Monoclonal antibody against pig cd82 protein, hybridoma cell strain and application

Abstract

The present application relates to the technical field of biology, in particular to a kind of anti-pig CD82 protein monoclonal antibody, hybridoma cell strain and application, monoclonal antibody is expressed and purified using prokaryotic expression system to CD82 (EC2) region, the recombinant protein after purification is prepared as immunogen, is screened by indirect ELISA and Flow Cytometry, obtains a monoclonal cell strain of IgG2a type antibody secretion, the antibody after purification is detected by indirect ELISA method, antibody concentration is to 31.25ng / ml Still have sensitive response, the PAM cell capable of expressing endogenous CD82 protein is detected by flow cytometry, and the results show that mAb has good marking ability to PAM cell, it is explained that CD82 protein can be used for the preparation of mAb of flow cytometry, and the function and mechanism of CD82 protein are further analyzed, and biological tool is provided.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a monoclonal antibody against porcine CD82 protein, a hybridoma cell strain and application. BACKGROUND

[0002] CD82 protein (KAI1) belongs to the four-transmembrane protein family, and its four transmembrane domains endow it with unique biological functions. It is widely expressed in various normal tissue cells and shows certain homology. The 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 response 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 response by regulating the activation, migration and function of immune cells. For example, CD82 protein can inhibit the excessive activation of T cells, prevent the occurrence of autoimmune diseases, and promote the maturation and antigen-presenting function of dendritic cells, thereby enhancing antigen-specific immune response. In addition, CD82 protein plays an important role in immunity and antiviral, and its mechanism involves the activation, migration and function regulation of immune cells, as well as the inhibition of migration, invasion and replication of virus-infected cells. Therefore, in-depth study of the function and mechanism of CD82 protein is of great significance for the development of new immunotherapy strategies and antiviral drugs. However, there is no commercial flow cytometry antibody that specifically binds to porcine CD82, so the preparation of monoclonal antibodies against porcine CD82 protein has important scientific significance and application value in the study of pig diseases.

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

[0004] The application expresses and purifies CD82 (EC2) regions by using a prokaryotic expression system, and uses the purified recombinant protein as an immunogen to prepare a monoclonal antibody, and finally obtains a monoclonal cell strain 11G3 capable of secreting an IgG2a type antibody through indirect ELISA and Flow Cytometry screening. The purified antibody is detected by an indirect ELISA method, and the results show that the antibody has a sensitive reaction when the concentration is 31.25 ng / ml. The PAM cells capable of expressing endogenous CD82 protein are detected by flow cytometry, and the results show that the mAb has good labeling ability for PAM cells. The function of CD82 protein in immune regulation and antiviral response in pigs can be further revealed, and a theoretical basis and experimental basis are provided for developing new immunotherapy strategies and antiviral drugs. SUMMARY

[0005] The primary object of the application is to provide an anti-porcine CD82 protein monoclonal antibody, which comprises a heavy chain constant region and a light chain constant region, a heavy chain variable region and a light chain variable region, the CDR of the heavy chain variable region of the monoclonal antibody comprises CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of which are shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 respectively; the CDR of the light chain variable region of the monoclonal antibody comprises CDR-L1, CDR-L2 and CDR-L3, the amino acid sequences of which are shown in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 respectively.

[0006] Preferably, 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.

[0007] The second object of the application is to provide a gene sequence encoding the monoclonal antibody, 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.

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

[0009] The fourth object of the application is to provide a hybridoma cell strain capable of secreting the monoclonal antibody.

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

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

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

[0013] The seventh object of the present application is to provide a flow cytometry reagent comprising the monoclonal antibody.

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

[0015] (2) the purified antibody is detected by indirect ELISA method, the results show that the antibody concentration is 31.25 ng / ml, and there is still a sensitive reaction, and 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, which indicates that the CD82 protein can be used for the preparation of mAb for flow cytometry, and provides a biological tool for further analyzing the function and mechanism of CD82 protein. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creative labor.

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

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

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

[0020] Figure 3 Immune mouse serum titer (A) and screening of monoclonal antibody positive cell lines (B)

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

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

[0023] Figure 6 Monoclonal antibody subtype detection

[0024] Figure 7 Monoclonal antibody purification (A), titer (B) and flow verification (C)

[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 DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present application clearer, the embodiments of the present application will be described in detail below with reference to the accompanying drawings. However, those skilled in the art can understand that in the embodiments of the present application, many technical details are proposed in order to make the reader 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 by the present application can be implemented.

