Anti-Ro52 human recombinant monoclonal antibody as well as preparation method and application thereof

Abstract

The invention provides an anti-Ro52 human recombinant monoclonal antibody and a preparation method and application thereof.The anti-Ro52 human recombinant monoclonal antibody is derived from B cells in blood of a self-immunization patient, is completely a human monoclonal antibody, and does not have species difference with a Ro52 human antigen, so that the functional problem caused by species specificity is avoided, and the application prospect is wide. The problems of rejection reaction, weak affinity and the like of heterologous expression are solved. The specific recognition of the Ro52 protein can be realized, the relatively good sensitivity, anti-interference capability and specificity are shown, and a raw material guarantee is provided for the downstream development of a high-quality diagnostic reagent.

Description

Technical Field

[0001] The present application relates to the field of cellular immunity technology, and in particular to an anti-Ro52 human recombinant monoclonal antibody and a preparation method and application thereof. Background Art

[0002] A positive anti-Ro-52 antibody usually indicates the presence of antibodies against the Ro-52 antigen in the human body. The appearance of such antibodies is associated with a variety of autoimmune diseases and is more common in Sjögren's syndrome. Currently, most anti-Ro52 antibodies on the market are derived from animal immunization. The preparation of anti-Ro52 antibodies usually includes the following steps: (1) Antigen selection: Select the Ro52 antigen as the immunogen; (2) Select a suitable animal model: Use transgenic or gene-edited animal models to simulate human autoimmune diseases in order to test the efficacy and safety of the prepared antibodies in vivo. (3) Immunization process: Through immunization procedures, such as multiple immunizations and booster immunizations, the animals are stimulated to produce specific antibodies. (4) Antibody extraction and purification: Antibodies are separated from blood samples and then purified using techniques such as protein A affinity chromatography.

[0003] However, there are certain disadvantages in obtaining humanized antibodies through animal immunization: (1) Immunogenicity issues: The complementary determining regions (CDRs) of mouse antibodies are transplanted into the variable regions of human antibodies. Although this can produce humanized antibodies, it may produce antibody reactions. (2) Decreased affinity: During the preparation of humanized antibodies, the complementary determining regions (CDRs) of the mouse origin are transplanted into the human antibody framework, which may cause the affinity of the antibody to decrease. This is because the mouse CDRs may not be completely compatible with the human antibody framework, affecting the binding efficiency of the antibody to the antigen. (3) Changes in structure and function: Humanized antibodies may not be able to completely mimic human antibodies in their natural state in terms of structure and function. This is because even if the complementary determining regions are transplanted, the amino acid sequences of other regions may change, affecting the overall function and stability of the antibody.

[0004] Therefore, existing animal-derived antibodies have functional problems due to species specificity. Summary of the Invention

[0005] The present invention provides a method for preparing a humanized recombinant monoclonal antibody against Ro52 and its application to solve the problems existing in the related art. The technical solution is as follows:

[0006] In a first aspect, the embodiments of the present application provide an anti-Ro52 human recombinant monoclonal antibody comprising a light chain variable region and a heavy chain variable region;

[0007] The heavy chain variable region includes complementarity determining regions VH CDR1, VH CDR2 and VH CDR3; the light chain variable region includes complementarity determining regions VK CDR1, VK CDR2 and VK CDR3;

[0008] The amino acid sequence of VH CDR1 is SEQ ID NO. 1: SYAMG; the encoding nucleotide sequence is SEQ ID NO. 13: agctatgcgatgggctaa.

[0009] The amino acid sequence of VH CDR2 is SEQ ID NO. 2: AISYDGSNKYYADSVKG; the encoding nucleotide sequence is SEQ ID NO. 14: gcgattagctatgatggcagcaacaaatattatgcggatagcgtgaaaggctaa.

[0010] The amino acid sequence of VH CDR3 is SEQ ID NO. 3: DHGYFDY; the encoding nucleotide sequence is SEQ ID NO. 15: gaycayggntayttygaytaytrr.

[0011] The amino acid sequence of VK CDR1 is SEQ ID NO.4: RASQDISNYLN; the encoding nucleotide sequence is SEQ ID NO.16: cgcgcgagccaggatattagcaactatctgaactaa.

