Novel epitope based on CD271 and application thereof
阅读说明：本技术 一种基于cd271的新型抗原表位及其应用 (Novel epitope based on CD271 and application thereof ) 是由 路慧丽 王紫嫣 杨慧 朱建伟 于 2021-02-04 设计创作，主要内容包括：本发明涉及一种基于CD271的新型的抗原表位肽及其应用,所述抗原表位肽为：(1)位于SEQ ID NO.1中；或者,(2)位于与(1)中SEQ ID NO.1序列具有至少80％同一性的氨基酸序列。且上述SEQ ID NO.1中包含一个或多个抗原表位,并且所述抗原表位肽的氨基酸序列长度为SEQ ID NO.1全长的5-70％。靶向该抗原肽的抗体、核酸适配体、疫苗、纳米粒子等能够与天然带有CD271蛋白的细胞具有稳定的结合。该抗原表位肽或含有该抗原表位肽的核酸、融合蛋白、载体或宿主细胞等在制备CD271相关的检测试剂、疾病治疗生物药物、疫苗等领域,具有重要应用意义。(The invention relates to a novel epitope peptide based on CD271 and application thereof, wherein the epitope peptide comprises the following components in parts by weight: (1) is located in SEQ ID NO. 1; or, (2) an amino acid sequence which is located at least 80% identical to the sequence of SEQ ID NO.1 of (1). And the above-mentioned SEQ ID NO.1 contains one or more antigen epitopes, and the amino acid sequence length of the antigen epitope peptide is 5-70% of the full length of SEQ ID NO. 1. Antibodies, aptamers, vaccines, nanoparticles and the like targeting the antigenic peptide can stably bind to cells naturally carrying the CD271 protein. The epitope peptide or nucleic acid, fusion protein, vector or host cell containing the epitope peptide has important application significance in the fields of preparation of CD271 related detection reagents, disease treatment biological drugs, vaccines and the like.)
1. A novel epitope peptide, which is characterized in that: the epitope peptide is:
(1) is SEQ ID NO.1 or is located in a polypeptide chain SEQ ID NO. 1; alternatively, the first and second electrodes may be,
(2) located in an amino acid sequence having at least 80% identity to the sequence of SEQ ID No.1 of (1);
(3) and the above-mentioned SEQ ID NO.1 contains one or more antigen epitopes, and the amino acid sequence length of the antigen epitope peptide is 5-70% of the full length of SEQ ID NO. 1.
2. The novel epitope peptide according to claim 1, characterized in that: the epitope peptide is a continuous amino acid sequence located in the 1 st to 35 th amino acid sequences in SEQ ID NO. 1.
3. The novel epitope peptide according to claim 2, characterized in that: the epitope peptide is a continuous amino acid sequence located in the 11 th to 35 th amino acid sequences in SEQ ID NO. 1.
4. The novel epitope peptide according to claim 3, characterized in that: the epitope peptide is a continuous amino acid sequence located in the 18 th to 27 th amino acid sequences in SEQ ID NO. 1.
5. The novel epitope peptide according to claim 4, characterized in that: the epitope peptide is an amino acid sequence which has more than 80 percent of overlapping degree with the continuous amino acids from 18 th to 27 th in SEQ ID NO. 1.
6. The novel epitope peptide according to claim 1, characterized in that: the amino acid sequence of the epitope peptide is shown in any one of SEQ ID NO. 3-11.
7. A fusion protein formed by fusing the epitope peptide according to any one of claims 1 to 6 with a carrier protein.
8. A nucleic acid sequence capable of encoding an epitope peptide, characterized in that: the nucleic acid sequence is a gene sequence or gene fragment capable of encoding the epitope peptide of any one of claims 1 to 6.
9. An expression vector comprising the nucleic acid sequence of claim 8.
10. A host cell comprising the expression vector of claim 9 or having integrated in its genome the nucleic acid sequence of claim 8.
11. An antibody, characterized by: the antibody is an antibody capable of binding to the epitope peptide of any one of claims 1 to 6.
12. The antibody of claim 10, wherein: the antibody is an antibody capable of inhibiting or reducing signal transmission related to the epitope peptide according to any one of claims 1 to 5, and the antibody is a monoclonal antibody or a polyclonal antibody, and the species source can be human, mouse, rabbit, monkey, cow, sheep or alpaca and other mammals.
13. A vaccine comprising the epitope peptide of any one of claims 1 to 6, the fusion protein of claim 7, the nucleic acid sequence of claim 8, the expression vector of claim 9, or the host cell of claim 10.
14. A composition comprising the epitope peptide according to any one of claims 1 to 6, the fusion protein according to claim 7, the nucleic acid sequence according to claim 8, the expression vector according to claim 9 or the host cell according to claim 10, the antibody according to any one of claims 11 to 12 or the vaccine according to claim 13, and an immunologically and pharmaceutically acceptable carrier and/or adjuvant.
15. Use of the epitope peptide of any one of claims 1 to 6, the fusion protein of claim 7, the nucleic acid sequence of claim 8, the expression vector of claim 9, or the host cell of claim 10:
(1) for preparing antibodies, aptamers, vaccines and nanoparticles against the epitope; and/or
(2) For the preparation of a medicament for the treatment of a disease associated with said epitope.
Technical Field
The invention relates to the technical field of biology, in particular to a novel epitope peptide of CD271 and application thereof.
Background
CD271 is a low affinity nerve growth factor receptor (LNGFR) belonging to the tumor necrosis factor receptor superfamily, with a relative molecular mass of 75kD, also known as p75NTR, and it contains 3 regions: a cysteine-rich extracellular domain, a transmembrane domain and an intracellular domain of 155 amino acid residues. CD271 is not only expressed in the nervous system and closely related to development, differentiation and survival of nerve cells, but also can identify tumor stem cells and mesenchymal stem cells. In the field of tumor treatment, CD271 is highly expressed on the surfaces of various tumor cells or tumor stem cells such as melanoma, and the monoclonal antibody targeting CD271 and a coupled drug thereof can play a role in targeted killing, so that the tumor is treated. In the field of mesenchymal stem cells, the CD271 antibody can perform magnetic sorting on the mesenchymal stem cells by flow sorting or coupling to magnetic beads, and the enriched cells have the advantages of high specificity, high purity, strong colony forming capability and the like. Therefore, the antibody for specifically recognizing the CD271 has wide application value, and the stable and efficient epitope has important significance for developing CD271 monoclonal antibodies or vaccines based on the CD271 and the like.
