the biotin to realize synchronous separation of the purification medium when the purification medium needs to be replaced, and then an activation site which can be recombined with a new avidin-purification medium covalent binding complex (such as a purification medium with a streptavidin label) is released, so that the purification performance of the magnetic bead can be quickly recovered, and the separation and purification cost of a target substance (such as an antibody) is greatly reduced. The process of eluting the biomagnetic microspheres modified with the purification medium and removing the avidin-purification medium covalent linkage compound to recover the biomagnetic microspheres modified with biotin or biotin analogues is called as the regeneration of the biotin magnetic microspheres. The regenerated biotin magnetic microspheres have released biotin active sites and can be recombined with avidin-purification medium covalent linkage complexes to obtain purification medium modified biomagnetic microspheres (corresponding to the regeneration of the biomagnetic microspheres), so that fresh purification medium can be provided, and new target binding sites can be provided. Therefore, the biotin magnetic microspheres of the invention can be recycled, namely, the purified medium can be replaced for reuse.

2.4. Purification substrate (preferably proteinaceous material)

The purification substrate of the present invention refers to the magnetic microspheres of the present invention for capturing the separated substance, and is not particularly limited as long as the purification substrate can specifically bind to the purification medium of the magnetic microspheres of the present invention.

When the purification substrate is a protein substance, the purification substrate is also referred to as a target protein.

2.4.1. Purification tags in proteins of interest

The target protein may not carry a purification tag, and in this case, the target protein itself should be captured by the purification medium in the magnetic microspheres. For example, the term "target protein, purification medium" refers to a combination of "antibody, antigen", "antigen, antibody", "avidin or its analog, biotin or its analog", and the like.

In some preferred embodiments, the protein of interest carries a purification tag that is capable of specifically binding to the purification medium. One, two or more purification tags per target protein molecule; when two or more purification tags are contained, the kinds of the purification tags are one, two or more. It should be noted that, as long as the amino acid sequences of the tags are different, the tags are regarded as different types of tags.

The purification tag in the protein of interest may be selected from the group including, but not limited to: a histidine tag, avidin, an avidin analog, a Streg tag (a tag comprising a WSHPQFEK sequence or variant thereof), a tag comprising a WRHPQFGG sequence or variant thereof, a tag comprising a RKAAVSHW sequence or variant thereof, a FLAG tag or variant thereof, a C tag and variant thereof, a Spot tag and variant thereof, a GST tag and variant thereof, an MBP tag and variant thereof, a SUMO tag and variant thereof, a CBP tag and variant thereof, an HA tag and variant thereof, an Avi tag and variant thereof, an affinity protein, an antibody-based tag, an antigen-based tag, and combinations thereof. It may also be selected from the purification tags disclosed in US6103493B2, US10065996B2, US8735540B2, US20070275416a1, including but not limited to Streg tags and variants thereof.

The purification tag may be fused via the N-terminus or C-terminus.

The histidine tag typically contains at least 5 histidine residues, such as a 5 × His tag, a 6 × His tag, an 8 × His tag, and the like.

The octapeptide WRHPQFGG can specifically bind to core streptavidin (core streptavidin).

A Streg tag capable of forming a specific binding interaction with avidin or an analog thereof, said Streg tag comprising WSHPQFEK or a variant thereof. By way of example, WSHPQFEK- (XaaYaWaaZaa)_n-WSHPQFEK, wherein Xaa, Yaa, Waa, Zaa are each independently any amino acid, Xaa yaawazaa comprises at least one amino acid and (Xaa yaawazaa)_nAt least 4 amino acids, wherein n is selected from 1-15 (such as 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15); (XaaYaWaaZaa)_nSpecific examples of (G)₈,(G)₁₂,GAGA,(GAGA)₂,(GAGA)₃,(GGGS)₂、(GGGS)₃. Streg tags such as WSHPQFEK, WSHPQFEK- (GGGS)_n-WSHPQFEK、WSHPQFEK-GGGSGGGSGGSA-WSHPQFEK、SA-WSHPQFEK-(GGGS)₂GGSA-WSHPQFEK, WSHPQFEK-GSGGG-WSHPQFEK-GL-WSHPQFEK, GGSA-WNHPQFEK-GGGSGSGGSA-WSHPQFEK-GS, GGGS-WSHPQFEK-GGGSGGGSGGSA-WSHPQFEK, etc.

The sequence of the FLAG tag is DYKDDDDK. Examples of variant sequences of the FLAG tag are DYKDHD-G-DYKDHD-I-DYKDDDDK.

The sequence of the Spot tag is PDRVRAVSHWSS.

The C tag comprises an EPEA sequence.

The GST tag is a glutathione S-transferase tag.

The MBP tag refers to a maltose binding protein tag.

The SUMO tag is a known Small molecule ubiquitin-like modifier (Small ubiquitin-like modifier), and is one of important members of the polypeptide chain superfamily of ubiquitin (ubiquitin). In the primary structure, SUMO has only 18% homology with ubiquitin, however, the tertiary structure and its biological function are very similar.

The sequence of the CBP tag is KRRWKKNFIAVSAANRFKKISSSGAL.

The sequence of the HA tag is YPYDVPDYA.

The Avi tag, a known small tag consisting of 15 amino acid residues, is specifically recognized by biotin ligase BirA.

Antibody-based tags, including but not limited to the complete structure (complete antibody), domains, subunits, fragments, heavy chains, light chains, single chain fragments (e.g., nanobodies, heavy chains lacking light chains, heavy chain variable regions, complementarity determining regions, etc.) of antibodies, and the like.

Antigenic class tags include, but are not limited to, the complete structure of an antigen (complete antigen), domains, subunits, fragments, heavy chains, light chains, single chain fragments (e.g., epitopes, etc.), and the like.

In some preferred embodiments, the target protein is linked to a purification tag at the N-terminus or C-terminus, or to both termini.

Various purification tags described in this section can be candidates for purification media in the magnetic microspheres of the invention.

2.4.2. Type of protein of interest

The target protein can be a natural protein or an altered product thereof, and can also be an artificially synthesized sequence. The source of the native protein is not particularly limited, including but not limited to: eukaryotic cells, prokaryotic cells, pathogens; wherein eukaryotic cell sources include, but are not limited to: mammalian cells, plant cells, yeast cells, insect cells, nematode cells, and combinations thereof; the mammalian cell source can include, but is not limited to, murine (including rat, mouse, guinea pig, hamster, etc.), rabbit, monkey, human, pig, sheep, cow, dog, horse, etc. The pathogens include viruses, chlamydia, mycoplasma, etc. The viruses include HPV, HBV, TMV, coronavirus, rotavirus, etc.

The types of the target protein include, but are not limited to, polypeptides ("target protein" in the present invention broadly includes polypeptides), fluorescent proteins, enzymes and corresponding zymogens, antibodies, antigens, immunoglobulins, hormones, collagens, polyamino acids, vaccines, etc., partial domains of any of the foregoing, subunits or fragments of any of the foregoing, and variants of any of the foregoing. The "subunit or fragment of any one of the aforementioned proteins" includes a subunit or fragment of "a partial domain of any one of the aforementioned proteins". The "variant of any one of the aforementioned proteins" includes a variant of "a partial domain of any one of the aforementioned proteins, a subunit or fragment of any one of the aforementioned proteins". Such "variants of any of the foregoing proteins" include, but are not limited to, mutants of any of the foregoing proteins. In the present invention, the meanings of two or more "preceding" cases in succession in other positions are similarly explained.

The structure of the target protein can be a complete structure, and can also be selected from corresponding partial domains, subunits, fragments, dimers, multimers, fusion proteins, glycoproteins and the like. Examples of incomplete antibody structures are nanobodies (heavy chain antibody lacking light chain, V)_HH, retains the full antigen binding ability of the heavy chain antibody), the heavy chain variable region, the Complementarity Determining Region (CDR), and the like.

For example, the target protein synthesized by the in vitro protein synthesis system of the present invention can be selected from the group consisting of, but not limited to, any one of the following proteins, fusion proteins in any combination, and compositions in any combination: luciferase (e.g., firefly luciferase), Green Fluorescent Protein (GFP), enhanced green fluorescent protein (eGFP), Yellow Fluorescent Protein (YFP), aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, Catalase (Catalase, e.g., murine Catalase), actin, antibody, variable region of antibody (e.g., single chain variable region of antibody, scFV), single chain of antibody and fragment thereof (e.g., heavy chain of antibody, nanobody, light chain of antibody), alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin and precursors thereof, glucagon-like peptide (GLP-1), interferon (including but not limited to interferon alpha, e.g., interferon alpha A, interferon beta, interferon gamma, etc.), interleukin (e.g., interleukin-1 beta, interleukin 2, interleukin 12, etc.),(s), Lysozyme, serum albumin (including but not limited to human serum albumin, bovine serum albumin), transthyretin, tyrosinase, xylanase, beta-galactosidase (β -galactosidase, LacZ, such as e.g. e.coli β -galactosidase), etc., a partial domain of any of the foregoing, a subunit or fragment of any of the foregoing, or a variant of any of the foregoing (as defined above, including mutants, such as, for example, luciferase mutants, eGFP mutants, which may also be homologous). Examples of the aminoacyl tRNA synthetase include human lysine-tRNA synthetase (lysine-tRNA synthetase), human leucine-tRNA synthetase (leucine-tRNA synthetase), and the like. Examples of the glyceraldehyde-3-phosphate dehydrogenase include Arabidopsis glyceraldehyde-3-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Reference may also be made to patent document CN 109423496A. The composition in any combination may comprise any one of the proteins described above, and may also comprise a fusion protein in any combination of the proteins described above.

In some preferred embodiments, the protein synthesis capacity of the in vitro protein synthesis system is evaluated by using a target protein having fluorescent properties, such as one of GFP, eGFP, mSCarlet, etc., or an analogous substance thereof, or a mutant thereof.

The application fields of the target protein include but are not limited to the fields of biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nano engineering, cosmetics, food additives, nutritional agents, agriculture, feed, living goods, washing, environment, chemical dyeing, fluorescent labeling and the like.

2.4.3. Mixed system containing target protein

The magnetic microspheres of the invention can be used for separating target protein from a mixed system thereof. The target protein is not limited to one substance, and may be a combination of substances as long as the purpose of purification is to obtain such a composition, or the form of such a composition may satisfy the purification requirements.

The mixed system containing the target protein is not particularly limited as long as the purification medium of the magnetic microspheres of the present invention can specifically bind to the target protein; it is also generally desirable that the purification medium does not have specific or non-specific binding to other substances than the target protein in the mixed system.

In the embodiment of the invention, the mixed system containing the target protein can be a natural source, and can also be an artificially constructed or obtained mixed system.

