Porcine reproductive and respiratory syndrome virus m protein antigen epitope peptide, monoclonal antibody thereof and application

Abstract

This invention discloses an antigenic epitope peptide of porcine reproductive and respiratory syndrome virus (PRRSV) M protein and its monoclonal antibody. The amino acid sequence of the antigenic epitope peptide is shown in SEQ ID No. 11; the heavy chain variable region of the monoclonal antibody includes CDR1-3 as shown in SEQ ID Nos. 1-3; the light chain variable region includes CDR1-3 as shown in SEQ ID Nos. 4-6; the monoclonal antibody reacts well with various PRRSV strains, including HP-PRRSV, NADC30PRRSV-like strains, and NADC34PRRSV-like strains; it can be obtained using conventional biological methods such as genetic engineering, avoiding the problem of easy loss of antibody genetic sequence due to long-term cryopreservation of traditional hybridoma cells; it also facilitates antibody optimization at the gene or protein level, further improving the antibody's specificity and affinity.

Description

Technical Field

[0001] This invention belongs to the fields of immunology and in vitro diagnostic technology, specifically relating to a porcine reproductive and respiratory syndrome virus M protein antigenic epitope peptide, its monoclonal antibody, and its applications. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a major infectious disease of animals caused by Porcine reproductive and respiratory syndrome virus (PRRSV), and is a significant animal disease threatening the global pig industry. PRRSV is one of the fastest-mutating RNA viruses known. Currently, my country is experiencing a mixed epidemic situation with multiple strains circulating, including NADC30-like PRRSV as the dominant strain, NADC34-like PRRSV and European PRRSV (PRRSV-1), and sporadic outbreaks of HP-PRRSV.

[0003] PRRSV belongs to the Arteritis Viridae family. Its genome is approximately 15 kb in length and contains at least ten open reading frames (ORFs) and two untranslated regions (UTRs) at the 5' and 3' ends. ORF1, further divided into ORF1a and ORF1b, accounts for about 75% of the entire genome and encodes two large replicase precursor proteins, pp1a and pp1ab. pp1a and pp1b are further hydrolyzed to produce at least 13 non-structural proteins (NSPs) related to viral replication. ORF2-ORF7 are located at the 3' end of the PRRSV genome, accounting for about 25% of the remaining genome, and encode eight structural proteins: GP2, E, GP3, GP4, GP5, GP5a, M, and N proteins. PRRSVM proteins are highly conserved non-glycosylated membrane structural proteins, totaling 174 amino acids. They contain multiple dominant antigenic epitopes, can rapidly induce antibody production, and serve as important viral targets for serological diagnostics and vaccine development. Therefore, monoclonal antibodies targeting the M protein play an important role in both basic theoretical research on PRRSV and the development of prevention and control products.

[0004] Gene-marked vaccines are crucial tools for eradicating major animal diseases, enabling serological differentiation between marker vaccine strains and circulating or vaccine strains. Currently, several pig farms have achieved complete PRV eradication using PRV gene-marked vaccines. However, current research indicates that PRRSV has no missing structural proteins; the deletion of any single structural protein prevents the production of infectious viral particles. Therefore, constructing a gene-marked vaccine strain by deleting the entire gene is not feasible for PRRSV. An alternative strategy is to use reverse genetics to silence key antigenic epitopes in the dominant PRRSV immune genes, preventing the induction of corresponding antibodies and thus distinguishing it from wild-type strains or currently commercially available vaccine strains, thereby promoting PRRSV eradication. Preparing broad-spectrum, highly effective antibodies against dominant PRRSV immune proteins such as the M protein, and identifying the key immune epitopes they recognize, can provide a crucial research foundation and materials for the development of PRRSV gene-engineered marker vaccines with missing key antigenic epitopes. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention, based on the NADC30-like PRRSV M protein, which is currently prevalent in my country, prepares a specific monoclonal antibody targeting the M protein. This monoclonal antibody not only recognizes NADC30-like PRRSV but also exhibits good reactivity with various PRRSV strains, including HP-PRRSV and NADC34-like PRRSV. Furthermore, the monoclonal antibody specifically recognizes and binds to 94-119 aa of the PRRSV M protein, providing a reliable tool for exploring the function of the PRRSV M protein and developing PRRSV diagnostic reagents. It also provides key research materials for the development of novel genetically engineered marker vaccines based on reverse genetics techniques. Specifically, it includes the following:

[0006] In a first aspect, the present invention provides a monoclonal antibody against the M protein of porcine reproductive and respiratory syndrome virus, wherein the monoclonal antibody comprises an antibody heavy chain and an antibody light chain;

[0007] The variable region CDR of the antibody heavy chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3;

[0008] The variable region CDR of the antibody light chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 4, CDR2 shown in SEQ ID No. 5, and CDR3 shown in SEQ ID No. 6.

