Human-mouse chimeric monoclonal antibody for resisting human MOG protein and application of human-mouse chimeric monoclonal antibody

Abstract

The invention belongs to the technical field of monoclonal antibody preparation, and particularly relates to a human-mouse chimeric monoclonal antibody for resisting human myelin sheath oligodendrocyte glycoprotein (MOG) and application of the human-mouse chimeric monoclonal antibody. Complementary determining regions in variable regions of a heavy chain and a light chain of the human-mouse chimeric monoclonal antibody of the anti-human MOG protein come from a mouse, parent activity is reserved to the maximum extent, a constant region is a human antibody constant region, immunogenicity is greatly reduced due to introduction of the human antibody constant region, the occurrence probability of a human anti-mouse antibody reaction is reduced, and the anti-human MOG protein is prepared. The specificity of the antibody is improved. The human-mouse chimeric monoclonal antibody resisting the human MOG protein has antigen binding specificity, the heterology of a mouse monoclonal antibody is greatly reduced, the biological activity of the human-mouse chimeric monoclonal antibody is good, and the human-mouse chimeric monoclonal antibody resisting the human MOG protein can specifically detect the MOG protein.

Description

Technical Field

[0001] The invention belongs to the technical field of monoclonal antibody preparation, and specifically relates to a human-mouse chimeric monoclonal antibody against human MOG protein and application thereof. 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 that may be caused by MOG-IgG. Their pathogenesis, clinical manifestations, efficacy, and prognosis are different from those of classic MS and AQP4 antibody-positive NMOSD. MOGAD has gradually become an independent disease. The pathogenesis of MOGAD is MOG antibody-mediated oligodendrocyte damage, secondary demyelination, and axonal injury. The pathological characteristics of this disease are that the optic nerve, spinal cord, pons, and thalamus are often involved, and T cells and macrophages infiltrate around small veins, accompanied by immune complex deposition. Studies have shown that anti-MOG antibodies may be pathogenic antibodies for acute dissemina tedencephalomyelitis (ADEM), multiphasic disseminated encephalomyelitis (MDEM), optic neuritis (ON), longitudinally extensive transverse myelitis (LETM), neuromyelitis optica spectrum disease (NMOSD), etc. The international diagnostic guidelines for anti-MOG antibody-associated diseases (MOGAD) recommend the cell-based immunofluorescence assay (CBA) to detect anti-MOG antibodies in serum and cerebrospinal fluid. In addition, the titer of anti-MOG antibodies in serum is related to the severity of the disease. The persistent presence of antibodies or the increase in titer indicates the possibility of recurrence. Timely, accurate and rapid diagnosis is conducive to a good prognosis and a reduction in disability rate.

[0004] The detection of MOG-IgG autoantibodies is inseparable from the MOG antigen. The MOG antigen coated with the common CBA test reagent is usually a complete full-length human MOG protein with the correct spatial conformation. At the same time, a positive control sample is required to control the quality of the test reagent. The positive control can usually be selected from positive patient serum, or a MOG human-mouse chimeric monoclonal antibody can be prepared, which can not only serve as a control for positive serum, perform quantitative determination of protein concentration, provide the positive control required by the kit for a long time and stably, but also identify the expression of MOG protein. However, positive patient serum is not easy to obtain, and it cannot be stably and continuously provided to meet the needs of the kit quality control product. The existing MOG monoclonal antibodies are mostly mouse or rabbit derived, and cannot be directly used as a substitute 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 is shown in SEQ ID NO:3, the amino acid sequence of the light chain complementary determining region CDR2 is shown in SEQ ID NO:4, the amino acid sequence of the light chain complementary determining region CDR3 is shown in SEQ ID NO:5, or on the basis of 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 comprises 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, wherein the engineered cell 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 a positive reference or quality control product in the kit.

[0021] The present invention also provides a kit for detecting anti-MOG antibodies, wherein the kit comprises 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 human-mouse chimeric monoclonal antibody against human MOG protein. The complementary determining regions in the variable regions of the heavy chain and light chain of the human-mouse chimeric monoclonal antibody against human MOG protein are from mice, which retains the parent activity to the maximum extent, and the constant region is a human antibody constant region. The introduction of the human antibody constant region greatly reduces the immunogenicity, reduces the probability of human anti-mouse antibody reaction, and improves the specificity of the antibody. The human-mouse chimeric monoclonal antibody against human MOG protein of the present invention has both antigen binding specificity and greatly reduces the heterogeneity of mouse monoclonal antibodies. At the same time, the human-mouse chimeric monoclonal antibody against human MOG protein of the present invention is easy to store and easy to control the quality of the antibody production process; it is also convenient to make a series of modifications to the antibody, conduct more in-depth research, and apply it more widely. 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 to be used in the embodiments are briefly introduced below.

