A human-mouse chimeric monoclonal antibody against human MOG protein and its application

Abstract

The present invention belongs to the technical field of monoclonal antibody preparation, and specifically relates to a human-mouse chimeric monoclonal antibody against human myelin oligodendrocyte glycoprotein (MOG) and its application. The variable regions of the heavy and light chains of the human-mouse chimeric monoclonal antibody against human MOG protein described in the present invention have complementarity-determining regions derived from mice, maximally retaining the parental activity, while the constant regions are human antibody constant regions. The introduction of human antibody constant regions greatly reduces immunogenicity, reduces the incidence of human anti-mouse antibody reactions, and improves the specificity of the antibody. The human-mouse chimeric monoclonal antibody against human MOG protein described in the present invention has both antigen-binding specificity and greatly reduces the heterogeneity of mouse monoclonal antibodies, resulting in better biological activity and the ability to specifically detect MOG protein.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibody preparation, and particularly relates to a human-mouse chimeric monoclonal antibody against human MOG protein and its application. Background Art

[0002] Human myelin oligodendrocyte glycoprotein (MOG) is a single-transmembrane protein present in the central nervous system. It is composed of 218 amino acids and is a member of the immunoglobulin superfamily. It is expressed relatively late in the development of the nervous system and is specifically expressed on the plasma membrane of oligodendrocytes in the central nervous system. It is an important surface marker of oligodendrocyte maturation and plays an important role in maintaining the integrity of the myelin sheath and promoting information exchange between cells.

[0003] MOG-IgG-associated disorders (MOGAD) are autoimmune diseases likely caused by MOG-IgG. Their pathogenesis, clinical manifestations, therapeutic efficacy, and prognosis differ from those of classic MS and AQP4 antibody-positive NMOSD. MOGAD is gradually becoming a distinct entity. The pathogenesis of MOGAD is MOG antibody-mediated oligodendrocyte damage, followed by demyelination and axonal injury. Pathologically, MOGAD often involves the optic nerves, spinal cord, pons, and thalamus, with T cell and macrophage infiltration around venules and immune complex deposition. Studies have shown that anti-MOG antibodies may be the causative antibodies for conditions such as acute dissemina tedencephalomyelitis (ADEM), multiphasic disseminated encephalomyelitis (MDEM), optic neuritis (ON), longitudinally extensive transverse myelitis (LETM), and neuromyelitis optica spectrum disease (NMOSD). International diagnostic guidelines for anti-MOG antibody-associated disease (MOGAD) recommend the use of a cell-based immunofluorescence assay (CBA) to detect anti-MOG antibodies in serum and cerebrospinal fluid. Furthermore, serum anti-MOG antibody titers correlate with disease severity, with persistent or elevated titers indicating the possibility of relapse. Timely, accurate, and rapid diagnosis is beneficial for a good prognosis and reduced disability.

[0004] Detecting MOG-IgG autoantibodies requires the MOG antigen. Common CBA assays typically coat the MOG antigen with intact, full-length human MOG protein in the correct spatial conformation. A positive control sample is also required for quality control of the assay. Positive controls typically include serum from positive patients, or a chimeric MOG monoclonal antibody can be prepared. This serves as a control for positive serum, allowing for quantitative protein concentration determination and providing a long-term, stable supply of the positive control required for the assay. It also allows for identification of MOG protein expression. However, positive patient serum is difficult to obtain and cannot be consistently supplied to meet the quality control requirements of the assay. Existing MOG monoclonal antibodies are mostly mouse or rabbit derived, making them inadequate substitutes for positive patient serum.

[0005] Therefore, the development of anti-human MOG monoclonal chimeric antibodies is of great significance for the study of MOG protein and the detection of MOGAD. Summary of the Invention

[0006] The object of the present invention is to provide a human-mouse chimeric monoclonal antibody against human MOG protein and its application. The human-mouse chimeric monoclonal antibody against human MOG protein has human properties and can be directly used as a substitute for positive patient serum. At the same time, the human-mouse chimeric monoclonal antibody against human MOG protein has high biological activity and specificity and can specifically detect MOG protein.

[0007] The present invention provides a human-mouse chimeric monoclonal antibody against human MOG protein, comprising a light chain and a heavy chain; the light chain comprises a light chain variable region and a light chain constant region; the light chain variable region comprises a light chain complementary determining region CDR1, a light chain complementary determining region CDR2, and a light chain complementary determining region CDR3, the amino acid sequence of the light chain complementary determining region CDR1 being as shown in SEQ ID NO:3, the amino acid sequence of the light chain complementary determining region CDR2 being as shown in SEQ ID NO:4, and the amino acid sequence of the light chain complementary determining region CDR3 being as shown in SEQ ID NO:5, or based on the amino acids shown in SEQ ID NO:5, the third amino acid is mutated from V to G and / or the sixth amino acid is mutated from Y to S; the light chain constant region is a human light chain constant region;

[0008] The heavy chain includes a heavy chain variable region and a heavy chain constant region; the heavy chain variable region includes a heavy chain complementary determining region CDR1, a heavy chain complementary determining region CDR2 and a heavy chain complementary determining region CDR3, and the amino acid sequences of the heavy chain complementary determining region CDR1, the heavy chain complementary determining region CDR2 and the heavy chain complementary determining region CDR3 are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQID NO: 12, respectively; the heavy chain constant region is a human heavy chain constant region.

[0009] Preferably, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0010] Preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9.

[0011] The present invention also provides a recombinant expression vector comprising a DNA sequence for expressing the human-mouse chimeric monoclonal antibody against human MOG protein described in the above technical solution.

[0012] Preferably, the recombinant expression vector includes a light chain recombinant expression vector and / or a heavy chain recombinant expression vector;

[0013] The light chain recombinant expression vector comprises a DNA sequence expressing a light chain variable region and a DNA sequence expressing a light chain constant region;

[0014] The heavy chain recombinant expression vector comprises a DNA sequence expressing a heavy chain variable region and a DNA sequence expressing a heavy chain constant region;

[0015] The DNA sequence expressing the light chain variable region comprises the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8;

[0016] The DNA sequence expressing the heavy chain variable region includes the nucleotide sequence shown in SEQ ID NO: 13.

[0017] Preferably, the backbone vector of the recombinant expression vector includes a plasmid vector.

[0018] The present invention also provides an engineered cell, which overexpresses the human-mouse chimeric monoclonal antibody against human MOG protein described in the above technical solution.

[0019] The present invention also provides the use of the human-mouse chimeric monoclonal antibody against human MOG protein described in the above technical solution in the preparation of a kit for detecting MOG protein;

[0020] Preferably, the human-mouse chimeric monoclonal antibody against human MOG protein is used as a positive reference or quality control product in the kit.