[0009] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID No. 7, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID No. 8.

[0010] In a second aspect, the present invention provides a nucleic acid that encodes the antibody heavy chain and antibody light chain of the monoclonal antibody described in the first aspect above.

[0011] Preferably, the nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID NO.9, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID NO.10.

[0012] Thirdly, the present invention provides an expression cassette, expression vector, and recombinant bacteria containing the nucleic acid described in the second aspect above.

[0013] Fourthly, the present invention provides the use of the monoclonal antibody described in the first aspect above in the preparation of reagents, test strips or kits for detecting porcine reproductive and respiratory syndrome virus.

[0014] Fifthly, the present invention provides the application of the monoclonal antibody described in the first aspect above in the detection of porcine reproductive and respiratory syndrome virus (PRRSV) infection for non-disease diagnosis purposes.

[0015] In a sixth aspect, the present invention provides the application of the monoclonal antibody described in the first aspect above in the functional study of the porcine reproductive and respiratory syndrome virus M protein for non-disease diagnosis purposes.

[0016] In a seventh aspect, the present invention provides a detection kit for porcine reproductive and respiratory syndrome virus, the kit comprising the monoclonal antibody described in the first aspect above.

[0017] Preferably, the kit further includes an enzyme-labeled plate, blocking solution, diluent, washing solution, chromogenic agent, and stop solution.

[0018] Eighthly, the present invention provides a method for preparing the monoclonal antibody described in the first aspect above. The method comprises: cloning the porcine reproductive and respiratory syndrome virus (PRRSV) ORF6 gene into the pET-21b vector to construct a prokaryotic expression vector; inducing expression; denaturing the protein with urea; purifying the protein by elution with different concentrations of imidazole; using the purified PRRSV M recombinant protein as an antigen to immunize Balb / c mice; fusing mouse spleen cells with myeloma cells SP2 / 0 to prepare hybridoma cells; verifying the cell supernatant by indirect ELISA and indirect immunofluorescence to screen for positive clones; after three subcloning processes, injecting the hybridoma cells into mice to prepare ascites; finally purifying the obtained ascites to obtain a monoclonal antibody against the porcine reproductive and respiratory syndrome virus (PRRSV) M protein.

[0019] In a ninth aspect, the present invention provides a porcine reproductive and respiratory syndrome virus (PRRSV) M protein antigenic epitope peptide, which specifically binds to the monoclonal antibody described in the first aspect above; the antigenic epitope peptide is located at 94-119aa of the PRRSV M protein, and its amino acid sequence is shown in SEQ ID No. 11.

[0020] In a tenth aspect, the present invention provides an application of silencing PRRSV M protein 94-119aa expression in the preparation of PRRSV genetically engineered marker vaccines.

[0021] The beneficial effects of this invention are as follows: This invention utilizes a prokaryotic expression system to express and purify recombinant PRRSV M protein, which is then used as an immunogen to immunize mice. Through cell fusion and subcellular screening, a monoclonal antibody targeting the PRRSV M protein was successfully obtained. The prepared monoclonal antibody can react with various PRRSV strains, including HP-PRRSV, NADC30-like PRRSV, and NADC30-like PRRSV, exhibiting good conservation. Through gradient truncated expression of the PRRSV ORF6 gene, it was found that the region of the antibody that specifically recognizes and binds to the PRRSV M protein is 26 amino acids from 94 to 119 aa, providing a reliable research tool for exploring the function of PRRSV, developing PRRSV diagnostic reagents, and developing and identifying novel genetically engineered marker vaccines based on reverse genetics techniques. The monoclonal antibody provided by this invention can be obtained using conventional genetic engineering or protein engineering methods, avoiding antibody loss during long-term cryopreservation of hybridoma cells. It also facilitates antibody optimization at the gene and protein levels, thereby improving antibody specificity and affinity. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 Results of pET21b-M protein expression, purification and identification;

