WO2022166806A1 - 一种基于cd271的新型抗原表位及其应用 - Google Patents

一种基于cd271的新型抗原表位及其应用 Download PDF

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WO2022166806A1
WO2022166806A1 PCT/CN2022/074573 CN2022074573W WO2022166806A1 WO 2022166806 A1 WO2022166806 A1 WO 2022166806A1 CN 2022074573 W CN2022074573 W CN 2022074573W WO 2022166806 A1 WO2022166806 A1 WO 2022166806A1
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epitope peptide
acid sequence
seq
antibody
amino acid
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路慧丽
王紫嫣
杨慧
朱建伟
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Shanghai Jiao Tong University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001116—Receptors for cytokines
    • A61K39/001117—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR] or CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biotechnology, in particular to a novel antigenic epitope peptide of CD271 and its application.
  • CD271 is a low-affinity nerve growth factor receptor (LNGFR), a member of the tumor necrosis factor receptor superfamily with a relative molecular mass of 75kD, also known as p75NTR, which contains 3 regions: cysteine-rich The extra-membrane domain, the transmembrane domain and the intracellular domain consisting of 155 amino acid residues.
  • LNGFR low-affinity nerve growth factor receptor
  • p75NTR a member of the tumor necrosis factor receptor superfamily with a relative molecular mass of 75kD, also known as p75NTR, which contains 3 regions: cysteine-rich The extra-membrane domain, the transmembrane domain and the intracellular domain consisting of 155 amino acid residues.
  • CD271 is not only expressed in the nervous system, but also closely related to the development, differentiation and survival of nerve cells, and can also identify tumor stem cells and mesenchymal stem cells. In the field of tumor treatment, CD271 is highly expressed on the surface of various
  • CD271 antibody can be used for magnetic sorting of mesenchymal stem cells by flow sorting or coupling to magnetic beads.
  • the enriched cells have the advantages of high specificity, high purity, and strong colony formation ability. . Therefore, antibodies that specifically recognize CD271 have a wide range of applications, and stable and efficient antigenic epitopes are of great significance for the development of CD271 monoclonal antibodies or CD271-based vaccines.
  • the extracellular structure of native CD271 includes four cysteine-rich CRD structures (CRD1-4) and a linker region (stalk region).
  • the existing CD271 antibody products are mainly various scientific research reagents, and their antigenic epitopes are mainly cells expressing CD271, such as melanoma cells, or the CRD region of CD271.
  • CD271 the CRD region will be hydrolyzed by protease and then fall off, so the binding of antibodies targeting the CRD region to CD271-positive cells will be affected, and even the expression of CD271 cannot be detected at a certain stage of cell metabolism.
  • New epitopes need to be developed to provide new tools for CD271-based antibody, vaccine research and related detection or disease treatment.
  • the present invention provides a CD271-based novel antigenic epitope peptide and its application to solve the deficiencies of the prior art.
  • a first aspect of the present invention provides an epitope peptide of CD271, and the epitope peptide is:
  • (2) is located in an amino acid sequence having at least 80% identity with the sequence of SEQ ID NO.1 in (1);
  • SEQ ID NO.1 contains one or more antigenic epitopes, and the amino acid sequence length of the antigenic 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.
  • epitope peptide is located in the continuous amino acid sequence in the amino acid sequence of positions 1 to 35 in SEQ ID NO.1.
  • epitope peptide is located in the continuous amino acid sequence in the amino acid sequence of positions 11 to 35 in SEQ ID NO.1.
  • epitope peptide is located in the continuous amino acid sequence in the amino acid sequence of positions 18 to 27 in SEQ ID NO.1.
  • the epitope peptide is an amino acid sequence with a degree of overlap of more than 80% with the consecutive amino acids at positions 18 to 27 in SEQ ID NO.1.
  • amino acid sequence of the epitope peptide is shown in any of SEQ ID NO.3-11.
  • an antigenic epitope peptide Peptide length: 19aa, sequence as shown in SEQ ID NO.3, specifically: TRSTPPEGSDSTAPSTQEPE.
  • the second aspect of the present invention provides a fusion protein, which is formed by fusing the antigenic epitope peptide described in any of the above with a carrier protein.
  • a third aspect of the present invention provides a nucleic acid sequence capable of encoding an antigenic epitope peptide, the nucleic acid sequence being a gene sequence or gene fragment capable of encoding the antigenic epitope peptide described above.
  • SEQ ID NO.2 the nucleotide sequence encoding SEQ ID NO.1 is shown in SEQ ID NO.2,
  • the fourth aspect of the present invention provides an expression vector, the expression vector contains the above-mentioned nucleic acid sequence.
  • a fifth aspect of the present invention provides a host cell, wherein the host cell contains the above-mentioned expression vector, or integrates the above-mentioned nucleic acid sequence into the genome.
  • the sixth aspect of the present invention provides an antibody, characterized in that: the antibody is an antibody capable of binding the epitope peptide according to any one of the above claims.
  • the antibody is an antibody capable of inhibiting or attenuating the signaling associated with the epitope peptide described in any one of the above.
  • the antibody is a monoclonal antibody or a polyclonal antibody
  • the species source can be mammals such as human, mouse, rabbit, monkey, cow, sheep or alpaca.
  • the sixth aspect of the present invention provides a vaccine, which contains the antigenic epitope peptide, fusion protein, nucleic acid sequence, expression vector or host cell described in any one of the above.
  • the seventh aspect of the present invention provides a composition, which is characterized in that it contains the antigenic epitope peptide, fusion protein, nucleic acid sequence, expression vector, host cell, antibody or vaccine described in any of the above, as well as immunological and pharmaceutical acceptable carriers and/or excipients.
  • the eighth aspect of the present invention provides the use of the antigenic epitope peptide, fusion protein, nucleic acid sequence, expression vector or host cell described in any of the above:
  • the antigenic epitope peptide of the present invention has immunogenicity, it can induce an immune response
  • the Epitope peptides or gene molecules, fusion proteins, vectors or host cells containing the epitope peptides can be used to prepare antibodies, nucleic acid aptamers, vaccines, nanoparticles targeting CD271, etc., and have important application significance.
  • the epitope peptide is selected from the sequence of CD271 target that is stable outside the membrane, has low homology, and has no glycosylation and phosphorylation sites. Cells have tighter and more stable associations.
  • the antigenic epitope peptide has specific binding with the antibody in the present application, has a strong affinity, the equilibrium dissociation constant can reach the nM level, and the binding is stable, and will not be affected by hydrolysis of the CRD region of the CD271 molecule on the cell surface. This plays an extremely important role for antigens or antibodies to play a drug role, which makes it have great potential value for preparation as diagnostic reagents or drugs, and can be used in stem cell tissue repair, nervous system diseases, tumors and other fields related to CD271.
  • Figure 1 is a schematic diagram of the construction of the expression vector plasmid pCD271T of the polypeptide chain SEQ ID NO.1 (named CD271T) where the antigenic epitope peptide is located.
  • the CD271T is fused and expressed with the sumo tag, and the vector is pET-28a(+);
  • Fig. 2 is a graph showing the optimized SDS-PAGE (Fig. 2A & 2B & 2C) and Western Blotting (Fig. 2D) of the SEQ ID NO.1 epitope peptide induced expression in E. coli.
  • the molecular weight of the fusion protein containing CD271T is about 37kDa.