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

[0027] Figure 2 The immunofluorescence results for detecting mouse antibody production in Example 2;

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

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

[0030] Figure 5 The immunofluorescence results 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 is shown in SEQ ID NO:3, the amino acid sequence of the light chain complementary determining region CDR2 is shown in SEQ ID NO:4, the amino acid sequence of the light chain complementary determining region CDR3 is shown in SEQ ID NO:5, or on the basis of 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); 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 shown 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 shown in SEQ ID NO: 5, specifically, the amino acid at position 3 is mutated from V to G and the amino acid at position 6 is mutated 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 parts are three light chain complementary determining regions, that is, SEQ ID NO:2 is SEQ ID The amino acid at position 116 of the amino acid sequence shown in NO: 1 is mutated from V to G, and the amino acid at position 119 is mutated 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'-ATGAGGTGCTCTCTTCAGTTCTTGGGGATGCTTATGTTCTGGATCTCTGGAGTCAGTGGGGATATTGTTATAACCCAGGATGAACTCTCCAATCCTGTCACTTCTGGAGAATCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATAAGGATGGGAAGACATACTTGAGTTGGTTTCTGCAGAGGCC AGGACAGTCTCCTCAGCTCCTGGTCTATTGGATGTCCACCCGTGCATCAGGAGTCTCAGACCGGTTTAGTGGCAGTGGGTCAGGAACAGATTTCACACTGGAAATCAGTGGAGTGAAGGCTGAGGATGTCGGTGTGTATCCTGTCAACAAGGTGTAGAGTCTCCGTATACGTTCGGATCGGGGACCAAGCTGGAAAATAAGA-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 complementary 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 as shown in SEQ ID NO: 15, specifically: ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; 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 of the present invention has complementary determining regions in the light chain variable region and complementary determining regions in the heavy chain variable region both from mice, which can retain the parent activity to the maximum extent; the light chain constant region and the heavy chain constant region are both humanized antibody constant regions, and the introduction of humanized antibody constant regions can reduce immunogenicity, reduce the probability of human anti-mouse antibody reaction, 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. As an embodiment, the backbone vector of the recombinant expression vector of the present invention is a plasmid vector; as 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 may be a light chain recombinant expression vector and / or a heavy chain recombinant expression vector.

[0047] As an embodiment, the light chain recombinant expression vector of the present invention is obtained by connecting a DNA sequence expressing a light chain variable region and a DNA sequence expressing a light chain constant region to a backbone vector. As an 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 embodiment, 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, specifically: 5'-ATGAGGTGCTCTCTTCAGTTCTTGGGGATGCTTATGTTCTGGATCTCTGGAGTCAGTGGGGATATTGTTATAACCCAGGATGAACTCTCCAATCCTGTCACTTCTGGAGAATCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAGTTGGTTTCTGCAGAGGCCAGGACAGTCTCCTCAGCTCCTGGTCTATTGGATGTCCACCCGTGCATCAGGAGTCTCAGACCGGTTTAGTGGCAGTGGGTCAGGAACAGATTTCACACTGGAAATCAGTGGAGTGAAGGCTGAGGATGTCGGTGTGTATTCCTGTCAACAAGTTGTAGAGTATCCGTATACGTTCGGATCGGGGACCAAGCTGGAAATAAGAAGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG-3' (SEQ ID NO: 21);or 5'-ATGAGGTGCTCTCTTCAGTTCTTGGGGATGCTTATGTTC TGGATCTCTGGAGTCAGTGGGGATATTGTTATAACCCAGGATGAACTCTCCAATCCTGTCACTTCTGGAGAATCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAGTTGGTTTCTGCAGAGGCCAGGACAGTCTCCTCAGCTCCTGGTCTATTGGATGTCCACCCGTGCATCAGGAGTCTCAGACCGGTTTAGTGGCAGTGGGTCAGGAACAGATTTCACACTGGAAATCAGTGGAGTGAAGGCTGAGGATGTCGGTGTGTATTCCTGTCAACAAGGTGTAGAGTCTCCGTATACGTTCGGATCGGGGACCAAGCTGGAAATAAGAAGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG-3'(SEQ ID NO:22).;

[0049] As an embodiment, the heavy chain recombinant expression vector of the present invention is obtained by connecting a DNA sequence expressing a heavy chain variable region and a DNA sequence expressing a heavy chain constant region to a backbone vector. As an 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, wherein the engineered cell overexpresses the human-mouse chimeric monoclonal antibody against human MOG protein described in the above technical solution. As an embodiment, the initial cell of the engineered cell of the present invention may be a eukaryotic cell; as another embodiment, the eukaryotic cell may be a 293 cell; in a specific embodiment, the cell is an Expi293F cell. As an 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 the cell for expression.

