Preparation and application of anti-CD22 rabbit recombinant monoclonal antibody

Abstract

The application relates to preparation and application of anti-CD22 rabbit recombinant monoclonal antibodies. The obtained antibodies have good specificity, and most importantly, the antibodies have high affinity and can recognize natural cell surface CD22 proteins, and can be used for flow and ELISA detection applications.

Description

Technical Field

[0001] This invention belongs to the field of antibody preparation in biotechnology, and in particular relates to a rabbit recombinant monoclonal antibody that can specifically bind to the CD22 protein and its applications. Background Technology

[0002] CD22 is a type I transmembrane glycoprotein, a member of the sialic acid-binding immunoglobulin-like lectin family, and an inhibitory co-receptor of the B cell receptor, playing a negative regulatory role in B cell activation signaling. CD22 specifically binds to glycoprotein ligands containing α-2,6-linked sialic acid, activating the B cell receptor (BCR) via antigen activation. It also rapidly phosphorylates tyrosine residues in the tyrosine inhibitory motif of the CD22 cytoplasmic immunoreceptor, activating downstream signaling molecules to inhibit calcium ion influx and weaken the BCR signal. CD22 is relatively specifically expressed on the surface of B cells and has become a promising target for regulating B cell immunity and treating certain B cell tumors.

[0003] Currently, some types of anti-CD22 antibodies have been developed in existing technologies. For example, Chinese patent application CN2020100889800 developed a chimeric antigen receptor targeting humanized CD22 and its applications. Although the above-mentioned antibodies have certain application prospects, the requirements for antibodies in flow cytometry and ELISA detection applications are higher, and the above-mentioned antibodies may not fully meet the current detection needs. Therefore, it is still necessary to develop monoclonal antibodies with stronger affinity and more epitopes. Summary of the Invention

[0004] The purpose of this invention is to provide a recombinant rabbit monoclonal antibody against CD22, selected from one or more of the following recombinant rabbit monoclonal antibodies:

[0005] The rabbit recombinant monoclonal antibody named 8B5 has the following amino acid sequences: CDR1, CDR2, and CDR3 of its heavy chain complementarity-determining regions are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the amino acid sequences of CDR1, CDR2, and CDR3 of its light chain complementarity-determining regions are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0006] The rabbit recombinant monoclonal antibody named 9A3 has the following amino acid sequences: CDR1, CDR2, and CDR3 of its heavy chain complementarity-determining regions are shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; and CDR1, CDR2, and CDR3 of its light chain complementarity-determining regions are shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively.

[0007] The rabbit recombinant monoclonal antibody named 10F9 has the following amino acid sequences in its heavy chain complementarity-determining regions CDR1, CDR2, and CDR3: as shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively, and the following amino acid sequences in its light chain complementarity-determining regions CDR1, CDR2, and CDR3: as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively.

[0008] Based on the above technical solution, a rabbit recombinant monoclonal antibody named 8B5 is developed, whose heavy chain variable region sequence is the amino acid sequence shown in SEQ ID NO:7 and whose light chain variable region sequence is the amino acid sequence shown in SEQ ID NO:8.

[0009] The rabbit recombinant monoclonal antibody named 9A3 has the heavy chain variable region sequence shown in SEQ ID NO:16 and the light chain variable region sequence shown in SEQ ID NO:17.

[0010] The rabbit recombinant monoclonal antibody named 10F9 has the following amino acid sequence: heavy chain variable region sequence as shown in SEQ ID NO:25 and light chain variable region sequence as shown in SEQ ID NO:26.

[0011] Based on the above technical solution, a rabbit recombinant monoclonal antibody named 8B5 was developed, and its SCFV sequence is the amino acid sequence shown in SEQ ID NO:9.

[0012] A rabbit recombinant monoclonal antibody named 9A3 has an SCFV sequence as shown in SEQ ID NO:18;

[0013] The rabbit recombinant monoclonal antibody named 10F9 has the SCFV sequence shown in SEQ ID NO:27.