  • Figure 3 shows the SDS-PAGE detection of the Sumo tag and CD271T protein released after the fusion protein was digested by Sumo protease: SDS-PAGE identification was performed after 1h, 2h, 4h and 6h of digestion at 37°C ( Figure 3A), showing that the protein was about 2h It has been completely digested, and the target band is the same size as SUMO tag and CD271T, and the target protein is further enriched in the later purification (Figure 3B);
  • Figure 4 is a graph showing the titer detection of the polyclonal antibody in mouse serum after immunization with CD271 antigen polypeptide by ELISA: the coating antigen concentration was 4 ⁇ g/mL, and the loading serum was diluted from 1:2000 to 1:256000, No. 1 and No. 2
  • the serum titers of mice No. 3, No. 4, and No. 5 are all above 1:128,000, and the sera of 5 mice all contain polyclonal antibodies that recognize the CD271 polypeptide antigen, which has a strong affinity for the antigen polypeptide;
  • FIG. 5 is the SDS-PAGE detection chart of ascites monoclonal antibody CD271T-2 screened by CD271 antigen epitope polypeptide immunized mice: Band 1 is a non-reduced sample, the antibody band is relatively simple, and band 2 is a reduced sample. 26kDa and 53kDa are the light chain LC and heavy chain HC of the antibody, respectively;
  • Figure 6 shows the ELISA identification of the purified CD271T-2 monoclonal antibody: the coating antigen is CD271 epitope polypeptide, the antigen concentration is 2 ⁇ g/mL, each well is 100 ⁇ L, and the loading antibody concentration gradient is 0.625 ⁇ g/mL, 1.25 ⁇ g/mL mL, 2.5 ⁇ g/mL, 5 ⁇ g/mL, 10 ⁇ g/mL, 20 ⁇ g/mL, 40 ⁇ g/mL, 100 ⁇ L per well, the results showed that the CD271T-2 antibody could recognize and bind the full-length human CD271 protein with native structure;
  • Fig. 7 is the antibody affinity map after verification and purification by ForteBio, it can be seen that the CD271 epitope polypeptide of the present invention has a strong affinity with the CD271T-2 monoclonal antibody obtained by immunizing mice;
  • Figure 8 is the antigenic epitope competitive inhibition ELISA detection of CD271T-2 monoclonal antibody: the coating antigen is CD271 protein, the antigen concentration is 4 ⁇ g/mL, each well is 100 ⁇ L, the dilution ratio of the antibody in the loading ascites is 1:600, and each polypeptide is The concentration was 16 ⁇ g/mL, 100 ⁇ L per well, and the ascites antibody was mixed with the polypeptide 1:1. The results showed that the polypeptide 2-4 had the effect of blocking the binding of the CD271T-2 monoclonal antibody to the CD271 protein, which was recognized by the monoclonal antibody. minimal epitope;
  • Figure 9 is the electrophoresis image of the heavy and light chain variable region gene clone PCR fragment of anti-human CD271T-2:
  • Figure 9A is the gene clone PCR fragment of the CD271T-2 hybridoma heavy and light chain variable region,
  • Figure 9B is the CD271T-2 heavy and light Chain variable region chimeric ScFv gene clone PCR fragment;
  • Figure 10 shows the expression of CD271 on the surface of mesenchymal stem cells detected by anti-human CD271 monoclonal antibody:
  • Figure 10A is the positive control antibody of APC mouse anti human CD271 from Biolegend, the immunogen comes from melanoma cells, which highly express CD271 on the cell surface molecule, this antibody has a slight migration compared with the negative control;
  • Figure 10B shows the Rabbit anti human CD271 control antibody derived from Abcam, the immunogen comes from the 350-450 amino acid region in the human CD271 cell membrane, and there is no shift compared with the negative control ;
  • Figure 10C the CD271T-2 monoclonal antibody has a higher degree of deviation compared with the positive and negative controls, indicating that the prepared anti-human CD271 monoclonal antibody has very high affinity and specificity for cells expressing CD271 molecules , superior to commercially available antibodies targeting other epitopes of CD271.
  • the design of the antigen needs to satisfy two points.
  • the antigen will not produce an excessively strong immune response, and at the same time, it can produce antibodies that have the ability to bind to the target protein.
  • Antibody The conformation of the protein will affect the interaction between the antibody and its recognition region. If the antibody recognition region is hidden inside the protein, the antibody will not be able to interact. Most antibodies are directed against continuous recognition regions, and antibodies can bind to such regions with high affinity.
  • the surface sequence is not inside the protein. Discontinuous recognition regions represent polypeptide sequences with certain folds, or antibody recognition regions that connect two separate polypeptides together.
  • antibodies against such a discontinuous recognition region can also be produced, but the antigenic polypeptide used for immunization must have a secondary structure similar to the discontinuous recognition region, and the length of the sequence needs to meet the relevant requirements.
  • the N and C ends are usually exposed on the surface of the protein, while the C-terminus of membrane proteins is too hydrophobic to be suitable for use as an antigen.
  • proteins derived from mammalian cells may have modifications such as glycosylation on the surface. Therefore, it is difficult to ensure that the antibodies screened by using immunogens derived from mammalian cells bind amino acid epitopes.
  • antibodies derived from E. coli expressed immunogens or synthetic polypeptides can recognize amino acid epitopes, but since immunogens may not have higher-order structures, or have higher-order structures that are different from the natural conformation, the Screened antibodies may not recognize native antigens, especially cell surface antigens. In order to obtain antibodies that can recognize cell surface antigens, we designed this immunization strategy.
  • the immunizing antigens are derived from polypeptide synthesis or prokaryotic expression, and the antigens are full-length molecules for expression in mammalian cells to ensure that their spatial structure is in a natural state.
  • polyclonal serum titer detection and hybridoma screening two antigens are used alternately, and hybridoma cell lines and antibodies that meet the requirements are finally obtained.
  • the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology.
  • “Pharmaceutically acceptable carriers and/or excipients” should be compatible with the active ingredient, ie be able to be blended with it without greatly reducing the effect of the drug under normal circumstances.
  • Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, Ethyl cellulose and methyl cellulose; tragacanth powder; 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 oils, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycols; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl
  • Vaccine compositions contain immunizing antigens, including recombinant proteins of the invention, and are typically combined with "pharmaceutically acceptable carriers” including any carrier that does not itself induce the production of antibodies detrimental to the individual receiving the composition.
  • Suitable carriers are usually large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and the like. These vectors are well known to those of ordinary skill in the art. Additionally, these carriers can act as immunostimulatory agents ("adjuvants").
  • CD271 protein The full-length amino acid sequence of CD271 protein was retrieved according to NCBI, and the antigenic determinant was analyzed by DNAStar software, IEDB website, etc., and finally the epitope peptide was determined to be located in SEQ ID NO. , a series of epitope peptides are designed to satisfy:
  • (2) is located in an amino acid sequence having at least 80% identity with the sequence of SEQ ID NO. 1 in (1).
  • CD271 protein and the amino acid sequence of CD271T were retrieved according to NCBI, and the prokaryotic expression codon was optimized by Suzhou Hongxun Co., Ltd., and cloned into the pET-28a(+) vector together with the Sumo tag to synthesize the CD271T antigen expression plasmid pCD271T, such as Figure 1.
  • the supernatant and pellet were added to 5 ⁇ reduced SDS-PAGE loading buffer respectively, heated at 95°C for 10min, and centrifuged at 12000rpm for 5min. Take 10 ⁇ L of the prepared samples for SDS-PAGE gel electrophoresis and Western Blotting detection to determine the final IPTG concentration, induction temperature and induction time.
  • the high-pressure homogenizer is sterilized and turned on in advance, rinsed with 20% ethanol and water successively, and then poured into the bacterial liquid.
  • Figures 2B and 2C show that after the engineered bacteria were cultured at two different induction temperatures and induction times at 20°C and 37°C, when the induction temperature was 20°C, with the increase of induction time, the target bands in the supernatant after the bacteria were broken became more and more It was obvious that the peak was reached after 12h, and the depth of the target band was not changed significantly after 12h (Fig. 2B); when the induction temperature was 37 °C, with the increase of the induction time, the bands in the supernatant after the bacteria were broken became more and more obvious.
  • the expression level of the target band tends to be stable after 4 hours of induction (2C);
  • Western Blotting analysis with anti-His tag antibody shows that the supernatant shows specific bands after the bacterial cells are broken at the same molecular weight position, and no precipitate is observed after the bacterial cells are broken. band, confirming the correct expression of CD271 protein in the supernatant after cell disruption (Fig. 2D).
  • the enzyme digestion system is shown in Table 1. The reaction was performed in a water bath at 30°C for 1h, 2h, 4h, and 6h, and SDS-PAGE was performed for identification.
  • Example 3 Screening of antibodies using the antigenic peptides in Example 1
  • Antigen preparation for secondary immunity and tertiary immunity take a 1 mL sterile EP tube, dilute 100 ⁇ g of immunogen with PBS to 100 ⁇ L and place it in the EP tube; shake the incomplete Freund’s adjuvant well to mix the precipitated bifidobacteria evenly ; Take 100 ⁇ L of incomplete Freund's adjuvant and add it to a 5 mL sterile EP tube, then add the diluted immunogen to make the volume ratio of antigen and adjuvant 1:1, and place the centrifuge tube in an ice box; prepare Good sonicator, put the centrifuge tube in the ice box to sonicate.
  • Ultrasonic conditions were total power 200W, total time 10min, working time 5s, and interval time 6s.
