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

Abstract

The present invention discloses an antigenic epitope peptide of the N protein of porcine reproductive and respiratory syndrome virus and a monoclonal antibody thereof. 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 shown in SEQ ID No. 1-3; the light chain variable region includes CDR1-3 shown in SEQ ID No. 4-6; the monoclonal antibody reacts with a variety of PRRSV strains, including classical PRRSV, HP-PRRSV, NADC30-like PRRSV, NADC34-like PRRSV, and European PRRSV, and has good conservatism; and can be obtained by conventional genetic engineering and other biological means, avoiding the problem of easy loss of antibody genetic sequences caused by long-term freezing of traditional hybridoma cells; and is also conducive to optimizing the antibody at the gene or protein level, further improving the specificity and affinity of the antibody.

Description

Technical Field

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

[0002] Porcine reproductive and respiratory syndrome (PRRS), caused by the porcine reproductive and respiratory syndrome virus (PRRSV), is a major zoonotic disease affecting the global swine industry. PRRSV is one of the fastest-mutating RNA viruses known. Currently, my country is experiencing a multi-strain epidemic, with NADC30-like PRRSV as the predominant strain, NADC34-like PRRSV and European PRRSV (PRRSV-1) becoming increasingly prevalent, and HP-PRRSV continuing to circulate.

[0003] PRRSV belongs to the family Arteriviridae. Its genome is approximately 15 kb long 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 approximately 75% of the 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) involved in viral replication. ORF2-ORF7, located at the 3' end of the PRRSV genome, account for approximately the remaining 25% of the genome and encode eight structural proteins: GP2, E, GP3, GP4, GP5, GP5a, M, and N. N is the nucleocapsid protein of PRRSV, consisting of 128 or 123 amino acids and approximately 15 kDa in size. The N protein is highly conserved, with greater than 96% identity within the same PRRSV genotype. The N protein is also the most abundant viral protein in PRRSV, accounting for over 40% of the total protein. Its immunogenicity and antigenicity are exceptional, making it the first viral protein to induce antibody production. Therefore, the N protein is a key viral protein for the early detection and diagnosis of PRRSV. Monoclonal antibodies targeting the N protein play a vital role in both fundamental PRRSV research and the development of prevention and control products.

[0004] Genetically marked vaccines are an important tool for eradicating major animal diseases, allowing serological differentiation of marked vaccine strains from circulating 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). To effectively control PRV, genetically marked PRV vaccine strains lacking the non-essential glycoprotein gene gI have been developed. This lack of the gI gene prevents piglets from producing antibodies against gI, and can serve as a distinguishing marker between vaccine strains and wild-type strains. Currently, several pig farms have achieved complete eradication of PRV using genetically marked PRV vaccines. Current research has shown that PRRSV lacks any structural proteins, and the absence of any single structural protein prevents the production of infectious virions. Therefore, constructing a genetically marked vaccine strain by deleting an entire gene is not feasible for PRRSV. An alternative strategy is to use reverse genetic manipulation to silence key antigenic epitopes in PRRSV's dominant immune genes, rendering them unable to induce corresponding antibodies, thereby distinguishing them from wild-type strains or currently commercialized vaccine strains and promoting PRRSV purification. Therefore, preparing broad-spectrum and highly effective antibodies against PRRSV's dominant immune proteins, such as the N protein, and identifying the key immune epitopes they recognize can provide a key research foundation and research materials for the development of genetically engineered PRRSV marker vaccines that lack key antigenic epitopes.

[0005] This invention uses the N protein of the NADC30-like PRRSV, currently prevalent in my country, as a basis for the preparation of a specific monoclonal antibody targeting the N protein. This monoclonal antibody not only recognizes the NADC30-like PRRSV, but also exhibits good reactivity with a variety of PRRSV strains, including classical PRRSV, HP-PRRSV, NADC34-like PRRSV, and European PRRSV. This monoclonal antibody specifically recognizes and binds to the 50-57 aa region 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. It also provides key research material for the development of novel genetically engineered marker vaccines based on reverse genetic manipulation techniques. Summary of the Invention

[0006] In response to the above technical problems, the purpose 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 an antigenic epitope peptide of the N protein of porcine reproductive and respiratory syndrome virus, the amino acid sequence of the antigenic epitope peptide being shown as SEQ ID No.11.

[0008] In a second aspect, the present invention provides the use of the antigenic 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 50-57aa of the PRRSV N protein in the preparation of a genetically engineered PRRSV marker vaccine.

