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

Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus N protein antigen epitope peptide and a monoclonal antibody thereof. The amino acid sequence of the antigen epitope peptide is as shown in SEQ ID No. 11; the heavy chain variable region of the monoclonal antibody comprises CDR1-3 as shown in SEQ ID No.1 to SEQ ID No.3; the light chain variable region comprises CDR1-3 as shown in SEQ ID No. 4 to 6; the monoclonal antibody reacts with a variety of PRRSV strains such as classical PRRSV, HP-PRRSV, NADC30PRRSV-like, NADC34PRRSV-like, European PRRSV and the like, and the conservative property is good; the antibody can be obtained by biological means such as conventional genetic engineering, and the problem that the genetic sequence of the antibody is easy to lose due to long-term cryopreservation of traditional hybridoma cells is avoided; optimization of the antibody on the gene or protein level is facilitated, and the specificity and affinity of the antibody are further improved.

Description

Technical Field

[0001] The invention belongs to the technical field of immunology and in vitro diagnosis, and specifically relates to a porcine reproductive and respiratory syndrome virus N protein antigen epitope peptide and a monoclonal antibody and application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an important animal infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), and is a major animal epidemic that threatens the global pig industry. PRRSV is one of the fastest-mutating RNA viruses known. Currently, my country is experiencing a mixed epidemic of multiple strains, with NADC30-like PRRSV as the dominant strain, NADC34-like PRRSV and European PRRSV (PRRSV-1) becoming more prevalent, and HP-PRRSV continuing to spread.

[0003] PRRSV belongs to the family of Arteriviridae. The genome is about 15kb in length and contains at least ten open reading frames (ORFs) and two untranslated regions (UTRs) at the 5' and 3' ends. ORF1, which can be divided into ORF1a and ORF1b, accounts for about 75% of the entire genome and encodes two large replicase precursor polyproteins pp1a and pp1ab. Pp1a and pp1b are further hydrolyzed to produce at least 13 non-structural proteins (NSPs) related to viral replication. ORF2-ORF7 is located at the 3' end of the PRRSV genome, accounting for about the remaining 25% of the genome, and encodes 8 structural proteins, including GP2, E, GP3, GP4, GP5, GP5a protein, M protein and N protein. N is the nucleocapsid protein of PRRSV, containing 128 or 123 amino acids and about 15KDa in size. The N protein is highly conserved, with a homology of more than 96% in the same genotype of PRRSV. N protein is also the most abundant viral protein among all PRRSV proteins, accounting for more than 40% of the total protein. N protein has outstanding immunogenicity and antigenicity, and is the earliest viral protein to induce the body to produce antibodies. Therefore, N protein is a key viral protein for early detection and diagnosis of PRRSV. Monoclonal antibodies against N 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 an important tool for purifying major animal diseases, and can serologically distinguish between marker vaccine strains and epidemic strains or vaccine strains. Porcine pseudorabies virus (PRV) contains multiple glycoproteins, including non-essential glycoproteins (gC, gE, gG, gI, gM, and gN) and essential glycoproteins (gB, gD, gH, gK, and gL). In order to achieve effective prevention and control of PRV, a PRV gene-marked vaccine strain lacking the non-essential glycoprotein gene gI came into being. This strain lacks the gI gene and will not induce piglets to produce antibodies against gI, which can be used as a marker for distinguishing vaccine strains from wild strains. At present, many pig farms have achieved thorough purification of PRV using PRV gene-marked vaccines. Current studies have shown that PRRSV has no missing structural proteins, and the absence of any structural protein cannot produce infectious virus particles. Therefore, the scheme of constructing a gene-marked vaccine strain by missing the entire gene cannot be implemented in PRRSV. An alternative strategy is to use reverse genetic manipulation technology to silence the key antigenic epitopes in the dominant immune genes of PRRSV, so that they cannot induce the corresponding antibodies, and then differentiate from wild strains or current commercial vaccine strains to promote PRRSV purification. Therefore, preparing broad-spectrum and highly effective antibodies against PRRSV dominant immune proteins such as N protein and identifying the key immune epitopes they recognize can provide a key research basis and research materials for the development of PRRSV genetic engineering marker vaccines that lack key antigenic epitopes.

