Anti-ro52 human recombinant monoclonal antibody and its preparation method and application

Abstract

The application 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 healthy person, is a human monoclonal antibody, and does not have species difference with a Ro52 human antigen, so that a functional problem caused by species specificity is avoided, and problems such as rejection and weak affinity of heterologous expression are solved. The anti-Ro52 human recombinant monoclonal antibody can specifically recognize the Ro52 protein, has good sensitivity, anti-interference ability and specificity, and provides raw material guarantee for downstream development of high-quality diagnostic reagents.

Description

Technical Field

[0001] This application relates to the field of cell immunology technology, and in particular to an anti-Ro52 human recombinant monoclonal antibody, its preparation method, and its application. Background Technology

[0002] A positive anti-Ro-52 antibody usually indicates the presence of antibodies against the Ro-52 antigen in the human body. The presence of such antibodies is associated with a variety of autoimmune diseases and is more common in Sjögren's syndrome. Currently, most anti-Ro-52 antibodies on the market are derived from animal immunization. The preparation of anti-Ro-52 antibodies usually includes the following steps: (1) Antigen selection: Selecting the Ro-52 antigen as the immunogen; (2) Selecting a suitable animal model: Using 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: Stimulating animals to produce specific antibodies through immunization procedures, such as multiple immunizations and booster immunizations; (4) Antibody extraction and purification: Separating antibodies from blood samples and then purifying the antibodies using techniques such as protein A affinity chromatography.

[0003] However, obtaining humanized antibodies through animal immunization also has certain drawbacks: (1) Immunogenicity issues: The complementarity-determining region (CDR) of mouse antibodies is transplanted into the variable region of human antibodies. Although this can produce humanized antibodies, it may lead to antibody reactions. (2) Decreased affinity: During the preparation of humanized antibodies, the complementarity-determining region (CDR) of mouse antibodies is transplanted into the framework of human antibodies, which may lead to a decrease in antibody affinity. This is because the CDR of mouse antibodies may not be fully compatible with the framework of human antibodies, affecting the binding efficiency of antibodies to antigens. (3) Changes in structure and function: Humanized antibodies may not be able to completely mimic human antibodies in their structure and function. This is because even if the complementarity-determining region is transplanted, the amino acid sequence 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] This application provides a method for preparing an anti-Ro52 human recombinant monoclonal antibody and its application, in order to solve the problems existing in related technologies. The technical solution is as follows: In a first aspect, embodiments of this application provide an anti-Ro52 human recombinant monoclonal antibody, comprising a light chain variable region and a heavy chain variable region; The variable region of the heavy chain includes complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3; the variable region of the light chain includes complementarity-determining regions VK-CDR1, VK-CDR2, and VK-CDR3. The amino acid sequence of VH-CDR1 is SEQ ID NO. 1: SYAMG; the encoding nucleotide sequence is SEQ ID NO. 13: agctatgcgatgggctaa.

[0006] The amino acid sequence of VH-CDR2 is SEQ ID NO. 2: AISYDGSNKYYADSVKG; the encoding nucleotide sequence is SEQ ID NO. 14: gcgattagctatgatggcagcaacaaatattatgcggatagcgtgaaaggctaa.

[0007] The amino acid sequence of VH-CDR3 is SEQ ID NO.3: DHGYFDY; the encoding nucleotide sequence is SEQ ID NO.15: gatcatggctattttgattat.

[0008] The amino acid sequence of VK-CDR1 is SEQ ID NO.4: RASQDISNYLN; the encoding nucleotide sequence is SEQ ID NO.16: cgcgcgagccaggatattagcaactatctgaactaa.

[0009] The amino acid sequence of VK-CDR2 is SEQ ID NO.5: YTSSLHS; the encoding nucleotide sequence is SEQ ID NO.17: tataccagcagcctgcatagctaa.

[0010] The amino acid sequence of VK-CDR3 is SEQ ID NO.6: QQYNSYPYT; the encoding nucleotide sequence is SEQ ID NO.18: cagcagtataacagctatccgtatacctaa.