  • the emulsification of the solution was observed in real time; the emulsified antigen was inhaled with a 1 ml disposable syringe and placed in an ice box for later use.
  • the immunizing antigen was inhaled with a 1 ml syringe, divided into 2-3 points, each point of about 0.1 mL, and subcutaneously injected into the back (or abdomen) of the mice; blood was collected on the third day after the three immunizations for antibody titer ELISA detection, the specific results are shown in the figure
  • the serum titers of mice No. 4, No. 1, No. 2, No. 3, No. 4 and No. 5 were all above 1:128,000, and the 5 mice had high reactivity to the antigen, indicating that anti-CD271 polyclonal antibodies were produced. .
  • Feeder cells can promote the growth and reproduction of single or a few scattered cells.
  • Cells Sp2/0-Ag14
  • medium DMEM+10% FBS
  • passaged 48 hours before fusion and the density should be about 80% of the bottom of the bottle covered after 48 hours.
  • mice Balb/c mice were killed by tail clipping and neck pulling, and immersed in 70% alcohol for 10 min.
  • an ELISA plate coated with full-length CD271 protein is used to detect the antibody level in the culture medium.
  • each nude mouse was intraperitoneally injected with 0.5 mL of autoclaved pristane (paraffin oil).
  • mice Female Balb/c mice were injected intraperitoneally for 8 weeks.
  • ascites begins to appear. Ascites can be drawn when the animal is the size of a pregnant female. With a 5mL syringe, ascites was drawn from the periphery of the abdominal cavity, about 2-3mL per animal.
  • the extraction can be repeated at intervals of 1-3 days according to the situation of ascites.
  • mice ascites was centrifuged immediately after collection, and the supernatant was frozen at -80°C. Before purification, thaw at room temperature or 4 degrees, centrifuge to take the supernatant, add 1/10 volume of 1M Tris-HCl, 0.5M NaCl, pH 8.0 to adjust the pH value, filter at 0.22 ⁇ m to be the sample to be loaded on the column, and sample S;
  • the purified ascites antibody CD271T-2 was subjected to SDS-PAGE protein electrophoresis analysis, and the specific results are shown in Figure 5.
  • the protein in all collection tubes was collected, and the protein concentration was determined after ultrafiltration and centrifugation.
  • the CD271T-2 was identified by ELISA, and it was confirmed that it could bind to the CD271 full-length protein. The specific results are shown in Figure 6.
  • the IEDB website was used to predict the antigenic epitope, and the polypeptides SEQ ID NO.3-11 located in SEQ ID NO.1 were designed and sent to Nanjing GenScript for synthesis.
  • the ELISA method was used to detect the binding of different polypeptide fragments in the serum of mice immunized with CD271T epitope peptides, and positive color development was observed, that is, these polypeptides could induce immune responses in animals, thereby producing corresponding polyclonal antibodies.
  • Coating Use the full-length CD271 protein as the coating antigen, dilute it with coating solution to 4 ⁇ g/mL, add 100 ⁇ L per well to the ELISA plate, and incubate at 37°C for 2 hours; Blocking: Discard the coating antigen and add 300 ⁇ L to each well ELISA washing solution (PBST) was washed 3 times, patted dry after washing, then added 100 ⁇ L 5% BSA (5 g BSA, 100 mL PBST) to each well, incubated at 37°C for 2 hours; sandwich incubation: discard the blocking solution and add 300 ⁇ L to each well The ELISA washing solution was washed three times, and patted dry after washing.
  • PBST ELISA washing solution
  • the full-length CD271 protein group was diluted 1:600 with PBS, and 100 ⁇ L was added to each well.
  • 50 ⁇ L of the diluted full-length CD271 was added to each well.
  • Protein and 50 ⁇ L of 16 ⁇ g/mL corresponding polypeptide (including SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 in Example 5), incubated at 37°C for 1 hour; secondary antibody incubation: discard the sandwich, each Add 300 ⁇ L of ELISA washing solution to the wells to wash 3 times, pat dry after washing, dilute the HRP-labeled goat anti-mouse secondary antibody with PBST at a ratio of 1:10000, add 100 ⁇ L to each well, and incubate at 37°C for 0.5 hours; : Discard the secondary antibody, add 300 ⁇ L of ELISA washing solution to each well to wash 3 times, pat dry after washing, add 100 ⁇ L of TMB single-component color developing solution to each well, and develop color at room temperature for 10 minutes in the dark; Add 50 ⁇ L of 0.2M H2SO4 stop solution to the well, and use a microplate reader to measure the absorbance (OD450) at a wavelength of 450 nm.
  • RNA extraction using Trizol one-step method, take about 10 6 hybridoma cells CD271T-2, add Trizol, extract RNA according to Kangwei Century Ultrapure RNA Kit CW0581S kit, and identify RNA integrity by nucleic acid electrophoresis; it should be noted that, Hybridoma cell CD271T-2 has an accession number of CGMCC 21494. It is preserved in the China General Microorganism Culture Collection and Management Center, and the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is January 25, 2021. The classification is named as hybridoma cell line CD271T-2;
  • Reverse transcription into cDNA (20 ⁇ L): take 1 ⁇ L of Oligo dT Primer (50 ⁇ M), 1 ⁇ L of dNTP Mixture (10 nM), 5 ⁇ g of template RNA, add water to 10 ⁇ L, incubate at 65°C for 5 min, and quickly cool on ice; add the above denaturation reaction solution 10 ⁇ L, 5x primerscript II Buffer 4 ⁇ L, RNase Inhibitor (40U/ ⁇ L) 0.5 ⁇ L, PrimeScirpt II RTase (200U/ ⁇ L) 1 ⁇ L, add water to 20 ⁇ L, react at 42°C for 60min, 70°C, 15min inactivation;
  • PCR amplification of heavy and light chain variable region genes of anti-CD271 antibody PCR amplification of light and heavy chain variable region genes was performed in Suzhou Hongxun Biotechnology Co., Ltd. The PCR results are shown in Figure 9A&9B;
  • PMD19-T vector was purchased from takara, the heavy and light chain variable region gene PCR products were recovered, and after ligation with the T vector, DH5 ⁇ transformation was performed, and positive clones were screened at a concentration of 100 ⁇ g/mL ampicillin, and sent for sequencing. Alignment with several conserved framework amino acids possessed by protein databases.
  • the obtained antibody includes a light chain and a heavy chain
  • the light chain complementarity determining region includes LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO.12, and the amino acid sequence of the LCDR2 is as shown in SEQ ID NO.13 shown, the amino acid sequence of the LCDR3 is shown in SEQ ID NO.14;
  • the heavy chain complementarity determining region includes HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO.15, the HCDR2
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO.16, and the amino acid sequence of HCDR3 is shown in SEQ ID NO.17.
  • Light chain complementarity determining region LCDR1 length: 10aa, amino acid sequence as shown in SEQ ID NO.12, specifically: QSVDYDGDSY.
  • Light chain complementarity determining region LCDR2 length: 3aa, amino acid sequence as shown in SEQ ID NO.13, specifically: AAS.
  • Heavy chain complementarity determining region HCDR2 length: 7aa, amino acid sequence as shown in SEQ ID NO.16, specifically: IWWDDDK.
  • the light chain variable region amino acid sequence is shown in SEQ ID NO.18, specifically:
  • the heavy chain variable region amino acid sequence is shown in SEQ ID NO.19, specifically:
  • Light chain complementarity determining region LCDR1 DNA, length: 30bp, nucleotide sequence as shown in SEQ ID NO.20, specifically: caaagtgttgattatgatggtgatagttat.
  • Light chain complementarity determining region LCDR2 DNA, length: 9bp, nucleotide sequence shown in SEQ ID NO.21, specifically: gctgcatcc.
  • Heavy chain complementarity determining region HCDR1 DNA, length: 30bp, nucleotide sequence shown in SEQ ID NO.23, specifically: gggttttcactgagcacttctggtatgggt.
  • Heavy chain complementarity determining region HCDR2 DNA, length: 21bp, nucleotide sequence as shown in SEQ ID NO.24, specifically: atttggtgggatgatgataag.
  • the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.26, specifically:
  • the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.27, specifically:
  • A375-S2 is a human melanoma cell line.
  • the CD271 molecule is highly expressed in the cell subset with tumor stem cell characteristics.
  • the detection method of Example 8 was used to investigate the ability of CD271T-2 monoclonal antibody to recognize the cell.