The extracellular structure of native CD271 comprises 4 cysteine-rich CRD structures (CRD1-4) and a linking region (talk region). The existing CD271 antibody products are mainly various scientific research reagents, and the epitope of the CD271 antibody is mainly a cell expressing CD271, such as a melanoma cell, or a CRD region of CD 271. However, after CD271 is expressed, CRD domain is hydrolyzed by protease and then shed off, so that the binding of the CRD domain-targeting antibody to CD271 positive cells is affected, and even at some stage of cell metabolism, the expression of CD271 cannot be detected, so that there is a need to develop a new epitope, which provides a new tool for CD 271-based antibody, vaccine research and related detection or disease treatment.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention provides a novel epitope peptide based on CD271 and its application, so as to solve the shortcomings of the prior art.
The purpose of the invention is realized by the following technical scheme:
the invention provides an epitope peptide of CD271, which is:
(1) in the polypeptide chain SEQ ID NO. 1; alternatively, the first and second electrodes may be,
(2) located in an amino acid sequence having at least 80% identity to the sequence of SEQ ID No.1 of (1);
(3) and the above-mentioned SEQ ID NO.1 contains one or more antigen epitopes, and the amino acid sequence length of the antigen epitope peptide is 5-70% of the full length of SEQ ID NO. 1.
Sequence shown in SEQ ID NO.1, length: 62aa, specifically:
EEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN。
further, the epitope peptide is located in a continuous amino acid sequence in amino acid sequences from 1 st to 35 th in SEQ ID NO. 1.
Furthermore, the epitope peptide is located in a continuous amino acid sequence in the amino acid sequence from 11 th position to 35 th position in SEQ ID NO. 1.
Further, the epitope peptide is located in a continuous amino acid sequence in amino acid sequences 18 th to 27 th in SEQ ID NO. 1.
Furthermore, the epitope peptide is an amino acid sequence which has more than 80% of overlapping degree with the continuous amino acids from 18 th to 27 th in SEQ ID NO. 1.
Optionally, the amino acid sequence of the epitope peptide is shown in any one of SEQ ID NO. 3-11.
Specifically, an epitope Peptide, length: 19aa, the sequence is shown as SEQ ID NO.3, and specifically comprises: TRSTPPEGSDSTAPSTQEPE are provided.
Another epitope Peptide, length: 19aa, the sequence is shown as SEQ ID NO.4, and specifically comprises: EPEAPPEQDLIASTVAGVV are provided.
Another epitope Peptide, length: 19aa, the sequence is shown as SEQ ID NO.5, and specifically comprises: VTTVMGSSQPVVTRGTTDN are provided.
Another epitope Peptide, length: 19aa, the sequence is shown as SEQ ID NO.6, and specifically comprises: LIASTVAGVVTTVMGSSQP are provided.
Another epitope Peptide, length: 19aa, the sequence is shown as SEQ ID NO.7, and specifically comprises: EEIPGRWITRSTPPEGSDS are provided.
Another epitope Peptide, length: 19aa, the sequence is shown as SEQ ID NO.8, and specifically comprises: DSTAPSTQEPEAPPEQDLI are provided.
Another epitope Peptide, length: 10aa, the sequence is shown as SEQ ID NO.9, and specifically comprises: GSDSTAPSTQ are provided.
Another epitope Peptide, length: 10aa, the sequence is shown as SEQ ID NO.10, and specifically comprises: DSTAPSTQEP are provided.
Another epitope Peptide, length: 7aa, the sequence is shown as SEQ ID NO.11, and specifically comprises: TPPEGSD.
In a second aspect, the present invention provides a fusion protein formed by fusing the epitope peptide described in any one of the above with a carrier protein.
In a third aspect, the invention provides a nucleic acid sequence capable of encoding an epitope peptide, wherein the nucleic acid sequence is a gene sequence or a gene fragment capable of encoding the epitope peptide.
For example, the nucleotide sequence encoding SEQ ID NO.1 is shown in SEQ ID NO.2,
specifically, the method comprises the following steps: 186bp
gaggagatccctggccgttggattacacggtccacacccccagagggctcggacagcacagcccccagcacccaggagcctgaggcacctccagaacaagacctcatagccagcacggtggcaggtgtggtgaccacagtgatgggcagctcccagcccgtggtgacccgaggcaccaccgacaac。
In a fourth aspect, the present invention provides an expression vector comprising a nucleic acid sequence as described above.
In a fifth aspect, the invention provides a host cell comprising an expression vector as described above, or having integrated into its genome a nucleic acid sequence as described above.
In a sixth aspect, the present invention provides an antibody characterized in that: the antibody is an antibody capable of binding to the epitope peptide according to any one of the above.
Further, the antibody is an antibody capable of inhibiting or attenuating signaling associated with the epitope peptide of any one of the above.
Furthermore, the antibody is a monoclonal antibody or a polyclonal antibody, and the species source can be human, mouse, rabbit, monkey, cow, sheep or alpaca and other mammals.
In a sixth aspect, the present invention provides a vaccine comprising an epitope peptide, a fusion protein, a nucleic acid sequence, an expression vector or a host cell as defined in any one of the above.
The seventh aspect of the present invention provides a composition comprising an epitope peptide, a fusion protein, a nucleic acid sequence, an expression vector, a host cell, an antibody or a vaccine of any one of the above, and an immunologically and pharmaceutically acceptable carrier and/or excipient.
The eighth aspect of the present invention provides the use of the epitope peptide, fusion protein, nucleic acid sequence, expression vector or host cell of any one of the above:
(1) for preparing antibodies, aptamers, vaccines and nanoparticles against the epitope; and/or
(2) For the preparation of a medicament for the treatment of a disease associated with said epitope.
As described above, a novel epitope peptide and its use based on CD271 are provided, which have at least the following beneficial effects: the epitope peptide has immunogenicity, can induce and generate immune response reaction, can be used for preparing antibodies, nucleic acid aptamers, vaccines, nanoparticles targeting CD271 and the like, and has important application significance.
The epitope peptide is selected from a sequence which is stable outside a CD271 target membrane, has low homology and does not contain glycosylation and phosphorylation sites, and molecules such as an antibody targeting the epitope peptide are combined with CD271 target cells more tightly and stably. The epitope peptide has specific binding with the antibody in the application, strong affinity, balanced dissociation constant reaching nM level, stable binding and no influence caused by the hydrolysis of the CRD region of the CD271 molecule on the cell surface. The polypeptide has extremely important function for the antigen or antibody to play a medicinal role, has great potential value for preparing diagnostic reagents or medicaments, and can be used in fields related to CD271, such as stem cell tissue repair, nervous system diseases, tumors and the like.