[0052] 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. As an embodiment, the human-mouse chimeric monoclonal antibody against human MOG protein is a positive reference or quality control product in the kit. As an embodiment, the method of specific detection can be detection by immunoassay technology; as another embodiment, the immunoassay technology includes but is not limited to radioimmunoassay, enzyme-labeled immunoassay, fluorescent-labeled immunoassay, chemiluminescent-labeled immunoassay, colloidal gold-labeled immunoassay technology or colored latex-labeled immunoassay.

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

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

[0055] Example 1

[0056] Expression and purification of MOG prokaryotic protein

[0057] The sequence number of the human MOG gene retrieved from the GenBank database 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, and a plasmid was constructed and named MOG-Qsp-6his. The constructed and correctly sequenced plasmid was transferred into Escherichia coli BL21 (DE3) expression competent cells; a single clone was picked and inoculated into LB medium and shaken at 37°C until OD 600 The value was 0.5-1, IPTG was added for induction, and the cells were expressed at 16°C overnight. The cells were collected, ultrasonically broken, 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 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 expressed and purified successfully.

[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 the recombinant human MOG-Qsp-6his protein purified in Example 1 was mixed with complete Freund's adjuvant in an amount of 40 μg at a volume ratio of 1:1, and 100 μL (40 μg antigen) of the mixed antigen was injected into the left hind calf muscle; a second immunization was performed at an interval of 14 days after the initial immunization, 40 μg antigen was mixed with incomplete Freund's adjuvant at a volume ratio of 1:1, and 100 μL (40 μg antigen) of the mixed antigen was injected into the right hind calf muscle, and a third immunization was performed at an interval of 14 days after the second immunization. During the third immunization, the dosage, method, and route of the injected antigen were exactly the same as those of 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 gene sequence of human MOG (gene sequence number: NM_001363610.2) was synthesized by Jinweizhi Company onto the pcDNA3.4 vector, with restriction sites of 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 respectively, and the serum was obtained by centrifugation. The serum of 5 immunized mice was diluted 1:32 with PBST buffer, incubated with MOG overexpressing cell slides, incubated at room temperature for 1 hour, and washed 3 times with PBST buffer, 5 minutes each time; AlexaFluor 594-labeled goat anti-mouse IgG secondary antibody (manufacturer: Jackson, catalog number: 115-585-146) diluted 1:200 was used, incubated at room temperature for 30 minutes, washed 3 times with PBST buffer, 5 minutes each time, and the results were observed under a fluorescence microscope, as shown in FIG. 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 were taken from the ascites of ordinary mice for culture as feeder cells. Feeder cells were obtained from the ascites of 4 to 6 mice and plated on 10 96-well plates. After 2 days of culture, hybridoma cells were prepared. Myeloma cells SP2 / 0 with good growth and a cell density of 90% in the logarithmic growth phase were collected, a total of 10 dishes. Centrifuge and discard the supernatant, resuspend the cells in DMEM medium and count them.

[0070] Preparation of spleen cells: Mouse No. 2 was boosted with intraperitoneal injection of 100 μL antigen (40 μg antigen). Three days later, blood was collected from the tail of the mouse 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 cut into pieces with sterile surgical scissors and ground using the piston handle of a 50 mL sterile syringe to obtain a single spleen cell suspension.

