Monoclonal antibody for resisting porcine CD82 protein, hybridoma cell strain and application

Abstract

The invention relates to the technical field of biology, in particular to an anti-porcine CD82 protein monoclonal antibody, a hybridoma cell strain and application, the monoclonal antibody is prepared by the following steps: expressing and purifying a CD82 (EC2) region by utilizing a prokaryotic expression system, preparing by taking the purified recombinant protein as an immunogen, and carrying out indirect ELISA (enzyme-linked immuno sorbent assay) and Flow Cytometry screening to obtain the monoclonal cell strain capable of secreting an IgG2a type antibody. The purified antibody is detected through an indirect ELISA method, sensitive reaction still exists when the antibody concentration reaches 31.25 ng / ml, PAM cells capable of expressing endogenous CD82 protein are detected through flow cytometry, and the result shows that the mAb has good marking capacity for the PAM cells, indicating that the mAb of the CD82 protein capable of being used for flow cytometry is successfully prepared. A biological tool is provided for further analyzing the function and mechanism of the CD82 protein.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a monoclonal antibody against porcine CD82 protein, a hybridoma cell line and applications thereof. Background Art

[0002] CD82 protein (KAI1) belongs to the tetraspanin family. Its four transmembrane domains endow it with unique biological functions. It is widely expressed in various normal tissue cells and shows a certain degree of homology. This protein is involved in various pathological and physiological reactions and has multiple biological functions. In recent years, the role of CD82 protein in immune regulation and antiviral responses has attracted much attention. It not only plays an important role in cell migration, adhesion and proliferation, but also participates in the maintenance of immune homeostasis and the regulation of antiviral immune responses by regulating the activation, migration and functions of immune cells. For example, CD82 protein can inhibit the over-activation of T cells, prevent the occurrence of autoimmune diseases, and promote the maturation and antigen presentation function of dendritic cells, enhancing antigen-specific immune responses. In addition, CD82 protein plays an important role in immunity and antiviral processes. Its mechanism involves the regulation of the activation, migration and functions of immune cells, as well as the inhibition of the migration, invasion and replication of virus-infected cells. Therefore, in-depth study of the functions and mechanisms of CD82 protein is of great significance for the development of new immunotherapy strategies and antiviral drugs. However, there is currently no commercial flow antibody that specifically binds to porcine CD82. Therefore, the preparation of monoclonal antibodies against porcine CD82 protein has important scientific significance and application value in porcine disease research.

[0003] The tetraspanin family consists of four highly hydrophobic and conserved transmembrane domains, TM1, TM2, TM3 and TM4. TM3 and TM4 are connected by a large extracellular loop (EC2), which mediates specific protein-protein interactions and is the main functional region of the tetraspanin family. Existing studies have shown that a 20-mer peptide derived from the large extracellular loop of CD82 has been proven to have anti-angiogenic properties and can block the formation of retinal neovascularization and the progression of breast cancer.

[0004] The present invention uses a prokaryotic expression system to express and purify the CD82 (EC2) region, and uses the purified recombinant protein as an immunogen to prepare monoclonal antibodies. After screening by indirect ELISA and Flow Cytometry, a monoclonal cell line 11G3 that can secrete IgG2a-type antibodies is finally obtained. The purified antibody is detected by the indirect ELISA method, and the results show that there is still a sensitive reaction when the antibody concentration reaches 31.25 ng / ml. The PAM cells that can express endogenous CD82 protein are detected by flow cytometry, and the results show that the mAb has good labeling ability for PAM cells. It can more deeply reveal the function of CD82 protein in pigs participating in immune regulation and antiviral responses, providing a theoretical basis and experimental foundation for the development of new immunotherapy strategies and antiviral drugs. Summary of the Invention

[0005] The primary object of the present invention is to provide a monoclonal antibody against porcine CD82 protein. The monoclonal antibody includes a heavy chain constant region, a light chain constant region, a heavy chain variable region, and a light chain variable region. The CDRs of the heavy chain variable region of the monoclonal antibody include CDR-H1 with the amino acid sequence shown in SEQ ID No.1, CDR-H2 with the amino acid sequence shown in SEQ ID No.2, and CDR-H3 with the amino acid sequence shown in SEQ ID No.3; the CDRs of the light chain variable region of the monoclonal antibody include CDR-L1 with the amino acid sequence shown in SEQ ID No.4, CDR-L2 with the amino acid sequence shown in SEQ ID No.5, and CDR-L3 with the amino acid sequence shown in SEQ ID No.6.