[0024] Figure 2 Results of serum titer assay in mice immunized with pET21b-M protein;

[0025] Figure 3 Results of titer assay for the prepared monoclonal antibody;

[0026] Figure 4Western blot analysis results of the reactivity of monoclonal antibodies to multiple PRRSV strains;

[0027] Figure 5 Preparation of monoclonal antibody subtype classification and identification results;

[0028] Figure 6 Schematic diagram of PRRSVM protein truncated form construction;

[0029] Figure 7 Identification results of monoclonal antibody recognition of PRRSVM protein sites. Detailed Implementation

[0030] The present invention will be further described in detail below through specific embodiments. However, those skilled in the art will understand that the following embodiments are only for illustration and explanation of the present invention and should not be regarded as limiting the scope of protection of the present invention. In addition, where specific technical operation steps or conditions are not specified in the embodiments, they are all carried out in accordance with the techniques or conditions described in general literature in the art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be obtained commercially.

[0031] Example 1: Construction and identification of the pET21b-PRRSV ORF6 recombinant plasmid

[0032] Sequence analysis of the 174 amino acids in the NADC30 PRRSV SX-YL1806 ORF6 gene revealed that amino acids 1-93 are transmembrane regions. After deleting this transmembrane region, a transmembrane peptide sequence was added to the N-terminus of the remaining gene sequence as the target gene, and optimized for *E. coli*. The optimized nucleotide sequence is shown in SEQ ID No. 12. The optimized sequence was amplified and ligated into the pET21b prokaryotic expression vector to construct the pET21b-PRRSV ORF6 recombinant plasmid. The recombinant plasmid was identified using double enzyme digestion. The specific procedures are as follows:

[0033] 1.1 Primer Design

[0034] The primers for PRRSV ORF6 amplification are shown in Table 1. The underlined sequences correspond to the restriction enzyme sites. After the primers were designed, they were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0035] Table 1. Primers for PRRSV ORF6 amplification

[0036]

[0037] 1.2 PCR amplification

[0038] Using the optimized pUC57-PRRSV-ORF6 plasmid as a template, the target gene was amplified by PCR using the primers described above. The PCR amplification system and reaction procedure are shown in Tables 2-3.

[0039] Table 2 PCR amplification system for the target gene

[0040] reagents Volume (μL) PrimeSTARGXL DNA Polymerase 2 dNTP 4 PrimeSTARGXL Buffer 10 template 1 PRRSV-ORF6-F 1.25 PRRSV-ORF6-R 1.25 <![CDATA[ddH2O]]> 30.5

[0041] Table 3 Reaction Procedure

[0042]

[0043] 1.3 Construction and Identification of Recombinant Vectors

[0044] The amplified target gene and pET21b empty vector were digested with two enzymes, and then the target gene was ligated into the digested vector and transformed into DH5α competent cells. Clones were selected and colony PCR was performed using the primers in Table 1. Positive bacteria were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Plasmids were extracted from correctly sequenced bacterial cultures and cryopreserved for later use.

[0045] Sequencing results showed that the recombinant plasmid sequence was consistent with expectations. Further digestion of the recombinant plasmid with both BamHI and Xho I restriction endonucleases confirmed the sequence was as expected.

[0046] Example 2 Expression and purification of PRRSV M protein

[0047] 2.1 Expression of PRRSV M protein

[0048] The pET21b-PRRSV ORF6 plasmid constructed in Example 1 was transformed into the prokaryotic expression strain BL21(DE3). A single colony was picked and cultured overnight in 1 mL of liquid LB medium containing kanamycin resistance with shaking. The next day, the bacterial culture was inoculated into 10 mL of fresh medium at a ratio of 1:100 and cultured at 37°C with shaking at 220 rpm. Samples were taken every hour to measure the OD of the bacterial culture. 600 Value, pending OD 600 When the value reaches 0.4–0.6, 1 mmol / L IPTG is added to induce expression. After 6 h of induction, the bacterial culture is collected, centrifuged at 8000 r / min for 5 minutes, and the precipitate is collected. The precipitate is resuspended in 10 mL PBS, the bacterial cells are sonicated and the precipitate and supernatant are collected separately after centrifugation. 2× loading buffer is added directly to the supernatant, and 2× loading buffer is added after dissolving the precipitate in 8 M urea solution. The samples are identified by SDS-PAGE and stained with Coomassie Brilliant Blue.