[0012] The amino acid sequence of VK CDR2 is SEQ ID NO.5: YTSSLHS; the encoding nucleotide sequence is SEQ ID NO.17: tataccagcagcctgcatagctaa.

[0013] The amino acid sequence of VK CDR3 is SEQ ID NO.6: QQYNSYPYT; the encoding nucleotide sequence is SEQ ID NO.18: cagcagtataacagctatccgtatacctaa.

[0014] In one embodiment, the amino acid sequence of the heavy chain variable region of the anti-Ro52 human recombinant monoclonal antibody is as shown in SEQ ID NO.7;

[0015] SEQ ID NO.7:

[0016] EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMGWFRQAPGKEREFVAAIS

[0017] YDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDHGYF

[0018] DYWGQGTQVTVSS

[0019] The encoding nucleotide sequence is shown in SEQ ID NO.11;

[0020] SEQ ID NO.11

[0021] gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctg

[0022] agctgcgcggcgagcggctttacctttaacagctatgcgatgggctggtttcgccaggcg

[0023] ccgggcaaagaacgcgaatttgtggcggcgattagctatgatggcagcaacaaatattat

[0024] gcggatagcgtgaaaggccgctttaccattagccgcgataacagcaaaaacaccctgtat

[0025] ctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgcgcgatcat

[0026] ggctattttgattattggggccagggcacccaggtgaccgtgagcagctaa

[0027] The amino acid sequence of the light chain variable region of the anti-Ro52 human recombinant monoclonal antibody is shown in SEQ ID NO.8;

[0028] SEQ ID NO.8:

[0029] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKVEIK

[0030] The encoding nucleotide sequence is shown in SEQ ID NO.12;

[0031] SEQ ID NO.12:

[0032] gatattcagatgacccagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgacc

[0033] attacctgccgcgcgagccaggatattagcaactatctgaactggtatcagcagaaaccg

[0034] ggcaaagcgccgaaactgctgatttattataccagcagcctgcatagcggcgtgccgagc

[0035] cgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcagccg

[0036] gaagattttgcgacctattattgccagcagtataacagctatccgtatacctttggccag

[0037] ggcaccaaagtggaaattaaataa.

[0038] In one embodiment, the sequence of the anti-Ro52 human recombinant monoclonal antibody is derived from a human sequence.

[0039] In one embodiment, the amino acid sequence of the heavy chain of the anti-Ro52 human recombinant monoclonal antibody is as shown in SEQ ID NO.9;

[0040] SEQ ID NO.9:

[0041] EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMGWFRQAPGKEREFVAAISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDHGYFDYWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK;

[0042] The coding nucleotide sequence is shown in SEQ ID NO. 19;

[0043] SEQ ID NO. 19:

[0044] gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctg

[0045] agctgcgcggcgagcggctttacctttaacagctatgcgatgggctggtttcgccaggcg

[0046] ccgggcaaagaacgcgaatttgtggcggcgattagctatgatggcagcaacaaatattat

[0047] gcggatagcgtgaaaggccgctttaccattagccgcgataacagcaaaaacaccctgtat

[0048] ctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgcgcgatcat

[0049] ggctattttgattattggggccagggcacccaggtgaccgtgagcagcgcgagcaccaaa

[0050] ggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcg

[0051] ctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggc

[0052] gcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagc

[0053] ctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaac

[0054] gtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgat

[0055] aaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgttt

[0056] ctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgc

[0057] gtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggc

[0058] gtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgc

[0059] gtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgc

[0060] aaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaataa。

[0061] The amino acid sequence of the light chain of the anti-Ro52 human recombinant monoclonal antibody is as shown in SEQ ID NO. 10;

[0062] SEQ ID NO.10:

[0063] DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0064] The encoding nucleotide sequence is as shown in SEQ ID NO.20;

[0065] SEQ ID NO.20:

[0066] gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgacc

[0067] attacctgccgcgcgagccaggatattagcaactatctgaactggtatcagcagaaaccg[[ID=**17**]] [[ID=**18**]]

[0068] [[ID=**19**]]ggcaaagcgccgaaactgctgatttattataccagcagcctgcatagcggcgtgccgagc[[ID=**20**]] [[ID=**21**]]