[0021] The present invention also provides a kit for detecting anti-MOG antibodies, which includes the human-mouse chimeric monoclonal antibody against human MOG protein described in the above technical solution.

[0022] Preferably, the kit is a kit prepared based on immunoassay technology.

[0023] Beneficial effects:

[0024] The present invention provides a chimeric monoclonal antibody against human MOG protein. The complementarity-determining regions in the variable regions of the heavy and light chains of this chimeric monoclonal antibody against human MOG protein are derived from mice, maximally retaining the parental activity. The constant regions are derived from human antibodies. The introduction of these human antibody constant regions significantly reduces immunogenicity, decreases the incidence of human anti-mouse antibody reactions, and improves the antibody's specificity. The chimeric monoclonal antibody against human MOG protein of the present invention exhibits antigen-binding specificity while significantly reducing the heterologous nature of mouse monoclonal antibodies. Furthermore, the chimeric monoclonal antibody against human MOG protein of the present invention is easy to store and facilitates quality control during the antibody production process. It also facilitates a series of antibody modifications, enabling more in-depth research and wider application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0026] Figure 1 The purification results of MOG prokaryotic protein in Example 1;

[0027] Figure 2 This is the immunofluorescence result of detecting mouse antibody production in Example 2;

[0028] Figure 3 The immunofluorescence screening results of the positive hybridoma cells in Example 2 are shown;

[0029] Figure 4 The purification results of the anti-human MOG human-mouse chimeric monoclonal antibody in Example 4;

[0030] Figure 5 This is the immunofluorescence result for identifying the function of the anti-human MOG human-mouse chimeric monoclonal antibody in Example 5;

[0031] Figure 6 This is the Western Blot result for identifying the function of the anti-human MOG human-mouse chimeric monoclonal antibody in Example 5. DETAILED DESCRIPTION

[0032] The present invention provides a human-mouse chimeric monoclonal antibody against human MOG protein, comprising a light chain and a heavy chain; the light chain comprises a light chain variable region and a light chain constant region; the light chain variable region comprises a light chain complementary determining region CDR1, a light chain complementary determining region CDR2, and a light chain complementary determining region CDR3, the amino acid sequence of the light chain complementary determining region CDR1 being as shown in SEQ ID NO:3, the amino acid sequence of the light chain complementary determining region CDR2 being as shown in SEQ ID NO:4, and the amino acid sequence of the light chain complementary determining region CDR3 being as shown in SEQ ID NO:5, or based on the amino acids shown in SEQ ID NO:5, the third amino acid is mutated from V to G and / or the sixth amino acid is mutated from Y to S; the light chain constant region is a human light chain constant region;

[0033] The heavy chain includes a heavy chain variable region and a heavy chain constant region; the heavy chain variable region includes a heavy chain complementary determining region CDR1, a heavy chain complementary determining region CDR2 and a heavy chain complementary determining region CDR3, and the amino acid sequences of the heavy chain complementary determining region CDR1, the heavy chain complementary determining region CDR2 and the heavy chain complementary determining region CDR3 are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQID NO: 12, respectively; the heavy chain constant region is a human heavy chain constant region.

[0034] In the present invention, the amino acid sequences of the three light chain complementary determining regions of the light chain variable region are as follows: light chain complementary determining region CDR1: RSSKSLLYKDGKTYLS (SEQ ID NO: 3); light chain complementary determining region CDR2: WMSTRAS (SEQ ID NO: 4); and light chain complementary determining region CDR3: QQVVEYPYT (SEQ ID NO: 5) or QQGVESPYT (SEQ ID NO: 6). The light chain complementary determining region CDR3 of the amino acids set forth in SEQ ID NO: 6 of the present invention is obtained by point mutation of the light chain complementary determining region CDR3 of the amino acids set forth in SEQ ID NO: 5, specifically, by mutating the amino acid at position 3 from V to G and the amino acid at position 6 from Y to S.

[0035] As an embodiment, the amino acid sequence of the light chain variable region of the present invention is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, wherein the amino acid sequence shown in SEQ ID NO: 1 is: MRCSLQFLGMLMFWISGVSGDIVITQDELSNP VTSGESVSISCRSSKSLLYKDGKTYLSWFLQRPGQSPQLLVYWMSTRASGVSDRFSGSGSGTDF TLEISGVKAEDVGVYSCQQVVEYPYTFGSGTKLEIR; the amino acid sequence shown in SEQ ID NO: 2 is: MR CSLQFLGMLMFWISGVSGDIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLSWFLQRPGQ SPQLLVYWMSTRASGVSDRFSGSGSGTDFTLEISGVKAEDVGVYSCQQGVESPYTFGSGTKLEI R; wherein the bold portion is three light chain complementary determining regions, that is, SEQ ID NO: 2 is SEQ ID The amino acid sequence shown in NO: 1 is obtained by mutating the amino acid at position 116 from V to G, and simultaneously mutating the amino acid at position 119 from Y to S.

[0036] As an embodiment, the nucleotide sequence encoding the light chain variable region shown in SEQ ID NO: 1 or SEQ ID NO: 2 is shown in SEQ ID NO: 7 or SEQ ID NO: 8, respectively, wherein the nucleotide sequence shown in SEQ ID NO: 7 is: 5'-ATGAG GTGCTCTCTTCAGTTCTTGGGGATGCTTATGTTCTGGATCTCTGGAGTCAGTGGGGATATTGTTATAACCCAGGATGAACTCTCCAATCCTGTCACTTCTGGAGAATCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAGTTGGTTTCTGCAGAGGCCAGGACAGTCT CCTCAGCTCCTGGTCTATTGGATGTCCACCCGTGCATCAGGAGTCTCAGACCGGTTTAGTGGCAGTGGGTCAGGAACAGATTTCACACTGGAAATCAGTGGAGTGAAGGCTGAGGATGTCGGTGTGTATTCCTGTCAACAAGTTGTAGAGTATCCGTATACGTTCGGATCGGGGACCAAGCTGGAAATAAGA-3'; SEQ ID The nucleotide sequence shown in NO:8 is: 5'-ATGAGGTGCTCTCTTCAGTTCTTGGGGATGCTTATGTTCTGGATCTCTGGAGTCAGTGGGGATATTGTTATAACCCAGGATGAACTCTCCAATCCTGTCACTTCTGGAGAATCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAGTTGGTTTCTGCAGAGGCC AGGACAGTCTCCTCAGCTCCTGGTCTATTGGATGTCCACCCGTGCATCAGGAGTCTCAGACCGGTTTAGTGGCAGTGGGTCAGGAACAGATTTCACACTGGAAATCAGTGGAGTGAAGGCTGAGGATGTCGGTGTGTATTCCTGTCAACAAGGTGTAGAGTCTCCGTATACGTTCGGATCGGGGACCAAGCTGGAAAATAAGA-3'; where SEQ The bold part in ID NO:8 is the encoding SEQ ID The base of the mutated amino acid in NO:2.