[0049] 2.2 Purification of PRRSV M protein

[0050] M protein was expressed in both the supernatant and the precipitate, but inclusion body protein was abundant and easily purified; therefore, inclusion body protein was selected for further purification. The bacterial cell precipitate after sonication was collected, resuspended in equilibration buffer, centrifuged at 4000 rpm for 10 min at 4°C, and the supernatant was collected and filtered through a 0.45 μm filter for purification.

[0051] (1) Add 2 mL of His-tagged protein purification resin (Ni-NTA Resin) to the purification tube, rinse the column once with 5 column volumes of distilled water, and then equilibrate the purification column twice with 5 column volumes of equilibration buffer (8 M Urea, 10 mM NaH2PO4, 10 mM Trisbase, pH = 8.6).

[0052] (2) Add the prepared sample to the affinity chromatography column and incubate overnight or bind for 6-8 hours at 4°C. Elute the target protein with elution buffers containing different concentrations of imidazole, such as 10mM, 50mM, 100mM, 250mM, 500mM, 1M, and 2M. Add 2× loading buffer and perform SDS-PAGE identification.

[0053] (3) The high-purity eluted proteins were refolded by gradient dialysis at 4°C using refolding solutions containing different concentrations of urea (4M, 2M, and 1M). The samples were collected and identified by SDS-PAGE.

[0054] (4) Add the dialyzed protein to a 3.0 kDa concentration column and concentrate at 4°C. Take a small amount of protein, mix it with G250 solution, and measure its OD. 595nm And calculate the protein concentration.

[0055] The results of Pet21b-M protein expression and purification identification are as follows: Figure 1 As shown, the PRRSV M protein was successfully expressed and purified, and its size met expectations.

[0056] Example 3: Preparation, Reactivity, and Variable Region Sequencing of Monoclonal Antibodies

[0057] 3.1 Preparation of Monoclonal Antibodies

[0058] (1) Animal Immunization: Five female BALB / c mice aged 6-8 weeks were selected, and the PRRSV M recombinant protein obtained above was used to immunize the mice three times according to the immunization cycle. For the first immunization, the PRRSV M recombinant protein was mixed with Bio-Drone water adjuvant at a ratio of 1:1 and administered subcutaneously to multiple sites at a dose of 50 μg / mouse. After an interval of 21 days, a second immunization was performed, in which the PRRSV M recombinant protein was mixed with Freund's incomplete adjuvant at a ratio of 1:1 and administered at a dose of 100 μg / mouse. Similarly, a third immunization was performed 14 days later. Blood was collected from the eyeballs of the mice 14 days after the third immunization, and serum was collected to determine the antibody titer. Before fusion, a booster immunization was performed, in which the mice to be subjected to cell fusion were sensitized by intraperitoneal injection of PRRSV M recombinant protein at a dose of 30 μg / mouse.

[0059] (2) Cell fusion: SP2 / 0 myeloma cells were revived and passaged three times to restore cell viability, and the cells were proliferated to meet the fusion conditions. One day in advance, peritoneal macrophages from 6-8 week old NC mice were used as feeder cells and seeded into five 96-well cell plates. Mice with high serum titers were selected for B cell isolation. The spleen of the mice was aseptically removed and repeatedly injected with 1640 medium containing 1% penicillin-streptomycin-amphotericidal (triple antibody) until the internal B cells were flushed out into the medium. The spleen was then placed on a cell sieve and ground through the sieve using a syringe plunger to obtain mouse B cells. The previously prepared SP2 / 0 myeloma cells were taken, the original culture medium was discarded, 1640 medium was added, the cells were tapped, and then transferred to the prepared B cells for cell fusion.

[0060] (3) Screening and identification of monoclonal hybridoma cells: The PRRSVM recombinant protein prepared above was coated onto an ELISA plate. The supernatant from the positive wells containing hybridoma cells was collected and added to the ELISA plate for titer determination. Wells with high reactivity were selected for subcloning. Subcloning was performed three times consecutively until well-reactive monoclonal hybridoma cells were selected. The high-titer, well-reactive monoclonal hybridoma cells were then propagated and cryopreserved.

[0061] ELISA potency assay results as follows Figure 2 As shown, serum titers gradually increased after immunization, with all five serum samples having titers ≥1:128000 and P / N>2, indicating high serum titers in the five mice. Mouse number 3 had the highest titer and was used for subsequent hybridoma cell fusion.