For example, a specific protein can be isolated and purified from commercially available serum.

For example, the target protein can be isolated from the reacted system of the in vitro protein synthesis system.

One embodiment of the in vitro protein synthesis system further includes, but is not limited to, for example, the cell-free E.coli-based protein synthesis system described in WO2016005982A 1. Other citations of the present invention, including but not limited to in vitro cell-free protein synthesis systems based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, as described in direct and indirect citations thereof, are also incorporated herein as embodiments of the in vitro protein synthesis system of the present invention. For example, the in vitro Cell-Free protein synthesis system described in the "Lu, Y.Advances in Cell-Free biosynthestic technology. Current Developments in Biotechnology and Bioengineering,2019, Chapter 2, 23-45" section, including but not limited to the "2.1 Systems and Advantages" section, pages 27-28, can be used as an in vitro protein synthesis system for carrying out the present invention. For example, documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN 36568472, CN 110568422, CN109971775A, CN 0720723672, CN 2019198813, CN2019112066163, CN2018112862093(CN 109971775A), CN 20191819181518, CN2020100693833, CN2020101796894, CN109971775A, CN2020102693382 and cited documents, and the in vitro cell-free protein amplification system, DNA template, and the method of the present invention can be used as the in vitro protein amplification system and the method of the present invention.

The source cell of the cell extract of the in vitro protein synthesis system is not particularly limited as long as the target protein can be expressed in vitro. The exogenous proteins disclosed in the prior art and suitable for in vitro protein synthesis systems derived from prokaryotic cell extracts and eukaryotic cell extracts (yeast cell extracts can be preferred, and kluyveromyces lactis can be more preferred), or the endogenous proteins suitable for prokaryotic cell systems and eukaryotic cell systems (yeast cell systems can be preferred, and kluyveromyces lactis can be more preferred) synthesized in cells can be synthesized by using the in vitro protein synthesis system disclosed by the invention, or synthesized by using the in vitro protein synthesis system provided by the invention.

One of the preferred modes of the in vitro protein synthesis system is the IVTT system. The liquid after the IVTT reaction (referred to as IVTT reaction liquid) contains not only the expressed target protein but also residual reaction materials in the IVTT system, and in particular, various factors derived from cell extracts (such as ribosomes, tRNA, translation-related enzymes, initiation factors, elongation factors, termination factors, and the like). The IVTT reaction liquid can provide a target protein for being combined with magnetic beads on one hand, and can also provide a mixed system for testing the separation effect of the target protein on the other hand.

The biomagnetic microspheres of the third aspect of the invention are also referred to as avidin magnetic microspheres or avidin magnetic beads.

The avidin or avidin analogue can be used as a purification medium, and can also be used as a connecting element to be further connected with other types of purification media. Wherein, the biotin or the biotin analogue and the avidin or the avidin analogue form affinity complex binding effect.

In some preferred embodiments, the biomagnetic microspheres provided by the first aspect of the present invention further include avidin bound to the biotin. Wherein, biotin and avidin form affinity complex binding action. Namely: the tail end of a branched chain of the polymer of the magnetic microsphere is connected with biotin; the purification medium is avidin, and forms affinity complex binding action with the biotin.

In some preferred embodiments, the avidin is any one of streptavidin, modified streptavidin, a streptavidin analog, or a combination thereof.

The biomagnetic microspheres of the fourth aspect of the invention are also referred to as affinity protein magnetic microspheres or affinity protein magnetic beads.

A typical structure of the biomagnetic microspheres is shown in FIG. 2.

In some preferred modes, on the basis of the biomagnetic microspheres provided by the second aspect of the present invention, the purification medium is an avidin, and the biomagnetic microspheres further comprise avidin linked to the avidin, and biotin bound to the avidin; wherein the purification medium is linked to the ends of the polymer arms by a linking element comprising an affinity complex of biotin and avidin.

In some preferred embodiments, the affinity protein is one of protein a, protein G, protein L, or a modified protein thereof. The corresponding biomagnetic microspheres can be respectively called protein A magnetic microspheres or protein A magnetic beads, protein G magnetic microspheres or protein G magnetic beads, protein L magnetic microspheres or protein L magnetic beads, and the like.

The biomagnetic microsphere provided by the fourth aspect of the present invention, taking the connection mode of biotin-avidin as an example, not only can firmly bind avidin to the end of the polymer branched chain on the outer surface of the magnetic bead, but also can realize synchronous detachment of avidin by eluting avidin (such as streptavidin) from the specific binding position of biotin when the avidin needs to be replaced, and further release an activation site capable of re-binding a new avidin-avidin covalent linkage complex E (such as avidin with a streptavidin tag), thereby realizing rapid recovery of the purification performance of the magnetic bead, and greatly reducing the cost of antibody separation and purification. And (3) eluting the biological magnetic microspheres modified with the avidin, and removing the avidin-avidin covalent connection compound E, so as to obtain the biotin-modified biological magnetic microspheres again, which is called as the regeneration of the biotin magnetic microspheres. The regenerated biotin magnetic microspheres have released biotin active sites, can be recombined with avidin-avidin covalent linkage complexes E, obtain avidin-modified biomagnetic microspheres F again (corresponding to the regeneration of the biomagnetic microspheres F), can provide fresh avidin and provide new antibody binding sites. Therefore, the biotin magnetic microspheres can be recycled, namely, the avidin can be replaced for reuse.

5. The fifth aspect of the present invention provides a preparation method of the biomagnetic microspheres provided by the first aspect of the present invention.

5.1. Preparation and principle of the biomagnetic microspheres provided by the first aspect

The invention provides a biotin magnetic microsphere modified with biotin or biotin analogues in a first aspect.

Take the modification with biotin as an example.

The biomagnetic microspheres provided by the first aspect can be prepared by the following steps: providing SiO₂Coated magnetic beads (commercially available or self-made), SiO₂Activated modification of (3), covalent attachment of Polymer to SiO₂(the polymer is covalently attached to the SiO through one end of the linear backbone₂And a plurality of side branches distributed along the polymer backbone), biotin is covalently attached to the ends of the branches of the polymer. It should be noted that the above-mentioned links are not required to be completely isolated, and two or three links are allowed to be combined into one link, for example, activated silica-coated magnetic beads (commercially available or home-made) may be directly provided. SiO 2₂For example, the step (1) of the preparation method of the biomagnetic microspheres according to the fifth to eighth aspects of the invention. Covalent attachment of polymers to SiO₂For example, the steps (2) and (3) of the preparation method of the biomagnetic microspheres according to the fifth to eighth aspects of the invention. Biotin is covalently linked to the end of the branched chain of the polymer, for example, step (4) of the preparation method of the biomagnetic microspheres of the fifth to eighth aspects provided by the invention.

The biomagnetic microspheres can be prepared by the following steps: (1) providing or preparing a magnetic microsphere body, wherein the outer surface of the magnetic microsphere body is provided with a reactive group R₁(ii) a (2) At the reactive group R₁Is linked to a polymer having a linear main chain and a plurality of branches, one end of the linear main chain being linked to the reactive group R₁Covalent attachment; (3) biotin or a biotin analogue is attached to the end of the branch.

With SiO₂The coated magnetic material is taken as an example of a magnetic microsphere body, and the preparation process of the biological magnetic microsphere can be prepared through the following steps: (1) providing SiO₂Encapsulated magnetic microspheres (commercially available or self-made)) On SiO₂Activated modification of (2) to form a reactive group R₁(ii) a (2) At the reactive group R₁Carrying out a polymerization reaction (for example, using acrylic acid or sodium acrylate as monomer molecules) to form a polymer having a linear main chain and a plurality of branches, and having a functional group F at the end of the branch₁(ii) a (3) Functional group F for linking biotin or biotin analogue to the end of the branch₁To (3). In this case, the polymer covalently bonded to the magnetic microsphere body has a linear main chain, one end of which is covalently fixed to the reactive group R₁And a plurality of pendant side chains distributed along the polymer backbone.

5.2. Typical examples

A typical method for preparing the biomagnetic microspheres (refer to fig. 3) comprises the following steps:

step (1): providing a magnetic microsphere body, carrying out chemical modification on the magnetic microsphere body, and introducing amino to the outer surface of the magnetic microsphere body to form the amino modified magnetic microsphere A.

In some preferred modes, the magnetic microsphere body is chemically modified by using a coupling agent.

In some preferred embodiments, the coupling agent is an aminosilicone coupling agent.

In some preferred modes, the magnetic microsphere body is SiO₂The wrapped magnetic material is prepared by chemically modifying a magnetic microsphere body by using a silane coupling agent; the silane coupling agent is in some preferred forms an amino silane coupling agent.

Step (2): covalently coupling acrylic acid molecules to the outer surface of the magnetic microsphere A by utilizing covalent reaction between carboxyl and amino, and introducing carbon-carbon double bonds to form a carbon-carbon double bond-containing magnetic microsphere B.

And (3): polymerizing acrylic monomer molecules (such as sodium acrylate) by utilizing the polymerization reaction of carbon-carbon double bonds to obtain a branched-chain acrylic polymer which has a linear main chain and contains a functional group F₁The polymer is covalently coupled on the outer surface of the magnetic microsphere B through one end of the linear main chain to form the acrylic polymer modified magnetic microsphere C. This step can be carried out without addition of a crosslinking agent.

The definition of the functional groups of the acrylic monomer molecules and the polymer branches is shown in the noun and term part.

In some preferred modes, the functional group F₁Is carboxyl, hydroxyl, amino, sulfhydryl, formate, ammonium salt, salt form of carboxyl, salt form of amino, formate group, or combination of the aforementioned functional groups; the "combination of functional groups" refers to the functional groups contained in all the branched chains of all the polymers on the outer surface of one magnetic microsphere, and the types of the functional groups can be one or more. The meaning of "combination of functional groups" as defined in the first aspect is identical.

In other preferred embodiments, the functional group is a specific binding site.

And (4): functional group F contained by a branch of the polymer₁Biotin or a biotin analogue is covalently coupled to the end of the polymer branch chain to obtain a biomagnetic microsphere (a biotin magnetic microsphere) to which biotin or a biotin analogue is bound. In the prepared biological magnetic microsphere, a large number of sites capable of being combined with biotin are provided by acrylic polymers (with polyacrylic acid skeletons).

5.3. Detailed description of the preferred embodiments

One specific embodiment of the preparation of the biotin magnetic microspheres is as follows.