Drawings
FIG. 1 is a schematic diagram of construction of an expression vector plasmid pCD271T of SEQ ID NO.1 (named CD271T) in which an epitope peptide is located, wherein the CD271T is expressed by fusion with a sumo tag, and the vector is pET-28a (+);
FIG. 2 is a graph of expression conditions optimized SDS-PAGE (FIGS. 2A &2B &2C) and Western Blotting (FIG. 2D) for the epitope peptide of SEQ ID NO.1 induced in E.coli, and the fusion protein containing CD271T has a molecular weight of about 37 kDa.
FIG. 3 is a SDS-PAGE map of the release of the Sumo tag from the Sumo protein cleavage of the fusion protein with CD271T protein: performing SDS-PAGE identification after enzyme digestion for 1h, 2h, 4h and 6h at 37 ℃ (FIG. 3A), showing that about 2h of protein is completely digested, the size of a target band is consistent with that of SUMO tag and CD271T, and further enriching the target protein by late purification (FIG. 3B);
FIG. 4 is a graph showing the ELISA detection of the potency of mouse serum polyclonal antibodies after immunization with the CD271 antigen polypeptide: the concentration of the coating antigen is 4 mug/mL, the sample serum is diluted from 1:2000 to 1:256000, and the serum titers of mice No.1, No.2, No.3, No.4 and No.5 are all 1: 128000 above, the serum of 5 mice contains polyclonal antibody which can recognize CD271 polypeptide antigen, and has strong affinity to antigen polypeptide;
FIG. 5 is a SDS-PAGE detection map of ascites monoclonal antibody CD271T-2 obtained by screening mice immunized with the CD271 epitope polypeptide: the band 1 is a non-reduction sample, the antibody band is single, the band 2 is a reduction sample, and the light chain LC and the heavy chain HC of the antibody are respectively at 26kDa and 53 kDa;
FIG. 6 is an ELISA identification of the purified CD271T-2 monoclonal antibody: the coating antigen is CD271 epitope polypeptide, the antigen concentration is 2 mug/mL, each hole is 100 mug L, the concentration gradient of the loading antibody is 0.625 mug/mL, 1.25 mug/mL, 2.5 mug/mL, 5 mug/mL, 10 mug/mL, 20 mug/mL, 40 mug/mL, each hole is 100 mug L, and the result shows that the CD271T-2 antibody can identify and bind to the full-length human CD271 protein with a natural structure;
FIG. 7 is an antibody affinity map of ForteBio after verification and purification, and it can be seen that the CD271 epitope polypeptide of the invention has strong affinity with the CD271T-2 monoclonal antibody obtained from the immunized mouse;
FIG. 8 is an epitope competitive inhibition ELISA assay for the CD271T-2 monoclonal antibody: the envelope antigen is CD271 protein, the antigen concentration is 4 mug/mL, each hole is 100 mug L, the dilution ratio of the loading ascites antibody is 1:600, the polypeptide concentration is 16 mug/mL, each hole is 100 mug L, the ascites antibody is mixed with the polypeptide 1:1, the result shows that the polypeptide 2-4 has the function of blocking the combination of the CD271T-2 monoclonal antibody and the CD271 protein, and is the minimum epitope recognized by the monoclonal antibody;
FIG. 9 is the electrophoresis diagram of the PCR fragment of the heavy chain variable region gene clone of anti-human CD 271T-2: FIG. 9A is the PCR fragment of the gene clone of the heavy chain variable region of CD271T-2 hybridoma, and FIG. 9B is the PCR fragment of the gene clone of the chimeric ScFv of the heavy chain variable region of CD 271T-2;
fig. 10 shows the detection of the expression of CD271 on the surface of mesenchymal stem cells by anti-human CD271 monoclonal antibody: FIG. 10A shows a positive control antibody derived from APC mouse anti human CD271 from Biolegend, the immunogen being from melanoma cells with high expression of CD271 molecules on the cell surface, the antibody migrating slightly compared to the negative control; FIG. 10B shows a Rabbit anti human CD271 control antibody derived from Abcam, the immunogen is from the 350-450 amino acid region within the human CD271 cell membrane, without any shift compared to the negative control; from FIG. 10C, the CD271T-2 monoclonal antibody has higher deviation degree compared with the positive and negative control, which indicates that the prepared anti-human CD271 monoclonal antibody has very high affinity and specificity to the cells expressing CD271 molecules, and is superior to the commercially available antibodies targeting other epitopes of CD 271.
Detailed Description
Selecting an antigen sequence to immunize an animal, fusing myeloma cells with immune animal spleen cells to form a monoclonal antibody which can secrete homogeneous high specificity and high affinity aiming at the antigen, and obtaining a monoclonal antibody hybridoma cell strain capable of stably expressing the antigen through subclone screening.
In order to obtain the best affinity effect and specificity of the selected monoclonal antibody, the design of the antigen needs to meet two requirements, firstly, the antigen does not produce over-strong immune reaction, and simultaneously, the antigen can produce the antibody with the binding capacity to the target protein. The conformation of the protein will affect the interaction between the antibody and its recognition region, and if the antibody recognition region is hidden inside the protein, the antibody will not interact. Most antibodies are directed against a continuous recognition domain, and the sequence in which the antibody binds to the surface with high affinity to such a domain is not within the protein. The discrete recognition region is a recognition region representing a polypeptide sequence that has a fold, or an antibody that binds two separate polypeptides together. In some cases, antibodies directed against such discontinuous recognition regions can be generated, except that the antigenic polypeptide used for immunization must have a secondary structure similar to that of the discontinuous recognition region, and the length of the sequence needs to meet the relevant requirements. In addition, in the intact protein, both ends of N and C are usually exposed on the surface of the protein, while the C end of the membrane protein is too hydrophobic to be suitable as an antigen.
In general, since proteins derived from mammalian cells may have modifications such as glycosylation on the surface, it is difficult to ensure that antibodies selected using immunogens derived from mammalian cells bind to amino acid epitopes. On the other hand, the screened antibody derived from an immunogen expressed in E.coli or a synthetic polypeptide can recognize an amino acid epitope, but since the immunogen may not have a high-order structure or a high-order structure different from a natural conformation, the screened antibody may not recognize a natural antigen, particularly a cell surface antigen. In order to obtain an antibody capable of recognizing a cell surface antigen, the immunization strategy is designed, the immunization antigen is derived from polypeptide synthesis or prokaryotic expression, and the mammalian cell expression with the antigen as a full-length molecule is screened so as to ensure that the spatial structure of the immunization antigen is in a natural state. In the stages of animal immunity, polyclonal serum titer detection and hybridoma screening, the two antigens are alternately used, and finally, the hybridoma cell strain and the antibody meeting the requirements are obtained.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.
The "pharmaceutically acceptable carrier and/or adjuvant" should be compatible with the active ingredient, i.e. capable of being blended therewith without substantially reducing the efficacy of the drug in the usual manner. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or adjuvants are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. These materials are used as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration.