[0005] The present invention is based on the NADC30-like PRRSV N protein, which is currently in a dominant epidemic position in my country, and prepares a specific monoclonal antibody against the N protein. The monoclonal antibody can not only recognize the NADC30-like PRRSV, but also has good reactivity with various PRRSV strains such as classical PRRSV, HP-PRRSV, NADC34-like PRRSV and European PRRSV. The monoclonal antibody specifically recognizes and binds to 50-57aa of the PRRSV N protein, providing a reliable tool for exploring the function of the PRRSV N protein and the development of PRRSV diagnostic reagents, and providing key research materials for the development of new genetic engineering marker vaccines based on reverse genetic manipulation technology. Summary of the invention

[0006] In view of the above technical problems, the object of the present invention is to provide a porcine reproductive and respiratory syndrome virus N protein antigen epitope peptide and its monoclonal antibody and application.

[0007] In a first aspect, the present invention provides a porcine reproductive and respiratory syndrome virus N protein antigenic epitope peptide, the amino acid sequence of the antigenic epitope peptide is shown as SEQ ID No.11.

[0008] In a second aspect, the present invention provides the use of the antigen epitope peptide described in the first aspect in the preparation of monoclonal antibodies against porcine reproductive and respiratory syndrome virus N protein.

[0009] In a third aspect, the present invention provides the use of silencing the expression of PRRSV N protein 50-57aa in the preparation of a PRRSV genetically engineered marker vaccine.

[0010] In a fourth aspect, the present invention provides a monoclonal antibody against porcine reproductive and respiratory syndrome virus N protein, wherein the monoclonal antibody comprises an antibody heavy chain and an antibody light chain;

[0011] The variable region CDR of the antibody heavy chain includes CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3;

[0012] The variable region CDR of the antibody light chain includes CDR1 shown in the amino acid sequence of SEQ ID No.4, CDR2 shown in SEQ ID No.5 and CDR3 shown in SEQ ID No.6.

[0013] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is shown as SEQ ID No.7, and the amino acid sequence of the variable region of the antibody light chain is shown as SEQ ID NOo.8.

[0014] In a fifth aspect, the present invention provides a nucleic acid encoding the antibody heavy chain and antibody light chain of the monoclonal antibody described in the fourth aspect.

[0015] Preferably, the nucleotide sequence encoding the variable region of the antibody heavy chain is shown as SEQ ID No.9, and the nucleotide sequence encoding the variable region of the antibody light chain is shown as SEQ ID No.10.

[0016] In a sixth aspect, the present invention provides an expression cassette, an expression vector, and a recombinant bacterium containing the nucleic acid described in the fifth aspect.

[0017] In a seventh aspect, the present invention provides the use of the monoclonal antibody described in the fourth aspect in the preparation of a reagent, a test strip or a kit for detecting porcine reproductive and respiratory syndrome virus.

[0018] In an eighth aspect, the present invention provides the use of the monoclonal antibody described in the fourth aspect in the detection of porcine reproductive and respiratory syndrome virus infectivity for non-disease diagnosis purposes, or in the study of the function of porcine reproductive and respiratory syndrome virus N protein.

[0019] In a ninth aspect, the present invention provides a porcine reproductive and respiratory syndrome virus detection kit, the kit comprising the monoclonal antibody described in the fourth aspect above.

[0020] Preferably, the kit further comprises an ELISA plate, a blocking solution, a diluent, a washing solution, a color developer, and a stop solution.

[0021] In the tenth aspect, the present invention provides a method for preparing the monoclonal antibody described in the fourth aspect, which is: cloning the porcine reproductive and respiratory syndrome virus ORF7 gene into the pET-28a vector to construct a prokaryotic expression vector, inducing expression, and purifying the protein by eluting with imidazole at different concentrations after urea denaturation; using the purified PRRSV N recombinant protein as an antigen to immunize Balb / c mice, taking the mouse spleen cells and fusing them with myeloma cells SP2 / 0 cells to prepare hybridoma cells; performing indirect ELISA and indirect immunofluorescence verification on the cell supernatant to screen for positive clones; after three subclonings, injecting the hybridoma cells into mice to prepare ascites, and finally purifying the obtained ascites to obtain anti-porcine reproductive and respiratory syndrome virus N protein monoclonal antibodies.