[0069] [[ID=**22**]]cgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcagccg[[ID=**23**]] [[ID=**24**]]

[0070] [[ID=**25**]]gaagattttgcgacctattattgccagcagtataacagctatccgtatacctttggccag[[ID=**26**]] [[ID=**27**]]

[0071] [[ID=**28**]]ggcaccaaagtggaaattaaacgcaccgtggcggcgccgagcgtgtttatttttccgccg[[ID=**29**]] [[ID=**30**]]

[0072] [[ID=**31**]]agcgatgaacagctgaaaagcggcaccgcgagcgtggtgtgcctgctgaacaacttttat[[ID=**32**]] [[ID=**33**]]

[0073] ccgcgcgaagcgaaagtgcagtggaaagtggataacgcgctgcagagcggcaacagccag

[0074] gaaagcgtgaccgaacaggatagcaaagatagcacctatagcctgagcagcaccctgacc

[0075] ctgagcaaagcggattatgaaaaacataaagtgtatgcgtgcgaagtgacccatcagggc

[0076] ctgagcagcccggtgaccaaaagctttaaccgcggcgaatgctaa.

[0077] In a second aspect, an embodiment of the present application provides a nucleic acid encoding any of the above-mentioned anti-Ro52 human recombinant monoclonal antibodies.

[0078] In one embodiment, the nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID NO.11; the nucleotide sequence encoding the light chain variable region is shown as SEQ ID NO.12.

[0079] In a third aspect, an embodiment of the present application provides a biomaterial, wherein the biomaterial comprises the nucleic acid described above; the biomaterial is an expression cassette, a vector or a transgenic cell.

[0080] In a fourth aspect, the embodiments of the present application provide a method for preparing the above-mentioned anti-Ro52 human recombinant monoclonal antibody, expressing the above-mentioned nucleic acid; or culturing the above-mentioned transgenic cells.

[0081] In a fifth aspect, the reagent or kit of the embodiment of the present application comprises any of the above-mentioned anti-Ro52 human recombinant monoclonal antibodies, or the above-mentioned nucleic acid, or the above-mentioned biological material.

[0082] In one embodiment, the reagent or kit is used to detect Ro52 antibodies.

[0083] The advantages or beneficial effects of the above technical solution include at least:

[0084] The anti-Ro52 human recombinant monoclonal antibody described in this application is derived from B cells in the blood of autoimmune patients. It is a completely human monoclonal antibody with no species differences from the human Ro52 antigen, thus avoiding functional issues caused by species specificity and resolving issues such as rejection reactions and weak affinity associated with heterologous expression. Furthermore, it can specifically recognize the Ro52 protein, demonstrating good sensitivity, anti-interference ability, and specificity, providing raw material support for the development of high-quality diagnostic reagents downstream.

[0085] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the multiple drawings represent the same or similar components or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0087] Figure 1 This is the SDS-PAGE image of Ro52 protein;

[0088] Figure 2 This is the electrophoresis diagram of variable region gene amplification of a single B cell. DETAILED DESCRIPTION

[0089] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0090] Example

[0091] Materials: mammalian cells; cell culture medium, transfection medium, imidazole; Ni-NTA filler; SDS-PAGE gel; sonicator; broth culture medium; dimethyl sulfoxide; biotin; streptavidin; flow cytometer; DAPI, IgM-BV241; transfection reagent, 96-well plate, BCA protein concentration assay kit, mouse anti-human IgG, TMB colorimetric solution, 2 M sulfuric acid stop solution.

[0092] method:

[0093] 1. Eukaryotic expression of Ro52 protein

[0094] The amino acid sequence of the Ro52 protein was obtained from the Uniprot database (UniPort ID: P19474). The full-length gene sequence of the Ro52 protein was selected as the expressed gene (encoding a protein sequence of 1-475 aa). The expressed sequence was codon-optimized for eukaryotic expression by Beijing Qingke Biotechnology Co., Ltd. and then gene synthesis was performed. The synthesized Ro52 protein gene fragment was cloned into a eukaryotic expression vector using the Nco I and Xho I restriction sites. The target protein was labeled with a 10*HIS tag at the N-terminus. The specific amino acid sequence and gene sequence are shown in SEQ ID NO. 21 and SEQ ID NO. 22.

