阅读说明：本技术 一种捕获水蛭素类多肽的探针及其应用 (Probe for capturing hirudin polypeptide and application thereof ) 是由 曹秀君 国锦琳 聂应 罗莎杰 张佳斌 于 2020-04-16 设计创作，主要内容包括：本发明公开了一种捕获水蛭素类多肽的生物识别探针的制备方法及应用。本发明通过在磁性固相载体表面修饰配基,间接固定凝血酶,极大程度保留其生物活性,所获得的探针可结合多种检测器实现水蛭素类多肽的提取和测定,具有选择性高、快速灵敏、稳定性好、适用性广、干扰小、重复利用率高和环境友好等优势。(The invention discloses a preparation method and application of a biological recognition probe for capturing hirudin polypeptides. According to the invention, the biological activity of thrombin is retained to a great extent by modifying the aglucon on the surface of the magnetic solid phase carrier and indirectly fixing the thrombin, and the obtained probe can be combined with various detectors to realize the extraction and determination of hirudin polypeptides, and has the advantages of high selectivity, rapidness, sensitivity, good stability, wide applicability, small interference, high repeated utilization rate, environmental friendliness and the like.)

1. A probe for capturing hirudin polypeptides, which is characterized in that: the probe consists of a magnetic solid phase carrier, thrombin ligand and thrombin;

wherein, the magnetic beads and the ligand are in covalent or non-covalent chemical connection through active groups on the surface of the magnetic solid phase carrier;

the thrombin ligand is non-covalently chemically linked to thrombin.

2. The probe of claim 1, wherein:

the nucleotide sequence of the ligand is shown in any one of SEQ ID NO. 1-6, or 6-18T basic groups are added at the 3 'or 5' tail end of SEQ ID NO. 1-6;

preferably, the nucleotide sequence of the ligand is shown as SEQ ID NO.7 or 8.

3. The probe of claim 1 or 2, wherein: the magnetic solid phase carrier takes a magnetic substance as a core, and the outer layer of the magnetic solid phase carrier is coated with polymer or biomacromolecule magnetic beads;

preferably, the magnetic solid phase carrier is: polystyrene magnetic beads, agarose magnetic beads, silica magnetic beads, MOF gold magnetic beads, MOF mercury magnetic beads, or MOF lead magnetic beads.

4. The probe of claim 1 or 2, wherein: the active group is carboxyl or activated carboxyl, amino, sulfydryl, tosylate or epoxy;

or the active group is streptavidin, biotin or pyridine disulfide.

5. The probe of claim 1 or 2, wherein: the diameter of the magnetic solid phase carrier is 0.2-25 μm.

6. The probe of claim 1 or 2, wherein: the hirudin polypeptide is hirudin, hirudin or recombinant hirudin.

7. A method for separating hirudin polypeptides is characterized in that: the method is a method for separating hirudin polypeptides from a solution using the probe according to any one of claims 1 to 6.

8. The method of claim 7, comprising the steps of:

1) contacting the probe of any one of claims 1 to 6 with a hirudin polypeptide;

2) separating the probe from the solution;

3) the hirudin polypeptides on the probe are eluted using a trifluoroacetic acid solution.

9. The method of claim 8, wherein: the concentration of the trifluoroacetic acid solution in the step 3) is 0.01M.

10. A method for detecting hirudin polypeptides is characterized in that: the method comprises the following steps: taking a sample to be detected, separating the hirudin polypeptides by the separation method of any one of claims 7 to 9, and detecting by methods such as chromatography, chromatography-mass spectrometry, thrombin titration, chromogenic substrate method, light scattering method, fibrinogen plate method or thrombin time method.

Technical Field

The invention belongs to the field of polypeptide detection.