[0037] As an embodiment, the light chain constant region of the present invention is a human kappa type light chain constant region. In a specific embodiment, the amino acid sequence of the human kappa type light chain constant region is shown in SEQ ID NO: 14, specifically RTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC; the nucleotide sequence encoding the human kappa type light chain constant region shown in SEQ ID NO: 14 is shown in SEQ ID NO: 16, specifically: 5'-AGAACTGTGGCTGCACCATCTGTCTTCATCTTCC CGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACA GCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG-3'.

[0038] In the present invention, the amino acid sequences of the three heavy chain complementarity determining regions of the heavy chain variable region are as follows:

[0039] Heavy chain complementarity determining region CDR1: DYWIE (SEQ ID NO: 10); heavy chain complementarity determining region CDR2: EFLPGSGNTHYNEKFMG (SEQ ID NO: 11); heavy chain complementarity determining region CDR3: LPFDY (SEQ ID NO: 12).

[0040] As an embodiment, the amino acid sequence of the heavy chain variable region of the present invention is shown in SEQ ID NO: 9, specifically: MEWTWVFLFLLSLTAGVHSQVQLQQSGAELMKPGASVKLSCKATGYTFTDYWIEWVKQRPGHGL EWIGEFLPGSGNTHYNEKFMGKATFTADTSSSTAYMQLSSLTTEDSAIYYCAILPFDYWGQGST LTVSS; the bold parts are three heavy chain complementary determining regions.

[0041] As an embodiment, the nucleotide sequence encoding the heavy chain variable region shown in SEQ ID NO: 9 is shown in SEQ ID NO: 13, specifically 5'-ATGGAGTGGACCTGGGTCTTTCTCTTCCTCCTGTCACTAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGCTTTCCTGCAAGGCTACTGGCTACACATTCACTGACTATTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAATTTTTACCTGGTAGTGGTAATACTCACTACAATGAGAAGTTCATGGGCAAGGCCACTTTCACTGCAGATACATCCTCCAGCACAGCCTATATGCAACTCAGCAGCCTGACAACTGAGGACTCTGCCATCTATTACTGTGCAATACTCCCTTTTGACTATTGGGGCCAAGGCTCCACTCTCACAGTCTCCTCA-3'.

[0042] As an embodiment, the heavy chain constant region of the present invention is a human IgG1 heavy chain constant region. In a specific embodiment, the amino acid sequence of the human IgG1 heavy chain constant region is shown in SEQ ID NO: 15, specifically: ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; encoding SEQ ID The nucleotide sequence of the human IgG1 heavy chain constant region shown in NO: 15 is shown in SEQ ID NO: 17, specifically: 5'-GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA-3'。

[0043] As an embodiment, the framework region of the light chain variable region of the present invention can be a mouse sequence, and the framework region of the heavy chain variable region can be a mouse sequence.

[0044] The human-mouse chimeric monoclonal antibody against human MOG protein described in the present invention has complementarity-determining regions in both the light chain variable region and the heavy chain variable region derived from mice, thereby retaining the parental activity to the greatest extent possible; the light chain constant region and the heavy chain constant region are both humanized antibody constant regions. The introduction of humanized antibody constant regions can reduce immunogenicity, lower the incidence of human anti-mouse antibody reactions, and improve the specificity of the antibody.

[0045] The present invention also provides a recombinant expression vector comprising a DNA sequence for expressing the human-mouse chimeric monoclonal antibody against human MOG protein described in the above technical solution. In one embodiment, the backbone vector of the recombinant expression vector of the present invention is a plasmid vector; in another embodiment, the backbone vector of the expression vector of the present invention is but not limited to pcDNA3.1, pBAD, pQE-12, pGEX, pBluescript, pET-series expression vectors, pCAI-n, pPOW3.0, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP1 One or more of 3CAT, pREP, pCEP4, pMC1neo, pXT1, pSG5, EBO-pSV2neo, pBPV-1, pFUSE, pRSVgpt, pRSVneo, pIZD35, pRc / CMV, pcDNA1, pcDNA3.1, pSPORT1, pGEMHE, pLXIN, pSIR, pIRES-EGFP, pEAK-10, pTriEx-Hygro, pCINeo, pAO815, pPIC9K, and pPIC3.5K. In a specific embodiment, the backbone vector of the recombinant expression vector is pcDNA3.1.

[0046] As an embodiment, the recombinant expression vector described in the present invention can be a light chain recombinant expression vector and / or a heavy chain recombinant expression vector.

[0047] In one embodiment, the light chain recombinant expression vector of the present invention is obtained by ligating a DNA sequence expressing a light chain variable region and a DNA sequence expressing a light chain constant region to a backbone vector. In one embodiment, the amino acid sequence of the light chain composed of the light chain variable region and the light chain constant region is as shown in SEQ ID NO: 18 or SEQ ID NO: 19, specifically: MRCSLQFLGMLM FWISGVSGDIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLSWFLQRPGQSPQLLVYWMSTRASGVSDRFSGSGSGTDFTLEISGVKAEDVGVYSCQQVVEYPYTFGSGTKLEIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18); GDIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLSWFLQRPGQSPQLLVYWMSTRASGVSDRFSGSGSGTDFTLEISGVKAEDVGVYSCQQGVESPYTFGSGTKLEI RRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:19).

[0048] As an implementation manner, the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO:18 and SEQ ID NO:19 are shown in SEQ ID NO:21 or SEQ ID NO:22 respectively, specifically: 5'-ATGAGGTGCTCTCTTCAGTTCTTGGGGATGCTTATGTTCTGGATCTCTGGAGTCAGTGGGGATATTGTTATAACCCAGGATGAACTCTCCAATCCTGTCACTTCTGGAGAATCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAGTTGGTTTCTGCAGAGGCCAGGACAGTCTCCTCAGCTCCTGGTCTATTGGATGTCCACCCGTGCATCAGGAGTCTCAGACCGGTTTAGTGGCAGTGGGTCAGGAACAGATTTCACACTGGAAATCAGTGGAGTGAAGGCTGAGGATGTCGGTGTGTATTCCTGTCAACAAGTTGTAGAGTATCCGTATACGTTCGGATCGGGGACCAAGCTGGAAATAAGAAGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG-3' (SEQ ID NO: 21);or 5'-ATGAGGTGCTCTCTTCAGTTCTTGGGGATGCTTATGTTC TGGATCTCTGGAGTCAGTGGGGATATTGTTATAACCCAGGATGAACTCTCCAATCCTGTCACTTCTGGAGAATCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAGTTGGTTTCTGCAGAGGCCAGGACAGTCTCCTCAGCTCCTGGTCTATTGGATGTCCACCCGTGCATCAGGAGTCTCAGACCGGTTTAGTGGCAGTGGGTCAGGAACAGATTTCACACTGGAAATCAGTGGAGTGAAGGCTGAGGATGTCGGTGTGTATTCCTGTCAACAAGGTGTAGAGTCTCCGTATACGTTCGGATCGGGGACCAAGCTGGAAATAAGAAGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG-3'(SEQ ID NO:22).;

