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

Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus M 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 plurality of PRRSV strains such as HP-PRRSV, NADC30PRRSV, NADC34PRRSV 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 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 M 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, which is 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. The PRRSV V proteins are highly conserved, non-glycosylated membrane proteins consisting of 174 amino acids. They contain multiple dominant antigenic epitopes, rapidly inducing antibody production and serving as important viral targets for serological diagnosis and vaccine development. Therefore, monoclonal antibodies against M protein play an important role in basic theoretical research on PRRSV and development of prevention and control products.

[0004] Genetically marked vaccines are a crucial tool for eradicating major animal diseases, enabling serological differentiation of marked vaccine strains from circulating or vaccine strains. Currently, several pig farms have achieved complete eradication of PRV using genetically marked vaccines. However, current research has shown that PRRSV lacks any missing 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 involves using reverse genetic manipulation to silence key antigenic epitopes within the PRRSV dominant immune gene, rendering them incapable of inducing antibodies. This, in turn, allows for differentiation from wild-type strains or currently commercialized vaccine strains, thereby facilitating PRRSV eradication. By generating broadly and highly effective antibodies against PRRSV dominant immune proteins, such as the M protein, and identifying the key immune epitopes they recognize, this strategy provides a critical research foundation and materials for the development of genetically engineered PRRSV vaccines lacking these key epitopes. Summary of the Invention

[0005] To address the above technical issues, the present invention uses the NADC30-like PRRSV M protein, currently prevalent in my country, as a basis to prepare a specific monoclonal antibody against the M protein. The monoclonal antibody not only recognizes NADC30-like PRRSV, but also exhibits good reactivity with multiple PRRSV strains, including HP-PRRSV and NADC34-like PRRSV. Furthermore, the monoclonal antibody specifically recognizes and binds to aa 94-119 of the PRRSV M protein, providing a reliable tool for exploring the function of the PRRSV M protein and the development of PRRSV diagnostic reagents, and providing key research materials for the development of new genetically engineered marker vaccines based on reverse genetic manipulation techniques. Specifically, the invention includes the following:

[0006] In a first aspect, the present invention provides a monoclonal antibody against porcine reproductive and respiratory syndrome virus M protein, 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 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 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.

[0009] 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 NO. 8.

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

[0011] 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.

[0012] In a third aspect, the present invention provides an expression cassette, an expression vector, and a recombinant bacterium containing the nucleic acid described in the second aspect.

[0013] In a fourth aspect, the present invention provides use of the monoclonal antibody described in the first aspect in the preparation of a reagent, a test strip or a kit for detecting porcine reproductive and respiratory syndrome virus.

[0014] In a fifth aspect, the present invention provides the use of the monoclonal antibody described in the first aspect in detecting porcine reproductive and respiratory syndrome virus infectivity for non-disease diagnosis purposes.

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

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

[0017] Preferably, the kit further comprises an ELISA plate, a blocking solution, a diluent, a washing solution, a color developer, and a stop solution.

[0018] In an eighth aspect, the present invention provides a method for preparing the monoclonal antibody described in the first aspect, which comprises: cloning the porcine reproductive and respiratory syndrome virus ORF6 gene into the pET-21b 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 M 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 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 a monoclonal antibody against porcine reproductive and respiratory syndrome virus M protein.

[0019] In the ninth aspect, the present invention provides an antigenic epitope peptide of the M protein of porcine reproductive and respiratory syndrome virus, 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 the expression of PRRSV M protein 94-119aa in preparing a PRRSV genetically engineered marker vaccine.

[0021] The present invention has the following beneficial effects: the present invention uses a prokaryotic expression system to express and purify recombinant PRRSV M protein, which is used as an immune source to immunize mice, and successfully obtains a monoclonal antibody against PRRSV M protein through cell fusion and subcellular screening; the prepared monoclonal antibody can react with multiple PRRSV strains such as HP-PRRSV, NADC30-like PRRSV, and NADC30-like PRRSV, and has good conservation; through gradient truncation expression of the PRRSV ORF6 gene, it is found that the region of the PRRSV M protein that the antibody can specifically recognize and bind to is 26 amino acids, 94-119aa, and provides a reliable research tool for exploring the function of PRRSV M, 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

[0022] 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.

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

[0024] Figure 2 The titer of serum from mice immunized with pET21b-M protein was determined;

[0025] Figure 3 The titer determination results of the prepared monoclonal antibodies;

[0026] Figure 4WB identification of the reactivity of monoclonal antibodies to various PRRSV strains;

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

[0028] Figure 6 Schematic diagram of the construction of PRRSVM protein truncations;

[0029] Figure 7 Identification results of the PRRSVM protein site recognized by monoclonal antibodies. DETAILED DESCRIPTION

[0030] 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.

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

[0032] Sequence analysis of the 174 amino acids of the NADC30 PRRSV SX-YL1806 ORF6 gene revealed that 1-93aa comprised the transmembrane region. After deleting the transmembrane domain gene sequence, a membrane-penetrating peptide sequence was added to the N-terminus of the remaining gene sequence as the target gene and optimized for Escherichia 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, which was then identified using double enzyme digestion. The specific steps are as follows:

[0033] 1.1 Primer design

[0034] The primers for PRRSV ORF6 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.

[0035] Table 1 PRRSV ORF6 amplification primers

[0036]

[0037] 1.2 PCR amplification

[0038] Using the optimized synthesized pUC57-PRRSV-ORF6 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 Table 2-3 respectively.