Background

The hirudin polypeptide is a bivalent direct thrombin inhibitor which has the same or similar chemical structure with natural hirudin and the molecular weight of 5-7 kDa, and can act on an active site and a substrate recognition site of thrombin simultaneously. The hirudin is a single-chain polypeptide compound consisting of 65-66 amino acids and separated and purified from leech saliva, comprises HV 1, HV 2 and HV 3 variants, is the most effective natural specific thrombin inhibitor, and is similar to the hirudin extracted from Poecilobdella manillensis and the hirudin separated from Hirudinaria manillensis. The natural hirudin has low yield, is not beneficial to clinical popularization, and the recombinant hirudin produced by the genetic engineering technology, such as lepirudin, disinuline and the like, has ideal curative effect in clinic. Research shows that the hirudin polypeptide has stronger anticoagulation and antithrombotic effect than heparin, and has great advantages in treating cases such as cardiovascular and cerebrovascular diseases, tumors, ophthalmic diseases, gynecological diseases, peripheral nerve injury and the like.

At present, the content determination of hirudin polypeptides basically adopts a biological activity method, mainly including a thrombin titration method, a chromogenic substrate method, a light scattering method, a fibrinogen plate method and a thrombin time method, and from the applicability of a drug quality control mode, a biological detection technology is closely related to safety and effectiveness, often has more practical value than chemical component detection, but has self defects: a) the quantitative sensitivity is low, and when the concentration of the target polypeptide is low, the titration method and the time method are difficult to detect; b) the quantitative repeatability is poor, and the determination result is easily influenced by factors such as free thrombin activity, environmental temperature, substrate concentration and the like. c) The pretreatment process is complicated and the automation degree is not high. The chromatographic-mass spectrometry technology is also adopted, but the impurity interference is large, and the anticoagulant activity of the hirudins cannot be evaluated.

The immunomagnetic bead enrichment technology is a technology in which magnetic beads (also referred to as "magnetic microspheres" or "magnetic microparticles") are used as solid phase carriers, and specific antibodies (or antigens) are bound thereto to separate a target substance from a liquid phase environment. The magnetic beads can be divided into 2 parts: 1) the core being a magnetic substance, e.g. gamma-Fe₂O₃、Fe₃O₄And MeFe₂O₃(ii) a 2) The outer layer is made of biomacromolecule or epoxy resin, polyacrylamide, polyacrylic acid or agaroseThe molecule is wrapped, the magnetic sealing performance is ensured to be good, and the magnetic leakage phenomenon is not easy to occur. The surface of the magnetic bead is also distributed with special active groups, common active groups comprise carboxyl or activated carboxyl, amino, sulfydryl, tosylate, epoxy and the like, and can be combined with an antibody (or antigen) in a covalent or non-covalent mode.

The immunomagnetic bead enrichment technology has high purity and almost no damage to the activity of a sample. At present, the immunomagnetic bead enrichment technology is widely applied to the separation and detection of specific cells, microorganisms, proteins and nucleic acid fragments. The method has good prospect when being used for separating and detecting the hirudin polypeptide.

The preparation of suitable magnetic beads for target substances is a key and difficult point for achieving effective enrichment. Generally, the process of preparing immunomagnetic beads requires attention to the size of the beads, the choice of surface active groups, the choice of ligands, the amount of coupling, the blocking substance, the blocking method, the preservation, and so on. It has been reported that thrombin is coupled to magnetic particles with amino terminal, and hirudin is separated and purified from the leachate of dried leech (the thrombin coupling magnetic particle technology is used for detecting the antithrombin activity in leech medicinal materials, China journal of medical biotechnology application, 2002), but the method has obvious defects: that is, the thrombin is linked to the amino terminal by a covalent bond (usually-CO-NH-), and the binding site of the thrombin to hirudin may be blocked or semi-blocked at the amino terminal of the magnetic particle, and thus the binding efficiency of the thrombin to hirudin is low.

Disclosure of Invention

The invention aims to solve the problems that: provides a probe with high efficiency of capturing hirudin polypeptides.