[0006] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No.9.

[0007] The second object of the present invention is to provide a gene sequence encoding the monoclonal antibody. The gene sequence encoding the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID No.8, and the gene sequence encoding the light chain variable region of the monoclonal antibody is as shown in SEQ ID No.10.

[0008] The third object of the present invention is to provide an expression cassette, an expression vector, and a recombinant bacterium containing the gene fragment.

[0009] The fourth object of the present invention is to provide a hybridoma cell line capable of secreting the monoclonal antibody.

[0010] Preferably, the monoclonal antibody is of the IgG2a type.

[0011] The fifth object of the present invention is to provide the use of the monoclonal antibody in specifically recognizing porcine CD82 protein.

[0012] The sixth object of the present invention is to provide the use of the monoclonal antibody in preparing a flow cytometry detection reagent.

[0013] The seventh object of the present invention is to provide a flow cytometry detection reagent comprising the monoclonal antibody described in the claims.

[0014] The beneficial effects of the present invention are as follows: (1) The present invention provides a monoclonal antibody against porcine CD82 protein. The monoclonal antibody is expressed and purified using a prokaryotic expression system for the CD82 (EC2) region, and the purified recombinant protein is used as an immunogen for preparation. After screening by indirect ELISA and Flow Cytometry, a monoclonal cell line 11G3 secreting IgG2a-type antibody is finally obtained.

[0015] (2) The purified antibody is detected by the indirect ELISA method. The results show that there is still a sensitive reaction when the antibody concentration reaches 31.25 ng / ml. The PAM cells capable of expressing endogenous CD82 protein are detected by flow cytometry. The results show that the mAb has good labeling ability for PAM cells, indicating that the CD82 protein can be successfully used for the preparation of mAb for flow cytometry, providing a biological tool for further analyzing the function and mechanism of CD82 protein. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained according to the provided drawings.

[0017] Figure 1 PCR amplification of CD82 gene (A), PCR identification of recombinant bacterial liquid (B), and restriction enzyme digestion identification of recombinant plasmid (C) Note: M: DNA Marker; 1-2: Gel electrophoresis of PCR amplification products of target gene and negative control; 3-4: Gel electrophoresis of PCR amplification products of positive bacterial liquid; 5: Gel electrophoresis of PCR amplification products of negative bacterial liquid; 6: Double digestion of pER-28a-CD82 plasmid; 7: pER-28a-CD82 plasmid

[0018] Figure 2 Prokaryotic expression (A, B) and purification effect (C) of CD82 recombinant protein

[0019] Note: M: Protein Marker; 1: Uninduced whole bacteria of pET-28a-CD82; 2: Induced whole bacteria of pET-28a-CD82; 3-4: Precipitate and supernatant after lysis of induced whole bacteria of pET-28a-CD82; 5: Purified CD82 recombinant protein.

[0020] Figure 3 Screening of serum titer (A) of immunized mice and positive cell lines of monoclonal antibodies (B)

[0021] Note: In Figure B, a: Negative control; b-h: Supernatant groups of cell lines 2F6, 4H12, 7B5, 8H7, 9A3, 9G5, and 11G3 respectively.