Specifically, taking an example in which an acrylic polymer provides a linear main chain and a large number of branches, the present invention provides one embodiment as follows: the ferroferric oxide magnetic beads coated by silicon dioxide are used as a body of the biological magnetic microsphere; firstly, chemically modifying a silicon dioxide-coated ferroferric oxide magnetic bead by using a coupling agent 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2, an aminated coupling agent, also a silane coupling agent, more specifically an aminated silane coupling agent), introducing amino to the outer surface of the magnetic bead to finish the SiO reaction₂Activating and modifying to obtain amino modified magnetic microspheres A; then the covalent reaction between carboxyl and amino is used to immobilize the molecule (propylene)Acid molecules, providing a carbon-carbon double bond and a reactive group carboxyl) to be covalently coupled to the outer surface of the magnetic bead, so that the carbon-carbon double bond is introduced to the outer surface of the magnetic bead to obtain a carbon-carbon double bond-containing magnetic microsphere B; then, polymerization reaction of carbon-carbon double bonds is utilized to carry out polymerization of acrylic monomer molecules (such as sodium acrylate), and the polymerization product is covalently coupled to the outer surface of the magnetic bead while the polymerization reaction is carried out, so that SiO is completed₂Connecting a polymer (covalent connection mode) to obtain the acrylic polymer modified magnetic microsphere C; the immobilized molecules are acrylic acid molecules, one immobilized molecule only leads out one polymer molecule, and simultaneously only leads out one polymer linear main chain; taking sodium acrylate as an example of a monomer molecule, the polymerization product is sodium polyacrylate, the main chain of the sodium polyacrylate is a linear polyolefin main chain, and a large number of side chain COONa are covalently connected along the main chain, and the functional group contained in the side chain is also COONa; in the polymerization reaction, a cross-linking agent such as N, N' -methylenebisacrylamide (CAS: 110-26-9) is not used, and molecular chains are prevented from being cross-linked with each other to form a network polymer, but a linear main chain is generated in the polymerization product under the condition of not adding the cross-linking agent. If the molecular chains are crosslinked into a network polymer, a porous structure is formed, and the elution efficiency of the target protein is influenced.

In some preferred modes, the amount of the acrylic acid used for preparing the magnetic microspheres B is 0.002-20 mol/L.

In some preferred modes, the amount of the sodium acrylate used for preparing the magnetic microspheres C is 0.53-12.76 mol/L.

The external surface of the biomagnetic microsphere can also adopt other activation modification modes besides amination. For example, the aminated biomagnetic microspheres (amino modified magnetic microspheres a) can further react with acid anhydride or other modified molecules, so as to implement chemical modification of the external surface carboxylation or other activation modes of the biomagnetic microspheres.

The immobilized molecules are small molecules which are used for covalently fixing the main chain of the polymer to the outer surface of the magnetic beads. The immobilized small molecule is not particularly limited as long as one end of the immobilized small molecule is covalently coupled to the outer surface of the magnetic bead, the other end of the immobilized small molecule can initiate polymerization reaction, including homopolymerization reaction, copolymerization reaction or polycondensation reaction, or the other end of the immobilized small molecule can be copolymerized with the end of the linear main chain of the coupled polymer.

The immobilized molecules allow for the extraction of only a single polymeric linear backbone, as well as two or more polymeric linear backbones, as long as they do not result in chain stacking and/or do not result in an increase in the retention ratio. Preferably, one immobilized molecule leads out only one polymer molecule and only one polymer linear backbone.

In some preferred embodiments, the immobilized molecule allows for the extraction of only a single polymeric linear backbone, as well as two or more polymeric linear backbones, as long as it does not result in chain stacking and/or does not result in an increase in the retention ratio. Preferably, one immobilized molecule leads out only one polymer molecule and only one polymer linear backbone.

In other preferred embodiments, the acrylic monomer molecule as a polymerized monomer unit may also be one of acrylic acid, acrylate, methacrylic acid, methacrylate type monomers or a combination thereof.

In another embodiment of the present invention, the acrylic polymer may be replaced with another polymer. The criteria chosen were: the formed polymer has a linear main chain, a large number of side branched chains are distributed along the main chain, and functional groups are carried on the side branched chains for subsequent chemical modification; namely, aiming at a binding site on the outer surface of the magnetic bead, a large number of functional groups are provided through branched chains distributed at the side end of a linear main chain of the polymer. Such as epsilon-polylysine chains, alpha-polylysine chains, gamma-polyglutamic acid, polyaspartic acid chains, aspartic acid/glutamic acid copolymers, and the like.

A method for introducing polymer molecules of other alternative ways of the above-mentioned polymers to the outer surface of the biomagnetic microspheres: according to the chemical structure of the polymer substitute and the type of the side chain active group thereof, selecting a proper activation modification mode of the external surface of the biomagnetic microsphere, the type of immobilized molecules and the type of monomer molecules, and carrying out proper chemical reaction to introduce a large amount of active groups positioned on the side chain into the external surface of the biomagnetic microsphere.

Covalently coupling acrylic polymer molecules (such as sodium polyacrylate linear molecular chains) to the outer surface of the magnetic beads, and then providing an activation site with a functional group at the tail end of a branched chain, or activating the branched chain functional group of the polymer molecules according to reaction requirements before connecting biotin or biotin analogue molecules to ensure that the polymer molecules have reaction activity and form the activation site; covalently coupling 1, 3-propanediamine to the activated sites of the polymer arms (each monomeric acrylic unit structure may provide one activated site) to form a new functional group (amino group), and then covalently coupling biotin or biotin analogue molecules to the new functional group at the end of the polymer arm by amidation covalent reaction between carboxyl and amino groups to complete covalent attachment of biotin or biotin analogue to the end of the polymer arm. Taking biotin as a purification medium as an example, obtaining biotin-modified biomagnetic microspheres D; a biotin molecule can provide a specific binding site. Taking the functional group of the polymer branch chain as COONa as an example, in this case, sodium acrylate is used as a monomer molecule, and before the covalent reaction with 1, 3-propane diamine, carboxyl activation can be performed first, and the existing carboxyl activation method can be used, for example: EDC. HCl and NHS were added.

5.3.1. Preparation of acrylic polymer modified magnetic microspheres

Preparing magnetic microspheres A: washing the magnetic microspheres with water solution of ferroferric oxide magnetic microspheres wrapped by silicon dioxide by using absolute ethyl alcohol, adding an ethanol solution of 3-aminopropyltriethoxysilane (APTES, coupling agent), reacting, washing, and introducing a large amount of amino groups on the outer surfaces of the magnetic microspheres.

Preparing magnetic microspheres B: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and N-hydroxysuccinimide (NHS) were added to an aqueous solution of acrylic acid to activate the carboxyl group, and then added to an aqueous solution containing magnetic microspheres A after activation. The activated carboxyl on the acrylic acid and the amino on the outer surface of the magnetic microsphere form covalent bond connection (amido bond), and a large amount of carbon-carbon double bonds are introduced into the outer surface of the magnetic microsphere.

Preparing magnetic microspheres C: and adding the aqueous solution of acrylic monomer molecules into the magnetic microspheres B, and adding an initiator to perform polymerization reaction of carbon-carbon double bonds. C-C double bonds in acrylic monomer molecules and C-C double bonds on the surfaces of the magnetic microspheres are subjected to open bond polymerization, and acrylic polymer molecules are covalently bonded to the outer surfaces of the magnetic microspheres, wherein the acrylic polymer contains carboxyl functional groups; the carboxyl functional group can exist in the form of carboxyl, formate, etc. In one of the preferred forms, sodium formate is present, in which case, for example, sodium acrylate or sodium methacrylate is used as monomer molecule. In another preferred embodiment, the monomer is present as a formate ester, in which case, for example, an acrylate or methacrylate ester is used as the monomer molecule. Formate and formate can obtain better reactivity after being activated by carboxyl.

5.3.2. Preparation of Biotin-modified biomagnetic microspheres D

Solution of magnetic microspheres C: adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC & HCl) and N-hydroxysuccinimide (NHS), activating carboxyl functional groups of side branches of polymer molecules on the outer surface of the microspheres, adding an aqueous solution of propylene diamine, performing a coupling reaction, grafting the propylene diamine at the positions of the side branches of the acrylic polymer molecules, and converting the functional groups of the side branches of the polymer into amino groups from the carboxyl groups.

Aqueous solution of biotin: adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to activate carboxyl in biotin molecules, then adding the activated carboxyl into an aqueous solution containing magnetic microspheres C, and covalently bonding biotin at the position of a nascent functional group (amino) of a side chain of a polymer on the outer surface of the magnetic microspheres C to obtain biomagnetic microspheres D with a large number of side chains of an acrylic polymer respectively connected with the biotin molecules.

5.3.3. Preferred embodiment(s) of the invention

In some preferred embodiments, the method for preparing the biomagnetic microspheres D comprises the following steps:

firstly, 0.5-1000 mL (20%, v/v) of aqueous solution of silicon dioxide-coated ferroferric oxide magnetic microspheres is measured, the magnetic microspheres are washed by absolute ethyl alcohol, 10-300 mL of ethanol solution (5% -50%, v/v) of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) is added into the washed magnetic microspheres, reaction is carried out for 2-72 hours, and the magnetic microspheres are washed by absolute ethyl alcohol and distilled water, so that amino modified magnetic microspheres A are obtained.

Removing 1.0X 10^-4About 1mol of acrylic acid is added into a solution X with the pH value of 4-6 (the solution X is an aqueous solution with the final concentration of 0.01-1 mol/L2-morpholine ethanesulfonic acid (CAS: 4432-31-9) and 0.1-2 mol/L NaCl), 0.001-0.5 mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl, CAS: 25952-53-8) and 0.001-0.5 mol of N-hydroxysuccinimide (NHS, CAS: 6066-82-6) are added, and the reaction is carried out for 3-60 min. And adding the solution into PBS buffer solution with the pH value of 7.2-7.5 mixed with 0.5-50 mL of magnetic microspheres A, reacting for 1-48 hours, and washing the magnetic microspheres with distilled water to obtain the carbon-carbon double bond modified magnetic microspheres B.

And (3) taking 0.5-50 mL of magnetic microsphere B, adding 0.5-200 mL of 5-30% (w/v) sodium acrylate solution, then adding 10-20 mL of 2-20% (w/v) ammonium persulfate solution and 1-1 mL of tetramethylethylenediamine, reacting for 3-60 minutes, and then washing the magnetic microsphere with distilled water to obtain the sodium polyacrylate modified magnetic microsphere C.