[0014] Based on the above technical solution, the light chain constant region of the rabbit recombinant monoclonal antibody is the κ chain, and the heavy chain constant region is of the IgG type.

[0015] The present invention also provides a nucleic acid molecule comprising a nucleic acid sequence of a heavy chain complementarity-determining region or a light chain complementarity-determining region capable of encoding a rabbit recombinant monoclonal antibody that binds to the CD22 protein.

[0016] The present invention also provides a carrier containing the above-mentioned nucleic acid molecules.

[0017] The present invention also provides a host cell containing the above-mentioned rabbit recombinant monoclonal antibody that binds to CD22 protein, the above-mentioned nucleic acid molecule, or the above-mentioned vector.

[0018] The present invention also provides a conjugate containing the above-mentioned antibody.

[0019] The present invention also provides a pharmaceutical composition comprising a main component and an excipient, wherein: the main component is one or more of the above-mentioned rabbit recombinant monoclonal antibody binding to CD22 protein, the above-mentioned nucleic acid molecule, the above-mentioned carrier, the above-mentioned host cell, and the above-mentioned conjugate; and the excipient is selected from pharmaceutically acceptable carriers or excipients, and optionally other bioactive substances.

[0020] The present invention also provides the use of the above-mentioned rabbit recombinant monoclonal antibody that binds to CD22 protein, the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned host cell, and the above-mentioned conjugate in the preparation of drugs for treating diseases or detection reagents.

[0021] The present invention also provides a kit comprising the above-described rabbit recombinant monoclonal antibody that binds to the CD22 protein.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] Currently, there are no rabbit-derived monoclonal antibodies targeting CD22. Rabbit antibodies have higher affinity than mouse antibodies, and the rabbit immunization process is more complex than that of mice, allowing for the production of antibodies targeting more epitopes. This invention utilizes the CD22 extracellular protein to immunize rabbits, and then prepares an anti-CD22 rabbit recombinant monoclonal antibody using B-cell cloning technology. Importantly, this anti-CD22 rabbit recombinant monoclonal antibody exhibits high affinity and good specificity, recognizing the native cell surface CD22 protein, and can be used for flow cytometry and ELISA detection applications. Furthermore, future development of other diagnostic and therapeutic reagents targeting CD22 is possible. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The image shows an SDS-PAGE image of the CD22 protein.

[0026] Figure 2 The results of PCR amplification of the heavy and light chains of the anti-CD22 rabbit recombinant monoclonal antibody are shown.

[0027] Figure 3The image shows an SDS-PAGE image of the purified CD22 rabbit recombinant monoclonal antibody.

[0028] Figures 4 - 6 This demonstrates the specificity of FACs in detecting recombinant monoclonal antibodies against CD22 in rabbits.

[0029] Figure 7 The binding affinity of FACs to the CD22 rabbit recombinant monoclonal antibody was demonstrated. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] The present invention will be further described below: Unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. Additionally, to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0032] The term "antibody" as used herein includes complete antibodies and any antigen-binding fragments (i.e., "antigen-binding moieties") or single chains thereof. An "antibody" is a glycoprotein, or its antigen-binding moieties, comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region and a heavy chain constant region. The proteins or fragments thereof involved in this invention may be naturally purified products, chemically synthesized products, or products generated from a eukaryotic host (e.g., mammalian cells) using recombinant technology. All raw materials and reagents used in this invention are commercially available.

[0033] The technical solution of the present invention will be further described below with reference to the embodiments.

[0034] Example:

[0035] (1) Preparation of CD22 protein: In this invention, a CD22 FC fusion tag protein eukaryotic expression plasmid was constructed. After transfecting 293F cells for 5 days, the cell culture supernatant was collected, purified using protein A resin, and the protein concentration was identified. The protein expression, purification, and identification results are shown in [Figure number missing]. Figure 1 .

[0036] (2) Obtaining peripheral blood mononuclear cells (PBMCs) from immunized animals: New Zealand white rabbits were selected as immunized animals. For the first immunization, 250 μg of CD22 protein was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple points on the back of the New Zealand white rabbits. A second immunization was performed 21 days later, with 120 μg of protein emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously into the back of the New Zealand white rabbits. A third immunization was performed 21 days later, with 120 μg of protein emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously into the back of the New Zealand white rabbits. Peripheral blood was aseptically collected one week after the third immunization.