[0095] SEQ ID NO.21:

[0096] MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRK HRDHAMVPLEEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISELDRRCHS SALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGLKKMLRTCAVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFDSYPMVLGAQHFHSGKHYWEVDVTGKEAWDLG VCRDSVRRKGHFLLSSKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPLNIGSQGSTDY.

[0097] SEQ ID NO.22:

[0098] ATGGCCAGCGCCGCCAGACTGACCATGATGTGGGAGGAGGTGACCTGCC

[0099] CCATCTGCCTGGACCCCTTCGTGGAGCCCGTGAGCATCGAGTGCGGCCAC

[0100] AGCTTCTGCCAGGAGTGCATCAGCCAGGTGGGCAAGGGCGGCGCAGCG

[0101] TGTGCCCCGTGTGCAGACAGAGATTCCTGCTGAAGAACCTGAGACCCAA

[0102] CAGACAGCTGGCCAACATGGTGAACAACCTGAAGGAGATCAGCCAGGAG

[0103] GCCAGAGAGGGCACCCAGGGCGAGAGATGCGCCGTGCACGGGCGAGAGA

[0104] CTGCACCTGTTCTGCGAGAAGGACGGCAAGGCCCTGTGCTGGGTGTGCG

[0105] CCCAGAGCAGAAAGCACAGAGACCACGCCATGGTGCCCCTGGAGGAGGC

[0106] CGCCCAGGAGTACCAGGAGAAGCTGCAGGTGGCCCTGGGCGAGCTGAGA

[0107] AGAAAGCAGGAGCTGGCCGAGAAGCTGGAGGTGGAGATCGCCATCAAG

[0108] AGAGCCGACTGGAAGAAGACCGTGGAGACCCAGAAGAGCAGAATCCAC

[0109] GCCGAGTTCGTGCAGCAGAAGAACTTCCTGGTGGAGGAGGAGCAGAGAC

[0110] AGCTGCAGGAGCTGGAGAAGGACGAGAGAGCAGCTGGAGAATCCTGG

[0111] GCGAGAAGGAGGCCAAGCTGGCCCAGCAGAGCCAGGCCCTGCAGGAGC

[0112] TGATCAGCGAGCTGGACAGAAGATGCCACAGCAGCGCCTGGAGCTGCT

[0113] GCAGGAGGTGATCATCGTGCTGGAGAGAAGCGAGAGCTGGAACCTGAAG

[0114] GACCTGGACATCACCAGCCCCGAGCTGAGAAGCGTGTGCCACGTGCCCG

[0115] GCCTGAAGAAGATGCTGAGAACCTGCGCCGTGCACATCACCCTGGACCC

[0116] CGACACCGCCAACCCCTGGCTGATCCTGAGCGAGGACAGAAGACAGGTG

[0117] AGACTGGGCGACACCCAGCAGAGCATCCCCGGCAACGAGGAGAGATTCG

[0118] ACAGCTACCCCATGGTGCTGGGCGCCCAGCACTTCCACAGCGGCAAGCA

[0119] CTACTGGGAGGTGGACGTGACCGGCAAGGAGGCCTGGGACCTGGGCGTG

[0120] TGCAGAGACAGCGTGAGAAGAAAGGGCCACTTCCTGCTGAGCAGCAAG

[0121] AGCGGCTTCTGGACCATCTGGCTGTGGAACAAGCAGAAGTACGAGGCCG

[0122] GCACCTACCCCCAGACCCCCCTGCACCTGCAGGTGCCCCCCTGCCAGGTG

[0123] GGCATCTTCCTGGACTACGAGGCCGGCATGGTGAGCTTCTACAACATCAC

[0124] CGACCACGGCAGCCTGATCTACAGCTTCAGCGAGTGCGCCTTCACCGGCC

[0125] CCCTGAGACCCTTCTTCAGCCCCGGCTTCAACGACGGCGGCAAGAACAC

[0126] CGCCCCCCTGACCCTGTGCCCCCTGAACATCGGCAGCCAGGGCAGCACCGACTAC.