Vaccine compositions comprise an immunizing antigen (including a recombinant protein of the invention), and are typically combined with a "pharmaceutically acceptable carrier", including any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and the like. Such vectors are well known to those of ordinary skill in the art. In addition, these carriers may act as immunostimulants ("adjuvants").
Example 1 design, expression, purification of epitope peptide plasmid of human CD271
1. Selection of human CD271 epitope peptides
The full-length amino acid sequence of the CD271 protein is searched according to NCBI, and the antigen determinant analysis is carried out on the full-length amino acid sequence by adopting DNAStar software, IEDB website and the like, so as to finally determine that the epitope peptide is positioned in SEQ ID NO.1 (named as CD271T) of the extramembranous stal region of the CD271, and a series of epitope peptides are designed, and the epitope peptides meet the following requirements:
(1) in the polypeptide chain SEQ ID NO. 1; alternatively, the first and second electrodes may be,
(2) an amino acid sequence which is located with at least 80% identity with the sequence SEQ ID NO.1 of (1).
Design of CD271T expression plasmid
The complete gene sequence of the CD271 protein and the amino acid sequence of the CD271T were retrieved according to NCBI, and prokaryotic expression codon optimization was performed by Suzhou Hongxn GmbH, and cloned into pET-28a (+) vector together with Sumo tag to synthesize CD271T antigen expression plasmid pCD271T, as shown in FIG. 1.
2, induced expression of CD271T antigen plasmid in escherichia coli and SDS-PAGE & WB detection
(1) Prokaryotic inducible expression
1) The resultant puncturing bacteria were selected and inoculated into 5mL of LB (0.1% Kana), and cultured overnight at 37 ℃ and 235 rpm.
2) The next day, 1mL of yesternight bacterial solution (1% inoculum size) was taken and placed in 100mL LB (0.1% Kana), cultured in a shaker at 37 ℃ and 235rpm until OD600 became 0.5, and IPTG concentration, induction temperature and induction time were optimized with controlled variables, and harvested by centrifugation at 6000 rpm. Wherein the IPTG concentration is 0.1mM and 0.05Mm respectively, the temperature is 20 ℃ and 37 ℃, 1.5mL of bacterial liquid is sampled every 2h and put in a 1.5mL EP tube, and the bacterial is collected by centrifugation at 6000 rpm.
3) Add 10% PBS of the culture volume to resuspend, sonicate: 300W, 5s of ultrasonic treatment and 6s of ultrasonic treatment are stopped each time, and 5min of ultrasonic treatment is carried out.
4) Centrifuging at 4 deg.C at 12000rpm for 10min, and collecting the supernatant and precipitate after ultrasonic treatment for identification.
(2) SDS-PAGE and WB detection
The supernatant and the pellet were added to a 5 × reduced SDS-PAGE loading buffer, heated at 95 ℃ for 10min, and centrifuged at 12000rpm for 5min, respectively. And taking 10 mu L of prepared samples, carrying out SDS-PAGE gel electrophoresis and Western Blotting detection, and determining the final IPTG concentration, induction temperature and induction time.
(3) Purification of human CD271T antigen protein His Trap FF column
1) Adding PMSF with the initial concentration of 100mM to the stock solution of the bacteria liquid until the final concentration is 1 mM;
2) breaking bacteria by using a high-pressure homogenizer, starting the homogenizer in advance, washing the homogenizer by using 20% ethanol and water in sequence, and then pouring bacterial liquid, wherein the conditions are as follows: 900bar, comparing the bacterial liquid before and after the bacterial liquid is clarified obviously, and collecting the clarified bacterial liquid;
3) centrifuging by a centrifuge, carrying out balancing, carrying out 12000rpm for 30min, and taking the supernatant for protein purification;
4) setting delta P as 0.3MPa, washing the system with ethanol until the system is stabilized, connecting the column, and adjusting to position 1;
5) washing the ethanol in the column with pure water, and washing A1 and B1 together, wherein each washing rate is 50% until the baseline is flat;
6) the column was then equilibrated with 5-10 Column Volumes (CV) of loading buffer (20mM PB, 20mM NaCl), 100% A1 and 0% B1, until the conductance and pH stabilized;
7) loading the expression supernatant by AKTA at the speed of 1 mL/min;
8) the column was equilibrated again with loading buffer at channel A1 until the baseline was flat;
9) gradient elution is set, elution is respectively carried out according to the proportion of 0.5M imidazole of 10%, 20%, 30%, 40%, 50%, 60% and 100% until the base line is stable, and samples need to be collected when ultraviolet absorption exists;
10) washing with purified water, and preserving the column with 20% ethanol.
Taking SEQ ID NO.1 as an example, the result is shown in FIG. 2, and as shown in FIG. 2A, after induction with 0.5mM IPTG and 1mM IPTG respectively for 12h, a protein expression band is evident at the position with a molecular weight of 37kDa, which is basically consistent with the theoretical molecular weight of the CD271T fusion protein, which indicates that prokaryotic expression of CD271 is successfully carried out, and the expression amount of CD271 is slightly increased when the concentration of IPTG is increased from 0.5mM to 1 mM. FIGS. 2B and 2C show that, after the engineering bacteria are cultured at two different induction temperatures of 20 ℃ and 37 ℃ and the induction temperature is 20 ℃, the target band in the supernatant after the bacteria are broken becomes more obvious along with the increase of the induction time, the peak is reached after 12h, and the depth change of the target band after 12h is not obvious (FIG. 2B); when the induction temperature is 37 ℃, the band in the supernatant after bacteria breaking becomes more obvious along with the increase of the induction time, and the expression quantity of the target band tends to be stable (2C) after the induction for 4 hours; western Blotting analysis was performed with anti-His tag antibody, and the results showed that the supernatant showed a specific band after disruption of the cells at the same molecular weight position, and that no band was precipitated after disruption of the cells, confirming the correct expression of CD271 protein in the supernatant after disruption of the cells (FIG. 2D).
[ example 2 ] protease cleavage reaction and purification of human CD271T antigen peptide
1. Protease digestion reaction and SDS-PAGE identification of human CD271T antigen peptide
(1) Enzyme digestion system
The enzyme digestion system is shown in Table 1, and the enzyme digestion system is subjected to SDS-PAGE identification after 1h, 2h, 4h and 6h of water bath reaction at 30 ℃.
TABLE 1
(2) SDS-PAGE identification
Sampling 20 μ L before enzyme digestion and 1h, 2h, 4h and 6h after enzyme digestion, adding 5 × reduced SDS-PAGE loading buffer, heating at 95 deg.C for 10min, and centrifuging at 12000rpm for 5 min. 10 μ L of each prepared sample was subjected to SDS-PAGE gel electrophoresis, and the results are shown in FIG. 3A.