[0037] (3) Obtaining CD22-specific B lymphocytes: PBMC cells were separated from the collected peripheral blood using lymphocyte separation solution; CD22 protein was coupled to the magnetic beads according to the instructions for using immunomagnetic beads; the mixture of CD22 protein-coupled magnetic beads and separated PBMC cells was incubated at room temperature for 50 min and then placed in a magnetic rack. After 5 min, all the magnetic beads sank to the bottom. The supernatant was discarded, and sterile PBS was added to wash the cells. The washing was repeated 3 times. The cells obtained in the end were CD22-specific B lymphocytes.

[0038] (4) Identification of B lymphocytes: The isolated B lymphocytes were diluted several times and placed in 96-well cell culture plates. 1640 medium containing 10% fetal bovine serum (FBS) and 2 μg / ml human IL2 was added, and the plates were cultured at 37℃ and 5% CO2 for 6 days. The supernatant of the medium was collected for antibody identification.

[0039] Indirect ELISA identification:

[0040] The CD22 protein at a concentration of 1 μg / ml was coated at 100 μl / well and incubated at 4℃ for 16 h. The next day, after discarding the coating solution, the cells were blocked with PBS containing 1% bovine serum albumin (BSA) at 150 μl / well and incubated at 37℃ for 1 h. The blocking solution was discarded, and B cell supernatant was added at 50 μl / well and incubated at 37℃ for 1 h. The B cell supernatant was discarded, and the cells were washed 5 times with PBS containing 0.5 wt.% Tween-20 for 2 min each time. Finally, goat anti-rabbit IgG-HRP secondary antibody diluted 5000 times was added and the cells were incubated at 37℃ for 1 h. The secondary antibody was discarded, and the cells were washed 5 times with phosphate-Tween buffer (PBST) for 2 min each time. The washing solution was discarded, the cells were patted dry, and the substrate was added for color development. B cell clones labeled 8B5, 9A3, and 10F9 were identified as positive by ELISA. The detection results are shown in Table 1.

[0041] Table 1. ELISA test results

[0042] Clone number OD450 8B5 1.93 9A3 2.12 10F9 1.86

[0043] (5) Cloning of antibody genes

[0044] B cells that tested positive were collected, and RNA was extracted using standard RNA extraction methods. The RNA was then reverse transcribed into cDNA. The antibody heavy chain gene primers were used for amplification: upstream primer 5'-CAGTCGCTGGAGGAGTCCGG-3' and downstream primer 5'-CCATTGGTGAGGGTGCCCGAG-3'. The antibody light chain gene primers were used for amplification: upstream primer 5'-GACATTGTGATGACCCAGAC-3' and downstream primer 5'-CCACCTCGGTCCCTTCGCCG-3'. The amplification conditions were: denaturation at 94℃ for 3 min, followed by 30 cycles of (95℃ for 1 min, 56℃ for 30 s, and 72℃ for 1 min), and a final extension at 72℃ for 10 min. (PCR amplification results are shown in [link to PCR results]). Figure 2 The PCR products were recovered using a DNA gel purification and recovery kit. The heavy and light chain genes of the rabbit recombinant monoclonal antibody were cloned into an expression vector and transformed. Single colonies were verified using colony PCR, and gene sequencing was performed on positive colonies to obtain the gene sequence of the specific antibody. The amino acid sequences of each region of the specific antibody were obtained by translating the gene sequence of the specific antibody according to the codons. The amino acid sequences of the specific antibodies finally obtained using B lymphocytes labeled 8B5, 9A3, and 10F9 are shown in Table 2.