  • the results showed that both APC Goat anti human CD271 positive control antibody and CD271T-2 monoclonal antibody were able to detect more than 50% positive, while Rabbit anti human CD271 antibody and negative control had no offset, indicating that CD271T-2 antibody had no effect on the expression of CD271
  • Molecular tumor cells have good affinity and can be used for tumor diagnosis, treatment and other applications.
  • Figure 10 The specific results are shown in Figure 10.
  • the figure shows that the positive control antibody of APC Goat anti human CD271 derived from Biolegend, the immunogen is derived from melanoma cells, whose cell surface highly expresses CD271 molecule, this antibody has a slight migration compared with the negative control ( Figure 10A); derived from Abcam's Rabbit anti human CD271 control antibody, the immunogen comes from the 350-450 amino acid region in the human CD271 cell membrane, and there is no offset compared with the negative control ( Figure 10B); CD271T-2 ascites purified antibody, and positive and negative Compared with the control, the degree of deviation was higher, indicating that the prepared anti-human CD271 monoclonal antibody had very high affinity and specificity with CD271 on the surface of mesenchymal cells (Fig. 10C).
  • the CD271T immune antigen epitope design method and preparation of the present invention can be applied to screen antibodies and nucleic acid aptamers with high affinity and high specificity against human CD271, and can be applied to develop nucleic acid aptamers that recognize CD271.

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Abstract

提供一种基于CD271的新型的抗原表位肽，该表位肽为：(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％。还提供了靶向该抗原肽的抗体、核酸适配体、疫苗、纳米粒子、含有抗原表位肽的核酸、融合蛋白、载体或宿主细胞，用于制备CD271相关的检测试剂、疾病治疗生物药物、疫苗等。

Description

一种基于CD271的新型抗原表位及其应用 技术领域
本发明涉及生物技术领域，特别是涉及一种CD271的新型的抗原表位肽及其应用。
背景技术
CD271是一种低亲和力的神经生长因子受体(LNGFR)，属于肿瘤坏死因子受体超家族成员，相对分子质量为75kD，又称为p75NTR，它包含3个区域：富含半胱氨酸的膜外区、跨膜区和155个氨基酸残基组成的胞内区。CD271不仅表达在神经系统，与神经细胞的发育、分化和存活密切相关，也能够识别肿瘤干细胞、间充质干细胞。在肿瘤治疗领域，CD271在黑色素瘤等多种肿瘤细胞或肿瘤干细胞表面高表达，靶向CD271的单克隆抗体及其偶联药物能够起到靶向杀伤作用，从而治疗肿瘤。在间充质干细胞领域，CD271抗体可以通过流式分选或者偶联至磁珠对间充质干细胞进行磁性分选，所富集的细胞具有特异性高、纯度高、集落形成能力强等优点。因此，特异性识别CD271的抗体具有广泛的应用价值，稳定、高效的抗原表位对开发CD271单克隆抗体或基于CD271的疫苗等具有重要意义。
天然CD271的胞外结构包括4个富含半胱氨酸的CRD结构(CRD1-4)以及一个连接区域(stalk区)。目前已有的CD271抗体产品，主要为各类科研试剂，其抗原表位主要是表达CD271的细胞，如黑色素瘤细胞，或CD271的CRD区域。然而，在CD271表达后，CRD区域会被蛋白酶水解从而脱落，因此靶向CRD区的抗体与CD271阳性细胞的结合会受到影响，甚至在细胞代谢的某个阶段无法检测到CD271的表达，因此亟需开发新的抗原表位，为基于CD271的抗体、疫苗研究及相关检测或疾病治疗提供新的工具。
发明内容
鉴于以上所述现有技术的缺点，本发明提供一种基于CD271的新型抗原表位肽及其应用，以解决现有技术的不足。
发明的目的是通过以下技术方案来实现：
本发明第一方面提供一种CD271的抗原表位肽，所述抗原表位肽为：
(1)位于多肽链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中；
(3)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％。
SEQ ID NO.1所示序列，长度：62aa，具体为：EEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN。
进一步地，所述抗原表位肽位于SEQ ID NO.1中第1位～35位氨基酸序列中的连续氨基酸序列。
进一步地，所述抗原表位肽位于SEQ ID NO.1中第11位～35位氨基酸序列中的连续氨基酸序列。
进一步地，所述抗原表位肽位于SEQ ID NO.1中第18位～27位氨基酸序列中的连续氨基酸序列。
进一步地，所述抗原表位肽为与SEQ ID NO.1中第18位～27位的连续氨基酸具有80％以上重叠度的氨基酸序列。
可选的，所述抗原表位肽的氨基酸序列如SEQ ID NO.3-11任一所示。
具体的，一种抗原表位肽Peptide，长度：19aa，序列如SEQ ID NO.3所示，具体为：TRSTPPEGSDSTAPSTQEPE。
另一种抗原表位肽Peptide，长度：19aa，序列如SEQ ID NO.4所示，具体为：EPEAPPEQDLIASTVAGVV。
另一种抗原表位肽Peptide，长度：19aa，序列如SEQ ID NO.5所示，具体为：VTTVMGSSQPVVTRGTTDN。
另一种抗原表位肽Peptide，长度：19aa，序列如SEQ ID NO.6所示，具体为：LIASTVAGVVTTVMGSSQP。
另一种抗原表位肽Peptide，长度：19aa，序列如SEQ ID NO.7所示，具体为：EEIPGRWITRSTPPEGSDS。
另一种抗原表位肽Peptide，长度：19aa，序列如SEQ ID NO.8所示，具体为：DSTAPSTQEPEAPPEQDLI。
另一种抗原表位肽Peptide，长度：10aa，序列如SEQ ID NO.9所示，具体为：GSDSTAPSTQ。
另一种抗原表位肽Peptide，长度：10aa，序列如SEQ ID NO.10所示，具体为：DSTAPSTQEP。
另一种抗原表位肽Peptide，长度：7aa，序列如SEQ ID NO.11所示，具体为：TPPEGSD。