[0062] (4) Preparation of ascites fluid: 10-12 week old BALB / c female mice were sensitized by intraperitoneal injection of 0.4 mL Freund's incomplete adjuvant. Seven days later, the selected monoclonal hybridoma cell line was immunized into the mice at a dose of 3 × 10⁻⁶. 6 ~5×10 6Cells / mouse. 7–10 days post-immunization, when the mouse abdomen is noticeably enlarged and fluctuates upon palpation, collect ascites fluid and freeze it.

[0063] (5) Purification of ascites fluid: The obtained ascites fluid was purified according to the instructions of the TRAN ProteinIso Protein G Resin Kit. The purified sample was identified by SDS-PAGE. The PRRSV M protein-specific antibody was named 5-F8-D11.

[0064] 3.2 Identification of the reactivity of monoclonal antibodies

[0065] (1) ELISA to identify the titer of monoclonal antibodies

[0066] The obtained PRRSV M protein was coated into ELISA plates at a rate of 100 ng / well, and incubated at 37°C for 2 h or 4°C overnight. After washing with PBS, 5% skim milk powder was added for blocking. The prepared PRRSV M monoclonal antibody 5-F8-D11 was serially diluted and added to the coated plates, and incubated at 37°C for 1 h, followed by washing with PBS 4 times. HRP-labeled goat anti-mouse IgG (1:10000 dilution) was added, and incubated at 37°C for 1 h, followed by washing with PBS 4 times. TMB was added for color development for 15 min, and then stop buffer was added. The plates were then used to measure OD using an ELISA reader. 450 Numerical value.

[0067] ELISA results as follows Figure 3 As shown, the prepared monoclonal antibody 5-F8-D11 can react with PRRSVM protein with high titer.

[0068] (2) Western blotting to identify the reactivity of monoclonal antibodies with various PRRSV strains

[0069] HP-PRRSV, NADC30-like PRRSV, and NADC34-like PRRSV were inoculated into PAMs, and cells were collected for Western blot identification after 24 hours. The primary antibody used was the previously prepared PRRSVM monoclonal antibody 5-F8-D11, and the secondary antibody was HRP-labeled goat anti-mouse antibody.

[0070] Reactivity test results as follows Figure 4 As shown in the WB results, the prepared monoclonal antibody can detect multiple PRRSV strains, including HP-PRRSV, NADC30-like PRRSV, and NADC34-like PRRSV, and has good specificity and conservation.

[0071] (3) Identification of monoclonal antibody subtypes

[0072] The PRRSV M protein monoclonal antibody was subclassed according to the instructions of the Proteintech Mouse Monoclonal Antibody Isotyping Kit.

[0073] The results are as follows Figure 5 As shown, the heavy chain constant region of monoclonal antibody 5-F8-D11 is of the IgG1 type, and the light chain constant region is of the Kappa type.

[0074] (4) Identification of protein epitopes bound by monoclonal antibodies: The PRRSV ORF6 gene was truncated in a gradient and ligated into a prokaryotic expression vector to construct a recombinant plasmid. The corresponding protein was induced to express. Western blot reactions were performed on the truncated protein samples using the prepared PRRSV M protein monoclonal antibody, gradually shortening the protein until the key antigenic epitopes bound to it were finally identified. The gradient truncation construction strategy is as follows: Figure 6 As shown.

[0075] Epitope identification results of monoclonal antibody 5-F8-D11 are as follows: Figure 7 As shown, the key region for the specific binding of the surface monoclonal antibody 5-F8-D11 to the PRRSV M protein is B1, specifically 94-119aa. 94 FITSRCRLCLLGRKYILAPAHHVESA 119 It contains 26 amino acids. The specific binding site sequence is shown in SEQ ID No. 11.

[0076] 3.3 Monoclonal antibody variable region gene sequencing

[0077] The previously cryopreserved 5-F8-D11 monoclonal hybridoma cells were revived, and genomic RNA was extracted using the Trizol method. The RNA was then reverse-transcribed into cDNA using the HiScript IIQ RT SuperMix for qPCR kit from Novizan Pharmaceuticals.