[0045] Table 2. Amino acid sequences of specific antibodies

[0046] SEQ ID NO. Designation Form / Source Type Sequence 1 8B5 VH CDR1 aa GIDLSSYA 2 VH CDR2 aa VTYAGSA 3 VH CDR3 aa ARGVDNFYTLPDAFDP 4 VL CDR1 aa QSISRF 5 VL CDR2 aa RAS 6 VL CDR3 aa QQGDTYINVDNP 7 VH aa QEQLKESGGGLVTPGGSLTLTCTVSGIDLSSYAMGWVRQAPGKGLEYIGIVTYAGSAYYASWARGRFTISKTSTTVDLKITSPTTEDTATYFCARGVDNFYTLPDAFDPWGPGTLVTVS 8 VL aa AYDMTQTPASVSAAVGGTVTINCQASQSISRFLSWYQQKPGQRPKLLIYRASTLASGVSSRFEGSGSGTQFTLTISGVECADAATYYCQQGDTYINVDNPFGGGTEVVVK 9 scfv aa QEQLKESGGGLVTPGGSLTLTCTVSGIDLSSYAMGWVRQAPGKGLEYIGIVTYAGSAYYASWARGRFTISKTSTTVDLKITSPTTEDTATYFCARGVDNFYTLPDAFDPWGPGTLVTVSAYDMTQTPASVSAAVGGTVTINCQASQSISRFLSWYQQKPGQRPKLLIYRASTLASGVSSRFEGSGSGTQFTLTISGVECADAATYYCQQGDTYINVDNPFGGGTEVVVK 10 9A3 VH CDR1 aa GIDLSSYW 11 VH CDR2 aa IWSDDNT 12 VH CDR3 aa ARGYVGDI 13 VL CDR1 aa QNIYSG 14 VL CDR2 aa GAS 15 VL CDR3 aa QQTYTSDNVDNP 16 VH aa QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYWMNWVRQAPGKGLEWIGIIWSDDNTYYANWAKGRFTISKTSSTTVDLTITSPTTEDTATYFCARGYVGDIWGPGTLITVS 17 VL aa ALVMTQTPSSVDVAVGGTVTIRCQASQNIYSGLAWYQQKPGQPPKLLIYGASNLASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQTYTSDNVDNPFGGGTEVVVE 18 scfv aa QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYWMNWVRQAPGKGLEWIGIIWSDDNTYYANWAKGRFTISKTSSTTVDLTITSPTTEDTATYFCARGYVGDIWGPGTLITVSALVMTQTPSSVDVAVGGTVTIRCQASQNIYSGLAWYQQKPGQPPKLLIYGASNLASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQTYTSDNVDNPFGGGTEVVVE 19 10F9 VH CDR1 aa GFSLNTDA 20 VH CDR2 aa IGSSGSA 21 VH CDR3 aa ARFTGYGGYGRGGMDP 22 VL CDR1 aa ESVHNNNW 23 VL CDR2 aa KAS 24 VL CDR3 aa QATYNSGGWYVA 25 VH aa QSVEESGGRLVTPGTPLTLTCTVSGFSLNTDAMIWVRQAPGKGLEYIGIIGSSGSAFFASWAKGRFTISKTSTTVDLTIISPTTEDTATYFCARFTGYGGYGRGGMDPWGPGTLVTVSS 26 VL aa AQVLTQTPSSVSAAVGGTVTINCQSSESVHNNNWLSWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCQATYNSGGWYVAFGGGTEVVVK 27 scfv aa QSVEESGGRLVTPGTPLTLTCTVSGFSLNTDAMIWVRQAPGKGLEYIGIIGSSGSAFFASWAKGRFTISKTSTTVDLTIISPTTEDTATYFCARFTGYGGYGRGGMDPWGPGTLVTVSSAQVLTQTPSSVSAAVGGTVTINCQSSESVHNNNWLSWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCQATYNSGGWYVAFGGGTEVVVK

[0047] (6) Production and identification of CD22 rabbit recombinant monoclonal antibody

[0048] The expression plasmids of the heavy and light chain genes of the antibody were co-transfected into 293 cells and cultured at 37℃ with 5% CO2 for 72 h. Cell supernatant was collected, and the antibody was purified using Protein A resin. The purified antibody was then identified by SDS-PAGE. The results are shown below. Figure 3 .