[0022] Figure 4 Results of RNA electrophoresis Note: M: Marker (5ul, 0.1mg / mL), molecular weights of Marker are 2000 / 1000 / 750 / 500 / 250 / 100bp; 1: 11G32D8 Figure 5 Results of cDNA electrophoresis Note: M: Marker (5ul, 0.1mg / mL), molecular weights of Marker are 2000 / 1000 / 750 / 500 / 250 / 100bp, 1: 11G32D8 cDNA-VH; 2: 11G32D8 cDNA-VL

[0023] Figure 6 Detection of monoclonal antibody subtypes

[0024] Figure 7 Purification (A), titer (B) and flow cytometry verification (C) of monoclonal antibodies

[0025] Note: In Figure A, M: Protein Marker; 1: Monoclonal antibody purification group;

[0026] In Figure C, a: Negative control; b: Monoclonal antibody group Detailed implementation manners

[0027] To make the objectives, technical solutions and advantages of the present invention clearer, the following will elaborate on each embodiment of the present invention with reference to the accompanying drawings. However, those of ordinary skill in the art can understand that in each embodiment of the present invention, many technical details are provided to help readers better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical solutions claimed in the present application can still be implemented.

[0028] Terms:

[0029] The term "loss of protein function" refers to the loss of the function of the protein encoded by a gene through knockout, mutation of the gene encoding the protein, or insertion of a partial gene into the gene fragment encoding the protein, resulting in a frameshift mutation in the protein encoded by the gene.

[0030] The term "gene silencing" refers to the phenomenon of low or no expression of a gene without damaging the original DNA, which mainly includes two aspects. One is transcriptional gene silencing caused by DNA methylation, heterochromatinization, and position effects, etc. The other is post-transcriptional gene silencing, that is, the inactivation of a gene by specifically inhibiting the target RNA at the post-transcriptional level of the gene, including antisense RNA, co-suppression, gene silencing, RNA interference, and translation inhibition mediated by microRNA, etc.

[0031] In this experimental study, the reagents and consumables used, unless otherwise specified, can be purchased commercially.

[0032] The following experiments are all standard molecular biology, cell biology, or virology operation methods, which can be easily understood and operated by researchers in this research field.

[0033] In the following examples, the porcine alveolar macrophage cells (PAM) and SP2 / 0 myeloma cell line are preserved by the Lanzhou Veterinary Research Institute; the PET-28a(+) plasmid is preserved by this laboratory; TOP10 cells are purchased from Beijing Kangrun Company; SPF-grade female BALB / C mice are uniformly purchased by the Lanzhou Veterinary Research Institute.

[0034] In the following examples, the DNA polymerase is purchased from Life technology Company; restriction endonucleases, DNA ligase, Protein marker, goat anti-mouse PE-fluorescent labeled IgG(H+L) are all purchased from Thermo Company; DNA Marker is purchased from TaKaRa Company; goat anti-mouse IgG(H+L)-HRP antibody is purchased from Jackson Company; PEG is purchased from MERCK Company; Tiangen DNA recovery kit is purchased from Gansu Comos Biotechnology Co., Ltd.; Yaenzyme PAGE rapid gel kit and Yaenzyme ECL ultrasensitive chemiluminescence detection kit are both purchased from Gansu Yishu Biotechnology Co., Ltd.; Omega plasmid extraction kit is purchased from Gansu Ruide Biotechnology Co., Ltd.; affinity chromatography column material is purchased from Huiyan Biology; BSA is purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0035] Flow Cytometry has a wide range of applications in biological research. Its advantages include multi-parameter analysis, rapid quantification, high sensitivity, and the ability to analyze at the single-cell level. Flow Cytometry is particularly important in immunological research and can be used to detect the phenotypes, functional states, and activation of intracellular signaling pathways of immune cells. In addition, Flow Cytometry also has extensive applications in antiviral research, such as detecting markers of virus-infected cells, virus replication levels, and dynamic changes in the host immune response.