[0022] The invention has the following beneficial effects: the invention uses a prokaryotic expression system to express and purify recombinant PRRSV N protein, uses the protein as an immune source to immunize mice, and successfully obtains a monoclonal antibody against PRRSV N protein through cell fusion and subcellular screening; the prepared monoclonal antibody can react with a variety of PRRSV strains such as classical PRRSV, HP-PRRSV, NADC34-like PRRSV and European PRRSV, and has good conservatism; the PRRSV ORF7 gene is truncated and expressed in a gradient manner, and it is found that the region in which the antibody can specifically recognize and bind to the PRRSV N protein is aa50-57, a total of 8 amino acids, which provides a reliable research tool for exploring the function of PRRSV N, PRRSV diagnostic reagents, and the development and differential diagnosis of a new genetic engineering marker vaccine based on reverse genetic manipulation technology; the monoclonal antibody provided by the invention can be obtained by conventional genetic engineering or protein engineering methods, thereby avoiding the loss of antibodies during long-term cryopreservation of hybridoma cells, and is also conducive to optimizing the antibodies at the gene and protein levels, thereby improving the specificity and affinity of the antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0024] Figure 1 .pET28a-N protein expression and purification identification results;

[0025] Figure 2 .The results of serum titer determination of mice immunized with pET28a-N protein;

[0026] Figure 3 .The titer test results of the prepared monoclonal antibodies;

[0027] Figure 4 .IFA identified the reactivity of monoclonal antibodies to various PRRSV strains;

[0028] Figure 5 . Prepare monoclonal antibody subtype classification and identification results;

[0029] Figure 6 .Schematic diagram of the construction of PRRSV N protein truncations;

[0030] Figure 7 .Identification results of the monoclonal antibody recognizing the PRRSV N protein site. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below by specific embodiments. However, it will be appreciated by those skilled in the art that the following examples are only used to illustrate and explain the present invention and should not be considered as limiting the scope of protection of the present invention. In addition, those who do not indicate specific technical operation steps or conditions in the embodiments are all carried out according to the technology or conditions described in the general literature in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained by commercial purchase.

[0032] Example 1 Construction and identification of pET28a-PRRSV ORF7 recombinant plasmid

[0033] The ORF7 gene of NADC30-like PRRSV SX-YL1806 was optimized for Escherichia coli, and the optimized nucleotide sequence is shown in SEQ ID No. 12. The optimized sequence was amplified and connected to the pET28a prokaryotic expression vector to construct the pET28a-PRRSV ORF7 recombinant plasmid, and the recombinant plasmid was identified by double restriction digestion. The specific operation is as follows:

[0034] 1.1 Primer design

[0035] The primers for amplifying PRRSV ORF7 are shown in Table 1 , where the underlined sequences correspond to restriction sites. The primers were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0036] Table 1 PRRSV ORF7 amplification primers

[0037]

[0038] 1.2 PCR amplification

[0039] Using the optimized synthetic pUC57-PRRSV-ORF7 plasmid as a template, the target gene was PCR amplified using the above primers. The PCR amplification system and reaction procedure are shown in Table 2-3 respectively.

[0040] Table 2 Target gene PCR amplification system

[0041]

[0042]

[0043] Table 3 Reaction procedure

[0044]

[0045] 1.3 Construction and identification of recombinant vectors

[0046] The target gene amplified above and the pET28a empty vector stored in the laboratory were digested with two enzymes, and then the target gene and the enzyme-cut vector were connected and transformed into DH5a competent cells. The selected clones were subjected to colony PCR using the primers in Table 1, and the positive bacteria were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The plasmids extracted from the bacterial solution with correct sequencing were frozen for later use.

[0047] The sequencing results showed that the recombinant plasmid sequence was consistent with the expected sequence. The recombinant plasmid was then double-digested with two restriction endonucleases, Nco I and Xho I, for restriction endonuclease identification, which was consistent with the expected sequence.

[0048] Example 2 Expression and purification of PRRSV N protein

[0049] 2.1 Expression of PRRSV N protein

[0050] The pET-28a-PRRSV ORF7 plasmid constructed in Example 1 was transformed into the prokaryotic expression strain BL21 (DE3), and a single clone colony was picked and placed in 1 mL of liquid LB medium containing kanamycin resistance and shaken and cultured overnight; the next day, the bacterial solution was inoculated into 10 mL of fresh culture medium at a ratio of 1:100, and shaken and cultured at 37°C, 220 r / min; a portion of the sample was taken every hour to measure the OD of the bacterial solution 600 Value, waiting for OD 600When the value reached 0.4-0.6, 1mmol / L IPTG was added to induce expression; after 6h of induction, the bacterial solution was collected, centrifuged at 8000r / min for 5 minutes, and the precipitate was collected; the precipitate was resuspended with 10mL PBS, the bacteria were ultrasonically broken, and the precipitate and supernatant were collected separately after centrifugation; the supernatant was directly added to 2× loading buffer, the precipitate was dissolved with 8M urea solution and then added to 2× loading buffer, the sample was identified by SDS-PAGE and stained with Coomassie Brilliant Blue.