[0049] The purified antibody underwent functional identification, as detailed below:

[0050] a) ELISA reaction

[0051] The CD22 protein at a concentration of 1 μg / ml was coated at 100 μl / well and incubated at 4℃ for 16 h. The next day, the coating solution was discarded, and the plate was blocked with PBS containing 1% BSA at 150 μl / well and incubated at 37℃ for 1 h. The blocking solution was discarded, and antibodies diluted at different fractions were added at 50 μl / well and incubated at 37℃ for 1 h. The antibodies were discarded, and the plate was washed 5 times with PBS containing 0.5% Tween-20 for 2 min each time. Finally, goat anti-rabbit IgG-HRP secondary antibody diluted 5000 times was added and incubated at 37℃ for 1 h. The secondary antibody was discarded, and the plate was washed 5 times with PBST for 2 min each time. The washing solution was discarded, the plate was patted dry, and substrate was added for color development. The ELISA results are shown in Table 2.

[0052] Table 2. ELISA Test Results

[0053] Dilution factor 8B5 9A3 10F9 1K Overflow 3.9542 Overflow 5K Overflow Overflow Overflow 25K 3.5169 3.1039 3.3671 125K 1.9231 1.3259 1.3943 625K 0.5284 0.3816 0.4051 3125k 0.2027 0.1796 0.1714 15625k 0.1459 0.1204 0.1246 blank 0.1162 0.1326 0.1123

[0054] b) Flow cytometry-based specific assay method

[0055] The CD22 full-length protein particle was transfected into 293 cells for testing. Transfected cells were collected in centrifuge tubes, washed twice with sterile PBS, and Fc receptor blocking buffer was used to block the cell surface Fc receptors. The cells were incubated at 4°C for 30 min. Cells were collected by centrifugation, washed twice with PBS containing 0.5 wt.% BSA, and then incubated at 4°C for 30 min with different concentrations of antibody. Cells were then washed twice again with PBS containing 0.5 wt.% BSA, and finally, goat anti-rabbit IgG-488 fluorescent secondary antibody was added and incubated at 4°C for 30 min. After washing twice with PBS containing 0.5 wt.% BSA, the cells were resuspended in 200 μl of PBS containing 0.5 wt.% BSA and analyzed by flow cytometry. Flow cytometry specificity results are shown below. Figures 4 - 6 The analysis results show that the screened antibodies can specifically recognize CD22 on the cell membrane.

[0056] (c) Flow cytometry-based method for determining binding force

[0057] Raji cells were used for testing. Cells were collected in centrifuge tubes, washed twice with sterile PBS, and Fc receptors on the cell surface were blocked using Fc receptor blocking solution. The cells were incubated at 4°C for 30 min. Cells were collected by centrifugation, washed twice with PBS containing 0.5 wt.% BSA, and then incubated at 4°C for 30 min with different concentrations of antibody. Cells were then washed twice more with PBS containing 0.5 wt.% BSA, and finally, goat anti-rabbit IgG-488 fluorescent secondary antibody was added and incubated at 4°C for 30 min. After washing twice with PBS containing 0.5 wt.% BSA, the cells were resuspended in 200 μl of PBS containing 0.5 wt.% BSA and analyzed by flow cytometry. The results of the flow cytometry binding affinity are shown below. Figure 7Flow cytometry binding affinity analysis showed that the screened antibodies had different binding affinity.