[0010] In a fourth aspect, the present invention provides a monoclonal antibody against the N protein of porcine reproductive and respiratory syndrome virus, 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 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 detecting porcine reproductive and respiratory syndrome virus infectivity for non-disease diagnosis purposes, or in studying 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, which comprises the monoclonal antibody described in the fourth aspect.

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

[0022] The present invention has the following beneficial effects: the present invention uses a prokaryotic expression system to express and purify recombinant PRRSV N protein, which is used 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 multiple PRRSV strains, such as classical PRRSV, HP-PRRSV, NADC34-like PRRSV, and European PRRSV, and has good conservation; the PRRSV ORF7 gene is gradiently truncated and expressed, and it is found that the region of the PRRSV N protein that the antibody can specifically recognize and bind to is aa50-57, a total of 8 amino acids, providing a reliable research tool for exploring the function of PRRSV N, PRRSV diagnostic reagents, and the development and differential diagnosis of new genetically engineered marker vaccines based on reverse genetic manipulation technology; 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 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

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

[0025] Figure 2 .The titer of serum from mice immunized with pET28a-N protein was determined;

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

[0027] Figure 4 IFA identification of 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 sites of PRRSV N protein recognized by monoclonal antibodies. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below by specific embodiments. However, it will be understood 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, where specific technical operation steps or conditions are not indicated in the examples, they are all performed according to the technology or conditions described in the general literature in this area or according to the product specifications. Reagents or instruments used that do not indicate the manufacturer are all conventional products that can be obtained commercially.

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

[0033] The ORF7 gene of the NADC30-like PRRSV SX-YL1806 strain was optimized for Escherichia coli. The optimized nucleotide sequence is shown in SEQ ID No. 12. The optimized sequence was amplified and ligated into the pET28a prokaryotic expression vector to construct the pET28a-PRRSV ORF7 recombinant plasmid. The recombinant plasmid was then identified using double enzyme digestion. The specific steps are as follows:

[0034] 1.1 Primer design

[0035] The primers for PRRSV ORF7 amplification are shown in Table 1 , where the underlined sequences correspond to restriction enzyme cleavage 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 synthesized pUC57-PRRSV-ORF7 plasmid as a template, the target gene was amplified by PCR using the above primers. The PCR amplification system and reaction procedures are shown in Tables 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 amplified target gene and laboratory-stored pET28a empty vector were digested with two enzymes. The target gene and digested vector were then ligated and transformed into DH5a competent cells. Colony PCR was performed on selected clones using the primers listed in Table 1. Positive clones were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Plasmids were extracted from the clones that were sequenced correctly and frozen for future use.

[0047] Sequencing results showed that the recombinant plasmid sequence was consistent with the expected sequence. The recombinant plasmid was then digested with two restriction endonucleases, Nco I and Xho I, for enzyme digestion verification, 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 was picked and cultured in 1 mL of liquid LB medium containing kanamycin resistance overnight; the next day, the bacterial solution was inoculated into 10 mL of fresh culture medium at a ratio of 1:100 and cultured with shaking at 37°C and 220 rpm; a portion of the bacterial solution was sampled every hour to measure the OD value. 600 Value, waiting for OD 600When the value reached 0.4-0.6, 1 mmol / L IPTG was added to induce expression; after 6 h of induction, the bacterial solution was collected and centrifuged at 8000 rpm for 5 minutes to collect the precipitate; the precipitate was resuspended in 10 mL PBS, the bacteria were ultrasonically disrupted, and the precipitate and supernatant were collected separately after centrifugation; the supernatant was directly added to 2× loading buffer, and the precipitate was dissolved in 8 M 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 inclusion body protein is large and easy to purify, so the inclusion body protein is selected for further purification. Collect the bacterial pellet after ultrasonic disruption, resuspend and dissolve in equilibrium buffer, centrifuge at 4°C, 4000 rpm, and 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 equilibrate the purification column twice with 5 column volumes of equilibration solution (8 M Urea, 10 mM NaH2PO4, 10 mM Trisbase, pH = 8.6);

[0054] (2) Add the prepared sample to the affinity chromatography column and incubate at 4°C overnight or for 6-8 hours. Elute the target protein with eluents containing different concentrations of imidazole, such as 10 mM, 50 mM, 100 mM, 250 mM, 500 mM, 1 M, and 2 M. Add 2× loading buffer and perform SDS-PAGE identification.