[0049] In one embodiment, the heavy chain recombinant expression vector of the present invention is obtained by ligating a DNA sequence expressing a heavy chain variable region and a DNA sequence expressing a heavy chain constant region to a backbone vector. In one embodiment, the amino acid sequence of the heavy chain composed of the heavy chain variable region and the heavy chain constant region is shown in SEQ ID NO: 20, specifically: MEWTWVFLFLLSLTAGVHSQVQLQ QSGAELMKPGASVKLSCKATGYTFTDYWIEWVKQRPGHGLEWIGEFLPGSGNTHYNEKFMGKATFTADTSSSTAYMQLSSLTTEDSAIYYCAILPFDYWGQGSTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:20).

[0050]

[0051] The present invention also provides an engineered cell that overexpresses the human-mouse chimeric monoclonal antibody against human MOG protein described in the above technical solution. In one embodiment, the initial cells of the engineered cell of the present invention can be eukaryotic cells; in another embodiment, the eukaryotic cells can be 293 cells; in a specific embodiment, the cells are Expi293F cells. In one embodiment, the engineered cell of the present invention is obtained by co-transfecting the light chain expression vector and the heavy chain expression vector into cells for expression.

[0052] The present invention also provides the use of the chimeric monoclonal antibody against human MOG protein described in the above technical solution in the preparation of a kit for detecting MOG protein. In one embodiment, the chimeric monoclonal antibody against human MOG protein is used as a positive reference or quality control in the kit. In one embodiment, the specific detection method can be an immunoassay; in another embodiment, the immunoassay technique includes but is not limited to radioimmunoassay, enzyme-labeled immunoassay, fluorescent-labeled immunoassay, chemiluminescent-labeled immunoassay, colloidal gold-labeled immunoassay, or colored latex-labeled immunoassay.

[0053] The present invention also provides a kit for detecting anti-MOG antibodies, comprising the human-mouse chimeric monoclonal antibody against human MOG protein described in the above technical solution. In one embodiment, the kit is prepared based on immunoassay technology. In another embodiment, the immunoassay technology includes one or more of the following immunoassay techniques: radioimmunoassay, enzyme-labeled immunoassay, fluorescent-labeled immunoassay, chemiluminescent-labeled immunoassay, colloidal gold-labeled immunoassay, and colored latex-labeled immunoassay. The present invention does not specifically limit the composition and source of the reagents in the immunoassay kit; these can be conventionally selected as needed.

[0054] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1

[0056] Expression and purification of MOG prokaryotic protein

[0057] The sequence number of the human MOG gene was retrieved from the GenBank database and was NM_001363610.2. A 6his tag sequence was added to the C-terminus of the gene, and the nucleotide sequence of the N-terminal signal peptide (MASLSRPSLPSCLCSFLLLLLLQVSSSYA, SEQ ID NO: 32) was removed. The obtained human MOG gene-6his sequence was entrusted to Jinweizhi Company to be cloned between BamHI and NotⅠ of the pET30a vector to construct a plasmid named MOG-Qsp-6his. The constructed and correctly sequenced plasmid was transformed into Escherichia coli BL21 (DE3) expression competent cells; a single clone was picked and inoculated into LB medium and shaken at 37°C until the OD 600 The value was 0.5 to 1, IPTG was added for induction, and expression was carried out at 16°C overnight. The cells were collected, ultrasonically disrupted, and the supernatant was collected after centrifugation. The precipitate was resuspended in 8M urea. The MOG protein expressed by pET-30a was purified by Ni column affinity chromatography. The purified protein was recorded as MOG-Qsp-6his. The purification results are shown in the figure. Figure 1 As shown; the purified protein was dialyzed and concentrated to obtain a high concentration of MOG-Qsp-6his protein as an antigen for later use.

[0058] Depend on Figure 1 It can be seen that the MOG-Qsp-6his protein was successfully expressed and purified.

[0059] Example 2

[0060] Preparation of mouse monoclonal antibody against human MOG

[0061] 2.1 Immunity

[0062] Five Balb / c female mice aged 6 to 8 weeks were selected, and 40 μg of the recombinant human MOG-Qsp-6his protein purified in Example 1 was mixed with complete Freund's adjuvant in a volume ratio of 1:1. 100 μL (40 μg antigen) of the mixed antigen was injected into the left hind calf muscle; a second immunization was performed 14 days after the initial immunization, in which 40 μg of antigen was mixed with incomplete Freund's adjuvant in a volume ratio of 1:1, and 100 μL (40 μg antigen) of the mixed antigen was injected into the right hind calf muscle. A third immunization was performed 14 days after the second immunization. During the third immunization, the dosage, method, and route of antigen injection were exactly the same as those for the second immunization.

[0063] 2.2 Immunofluorescence detection of mouse antibody production

[0064] On the 14th day after the second immunization and the 14th day after the third immunization, blood was collected from the tail of the mice, and serum was obtained by centrifugation. The production of mouse antibodies was detected by immunofluorescence. The specific steps are as follows:

[0065] Preparation of MOG overexpression slides: The full-length human MOG gene sequence (gene sequence number NM_001363610.2) was synthesized by Jinweizhi Company and inserted into the pcDNA3.4 vector, with the restriction sites NheⅠ and NotⅠ. The MOG-pcDNA3.4 recombinant vector and empty pcDNA3.4 were transfected into 293T adherent cells using PEI transfection reagent, and cultured. After 48 hours, the cells were fixed with 0.5% formaldehyde to prepare MOG overexpression cell slides and pcDNA3.4 control slides.

[0066] Immunofluorescence detection: On the 14th day after the second immunization and the 14th day after the third immunization, the tail blood was collected from the mice, and the serum was obtained by centrifugation. The serum of the five immunized mice was diluted 1:32 with PBST buffer and incubated with MOG-overexpressing cell slides. The slides were incubated at room temperature for 1 hour and washed 3 times with PBST buffer, each time for 5 minutes. The slides were incubated with AlexaFluor 594-labeled goat anti-mouse IgG secondary antibody (manufacturer: Jackson, catalog number: 115-585-146) diluted 1:200, incubated at room temperature for 30 minutes, and washed 3 times with PBST buffer, each time for 5 minutes. The results were observed under a fluorescence microscope, as shown in Figure 2. Figure 2 As shown, Figure 2 The scale bar is 300 μm.