[0078] Nested PCR was used to amplify the antibody variable region gene. Using the above cDNA as a template, the antibody variable region gene was amplified using the first round of mouse antibody IgG1 and κ light chain primers. Then, using the first round product as a template, the antibody variable region gene was amplified using the second round of mouse antibody IgG1 and κ light chain primers. Primer synthesis reference (von Boehmer L, Liu C, Ackerman S, Gitlin AD, Wang Q, Gazumyan A, Nussenzweig MC. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat Protoc. 2016 Oct; 11(10): 1908-1923. doi: 10.1038 / nprot. 2016.102. Epub 2016 Sep 15. PMID: 27658009.).

[0079] After amplification, the target fragment was ligated into the pMD-19T vector to construct a sequencing plasmid, which was then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed using the NCBI and IMGT gene libraries to identify the antibody variable region sequence.

[0080] Sequencing results showed that the amplified sequences were the complementarity determining regions (CDRs) of the heavy chain and light chain variable regions of the monoclonal antibody, as shown in Table 4.

[0081] Table 4 Antibody variable region sequences

[0082]

[0083] The amino acid sequence of the heavy chain variable region is as follows:

[0084] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYIRYSGNT DYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAGPVWGQGTTLTVSS;

[0085] The amino acid sequence of the light chain variable region is as follows:

[0086] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGNSYMNWYQQKPGQPPKLLIYAASNL ESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQTIGVPWTFGGGTKLEIK;

[0087] The gene sequence encoding the heavy chain variable region is:

[0088] GATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAGGTACAGTGGTAACACTGACTACAACCCATCTCTCAAAAGTCGCATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCGGGGCCTGTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA;

[0089] The gene sequence encoding the light chain variable region is:

[0090] GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTAATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAACTATTGGGGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA。

[0091] The M protein monoclonal antibody 5-F8-D11 can specifically recognize the region of the porcine reproductive and respiratory syndrome virus M protein as: FITSRCRLCLLGRKYILAPAHHVESA.

[0092] In summary, this invention provides a monoclonal antibody against the M protein of porcine reproductive and respiratory syndrome virus (PRRSV). This monoclonal antibody specifically recognizes and binds to the 94-119 aa of the M protein and reacts with multiple strains, including HP-PRRSV, NADC30-like PRRSV, and NADC34-like PRRSV, exhibiting good reactivity and conservation. The monoclonal antibody provided by this invention can be obtained using conventional genetic engineering or protein engineering methods, avoiding antibody loss during long-term cryopreservation of hybridoma cells. It also facilitates antibody optimization at the gene and protein levels, thereby improving antibody specificity and affinity. The monoclonal antibody prepared by this invention provides a reliable tool for studying the pathogenic mechanism of PRRSV, developing PRRSV diagnostic kits, and developing and identifying novel genetically engineered marker vaccines based on reverse genetics techniques.

[0093] The embodiments described above are only some embodiments of the present invention and are not intended to limit the scope of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the patent claims of the present invention should be included within the scope of the patent claims of the present invention.

Claims

1. A monoclonal antibody against the M protein of porcine reproductive and respiratory syndrome virus, characterized in that, The monoclonal antibody comprises an antibody heavy chain and an antibody light chain; The variable region CDR of the antibody heavy chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3; The variable region CDR of the antibody light chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 4, CDR2 shown in AAS, and CDR3 shown in SEQ ID No.

6.

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

8.

3. A nucleic acid, characterized in that, The nucleic acid encodes the antibody heavy chain and antibody light chain of the monoclonal antibody of claim 1 or 2.

4. The nucleic acid as described in claim 3, characterized in that, The nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID NO.9, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID NO.

10.

5. The use of the monoclonal antibody as described in claim 1 or 2 in the preparation of reagents for detecting porcine reproductive and respiratory syndrome virus.

6. The application of the monoclonal antibody as described in claim 1 or 2 in the detection of porcine reproductive and respiratory syndrome virus (PRRSV) infection for non-disease diagnosis purposes.

7. The application of the monoclonal antibody as described in claim 1 or 2 in the functional study of the M protein of porcine reproductive and respiratory syndrome virus for non-disease diagnosis purposes.

8. A detection kit for porcine reproductive and respiratory syndrome virus, characterized in that, The kit includes the monoclonal antibody as described in claim 1 or 2.

9. A porcine reproductive and respiratory syndrome virus (PRRSV) M protein antigenic epitope peptide, wherein the antigenic epitope peptide specifically binds to the monoclonal antibody according to claim 1 or 2; characterized in that, The antigenic epitope peptide is located at 94-119aa of the PRRSV M protein, and its amino acid sequence is shown in SEQ ID No. 11.

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