[0051] 2.2 Purification of PRRSV N protein

[0052] N protein is expressed in both the supernatant and the precipitate, but the amount of inclusion body protein is large and easy to purify, so the inclusion body protein is selected for further purification. Collect the bacterial precipitate after ultrasonic disruption, resuspend and dissolve with equilibrium buffer, centrifuge at 4°C, 4000r / min, 10min, collect the supernatant and filter through a 0.45μm filter for purification:

[0053] (1) Add 2 mL of His-tagged protein purification resin (Ni-NTA Resin) to the purification tube, wash the column once with 5 column volumes of distilled water, and then wash with 5 column volumes of equilibration solution (8 M Urea, 10 mM NaH 2 PO 4 , 10 mM Trisbase, pH = 8.6) to equilibrate the purification column twice;

[0054] (2) Add the prepared sample to the affinity chromatography column, 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 10 mM, 50 mM, 100 mM, 250 mM, 500 mM, 1 M, and 2 M, and perform SDS-PAGE identification after adding 2× loading buffer;

[0055] (3) The eluted protein with high purity was renatured by gradient dialysis at 4°C using renaturation solutions containing 4M, 2M, 1M urea at different concentrations. Samples were collected for SDS-PAGE identification.

[0056] (4) Add the dialyzed protein to a 3.0 kDa concentration column and concentrate at 4°C. Take a small amount of protein and mix it with the G250 solution and measure its OD 595nm And calculate the protein concentration.

[0057] The results of pET28a-N protein expression, purification and identification are as follows Figure 1 As shown, the PRRSV N protein was successfully expressed and purified, and the size was consistent with the expected value.

[0058] Example 3 Preparation, reactivity and variable region sequence determination of monoclonal antibodies

[0059] 3.1 Preparation of monoclonal antibodies

[0060] (1) Animal immunization: Five female BALB / c mice aged 6 to 8 weeks were selected, and the PRRSV N recombinant protein obtained above was immunized three times according to the immunization cycle. For the first immunization, the PRRSV N recombinant protein was mixed with Biolon water adjuvant in a ratio of 1:1, and the mice were subcutaneously immunized at multiple points at a dose of 50 μg / mouse; a second immunization was performed 21 days later, and the PRRSV N recombinant protein was mixed with Freund's incomplete adjuvant in a ratio of 1:1; 100 μg / mouse was immunized; similarly, a third immunization was performed 14 days later. 14 days after the third immunization, blood was collected from the mice's eyeballs, and serum was taken to determine the antibody titer. A booster immunization was performed before fusion, and the PRRSV N recombinant protein was injected intraperitoneally at 30 μg / mouse into the mice to be subjected to cell fusion for sensitization.

[0061] (2) Cell fusion: Resuscitate SP2 / 0 myeloma cells, subculture them three times to restore cell viability, and expand the cells until they reach fusion conditions. One day in advance, peritoneal macrophages from 6-8 week old NC mice were used as feeder cells and spread into 5 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 and rinsed with 1640 culture medium containing 1% penicillin-streptomycin-amphotericin (triple antibodies) until the internal B cells were flushed out into the culture medium; then the spleen was placed on a cell sieve and ground and sieved with a syringe piston to obtain mouse B cells. Take the SP2 / 0 myeloma cells prepared in the early stage, discard the original culture medium, add 1640 culture medium, take a picture of the cells and transfer them to the prepared B cells for cell fusion.

[0062] (3) Screening and determination of monoclonal hybridoma cells: The PRRSV N recombinant protein prepared above is coated on an ELISA reaction plate, and the positive wells with hybridoma cells are selected and the supernatant is collected and added to the ELISA reaction plate for titer determination. The wells with higher reactivity are selected for subcloning, and subcloning is performed three times in succession until monoclonal hybridoma cells with good reactivity are screened. Monoclonal hybridoma cells with high titer and good reactivity are expanded and frozen.