[0067] Depend on Figure 2 It can be concluded that on the 14th day after the second immunization and the 14th day after the third immunization, the fluorescence signal of the serum of mouse No. 2 was the strongest, so mouse No. 2 was selected for subsequent experiments.

[0068] 2.3 Fusion:

[0069] Preparation of feeder cells and myeloma cells: Macrophages from the ascites of normal mice are cultured as feeder cells. Feeder cells obtained from the ascites of 4-6 mice can be plated onto 10 96-well plates. After 2 days of culture, hybridoma cells can be prepared. Properly growing SP2 / 0 myeloma cells in the logarithmic growth phase with a cell density of 90% are collected, totaling 10 plates. Centrifuge and discard the supernatant. Resuspend the cells in DMEM medium and count them.

[0070] Preparation of splenocytes: Mouse No. 2 was boosted with an intraperitoneal injection of 100 μL of antigen (40 μg of antigen). Three days later, tail blood was collected to separate the serum, which was rinsed with running water and placed in a 75% ethanol solution for 10 min. The spleen of the mouse was aseptically removed and placed in a 200-mesh disposable sterile filter. The spleen was minced with sterile surgical scissors and ground using the piston handle of a 50 mL sterile syringe to obtain a single splenocyte suspension.

[0071] Cell fusion: Preheat a 37°C water bath in a clean hood. Add pre-cultured SP2 / 0 cells and spleen cells at a 1:10 ratio (90% cell density) to a 50mL centrifuge tube and mix thoroughly. Centrifuge at 500g for 10 minutes, aspirate the supernatant, and gently tap the bottom of the tube to loosen the cell pellet. Add 1mL of 45% PEG-1450 solution, pre-warmed to 37°C, dropwise over 90 seconds, while gently shaking the tube. Keep the tube in a 37°C water bath throughout the process. Add DMEM medium to the cell mixture at a constant rate: 1mL dropwise over the first minute, 2mL over the second minute, 3mL over the third minute, 4mL over the fourth minute, and 5mL over the fifth minute, while shaking in a 37°C water bath. Incubate at 37°C for 15 minutes, centrifuge at 500g for 5 minutes, and discard the supernatant. Add 5 mL of DMEM medium containing HAT to gently resuspend the pelleted cells. Finally, top up with DMED medium containing HAT to approximately 100 mL. Aliquot 100 μL / well into a 96-well cell culture plate containing feeder cells. Incubate the plates at 37°C in a 5% CO2 incubator. Add 100 μL of HAT medium to each well the day after fusion. Perform a half-change of HAT medium every 2-3 days. Culture for approximately 2 weeks before screening.

[0072] 2.4 Screening of Positive Hybridoma Cells: Observe the growth of hybridoma cells. After 7-10 days, when the cell culture supernatant turns yellow, remove an appropriate amount of supernatant for immunofluorescence detection of antibodies. Equal volumes of supernatant from each row of 12 wells on a 96-well plate are mixed. Tween-20 is added to a final concentration of 0.2% and then tested. Based on the immunofluorescence results, a row of 12 wells corresponding to the sample with a strong positive signal is selected for further culture and screening of mother clones. After another 7-10 days of culture, the sample from each well with a strong positive signal is mixed, and the supernatant from each well is individually tested for immunofluorescence detection of antibodies. Finally, 13D5, 49G9, and 51E6 are screened as positive wells. The medium of these selected positive wells is exchanged for verification again, and limiting dilution is performed to culture approximately one cell per well. After 7-10 days of culture, three clones with strong positive signals are selected for further immunofluorescence verification. Finally, three positive clones are screened and designated: 13D5, 49G9, and 51E6 hybridoma cell lines.

[0073] The monoclonal antibodies secreted by the cells were named 13D5, 49G9, and 51E6 mouse monoclonal antibodies. The immunofluorescence results of the three screenings are as follows Figure 3 The results shown are those observed under a 10x objective lens.

[0074] Depend on Figure 3 It can be seen that the fluorescence intensity of the 13D5 hybridoma cell line is the strongest, that is, the cell viability is the strongest, and it is the best hybridoma cell line.

[0075] Example 3

[0076] Identification of mouse monoclonal antibodies

[0077] 3.1 Binding of anti-human MOG mouse monoclonal antibody to recombinant human MOG protein

[0078] The purified MOG-Qsp-6his protein was coated as an antigen to detect the titer of 13D5, 49G9 and 51E6 mouse monoclonal antibodies. The antigen coating concentration was 100 ng / well and the coating was carried out overnight at 4°C. The next day, the cells were washed three times with 0.1 M BST buffer for 3 minutes each time and patted dry. Blocking protein (2% BSA solution) was added at 200 μL / well and incubated at 37°C for 1 hour. 0.1 M Wash 3 times with PBST buffer, 3 minutes each time, and pat dry; dilute the supernatant of monoclonal 13D5, 49G9, and 51E6 cells with PBS at a ratio of 1:1, 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, and 1:2187 of the original solution, 100 μL / well, and use the tail blood of No. 2 mouse and the tail blood of normal mouse at a volume ratio of 1:1000 as the initial concentration. After serial dilution, 100 μL of sample was added to each well and incubated at 37°C for 1 hour; wash 3 times with 0.1M PBST, 3 minutes each time, and pat dry; use HRP-labeled IgG secondary antibody, 1:5000 dilution (manufacturer: Jackson), and incubate at 37°C for 30 minutes; wash 3 times with 0.1M PBST buffer, 3 minutes each time, and pat dry; add TMB for color development for 5 minutes, and use 2M The color development was stopped by H2SO4 and the absorbance was measured at OD450nm. The experimental results are shown in Table 1, which are the titers of the mouse monoclonal antibodies 13D5, 49G9 and 51E6.

[0079] Table 11 Results of titer determination of 3D5, 49G9 and 51E6 mouse monoclonal antibodies

[0080] Dilution ratio 13D5 49G9 51E6 Positive control Negative control Original solution 1.986 0.048 1.309 1.839 0.047 1:3 2.096 0.046 1.360 1.944 0.049 1:9 1.857 0.045 1.345 1.544 0.045 1:27 1.316 0.049 1.119 0.744 0.044 1:81 0.813 0.047 0.814 0.493 0.048 1:243 0.566 0.044 0.369 0.25 0.046 1:729 0.307 0.043 0.168 0.125 0.043 1:2187 0.124 0.057 0.084 0.078 0.045

[0081] It can be concluded from Table 1 that the titer of 13D5 mouse monoclonal antibody is the highest, reaching 1:2187, the titer of 51E6 mouse monoclonal antibody can reach 1:729, and 49G9 mouse monoclonal antibody is eliminated as a false positive.