[0058] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. sequence list <110> Suzhou Dima Biotechnology Co., Ltd. <120> Preparation and application of anti-CD22 rabbit recombinant monoclonal antibody <150> 2021106279988 <151> 2021-06-05 <160> 27 <170> SIPOSequenceListing 1.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <400> 1 Gly Ile Asp Leu Ser Ser Tyr Ala 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <400> 2 Val Thr Tyr Ala Gly Ser Ala 1 5 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <400> 3 Ala Arg Gly Val Asp Asn Phe Tyr Thr Leu Pro Asp Ala Phe Asp Pro 1 5 10 15 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <400> 4 Gln Ser Ile Ser Arg Phe 1 5 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <400> 5 Arg Ala Ser 1<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 50 55 60 Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly 85 90 95 Val Asp Asn Phe Tyr Thr Leu Pro Asp Ala Phe Asp Pro Trp Gly Pro 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 8 <211> 110 <212> PRT <213> Artificial Sequence <400> 8 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Ile Ser Arg Phe 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Glu Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Thr Tyr Ile Asn 85 90 95 Val Asp Asn Pro Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 9 <211> 229 <212> PRT <213> Artificial Sequence <400> 9 Gln Glu Gln Leu Lys Glu Ser Gly Gly Gly Leu Val Thr Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Ile Val Thr Tyr Ala Gly Ser Ala Tyr Tyr Ala Ser Trp Ala Arg 50 55 60 Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly 85 90 95 Val Asp Asn Phe Tyr Thr Leu Pro Asp Ala Phe Asp Pro Trp Gly Pro 100 105 110 Gly Thr Leu Val Thr Val Ser Ala Tyr Asp Met Thr Gln Thr Pro Ala 115 120 125 Ser Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala 130 135 140 Ser Gln Ser Ile Ser Arg Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly 145 150 155 160 Gln Arg Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly 165 170 175 Val Ser Ser Arg Phe Glu Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu 180 185 190 Thr Ile Ser Gly Val Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln 195 200 205 Gln Gly Asp Thr Tyr Ile Asn Val Asp Asn Pro Phe Gly Gly Gly Thr 210 215 220 Glu Val Val Val Lys 225 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <400> 10 Gly Ile Asp Leu Ser Ser Tyr Trp 1 5 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <400> 11 Ile Trp Ser Asp Asp Asn Thr 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <400> 12 Ala Arg Gly Tyr Val Gly Asp Ile 1 5 <210> 13 <211> 6 <212> PRT <213> Artificial Sequence <400> 13 Gln Asn Ile Tyr Ser Gly 1 5 <210> 14 <211> 3 <212> PRT <213> Artificial Sequence <400> 14 Gly Ala Ser 1 <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <400> 15 Gln Gln Thr Tyr Thr Ser Asp Asn Val Asp Asn Pro 1 5 10 <210> 16 <211> 111 <212> PRT <213> Artificial Sequence <400> 16 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr Trp 20 25 30 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Trp Ser Asp Asp Asn Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Thr Ile 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly 85 90 95 Tyr Val Gly Asp Ile Trp Gly Pro Gly Thr Leu Ile Thr Val Ser 100 105 110 <210> 17 <211> 110 <212> PRT <213> Artificial Sequence <400> 17 Ala Leu Val Met Thr Gln Thr Pro Ser Ser Val Asp Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Arg Cys Gln Ala Ser Gln Asn Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Ser Asp Asn 85 90 95 Val Asp Asn Pro Phe Gly Gly Gly Thr Glu Val Val Val Glu 100 105 110 <210> 18 <211> 221 <212> PRT <213> Artificial Sequence <400> 18 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro<着 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr Trp 20 25 30 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Trp Ser Asp Asp Asn Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 It should be noted that there seems to be a misspelling in "着0000525" in the original text. It is likely a typo and should probably be " " as in other parts of the text. This has been left as is in the translation to maintain consistency with the original. Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Thr Ile 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly 85 90 95 Tyr Val Gly Asp Ile Trp Gly Pro Gly Thr Leu Ile Thr Val Ser Ala 100 105 110 Leu Val Met Thr Gln Thr Pro Ser Ser Val Asp Val Ala Val Gly Gly 115 120 125 Thr Val Thr Ile Arg Cys Gln Ala Ser Gln Asn Ile Tyr Ser Gly Leu 130 135 140 Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 145 150 155 160 Gly Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly Ser 165 170 175 Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys Ala 180 185 190 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Ser Asp Asn Val 195 200 205 Asp Asn Pro Phe Gly Gly Gly Thr Glu Val Val Val Glu 210 215 220 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <400> 19 Gly Phe Ser Leu Asn Thr Asp Ala 1 5 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <400> 20 Ile Gly Ser Ser Gly Ser Ala 1 5 <210> twenty one <211> 16 <212> PRT <213> Artificial Sequence <400> twenty one Ala Arg Phe Thr Gly Tyr Gly Gly Tyr Gly Arg Gly Gly Met Asp Pro 1 5 10 15 <210> twenty two <211> 8 <212> PRT <213> Artificial Sequence <400> twenty two Glu Ser Val His Asn Asn Asn Trp 1 5 <210> twenty three <211> 3 <212> PRT <213> Artificial Sequence <400> twenty three Lys Ala Ser 1 <210> twenty four <211> 12 <212> PRT <213> Artificial Sequence <400> 24 Gln Ala Thr Tyr Asn Ser Gly Gly Trp Tyr Val Ala 1 5 10 <210> 25 <211> 119 <212> PRT <213> Artificial Sequence <400> 25 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Thr Asp Ala 20 25 30 Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly[[ID=二十九]] 35 40 45 Ile Ile Gly Ser Ser Gly Ser Ala Phe Phe Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Thr Ile Ile 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Phe Thr 85 90 95 Gly Tyr Gly Gly Tyr Gly Arg Gly Gly Met Asp Pro Trp Gly Pro Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 26 <211> 112 <212> PRT <213> Artificial Sequence <400> 26 Ala Gln Val Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Glu Ser Val His Asn Asn 20 25 30 Asn Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Ala Thr Tyr Asn Ser 85 90 95 Gly Gly Trp Tyr Val Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 27 <211> 231 <212> PRT <213> Artificial Sequence <400> 27 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Thr Asp Ala 20 25 30 Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Ile Ile Gly Ser Ser Gly Ser Ala Phe Phe Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Thr Ile Ile 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Phe Thr 85 90 95 Gly Tyr Gly Gly Tyr Gly Arg Gly Gly Met Asp Pro Trp Gly Pro Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Gln Val Leu Thr Gln Thr Pro Ser 115 120 125 Ser Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ser 130 135 140 Ser Glu Ser Val His Asn Asn Asn Trp Leu Ser Trp Tyr Gln Gln Lys 145 150 155 160 Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Lys Ala Ser Thr Leu Ala 165 170 175 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe 180 185 190 Thr Leu Thr Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr 195 200 205 Cys Gln Ala Thr Tyr Asn Ser Gly Gly Trp Tyr Val Ala Phe Gly Gly 210 215 220 Gly Thr Glu Val Val Val Lys 225 230