[0028] Terms:

[0029] The term "loss of protein function" refers to the loss of function of a protein by knocking out, mutating or inserting a partial gene in a gene segment encoding the protein, so that the gene encoding the protein is subjected to a frame shift mutation, resulting in the loss of function of the protein encoded by the gene.

[0030] The term "gene silencing" refers to the phenomenon of low expression or non-expression of a gene without damaging the original DNA, mainly including two aspects, one is the transcription level gene silencing caused by DNA methylation, heterochromatinization and position effect, etc.; the other is post-transcriptional gene silencing, i.e. inactivating a gene by specifically inhibiting the target RNA after transcription, including antisense RNA, co-suppression, gene suppression, RNA interference and microRNA-mediated translation inhibition, etc.

[0031] The reagents and consumables used in the present experimental study can be purchased from commercial channels, unless otherwise specified.

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

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

[0034] In the following examples, the DNA polymerase was purchased from Life technology company; the restriction endonuclease, DNA ligase, Protein marker, goat anti-mouse PE fluorescent labeled IgG(H+L) were all purchased from Thermo company; the DNA marker was purchased from TaKaRa company; the goat anti-mouse IgG(H+L)-HRP antibody was purchased from Jackson company; the PEG was purchased from MERCK company; the Tiangen DNA recovery kit was purchased from Gansu Komus Biotechnology Co., Ltd.; the Yezhen PAGE rapid gel reagent kit and Yezhen ECL ultra-sensitive chemiluminescence detection kit were both purchased from Gansu Yikeshu Biotechnology Co., Ltd.; the Omega plasmid extraction kit was purchased from Gansu Ruide Biotechnology Co., Ltd.; the affinity chromatography column material was purchased from Huiran Biotechnology; the BSA was 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 single-cell level analysis capabilities. Flow cytometry is particularly important in immunology research, and can be used to detect the phenotype, functional status, and activation of intracellular signaling pathways of immune cells. In addition, flow cytometry has a wide range of applications in antiviral research, such as detecting markers of virus-infected cells, virus replication levels, and dynamic changes in host immune responses.

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

[0040] The CD82 protein (109-226 aa) sequence was synthesized by Jinweizhi Biotechnology Co., Ltd. as a template for PCR amplification, purified by agarose gel electrophoresis, and the target band was recovered. The recovered PCR product and pET-28a(+) vector were double-digested with BamH I and Xho I, and then ligated with DNA ligase. The ligation product was then transformed into TOP10 cells, and the colony PCR product was verified by agarose gel electrophoresis. The positive bacterial solution was extracted to obtain the plasmid, which was then double-digested for detection. The positive plasmid was sequenced by Jinweizhi Biotechnology Co., Ltd., and the positive plasmid with correct sequencing results was named pET-28a-CD82 and stored at -20°C.

[0041] 1.2 Expression and purification of CD82 protein

[0042] The positive colonies with correct plasmid sequencing were expanded, and the protein expression was induced by IPTG. The protein was purified by Ni-NTA affinity chromatography column and analyzed by SDS-PAGE.

[0043] 1.3 Preparation of monoclonal antibody

[0044] Six 6-week-old SPF 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 then injected subcutaneously into each mouse at multiple sites with 50 μg of protein. The same method was used for the second to fourth immunizations using Freund's incomplete adjuvant, with an interval of 14 days between each immunization. Fourteen days after the fourth immunization, the mouse blood was collected, and the serum was separated by centrifugation. The antibody titer in the serum was detected by indirect ELISA. Three days before cell fusion, the BALB / c mouse with the highest antibody titer was injected intraperitoneally 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. ELISA and Flow Cytometry were used to screen positive clones of the fused cells. Finally, a cell strain that could stably secrete CD82 mAb was obtained, and it was expanded and cryopreserved.

[0045] Ten-week-old SPF female BALB / c mice were selected and injected intraperitoneally with 500 μl of Freund's incomplete adjuvant. 1 x 10 6 The 11G3 hybridoma cells were injected into the mouse abdominal cavity, and 7 days later, the mouse ascites were collected. The collected ascites were purified by Protein G affinity chromatography column, and then analyzed by SDS-PAGE to verify the purity of the antibody. The purified antibody was divided and stored at -80°C.