[0039] Table 2 Target gene PCR amplification system

[0040] Reagents Volume (μL) PrimeSTARGXLDNA Polymerase 2 dNTP 4 PrimeSTARGXLBuffer 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. The target gene and the 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.

[0045] Sequencing results showed that the recombinant plasmid sequence was consistent with the expected sequence. The recombinant plasmid was then digested with two restriction endonucleases, BamHI and Xho I, for enzyme digestion verification, and the result was consistent with the expected sequence.

[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), 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 600 When 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.

[0049] Purification of PRRSV M protein

[0050] M 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 precipitate after ultrasonic disruption, resuspend and dissolve it in the equilibrium buffer, centrifuge at 4°C, 4000 rpm, and collect the supernatant. Filter it through a 0.45μm filter and purify it:

[0051] (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);

[0052] (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.

[0053] (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.

[0054] (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.

[0055] The results of Pet21b-M protein expression, purification and identification are as follows Figure 1 As shown, the PRRSV M protein was successfully expressed and purified, and the size was consistent with the expected value.

[0056] Example 3 Preparation, Reactivity and Variable Region Sequence Determination of Monoclonal Antibodies

[0057] 3.1 Preparation of monoclonal antibodies

[0058] (1) Animal immunization: Five female BALB / c mice aged 6 to 8 weeks were selected and the PRRSV M recombinant protein obtained above was immunized three times according to the immunization cycle. For the first immunization, the PRRSV M 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 M 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 M recombinant protein was injected intraperitoneally at a dose of 30 μg / mouse into the mice to be subjected to cell fusion for sensitization.

[0059] (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.

[0060] (3) Screening and determination of monoclonal hybridoma cells: The PRRSVM recombinant protein prepared above was coated on an ELISA reaction plate, and the supernatant of the positive wells containing hybridoma cells was collected and added to the ELISA reaction plate for titer determination. The wells with higher reactivity were selected for subcloning. Subcloning was performed three times in succession until monoclonal hybridoma cells with good reactivity were screened. Monoclonal hybridoma cells with high titer and good reactivity were expanded and frozen.

[0061] 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.

[0062] (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 67-10 days after immunization, when the mouse abdomen becomes noticeably heavy and fluctuates when touched, collect ascites and freeze.

[0063] (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 M protein-specific antibody was named 5-F8-D11.

[0064] 3.2 Identification of Monoclonal Antibody Reactivity

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

[0066] The PRRSV M 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 M monoclonal antibody 5-F8-D11 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.

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

[0068] (2) Western blot analysis of the reactivity of monoclonal antibodies with various PRRSV strains

[0069] HP-PRRSV, NADC30-like PRRSV, and NADC34-like PRRSV were inoculated into PAMs. Cells were harvested 24 hours later for Western blotting. The primary antibody used was the previously prepared PRRSV monoclonal antibody 5-F8-D11, and the secondary antibody was HRP-conjugated goat anti-mouse.

[0070] The results of the reactivity test were as follows Figure 4 As shown, WB results showed that the prepared monoclonal antibodies could detect multiple PRRSV strains such as HP-PRRSV, NADC30-like PRRSV and NADC34-like PRRSV, with good specificity and conservation.

[0071] (3) Identification of monoclonal antibody subtypes

[0072] The subtype identification of the obtained PRRSV M protein monoclonal antibody was performed according to the operating instructions of the Mouse Monoclonal Antibody Lsotyping Kit antibody subtype identification kit of Proteintech.

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

[0074] (4) Identification of protein epitopes bound by monoclonal antibodies: The PRRSV ORF6 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 M protein monoclonal antibody was used to perform Western blot reaction with the truncated protein samples, and the protein epitopes were gradually shortened until the key antigen epitopes to which it binds were finally identified. The gradient truncation construction strategy is as follows: Figure 6 shown.

[0075] The epitope identification results of monoclonal antibody 5-F8-D11 are as follows Figure 7 As shown, the key region of the surface monoclonal antibody 5-F8-D11 that specifically binds to the PRRSV M protein is B1, i.e., 94-119aa. 94 FITSRCRLCLLGRKYILAPAHHVESA 119 , a total of 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 frozen 5-F8-D11 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.

[0078] 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 IgG1 and κ 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 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 sequences.

[0080] 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.

[0081] Table 4 Antibody variable region sequences

[0082]

[0083] Among them, the amino acid sequence of the heavy chain variable region is:

[0084] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYIRYSGNT DYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAGPVWGQGTTLTVSS;

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

[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 M protein of porcine reproductive and respiratory syndrome virus: FITSRCRLCLLGRKYILAPAHHVESA.

[0092] In summary, the present invention provides a monoclonal antibody against the M protein of porcine reproductive and respiratory syndrome virus. The monoclonal antibody can specifically recognize and bind to 94-119aa of the M protein, and can react with multiple strains such as HP-PRRSV, NADC30-like PRRSV, and NADC34-like 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.

[0093] 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 M 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.

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. Use of the monoclonal antibody according to claim 1 or 2 in the study of the function of porcine reproductive and respiratory syndrome virus M protein for non-disease diagnosis purposes.

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

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

11.

10. Application of silencing the expression of PRRSV protein 94-119aa in the preparation of PRRSV genetically engineered marker vaccine.

Images