Transferring 0.5-50 mL of magnetic microsphere C into a solution X with the pH value of 4-6, adding 0.001-0.5 mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.001-0.5 mol of N-hydroxysuccinimide (NHS), and reacting for 3-60 min. Then adding PBS buffer solution with 0.0001-1 mol of 1, 3-propane diamine and pH7.2-7.5, and reacting for 1-48 hours. Washing with distilled water, adding PBS buffer solution, and converting COONa of a side branch chain of a polymer in the magnetic microsphere C into an amino functional group; weighing 1.0 × 10^-6～3.0×10^-4Adding biotin into the solution X in mol, adding 2.0X 10^-6～1.5×10^- ³mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2.0X 10^-6～1.5×10^-3And (3) mol of N-hydroxysuccinimide, and reacting for 3-60 min. Then adding the magnetic microspheres into the cleaned magnetic microsphere solution, reacting for 1-48 hours, and cleaning with distilled water to obtain the magnetic microsphere solutionBiotin-modified magnetic microspheres D.

5.4. The biomagnetic microspheres provided by the third aspect of the invention can be obtained by directly reacting the biomagnetic microspheres provided by the first aspect with avidin or avidin-like substances.

6. The sixth aspect of the present invention provides a method for preparing the biomagnetic microspheres provided by the second aspect of the present invention, comprising the following steps: (i) providing the biomagnetic microspheres of claim 1; the production can be carried out by the steps (1) to (4) of the fifth aspect. (ii) And (3) connecting a purification medium with biotin or biotin analogues at the tail ends of the polymer branched chains of the biomagnetic microspheres.

In some preferred modes, the biomagnetic microspheres are prepared by the following steps: the steps (1) to (4) are the same as in the fifth aspect; and (5) connecting a purification medium with biotin at the tail end of a polymer branched chain of the biomagnetic microsphere.

7. The seventh aspect of the present invention provides a method for preparing the biomagnetic microspheres provided by the second aspect of the present invention, comprising the following steps: (i) providing the biomagnetic microspheres of claim 1; the production can be carried out by the steps (1) to (4) of the fifth aspect. (ii) A covalent connection complex of avidin or an avidin analogue and a purification medium (such as an avidin-purification medium covalent connection complex) is used as a raw material for providing the purification medium, the covalent connection complex is bonded to the tail end of a branched chain of a polymer, and the biotin or the biotin analogue and the avidin or the avidin analogue form the binding action of the affinity complex, so that the biomagnetic microspheres with the purification medium are obtained.

Independently and optionally, comprises (6) magnet sedimentation of the biomagnetic microspheres, liquid phase removal and washing;

independently optionally, including replacement of the purification medium, may be achieved by eluting the covalently linked complex of avidin or avidin analogue to the purification medium under appropriate conditions.

In some preferred modes, the biomagnetic microspheres are prepared by the following five steps: the steps (1) to (4) are the same as in the fifth aspect; step (5) is the same as step (ii) described above.

8. The eighth aspect of the present invention provides a method for preparing the biomagnetic microspheres provided by the fourth aspect of the present invention (for example, fig. 3).

The fourth aspect of the invention provides an affinity protein magnetic microsphere.

The biomagnetic microspheres provided by the fourth aspect of the invention can be obtained by using the biomagnetic microspheres provided by the first aspect as a raw material and then combining covalent connection complexes of avidin or analogues thereof and avidin, wherein the avidin is loaded on the polymer branched chains of the biomagnetic microspheres through the affinity complex action between the biotin or analogues thereof and the avidin or analogues thereof.

In a preferred embodiment, the biomagnetic microspheres provided by the fourth aspect of the present invention can be obtained by using the biomagnetic microspheres provided by the first aspect as a raw material and then combining with avidin-avidin covalent linkage complex E.

Avidin-avidin covalent linkage complex E: also called as avidin-avidin complex E, a complex formed by covalent linkage, in which one end is avidin or its analog and the other end is avidin, and the two are directly linked by a covalent bond or indirectly linked by a covalent linking member. The covalent linking group includes a covalent bond, a linking peptide, and the like. The avidin-avidin complex E is exemplified by streptavidin-bearing avidin, wherein the avidin is selected from the group consisting of, but not limited to, protein a, protein G, and/or protein L, and the like. Examples of avidin-avidin complexes E also include: Streptavidin-Protein a complex, Streptavidin-Protein a fusion Protein, Streptavidin-enhanced green fluorescent Protein-Protein a fusion Protein (Protein a-eGFP-Streptavidin), Protein a-eGFP-Tamavidin2, Protein a-eGFP-Tamavidin1, and the like; wherein the eGFP broadly comprises an eGFP mutant, Streptavidin is Streptavidin, and Tamavidin1 and Tamavidin2 are both avidin analogues.

Avidin binds specifically to biotin to form an affinity complex. The binding effect of the affinity complex between avidin and biotin may be replaced with the binding effect of another affinity complex, and the effect of reusing the affinity protein may be similarly achieved. But more preferably the affinity complex provided by avidin and biotin; this is because the two proteins have high specificity and high affinity, biotin has an extra carboxyl group for bonding in addition to the binding domain of avidin, and avidin is also easily prepared as a fusion protein with avidin.

Affinity complex selection criteria: has good specificity and strong affinity, and also provides a site for chemical bonding, so that the covalent bonding can be covalently connected to the tail end of the polymer branch chain or can be covalently connected to the tail end of the polymer branch chain after chemical modification.

8.1. Preparation process

In some embodiments, the following step (5) is performed on the basis of the biomagnetic microspheres (a biotin magnetic microspheres) prepared in the fifth aspect to obtain a magnetic microsphere system with affinity protein as a purification medium.

And (5): a covalent linking complex that binds avidin or an avidin analog to an affinity protein (e.g., avidin-affinity protein covalent linking complex E). The covalent connection complex (such as avidin-avidin covalent connection complex E) is combined to the tail end of the branched chain of the polymer through the specific binding action between biotin or an analogue thereof and avidin or an analogue thereof, and the binding action of the affinity complex is formed between the biotin or the analogue thereof and the avidin or the analogue thereof, so that the avidin magnetic microsphere is obtained.

For example: an avidin-avidin covalent linking complex E (for example, streptavidin-bearing avidin, wherein the avidin is selected from the group consisting of but not limited to protein A, protein G and/or protein L, and the like) is added to the system of the biotin-modified biomagnetic microsphere D, the avidin is non-covalently linked to the polymer branch ends on the outer surface of the magnetic beads by utilizing the extremely strong specific affinity between biotin and avidin (such as streptavidin), thereby obtaining avidin-modified magnetic beads which can be used for separating and purifying antibody substances, and the avidin serves as a purification medium to provide a binding site for capturing a target protein.

8.2. Examples of the preparation procedure

The biomagnetic microspheres provided by the fourth aspect of the invention, taking the coupling to the ends of the polymer branched chains by the biotin-avidin manner as an example, can be prepared by the following steps:

In one preferred embodiment, the magnetic microsphere body is chemically modified by a coupling agent.

(3) Under the condition of not adding a cross-linking agent, polymerizing acrylic monomer molecules (such as sodium acrylate) by utilizing the polymerization reaction of carbon-carbon double bonds to obtain an acrylic polymer chain which has a linear main chain and a branched chain containing functional groups, wherein the polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain to form the acrylic polymer modified magnetic microsphere C.

One of the preferred embodiments of the functional group is a specific binding site.

Other preferred modes of the functional group are in accordance with the above first aspect.

(4) And covalently coupling biotin through functional groups contained in branched chains of the polymer to obtain the biotin-modified biomagnetic microsphere D.

(5) And (3) binding the avidin-avidin covalent connection compound E to the tail end of the branched chain of the polymer through the specific binding action between biotin and avidin, and forming the binding action of an affinity compound between the biotin and the avidin to obtain the avidin modified biomagnetic microspheres (avidin magnetic microspheres).

Optionally (6) magnetic sedimentation of affinity protein magnetic microspheres, removal of liquid phase, and washing.

Also independently optionally comprising step (7), replacing said avidin-avidin covalent linkage complex E, which may be achieved by eluting said avidin-avidin covalent linkage complex E under suitable conditions.

8.3. Biological magnetic microspheres: preparation of avidin-protein A-bound biomagnetic microspheres F

(affinity protein-bound biomagnetic microspheres F, protein A-modified magnetic microspheres)

The biotin-modified biomagnetic microspheres D are added to a fusion protein solution of an avidin-protein A linked complex E (e.g., ProteinA-eGFP-Streptavidin, ProteinA-eGFP-Tamvavidin2), and mixed for incubation. The protein A is fixed on the terminal group of the polymer branch chain on the outer surface of the biomagnetic microsphere D by the specific combination of avidin (such as Streptavidin or Tamvavidin2) and biotin, so as to obtain the biomagnetic microsphere F combined with avidin-protein A. In the structure of the obtained biomagnetic microsphere F, a side chain of an acrylic polymer contains an affinity complex structure of biotin-avidin-protein A, the side chain is covalently connected to a linear main chain branch point of the polymer through a biotin end, a non-covalent strong specific binding effect of the affinity complex is formed between the biotin and the avidin, the avidin and the protein A are covalently connected, a fluorescent label can be inserted between the avidin and the protein A, and other connecting peptides can be inserted.

Among them, avidin-protein A fusion proteins, such as ProteinA-eGFP-Streptavidin fusion protein and ProteinA-eGFP-Tamvavidin2 fusion protein, can be obtained by in vitro cell-free protein synthesis through IVTT reaction. At the moment, supernatant obtained after the reaction of the biomagnetic microspheres D and the IVTT is mixed, and the binding of the affinity protein A is realized through the specific binding action between the biotin on the outer surfaces of the biomagnetic microspheres D and the avidin fusion protein in the solution.

8.4. The binding capacity of the affinity protein on the outer surface of the biomagnetic microsphere can be determined by the following method (taking a fluorescent protein eGFP as an example):

first, after the binding reaction between the solution of the affinity protein and the magnetic beads is completed, the biomagnetic microspheres bound with the affinity protein are adsorbed and settled by a magnet. The liquid phase was then collected separately and noted as flow-through. At this time, the concentration of the affinity protein in the liquid phase decreases. The fluorescence intensity of the fluorescent protein eGFP bound on the biological magnetic microspheres is calculated by measuring the change value of the fluorescent protein eGFP in the supernatant obtained by IVTT reaction before and after binding the biological magnetic microspheres, and the concentration of the affinity protein is obtained by conversion. When the concentration of the affinity protein in the flow-through liquid is basically not changed compared with the concentration of the affinity protein in the IVTT solution before the biological magnetic microsphere is incubated, the adsorption of the biological magnetic microsphere to the affinity protein is saturated, and the fluorescence value of the corresponding fluorescent protein eGFP is not obviously changed. The pure product of the eGFP can be used for establishing a standard curve of the fluorescence value and the concentration of the eGFP so as to quantitatively calculate the content and the concentration of the avidin-avidin (such as streptavidin-protein A) bound on the biomagnetic microspheres.