[0046] 1.4 Sequence determination of antibody

[0047] Sample pretreatment: After blowing and centrifuging the cultured cell strain, the supernatant was discarded, then washed twice with sterilized 1*PBS, and the supernatant PBS was discarded after centrifugation;

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

[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 then allowed to stand at room temperature until a clear layer appeared in the EP tube. Centrifugation was performed at 4°C, 12000 rpm, for 15 min;

[0050] RNA precipitation (30 min): The supernatant was removed and isopropanol was added in an equal volume to the supernatant. After mixing well, the EP tube was inserted into ice for 30 min to precipitate the RNA. Then, the tube was centrifuged at 12000 rpm for 10 min at 4°C

[0051] 30 min;

[0052] RNA washing (5 min): The supernatant was removed and the RNA precipitate was washed with 1 ml of 75% ethanol. After centrifugation at 12000 rpm for 5 min at 4°C, the supernatant was removed and the precipitate was retained;

[0053] RNA dissolution: 50 ul of double distilled water was added to the RNA precipitate;

[0054] RNA gel running (30 min): Agarose gel electrophoresis was used to identify the purity and quantity of the RNA;

[0055] RNA denaturation (20 min): 15 ul of the dissolved RNA was aliquoted into PCR tubes according to the reverse transcription system (total system 20 ul). The PCR tubes were placed in a PCR instrument at 65°C for 5 min for denaturation;

[0056] Reverse transcription to prepare single-stranded cDNA (20 min): 6 ul of the reverse transcription reaction system was added to the denatured PCR tube. The reverse transcription reaction time was set as follows: 37°C for 15 min, 98°C for 5 min. Double-stranded cDNA amplification (2 h): The total PCR reaction system was prepared. The mouse IgG VH VL primer library designed by the company was used to amplify the above cDNA 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; 30 cycles, then 72°C for 6 min for extension

[0057] Double-stranded cDNA quality control: The PCR product was subjected to agarose gel electrophoresis quality control;

[0058] Table 1 Enzyme digestion conditions of vectors and target genes

[0059]

[0060] Table 2 Connection conditions of vectors and target genes

[0061]

[0062] Transformation:

[0063] 1) Add the DNA fragment to be transformed into the tube containing the TOP10 competent cells (25 ng DNA for 50 μl competent cells), the volume should not exceed 5% of the competent cells, mix the contents gently by rotating several times, and then place the tube in ice bath for 30 min;

[0064] 2) Place the centrifuge tube mixture into the circulating water heated to 42°C, heat shock for 90 s, and do not shake the tube;

[0065] 3) Quickly transfer the tube to the 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, and then transfer the tube to a shaker set at 37°C, and incubate at 220 rpm for 45 min

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

[0068] 6) Invert the plate and incubate at 37°C, and bacterial colonies will appear after 12-16 hours

[0069] Colony PCR verification and sequencing:

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

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

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

[0073] 1.6 Flow detection of the monoclonal antibody

[0074] Use 200 ul PBS (containing 0.1% BSA) to dilute 1 x 10 5PAM cells were resuspended, incubated with 15 ug / ml mAb as primary antibody at 4°C for 1 h, washed with 1 ml PBS (containing 0.1% BSA) for 3 times, resuspended with 200 ul PBS (containing 0.1% BSA), incubated with 3 ul PE fluorescent labeled goat anti-mouse IgG (H+L) antibody as secondary antibody at 4°C for 1 h, washed with 1 ml PBS (containing 0.1% BSA) for 3 times again and resuspended with 400 ul for detection by flow cytometer.

[0075] 1.7 Determination of the titer of monoclonal antibody

[0076] The method was the same as the determination of the titer of serum. The mAb was diluted by 1 ug / ml to 0.977 ng / ml as primary antibody, and the OD value at 450 nm was detected.

[0077] 1.8 Identification of the subtype of monoclonal antibody

[0078] The supernatant of the monoclonal cells in large-scale culture was collected, and the mAb subtype identification kit was used for subtype identification.

[0079] 2 Results

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

[0081] The pig CD82 gene was amplified by PCR, and the product was analyzed by 1% agarose gel electrophoresis. The results showed that the target band of 412 bp was successfully amplified ( Figure 1 A). The PCR product was treated with BamH I and Xho I double enzyme digestion, followed by ligation with DNA ligase to construct the pET-28a-CD82 recombinant expression plasmid. PCR amplification of the bacterial solution showed that the target band of 412 bp was amplified ( Figure 1 B). The plasmid was extracted and treated with BamH I and Xho I double enzyme digestion, and the enzyme digestion product was also detected by electrophoresis. The target band was about 372 bp ( Figure 1 C), which was consistent with the expected result. The sequencing result showed that the sequence of the inserted fragment was completely consistent with the sequence of the CD82 target gene, and the recombinant expression vector was successfully constructed.