[0127] The Ro52 protein expression plasmid was transformed into competent E. coli, and single clones were picked and the plasmid was amplified. After the transfection of a certain volume of cell culture system, the cells were collected by centrifugation, the culture medium was discarded, and the cells were resuspended with pre-cooled PBS and then added with cell lysis buffer. The cells were broken using an ultrasonic disruptor (output power 60%, working 4s, stopping 7s as a cycle, cumulative time 20min); 12000g, 30min centrifugation to collect the supernatant, and the supernatant was purified by affinity chromatography through Ni-NTA affinity resin. First, 10ml Native Binding Buffer (10mM imidazole) was used to equilibrate 1ml Ni-NTA, and then the cell supernatant was slowly loaded and passed through Ni-NTA, and 10ml Native Wash Buffer (containing imidazole from low to high) was used to wash the impurities bound to Ni-NTA, and finally 20ml Native Elution Buffer (high concentration imidazole) was used to elute the target protein. The eluted target protein was concentrated and replaced with PBS buffer through an ultrafiltration tube, and then identified by SDS-PAGE. The electrophoresis pattern is shown as follows. Figure 1 The molecular weight of Ro52 protein is about 52 kDa and the purity is >90%.

[0128] 2. Ro52 protein coupled to fluorescein

[0129] Biotin can be used to simply and effectively label antibodies, proteins, and other macromolecules containing primary amines in solution. Dissolve biotin in dimethyl sulfoxide (DMSO) and prepare a 10mM biotin solution. First, conjugate biotin to Ro52 protein, and then use flow cytometry to link it to biotin. Then add an excess of biotin reagent to label Ro52 protein and incubate on ice for 2 hours. Use a desalting column to purify the labeled Ro52 protein and remove unreacted biotin. According to the binding molar ratio of SA:biotin = 1:4, mix the biotinylated Ro52 protein with flow cytometry fluorescein, incubate on ice for 30 minutes, and then proceed directly to the next step of cell labeling.

[0130] 3. Flow cytometry sorting of single B cells

[0131] Peripheral blood was collected from several patients with autoimmune diseases in the hospital, and PBMCs were separated using human lymphocyte separation medium. 6The frozen PBMCs were placed in a 37°C water bath for rapid thawing and resuscitation. The resuscitated PBMCs were resuspended in FACS buffer (10mM PBS, 1mM EDTA, 2% FBS). 100μL of the resuspended cells (1*10 6 ) Fluorescent markers, DAPI and IgM-BV241, were added sequentially and incubated on ice for 30 minutes. PBMCs were sorted using a BD FACSAria III flow cytometer, and the DAPI- / IgM- and Ro52 protein-double-positive cell population was selected to obtain 54 single B cells specific for the Ro52 protein.

[0132] 4. Amplify the variable region genes of single B cells,

[0133] Amplification primers and amplification conditions refer to the method in the literature (Smith, K., Garman, L., Wrammert, J. et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinatingantigen. Nat Protoc 4, 372-384 (2009). https: / / doi.org / 10.1038 / nprot.2009.3). First, RT-PCR is performed using a mixture of nine primers (see Table 1) to cover all possible variable region (V) genes. PCR enriches the variable region genes and amplifies the DNA to a degree sufficient to obtain the heavy chain and light chain variable gene sequences. Cloning PCR uses highly specific primers for each variable gene family to amplify, aiming to integrate cloning restriction sites and place the variable region heavy chain or variable region light chain gene in frame with the signal peptide sequence and constant region gene in the respective cloning vector. The gene amplification results are shown in Figure 2. Figure 2 As shown, heavy chain 44 / 88 (50%), light chain Kappa chain (47 / 88, 53%), light chain Lambda chain (34 / 88, 39%).