The separation and purification of the antibody are carried out by adopting the protein A modified biological magnetic microsphere, and the antibody binding capacity (taking bovine serum antibody as an example) can be calculated by the following method: incubating the protein A modified magnetic beads with a bovine serum antibody solution (obtained by expressing the antibody by using an in vitro protein synthesis system or obtained by the market), eluting the bovine serum antibody from the magnetic beads by using an elution buffer solution after the reaction is finished, and allowing the separated bovine serum antibody to exist in an eluent. The concentration of bovine serum albumin in the eluate was determined by the Bradford method. Meanwhile, BSA is used as a standard protein to carry out enzyme-labeling instrument test, the standard protein is used as a reference, the protein concentration of the purified antibody can be calculated, and the yield of separation and purification are further calculated.

8.5. Regeneration of the biomagnetic microspheres: replacement of purification Medium (example of affinity protein not protein A)

Elution Replacing protein A: the synchronous separation of protein A is realized while the avidin is eluted, so that the avidin-protein A is replaced.

For example: adding a denaturation buffer solution (containing urea and sodium dodecyl sulfate) into the protein A modified biological magnetic microspheres F, incubating in a metal bath at 95 ℃, eluting avidin-protein A fusion proteins (such as SPA-eGFP-Tamvavidin2) combined with biotin on the biological magnetic microspheres D to obtain regenerated biological magnetic microspheres D (releasing binding sites of the biotin at the tail ends of polymer branches), adding a fresh avidin-protein A fusion protein-containing solution (such as supernatant obtained after IVTT reaction of SPA-eGFP-Tamvavidin2) into the regenerated biological magnetic microspheres D, allowing the released biological magnetic microspheres D to be recombined with new avidin-protein A (such as SPA-eGFP-Tamvavidin2) at the biotin binding sites, and forming noncovalent specific binding action between the biotin and the avidin (such as Tamvavidin2), thereby realizing the replacement of the protein A and obtaining the regenerated biological magnetic microsphere F.

9. Position control of magnetic microspheres

After the biomagnetic microspheres (including but not limited to the biomagnetic microspheres D and the biomagnetic microspheres F) according to the first to fourth aspects of the invention are prepared, the magnetic microspheres can be simply settled by using a magnet, the liquid phase is removed, and the adsorbed foreign proteins or/and other impurities are removed by washing.

By controlling the size of the magnetic microspheres and the chemical and structural parameters of the polymer, the magnetic microspheres can be stably suspended in a liquid phase and can not settle within two days or even longer. And can be stably suspended in a liquid system without continuous stirring. On one hand, the magnetic microsphere can be controlled to be in a nanometer size of several micrometers or even less than 1 micrometer, on the other hand, the grafting density of the polymer on the outer surface of the magnetic microsphere can be adjusted, and the characteristics of the hydrophilicity, the structure type, the hydrodynamic radius, the chain length, the number of branched chains, the length of the branched chains and the like of the polymer can be adjusted, so that the suspension performance of the magnetic microsphere system in the system can be better controlled, and the full contact between the magnetic microsphere system and an in vitro protein synthesis reaction mixed system can be realized. One preferred size of the magnetic microspheres is about 1 micron.

10. The ninth aspect of the invention provides the application of the biomagnetic microspheres of the first to fourth aspects of the invention in separation and purification of protein substances.

In a preferred mode, the biological magnetic microsphere is applied to separation and purification of antibody substances.

The definition of antibody class refers to the nomenclature section.

The invention particularly provides application of the biomagnetic microspheres in separation and purification of antibodies, antibody fragments, antibody fusion proteins and antibody fragment fusion proteins.

11. The tenth aspect of the present invention provides an application of the biomagnetic microspheres of the second aspect or the fourth aspect of the present invention in separation and purification of antibody substances, particularly an application in separation and purification of antibodies, antibody fragments, antibody fusion proteins, and antibody fragment fusion proteins.

The purification medium is an affinity protein.

Preferably, the affinity protein is linked to the polymer branches in the manner of a biotin-avidin-affinity protein.

In the applied biomagnetic microspheres, the binding effect of an affinity compound exists in a branched chain skeleton between the affinity protein and a polymer linear main chain, the affinity protein can be optionally replaced and then reused, and the biomagnetic microspheres can be recycled. Preferably, the biotin-avidin affinity complex binding effect exists in the branched chain skeleton between the avidin and the linear polymer main chain, that is, the avidin is connected to the polymer branched chain in the biotin-avidin manner, and at this time, the biological magnetic microspheres can be reused by eluting and replacing the avidin-avidin.

12. The eleventh aspect of the invention provides a biomagnetic microsphere. The magnetic microsphere comprises a magnetic microsphere body and is characterized in that the outer surface of the magnetic microsphere body is provided with at least one polymer with a linear main chain and a branched chain, one end of the linear main chain is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, the tail end of the branched chain of the polymer of the magnetic microsphere is connected with a purification medium, and the purification medium is selected from an avidin type label, a polypeptide type label, a protein type label, an antibody type label, an antigen type label or a combination.

In one preferred form, the avidin-type tag is avidin, an avidin analog that binds biotin, an avidin analog that binds a biotin analog, or a combination thereof.

Preferably, the avidin is streptavidin, modified streptavidin, a streptavidin analog, or a combination thereof.

In a preferred embodiment, the polypeptide-type tag is selected from any one of the following tags or variants thereof: a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag comprising a WRHPQFGG sequence, a tag comprising a variant sequence of WRHPQFGG, a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, or a combination thereof; the Streg tag contains WSHPQFEK and variants thereof.

In a preferred embodiment, the protein-based tag is selected from any one of the following tags or variants thereof: affinity proteins, SUMO tags, GST tags, MBP tags and combinations thereof.

Preferably, further, the affinity protein has a binding effect of an affinity complex to a branched backbone between the linear backbone of the polymer.

The protein factor system based on the in vitro cell-free protein synthesis method (D2P technology) was used in the following examples 2-6. The in vitro protein synthesis system (IVTT system) used in the in vitro cell-free protein synthesis methods of examples 2-6 below included the following components (final concentrations): 9.78mM Tris-HCl pH8.0, 80mM potassium acetate, 5mM magnesium acetate, 1.8mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a concentration of 1.8mM), 0.7mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, each at a concentration of 0.1mM), 15mM glucose, 320mM maltodextrin (molar concentration in glucose units, corresponding to about 52mg/mL), 24mM tripotassium phosphate, 2% (w/v) polyethylene glycol 8000, finally, 50% by volume of cell extract (in particular yeast cell extract, more in particular kluyveromyces lactis cell extract) is added.

Wherein the Kluyveromyces lactis extract comprises endogenously expressed T7 RNA polymerase. The Kluyveromyces lactis extract is modified in the following way: adopting a modified strain based on a Kluyveromyces lactis strain ATCC 8585; integrating a coding gene of T7 RNA polymerase into a genome of Kluyveromyces lactis by adopting the method described in CN109423496A to obtain a modified strain, so that the modified strain can endogenously express T7 RNA polymerase; culturing cell material with the modified strain, and preparing cell extract. The preparation process of the kluyveromyces lactis cell extract adopts conventional technical means, and refers to the method recorded in CN 109593656A. The preparation steps, in summary, include: providing appropriate amount of raw materials of Kluyveromyces lactis cells cultured by fermentation, quickly freezing the cells with liquid nitrogen, crushing the cells, centrifuging, and collecting supernatant to obtain cell extract. The protein concentration of the obtained kluyveromyces lactis cell extract is 20-40 mg/mL.

IVTT reaction: and adding a 15 ng/mu L DNA template (the coded protein contains a fluorescent label) into the in-vitro protein synthesis system to perform in-vitro protein synthesis reaction, uniformly mixing, and placing in an environment with the temperature of 25-30 ℃ for reaction for 6-18 h. Synthesizing the protein coded by the DNA template to obtain IVTT reaction liquid containing the protein. The RFU value is measured by adopting an ultraviolet absorption method, and the content of the protein can be calculated by combining a standard curve of the concentration and the RFU value.

EXAMPLE 1 preparation of biomagnetic microspheres D (conjugated Biotin)

Preparation of silica-coated magnetic microspheres (also known as magnetic microsphere bodies, magnetic beads, glass beads)

20g of Fe₃O₄The microspheres are put into a mixed solvent of 310mL of ethanol and 125mL of water, 45mL of 28 percent (wt) ammonia water is added, 22.5mL of tetraethoxysilane is added dropwise, the mixture is stirred and reacted for 24 hours at room temperature, and the mixture is washed by ethanol and water after the reaction. Ferroferric oxide microspheres with different particle sizes (about 1 micron, 10 microns and 100 microns) are used as raw materials, and the particle size of the obtained glass beads is controlled. The ferroferric oxide microspheres with different particle sizes can be prepared by a conventional technical means.

The magnetic microspheres produced are used as a base material for modifying purification media or connecting elements-purification media and are therefore also referred to as magnetic microsphere bodies.

The prepared magnetic microsphere has a magnetic core, can be subjected to position control under the action of magnetic force, and realizes operations such as movement, dispersion, sedimentation and the like, so that the magnetic microsphere is a generalized magnetic bead.

The prepared magnetic microsphere has a coating layer of silicon dioxide, so the magnetic microsphere is also called as glass bead, and can reduce the adsorption of the magnetic core on the following components or components: polymer, purification medium, components of in vitro protein synthesis system, nucleic acid template, protein expression product, etc.

Multiple experiments show that the magnetic microsphere has the best suspension property, suspension durability and protein combination efficiency when the particle size is about 1 mu m. The IVTT reaction liquid is used for providing a mixed system of target protein, and for the combination efficiency of the target protein, when the grain size of the magnetic microsphere is about 1 mu m, the grain size can be improved by more than 50% compared with 10 mu m, and can be improved by more than 80% compared with 100 mu m.

The magnetic microsphere coated with silicon dioxide is used for preparing biotin magnetic beads through the following steps.

Firstly, 50mL of aqueous solution of silicon dioxide-coated ferroferric oxide magnetic microspheres (the particle size of the magnetic microspheres is about 1 μm) with solid content of 20% (v/v) is measured, the magnetic microspheres are settled by a magnet, liquid phase is removed, 60mL of absolute ethyl alcohol is used for cleaning the magnetic microspheres each time, and the total cleaning is carried out for 5 times. 100mL of an excessive ethanol solution (25%, v/v) of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) was added to the washed magnetic microspheres, and the mixture was mechanically stirred in a water bath at 50 ℃ for 48 hours, then in a water bath at 70 ℃ for 2 hours, the magnetic microspheres were settled with a magnet, the liquid phase was removed, the magnetic microspheres were washed with 60mL of absolute ethanol each time, 2 times in total, then with 60mL of distilled water each time, and the washing was repeated 3 times to obtain magnetic microspheres A.