[0082] 2.2 Expression and purification of CD82 recombinant protein

[0083] The recombinant bacteria induced at 16°C and 140 r / min for 16 h were centrifuged, ultrasonically broken and subjected to SDS-PAGE analysis. The results showed that the induced recombinant plasmid obviously appeared a target band of 17.09 kDa in size ( Figure 2A), which was consistent with the expectation. In addition, the CD82 recombinant protein was mainly expressed in the form of inclusion body, and was subjected to inclusion body dissolution, renaturation and purification by Ni-NTA affinity chromatography column. After purification, the purified CD82 recombinant protein was analyzed by SDS-PAGE, and the results 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 strain

[0085] The blood of the mice was collected 14 days after the third immunization, and the antibody titers of the sera of the 6 immunized mice were determined by indirect ELISA to be 1:128000 Figure 3 A), the mouse with the highest antibody titer was selected for cell fusion, and 7 subcloned cells were selected, and the supernatants thereof were subjected to Flow Cytometry detection, and 3 hybridoma cell strains capable of stably secreting CD82 monoclonal antibody were obtained Figure 3 B), and were named 2F6, 9G5 and 11G3, respectively. The cell strain 11G3 with the best flow cytometry effect was selected for preparation of monoclonal antibody.

[0086] 2.4 Determination of monoclonal antibody variable region sequence

[0087] According to the results of gel electrophoresis, as shown in Figure 4 and Figure 5 , it can be seen that the quality of the hybridoma RNA extraction is qualified, and the cells after amplification of light and heavy chains have obvious bands at about 700bp, which is 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] CAGATCCAGTTGGTGCAGTCTGGACCTGAGTTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCGTCTGGATATACCTTCACAAAATATGGAATGAGCTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACATACACTGGAGAGCCAACATATACTAATGACTTCAAGGGACGGTTTGTCTTCTCTTTGGAAACCTCTGCCAGAACTGCCTATTTGCAGATCAACAGCCTCAAAAATGAGGACATGTCTACTTATTTCTGTGCAAGAGGGAGGTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID No. 8) amino acid sequence of the heavy chain variable region:

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

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

[0094] GACATTGTGATGACACAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAAGTGTTGATCTACGGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAAGCGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATCATAGTTACCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG (SEQ ID No. 10)

[0095] 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 2F6, 9G5, 11G3 cell supernatant was subjected to ELISA detection using a kit, and the results showed that the antibodies secreted by the 2F6 and 9G5 cell strains were IgG2b type, and the antibodies secreted by the 11G3 cell strain were IgG2a type. Figure 6 ).

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

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

[0101] In summary, the present application provides a monoclonal antibody against pig CD82 protein, a hybridoma cell strain and application. The monoclonal antibody is expressed and purified against the CD82 (EC2) region using a prokaryotic expression system, and the purified recombinant protein is used as an immunogen for preparation. After indirect ELISA and Flow Cytometry screening, a monoclonal cell strain secreting IgG2a type antibody is obtained. The purified antibody is detected by indirect ELISA method, and the antibody still has a sensitive reaction when the concentration is 31.25 ng / ml. The PAM cells capable of expressing endogenous CD82 protein are detected by flow cytometry, and the results show that the mAb has good labeling ability for PAM cells, indicating that the CD82 protein can be used for the preparation of mAb by flow cytometry. The present application provides 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 includes a heavy chain constant region and a light chain constant region, a heavy chain variable region and a light chain variable region. The CDR of the heavy chain variable region of the monoclonal antibody includes the amino acid sequences CDR-H1 as shown in SEQ ID No. 1, CDR-H2 as shown in SEQ ID No. 2 and CDR-H3 as shown in SEQ ID No.

3. The CDR of the light chain variable region of the monoclonal antibody includes the amino acid sequences CDR-L1 as shown in SEQ ID No. 4, CDR-L2 as shown in SEQ ID No. 5 and CDR-L3 as shown in SEQ ID No.

6.

2. The monoclonal antibody as described in 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 encoding the monoclonal antibody of 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 bacteria containing the gene described in claim 3.

5. The monoclonal antibody as described in claim 1 or 2, characterized in that, The monoclonal antibody mentioned is of type IgG2a.

6. The use of the monoclonal antibody as described in claim 1 or 2 in the preparation of flow cytometry detection reagents.

7. A flow cytometry assay kit comprising the monoclonal antibody as described in claim 1 or 2.

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