[0134] Table 1

[0135] Primer Sequence 1F ATGAGGCCTGCTCAGCTTCTGG 1R RAGGTGTGCACGCCGCTGGTC; 2F ATGGAAGCCCCAGCTCAGCTTC 2R GTTTCTCGTAGTCTGCTTTGCTCA 3F CCCAGCTCAGCTTCTCTTCCTCCTG 3R CACCAGTGTGGCCTTGTTGGCTTG 4F TGGTGTTGCAGACCCAGGTCTTCATTTC 4R GTTCGGGGAAGTAGTCCTTGAC 5F GTCCCAGGTTCACCTCCTCAGCTTC 5R GTGCTGTCCTTGCTGTCCTGCT 6F GCCATCACAACTCATTGGGTTTCTGCTG 6R CTCCTCACTCGAGGGTGGGAACAGAGTG 7F TCCCTGCTCAGCTCCTGGG 7R GGGTGCAGGGGGAAGACCGATGGGCCCTTGGTCGAGGC 8F CCTGGGACTCCTGCTGCTCTG 8R CTCATCAGATGGCGGGAAGATGAAGACAGATGGTGCAGCCACCGTACG 9F CCCTGGGTCATGCTCCTCCTGAAATC 9R GAAGCTCCTCACTCGAGGGYGGGAACAGAGTA

[0136] 5. Antibody expression plasmid construction

[0137] First, double-digest the gamma, kappa, or lambda cloning vector with restriction endonucleases Age I & Sal I, Age I & BsiW I, or Age I & Xho I, respectively. Refer to the reference for cloning vector sequences (Smith, K., Garman, L., Wrammert, J. et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc 4, 372-384 (2009). https: / / doi.org / 10.1038 / nprot.2009.3) at 37°C for 2 hours. Purify and recover the gamma, kappa, or lambda chain variable region gene product amplified in step 4 using a standard DNA purification kit. Submit a sample of the double-digested cloning vector for electrophoresis on a 1% agarose gel. The vector is approximately 5700 bp in length. The variable region gene fragment and the vector fragment were homologously recombined using the ClonExpress II One Step Cloning Kit and reacted at 37°C for 30 minutes. The recombinant product was transformed into E. coli competent cells, evenly spread on a plate containing Amp resistance, and cultured at 37°C overnight. The next day, single clones from three plates were picked using a pipette tip and placed in 500 μL of LB medium containing Amp resistance and cultured at 37°C for 4-6 hours. 2× Taq Master Mix was used to perform bacterial liquid PCR verification to confirm whether the variable region was successfully inserted into the vector. Positive clones were sequenced to confirm the accuracy of the variable region sequence.

[0138] 6. Antibody small batch expression

[0139] Transfect mammalian cells with the recombinant antibody plasmid for small-scale expression. 24 hours before transfection, seed 293T cells into a 96-well plate, maintaining a confluency of 90% before transfection. Gently aspirate the complete medium from the cells in the 96-well plate and replace it with serum-free medium. Prepare a separate sterile 96-well plate, take 30 ml of DMEM medium, add 500 μL of transfection reagent, mix thoroughly, and add 300 μL / well to the 96-well plate. Then, add 10 ng of each light and heavy chain to the 96-well plate, incubate at room temperature for 15 minutes, and then add to the corresponding wells. After culturing at 37°C, 5% CO2 for 3 days, the cell supernatant was analyzed by ELISA.

[0140] 7. Antibody ELISA specificity screening

[0141] Dilute the Ro52 protein antigen to a specific concentration using phosphate-coating buffer. Add 100 μL to each well of the ELISA plate and incubate at 37°C for 2 hours. Shake off the antigen, tap the plate gently, add 200 μL of 5% skim milk to each well, and block at 37°C for 1 hour. Wash the plate three times with 300 μL of PBST for 3 minutes each time, add 100 μL of cell supernatant to each well, and incubate at 37°C for 1 hour. Wash the plate three times with 300 μL of PBST for 3 minutes each time, add 100 μL of the specified concentration of secondary antibody (diluted to the specified concentration with 5% skim milk) to each well, and block at 37°C for 40 minutes. Wash the plate three times with 300 μL of PBST for 3 minutes each time, add 100 μL of TMB chromogenic substrate to each well, and incubate at room temperature in the dark for 10 minutes. Add 50 μL of 2 M sulfuric acid stop solution to each well to stop the color reaction. Place the plate on a microplate reader, measure the absorbance at 450 nm, and record the OD value. The results showed that 23 positive antibodies with an OD > 2 were found in 88 samples. The results are shown in Table 2.