[0082] 3.2 Identification of anti-human MOG mouse monoclonal antibody typing

[0083] Re-coat the purified MOG-Qsp-6his protein as an antigen to detect the typing of 13D5 and 51E6 mouse monoclonal antibodies. The antigen coating concentration was 100 ng / well and the coating was carried out at 4°C overnight. The next day, the cells were washed three times with 0.1 M PBST buffer for 3 minutes each time and patted dry. Blocking protein (2% BSA solution) was added at 200 μL / well and incubated at 37°C for 1 hour. The cells were washed three times with 0.1 M PBST buffer for 3 minutes each time and patted dry. 100 μL / well of monoclonal 13D5 or 51E6 cell supernatant was added and a 1:1000 dilution of mouse tail blood No. 2 was used as a positive control. 100 μL was added to each well and incubated at 37°C for 1 hour. 0.1 M The cells were washed three times with PBST buffer (3 min each) and patted dry. HRP-conjugated secondary antibodies (total IgG / IgG1 / IgG2a / IgG2b / IgG3 / IgM / Igκ / Igλ) were applied at a 1:5000 dilution (Jackson) and incubated at 37°C for 30 min. The cells were washed three times with 0.1 M PBST buffer (3 min each) and patted dry. TMB was added for color development for 5 min, and development was stopped with 2 M H2SO4. The absorbance was measured at OD450 nm. The results are shown in Table 2.

[0084] Table 2 Typing results of 13D5 and 51E6 mouse monoclonal antibodies

[0085]

[0086]

[0087] Table 2 shows that the 13D5 mouse monoclonal antibody has an IgG2b heavy chain and an Igκ light chain; the 51E6 mouse monoclonal antibody has an IgG1 heavy chain and an Igκ light chain. Based on this comprehensive comparison, the 13D5 hybridoma cells were selected for further experiments. The 13D5 mouse monoclonal antibody was purified from the culture supernatant of the positive hybridoma cells as a control. The protein concentration was determined by BCA assay to be 0.9 mg / mL.

[0088] 3.3 Heavy and light chain variable region sequencing

[0089] The selected 13D5 hybridoma cells were cultured and lysed with Trizol. Total RNA was extracted from the hybridoma cell lysate (Quick-RNA MicroPrep Kit). RNA was separated by agarose gel electrophoresis. The first-chain cDNA was synthesized by 5'RACE using RNA as a template (Clontech SMARTerRACE 5' / 3'kit). PCR amplification was performed using the RNA as a template (PrimeSTAR Max DNA Polymerase) was used to cross-link the heavy chain variable region (VH) and light chain variable region (VL) genes of the tumor cell monoclonal antibody, and the antibody light chain / heavy chain variable region cDNA was extracted. The cDNA was sequenced by a sequencing company to obtain the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence of the 13D5 mouse monoclonal antibody. The CDR region of the 13D5 mouse monoclonal antibody amino acid sequence was marked using the Kabat method.

[0090] Example 4

[0091] Construction and expression of recombinant vector of human-mouse chimeric monoclonal antibody against human MOG

[0092] 4.1 Design of light and heavy chain sequences of anti-human MOG human-mouse chimeric monoclonal antibody

[0093] Using conventional molecular biology methods, the antibody sequence was amplified using Fast PfuDNA Polymerase (Full Gold) to construct a mutant of the antibody sequence. The amino acid V at position 116 of the light chain variable region of the 13D5 murine monoclonal antibody shown in SEQ ID NO. 1 was mutated to G, and the amino acid Y at position 119 was mutated to S. The resulting amino acid sequence is SEQ ID NO. 2, and the corresponding nucleotide sequence is shown in SEQ ID NO. 8. The light chain of the designed anti-human MOG human-mouse chimeric monoclonal antibody consists of the 13D5 murine light chain variable region (amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2) and the human kappa type light chain constant region (amino acid sequence shown in SEQ ID NO.14). The complete light chain amino acid sequence of the anti-human MOG human-mouse chimeric monoclonal antibody is shown in SEQ ID NO:18 or SEQ ID NO:19. The heavy chain of the designed anti-human MOG human-mouse chimeric monoclonal antibody consists of the 13D5 murine heavy chain variable region (amino acid sequence shown in SEQ ID NO.9) and the human IgG1 heavy chain constant region (amino acid sequence shown in SEQ ID NO.15). The complete heavy chain nucleotide sequence of the anti-human MOG human-mouse chimeric monoclonal antibody is shown in SEQ ID NO:20. The specific anti-human MOG human-mouse chimeric monoclonal antibodies obtained have the following combinations as shown in Table 3, wherein the antibody consisting of the complete light chain amino acid sequence of SEQ ID NO:18 and the complete heavy chain amino acid sequence of SEQ ID NO:20 is named 1820, and the antibody consisting of the complete light chain amino acid sequence of SEQ ID NO: The antibody consisting of NO:19 and the complete heavy chain amino acid sequence SEQ ID NO:20 was named 1920.

[0094] Table 3 Combination of anti-human MOG human-mouse chimeric monoclonal antibodies

[0095] Antibody name 1820 1920 Complete light chain amino acid sequence SEQ ID NO: 18 SEQ ID NO: 19 Light chain variable region amino acid sequence SEQ ID NO: 1 SEQ ID NO:2 Light chain constant region amino acid sequence SEQ ID NO:14 SEQ ID NO:14 Complete heavy chain amino acid sequence SEQ ID NO:20 SEQ ID NO:20 Heavy chain variable region amino acid sequence SEQ ID NO:9 SEQ ID NO:9 Heavy chain constant region amino acid sequence SEQ ID NO:15 SEQ ID NO:15

[0096] 4.2 Construction of recombinant vector of anti-human MOG human-mouse chimeric monoclonal antibody

[0097] The nucleotide sequence of the light chain variable region of the 13D5 murine monoclonal antibody (SEQ ID NO:7 or SEQ ID NO:8) and the nucleotide sequence of the human kappa chain constant region (SEQ ID NO:16) were amplified. The light chain variable and constant region fragments were ligated into the pcDNA3.1 vector using homologous recombination (Manufacturer: Quanshijin, Catalog No. CU201-02). The variable region amplified fragment and the constant region amplified fragment were ligated sequentially from 5' to 3', and inserted into the multiple cloning site of the pcDNA3.1 vector. These fragments were labeled pcDNA3.1-MOG-VL or pcDNA3.1-MOG-VL', respectively. The ligated recombinant plasmids were sent to Sangon Biotechnology for sequencing. The sequenced recombinant plasmids were then amplified and used for cell transfection to produce antibodies.

[0098] The primers used to amplify the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 are as follows:

[0099] Upstream primer F (SEQ ID NO: 24): 5′-ctatagggagacccaagctggctagcATGAGGTGCTCTCTTCAGTT-3′;

[0100] Downstream primer R (SEQ ID NO: 25): 5′-agacagatggtgcagccacagttctTCTTATTTCCAGCTTGGTCC-3′;

[0101] The program used for amplifying the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 was as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 58°C annealing for 20 s, 72°C extension for 20 s, 30 cycles; and 72°C extension for 3 min.