本发明第二方面提供一种融合蛋白，所述融合蛋白是由以上任一项所述的抗原表位肽与载体蛋白融合所形成的。
本发明第三方面提供一种能够编码抗原表位肽的核酸序列，所述核酸序列为能够编码以上任一项所述的抗原表位肽的基因序列或基因片段。
例如，编码SEQ ID NO.1的核苷酸序列如SEQ ID NO.2所示，
具体的：186bp
Figure PCTCN2022074573-appb-000001
本发明第四方面提供一种表达载体，所述表达载体含有以上所述的核酸序列。
本发明第五方面提供一种宿主细胞，所述的宿主细胞含有以上所述的表达载体，或者在基因组中整合有以上所述的核酸序列。
本发明第六方面提供一种抗体，其特征在于：所述抗体为能够结合权利以上任一项所述的抗原表位肽的抗体。
进一步地，所述抗体为能够抑制或减弱与以上任一项所述的抗原表位肽相关的信号传递的抗体。
进一步地，所述抗体为单克隆抗体或多克隆抗体，种属来源可以为人，鼠，兔，猴，牛，羊或羊驼等哺乳动物。
本发明第六方面提供一种疫苗，所述疫苗含有以上任一项所述的抗原表位肽、融合蛋白、核酸序列、表达载体或者宿主细胞。
本发明第七方面提供一种组合物，其特征在于，含有以上任一项所述的抗原表位肽、融合蛋白、核酸序列、表达载体、宿主细胞、抗体或者疫苗，以及免疫学和药学上可接受的载体和/或辅料。
本发明第八方面提供以上任一项所述的抗原表位肽、融合蛋白、核酸序列、表达载体或者宿主细胞的用途：
(1)用于制备针对所述抗原表位的抗体、核酸适配体、疫苗、纳米粒子；和/或
(2)用于制备治疗与所述抗原表位相关的疾病的药物。
如上所述，供了一种与基于CD271的新型的抗原表位肽及其应用，其至少具有以下有益效果：本发明的抗原表位肽具有免疫原性，其可诱导产生免疫应答反应，该抗原表位肽或含有该抗原表位肽的基因分子、融合蛋白、载体或宿主细胞等可用来制备抗体、核酸适配体、疫苗、靶向CD271的纳米颗粒等，具有重要应用意义。
该抗原表位肽选取自CD271靶点膜外稳定、同源性低、无糖基化和磷酸化位点的序列，靶向该抗原表位肽的抗体等分子与天然带有CD271靶点细胞具有更加紧密和稳定的结合。该 抗原表位肽与本申请中的抗体具有特异性结合，亲和力强，平衡解离常数可达到nM级别，而且结合稳定，不会因细胞表面CD271分子的CRD区水解而受影响。这对于抗原或抗体发挥药物作用具有极其重要的作用，使其具有极大的制备成为诊断试剂或药物的潜在价值，能够用于干细胞组织修复、神经系统疾病、肿瘤等与CD271相关的领域。
附图说明
图1是抗原表位肽所在的多肽链SEQ ID NO.1(命名为CD271T)表达载体质粒pCD271T构建示意图，该CD271T与sumo标签融合表达，载体为pET-28a(+)；
图2是SEQ ID NO.1抗原表位肽在大肠杆菌中诱导表达条件优化SDS-PAGE(图2A&2B&2C)及Western Blotting(图2D)检测图，含有CD271T的融合蛋白分子量约37kDa。
图3是融合蛋白经Sumo蛋白酶切后释放出Sumo标签与CD271T蛋白的SDS-PAGE检测图：37℃酶切1h、2h、4h、6h后进行SDS-PAGE鉴定(图3A)，显示2h左右蛋白已经被酶切完全，目的条带与SUMO tag和CD271T大小一致，后期纯化进一步富集目的蛋白(图3B)；
图4是ELISA检测CD271抗原多肽免疫后小鼠血清多克隆抗体的效价检测图：包被抗原浓度为4μg/mL，上样血清从1:2000稀释到1:256000稀释，1号、2号、3号、4号、5号小鼠血清效价都在1：128000以上，5只小鼠血清中均含有识别CD271多肽抗原的多克隆抗体，对抗原多肽有很强的亲和力；
图5是CD271抗原表位多肽免疫小鼠筛选得到的腹水单克隆抗体CD271T-2的SDS-PAGE检测图：条带1为非还原样品，抗体条带较为单一，条带2为还原样品，在26kDa和53kDa处分别是抗体的轻链LC和重链HC；
图6是纯化后CD271T-2单克隆抗体的ELISA鉴定：包被抗原为CD271抗原表位多肽，抗原浓度为2μg/mL，每孔100μL,上样抗体浓度梯度为0.625μg/mL、1.25μg/mL、2.5μg/mL、5μg/mL、10μg/mL、20μg/mL、40μg/mL，每孔100μL，结果表明CD271T-2抗体能够识别结合具有天然结构的全长人CD271蛋白；
图7是ForteBio验证纯化后抗体亲和力图谱，可见本发明的CD271抗原表位多肽与其免疫小鼠所获得的CD271T-2单克隆抗体具有很强的亲和力；
图8是CD271T-2单克隆抗体的抗原表位竞争性抑制ELISA检测：包被抗原为CD271蛋白，抗原浓度为4μg/mL，每孔100μL，上样腹水抗体稀释比例为1:600，各多肽浓度均为16μg/mL，每孔100μL，腹水抗体与多肽1:1混合，结果表明，多肽2-4具有阻断CD271T-2单克隆抗体与CD271蛋白结合的作用，为该单克隆抗体识别的最小表位；
图9是抗人CD271T-2的重轻链可变区基因克隆PCR片段电泳图：图9A为CD271T-2杂交瘤重轻链可变区的基因克隆PCR片段，图9B为CD271T-2重轻链可变区嵌合ScFv基因克隆PCR片段；
图10是抗人CD271单克隆抗体检测间充质干细胞表面CD271的表达情况：图10A为来源于Biolegend的APC mouse anti human CD271的阳性对照抗体，免疫原来自黑色素瘤细胞，其细胞表面高表达CD271分子，此抗体与阴性对照相比有略微迁移；图10B为来源于Abcam的Rabbit anti human CD271对照抗体，免疫原来自人CD271细胞膜内350-450氨基酸区域，与阴性对照相比，无任何偏移；从图10C为CD271T-2单克隆抗体，与阳性和阴性对照相比，其偏移程度较高，说明制备的抗人CD271单克隆抗体对表达CD271分子的细胞具有非常高的亲和力和特异性，优于市售的靶向CD271其他抗原表位的抗体。
具体实施方式
选取抗原序列进行免疫动物，将骨髓瘤细胞与免疫的动物脾细胞融合，形成能分泌针对该抗原的均质的高特异性高亲和性的单克隆抗体，经过亚克隆筛选得到能够稳定表达针对于该抗原的单克隆抗体杂交瘤细胞株。
为了使筛选出的单克隆抗体获得最佳亲和效果和特异性，抗原的设计需要满足两点，首先该抗原不会产生过强的免疫反应，同时又能产生出对目标蛋白有结合能力的抗体。蛋白的构象将会影响抗体与其识别区域之间的相互作用，若抗体识别区藏在蛋白的内部，抗体将无法产生相互作用。大多数抗体是针对连续识别区域的，抗体能与这类区域以很高的亲和力相结合表面这段序列不在蛋白内部。不连续的识别区域是代表有一定折叠的多肽序列，或是将两段分离开的多肽连在一起的抗体识别区域。在某些情况下，针对这样不连续识别区域的抗体也能产生，只是用来免疫的抗原多肽必须具备与该不连续识别区域相似的二级结构，而序列的长度需要符合相关的要求。另外在完整的蛋白中，N，C两端通常是暴露在蛋白表面的，而膜蛋白的C端疏水性太强，不适合作为抗原。
通常情况下，来源于哺乳动物细胞的蛋白可能表面会有糖基化等修饰，因此使用哺乳动物细胞来源的免疫原所筛选到的抗体，难以保证所筛选得到的抗体是结合氨基酸表位的。另一方面，来源于大肠杆菌表达的免疫原，或者合成多肽，所筛选到的抗体能够识别氨基酸表位，但是由于免疫原可能不具备高级结构，或者具有与自然构象不同的高级结构，因此所筛选的抗体有可能不识别天然抗原，尤其是细胞表面抗原。为了获得能够识别细胞表面抗原的抗体，我们设计了该免疫策略，免疫抗原来源于多肽合成或者原核表达，筛选抗原为全长分 子的哺乳动物细胞表达，以保证其空间结构为天然状态。在动物免疫、多克隆血清滴度检测以及杂交瘤筛选阶段，交替使用两种抗原，最终获得满足要求的杂交瘤细胞株及抗体。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。
“药学上可接受的载体和/或辅料”应当与所述有效成分相容，即能与其共混而不会在通常情况下大幅度降低药物的效果。可作为药学上可接受的载体或辅料的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。这些物质根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味。
疫苗组合物包含免疫性抗原(包括本发明重组蛋白)，并且通常与“药学上可接受的载体”组合，这些载体包括本身不诱导产生对接受该组合物的个体有害的抗体的任何载体。合适的载体通常是大的、代谢缓慢的大分子，如蛋白质、多糖、聚乳酸、聚乙醇酸、氨基酸聚 合物、氨基酸共聚物、脂质凝集物(如油滴或脂质体)等。这些载体是本领域普通技术人员所熟知的。另外，这些载体可起免疫刺激剂(“佐剂”)作用。