[0036] Example 1: Preparation and Identification of Monoclonal Antibodies

[0037] 1 Materials and Methods

[0038] 1.1 Construction and Identification of pET-28a-CD82 Truncated Recombinant Plasmid

[0039] Download the sequence of Gene ID: 100270689 in GenBank. The amino acid sequence of the CD82 protein is shown as SEQ ID No. 11, and the nucleotide sequence is shown as SEQ ID No. 12. According to the prediction results of transmembrane proteins in Uniprot ID: B7ZEQ3, the EC2 structure (109-226aa) sequence of the CD82 protein was optimized for E. coli codon preference and primers were designed. F: GTGGACAGCAAATGGGTCGC GGATCC ATGAATATGGGTAAGCTGAAGCAGGAAATGGGT (the underlined part is the BamH I restriction site) (SEQ ID No. 13), R: CAGTGGTGGTGGTGGTGGTG CTCGAG TTAATTTTCCTGCAGCCATGCCTGCACTTTTTC (the underlined part is Xho I) (SEQ ID No. 14), and the length of the amplification product is 412bp.

[0040] The CD82 protein (109-226aa) sequence was synthesized by GenScript Biotech Corporation as a template for PCR amplification. It was purified by agarose gel electrophoresis, and the target band was recovered from the gel. The PCR product recovered from the gel and the pET-28a(+) vector were double-digested with BamH I and Xho I enzymes and then ligated with DNA ligase. Subsequently, the ligation product was transformed into TOP10 cells. The colony PCR product was verified by agarose gel electrophoresis. The plasmid was extracted from the positive bacterial solution and then detected by double digestion. The positive plasmid with correct sequencing results was named pET-28a-CD82 and stored in a -20°C refrigerator.

[0041] 1.2 Expression and Purification of CD82 Protein

[0042] The positive colonies with correct plasmid sequencing were expanded in culture, IPTG was added to induce protein expression, purification was carried out using a Ni-NTA affinity chromatography column, and SDS-PAGE analysis was performed.

[0043] 1.3 Animal immunization and preparation of monoclonal antibodies

[0044] Six 6-week-old SPF-grade female BALB / c mice were selected for primary immunization. The purified pET-28a-CD82 recombinant protein was mixed with Freund's complete adjuvant at a volume ratio of 1:1, emulsified, and 50 μg of the protein was injected subcutaneously at multiple points in each mouse. The second to fourth immunizations were carried out using Freund's incomplete adjuvant in the same method, with an interval of 14 days between each immunization. Fourteen days after the fourth immunization, the blood of the mice was collected, the serum was separated by centrifugation, and the antibody titer in the serum was detected by the indirect ELISA method. Three days before cell fusion, the BALB / c mouse with the highest antibody titer was intraperitoneally injected with 50 μg of purified protein (without adjuvant) for booster immunization. Three days later, single spleen cells were taken and immediately fused with SP2 / 0 cells using PEG. Positive clone screening of the fused cells was carried out by ELISA and Flow Cytometry. Finally, a cell line that could stably secrete CD82 mAb was obtained, and it was expanded in culture and cryopreserved.

[0045] Ten-week-old SPF-grade female BALB / c mice were selected and intraperitoneally injected with 500 μl of Freund's incomplete adjuvant. 1×10 6 11G3 hybridoma cells were injected into the peritoneal cavity of the mice. Seven days later, the ascites of the mice was collected. The collected ascites was purified by a ProteinG affinity chromatography column, and then SDS-PAGE analysis was performed to verify the purity of the antibody. The purified antibody was aliquoted and stored at -80 °C.

[0046] 1.4 Sequencing of the antibody

[0047] Sample pretreatment: After culturing the cell line, it was pipetted and centrifuged, the supernatant was discarded, and then it was washed twice with sterilized 1*PBS and centrifuged to discard the supernatant PBS;

[0048] Sample lysis: 1 ml of Trizol was added to the centrifuged cells and pipetted and mixed well;

[0049] RNA isolation (15 min): Chloroform was added at a ratio of 0.2 ml per 1 ml of Trizol, shaken vigorously for 15 seconds, and left standing at room temperature until obvious stratification occurred in the EP tube. Then, it was centrifuged at 4 °C, 12,000 rpm for 15 min;

[0050] RNA precipitation (30 min): Transfer the upper water sample layer to a new EP tube, add isopropanol with the same volume as the supernatant, mix well, insert the EP tube into ice for 30 min to precipitate RNA, then centrifuge at 4°C, 12,000 rpm for 10 min