[0102] The primers used to amplify the sequence shown in SEQ ID NO: 16 are as follows:

[0103] Upstream primer F (SEQ ID NO: 26): 5′-atcggggaccaagctggaaataagaAGAACTGTGGCTGCACCATC-3′;

[0104] Downstream primer R (SEQ ID NO: 27): 5′-aacgggccctctagactcgagcggccgcCTAACACTCTCCCCTGTTGA-3′;

[0105] The program used to amplify the sequence shown in SEQ ID NO: 16 was: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 62°C for 20 s, extension at 72°C for 20 s, 30 cycles; and extension at 72°C for 3 min.

[0106] The nucleotide sequence of the 13D5 murine monoclonal antibody heavy chain variable region (SEQ ID NO: 13) was amplified, and the human IgG1 heavy chain constant region sequence (SEQ ID NO: 17) was amplified. The two fragments were ligated into the pcDNA3.1 vector using homologous recombination (Manufacturer: Quanshijin Part No. CU201-02). The variable region amplified fragment and the constant region amplified fragment were ligated sequentially from the 5'-3' direction and inserted into the multiple cloning site of the pcDNA3.1 vector, labeled pcDNA3.1-MOG-VH. The ligated recombinant plasmid was sent to Sangon Biotechnology for sequencing. The sequenced recombinant plasmid was then amplified and used for cell transfection to produce antibodies.

[0107] The primers used to amplify the sequence shown in SEQ ID NO: 13 are as follows:

[0108] Upstream primer F (SEQ ID NO: 28): 5′-ctatagggagacccaagctggctagcATGGAGTGGACCTGGGTCTT-3′;

[0109] Downstream primer R (SEQ ID NO: 29): 5′-agaccgatgggcccttggtggaggcTGAGGAGACTGTGAGAGTGG-3′;

[0110] The program used to amplify the sequence shown in SEQ ID NO: 13 was: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 62°C for 20 s, extension at 72°C for 20 s, 30 cycles; and extension at 72°C for 3 min.

[0111] The primers used to amplify the sequence shown in SEQ ID NO: 17 are as follows:

[0112] Upstream primer F (SEQ ID NO: 30): 5′-aggctccactctcacagtctcctcaGCCTCCACCAAGGGCCCATC-3′;

[0113] Downstream primer R (SEQ ID NO: 31): 5′-aacgggccctctagactcgagcggccgcTCATTTACCCGGAGACAGG GA-3′;

[0114] The program used to amplify the sequence shown in SEQ ID NO: 17 was: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 64°C for 20 s, extension at 72°C for 20 s, 30 cycles; and extension at 72°C for 3 min.

[0115] 4.3 Expression and purification of anti-human MOG human-mouse chimeric monoclonal antibody

[0116] The following two groups of recombinant vectors were co-transfected into Expi293F cells (thermo) using the transfection reagent PEI (manufacturer: thermo) for transient expression: Group 1 (pcDNA3.1-MOG-VL and pcDNA3.1-MOG-VH recombinant vectors) expressed antibodies corresponding to the anti-human MOG protein human-mouse chimeric antibody 1820, and Group 2 (pcDNA3.1-MOG-VL' and pcDNA3.1-MOG-VH recombinant vectors) expressed antibodies corresponding to the anti-human MOG protein human-mouse chimeric antibody 1920. Three days after transfection, the supernatants were collected and purified using protein A filler. The protein concentrations were determined using a BCA kit. The concentrations of the anti-human MOG protein human-mouse chimeric antibody 1820 were 0.7 mg / mL and 1920 were 0.8 mg / mL. The antibody purification results are shown in Figure 2. Figure 4 shown.

[0117] Depend on Figure 4 It can be seen that the expression and purification of anti-human MOG human-mouse chimeric monoclonal antibody were successful.

[0118] 4.4 Binding of anti-human MOG human-mouse chimeric monoclonal antibody to MOG antigen

[0119] ELISA detection: ELISA plates were coated with purified MOG-Qsp-6his protein at 100 ng / well at 4°C overnight (the coating solution was 25 mL carbonate buffer, pH 9.6); the next day, the plates were washed three times with PBST buffer for 3 minutes each time, and patted dry each time; 2% BSA was added to the wells for blocking, and the plates were incubated at 37°C for 1 hour; after incubation, the plates were washed three times with PBST buffer for 3 minutes each time, and the plates were patted to remove excess water each time; the two groups of anti-human MOG human-mouse chimeric antibodies 1820 and 1920 prepared in 4.3 and the 13D5 mouse monoclonal antibody prepared in Example 2 were incubated. Long antibody was used as a positive control, and normal control mouse serum was used as a negative control. The stock solution was diluted with PBS at ratios of 1:1, 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, and 1:2187, respectively. 100 μL of these dilutions were added to each well, and the plates were incubated at 37°C for 1 hour. The plates were washed three times with PBST buffer for 3 minutes each, patting dry after each wash. Goat anti-human / goat anti-mouse IgG-HRP secondary antibodies were diluted 1:5000 and added to the plates. The plates were incubated at 37°C for 30 minutes. After incubation, the plates were washed three times with PBST buffer, patted dry, and color was developed with TMB for 3 minutes. The plates were terminated with 2 M H₂SO₄, and absorbance was measured at OD450 nm. The results are shown in Table 4.

[0120] Table 4 Binding effect of anti-human MOG human-mouse chimeric monoclonal antibody and MOG antigen

[0121] Dilution ratio 1820 chimeric antibody 1920 chimeric antibody 13D5 mouse monoclonal antibody Negative control Original solution 2.324 2.613 2.529 0.045 1:3 2.308 2.241 2.552 0.046 1:9 2.587 2.135 2.548 0.044 1:27 2.515 1.791 2.171 0.04 1:81 2.724 0.713 1.602 0.043 1:243 2.325 0.493 1.106 0.047 1:729 2.219 0.252 0.44 0.046 1:2187 2.058 0.173 0.181 0.047

[0122] It can be concluded from Table 4 that at a dilution of 1:2187, the titer of the chimeric antibody was still more than twice the titer of the negative control at the same dilution ratio, indicating that the two groups of anti-human MOG human-mouse chimeric antibodies 1820 and 1920 after modification can effectively recognize MOG antigen, and their activity is basically consistent with that of the 13D5 mouse monoclonal antibody before modification.