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

[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 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 Biolong water adjuvant in a ratio of 1:1 and the mice were immunized subcutaneously 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 and 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 collected to determine the antibody titer. Before fusion, a booster immunization was performed, and the PRRSV N recombinant protein was injected intraperitoneally at a dose of 30 μg / mouse into the mice to be subjected to cell fusion for sensitization.

[0061] (2) Cell fusion: Resuscitate SP2 / 0 myeloma cells, passage 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 plated into 5 96-well cell plates. Mice with high serum titers were selected for B cell isolation. The spleens of the mice were aseptically removed and repeatedly injected and flushed with 1640 culture medium containing 1% penicillin-streptomycin-amphotericin (triple antibodies) until the internal B cells were flushed into the culture medium; the spleens were then 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 advance, discard the original culture medium, add 1640 culture medium, photograph 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 was coated on an ELISA reaction plate. The positive wells containing hybridoma cells were selected and the supernatant was collected and added to the ELISA reaction plate for titer determination. The wells with high reactivity were selected for subcloning. Subcloning was repeated three times until monoclonal hybridoma cells with good reactivity were screened. Monoclonal hybridoma cells with high titer and good reactivity were expanded and frozen.

[0063] ELISA titer determination results Figure 2 As shown, the serum titers gradually increased after immunization, with all five serum titers ≥1:128,000 and a P / N ratio >2, indicating that the serum titers of the five mice were 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 mouse abdomen becomes noticeably heavy and fluctuates when touched, collect ascites and freeze.

[0065] (5) Purification of Ascites: The ascites was purified according to the instructions of the Protein Iso Protein G Resin kit from TRAN. The purified sample was identified by SDS-PAGE. The resulting 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 onto an ELISA reaction plate at 100 ng / well and incubated at 37°C for 2 hours or at 4°C overnight. After washing with PBS, 5% skim milk powder was added for blocking. The PRRSV N monoclonal antibody 3B3 prepared above was serially diluted and added to the coated plate. The plate was reacted at 37°C for 1 hour and washed four times with PBS. HRP-labeled goat anti-mouse IgG (1:10,000 dilution) was added and the plate was reacted at 37°C for 1 hour and washed four times with PBS. TMB color development was performed for 15 minutes, and then the stop solution was added. The OD was measured on a microplate 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. PAMs were inoculated with NADC34-like PRRSV and European PRRSV. Cells were harvested 24 hours later for IFA analysis. The primary antibody used was the previously prepared PRRSV N monoclonal antibody 3B3, and the secondary antibody was FITC-conjugated 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, including 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] The subtype identification of the obtained PRRSV N protein monoclonal antibody was performed according to the operating instructions of the Mouse Monoclonal Antibody Isolation Kit (Proteintech).

[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 in a gradient manner and then 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 sequence was gradually shortened until the key antigen epitope to which it binds was 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 Monoclonal Antibody Variable Region Gene Sequencing

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

[0080] Nested PCR was used to amplify the variable region genes of the antibody. Using the above cDNA as a template, the variable region genes of the antibody were amplified using the first round of mouse antibody IgG2a and kappa light chain primers. Then, using the first round product as a template, the variable region genes of the antibody were amplified using the second round of mouse antibody IgG2a and kappa 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.).

[0081] 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 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. The monoclonal antibody can specifically recognize and bind to 50-57aa of the N protein, and can react with multiple strains such as classical PRRSV, HP-PRRSV, NADC30-like PRRSV, NADC34-like PRRSV and European PRRSV, with good reactivity and conservatism. The monoclonal antibody provided by the present invention can be obtained using conventional genetic engineering or protein engineering methods, avoiding the loss of antibodies during long-term cryopreservation of hybridoma cells. It is also beneficial to optimize the antibodies at the gene and protein levels, thereby improving the specificity and affinity of the antibodies. The monoclonal antibodies prepared by the present invention provide 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 genetically engineered marker vaccines based on reverse genetic manipulation technology.

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

Claims

1. 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 the amino acid sequence of 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.

2. The monoclonal antibody according to claim 1, wherein 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 according to claim 1 or 2.

4. The nucleic acid according to claim 3, 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.

5. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a reagent for detecting porcine reproductive and respiratory syndrome virus.

6. Use of the monoclonal antibody according to claim 1 or 2 in detecting porcine reproductive and respiratory syndrome virus infectivity for non-disease diagnosis purposes.

7. A porcine reproductive and respiratory syndrome virus detection kit, characterized in that: The kit comprises the monoclonal antibody according to claim 1 or 2.

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