[0051] 30 min;

[0052] RNA washing (5 min): Discard the supernatant, add 1 ml of 75% ethanol to wash the RNA precipitate, centrifuge at 4°C, 12,000 rpm for 5 min, then discard the supernatant and retain the precipitate;

[0053] RNA dissolution: Add 50 μl of double-distilled water to the RNA precipitate;

[0054] RNA gel electrophoresis (30 min): Identify the purity and quantify by agarose gel electrophoresis;

[0055] RNA denaturation (20 min): Take 15 μl of the dissolved RNA according to the reverse transcription system (total system 20 μl) and dispense it into PCR tubes respectively, then place the PCR tubes in a PCR instrument at 65°C for 5 min for denaturation;

[0056] Reverse transcription to prepare single-stranded cDNA (20 min): Take 6 μl from the prepared reverse transcription reaction system and add it to the PCR tube with RNA denaturation, set the reverse transcription reaction time of the PCR instrument, 37°C for 15 min, 98°C for 5 min; Double-stranded cDNA amplification (2 h) Prepare the total PCR reaction system, use the mouse IgG VH VL primer library designed by our company to amplify the above cDNA respectively, and perform PCR reaction according to the following conditions, 98°C for 5 min for pre-denaturation; 94°C for 30 s for denaturation; 54°C for 30 s for annealing; 72°C for 2 min for extension; After 30 cycles, extend at 72°C for 6 min

[0057] Quality control of double-stranded cDNA: Perform agarose gel electrophoresis quality control on the above PCR products;

[0058] Table 1 Restriction enzyme digestion conditions of vector and target gene

[0059]

[0060] Table 2 Ligation conditions of vector and target gene

[0061]

[0062] Transformation:

[0063] 1) Add the DNA fragment to be transformed into the tube containing TOP10 competent cells (25 ng of DNA is required for 50 μl of competent cells), and the volume should not exceed 5% of the competent cells. Gently rotate the tube several times to mix the contents evenly, and incubate on ice for 30 min;

[0064] 2) Place the centrifuge tube mixture in a circulating water bath heated to 42 °C and perform heat shock for 90 s without shaking the tube;

[0065] 3) Quickly transfer the tube to an ice bath to cool the cells for 1 - 2 min;

[0066] 4) Add 200 μl of SOC liquid medium to each tube, warm the medium to 37 °C using a water bath, then transfer the tubes to a shaker set at 37 °C and culture at 220 rpm for 45 min

[0067] 5) Transfer an appropriate volume of the transformed competent cells to an LB medium containing the corresponding antibiotic

[0068] 6) Invert the plate and incubate at 37 °C. Colonies can appear after 12 - 16 hours

[0069] Colony PCR verification and sequencing:

[0070] After colonies grow on the plate, randomly pick several colonies for colony PCR verification. After detecting the transformants, send them to the company's sequencing platform for sequencing verification. The sequencing primer is M13 - F: GTAAAACGACGGCCAG (SEQ ID No.15).

[0071] 1.5 Determination of the titer of immune mouse serum

[0072] The titer of the monoclonal antibody was detected by the indirect ELISA method. The enzyme - linked immunosorbent assay (ELISA) plate was coated with 1 μg / ml of pET - 28a - CD82 purified protein and serially diluted 1:1000. The immune mouse serum was diluted to 1:128000 as the primary antibody, and HRP - labeled goat anti - mouse IgG (H + L) was used as the secondary antibody to detect the OD value at 450 nm.

[0073] 1.6 Flow cytometry detection of monoclonal antibody

[0074] Use 200 μl of PBS (containing 0.1% BSA) for 1×10 5Resuspend the PAM cells, incubate them with 15 μg / ml mAb as the primary antibody at 4°C for 1 h, wash the cells 3 times with 1 ml PBS (containing 0.1% BSA), then resuspend the cells with 200 μl PBS (containing 0.1% BSA), incubate them with 3 μl PE-fluorescently labeled goat anti-mouse IgG (H+L) antibody as the secondary antibody at 4°C in the dark for 1 h, wash them 3 times again with 1 ml PBS (containing 0.1% BSA) and resuspend with 400 μl for flow cytometry detection.