2. Purification of human CD271 immune antigen
The enzyme digestion system is expanded according to the reaction system, the reaction is carried out in water bath at 20 ℃ for 2h, a sample is filtered by a 0.45nm filter membrane, then HisTrap FF is used for purification, flow-through is collected for identification (figure 3, B), PBS buffer is replaced by ultrafiltration concentration, and the protein concentration is determined.
[ example 3 ] screening of antibodies Using the antigenic peptide of example 1
1. Antigen preparation
1) Preparation of a priming antigen: taking a 1mL sterile EP tube, diluting 100 mu g of immunogen with PBS to 100 mu L, and placing the diluted immunogen in the EP tube; fully shaking the Freund's complete adjuvant to uniformly mix the deposited bifidobacteria; 100 μ L of freund's complete adjuvant was added to a 5mL sterile EP tube, and the diluted immunogen was added so that the ratio of the volume of antigen to the volume of adjuvant was 1:1, placing a centrifugal tube in an ice box; preparing an ultrasonic crusher, and placing the centrifugal tube in an ice box for ultrasonic treatment. The ultrasonic conditions are total power of 200W, total time of 10min, working time of 5 seconds and interval time of 6 seconds. Observing the emulsification condition of the solution in real time; the emulsified antigen was aspirated with a 1ml disposable syringe and placed in an ice box for future use.
2) Preparing a secondary immune antigen and a tertiary immune antigen: taking a 1mL sterile EP tube, diluting 100 mu g of immunogen with PBS to 100 mu L, and placing the diluted immunogen in the EP tube; fully shaking up Freund's incomplete adjuvant to uniformly mix the deposited Bifidobacterium; 100 mul Freund's incomplete adjuvant was added to a 5mL sterile EP tube, and the diluted immunogen was added to make the ratio of the volume of antigen to the volume of adjuvant 1:1, placing a centrifugal tube in an ice box; preparing an ultrasonic crusher, and placing the centrifugal tube in an ice box for ultrasonic treatment. The ultrasonic conditions are total power 200W, total time 10min, working time 5s and interval time 6 s. Observing the emulsification condition of the solution in real time; the emulsified antigen was aspirated with a 1ml disposable syringe and placed in an ice box for future use.
3) Preparing a booster immune antigen: a1 mL sterile EP tube was taken, 200. mu.g of immunogen was diluted to 400. mu.L with PBS, aspirated with a 1mL disposable syringe, and placed in an ice box for use.
4) Mouse immunization method
Sucking immune antigen by a 1mL syringe, dividing into 2-3 points, each point is about 0.1mL, and carrying out back (or abdomen) subcutaneous injection on the mouse; blood is collected on the third day after the third immunization for antibody titer ELISA detection, the specific results are shown in figure 4, and the serum titers of mice No.1, No.2, No.3, No.4 and No.5 are all 1: 128000 above, 5 mice all had higher reactivity to antigen, indicating that polyclonal antibodies against CD271 were produced.
2. Preparation of feeder cells
Feeder cells promote the growth and propagation of individual or a small number of discrete cells.
1) Sterile surgical instruments and glass plates were prepared, 96-well plates, 5mL disposable syringes, 300mL 70% alcohol, Balb/c mice, serum-free DMEM medium, and sterile PBS.
2) Balb/c mice were sacrificed by pulling their necks and immersed in 70% alcohol for 10 min.
3) The mice were removed and the excess alcohol was drained and placed on a sterilized glass plate.
4) The skin of the mouse abdomen was cut open and separated (without cutting the muscle layer). 5mL of DMEM serum-free culture solution was aspirated by a 5mL syringe, injected into the abdomen of the mouse, and the body of the mouse was gently massaged and shaken to thoroughly mix macrophages into the culture solution. The culture was carefully aspirated back through a syringe and placed into a 15mL centrifuge tube.
5) Repeating the above method for 1-2 times to obtain macrophage as much as possible.
6) Centrifuging at 1500rpm for 10min, and discarding the supernatant. Prepared by suspending the culture solution, about 10 can be taken out of 1 adult Balb/c mouse63-4 pieces of 96-well plates can be paved on each abdominal cavity cell.
PEG-mediated fusion of rat spleen B cells and mouse myeloma cells
1) Cell: sp2/0-Ag14, medium: DMEM and 10% FBS, passage is carried out for 48 hours before fusion, and the bottle bottom is covered by about 80% after 48 hours according to the density.
2) The Balb/c mice were sacrificed by tail-cutting and neck-pulling, and immersed in 70% alcohol for 10 min.
3) The left upper abdomen was cut to the left back of the mouse, the spleen was isolated, connective tissue was removed from the spleen, placed in a clean plate, and a small amount of DMEM basal medium (without serum) was added.
4) The mouse spleen was cut into 4-5 pieces, ground with a 5mL syringe, stoppered at the top, and a small amount of DMEM was added while grinding the edges, and the ground spleen cells were washed into a plate.
5) And (3) transferring all the cells in the plate into a 15mL centrifuge tube, adding a proper amount of DMEM basic culture medium, uniformly mixing, centrifuging for 200g, and carrying out 5 min.
6) Washed twice with PBS, centrifuged, 200g, 5 min.
7) All Sp2/0-Ag14 cells were harvested and washed once with PBS and counted.
8) Will 108Spleen cells and 107The individual myeloma cells were mixed in a 50mL centrifuge tube (the number of myeloma cells was increased depending on the number of harvested cells), 30-40mL serum-free DMEM was added thereto, the mixture was thoroughly mixed, centrifuged at 1500rpm for 3min, and the supernatant was removed.
9) The bottom of the centrifuge tube was tapped to break up the pellet sufficiently.
10) The cells were placed in a37 ℃ water bath, and PEG was slowly dropped in the following manner.
11) Slowly dripping 1mL of PEG solution along the tube wall with a dropper, lasting for 1min, gently oscillating while adding, centrifuging for 2min at 100 g;
12) dripping 4.5mL of DMEM basal medium (preheated and serum-free) by the same method for 3min while gently oscillating;
13) dripping 5mL of DMEM basal medium (preheated and serum-free) by the same method for 2min, and slightly oscillating for 30s after finishing the dripping;
14) slowly adding 45mL of DMEM basal medium (preheating and serum-free),
15) centrifuge at 100g for 5min, remove supernatant, tap the bottom.
16) HAT-containing DMEM complete medium (20% FBS) was added, resuspended gently, cell counted using trypan blue, 100. mu.L/well, and plated into 5 96-well plates.
17)37℃，5％CO2The incubator is used for culturing without shaking the culture plate during culturing.