[0142] Table 2 Ro52 protein ELISA specificity screening results

[0143]

[0144]

[0145] 8. Specific Antibody Expression

[0146] Use mammalian cells to express the specific antibodies obtained by screening on a small scale. The day before transfection, the cells were subcultured. On the day of transfection, the cell activity was required to be >90% and the cell density was 3-4*10 6 cells / mL. During transfection, the cell density was adjusted to 1.0*10 6 cells / mL, a transfection reagent and 80 μg of plasmid DNA (H:L = 1:1) were mixed and incubated at room temperature for 10 minutes to form a cationic polymer, which was then slowly added to the cells. 80 ml of cells were transfected with each antibody, and the cell supernatant was collected 6 days later for purification. The antibody was purified using AT Protein A from the supernatant after expression. The antibody was washed with 10 ml of equilibration buffer and then eluted with 5 ml of 0.1 M glycine. The eluted antibody solution was immediately neutralized with 500 μL of buffer and dialyzed against PBS at 4°C, with the dialysate replaced every 2 hours for three cycles. Antibody purity and concentration were determined by SDS-PAGE and BCA assays.

[0147] Monoclonal antibody 5E1 was sequenced, and the heavy chain variable region sequence is shown as SEQ ID NO.7, and the light chain variable region sequence is shown as SEQ ID NO.8;

[0148] SEQ ID NO.7:

[0149] EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMGWFRQAPGKEREFVAAISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDHGYFDYWGQGTQVTVSS.

[0150] SEQ ID NO.8:

[0151] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKVEIK.

[0152] It contains the light chain variable region and the heavy chain variable region;

[0153] VH variable region complementarity determining region:

[0154] CDR1: amino acid sequence SYAMG (SEQ ID NO. 1); nucleotide sequence agctatgcgatgggctaa (SEQ ID NO. 13);

[0155] CDR2: amino acid sequence AISYDGSNKYYADSVKG (SEQ ID NO. 1=2); nucleotide sequence gcgattagctatgatggcagcaacaaatattatgcggatagcgtgaaaggctaa (SEQ ID NO. 14);

[0156] CDR3: amino acid sequence DHGYFDY (SEQ ID NO. 3); nucleotide sequence: gaycayggntayttygaytaytrr (SEQ ID NO. 15);

[0157] VK variable region complementarity determining region

[0158] CDR1: amino acid sequence RASQDISNYLN (SEQ ID NO. 4); nucleotide sequence cgcgcgagccaggatattagcaactatctgaactaa (SEQ ID NO. 16);

[0159] CDR2: amino acid sequence YTSSLHS (SEQ ID NO. 5); nucleotide sequence tataccagcagcctgcatagctaa (SEQ ID NO. 17);

[0160] CDR3: amino acid sequence QQYNSYPYT (SEQ ID NO. 6); nucleotide sequence cagcagtataacagctatccgtatacctaa (SEQ ID NO. 18).

[0161] 9. Monoclonal antibody performance testing

[0162] Specificity detection: The monoclonal antibody 5E1 obtained by the previous step was prepared into 1 μg / mL with 5% skim milk for standby use. Ro52 antigen, Jo-1 antigen, CENPB antigen, P0 antigen, and Scl-70 antigen were diluted into 1 μg / mL solution with 10 mM PBS buffer respectively; 100 μL of the above antigens were added to each well of a 96-well plate, and 3 wells of each antigen were paralleled. The plates were incubated at 37°C for 1 hour, and the antigen solution was discarded. The plate was gently tapped and 200 μL of 5% skim milk was added for blocking at 37°C for 1 hour. The plate was then washed twice with PBST for 3 minutes each time. 100 μL of 5E1 monoclonal antibody solution was added and incubated at 37°C for 1 hour. The plate was then washed three times with PBST for 3 minutes each time. Mouse anti-human HRP-labeled IgG secondary antibody (1:50,000 diluted in 5% skim milk) was added and incubated at 37°C for 40 minutes. The plate was then washed four times with PBST for 3 minutes each time. 100 μL of TMB colorimetric solution was added to each well and the reaction was carried out in the dark for 10 minutes. 50 μL of 2M sulfuric acid stop solution was added to each well and the absorbance was immediately detected at 450 nm on a microplate reader. The specificity results are shown in Table 3; the sensitivity results are shown in Table 4; and the stability results are shown in Table 5.