[0011] In one embodiment, the heavy chain variable region amino acid sequence of the anti-Ro52 human recombinant monoclonal antibody is shown in SEQ ID NO.7; SEQ ID NO.7: EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMGWFRQAPGKEREFVAAISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDHGYFDYWGQGTQVTVSS The encoding nucleotide sequence is shown in SEQ ID NO. 11; SEQ ID NO. 11: gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggctttacctttaacagctatgcgatgggctggtttcgccaggcgccgggcaaagaacgcgaatttgtggcggcgattagctatgatggcagcaacaaat attatgcggatagcgtgaaaggccgctttaccattagccgcgataacagcaaaaacaccctgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgcgcgatcatggctattttgattattggggccagggcacccaggtgaccgtgagcagctaa 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; SEQ ID NO.8: DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKVEIK The encoding nucleotide sequence is shown in SEQ ID NO. 12; SEQ ID NO.12 gatattcagatgacccagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgccgcgcgagccaggatattagcaactatctgaactggtatcagcagaaaccgggcaaagcgccgaaactgctgatttattataccagcagcctg catagcggcgtgccgagccgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcagccggaagattttgcgacctattattgccagcagtataacagctatccgtatacctttggccagggcaccaaagtggaaattaaataa.

[0012] In one embodiment, the sequence of the anti-Ro52 human recombinant monoclonal antibody is derived from a human sequence.

[0013] In one embodiment, the heavy chain amino acid sequence of the anti-Ro52 human recombinant monoclonal antibody is shown in SEQ ID NO. 9; SEQ ID NO.9: EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMGWFRQAPGKEREFVAAISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDHGYFDYWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK; The encoding nucleotide sequence is shown in SEQ ID NO. 19; SEQ ID NO.19:

[0014] The light chain amino acid sequence of the anti-Ro52 human recombinant monoclonal antibody is shown in SEQ ID NO. 10; SEQ ID NO.10: DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0015] The encoding nucleotide sequence is shown in SEQ ID NO. 20; SEQ ID NO.20: .

[0016] Secondly, embodiments of this application provide a nucleic acid that encodes any of the aforementioned anti-Ro52 human recombinant monoclonal antibodies.

[0017] In one embodiment, the nucleotide sequence for encoding the heavy chain variable region is shown in SEQ ID NO. 11; and the nucleotide sequence for encoding the light chain variable region is shown in SEQ ID NO. 12.

[0018] Thirdly, embodiments of this application provide a biological material comprising the nucleic acid described above; the biological material is an expression cassette, a vector, or a transgenic cell.

[0019] Fourthly, this application provides a method for preparing the above-mentioned anti-Ro52 human recombinant monoclonal antibody, which involves gene expression of the above-mentioned nucleic acid; or culturing the above-mentioned transgenic cells.

[0020] Fifthly, the reagents or kits in the embodiments of this application include any of the above-described anti-Ro52 human recombinant monoclonal antibodies, or the above-described nucleic acids, or the above-described biological materials.

[0021] In one embodiment, the reagent or kit is used to detect the Ro52 antigen.

[0022] The advantages or beneficial effects of the above technical solutions include at least the following: The anti-Ro52 human recombinant monoclonal antibody of this application is derived from B cells in the blood of autoimmune patients. It is a completely human monoclonal antibody, with no species difference from the Ro52 human antigen, thus avoiding functional problems caused by species specificity and solving issues such as rejection reactions and weak affinity associated with heterologous expression. Furthermore, it specifically recognizes the Ro52 protein, exhibiting good sensitivity, anti-interference ability, and specificity, providing a raw material guarantee for the downstream development of high-quality diagnostic reagents.

[0023] The above overview 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 this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0024] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0025] Figure 1 SDS-PAGE image of Ro52 protein; Figure 2 Electrophoresis diagram of variable region gene amplification in a single B cell. Detailed Implementation

[0026] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0027] Example Materials: Mammalian cells; cell culture medium, transfection medium, imidazole; Ni-NTA packing material; 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 chromogenic solution, 2M sulfuric acid stop solution.