【实施例1】人CD271抗原表位肽质粒的设计、表达、纯化
1.人CD271抗原表位肽的选择
根据NCBI检索CD271蛋白的全长氨基酸序列，采用DNAStar软件、IEDB网站等对其进行抗原决定簇分析，最终确定抗原表位肽位于CD271的膜外Stalk区的SEQ ID NO.1(命名为CD271T)中，设计一系列抗原表位肽，满足：
(1)位于多肽链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。
2.CD271T表达质粒的设计
根据NCBI检索CD271蛋白的全基因序列以及CD271T的氨基酸序列，由苏州泓迅有限公司进行原核表达密码子优化，与Sumo标签一起克隆到pET-28a(+)载体，合成CD271T抗原表达质粒pCD271T，如图1所示。
2.CD271T抗原质粒在大肠杆菌中的诱导表达及SDS-PAGE&WB检测
(1)原核诱导表达
1)挑取合成的穿刺菌，接种于5mL LB(0.1％Kana)中，37℃，235rpm培养过夜。
2)第二天，取昨夜的菌液1mL(1％接种量)于100mL LB(0.1％Kana)中，在摇床中37℃，235rpm培养至OD600＝0.5，分别对IPTG浓度、诱导温度、诱导时间进行控制变量优化，6000rpm离心收菌。其中IPTG浓度分别为0.1mM和0.05Mm，温度分别为20℃和37℃，每隔2h取样1.5mL菌液于1.5mL EP管中，6000rpm离心收菌。
3)加入培养基体积10％的PBS重悬，超声破碎：300W，每次超声5s，停6s，共超声5min。
4)4℃高速离心，12000rpm，10min，取超声后的上清和沉淀备用进行鉴定。
(2)SDS-PAGE及WB检测
将上清和沉淀分别加入5×reduced SDS-PAGE loading buffer，95℃，加热10min，12000rpm离心5min。各取制备好的样品10μL进行SDS-PAGE凝胶电泳及Western Blotting检测，确定最终的IPTG浓度、诱导温度及诱导时间。
(3)人CD271T抗原蛋白His Trap FF柱纯化
1)加初浓度为100mM的PMSF于菌液原液至其终浓度为1mM；
2)高压均质仪破菌，提前开机，先用20％乙醇、水先后进行冲洗，后倒入菌液，条件：900bar，对比前后菌液，发现其明显澄清，收集即可；
3)离心机离心，注意配平，12000rpm，30min，取上清进行蛋白纯化；
4)设置△P＝0.3MPa，用乙醇冲洗系统至稳定后接柱，调至position1；
5)用纯水将柱中的乙醇冲干净，A1、B1一起冲洗，各50％，至基线平；
6)之后用5-10倍柱体积(CV)的loading buffer(20mM PB，20mM NaCl)平衡柱子，A1为100％，B1为0％，直到电导和pH值稳定；
7)将表达上清以1mL/min的速度通过AKTA上样；
8)重新用loading buffer于A1通道平衡柱子，直至基线平；
9)设置梯度洗脱，以0.5M咪唑比例为10％、20％、30％、40％、50％、60％、100％分别进行洗脱，直至基线平稳，注意在有紫外吸收时需要进行收样；
10)用纯水冲洗，最后用20％的乙醇保存柱子。
以SEQ ID NO.1为例，结果如图2所示，如图2A所示，分别以0.5mM IPTG和1mM IPTG诱导12h后，在分子量37kDa处有明显蛋白表达条带，与CD271T融合蛋白理论分子量基本一致，这表明成功进行了CD271的原核表达，IPTG浓度从0.5mM升高至1mM时，CD271表达量有略微提高。图2B及2C显示，工程菌经20℃和37℃两种不同的诱导温度和诱导时间培养后，诱导温度为20℃时，随着诱导时间的增加，破菌后上清中目标条带愈发明显，在12h后到达顶峰，并且12h之后目标条带的深浅变化不明显(图2B)；诱导温度为37℃时，随着诱导时间的增加，破菌后上清中条带愈发明显，诱导4h后目标条带表达量趋于稳定(2C)；用抗His标签抗体进行Western Blotting分析，结果表明在相同分子量位置菌体破碎后上清显现特异性条带，菌体破碎后沉淀无条带，证实了CD271蛋白于菌体破碎后上清中的正确表达(图2D)。
【实施例2】人CD271T抗原肽的蛋白酶切反应、纯化
1.人CD271T抗原肽的蛋白酶切反应及SDS-PAGE鉴定
(1)酶切体系
酶切体系见表1，在30℃水浴反应1h、2h、4h、6h，进行SDS-PAGE鉴定。
表1
Figure PCTCN2022074573-appb-000002
Figure PCTCN2022074573-appb-000003
(2)SDS-PAGE鉴定
酶切前与酶切后1h、2h、4h、6h分别取样20μL，加入5×reduced SDS-PAGE loading buffer，95℃，加热10min，12000rpm离心5min。各取制备好的样品10μL进行SDS-PAGE凝胶电泳检测，具体结果见图3中的A图。
2.人CD271免疫抗原纯化
按照上述反应体系扩大酶切体系，在20℃水浴反应2h，0.45nm滤膜过滤样品，再用His Trap FF进行纯化，收集流穿进行鉴定(图3，B)，超滤浓缩置换PBS buffer，测定蛋白浓度。
【实施例3】利用实施例1中的抗原肽筛选抗体
1.抗原制备
1)初免抗原制备：取1mL无菌EP管，将免疫原100μg用PBS稀释至100μL放于EP管中；充分摇匀弗氏完全佐剂，使沉淀的双歧杆菌均匀混合；取100μL弗氏完全佐剂加入5mL的无菌EP管，再加入稀释好的免疫原，使抗原的体积与佐剂的体积比为1：1，将离心管置于冰盒中；准备好超声破碎仪，将离心管置于冰盒中超声。超声条件是总功率200W，总时间10min，工作时间5秒，间隔时间6秒。实时观察溶液乳化的情况；用1毫升一次性注射器吸入乳化好的抗原，置于冰盒中备用。
2)二免、三免抗原制备：取1mL无菌EP管，将免疫原100μg用PBS稀释至100μL放于EP管中；充分摇匀弗氏不完全佐剂，使沉淀的双歧杆菌均匀混合；取100μL弗氏不完全佐剂加入5mL的无菌EP管，再加入稀释好的免疫原，使抗原的体积与佐剂的体积比为1：1，将离心管置于冰盒中；准备好超声破碎仪，将离心管置于冰盒中超声。超声条件是总功率200W，总时间10min，工作时间5s，间隔时间6s。实时观察溶液乳化的情况；用1毫升一次性注射器吸入乳化好的抗原，置于冰盒中备用。
3)加强免疫抗原制备：取1mL无菌EP管，将免疫原200μg用PBS稀释至400μL，用1毫升一次性注射器吸入，置于冰盒备用。
4)小鼠免疫方法
用1毫升注射器吸入免疫抗原，分2-3点，每点0.1mL左右，给小鼠进行背部(或腹部)皮下注射；三免后第三天采血进行抗体滴度ELISA检测，具体结果见图4，1号、2号、3号、4号、5号小鼠血清效价都在1：128000以上，5只小鼠对抗原的反应性都较高，表明产生了抗CD271的多克隆抗体。
2.饲养细胞的准备
饲养细胞可促进单个或少数分散细胞的生长繁殖。
1)准备好灭菌的手术器具和玻璃平皿，96孔板，5mL一次性注射器，300mL 70％的酒精，Balb/c小鼠若干，无血清DMEM培养液，灭菌的PBS。
2)拉颈处死Balb/c小鼠，浸入70％酒精10min。
3)拿出小鼠沥去多余的酒精，置于灭菌的玻璃平皿上。
4)剪开并分离小鼠腹部的皮肤(勿剪破肌肉层)。用5mL注射器吸取5mL DMEM无血清培养液，注入小鼠腹部，轻轻按摩并摇动小鼠身体，使巨噬细胞充分混入培养液。用注射器小心吸回培养液，放入15mL离心管。
5)按上述方法重复1-2次，以尽量多的获得巨噬细胞。
6)1500rpm，离心10min，弃上清。用培养液悬浮备，1只成年Balb/c小鼠可取约10 6个腹腔细胞，可铺3-4块96孔板。
3.PEG介导大鼠脾脏B细胞和小鼠骨髓瘤细胞融合
1)细胞：Sp2/0-Ag14，培养基：DMEM+10％FBS，融合前48小时传代，密度视48小时后覆盖瓶底80％左右为宜。
2)小鼠剪尾拉颈处死Balb/c小鼠，浸入70％酒精10min。
3)剪开左侧上腹部直至小鼠左背部，分离出脾脏，除去脾脏上的结缔组织，放入干净平皿中，加少量DMEM基础培养基(无血清)。
4)将小鼠脾脏剪成4-5小段，用5mL注射器内塞顶部研磨，边磨边加入少量DMEM，将磨出的脾脏细胞冲入平皿。
5)将平皿中的细胞全部移入15mL离心管，加入适量的DMEM基础培养基，混匀离心，200g，5min。
6)用PBS洗涤二遍，离心，200g，5min。