18) ELISA was performed 11-13 days after the fusion, and the cell growth was measured, and the amount of antibody in the supernatant was sufficiently accumulated without changing the solution 2-3 days before the measurement.
4. Limiting dilution screening of monoclonal
1) Taking out the antibody positive hole cells, and preparing the cell suspension by using HT culture solution. Samples were taken for trypan blue staining and counted.
2) Dispersing 320 cells in 6.4mL of HT culture solution, fully and uniformly mixing, adding into the 1 st to 4 th rows of a 96-well plate by using a discharging gun, wherein each hole is 100 mu L, and enabling 5 cells to be in each hole; adding 3.2mL of HT culture solution into the rest cell sap, mixing, adding 5-8 rows per 100 μ L well to obtain 2.5 cells per well; the remaining cell fluid (1 mL) was added with 2mL of the culture medium, and after mixing well, 9-12 rows were added to 100. mu.L of each well so that 0.625 cells per well were obtained.
3) Placing 96-well plates in CO2The culture was carried out in an incubator at 37 ℃.
4) On the fifth day, the growth of the cells was observed under a microscope, and the wells in which the cells grew were recorded.
5) After cloning and mass propagation, when 1/3-1/2 of the bottom of a hole is filled, an ELISA plate coated by full-length CD271 protein is used for detecting the antibody level in a culture solution.
6) Selecting the cells in the wells with highest Elisa positivity and good growth state from the wells with 0.625 cell per well, and performing the next round of limiting dilution; simultaneously, another well of the same condition is selected and transferred to one well of a 24-well plate containing feeder cells, and then frozen and preserved after amplification.
7) After three rounds of limiting dilution, cells which are screened and meet the strain building conditions are subjected to amplification culture and frozen storage.
5. Preparation method of mouse ascites generated by hybridoma cells
1) 7-10 days before cell injection, 0.5mL of autoclaved pristane (paraffin oil) was injected intraperitoneally into each nude mouse.
2) Recovering the cell strain of the strain to culture and passage, measuring ELISA (enzyme-Linked immuno sorbent assay) on the supernatant, and centrifugally collecting cells when the supernatant is positive, and performing trypan blue staining technology; according to each Balb/c 5X 105Cells were resuspended in 0.2mL PBS.
3) 8 weeks female Balb/c mice were injected intraperitoneally.
4) Ascites began to appear about 1-2 weeks. Ascites extraction can be initiated when the animal body is imaged as a pregnant female mouse. Ascites was withdrawn from the periphery of the abdominal cavity with 5mL syringes, each approximately 2-3 mL.
5) Ascites fluid was centrifuged at 3000rpm (1500g) for 10 minutes, and the supernatant was aliquoted and stored at-70 ℃.
6) Under the condition of keeping the health of the nude mice, the nude mice can be repeatedly extracted at intervals of 1-3 days according to the generation condition of ascites.
Example 4 mouse ascites purification and identification and ForteBio validation of purified antibody affinity
1. Mouse ascites Mab Select SureTMPurification of
1) Ascites from mice were immediately centrifuged after collection and the supernatant was frozen at-80 ℃. Unfreezing at room temperature or 4 ℃ before purification, centrifuging to obtain a supernatant, adding 1/10 volumes of 1M Tris-HCl and 0.5M NaCl, adjusting the pH value to 8.0, filtering by 0.22 mu M to obtain a sample to be loaded on a column, and sampling S;
2) the column was equilibrated with 10CV of 1M Tris-HCl, 0.5M NaCl, pH 8.0;
3) sampling, collecting all flow-through and sampling FL;
4)Mab Select SureTMwashing with 10CV of 100mM Tris-HCl,50mM NaCl, pH 8.0;
5)Mab Select SureTMwashing with 10CV of 10mM Tris-HCl,5mM NaCl, pH 8.0;
6) the collection tube is added with 0.1CV of 1M Tris-HCl pH 9.0 in advance, 0.5CV of 100mM citrate buffer solution pH3.0 is added each time during elution, and the mixture is mixed immediately; elute the Elute of the Elute 1, 2, 3 … tube and sample in half.
2. Purified antibody SDS-PAGE and ELISA identification
1) SDS-PAGE identification
The ascites antibody CD271T-2 obtained by purification was subjected to SDS-PAGE protein electrophoresis analysis, and the specific results are shown in FIG. 5.
2) ELISA identification
All the collected tubes were collected, and after ultrafiltration and centrifugation, protein concentration was determined, and CD271T-2 was identified by ELISA and confirmed to bind to the full-length CD271 protein, with the specific results shown in fig. 6.
Validation of antibody affinity after purification by ForteBio
Example 5 Synthesis and immunological identification of epitope peptide of CD271T-2
An IEDB website is used for predicting antigen epitope, polypeptide SEQ ID NO.3-11 in SEQ ID NO.1 is designed, and the polypeptide is synthesized by Nanjing Kingsler company. The ELISA method is adopted to detect the combination of mouse serum to different polypeptide fragments after the immunization of the CD271T epitope peptide, positive color development can be seen, namely, the polypeptides can cause animal immune reaction, thereby generating corresponding polyclonal antibodies.
Example 6 competitive inhibition ELISA for detection of the minimal recognition epitope of the CD271T-2 monoclonal antibody
Coating: diluting the full-length CD271 protein serving as a coating antigen to 4 mu g/mL by using a coating solution, adding 100 mu L of the coating solution into an ELISA plate per well, and incubating for 2 hours at 37 ℃; and (3) sealing: discarding the envelope antigen, adding 300. mu.L of ELISA washing solution (PBST) to each well for washing 3 times, beating to dry after washing, adding 100. mu.L of 5% BSA (5g BSA, 100mL PBST) to each well, and incubating at 37 ℃ for 2 hours; and (3) sandwich incubation: discarding the confining liquid, adding 300. mu.L of ELISA washing liquid into each well, washing for 3 times, beating to dry after washing, diluting the full-length CD271 protein group with PBS according to a ratio of 1:600, adding 100. mu.L into each well, mixing the full-length CD271 protein with the polypeptide, adding 50. mu.L of the diluted full-length CD271 protein and 50. mu.L of the polypeptide (including SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11 in example 5) corresponding to 16. mu.g/mL into each well, and incubating for 1 hour at 37 ℃; and (3) secondary antibody incubation: discarding the sandwich, adding 300 mu L of ELISA washing solution into each hole, washing for 3 times, drying after washing, diluting HRP-labeled goat-anti-mouse secondary antibody with PBST at a ratio of 1:10000, adding 100 mu L of HRP-labeled goat-anti-mouse secondary antibody into each hole, and incubating for 0.5 hour at 37 ℃; developing with a developing solution: discarding the secondary antibody, adding 300 mu L of ELISA washing solution into each hole, washing for 3 times, beating to dry after washing, adding 100 mu L of TMB single-component color development solution into each hole, and developing for 10min at room temperature in a dark place; reading: after the completion of the color development, 50. mu.L of 0.2M H2SO4 stop solution was added to each well, and the absorbance (OD450) was measured at a wavelength of 450nm using a microplate reader, and the results are shown in FIG. 8. The group to which the polypeptide of SEQ ID NO.10 was added had the lowest OD450, which was the smallest epitope recognized by CD271T-2, compared to the other groups.