[0028] method: 1. Eukaryotic expression of Ro52 protein The Ro52 protein amino acid sequence was obtained from the Uniport database (UniPort ID: P19474). The full-length Ro52 protein gene sequence (encoding 1-475 amino acids) was selected as the expression gene. Codon optimization for the eukaryotic expression system was performed using Beijing Qingke Biotechnology Co., Ltd., and the gene was synthesized. The synthesized Ro52 protein gene fragment was cloned into a eukaryotic expression vector via Nco I and Xho I restriction enzyme sites. The target protein has a 10-1 amino acid sequence at its N-terminus. The HIS tag, with specific amino acid and gene sequences as shown in SEQ ID NO.21 and SEQ ID NO.22; SEQ ID NO.21: MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRK HRDHAMVPLEEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISELDRRCHS SALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGLKKMLRTCAVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFDSYPMVLGAQHFHSGKHYWEVDVTGKEAWDLG VCRDSVRRKGHFLLSSKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPLNIGSQGSTDY.

[0029] SEQ ID NO.22:

[0030] The Ro52 protein expression plasmid was transformed into competent E. coli cells, and single clones were picked and the plasmid amplified. After transfection with a certain volume of cell culture system, cells were collected by centrifugation, the culture medium was discarded, and the cells were resuspended in pre-cooled PBS. Cell lysis buffer was added, and cells were sonicated (output power 60%, working for 4 seconds, stopping for 7 seconds as one cycle, total time 20 min). The supernatant was collected by centrifugation at 12000g for 30 min and purified by affinity chromatography using Ni-NTA affinity resin. First, 1 ml of Ni-NTA was equilibrated with 10 ml of Native Binding Buffer (10 mM imidazole). Then, the cell supernatant was slowly loaded through Ni-NTA. Impurities bound to Ni-NTA were washed with 10 ml of Native Wash Buffer (containing imidazole from low to high concentrations). Finally, the target protein was eluted with 20 ml of Native Elution Buffer (high concentration imidazole) to obtain the target protein. The eluted target protein was concentrated by ultrafiltration to replace the PBS buffer, and then identified by SDS-PAGE. The electrophoresis image is shown below. Figure 1 The Ro52 protein has a molecular weight of approximately 52 kDa and a purity of >90%.

[0031] 2. Ro52 protein-coupled luciferin Biotin provides a simple and effective method for biotinylation of antibodies, proteins, and other macromolecules containing primary amines in solution. A 10 mM biotin solution was prepared by dissolving biotin in dimethyl sulfoxide (DMSO). First, biotin was conjugated to the Ro52 protein, followed by flow cytometry-based binding with streptomycin. An excess of biotin reagent was then added to label the Ro52 protein, and the mixture was incubated on ice for 2 hours. The labeled Ro52 protein was purified using a desalting column to remove unreacted biotin. Following a binding molar ratio of SA:biotin = 1:4, the biotinylated Ro52 protein was mixed with flow cytometry-based binding streptomycin and incubated on ice for 30 minutes before proceeding to the next cell labeling step.

[0032] 3. Flow cytometry sorting of single B cells Peripheral blood was collected from several patients with autoimmune diseases at the hospital, and PBMCs were obtained by separating them using human lymphocyte separation medium. 10 6 Cells / tubes were cryopreserved in liquid nitrogen for later use. After cryopreservation, PBMCs were rapidly thawed in a 37°C water bath. The thawed PBMCs were resuspended in FACS buffer (10mM PBS, 1mM EDTA, 2% FBS). 100 μL of the resuspended cells (1...) 10 6The fluorescent markers DAPI and IgM-BV241 were added sequentially and incubated on ice for 30 min. PBMCs were sorted using a BD FACSAria III flow cytometer, and cell populations that were double-positive for DAPI- / IgM- and Ro52 protein were selected, yielding 54 individual B cells specifically targeting Ro52 protein.

[0033] 4. Amplify the variable region genes of single B cells. The amplification primers and conditions followed the method described in the reference (Smith, K., Garman, L., Wrammert, J. et al. Rapid generation of fully human monoclonal antibodies specific to avaccinating antigen. Nat Protoc 4, 372-384 (2009). https: / / doi.org / 10.1038 / nprot.2009.3). First, RT-PCR was performed using a mixture of nine primers (see Table 1) designed to cover all possible variable region (V) genes. PCR enrichment of variable region genes amplified DNA to a level sufficient to obtain heavy and light chain variable gene sequences. Cloning PCR was performed using highly specific primers for each variable gene family, designed to integrate cloning restriction sites and frame the variable region heavy or light chain genes with the signal peptide sequences and constant region genes within their respective cloning vectors. Gene amplification results are shown below. Figure 2 As shown, the heavy chain is 44 / 88 (50%), the light chain is Kappa (47 / 88, 53%), and the light chain is Lambda (34 / 88, 39%).