Claims

1. A rabbit recombinant monoclonal antibody that binds to the CD22 protein, characterized in that: The antibody is named 8B5 rabbit recombinant monoclonal antibody. The amino acid sequences of its heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the amino acid sequences of its light chain complementarity-determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

2. The rabbit recombinant monoclonal antibody binding to CD22 protein according to claim 1, characterized in that: The rabbit recombinant monoclonal antibody named 8B5 has a heavy chain variable region sequence as shown in SEQ ID NO:7 and a light chain variable region sequence as shown in SEQ ID NO:

8.

3. The rabbit recombinant monoclonal antibody binding to CD22 protein according to claim 1 or 2, characterized in that: The rabbit recombinant monoclonal antibody named 8B5 has an SCFV sequence that is the amino acid sequence shown in SEQ ID NO:

9.

4. The rabbit recombinant monoclonal antibody binding to CD22 protein according to claim 1, characterized in that: The rabbit recombinant monoclonal antibody has a light chain constant region of κ chain and a heavy chain constant region of IgG type.

5. A nucleic acid molecule, characterized in that: It encodes the rabbit recombinant monoclonal antibody that binds to the CD22 protein as described in any one of claims 1-4.

6. A vector, characterized by: It contains the nucleic acid molecule as described in claim 5.

7. A host cell, characterized in that: The host cell contains the rabbit recombinant monoclonal antibody that binds to the CD22 protein as described in any one of claims 1 to 4, the nucleic acid molecule as described in claim 5, or the vector as described in claim 6.

8. The use of the rabbit recombinant monoclonal antibody binding CD22 protein according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, and the host cell according to claim 7 in the preparation of a reagent for detecting CD22 protein.

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