Example 7 cloning of heavy and light chain variable region Gene of anti-human CD271T-2 and construction of chimeric antibody 2
RNA extraction: adopting Trizol one-step method to obtain hybridoma cell CD271T-2 about 106Adding Trizol, extracting RNA according to an Ultrapure RNA Kit CW0581S Kit in the Kangji century, and identifying the integrity of the RNA by nucleic acid electrophoresis; the preservation number of the hybridoma CD271T-2 is CGMCC 21494. Is preserved in China general microbiological culture collection management center with the preservation address of No.3 Xilu No.1 Beijing north Chen of the rising area. The preservation date is 2021 year, 1 month and 25 days. Classified and named as hybridoma cell strain CD 271T-2;
2. reverse transcription to cDNA (20 μ L): taking Oligo dT Primer (50 muM) 1 muL, dNTP mix (10nM)1 muL and template RNA 5 mug, adding water to 10 muL, keeping the temperature at 65 ℃ for 5min, and rapidly cooling on ice; adding 10 μ L of the above denatured reaction solution, 4 μ L of 5 × primescript II Buffer, 0.5 μ L of RNase Inhibitor (40U/μ L), 1 μ L of PrimeScirpt II RTase (200U/μ L), adding water to 20 μ L, reacting at 42 deg.C for 60min, and inactivating at 70 deg.C for 15 min;
PCR amplification of heavy and light chain variable region genes of anti-CD 271 antibody: the light and heavy chain variable region gene PCR amplification is operated in Hongxin biotechnology company of Suzhou, and the PCR result is shown in FIGS. 9A and 9B;
construction and sequencing of sequencing vector: PMD19-T vector was purchased from takara, heavy and light chain variable region gene pcr products were recovered, ligated with T vector, DH5 α transformed, positive clones were selected at 100 μ g/mL ampicillin concentration, sequenced and aligned with several conserved framework amino acids in the protein database. The obtained antibody comprises a light chain and a heavy chain, wherein the light chain complementarity determining region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is shown in SEQ ID NO.12, the amino acid sequence of the LCDR2 is shown in SEQ ID NO.13, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO. 14; the heavy chain complementarity determining region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown as SEQ ID NO.15, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.16, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 17.
In particular, the method comprises the following steps of,
light chain complementarity determining region LCDR1, length: 10aa, the amino acid sequence is shown as SEQ ID NO.12, and specifically comprises: QSVDYDGDSY are provided.
Light chain complementarity determining region LCDR2, length: 3aa, the amino acid sequence is shown as SEQ ID NO.13, and specifically comprises: and (5) AAS.
Light chain complementarity determining region LCDR3, length: 9aa, the amino acid sequence is shown as SEQ ID NO.14, and specifically comprises: QQSNEDPFT are provided.
Heavy chain complementarity determining region HCDR1, length: 10aa, the amino acid sequence is shown as SEQ ID NO.15, and specifically comprises: GFSLSTSGMG are provided.
Heavy chain complementarity determining region HCDR2, length: 7aa, the amino acid sequence is shown as SEQ ID NO.16, and specifically comprises: IWWDDDK.
Heavy chain complementarity determining region HCDR3, length: 13aa, the amino acid sequence is shown as SEQ ID NO.17, and specifically comprises: ARRDYGNYYAMDY are provided.
The light chain variable region amino acid sequence is shown as SEQ ID NO.18, and specifically comprises the following components:
DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPFTFGSGTKLEIK。
the heavy chain variable region amino acid sequence is shown as SEQ ID NO.19, and specifically comprises the following components:
QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTAGTATYYCARRDYGNYYAMDYWGQGTSVTVS。
light chain complementarity determining region LCDR1, DNA, length: 30bp, the nucleotide sequence is shown as SEQ ID NO.20, and the nucleotide sequence specifically comprises: caaagtgttgattatgatggtgatagttat are provided.
Light chain complementarity determining region LCDR2, DNA, length: 9bp, the nucleotide sequence is shown as SEQ ID NO.21, and the nucleotide sequence specifically comprises: gctgcatcc.
Light chain complementarity determining region LCDR3, DNA, length: 27bp, the nucleotide sequence is shown as SEQ ID NO.22, and the nucleotide sequence specifically comprises: cagcaaagtaatgaggatccattcacg are provided.
Heavy chain complementarity determining region HCDR1, DNA, length: 30bp, the nucleotide sequence is shown as SEQ ID NO.23, and the nucleotide sequence specifically comprises the following components: gggttttcactgagcacttctggtatgggt are provided.
Heavy chain complementarity determining region HCDR2, DNA, length: 21bp, the nucleotide sequence is shown as SEQ ID NO.24, and the nucleotide sequence specifically comprises: atttggtgggatgatgataag are provided.
Heavy chain complementarity determining region HCDR3, DNA, length: 39bp, the nucleotide sequence is shown as SEQ ID NO.25, and the nucleotide sequence specifically comprises the following components: gctcgaagggactatggtaactactatgctatggactac are provided.
The nucleotide sequence of the light chain variable region is shown as SEQ ID NO.26, and specifically comprises the following steps:
gacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatatgaactggtaccaacagaaaccaggacagccacccaaactcctcatctatgctgcatccaatctagaatctgggatcccagccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcaaagtaatgaggatccattcacgttcggctcggggacaaagttggaaataaaa。
the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.27, and specifically comprises the following steps:
caagttactctaaaagagtctggccctgggatattgaagccctcacagaccctcagtctgacttgttctttctctgggttttcactgagcacttctggtatgggtgtaggctggattcgtcagccttcagggaagggtctggagtggctggcacacatttggtgggatgatgataagtactataacccatccctgaagagccagctcacaatctccaaggatacctccagaaaccaggtattcctcaagatcaccagtgtggacactgcagatactgccacttactactgtgctcgaagggactatggtaactactatgctatggactactggggtcaaggaacctcagtcaccgtctcctcag。
4. the light chain LCDR 1-3 and the heavy chain HCDR 1-3 are adopted to be fused with a human IgG molecule constant region to construct plasmids, a CMV promoter is adopted to perform transfection and expression in HEK293 cells and CHO cells, protein A affinity chromatography is performed, a humanized CD271 monoclonal antibody is obtained through purification, the engineered CD271 antibody and the CD271 protein have good affinity through Fortbio detection, and the equilibrium dissociation constant reaches the nM level.