The technical scheme of the invention is as follows:

a probe for capturing hirudin polypeptides is composed of a magnetic solid phase carrier, thrombin ligand and thrombin;

wherein, the magnetic beads and the ligand are in covalent or non-covalent chemical connection through active groups on the surface of the magnetic solid phase carrier;

the thrombin ligand is non-covalently chemically linked to thrombin.

The probe is characterized in that the nucleotide sequence of the ligand is shown in any one of SEQ ID NO. 1-6, or 6-18T basic groups are added at the 3 'or 5' tail end of SEQ ID NO. 1-6.

Preferably, the nucleotide sequence of the ligand is shown as SEQ ID NO.7 or 8.

The corresponding relation between SEQ ID NO. 1-8 and the sequence is as follows:

SEQ ID NO.1	5’-GGTTGGTGTGGTTGG-3’
		SEQ ID NO.2	5’-TGGTTGGTGTGGTTGG-3’
SEQ ID NO.3	5’-TGGTTGGTGTGGTTGGT-3’
		SEQ ID NO.4	5’-GGTTGGTGTGGTTGGT-3’
SEQ ID NO.5	5’-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3’
		SEQ ID NO.6	5’-TAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3’
SEQ ID NO.7	5’-TTTTTTTGGTTGGTGTGGTTGGT-3’
		SEQ ID NO.8	5’-TTTTTTTTTTTTTGGTTGGTGTGGTTGGT-3’

in the probe, the magnetic solid-phase carrier is a magnetic bead with a magnetic substance as a core and a polymer or biomacromolecule coated on an outer layer;

preferably, the magnetic solid phase carrier is: polystyrene magnetic beads, agarose magnetic beads, silica magnetic beads, MOF gold magnetic beads, MOF mercury magnetic beads, or MOF lead magnetic beads.

The probe as described above, wherein the active group is a carboxyl group or an activated carboxyl group, an amino group, a mercapto group, a tosylate group or an epoxy group;

or the active group is streptavidin, biotin or pyridine disulfide.

The probe as described above, the diameter of the magnetic solid phase carrier is 0.2 to 25 μm.

The probe is characterized in that the hirudin polypeptide is hirudin, hirudin or recombinant hirudin.

A method for separating a hirudin polypeptide from a solution by using the probe is provided.

The method comprises the following steps:

1) the probe is contacted with the hirudin polypeptide;

2) separating the probe from the solution;

3) the hirudin polypeptides on the probe are eluted using a trifluoroacetic acid solution.

As in the previous method, the concentration of the trifluoroacetic acid solution in the step 3) is 0.01M.

A method for detecting hirudin polypeptide comprises the following steps: taking a sample to be detected, separating the hirudin polypeptides by the separation method, and detecting by methods such as chromatography, chromatography-mass spectrometry, thrombin titration, chromogenic substrate method, light scattering method, fibrinogen plate method or thrombin time method.

The term "activation" refers to: groups are chemically modified to enable direct attachment to other groups. For example, the common manner of carboxyl activation in the art: EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) and Sulfo-NHS (N-hydroxy thiosuccinimide) are used together to activate carboxyl to obtain an active group, and the active group can directly generate amido bond with free amino.

Has the advantages that:

the probe of the invention, through modifying the aglucon on the surface of the magnetic solid phase carrier, indirectly fixes the thrombin, greatly retains the biological activity of the thrombin, has high efficiency of combining the hirudin polypeptides, can be applied to the detection of trace amount of the hirudin polypeptides, and has good application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a schematic diagram of a probe.

FIG. 23 μm scanning electron micrograph of polystyrene carboxyl magnetic beads.

FIG. 310 μm scanning electron micrograph of Sepharose carboxyl magnetic beads.

FIG. 425 μm agarose NHS beads scanning electron micrograph.

FIG. 5 comparison of the capturing effect of recombinant hirudin r-HV 2.

Detailed Description

Description of the drawings: the reagents and magnetic beads in the following examples are commercially available.