[0075] 1.7 Determination of the titer of monoclonal antibody

[0076] The method is the same as the determination of serum titer. Dilute the mAb serially at 1 μg / ml to 00.977 ng / ml as the primary antibody and detect the OD value at 450 nm.

[0077] 1.8 Identification of the subtype of monoclonal antibody

[0078] Collect the supernatant of the expanded monoclonal cells and use the mAb subclass identification kit for subclass identification.

[0079] 2 Results

[0080] 2.1 Construction and identification of the pET-28a-CD82 recombinant expression vector

[0081] PCR amplify the porcine CD82 gene, analyze its product by 1% agarose gel electrophoresis, and the result shows that the target band of 412 bp is successfully amplified ( Figure 1 A). Digest the PCR product and pET-28a(+) with BamH I and Xho I double enzymes respectively, then ligate them with DNA ligase to construct the pET-28a-CD82 recombinant expression plasmid. Perform PCR amplification on the bacterial solution, and the result shows that the target band of 412 bp is amplified ( Figure 1 B). Extract its plasmid and perform double digestion with BamH I and Xho I, and also perform electrophoresis detection on the digestion products. The target band is about 372 bp ( Figure 1 C), which is consistent with the expected result. The sequencing result shows that the sequence of the inserted fragment is completely consistent with the CD82 target gene sequence, and the recombinant expression vector is successfully constructed.

[0082] 2.2 Expression and purification of CD82 recombinant protein

[0083] Centrifuge the recombinant bacterial solution induced at 16°C and 140 r / min for 16 h, perform SDS-PAGE analysis after ultrasonic disruption. The result shows that the target band of 17.09 kDa size appears significantly in the induced recombinant plasmid ( Figure 2A), which is consistent with the expectation. In addition, the CD82 recombinant protein was mainly expressed in the form of inclusion bodies. The inclusion bodies were dissolved and refolded, and then purified by Ni-NTA affinity chromatography column. After purification, SDS-PAGE analysis showed that the purification effect of the CD82 recombinant protein was good( Figure 2 B, C).

[0084] 2.3 Screening of CD82 protein monoclonal antibody cell lines

[0085] Blood was collected 14 days after the third immunization of mice. By indirect ELISA determination, the serum antibody titers of 6 immunized mice all reached 1:128000( Figure 3 A). The mouse with the highest antibody titer was selected for cell fusion, and 7 subcloned cells were selected. The supernatant was detected by Flow Cytometry, and 3 hybridoma cell lines that could stably secrete CD82 monoclonal antibody were obtained( Figure 3 B), which were named 2F6, 9G5, and 11G3 respectively. The cell line 11G3 with the best flow cytometry effect was selected for the preparation of monoclonal antibody.

[0086] 2.4 Determination of monoclonal antibody variable region sequence

[0087] According to the gel electrophoresis running results as Figure 4 and Figure 5 , it can be seen that the quality of hybridoma RNA extraction is qualified, and there are obvious bands at about 700 bp after amplification of heavy and light chains, which are consistent with the size of the target band.

[0088] Table 3 Monoclonal antibody variable region sequence

[0089]

[0090] Nucleic acid sequence of heavy chain variable region:

[0091] The amino acid sequence of the heavy chain variable region: CAGATCCAGTTGGTGCAGTCTGGACCTGAGTTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCGTCTGGATATACCTTCACAAAATATGGAATGAGCTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACATACACTGGAGAGCCAACATATACTAATGACTTCAAGGGACGGTTTGTCTTCTCTTTGGAAACCTCTGCCAGAACTGCCTATTTGCAGATCAACAGCCTCAAAAATGAGGACATGTCTACTTATTTCTGTGCAAGAGGGAGGTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID No.8)