[0063] ELISA titer test results Figure 2 As shown, the serum titer gradually increased after immunization, and the titers of the five sera were all ≥1:128000, with P / N>2, indicating that the serum titers of the five mice were relatively high. Among them, mouse No. 3 had the highest titer and was used for subsequent hybridoma cell fusion.

[0064] (4) Preparation of ascites: 10-12 week old BALB / c female mice were sensitized by intraperitoneal injection of 0.4 mL of Freund's incomplete adjuvant. Seven days later, the selected monoclonal hybridoma cell lines were immunized into the mice. 3×10 6 ~5×10 6 7-10 days after immunization, when the abdomen of the mouse is obviously heavy and fluctuates when touched, the ascites is collected and frozen.

[0065] (5) Purification of ascites: The ascites was purified according to the instructions of the ProteinIso Protein G Resin kit of TRAN Company, and the purified samples were identified by SDS-PAGE. The obtained PRRSV N protein-specific antibody was named 3B3.

[0066] 3.2 Identification of monoclonal antibody reactivity

[0067] (1) ELISA to identify the titer of monoclonal antibodies

[0068] The PRRSV N protein obtained above was coated on the ELISA reaction plate at 100 ng / well, coated at 37°C for 2 hours or at 4°C overnight, washed with PBS'T and blocked with 5% skim milk powder. The PRRSV N monoclonal antibody 3B3 prepared above was graded diluted and added to the coated plate, reacted at 37°C for 1 hour, and washed 4 times with PBS'T; HRP-labeled goat anti-mouse IgG (1:10000 dilution) was added, reacted at 37°C for 1 hour, and washed 4 times with PBS'T; TMB color development was performed for 15 minutes, and then the stop solution was added, and the OD was measured on an ELISA reader. 450 Numeric value.

[0069] ELISA results Figure 3 As shown, the prepared monoclonal antibody 3B3 can react with PRRSV N protein with high titer.

[0070] (2) IFA identification of the reactivity of monoclonal antibodies with various PRRSV strains

[0071] MARC-145 cells were infected with classical PRRSV, HP-PRRSV and NADC30-like PRRSV, and PAMs were inoculated with NADC34-like PRRSV and European PRRSV. After 24 hours, the cells were collected for IFA identification. The primary antibody used was the PRRSV N monoclonal antibody 3B3 prepared above, and the secondary antibody was FITC-labeled anti-mouse IgG.

[0072] The results of the reactivity test were as follows Figure 4As shown, the IFA results showed that the prepared monoclonal antibodies could react with various PRRSV strains currently prevalent in my country, such as classical PRRSV, HP-PRRSV, NADC30-like PRRSV, NADC34-like PRRSV and European PRRSV, but did not react with the NC pure cell group, showing good specificity and conservation.

[0073] (3) Identification of monoclonal antibody subtypes

[0074] According to the operating instructions of the Mouse Monoclonal Antibody Lsotyping Kit of Proteintech Company, the subtype identification of the obtained PRRSV N protein monoclonal antibody was performed.

[0075] The results are as follows Figure 5 As shown, the heavy chain constant region of monoclonal antibody 3B3 is of IgG2a type, and the light chain constant region is of Kappa type.

[0076] (4) Identification of protein epitopes bound by monoclonal antibodies: The PRRSV ORF7 gene was truncated and connected to a prokaryotic expression vector to construct a recombinant plasmid, and the corresponding protein was induced to express. The prepared PRRSV N protein monoclonal antibody was used to perform Western blot reaction with the truncated protein samples, and the epitopes were gradually shortened until the key antigen epitopes bound by it were finally identified. The gradient truncation construction strategy is as follows: Figure 6 shown.

[0077] The epitope identification results of monoclonal antibody 3B3 are as follows Figure 7 As shown, the key region of the surface monoclonal antibody 3B3 that specifically binds to the PRRSV N protein is N13, i.e., 50-57aa. 50 PEKPHFPL 57 , a total of 8 amino acids. The specific binding site sequence is shown in SEQ ID No.11.

[0078] 3.3 Determination of monoclonal antibody variable region gene sequence

[0079] The frozen 3B3 monoclonal hybridoma cells were revived, genomic RNA was extracted using the Trizol method, and RNA was reversed into cDNA using the reverse transcription kit HiScript IIQ RT SuperMix for qPCR produced by Nanjing Novozymes.