[0163] Table 3 Anti-Ro52 antibody specificity detection

[0164] Ro52 antibody Ro52 antigen Jo-1 antigen CENPB antigen P0 antigen Scl-70 antigen Absorbance value 3.21 0.0879 0.0768 0.1097 0.0901

[0165] As can be seen from Table 3, the Ro52 antibody only reacted with the wells coated with the Ro52 antigen with a higher absorbance value, but had no obvious reaction with other antibodies, indicating that the prepared Ro52 monoclonal antibody had good specificity.

[0166] Table 4 Anti-Ro52 antibody sensitivity detection

[0167]

[0168] As shown in Table 4, the Ro52 antibody still had an absorbance value when reacting with the antigen at a concentration of 0.000001 μg / mL, indicating that the antibody had high sensitivity.

[0169] Table 5. Anti-Ro52 antibody stability test

[0170]

[0171]

[0172] As shown in Table 5, the Ro52 antibody specifically recognized the Ro52 antigen and did not recognize other antigens after accelerated culture at 37°C for 1, 3, and 7 days. It also specifically recognized the Ro52 antigen and did not recognize other antigens after four freeze-thaw cycles at -80°C and room temperature, demonstrating excellent stability.

[0173] In the description of this specification, the reference terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials, or characteristics described may be combined in any appropriate manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification, as well as features of different embodiments or examples, unless they are mutually inconsistent.

[0174] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0175] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any person skilled in the art can easily conceive of various modifications or substitutions within the technical scope disclosed in this application, and such modifications or substitutions should be included within the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. Anti-Ro52 human recombinant monoclonal antibody, characterized in that comprising a light chain variable region and a heavy chain variable region; The heavy chain variable region includes complementarity determining regions VH CDR1, VH CDR2 and VH CDR3; the light chain variable region includes complementarity determining regions VK CDR1, VK CDR2 and VK CDR3; The amino acid sequence of VH CDR1 is shown in SEQ ID NO. 1, the amino acid sequence of VH CDR2 is shown in SEQ ID NO. 2, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO. 3; The amino acid sequence of VK CDR1 is shown in SEQ ID NO.4, the amino acid sequence of VK CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of VK CDR3 is shown in SEQ ID NO.

6.

2. The anti-Ro52 human recombinant monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-Ro52 human recombinant monoclonal antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The anti-Ro52 human recombinant monoclonal antibody according to claim 1, characterized in that The sequence of the anti-Ro52 human recombinant monoclonal antibody is derived from a human sequence.

4. The anti-Ro52 human recombinant monoclonal antibody according to claim 1, characterized in that The heavy chain amino acid sequence of the anti-Ro52 human recombinant monoclonal antibody is shown in SEQ ID NO.9; the light chain amino acid sequence is shown in SEQ ID NO.

10.

5. A nucleic acid, characterized in that The nucleic acid encodes the anti-Ro52 human recombinant monoclonal antibody according to any one of claims 1 to 4.

6. The nucleic acid according to claim 5, characterized in that The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.11; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.

12.

7. A biomaterial, characterized in that The biological material comprises the nucleic acid according to claim 5 or 6; the biological material is an expression cassette, a vector or a transgenic cell.

8. The method for preparing the anti-Ro52 human recombinant monoclonal antibody according to any one of claims 1 to 4, characterized in that: Gene expression of the nucleic acid according to claim 5 or 6; or culturing the transgenic cell according to claim 7.

9. A reagent, characterized in that The reagent is used to detect Ro52 antibodies, and the reagent includes the anti-Ro52 human recombinant monoclonal antibody according to any one of claims 1 to 4, or the nucleic acid according to claim 5 or 6, or the biological material according to claim 7.

10. A kit, characterized in that The kit comprises the anti-Ro52 human recombinant monoclonal antibody according to any one of claims 1 to 4, or the nucleic acid according to claim 5 or 6, or the biological material according to claim 7, or the reagent according to claim 9; the reagent or kit is used to detect Ro52 antibodies.

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