[0123] Example 5

[0124] Application of human-mouse chimeric monoclonal antibodies against human MOG

[0125] 5.1 Immunofluorescence analysis to identify the function of anti-human MOG human-mouse chimeric monoclonal antibodies

[0126] The two groups of anti-human MOG chimeric antibodies 1820 and 1920 purified in Example 4 and the commercial MOG antibody (manufacturer: Wuhan Sanying 12690-1-AP) were respectively diluted with PBST solution at a ratio of 1:10 / 1:100, and the commercial MOG antibody was diluted at a ratio of 1:100. The MOG-positive patient serum was diluted with PBST solution at a ratio of 1:10 and incubated on the MOG-pcDNA3.4 cell slides and pcDNA3.4 control slides prepared in Example 1, respectively. The cells were incubated at room temperature for 1 hour, washed with PBST three times for 5 minutes each, and 1:200 diluted FITC-labeled goat anti-human / AlexaFluor 594-labeled goat anti-rabbit IgG (manufacturer: Jackson) was added. The cells were incubated at room temperature for 30 minutes, and washed with PBST buffer three times for 5 minutes each. The results were observed under a microscope. Figure 5 As shown, the scale bar is 150 μm.

[0127] according to Figure 5 The results showed that, compared with commercially available antibodies, the two anti-MOG chimeric antibodies 1820 and 1920 could be incubated with the same secondary antibody conditions as the test samples (all labeled secondary antibodies were FITC-labeled goat anti-human IgG), demonstrating a clear advantage in humanization and making them more suitable for use as positive references. Furthermore, under the same incubation conditions as MOG patient serum, the typical positive cell morphology and fluorescence signal intensity of anti-MOG chimeric antibodies 1820 and 1920 were consistent, making them suitable alternatives to positive serum / cerebrospinal fluid for diagnostic kits. Anti-MOG chimeric antibodies 1820 and 1920 also enabled accurate protein quantification and antibody titer determination, ensuring long-term product quality control. These chimeric antibodies enrich the repertoire of MOG monoclonal antibodies and provide a foundation for the auxiliary diagnosis of MOGAD. These chimeric antibodies can supplement immunofluorescence experiments with more typical positive cell morphology and increase the number of positive images, enabling more accurate interpretation of indirect immunofluorescence images using computer artificial intelligence technology.

[0128] 5.2 Western Blot Identification of the Function of Anti-Human MOG Human-Mouse Chimeric Monoclonal Antibody

[0129] One dish of MOG-overexpressing 293T cells and one dish of empty pcDNA3.4 cells were collected, lysed with RIPA lysis buffer, centrifuged to obtain the supernatant, and then subjected to gel electrophoresis. After the electrophoresis, wet transfer was performed at 300 mA for 90 min; 5% skim milk powder was blocked at room temperature for 1 h; 1:2000 dilution of anti-human MOG protein human-mouse chimeric antibody 1820 (concentration of 0.7 mg / mL) and commercial MOG (manufacturer: Wuhan Sanying, catalog number: 12690-1-AP) antibody were incubated on the transferred PVDF membrane, and incubated overnight at 4°C; the next day, TBST was washed 3 times, 5 min each time; HRP-labeled goat anti-human / goat anti-rabbit secondary antibody (manufacturer: Jackson) was added and incubated at room temperature for 1 h; TBST was washed 3 times, 5 min each time; ECL chemiluminescence solution was added for color development and photography, and the results are as follows Figure 6 shown.

[0130] Depend on Figure 6 The results show that compared with commercial antibodies, the recombinant anti-human MOG protein human-mouse chimeric antibody 1820 can also specifically recognize MOG protein in overexpressed cells, and the size of the antigen band recognized by the commercial antibody is consistent with that of the commercial antibody (although the band recognized by the commercial antibody is thicker and darker than the band recognized by 1820, the pcDNA3.1 control protein also has some non-specific bands, while the 1820 monoclonal antibody does not have non-specific bands in this area).

[0131] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A human-mouse chimeric monoclonal antibody against human MOG protein, characterized in that: comprising a light chain and a heavy chain; the light chain comprising a light chain variable region and a light chain constant region; the light chain variable region comprising a light chain complementary determining region CDR1, a light chain complementary determining region CDR2, and a light chain complementary determining region CDR3, the amino acid sequence of the light chain complementary determining region CDR1 being as shown in SEQ ID NO:3, the amino acid sequence of the light chain complementary determining region CDR2 being as shown in SEQ ID NO:4, and the amino acid sequence of the light chain complementary determining region CDR3 being as shown in SEQ ID NO:5, or based on the amino acid sequence of SEQ ID NO:5, wherein the third amino acid is mutated from V to G, and the sixth amino acid is mutated from Y to S; The light chain constant region is a human light chain constant region; The heavy chain includes a heavy chain variable region and a heavy chain constant region; the heavy chain variable region includes a heavy chain complementary determining region CDR1, a heavy chain complementary determining region CDR2 and a heavy chain complementary determining region CDR3, and the amino acid sequences of the heavy chain complementary determining region CDR1, the heavy chain complementary determining region CDR2 and the heavy chain complementary determining region CDR3 are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the heavy chain constant region is a human heavy chain constant region.

2. The human-mouse chimeric monoclonal antibody against human MOG protein according to claim 1, characterized in that: The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

3. The human-mouse chimeric monoclonal antibody against human MOG protein according to claim 1, characterized in that: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

9.

4. A recombinant expression vector, characterized in that: The invention comprises a DNA sequence for expressing the human-mouse chimeric monoclonal antibody against human MOG protein according to any one of claims 1 to 3.

5. The recombinant expression vector according to claim 4, characterized in that The recombinant expression vector includes a light chain recombinant expression vector and / or a heavy chain recombinant expression vector; The light chain recombinant expression vector comprises a DNA sequence expressing a light chain variable region and a DNA sequence expressing a light chain constant region; The heavy chain recombinant expression vector comprises a DNA sequence expressing a heavy chain variable region and a DNA sequence expressing a heavy chain constant region.

6. The recombinant expression vector according to claim 5, characterized in that The DNA sequence expressing the light chain variable region comprises the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8; The DNA sequence expressing the heavy chain variable region includes the nucleotide sequence shown in SEQ ID NO:

13.

7. The recombinant expression vector according to claim 4, characterized in that The backbone vector of the recombinant expression vector includes a plasmid vector.

8. An engineered cell, characterized in that The engineered cells overexpress the human-mouse chimeric monoclonal antibody against human MOG protein according to any one of claims 1 to 3.

9. Use of the human-mouse chimeric monoclonal antibody against human MOG protein according to any one of claims 1 to 3 in the preparation of a kit for detecting MOG protein.

10. The use according to claim 9, characterized in that The human-mouse chimeric monoclonal antibody against human MOG protein is used as a positive reference or quality control product in the kit.

11. A kit for detecting anti-MOG antibodies, characterized in that: The kit comprises the human-mouse chimeric monoclonal antibody against human MOG protein according to any one of claims 1 to 3.

12. The kit according to claim 11, characterized in that The kit is prepared based on immunoassay technology.

Images