[0080] Nested PCR was used to amplify the variable region gene of the antibody. The above cDNA was used as a template to amplify the variable region gene of the antibody using the first round of mouse antibody IgG2a and κ light chain primers, and then the first round product was used as a template to amplify the variable region gene of the antibody using the second round of mouse antibody IgG2a and κ light chain primers. The primer synthesis reference is (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.).

[0081] After amplification, the target fragment was connected to the pMD-19T vector to construct a sequencing plasmid and sent to Beijing Qingke Biotechnology Co., Ltd. for sequence determination. The sequencing results were analyzed using the NCBI and IMGT gene libraries to identify the antibody variable region sequences.

[0082] The sequencing results showed that the amplified sequences were the complementarity determining region (CDR) sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody as shown in Table 4.

[0083] Table 4 Antibody variable region sequences

[0084]

[0085] Among them, the amino acid sequence of the heavy chain variable region is:

[0086] GVQLQESGAELVRPGASVTLSCKASGFTFTDYEMHWVKQTPLHGLDWIGAIDPETGG TAYNQKFKGKATLTADKSSSTASMELRSLTSEDSAVYYCTRGGYAMDYWGQGTSVTVSS;

[0087] The amino acid sequence of the light chain variable region is:

[0088] DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGG;

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

[0090] GGAGTGCAGCTGCAGGAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTTCACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTCTGCATGGCCTGGACTGGATTGGAGCTATTGATCCTGAAACTGGTGGTACT GCCTACAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTCCATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGGGGGGGGTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA;

[0091] The gene sequence encoding the light chain variable region is:

[0092] GACATTTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATC TATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGGA.

[0093] The N protein monoclonal antibody 3B3 can specifically recognize the region of the porcine reproductive and respiratory syndrome virus N protein: PEKPHFPL.

[0094] In summary, the present invention provides a monoclonal antibody against the N protein of porcine reproductive and respiratory syndrome virus, which can specifically recognize and bind to 50-57aa of the N protein, and can react with a variety of strains such as classical PRRSV, HP-PRRSV, NADC30-like PRRSV, NADC34-like PRRSV and European PRRSV, and has good reactivity and conservatism. The monoclonal antibody provided by the present invention can be obtained by conventional genetic engineering or protein engineering methods, avoiding the loss of antibodies during long-term cryopreservation of hybridoma cells, and is also conducive to optimizing antibodies at the gene and protein levels, thereby improving the specificity and affinity of the antibodies. The monoclonal antibody prepared by the present invention provides a reliable tool for the study of the pathogenic mechanism of PRRSV, the development of PRRSV diagnostic kits, and the development and differential diagnosis of new genetic engineering marker vaccines based on reverse genetic manipulation technology.

[0095] The embodiments described above are only partial embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structures, features and principles described in the patent scope of the present invention should be included in the patent application scope of the present invention.

Claims

1. A porcine reproductive and respiratory syndrome virus N protein antigen epitope peptide, characterized in that: The antigen epitope peptide is located at 50-57aa of the PRRSV N protein, and its amino acid sequence is shown in SEQ ID No.

11.

2. Use of the antigen epitope peptide as claimed in claim 1 in the preparation of monoclonal antibodies against porcine reproductive and respiratory syndrome virus N protein.

3. Application of silencing the expression of PRRSV N protein 50-57aa in the preparation of PRRSV genetically engineered marker vaccine.

4. A monoclonal antibody against porcine reproductive and respiratory syndrome virus N protein, 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 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 CDR1 shown in the amino acid sequence of SEQ ID No.4, CDR2 shown in SEQ ID No.5 and CDR3 shown in SEQ ID No.

6.

5. The monoclonal antibody according to claim 4, 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.

6. A nucleic acid, characterized in that The nucleic acid encodes the antibody heavy chain and antibody light chain of the monoclonal antibody according to claim 4 or 5.

7. The nucleic acid according to claim 6, wherein 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.

8. Use of the monoclonal antibody according to claim 4 or 5 in the preparation of a reagent for detecting porcine reproductive and respiratory syndrome virus.

9. Use of the monoclonal antibody according to claim 4 or 5 in detecting the infectivity of porcine reproductive and respiratory syndrome virus for non-disease diagnosis purposes, or in studying the function of porcine reproductive and respiratory syndrome virus N protein.

10. A porcine reproductive and respiratory syndrome virus detection kit, characterized in that: The kit comprises the monoclonal antibody according to claim 4 or 5.

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