Example 8 application of the CD271T-2 monoclonal antibody to flow detection of tumor cells
A375-S2 is a human melanoma cell line, and the CD271 molecule is highly expressed in a cell subset with characteristics of tumor stem cells, and the recognition ability of the CD271T-2 monoclonal antibody on the cells is examined by the detection method of example 8. The result shows that the APC coat anti human CD271 positive control antibody and the CD271T-2 monoclonal antibody can detect more than 50% of positivity, while the Rabbit anti human CD271 antibody and the negative control have no deviation, which indicates that the CD271T-2 antibody has good affinity for tumor cells expressing CD271 molecules, and can be used for diagnosis, treatment and other applications of tumors.
Example 9 application of CD271T-2 monoclonal antibody to mesenchymal stem cell flow assay
Taking properly well-grown mesenchymal stem cells Hu-MSC, carrying out trypsinization centrifugation collection, carrying out heavy suspension washing by using FACS (PBS + 2% FBS), centrifuging at 1000rpm for 3min, and discarding the supernatant; add primary antibody according to table 3, incubate in dark at room temperature for 30min, resuspend wash with FACS (PBS + 2% FBS), centrifuge 1000rpm, 3min, repeat 3 times, discard supernatant, add secondary antibody according to table 3, photophobic at room temperature for 30min, resuspend wash with FACS (PBS + 2% FBS), centrifuge 1000rpm, 3min, repeat 3 times, add 300 μ L FACS to resuspend cells, detect with flow cytometer, see figure 10 for specific results. It is shown that the positive control antibody derived from APC, Goat anti human CD271 from Biolegend, the immunogen is from melanoma cells, the cell surface of which highly expresses CD271 molecules, and this antibody slightly migrates compared to the negative control (FIG. 10A); the Rabbit anti human CD271 control antibody derived from Abcam, the immunogen from the 350-450 amino acid region in the human CD271 cell membrane, did not have any shift compared to the negative control (FIG. 10B); the ascites purified antibody to CD271T-2 was more highly deviated than the positive and negative controls, indicating that the prepared anti-human CD271 monoclonal antibody has very high affinity and specificity to CD271 on the surface of mesenchymal cells (fig. 10C).
TABLE 3
The design method and preparation of the CD271T immune epitope can be applied to screening anti-human CD271 antibodies and aptamers with high affinity and high specificity, and can be applied to developing aptamers for identifying CD 271.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Sequence listing
<110> Shanghai university of transportation
<120> novel epitope based on CD271 and application thereof
<160> 27
<170> SIPOSequenceListing 1.0
<210> 1
<211> 62
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Glu Glu Ile Pro Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly
1 5 10 15
Ser Asp Ser Thr Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu
20 25 30
Gln Asp Leu Ile Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met
35 40 45
Gly Ser Ser Gln Pro Val Val Thr Arg Gly Thr Thr Asp Asn
50 55 60
<210> 2
<211> 186
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gaggagatcc ctggccgttg gattacacgg tccacacccc cagagggctc ggacagcaca 60
gcccccagca cccaggagcc tgaggcacct ccagaacaag acctcatagc cagcacggtg 120
gcaggtgtgg tgaccacagt gatgggcagc tcccagcccg tggtgacccg aggcaccacc 180
gacaac 186
<210> 3
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Thr Arg Ser Thr Pro Pro Glu Gly Ser Asp Ser Thr Ala Pro Ser Thr
1 5 10 15
Gln Glu Pro Glu
20
<210> 4
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Glu Pro Glu Ala Pro Pro Glu Gln Asp Leu Ile Ala Ser Thr Val Ala
1 5 10 15
Gly Val Val
<210> 5
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Val Thr Thr Val Met Gly Ser Ser Gln Pro Val Val Thr Arg Gly Thr
1 5 10 15
Thr Asp Asn
<210> 6
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Leu Ile Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met Gly Ser
1 5 10 15
Ser Gln Pro
<210> 7
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Glu Glu Ile Pro Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly
1 5 10 15
Ser Asp Ser
<210> 8
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Asp Ser Thr Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu Gln
1 5 10 15
Asp Leu Ile
<210> 9
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Gly Ser Asp Ser Thr Ala Pro Ser Thr Gln
1 5 10
<210> 10
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Asp Ser Thr Ala Pro Ser Thr Gln Glu Pro
1 5 10
<210> 11
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Thr Pro Pro Glu Gly Ser Asp
1 5
<210> 12
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr
1 5 10
<210> 13
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Ala Ala Ser
1
<210> 14
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Gln Gln Ser Asn Glu Asp Pro Phe Thr
1 5
<210> 15
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gly Phe Ser Leu Ser Thr Ser Gly Met Gly
1 5 10
<210> 16
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Ile Trp Trp Asp Asp Asp Lys
1 5
<210> 17
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Ala Arg Arg Asp Tyr Gly Asn Tyr Tyr Ala Met Asp Tyr
1 5 10
<210> 18
<211> 111
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 19
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30
Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu
35 40 45
Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val
65 70 75 80
Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Gly Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Arg Asp Tyr Gly Asn Tyr Tyr Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Ser Val Thr Val Ser
115 120
<210> 20
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
caaagtgttg attatgatgg tgatagttat 30
<210> 21
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gctgcatcc 9
<210> 22
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
cagcaaagta atgaggatcc attcacg 27
<210> 23
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gggttttcac tgagcacttc tggtatgggt 30
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
atttggtggg atgatgataa g 21
<210> 25
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gctcgaaggg actatggtaa ctactatgct atggactac 39
<210> 26
<211> 333
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60
atctcctgca aggccagcca aagtgttgat tatgatggtg atagttatat gaactggtac 120
caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa tctagaatct 180
gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240
cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtaatga ggatccattc 300
acgttcggct cggggacaaa gttggaaata aaa 333
<210> 27
<211> 364
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
caagttactc taaaagagtc tggccctggg atattgaagc cctcacagac cctcagtctg 60
acttgttctt tctctgggtt ttcactgagc acttctggta tgggtgtagg ctggattcgt 120
cagccttcag ggaagggtct ggagtggctg gcacacattt ggtgggatga tgataagtac 180
tataacccat ccctgaagag ccagctcaca atctccaagg atacctccag aaaccaggta 240
ttcctcaaga tcaccagtgt ggacactgca gatactgcca cttactactg tgctcgaagg 300
gactatggta actactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 360
tcag 364