Secondly, 0.01mol of acrylic acid is transferred and added into 100mL of solution X (solution X: the aqueous solution of 2-morpholine ethanesulfonic acid (CAS: 4432-31-9) with the final concentration of 0.1mol/L and NaCl 0.5 mol/L), 0.04mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (CAS: 25952-53-8) and 0.04mol of N-hydroxysuccinimide (CAS: 6066-82-6) are added, stirred and mixed evenly at room temperature, stirred and reacted for 15min, NaHCO is used for reaction₃Adjusting the pH of the solution to 7.2 by using solid powder, adding the solution with the adjusted pH into 100mL of PBS buffer solution added with 10mL of magnetic microspheres A, mechanically stirring for 20 hours in a water bath at 30 ℃, settling the magnetic microspheres by using a magnet, removing a liquid phase, washing the magnetic microspheres by using 60mL of distilled water each time, and repeatedly washing for 6 times to obtain magnetic microspheres B.

Thirdly, taking 1mL of the magnetic microsphere B, adding 12mL of 15% (w/v) sodium acrylate solution, adding 450 muL of 10% ammonium persulfate solution and 45 muL of tetramethylethylenediamine, reacting for 30 minutes at room temperature, settling the magnetic microsphere by using a magnet, removing a liquid phase, washing the magnetic microsphere by using 10mL of distilled water each time, and washing for 6 times in total to obtain the magnetic microsphere C (the magnetic microsphere C modified by the acrylic polymer).

Fourthly, transferring the synthesized magnetic microspheres C into 10mL of solution X, adding 0.004mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.004mol of N-hydroxysuccinimide, stirring and uniformly mixing at room temperature, stirring for reacting for 15min, settling the magnetic microspheres by using a magnet, removing a liquid phase, and washing 3 times by using 10mL of distilled water each time; removing 4.0X 10^-4Dissolving mol 1, 3-propanediamine in 10mL PBS buffer solution, adding into the washed magnetic microspheres, mechanically stirring for 20 hours in a water bath at 30 ℃, settling the magnetic microspheres by using a magnet, removing a liquid phase, washing for 6 times by using 10mL distilled water each time, and adding into 10mL PBS buffer solution; weighing 2.5X 10^-4mol biotin, 10mL of solution X, 1.0X 10^-3mixing mol 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.001mol N-hydroxysuccinimide at room temperature, stirring for reaction for 15min, and reacting with NaHCO₃Adjusting the pH value of the solution to 7.2, adding the solution into the washed magnetic microspheres containing 10mL of PBS buffer solution, mechanically stirring the solution in a water bath at 30 ℃ for 20 hours, settling the magnetic microspheres by using a magnet, removing a liquid phase, and washing the magnetic microspheres 10 times by using 10mL of distilled water each time to obtain the biotin-modified biomagnetic microspheres D.

Example 2 preparation of avidin-protein A-binding biomagnetic microspheres F (an avidin-binding biomagnetic microspheres F) (protein A as purification medium, biotin-avidin affinity complex as linker)

2.1 Synthesis of ProteinA-eGFP-avidin fusion proteins

DNA sequences consisting of three segments of genes of fusion proteins ProteinA-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2 were constructed, respectively.

Wherein, the sequence of protein A comes from Staphylococcus Aureus, SPA for short. The amino acid sequence of SPA is 516 amino acid residues in total length, and amino acids 37-327 are selected as gene sequences used for constructing fusion protein, namely antibody binding domain of SPA. The nucleotide sequence is obtained after the sequence is optimized by an optimization program, and the nucleotide sequence is shown in SEQ ID No. 1.

Tamvavidin2, an avidin analog, is a protein with the ability to bind biotin. Yamamoto et al found in 2009 (2009_ FEBS _ Yamanoo T _ Tamavidins- -novel avidin-like biotin-binding proteins from the Tamogitake mushroom, a translation: a novel biotin-binding avidin-analog protein from Tamogitake mushroom) that it has a strong biotin affinity similar to streptavidin, and in addition, its thermal stability is superior to streptavidin.

The amino acid sequence of Tamavidin2 can be retrieved from a relational database, such as UniProt B9A0T7, which contains 141 amino acid residues in total, and is optimized by a codon conversion and optimization program to obtain a DNA sequence, wherein the optimized nucleotide sequence is shown as SEQ ID No. 2.

In addition, the nucleotide sequence of the enhanced fluorescent protein eGFP related to the embodiment is shown as SEQ ID No. 3, is an A206K mutant of the eGFP, and is also marked as mEGFP.

DNA templates of two fusion proteins, namely Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2, are respectively constructed by adopting a recombinant PCR method. Then, two fusion proteins, namely Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2, are synthesized respectively by an in vitro cell-free Protein synthesis method (by adopting a Protein factor system based on an in vitro cell-free Protein synthesis method (D2P technology)). For in vitro cell-free protein synthesis methods, patent documents CN 201610868691.6, WO2018161374a1, KR20190108180A, CN108535489B and the like are referred to. The IVTT system is used for expressing SPA-eGFP-Streptavidin and SPA-eGFP-Tamavidin2 respectively. In summary, the IVTT system is added with cell extract (containing RNA polymerase expressed by genome integration), DNA template, energy system (such as phosphocreatine-phosphocreatine kinase system), magnesium ion, sodium ion, polyethylene glycol and other components, and the reaction is carried out at 28-30 ℃. And finishing the reaction after 8-12 hours. The resulting IVTT reaction solution contains ProteinA-eGFP-avidin fusion protein corresponding to the DNA template.

Obtaining IVTT reaction liquid of Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2 respectively.

The IVTT system was used to express SPA-eGFP-Streptavidin (corresponding to "1" in FIG. 4) and SPA-eGFP-Tamavidin2 (corresponding to "2" in FIG. 4), respectively, and the RFU values of the synthesized proteins were determined, and the results are shown as "total" in FIG. 4.

The reaction solution of IVTT of ProteinA-eGFP-Streptavidin and ProteinA-eGFP-Tamavidin2 was centrifuged at 4000rpm and 4 ℃ for 10min, and the supernatant was retained. Record as IVTT supernatant.

2.2 preparation of protein A-binding biomagnetic microspheres F

The obtained IVTT supernatant of two fusion proteins, namely protein A-eGFP-Streptavidin and protein A-eGFP-Tamavidin2, is incubated with the biomagnetic microspheres D obtained in example 1 respectively, the reaction is carried out for 1 hour, the content of the fusion protein in the biomagnetic microspheres F is determined according to the method given above, the binding capacity of the two fusion proteins is compared, and the result is shown in the figure 4 'supernatatant'.

Aspirate 30. mu.L of a 10% (v/v) suspension of biomagnetic microspheres D and mix with binding/washing buffer (10mM Na)₂HPO₄pH 7.4，2mM KH₂PO₄140mM NaCl, 2.6mM KCl) for use after 3 washes.

And (3) taking 2mL of IVTT supernatant containing the avidin-protein A fusion protein and the biomagnetic microspheres D to perform rotary incubation for 1 hour at the temperature of 4 ℃, collecting unbound supernatant, namely flow-through liquid, and repeating the step three times to obtain three different flow-through liquids. Namely, the same batch of the biomagnetic microspheres D is incubated with the avidin-protein A for three times continuously. 2mL of the IVTT supernatant described in step 2.1 was used each time, and the serial numbers of the obtained flow-through solutions were numbered 1,2 and 3. The fluorescent values of eGFP of the supernatant of IVTT and the three flowthrough were measured by fluorometry, and the results are shown in fig. 5, where the excitation wavelength (Ex) was 488nm and the emission wavelength (Em) was 507 nm. The closer the RFU value of the triple flow-through and the IVTT supernatant, the more saturated the binding of biomagnetic microspheres D to avidin-avidin a, and the corresponding RFU values are shown in table 1 below:

TABLE 1 comparison of eGFP fluorescence values of IVTT supernatants not treated with biomagnetic microspheres D and treated triple flow-through

RFU values of SPA-eGFP-Streptavidin and SPA-eGFP-Tamvavidin2 bound to biomagnetic microspheres D are shown in tables 2 and 3 below, respectively. Wherein for Streptavidin, the first binding is already saturating the protein A binding capacity of biomagnetic microsphere D. For Tamvavidin2, the protein A binding capacity of biomagnetic microspheres D was substantially saturated after the second binding. The first binding capacity was calculated by subtracting the amount of protein in the supernatant from the amount of protein in the flow-through1, the second binding capacity was calculated by subtracting the amount of protein in the supernatant from the amount of protein in the flow-through2, and the third binding capacity was calculated by subtracting the amount of protein in the supernatant from the amount of protein in the flow-through3, as shown in tables 2 and 3 below. Wherein, the binding force refers to the protein A fusion protein/biomagnetic microspheres D, the mass-to-volume ratio, and the unit (mg/mL).

TABLE 2 SPA-eGFP-Streptavidin for flow-through physico-chemical comparison of biomagnetic microsphere D treatment

TABLE 3 physicochemical comparison of flow-through solutions treated with SPA-eGFP-Tamvavidin2 with biomagnetic microspheres D

The concentration of protein per binding was calculated from a standard curve of fluorescence values versus mass concentration of protein, and the total amount of protein per binding was calculated from the incubation volume (2 mL).

Drawing up a standard curve according to the purified eGFP, and converting the calculated RFU value of the eGFP into a formula of protein mass concentration, wherein the formula is as follows:

wherein X is the protein mass concentration (μ g/mL), Y is the RFU fluorescence reading, M is the molecular weight of eGFP (26.7kDa), and N is the molecular weight of SPA-eGFP-Streptavidin (77.3kDa) or the molecular weight of SPA-eGFP-Tamvavidin2 (79.4 kDa).

The measured RFU fluorescence value is substituted, and the mass concentration (mu g/mL) of the target protein SPA-eGFP-Streptavidin or SPA-eGFP-Tamvavidin2 can be converted.

Wherein, the RFU value of IVTT supernatant fluid and flow-through fluid of the avidin-protein A fusion protein is Y, the numerical value of Y is substituted into the formula, the obtained X is the mass concentration of the corresponding fusion protein, and the total protein content of the avidin-protein A fusion protein can be obtained by multiplying the mass concentration of the protein A solution by the volume of the protein A solution. And (3) subtracting the protein amount of the avidin-protein A in the flow-through liquid from the protein amount of the avidin-protein A in the IVTT supernatant to obtain a difference value, namely the protein amount W of the avidin-protein A combined by the biomagnetic microspheres. Dividing W by the bed volume of the magnetic microspheres, and calculating to obtain the mass of the avidin-protein A fusion protein combined by the magnetic microspheres in unit volume, namely the binding force in mg/mL.