[0071] Cell fusion: Prepare a 37℃ water bath in the clean bench and preheat it in advance. Add the SP2 / 0 cells and spleen cells with a cell density of 90% and the ratio of 1:10 that have been cultured in advance to a 50mL centrifuge tube and mix well. Centrifuge at 500g for 10min, aspirate the supernatant, flick the bottom of the centrifuge tube to loosen the cell pellet slightly; slowly drip 1mL of 45% PEG1450 solution preheated to 37℃ within 90s; and shake the centrifuge tube gently continuously; the centrifuge tube is in a 37℃ water bath during the whole process. Then add DMEM culture medium to the cell mixture at a uniform speed, drip 1mL drop by drop in the first minute, drip 2mL drop by drop in the second minute, drip 3mL drop by drop in the third minute, drip 4mL drop by drop in the fourth minute, drip 5mL drop by drop in the fifth minute, and shake while adding in a 37℃ water bath. Then incubate at 37℃ for 15min, centrifuge at 500g for 5min, and remove the supernatant. Add 5mL of DMEM medium containing HAT, gently suspend the precipitated cells, and finally add DMED medium containing HAT to about 100mL. Dispense into a 96-well cell culture plate with feeder cells, 100μL / well, and then place the culture plate in a 37℃, 5% CO2 incubator for culture. Add 100μL of HAT culture medium to each well on the second day after fusion, and then half-change the HAT culture medium every 2-3 days, and screen after about 2 weeks of culture.

[0072] 2.4 Screening of positive hybridoma cells: Observe the growth of hybridoma cells. After 7 to 10 days, when the cell culture supernatant turns yellow, take an appropriate amount of cell supernatant for immunofluorescence detection of antibodies. Mix the cell supernatants of 12 wells in each row of 96-well plates in equal volumes, add Tween-20 to a final concentration of 0.2% and then perform mixed tests. According to the immunofluorescence results, select a row of 12 wells corresponding to the mixed sample with a strong positive signal to continue culturing and further screen the mother clone. After continuing to culture for 7 to 10 days, mix the samples of a row with a strong positive signal, and perform immunofluorescence detection of antibodies in each well of the cell supernatant. Finally, 13D5, 49G9, and 51E6 were screened as positive wells. The screened positive wells were replaced with liquid for verification again, and limited dilution was performed to culture about 1 cell per well. After 7 to 10 days of culture, three clones with strong positive signals were selected for further immunofluorescence verification. Finally, three positive clone cell lines were screened and named: 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 shown in Figure 3 The results are shown in Figure 1, which are 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 has the strongest activity and is the best hybridoma cell.

[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 0.1M BST buffer for 3 min each time and patted dry. Blocking protein (2% BSA solution) was added at 200 μL / well and incubated at 37°C for 1 h. 0.1M Wash with PBST buffer for 3 times, 3 min each time, and pat dry; dilute the supernatant of monoclonal 13D5, 49G9, and 51E6 cells with PBS at 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 mice diluted at a volume ratio of 1:1000 as the initial concentration, and use them as positive control wells / negative control wells after multiple dilutions, add 100 μL of sample to each well, and incubate at 37°C for 1h; wash with 0.1M PBST for 3 times, 3 min each time, and pat dry; use HRP-labeled IgG secondary antibody, antibody diluted 1:5000 (manufacturer: Jackson), incubate at 37°C for 30min; wash with 0.1M PBST buffer for 3 times, 3 min each time, and pat dry; add TMB for color development for 5min, and use 2M The color development was terminated by H2SO4, and the absorbance was measured at OD450nm. The experimental results are shown in Table 1, which are the titers of 13D5, 49G9 and 51E6 mouse monoclonal antibodies.

[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, which can reach 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 coated overnight at 4°C. The next day, wash 3 times with 0.1 M PBST buffer for 3 min each time and pat dry. Add blocking protein (2% BSA solution) at 200 μL / well and incubate at 37°C for 1 h. Wash 3 times with 0.1 M PBST buffer for 3 min each time and pat dry. Add 100 μL / well of monoclonal 13D5 or 51E6 cell supernatant and use No. 2 mouse tail blood diluted 1:1000 as a positive control, add 100 μL to each well and incubate at 37°C for 1 h. Wash 3 times with PBST buffer, 3 minutes each time, and pat dry; use HRP-labeled secondary antibodies (total IgG / IgG1 / IgG2a / IgG2b / IgG3 / IgM / Igκ / Igλ), antibody 1:5000 dilution (manufacturer: Jackson), 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, use 2M H2SO4 to stop color development, and measure the absorbance at OD450nm. The experimental results are shown in Table 2.