[0092] QIQLVQSGPELKKPGETVKISCKASGYTFTKYGMSWVKQAPGKGLKWMGWINTYTG EPTYTNDFKGRFVFSLETSARTAYLQINSLKNEDMSTYFCARGRYWGQGTLVTVSA (SEQ ID No.7)

[0093] The nucleic acid sequence of the light chain variable region:

[0094] GACATTGTGATGACACAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAAGTGTTGATCTACGGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAAGCGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATCATAGTTACCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG (SEQ ID No.10)

[0095] The amino acid sequence of the light chain variable region:

[0096] DIVMTQSPSSLSVSAGEKVTMSCKSSQSLKNYLAWYQQKPGQPPKVLIYGASTRESGV PDRFTGSGSGSDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLELKR(SEQ ID No.9)

[0097] 2.5 Identification of mAb subtypes

[0098] The cell supernatants of 2F6, 9G5, and 11G3 were detected by ELISA using a kit. The results showed that the antibodies secreted by the 2F6 and 9G5 cell lines were of the IgG2b type, and the antibody secreted by the 11G3 cell line was of the IgG2a type( Figure 6 ).

[0099] 2.6 Preparation of monoclonal antibody and verification of antibody

[0100] The 11G3 cell line was injected into the abdominal cavity of mice to prepare ascites, which was purified using a Protein G column. SDS-PAGE analysis of the purified protein showed that the purification effect of the mAb was good( Figure 7 A). Using the mAb as the primary antibody, the titer of the mAb was detected by the ELISA method established in this experiment. The results showed that there was still a sensitive reaction when the mAb was diluted to 31.25 ng / ml( Figure 7 B). At the same time, using the mAb as the primary antibody, the antibody was verified according to the flow cytometry detection method established in this experiment. The results showed that the mAb had good labeling ability for PAM cells, indicating that the antibody had good effect.

[0101] In summary, the present invention provides a monoclonal antibody against porcine CD82 protein, a hybridoma cell line and its application. The monoclonal antibody is expressed and purified using a prokaryotic expression system against the CD82 (EC2) region. The purified recombinant protein is used as an immunogen for preparation. After screening by indirect ELISA and Flow Cytometry, a monoclonal cell line secreting IgG2a type antibody is obtained. The purified antibody is detected by the indirect ELISA method. There is still a sensitive reaction when the antibody concentration reaches 31.25 ng / ml. Flow cytometry is used to detect PAM cells that can express endogenous CD82 protein. The results show that the mAb has good labeling ability for PAM cells, indicating that the CD82 protein can be successfully used for the preparation of mAb in flow cytometry, providing a biological tool for further analyzing the function and mechanism of CD82 protein.

Claims

1. A monoclonal antibody against porcine CD82 protein, characterized in that: The monoclonal antibody comprises a heavy chain constant region and a light chain constant region, a heavy chain variable region and a light chain variable region, wherein the CDR of the heavy chain variable region of the monoclonal antibody comprises a CDR-H1 as shown in SEQ ID No.1, a CDR-H2 as shown in SEQ ID No.2 and a CDR-H3 as shown in SEQ ID No.3; the CDR of the light chain variable region of the monoclonal antibody comprises a CDR-L1 as shown in SEQ ID No.4, a CDR-L2 as shown in SEQ ID No.5 and a CDR-L3 as shown in SEQ ID No.

6.

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

9.

3. The gene sequence encoding the monoclonal antibody according to claim 2, characterized in that: The gene sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No.8, and the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

10.

4. An expression cassette, expression vector, or recombinant bacterium containing the gene sequence of claim 3.

5. A hybridoma cell line capable of secreting the monoclonal antibody according to claim 1 or 2.

6. The monoclonal antibody according to claim 1 or 2, characterized in that The monoclonal antibody is of IgG2a type.

7. Use of the monoclonal antibody according to claim 1 or 2 in specifically recognizing porcine CD82 protein.

8. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of flow cytometry detection reagents.

9. A flow cytometry detection reagent comprising the monoclonal antibody according to claim 1 or 2.

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