7)收集所有的Sp2/0-Ag14细胞，并用PBS洗涤一遍待用并计数。
8)将10 8个脾脏细胞和10 7个骨髓瘤细胞混合在50mL离心管中(骨髓瘤细胞数可视收获细 胞再提高)，加入30-40mL无血清的DMEM，充分混匀，1500rpm离心3min，去上清。
9)轻敲离心管的底部，使沉淀物充分打散。
10)将细胞放入37℃水浴，按下列方法缓慢滴入PEG。
11)用滴管沿管壁缓慢滴入1mL PEG溶液，持续时间1min，边加边轻轻振荡，100g，离心2min；
12)用同样的方法滴入4.5mL DMEM基础培养基(预热、无血清)，持续时间3min，边加边轻轻振荡；
13)用同样的方法滴入5mL DMEM基础培养基(预热、无血清)，持续时间2min，加完后轻轻振荡30s；
14)慢慢加入45mL DMEM基础培养基(预热、无血清)，
15)100g离心5min，去上清，轻敲底部。
16)加入含HAT的DMEM完全培养基(含20％FBS)，轻轻重悬，用台盼蓝进行细胞计数，100μL/孔，种入5块96孔板。
17)37℃，5％CO 2培养箱培养，培养期间不要晃动培养板。
18)融合后11-13天进行ELISA鉴定，并根据细胞生长情况进行测定，在测定之前的2-3天内不要换液，使上清中抗体的量充分蓄积。
4.有限稀释筛选单克隆
1)取出抗体阳性孔细胞，用HT培养液制成细胞悬液。并取样进行台盼兰染色，计数。
2)取320个细胞分散在6.4mL的HT培养液中，充分混匀后用排枪加入96孔板的第1-4列，每孔100μL，使每孔5个细胞；剩下的细胞液再加入3.2mL的HT培养液，充分混匀后，100μL每孔加入5-8列，使每孔2.5个细胞；取剩下的细胞液1mL，再加入2mL培养液，充分混匀后，100μL每孔加入9-12列，使每孔0.625个细胞。
3)将96孔板放入CO 2培养箱，37℃培养。
4)第五天，显微镜下观察细胞的生长情况，记录细胞生长的孔。
5)克隆大量繁殖后，布满孔底的1/3～1/2时，用全长CD271蛋白包被的ELISA板检测培养液中抗体水平。
6)在每孔0.625细胞的孔中选取Elisa阳性最高、生长状态良好的孔中的细胞，做下一轮有限稀释；同时选取相同情况的另一孔转移到含有饲养细胞的24孔板中的一孔扩增后冻存。
7)三轮有限稀释后将经过筛选并符合建株条件的细胞扩增培养冻存。
5.杂交瘤细胞产生小鼠腹水的制备方法
1)细胞注射7-10天之前，每只裸鼠腹腔注射0.5mL高压灭菌的降植烷(石蜡油)。
2)复苏建株细胞株培养传代，上清测ELISA，确定为阳性时，离心收集细胞，台盼蓝染色技术；按每只Balb/c 5×10 5个细胞，重悬在0.2mL PBS中。
3)腹腔注射8周雌性Balb/c小鼠。
4)1-2星期左右，腹水开始出现。当动物的体型像怀孕的雌鼠时，可开始抽取腹水。用5mL注射器从腹腔的外周抽取腹水，每只约2-3mL左右。
5)将腹水3000rpm(1500g)离心10分钟，上清分装后-70℃保存。
6)保持裸鼠健康的情况下，可根据腹水产生的情况，间隔1-3天反复抽取。
【实施例4】小鼠腹水纯化鉴定及ForteBio验证纯化抗体亲和力
1.小鼠腹水Mab Select Sure TM纯化
1)小鼠腹水在采集后立刻离心，上清冻存于-80℃。纯化前室温或4度解冻，离心取上清，加入1/10体积1M Tris-HCl，0.5M NaCl，pH 8.0调pH值，0.22μm过滤即为待上柱样品，取样S；
2)柱子用10CV的1M Tris-HCl，0.5M NaCl,pH 8.0平衡；
3)上样，收集所有流穿，取样FL；
4)Mab Select Sure TM用10CV的100mM Tris-HCl,50mM NaCl,pH 8.0洗涤；
5)Mab Select Sure TM用10CV的10mM Tris-HCl,5mM NaCl,pH 8.0洗涤；
6)收集管中预先加入0.1CV的1M Tris-HCl pH 9.0，洗脱时每次加入0.5CV的100mM柠檬酸盐缓冲液pH3.0，并立即混合；洗脱Elute 1，2，3…管，分半取样制备样品。
2.纯化抗体SDS-PAGE及ELISA鉴定
1)SDS-PAGE鉴定
对纯化得到的腹水抗体CD271T-2进行SDS-PAGE蛋白电泳分析，具体结果见图5。
2)ELISA鉴定
收集所有收集管的蛋白，超滤离心后测定蛋白浓度，对CD271T-2进行ELISA鉴定，证实其能够结合CD271全长蛋白，具体结果见图6。
3.ForteBio验证纯化后抗体亲和力
【实施例5】CD271T-2抗原表位肽的合成及免疫鉴定
利用IEDB网站对抗原表位进行预测，设计位于SEQ ID NO.1中的多肽SEQ ID NO.3～11，送南京金斯瑞公司合成。采用ELISA方法检测CD271T抗原表位肽免疫后小鼠血清对不同多肽片段的结合，均可见阳性显色，即这些多肽均可引起动物免疫反应，从而产生相应的多克隆抗体。
【实施例6】竞争性抑制ELISA检测CD271T-2单克隆抗体的最小识别表位
包被：将全长CD271蛋白作为包被抗原，用包被液稀释成4μg/mL，按每孔100μL加入ELISA板中，37℃孵育2小时；封闭：弃去包被抗原，每孔加入300μL ELISA洗涤液(PBST)洗涤3次，洗涤完后拍干，然后每孔加入100μL 5％BSA(5g BSA，100mL PBST)，37℃孵育2小时；夹心孵育：弃去封闭液，每孔加入300μL ELISA洗涤液洗涤3次，洗涤完后拍干，全长CD271蛋白组按1:600用PBS稀释，每孔加入100μL，全长CD271蛋白与多肽混合组，每孔加入50μL稀释好的全长CD271蛋白以及50μL 16μg/mL对应的多肽(包括实施例5中的SEQ ID NO.9，SEQ ID NO.10，SEQ ID NO.11)，37℃孵育1小时；二抗孵育：弃去夹心，每孔加入300μL ELISA洗涤液洗涤3次，洗涤完后拍干，用PBST将HRP标记羊抗鼠二抗以1:10000比例进行稀释，每孔加入100μL，37℃孵育0.5小时；显色液显色：弃去二抗，每孔加入300μL ELISA洗涤液洗涤3次，洗涤完后拍干，每孔加入100μL TMB单组份显色液，室温避光显色10min；读数：显色完毕后，每孔加入50μL0.2M H2SO4终止液，使用酶标仪在450nm波长下测定吸光值(OD450)，结果见图8。与其他组相比，加入SEQ ID NO.10多肽的组OD450最低，为CD271T-2所识别的最小抗原表位。
【实施例7】抗人CD271T-2的重轻链可变区基因克隆及嵌合抗体2的构建
1.RNA提取：采用Trizol一步法，取杂交瘤细胞CD271T-2约10 6个，加入Trizol，按照康为世纪Ultrapure RNA Kit CW0581S试剂盒提取RNA，核酸电泳鉴定RNA完整性；需要说明的是，杂交瘤细胞CD271T-2的保藏号为CGMCC 21494。保藏于中国普通微生物菌种保藏管理中心，保藏地址为北京市朝阳区北辰西路1号院3号。保藏日期为2021年1月25日。分类命名为杂交瘤细胞株CD271T-2；
2.逆转录为cDNA(20μL)：取Oligo dT Primer(50μM)1μL，dNTP Mixture(10nM)1μL，模板RNA 5μg，加水至10μL，65℃保温5min，冰上迅速冷却；加入上述变性后反应液10μL，5x primerscript II Buffer 4μL，RNase Inhibitor(40U/μL)0.5μL，PrimeScirpt II RTase(200U/μL)1μL，加水至20μL，42℃反应60min，70℃，15min 失活；
3.PCR扩增抗CD271抗体的重轻链可变区基因：轻、重链可变区基因PCR扩增于苏州泓迅生物技术公司操作，PCR结果见图9A&9B；
测序载体的构建和测序：PMD19-T载体购自takara，将重轻链可变区基因pcr产物回收，与T载体连接后，进行DH5α转化，以100μg/mL氨苄浓度筛选阳性克隆，送测序，与蛋白数据库所具有的若干保守的框架氨基酸比对。所得抗体包括轻链和重链，所述轻链互补决定区包括LCDR1、LCDR2和LCDR3，所述LCDR1的氨基酸序列如SEQ ID NO.12所示，所述LCDR2的氨基酸序列如SEQ ID NO.13所示，所述LCDR3的氨基酸序列如SEQ ID NO.14所示；所述重链互补决定区包括HCDR1、HCDR2和HCDR3，所述HCDR1的氨基酸序列如SEQ ID NO.15所示，所述HCDR2的氨基酸序列如SEQ ID NO.16所示，所述HCDR3的氨基酸序列如SEQ ID NO.17所示。