In this example, 30. mu.L of 10% (v/v) suspension of the biomagnetic microspheres D was used, and the binding amounts of the biomagnetic microspheres D are shown in tables 2 and 3 as converted to 1mL of 100%. Wherein the binding force of the Tamvavidin2 and the biomagnetic microspheres D is slightly stronger than that of Streptavidin.

Example 3 protein A modified biomagnetic microspheres F for serum antibody purification

Different volumes of the biological magnetic microspheres combined with SPA-eGFP-Streptavidin fusion protein were incubated with 1mL of newborn bovine serum at 4 ℃ for 1 hour with rotation, and then washed 3 times with 1mL of binding/washing buffer, and incubated for 10 minutes at 4 ℃ for each wash. The antibody was eluted with 100. mu.L of 0.1M glycine pH2.8, the eluted antibody was present in the eluent, and one-tenth volume (10. mu.L) of 1M Tris-HCl pH8.0 was immediately added to the eluent. The control was a raw Protein A agarose column (cat # C600957-0005).

mu.L of the eluate was added to 10. mu.L of 5 XSDS loading buffer (containing no reducing agent), and subjected to metal bath at 95 ℃ for 10 minutes, followed by SDS-PAGE electrophoresis, and the results are shown in FIG. 6.

As shown in FIG. 6, the use of 6. mu.l of column bed volume of the biomagnetic microspheres combined with SPA-eGFP-Streptavidin fusion protein can achieve separation and purification effects superior to those of commercially available products.

Washing the eluted biomagnetic microspheres for 3 times by using 1mL of binding/washing buffer solution, and repeating the steps of incubation, washing and elution of the antibody; namely, after the first antibody incubation, washing and eluting the antibody to release the combined antibody, obtaining the protein A modified magnetic microsphere again, incubating, washing and eluting the protein A modified magnetic microsphere again with the antibody, obtaining the protein A modified magnetic microsphere again, and then incubating, washing and eluting the protein A modified magnetic microsphere for the third time. After incubating the biomagnetic microspheres with the antibody for 3 times in total, 40 μ L of the antibody-containing eluate obtained after the incubation with the antibody for the third time was taken, 10 μ L of 5 × loading buffer (containing no reducing agent) was added, and after metal bath at 95 ℃ for 10 minutes, SDS-PAGE electrophoresis was performed, and the detection results are shown in FIG. 7.

As can be seen from fig. 6 and 7, the biomagnetic microspheres F combined with SPA-eGFP-Streptavidin can be repeatedly used for multiple times, and after antibody incubation and antibody elution, the antibodies in the bovine serum can still be extracted by incubating with bovine serum for the second time and the third time, and the overall effect is not inferior to that of ProteinA a sepharose beads of the pharmaceutical company. The invention also has a better effect for a bed volume of 6 microlitres). The biomagnetic microspheres F combined with the affinity protein (here, protein A) provided by the invention can be repeatedly used for many times.

Example 4 recyclability of the binding of biomagnetic microspheres D to affinity complexes E (avidin-protein A)

4.1 incubation

And (3) incubating to prepare the SPA-eGFP-Tamvavidin 2-combined biomagnetic microspheres F:

after 50 μ L of 10% biomagnetic microsphere D suspension (volume 5 μ L) was washed 3 times with 2mL binding/washing buffer, 2mL of IVTT supernatant of SPA-eGFP-Tamvavidin2 was added and incubated at 4 ℃ for 1 hour with rotation, the supernatant was discarded (supernatant was collected to obtain flow-through solution 1), fresh IVTT supernatant containing SPA-eGFP-Tamvavidin2 was added repeatedly, incubation was performed at 4 ℃ for 1 hour with rotation, the supernatant was discarded (supernatant was collected to obtain flow-through solution 2), fresh IVTT supernatant containing SPA-eGFP-Tamvavidin2 was added for the third time, incubation was performed at 4 ℃ for 1 hour with rotation, and the supernatant was discarded (supernatant was collected to obtain flow-through solution 3). RFU values of IVTT supernatant and triplicate flow-through of SPA-eGFP-Tamvavidin2 were measured, and the remaining amount of SPA-eGFP-Tamvavidin2 in the solution was calculated according to the above formula, with the results shown in fig. 8 and table 4.

A standard curve is drawn according to the purified eGFP, and the calculated RFU value of the eGFP is converted into the protein mass concentration by the calculation formula:

wherein X is the protein mass concentration (μ g/mL), Y is the RFU fluorescence reading, M is the molecular weight of eGFP (26.7kDa), and N is the molecular weight of SPA-eGFP-Tamvavidin2 (79.4 kDa).

The mass concentration (. mu.g/mL) of the target protein SPA-eGFP-Tamvavidin2 was obtained by substituting the measured fluorescence value of RFU into the calculation formula. For example: when the value of Y is 1330.3, the corresponding value of X is 247.6 calculated by the above calculation formula, as shown in table 4.

4.2 elution and exchange

Elution and replacement of avidin-avidin complex E: SPA-eGFP-Tamvavidin 2.

After washing the biomagnetic microspheres F with 2mL of binding/washing buffer solution for 3 times, 200. mu.L of denaturation buffer solution (1M Urea and 10% SDS) was added, and the mixture was incubated in a metal bath at 95 ℃ for 10 minutes to elute the avidin-protein A bound to the biomagnetic microspheres. After 20. mu.L of the eluate was added to 5. mu.L of 5 XSDS loading buffer, SDS-PAGE electrophoresis was performed to obtain a band shown in lane 1 in FIG. 9. And (3) washing the eluted biomagnetic microspheres D for 3 times by using 2mL of binding/washing buffer solution to obtain the first regenerated biomagnetic microspheres D, wherein the binding sites of biotin of the polymer branched chains on the outer surfaces of the magnetic microspheres are released.

4.3 second incubation (regeneration)

The process of incubating SPA-eGFP-Tamvavidin2 and eluting the avidin-protein A is repeated to realize the first regeneration of the biomagnetic microspheres F, namely the first replacement of the avidin-protein A. The first regenerated biomagnetic microspheres D were combined with SPA-eGFP-Tamvavidin2, and the RFU values of the corresponding IVTT supernatant and the three-pass flow-through were measured, to obtain the data in Table 5. And washing the eluted biomagnetic microspheres D for 3 times by using 2mL of binding/washing buffer solution to obtain second regenerated biomagnetic microspheres D.

4.4 third incubation (second regeneration)

And repeating the processes of incubating the SPA-eGFP-Tamvavidin2 and eluting the avidin-protein A again to realize the second regeneration of the biomagnetic microspheres F, namely the second replacement of the avidin-protein A. The second regenerated biomagnetic microspheres D are combined with SPA-eGFP-Tamvavidin2, and the RFU values of the corresponding IVTT supernatant and the two-time flow-through liquid are measured, so as to obtain the data in Table 6. In tables 4-6, binding force refers to ProteinA fusion protein/biomagnetic microspheres D, mass to volume ratio, in units (mg/mL).

TABLE 4 calculation of the Loading of the first biomagnetic microsphere D-binding Protein A fusion Protein

TABLE 5 calculation of the load of the first regenerated biomagnetic microspheres D binding Protein A fusion Protein

TABLE 6 calculation of the load of the second regenerated biomagnetic microspheres D binding Protein A fusion Protein

The regenerated biomagnetic microspheres D were tested for their ability to rebind to protein a-eGFP-avidin.

The first prepared biological magnetic microsphere D is saturated and combined with SPA-eGFP-Tamvavidin2 for the first time, the first regenerated biological magnetic microsphere D is saturated and combined with SPA-eGFP-Tamvavidin2 for the second time, the second regenerated biological magnetic microsphere D is saturated and combined with SPA-eGFP-Tamvavidin2 for the third time, protein is eluted by denatured buffer solutions respectively, and three eluates containing SPA-eGFP-Tamvavidin2 are collected respectively for SDS-PAGE. The results are shown in FIG. 9. Lanes 1,2, and 3 represent electrophoretic bands of three eluates containing SPA-eGFP-tamvadin 2, which were obtained by eluting proteins from the first prepared biomagnetic microspheres F, the first regenerated biomagnetic microspheres F, and the second regenerated biomagnetic microspheres F, respectively, after the binding protein a-eGFP-avidin was saturated for the first time, the second time, and the third time, respectively.

4.5 detection of capability of regenerated biomagnetic microspheres F in combination with bovine serum antibody

When the biomagnetic microspheres D are combined and saturated with SPA-eGFP-Tamvavidin2 for the third time, the biomagnetic microspheres F regenerated for the second time are obtained. 2mL of newborn bovine serum (commercially available product, the same applies hereinafter) was added to the obtained second regenerated biomagnetic microspheres F, and the mixture was subjected to rotary incubation at 4 ℃ for 1 hour, and after discarding the supernatant (collecting the supernatant to obtain flow-through solution 1), 2mL of newborn bovine serum was added again, and the mixture was subjected to rotary incubation at 4 ℃ for 1 hour, and then the supernatant was discarded (collecting the supernatant to obtain flow-through solution 2). Wash 3 times with 1mL binding/wash buffer, incubate at 4 ℃ for 10min for each wash. Bovine serum antibody (antibody bound to protein A terminal in magnetic microspheres) was eluted with 100. mu.L of 0.1M glycine solution at pH2.8, and 1/10 volumes (i.e., 10. mu.L) of 1M Tris-HCl solution at pH8.0 was immediately added to the eluted bovine serum antibody solution to obtain 110. mu.L of eluate.

mu.L of the eluate was added to 10. mu.L of 5 XSDS loading buffer (no reducing agent), mixed, and subjected to metal bath at 95 ℃ for 10 minutes, followed by SDS-PAGE electrophoresis, and the results are shown in FIG. 9. According to the experimental results of fig. 7, fig. 9 and table 6, it can be known that the combination of the biomagnetic microspheres D and SPA-eGFP-Tamvavidin2 has reproducibility, and the biomagnetic microspheres F provided by the invention can be reused. And the step of heating the denaturing liquid is added, so that the binding efficiency of the biomagnetic microspheres is not influenced.

EXAMPLE 5 preparation of protein G-binding biomagnetic microspheres G (protein G as purification medium and biotin-avidin affinity complex as linker)

5.1. Synthesis of ProteinG-eGFP-avidin fusion protein (an avidin-avidin covalently linked Complex E)

Using the method of example 2, protein-eGFP-Tamvavidin 2 fusion protein (also abbreviated as protein fusion protein) was synthesized, and IVTT reaction solution and IVTT supernatant containing protein-eGFP-Tamvavidin 2 fusion protein were prepared in this order.