[0034] Table 1

[0035] 5. Construction of antibody expression plasmids First, the gamma, kappa, or lambda cloning vectors were double-digested using restriction endonucleases Age I & Sal I, Age I & BsiWI, and Age I & Xho I, respectively. The cloning vector sequences are referenced in the literature (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). Digestion was performed at 37°C for 2 hours. The variable region gene products of the gamma, kappa, or lambda chains amplified in step 4 were purified and recovered using a standard DNA purification kit. The double-digested cloning vector samples were subjected to 1% agarose gel electrophoresis; the vector length was approximately 5700 bp. Homologous recombination of the variable region gene fragment and the vector fragment was performed using the ClonExpress II One Step Cloning Kit at 37°C for 30 min. The recombinant product was transformed into competent *E. coli* cells and evenly plated on plates containing Amp resistance, then incubated overnight at 37°C. The next day, single colonies from three plates were picked using a pipette tip and placed in 500 μL of LB medium containing Amp resistance, and incubated at 37°C for 4–6 h. Colony PCR was performed using 2 × TaqMaster Mix to confirm successful insertion of the variable region into the vector. Positive clones were sequenced to confirm the accuracy of the variable region sequence.

[0036] 6. Small-batch expression of antibodies Recombinant antibody plasmids were transfected into mammalian cells for low-level expression. 24 hours before transfection, 293T cells were seeded into 96-well plates to maintain 90% confluence. The complete culture medium in the 96-well plates was gently aspirated and replaced with serum-free medium. Separately, sterile 96-well plates were prepared. 30 ml of DMEM medium was added to 500 μL of transfection reagent and mixed thoroughly. This mixture was then added to each well at a rate of 300 μL / well. 10 ng each of light and heavy chain antibodies were added to the 96-well plates, and the plates were incubated at room temperature for 15 min. The mixture was then added to the corresponding wells. After culturing at 37°C and 5% CO2 for 3 days, the cell supernatant was analyzed by ELISA.

[0037] 7. Antibody ELISA specific screening Dilute Ro52 protein antigen to a specific concentration with phosphate-coated buffer, add 100 μL to each well of an ELISA plate, and incubate at 37°C for 2 hours. Discard the antigen from the plate, gently tap the plate, 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 a specific concentration of secondary antibody (diluted to a specific 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 2M sulfuric acid stop solution to each well to stop the chromogenic reaction. Place the ELISA plate on a microplate reader and measure the absorbance at 450 nm, recording the OD value. The results showed that 23 positive antibodies with OD>2 were screened out from 88 samples. The results are shown in Table 2.

[0038] Table 2 Results of Ro52 protein ELISA specificity screening

[0039] 8. Specific antibody expression The selected specific antibodies were expressed on a small scale using mammalian cells. Cells were passaged one day before transfection, and on the day of transfection, cell viability was required to be >90%, and cell density to be 3-4 cells / day. 10 6 cells / mL. Adjust the cell density to 1.0 at transfection time. 10 6 Cells / mL, using transfection reagent and 80 μg plasmid DNA (H:L=1:1), incubated at room temperature for 10 min to form a cationic polymer, which was then slowly added to the cells. Each antibody transfected 80 mL of cells. After 6 days, the cell supernatant was collected for purification. The cell supernatant after expression was purified using AT Protein A. The cells were washed with 10 mL of equilibration buffer, followed by antibody elution with 5 mL of 0.1 M glycine. The eluted antibody solution was immediately neutralized with 500 μL buffer and dialyzed against PBS at 4 °C. The dialysate was changed every 2 h, and dialyzed 3 times. The purity and concentration of the antibody were identified by SDS-PAGE and BCA assay.

[0040] The monoclonal antibody 5E1 was sequenced, and the heavy chain variable region sequence is shown in SEQ ID NO.7, and the light chain variable region sequence is shown in SEQ ID NO.8. SEQ ID NO.7: EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMGWFRQAPGKEREFVAAISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDHGYFDYWGQGTQVTVSS.

[0041] SEQ ID NO.8: DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKVEIK.

[0042] It includes variable regions for light chains and variable regions for heavy chains; VH variable region complementarity determining region: CDR1: Amino acid sequence SYAMG (SEQ ID NO. 1); Nucleotide sequence agctatgcgatgggctaa (SEQ ID NO. 13); CDR2: Amino acid sequence AISYDGSNKYYADSVKG (SEQ ID NO. 2); Nucleotide sequence gcgattagctatgatggcagcaacaaatattatgcggatagcgtgaaaggctaa (SEQ ID NO. 14); CDR3: Amino acid sequence DHGYFDY (SEQ ID NO. 3); Nucleotide sequence: gatcatggctattttgattat (SEQ ID NO. 15); VK variable region complementarity determining region: CDR1: Amino acid sequence RASQDISNYLN (SEQ ID NO. 4); Nucleotide sequence cgcgcgagccaggatattagcaactatctgaactaa (SEQ ID NO. 16); CDR2: Amino acid sequence YTSSLHS (SEQ ID NO. 5); Nucleotide sequence tataccagcagcctgcatagctaa (SEQ ID NO. 17); CDR3: Amino acid sequence QQYNSYPYT (SEQ ID NO. 6); Nucleotide sequence cagcagtataacagctatccgtatacctaa (SEQ ID NO. 18).

[0043] 9. Monoclonal antibody performance testing Specificity assay: The monoclonal antibody 5E1 obtained in the previous screening step was prepared to a concentration of 1 μg / mL using 5% skim milk. Ro52 antigen, Jo-1 antigen, CENPB antigen, P0 antigen, and Scl-70 antigen were diluted to 1 μg / mL solutions using 10 mM PBS buffer. 100 μL of each antigen was added to each well of a 96-well plate, with three replicates per well. The plates were incubated at 37°C for 1 hour. The antigen solution was then discarded, the plates were gently tapped, and 200 μL of 5% skim milk was added for blocking at 37°C for 1 hour. The plates were then washed twice with PBST for 3 minutes each time. Add 100 μL of 5E1 monoclonal antibody solution and incubate at 37°C for 1 h. Wash the plate three times with PBST for 3 min each time. Add mouse anti-human HRP-labeled IgG secondary antibody (1:50000 diluted in 5% skim milk) and incubate at 37°C for 40 min. Wash the plate four times with PBST for 3 min each time. Add 100 μL of TMB chromogenic solution to each well and react in the dark for 10 min. Add 50 μL of 2M sulfuric acid stop solution to each well and immediately measure the absorbance at 450 nm using 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.

[0044] Table 3. Specificity detection of anti-Ro52 antibody

[0045] As can be seen from Table 3, the Ro52 antibody only reacts with the wells coated with Ro52 antigen, exhibiting a high absorbance value, while showing no significant reaction with other antibodies, indicating that the prepared Ro52 monoclonal antibody has good specificity.

[0046] Table 4 Sensitivity detection of anti-Ro52 antibody

[0047] As shown in Table 4, the Ro52 antibody still showed absorbance values ​​when reacting with the antigen at a concentration of 0.000001 μg / mL, indicating that the antibody has high sensitivity.

[0048] Table 5. Stability test of anti-Ro52 antibody

[0049] As shown in Table 5, the Ro52 antibody specifically recognized the Ro52 antigen after 1, 3, and 7 days of accelerated treatment at 37°C, without recognizing other antigens. It also specifically recognized the Ro52 antigen after four freeze-thaw cycles at -80°C and room temperature, without recognizing other antigens. This demonstrates excellent stability.

[0050] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0051] 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 indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0052] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A human recombinant monoclonal antibody against Ro52, characterized in that, It includes variable regions for light chains and variable regions for heavy chains; The variable region of the heavy chain includes complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3; the variable region of the light chain 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 1-4.

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

12.

7. A biomaterial, characterized in that, The biomaterial comprises the nucleic acid as described in claim 5 or 6; the biomaterial is an expression cassette, vector, or transgenic cell.

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

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

10. A reagent kit, characterized in that, The kit comprises the anti-Ro52 human recombinant monoclonal antibody according to any one of claims 1-4, 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 the Ro52 antigen.

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