具体的，
轻链互补决定区LCDR1，长度：10aa，氨基酸序列如SEQ ID NO.12所示，具体为：QSVDYDGDSY。
轻链互补决定区LCDR2，长度：3aa，氨基酸序列如SEQ ID NO.13所示，具体为：AAS。
轻链互补决定区LCDR3，长度：9aa，氨基酸序列如SEQ ID NO.14所示，具体为：QQSNEDPFT。
重链互补决定区HCDR1，长度：10aa，氨基酸序列如SEQ ID NO.15所示，具体为：GFSLSTSGMG。
重链互补决定区HCDR2，长度：7aa，氨基酸序列如SEQ ID NO.16所示，具体为：IWWDDDK。
重链互补决定区HCDR3，长度：13aa，氨基酸序列如SEQ ID NO.17所示，具体为：ARRDYGNYYAMDY。
轻链可变区氨基酸序列如SEQ ID NO.18所示，具体为：
Figure PCTCN2022074573-appb-000004
重链可变区氨基酸序列如SEQ ID NO.19所示，具体为：
Figure PCTCN2022074573-appb-000005
轻链互补决定区LCDR1，DNA，长度：30bp，核苷酸序列如SEQ ID NO.20所示，具体为：caaagtgttgattatgatggtgatagttat。
轻链互补决定区LCDR2，DNA，长度：9bp，核苷酸序列如SEQ ID NO.21所示，具体为：gctgcatcc。
轻链互补决定区LCDR3，DNA，长度：27bp，核苷酸序列如SEQ ID NO.22所示，具体为：cagcaaagtaatgaggatccattcacg。
重链互补决定区HCDR1，DNA，长度：30bp，核苷酸序列如SEQ ID NO.23所示，具体为：gggttttcactgagcacttctggtatgggt。
重链互补决定区HCDR2，DNA，长度：21bp，核苷酸序列如SEQ ID NO.24所示，具体为：atttggtgggatgatgataag。
重链互补决定区HCDR3，DNA，长度：39bp，核苷酸序列如SEQ ID NO.25所示，具体为：gctcgaagggactatggtaactactatgctatggactac。
轻链可变区的核苷酸序列如SEQ ID NO.26所示，具体为：
Figure PCTCN2022074573-appb-000006
重链可变区的核苷酸序列如SEQ ID NO.27所示，具体为：
Figure PCTCN2022074573-appb-000007
4.采用上述轻链LCDR1～3及重链HCDR1～3，与人IgG分子恒定区进行融合，构建质粒，采用CMV启动子在HEK293细胞及CHO细胞进行转染和表达，并通过protein A亲和层析，纯化获得人源化CD271单克隆抗体，经Fortbio检测，该工程化CD271抗体与CD271蛋白具有良好的亲和力，平衡解离常数达到nM级。
【实施例8】CD271T-2单克隆抗体应用于肿瘤细胞的流式检测
A375-S2是人黑色素瘤细胞系，具有肿瘤干细胞特性的细胞亚群中CD271分子是高表达的，采用实施例8的检测方法，考察CD271T-2单克隆抗体对该细胞的识别能力。结果发现，APC  Goat anti human CD271阳性对照抗体和CD271T-2单克隆抗体均能够检测到50％以上的阳性，而Rabbit anti human CD271抗体和阴性对照均无偏移，说明CD271T-2抗体对表达CD271分子的肿瘤细胞具有良好的亲和力，可用于肿瘤的诊断、治疗等应用。
【实施例9】CD271T-2单克隆抗体应用于间充质干细胞流式检测
取适当生长良好的间充质干细胞Hu-MSC细胞，胰酶消化离心收集，用FACS(PBS+2％FBS)重悬洗涤，1000rpm，3min离心，弃上清；按表3加入一抗，室温避光孵育30min，用FACS(PBS+2％FBS)重悬洗涤，离心1000rpm，3min，重复3次，弃上清，按表3加入二抗，室温避光30min，用FACS(PBS+2％FBS)重悬洗涤，离心1000rpm，3min，重复3次，加入300μL FACS重悬细胞，流式细胞仪检测，具体结果见图10。图中表明，来源于Biolegend的APC Goat anti human CD271的阳性对照抗体，免疫原来自黑色素瘤细胞，其细胞表面高表达CD271分子，此抗体与阴性对照相比有略微迁移(图10A)；来源于Abcam的Rabbit anti human CD271对照抗体，免疫原来自人CD271细胞膜内350-450氨基酸区域，与阴性对照相比，也无任何偏移(图10B)；CD271T-2的腹水纯化抗体，与阳性和阴性对照相比，其偏移程度较高，说明制备的抗人CD271单克隆抗体与间充质细胞表面的CD271具有非常高的亲和力和特异性(图10C)。
表3
Figure PCTCN2022074573-appb-000008
本发明的CD271T免疫抗原表位的设计方法及其制备可应用筛选抗人CD271高亲和力高特异性的抗体、核酸适配体，并可应用于开发识别CD271的核酸适配体。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实 施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。

Claims (15)

  1. 一种新型的抗原表位肽，其特征在于：所述抗原表位肽为：
    (1)为SEQ ID NO.1，或位于多肽链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中；
    (3)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％。
  2. 根据权利要求1所述的新型的抗原表位肽，其特征在于：所述抗原表位肽为位于SEQ ID NO.1中第1位～35位氨基酸序列中的连续氨基酸序列。
  3. 根据权利要求2所述的新型的抗原表位肽，其特征在于：所述抗原表位肽为位于SEQ ID NO.1中第11位～35位氨基酸序列中的连续氨基酸序列。
  4. 根据权利要求3所述的新型的抗原表位肽，其特征在于：所述抗原表位肽为位于SEQ ID NO.1中第18位～27位氨基酸序列中的连续氨基酸序列。
  5. 根据权利要求4所述的新型的抗原表位肽，其特征在于：所述抗原表位肽为与SEQ ID NO.1中第18位～27位的连续氨基酸具有80％以上重叠度的氨基酸序列。
  6. 根据权利要求1所述的新型的抗原表位肽，其特征在于：所述抗原表位肽的氨基酸序列如SEQ ID NO.3-11任一所示。
  7. 一种融合蛋白，所述融合蛋白是由权利要求1-6中任一项所述的抗原表位肽与载体蛋白融合所形成的。
  8. 一种能够编码抗原表位肽的核酸序列，其特征在于：所述核酸序列为能够编码权利要求1-6中任意一项所述的抗原表位肽的基因序列或基因片段。
  9. 一种表达载体，所述表达载体含有权利要求8所述的核酸序列。
  10. 一种宿主细胞，所述的宿主细胞含有权利要求9所述的表达载体，或者在基因组中整合有权利要求8所述的核酸序列。
  11. 一种抗体，其特征在于：所述抗体为能够结合权利要求1-6中任一项所述的抗原表位肽的抗体。
  12. 根据权利要求10所述的抗体，其特征在于：所述抗体为能够抑制或减弱与权利要求1-5中任一项所述的抗原表位肽相关的信号传递的抗体，且所述抗体为单克隆抗体或多克隆抗体，种属来源可以为人，鼠，兔，猴，牛，羊或羊驼等哺乳动物。
  13. 一种疫苗，所述疫苗含有权利要求1-6中任一项所述的抗原表位肽、权利要求7所述的融合蛋白、权利要求8所述的核酸序列、权利要求9所述的表达载体或者权利要求10所 述的宿主细胞。
  14. 一种组合物，其特征在于，含有权利要求1-6中任一项所述的抗原表位肽、权利要求7所述的融合蛋白、权利要求8所述的核酸序列、权利要求9所述的表达载体或者权利要求10所述的宿主细胞，权利要求11-12任一所述的抗体，或者权利要求13所述的疫苗，以及免疫学和药学上可接受的载体和/或辅料。
  15. 权利要求1-6中任一项所述的抗原表位肽、权利要求7所述的融合蛋白、权利要求8所述的核酸序列、权利要求9所述的表达载体或者权利要求10所述的宿主细胞的用途：
    (1)用于制备针对所述抗原表位的抗体、核酸适配体、疫苗、纳米粒子；和/或
    (2)用于制备治疗与所述抗原表位相关的疾病的药物。
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