First, the DNA sequence of the protein G-eGFP-Tamvavidin2 fusion protein including three gene sequences was constructed. Wherein the nucleotide sequences of the eGFP segment and the Tamvavidin2 segment are the same as those of the embodiment 2, and are respectively shown in SEQ ID No. 3 and SEQ ID No. 2. Wherein the nucleotide sequence of the "ProteinG" segment (SEQ ID No.:4) is derived from Streptococcus G (Streptococcus sp. group G), and the gene sequence of the antibody binding region thereof is selected.

A DNA template of the protein G-eGFP-Tamvavidin2 fusion protein is constructed by adopting a recombinant PCR method. RCA method is adopted for in vitro amplification. The protein-eGFP-Tamvavidin 2 fusion protein is synthesized by adopting the in vitro cell-free protein synthesis method (adopting a protein factor system based on the in vitro cell-free protein synthesis method (D2P technology), and specifically adopting a cell extract based on Kluyveromyces lactis).

And carrying out IVTT reaction to obtain IVTT reaction liquid containing Protein G-eGFP-Tamvavidin2 fusion Protein and IVTT supernatant in sequence.

5.2. Preparation of protein G-binding biomagnetic microsphere G

Aspirate 30. mu.L of 10% (w/v) biotin-modified biomagnetic microspheres D prepared in example 1 with binding/washing buffer (10mM Na)₂HPO₄ pH 7.4，2mM KH₂PO₄140mM NaCl, 2.6mM KCl) for use after 3 washes.

Taking 2mL of IVTT supernatant containing protein-eGFP-Tamvavidin 2 fusion protein and the biomagnetic microspheres D for rotary incubation for 1 hour at 4 ℃, and collecting the supernatant, namely flow-through liquid, wherein the flow-through liquid contains residual fusion protein which is not combined by the microspheres; this procedure was repeated three times, each time a new supernatant of IVTT was incubated with biomagnetic microspheres D to obtain three different flow-through solutions. Namely, the same batch of biomagnetic microspheres D is incubated with protein-eGFP-Tamvavidin 2 fusion protein in the supernatant of IVTT three times continuously. Each 2mL of the supernatant of IVTT obtained in step 5.1 was taken, and the three flow-through solutions were sequentially designated as flow-through solution 1(FT1), flow-through solution 2(FT2) and flow-through solution 3(FT 3). The fluorescent values of eGFP (measured as RFU values) were measured using the fluorometry method on the IVTT supernatant and the three flowthrough, and the results are shown in fig. 10.

The closer the RFU value of the flow-through and the IVTT supernatant, the more saturated the binding of biomagnetic microspheres D to protein-eGFP-Tamvavidin 2 fusion protein, and the corresponding RFU values are shown in table 6 below. The first binding capacity was calculated as the amount of protein in the supernatant minus the amount of protein in the flow-through1, the second binding capacity was calculated as the amount of protein in the supernatant minus the amount of protein in the flow-through2, and the third binding capacity was calculated as the amount of protein in the supernatant minus the amount of protein in the flow-through 3. The concentration of protein-eGFP-Tamvavidin 2 fusion protein was calculated according to the following formula using the method of example 2.

Wherein, X is the mass concentration (mu g/mL) of the protein G-eGFP-Tamvavidin2 fusion protein, Y is the RFU fluorescence reading, M is the molecular weight (26.7kDa) of eGFP, and N is the molecular weight of the protein G-eGFP-Tamvavidin2 fusion protein.

TABLE 6 eGFP fluorescence data of IVTT supernatant and triple flow-through containing ProteinG-eGFP-Tamvavidin2 fusion protein

Wherein, the binding force is the mass of the ProteinG fusion protein bound by the magnetic beads and the volume of the magnetic beads.

The biomagnetic microspheres G combined with ProteinG in an affinity complex (biotin-avidin) connection mode are obtained by the process of incubating the biomagnetic microspheres D and IVTT supernate containing ProteinG fusion protein, and are also marked as ProteinG magnetic beads.

ProteinG magnetic bead purification of serum antibodies (IgG antibodies as target proteins)

Different volumes of ProteinG magnetic beads with ProteinG-eGFP-Tamvavidin2 fusion protein bound thereto were incubated with 1mL of newborn bovine serum for 1 hour at 4 ℃ followed by 1mL wash (3 times) with binding/washing buffer and 10 minutes at 4 ℃ for each wash. The antibody was eluted with 100. mu.L of 0.1M glycine pH2.8 and one-tenth volume (10. mu.L) of 1M Tris-HCl pH8.0 was immediately added to the eluate. The purity of IgG antibody in the eluate was checked by SDS-PAGE, and the results are shown in FIG. 11. The purity was greater than 95% by quantitative analysis based on grey scale values.

Example 6 preparation of biomagnetic microspheres H binding to Nanobody anti-eGFP (Nanobody anti-eGFP as purification media, biotin-avidin affinity complexes as linker elements)

6.1. Synthesis of anti EGFP-mScelet-avidin fusion protein (anti EGFP-mScelet-Tamvavidin 2 fusion protein, a fusion protein of Nano antibody)

An anti EGFP-mScarlet-Tamvavidin2 fusion protein (also abbreviated as anti EGFP fusion protein, molecular weight 59kDa) was synthesized by the method of example 2, and IVTT reaction solution and IVTT supernatant containing the anti EGFP-mScarlet-Tamvavidin2 fusion protein were prepared in this order.

First, the DNA sequence of an anti EGFP-mScarlet-Tamvavidin2 fusion protein including three gene sequences was constructed.

Wherein the nucleotide sequence of segment "Tamvavidin 2" is the same as in example 2, and is referred to SEQ ID No. 2.

Wherein, the anti EGFP is a nano antibody with the amino acid sequence shown as SEQ ID No. 5.

Wherein the mScarlet is a bright red fluorescent protein and the corresponding nucleotide sequence is SEQ ID No. 6.

A DNA template of the anti EGFP-mScarlet-Tamvavidin2 fusion protein is constructed by adopting a recombinant PCR method. RCA method is adopted for in vitro amplification. The anti EGFP-mScarlet-Tamvavidin2 fusion protein was synthesized by the in vitro cell-free protein synthesis method (protein factor system based on the in vitro cell-free protein synthesis method (D2P technology)) indicated above.

And carrying out IVTT reaction to obtain IVTT reaction liquid containing the anti EGFP-mScarlet-Tamvavidin2 fusion protein and IVTT supernate in sequence.

6.2. Preparation of biomagnetic microsphere H combined with nano antibody anti-eGFP

2mL of IVTT supernatant (RFU value of 2400) containing the anti EGFP-mScarlet-Tamvavidin2 fusion protein and the biomagnetic microspheres D are subjected to rotary incubation for 1 hour at 4 ℃, and the supernatant is collected, namely the flow-through solution (RFU value of 1700). The flow-through contains the remaining fusion protein unbound by the microspheres. The test conditions for RFU values were: the excitation wavelength (Ex) was 569nm and the emission wavelength (Em) was 593 nm.

Through the process of incubating the biomagnetic microsphere D and the IVTT supernatant containing the anti EGFP fusion protein, the incubated magnetic beads are combined with a nano antibody anti-eGFP in an affinity complex (biotin-Tamvavidin 2) connection mode and are marked as biomagnetic microspheres H and anti EGFP magnetic beads (nano antibody magnetic beads).

6.3 Loading test of anti EGFP magnetic beads bound to eGFP protein (eGFP as target protein, eGFP excess)

mu.L of 10% (w/v) anti EGFP magnetic beads prepared in this example 6.2 were pipetted and washed 3 times with binding/washing buffer for use.

The fluorescence value of 2mL of IVTT reaction solution of eGFP protein (the nucleotide sequence of eGFP in the DNA template is shown in SEQ ID No.:3) was measured and recorded as the fluorescence value of Total. This was mixed with the previously washed 3. mu.L of anti EGFP magnetic beads, and the mixture was incubated for 1 hour by rotation, and the supernatant which was not bound to the magnetic beads was designated as Flow-through, and the fluorescence value was measured. According to the fluorescence value test results in table 7, the amount of eGFP protein was calculated by using the eGFP calculation formula, and the loading capacity of the anti eGFP magnetic beads was calculated to be 17.7mg/mL (mass of eGFP protein bound to each mL of anti eGFP magnetic beads).

TABLE 7 results of the loading test of anti EGFP magnetic beads combined with eGFP protein

Purification of eGFP binding efficiency with anti EGFP magnetic beads (eGFP as target protein, magnetic bead excess)

The anti EGFP magnetic beads prepared in example 6.2 were washed 3 times with binding/washing buffer for use.

1mL of IVTT reaction solution of eGFP protein (the nucleotide sequence of the DNA template coding eGFP is shown as SEQ ID No.:3) was taken, and the fluorescence value was measured and recorded as Total. This was mixed with an excess of anti EGFP magnetic beads, incubated for 1 hour with rotation, and the clear solution was collected and recorded as Flow-through and the fluorescence was measured. The magnetic beads were washed 2 times with 1mL of binding/Washing buffer, and the beads were incubated and rotated at 4 ℃ for 10 minutes for each Washing, and the Washing solutions were recorded as Washing1 and Washing2, and the fluorescence values thereof were measured. The incubated magnetic beads bound the target protein eGFP. The measurement results of the fluorescence values are shown in fig. 12 and table 8. The result shows that the magnetic beads for resisting the eGFP prepared by the scheme can be effectively combined and eluted to obtain the eGFP. And calculating the binding efficiency of the anti EGFP magnetic beads to the target protein eGFP after the incubation for 1 hour according to the fluorescence values of the IVTT supernatant and the flow-through liquid to be 98.2%.

Table 8 binding efficiency test for anti eGFP magnetic bead purified eGFP protein

eGFP was eluted with 100. mu.L of 0.1M glycine, pH2.8, and one-tenth volume (10. mu.L) of 1M Tris-HCl pH8.0 was immediately added to the eluate. The fluorescence value of the eluate was measured and recorded as "Elution" and the purity was checked by SDS-PAGE, and the purity was about 95% as shown in FIG. 13.

It should be understood that the above description is only a partial description of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made which will achieve the same technical effects within the spirit or scope of the invention and the scope of the invention is to be determined by the appended claims.

Sequence listing

<110> Kangma (Shanghai) Biotech Co., Ltd

<120> biological magnetic microsphere, preparation method and application thereof

<130> 2020

<141> 2020-11-27

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Biological magnetic microsphere and preparation method and application thereof

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