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

[0085]

[0086]

[0087] It can be concluded from Table 2 that the heavy chain of 13D5 mouse monoclonal antibody is IgG2b, and the light chain is Igκ; the heavy chain of 51E6 mouse monoclonal antibody is IgG1, and the light chain is Igκ. After comprehensive comparison, 13D5 hybridoma cells were selected for subsequent experiments. 13D5 mouse monoclonal antibody was purified from the culture supernatant of positive hybridoma cells as a control, and the protein concentration measured by BCA method was 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 to extract total RNA from the hybridoma cell lysate (Quick-RNA MicroPrep Kit). RNA was separated by agarose gel electrophoresis and 5'RACE was performed using RNA as a template to synthesize the first-chain cDNA (Clontech SMARTerRACE 5' / 3'kit). PCR amplification was performed using the RNA as a template (PrimeSTAR Max DNA Polymerase) was used to cross-link tumor cell monoclonal antibody heavy chain variable region (VH) gene and light chain variable region (VL) gene, and antibody light chain / heavy chain variable region cDNA was extracted. The heavy chain variable region amino acid sequence and light chain variable region amino acid sequence of 13D5 mouse monoclonal antibody were obtained, and 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 sequence against human MOG

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

[0093] The antibody sequence was amplified using Fast PfuDNA Polymerase (Full Gold) by conventional molecular biological methods to construct a mutant of the antibody sequence. The amino acid V at position 116 of the amino acid sequence of the light chain variable region of the 13D5 mouse 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 new amino acid sequence after mutation 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 is composed of the 13D5 mouse 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), and 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 is composed of the 13D5 mouse 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), and 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 composed of the complete light chain amino acid sequence SEQ ID NO:18 and the complete heavy chain amino acid sequence SEQ ID NO:20 is named 1820, and the antibody composed of the complete light chain amino acid sequence 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-MOG human-mouse chimeric monoclonal antibody

[0097] The nucleotide sequence of the light chain variable region of the 13D5 mouse monoclonal antibody (SEQ ID NO: 7 or SEQ ID NO: 8) was amplified, and the nucleotide sequence of the human Kappa chain constant region (SEQ ID NO: 16) was amplified. The light chain variable region and light chain constant region fragments were connected to the vector pcDNA3.1 using homologous recombination (manufacturer: Quanshijin, item number: CU201-02). The variable region amplified fragment and the constant region amplified fragment were connected in sequence from 5'-3', and the insertion position was the multiple cloning site of the vector pcDNA3.1. They were marked as pcDNA3.1-MOG-VL or pcDNA3.1-MOG-VL', respectively, and the connected recombinant plasmid was sent to Sangon Biotechnology for sequencing. The sequenced recombinant plasmid was extracted and used for cell transfection to prepare 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: 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 another 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 for amplifying 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 another 3 min.

[0106] The nucleotide sequence of the heavy chain variable region of the 13D5 mouse monoclonal antibody (SEQ ID NO: 13) was amplified, and the heavy chain constant region sequence of the human IgG1 (SEQ ID NO: 17) was amplified. The two fragments were connected to the vector pcDNA3.1 using homologous recombination (manufacturer: Quanshijin Part No.: CU201-02). The variable region amplified fragment and the constant region amplified fragment were connected in sequence from 5' to 3'. The insertion position was the multiple cloning site of the vector pcDNA3.1, marked as pcDNA3.1-MOG-VH, and the connected recombinant plasmid was sent to Sangon Biotechnology for sequencing. The sequenced recombinant plasmid was extracted and used for cell transfection to prepare 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 for amplifying the sequence shown in SEQ ID NO: 13 was: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 62°C annealing for 20 s, 72°C extension for 20 s, 30 cycles; and 72°C extension for another 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 for amplifying the sequence shown in SEQ ID NO: 17 was: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 64°C annealing for 20 s, 72°C extension for 20 s, 30 cycles; and 72°C extension for another 3 min.

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

[0116] The following two groups of recombinant vectors were co-transfected into Expi293F cells (thermo company) using transfection reagent PEI (manufacturer: thermo) for transient expression: the antibody expressed by group 1 (pcDNA3.1-MOG-VL and pcDNA3.1-MOG-VH recombinant vectors) corresponds to the anti-human MOG protein human-mouse chimeric antibody 1820, and the antibody expressed by group 2 (pcDNA3.1-MOG-VL' and pcDNA3.1-MOG-VH recombinant vectors) corresponds to the anti-human MOG protein human-mouse chimeric antibody 1920. The supernatant was collected 3 days after transfection, and purified using protein A filler, and the protein concentration was determined using a BCA kit. The concentration of anti-human MOG protein human-mouse chimeric antibody 1820 was 0.7 mg / mL, and the concentration of 1920 was 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-MOG human-mouse chimeric monoclonal antibody to MOG antigen

[0119] ELISA detection: Use the purified MOG-Qsp-6his protein to coat the ELISA plate, 100 ng / well, and coat overnight at 4°C (the coating solution is 25 mL carbonate buffer, pH 9.6); wash 3 times with PBST buffer the next day, 3 minutes each time, and pat dry each time; add 2% BSA to cover the wells and incubate at 37°C for 1 hour; after incubation, wash 3 times with PBST buffer, 3 minutes each time, and pat the plate to remove excess water each time; incubate 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 Long antibody was used as a positive control, and normal control mouse serum was used as a negative control. The dilution ratios were 1:1, 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, and 1:2187 of the original solution. 100 μL of the above dilution was added to each well, incubated at 37°C for 1 hour, washed 3 times with PBST buffer, 3 minutes each time, and patted dry each time; the goat anti-human / goat anti-mouse IgG-HRP secondary antibody was diluted at a ratio of 1:5000, added to the plate, and incubated at 37°C for 30 minutes; after incubation, it was washed 3 times with PBST buffer, patted dry, TMB was added for color development for 3 minutes, 2M H2SO4 was terminated, and the absorbance value was measured at OD450nm. The experimental results are shown in Table 4.

[0120] Table 4 Binding effect of anti-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: based on the fact that at a dilution of 1:2187, the titer of the chimeric antibody can still reach more than twice the titer of the negative control at the same dilution ratio, it can be seen 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 antibody against human MOG

[0125] 5.1 Identification of the function of anti-MOG human-mouse chimeric monoclonal antibody by immunofluorescence

[0126] The two groups of anti-human MOG human-mouse chimeric antibodies 1820 and 1920 purified in Example 4 were respectively diluted with a commercial MOG antibody (manufacturer: Wuhan Mitaka 12690-1-AP) using PBST buffer, and the concentrations were adjusted to be consistent. The two groups of MOG-13D5 human-mouse chimeric antibodies were 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 with the MOG-pcDNA3.4 cell slides and pcDNA3.4 control slides prepared in Example 1, respectively, incubated at room temperature for 1 hour, washed 3 times with PBST, each time for 5 minutes, and 1:200 diluted FITC-labeled goat anti-human / AlexaFluor 594-labeled goat anti-rabbit IgG (manufacturer: Jackson) was added, incubated at room temperature for 30 minutes, and washed 3 times with PBST buffer, each time for 5 minutes; observed under a microscope, the results are as follows Figure 5 As shown, the scale bar is 150 μm.

[0127] according to Figure 5 The results show that compared with commercial antibodies, the two groups of anti-human MOG human-mouse chimeric antibodies 1820 and 1920 can be incubated with the same secondary antibody as the sample to be tested (the labeled secondary antibodies used are all FITC-labeled goat anti-human IgG), have more obvious humanized species advantages, and are more suitable for the role of positive reference products; and the anti-human MOG human-mouse chimeric antibodies 1820 and 1920 have the same typical morphology of positive cells and basically the same fluorescence signal intensity as MOG patient serum under the same incubation conditions, and can be used as substitutes for positive serum / cerebrospinal fluid to meet the needs of diagnostic kits; anti-human MOG human-mouse chimeric antibodies 1820 and 1920 can accurately quantify protein and determine antibody titers, which can meet long-term product quality control. This chimeric antibody enriches the types of MOG monoclonal antibodies and lays a certain foundation for the auxiliary diagnosis of MOGAD disease. This chimeric antibody can supplement more typical positive cell morphology for immunofluorescence experiments, and can also enrich the number of positive images, so that computer artificial intelligence technology can obtain more accurate interpretation results when processing indirect immunofluorescence images.

[0128] 5.2 Western-Blot Identification of the Function of Anti-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, the membrane was transferred using the wet transfer condition of 300 mA for 90 min; 5% skim milk powder was blocked at room temperature for 1 h; 1:2000 diluted anti-human MOG protein human-mouse chimeric antibody 1820 (concentration of 0.7 mg / mL) and commercial MOG (manufacturer: Wuhan Mitaka, 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 chemiluminescent solution was added for color development and photography, and the results are shown 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 deeper than that recognized by 1820, the pcDNA3.1 control protein also has some non-specific bands, while the 1820 monoclonal antibody has no non-specific bands in this area).

[0131] Although the above embodiment describes the present invention in detail, 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 protection scope 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, 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; 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 comprises 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; Preferably, 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.

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

7. 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.

8. 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; Preferably, the human-mouse chimeric monoclonal antibody against human MOG protein is a positive reference or quality control product in the kit.

9. 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.

10. The kit according to claim 9, characterized in that The kit is a kit prepared based on immunoassay technology.

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