Anti-PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) Nsp4 protein monoclonal antibody with inhibition effect and application thereof

Abstract

The invention provides an anti-PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) Nsp4 protein monoclonal antibody with an inhibition effect and application thereof, a hybridoma cell strain capable of stably secreting the anti-Nsp4 protein monoclonal antibody is prepared, and a new antigen epitope is identified in nsp4 through expressed overlapping truncated protein. Wherein the 123ITEAGELV130 identified by the monoclonal antibody 1C11 shows the potential of universally identifying different types of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus). Meanwhile, expression of Nsp4 protein can be detected in the early stage of PRRSV infection by utilizing the monoclonal antibody 1C11, the monoclonal antibody has a blocking effect on PRRSV positive serum, and it is found that the monoclonal antibody 1C11 can inhibit proliferation of PRRSV in vitro. Research results increase the understanding of PRRSV Nsp4 protein structure and antigen characteristics, and provide a basis for PRRSV early antigen detection.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a monoclonal antibody against PRRSV Nsp4 protein with inhibitory effect and its application, as well as its application in the study of the relevant mechanisms of PRRSV neutralization. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which severely impacts the global pig industry. Clinically, it can cause reproductive disorders in sows and respiratory distress in pigs at all growth stages, resulting in enormous economic losses to the global pig industry. PRRSV belongs to the order Nidovirales, family Arterioviridae, and genus Arteriovir, and is an enveloped, single-stranded, positive-sense RNA virus. PRRSV can be divided into two genotypes: the European type (PRRSV-1) and the North American type (PRRSV-2). The predominant strain in China is PRRSV-2, mainly including strains of lineages 1, 3, 5, and 8. Due to the wide antigenic variation of PRRSV clinically, the genetic diversity of strains poses challenges to the accurate diagnosis and vaccination strategies for PRRSV.

[0003] The PRRSV genome is approximately 15 kb long and contains at least 11 open reading frames (ORFs). ORF1a and ORF1b encode the multiprotein pp1a, which, after frameshifting, produces pp1ab. This pp1ab is then cleaved by the viral PL and 3CL proteases into at least 13 mature non-structural proteins (Nsps), participating in the regulation of the viral replication lifecycle. Among these, Nsp4, a 3C-like protease (3CLpro) encoded by PRRSV, is considered the most important viral protease, responsible for cleaving nsp3–nsp12 into mature Nsps, promoting viral particle assembly and maturation. Similarly, similar protease structures exist in various RNA viruses, including coronaviruses, caliciviruses, and alphaviruses, capable of processing viral pp1a / pp1ab into mature Nsps. This indicates that the Nsp4 protein plays an irreplaceable role in viral replication. Furthermore, studies have confirmed that the Nsp4 protein participates in regulating the host's innate immune response; Nsp4 can antagonize the host's antiviral immune response by cleaving molecules such as VISA, NEMO, ZAP, DCP1a, and GBP1. Nsp4 has been found to play an important role in host metabolic reprogramming. Other studies have reported that Nsp4 is also involved in regulating biological processes such as apoptosis, endoplasmic reticulum stress, and autophagy. However, current information on Nsp4 protein-induced immune responses remains limited. Summary of the Invention

[0004] This study prepared a monoclonal antibody using Nsp4 protein as an immunogenic antigen and identified a new antigenic epitope in Nsp4 protein, revealing the potential of monoclonal antibodies against Nsp4 protein for the differential diagnosis of PRRSV. This provides an effective tool for understanding the antigenic characteristics of Nsp4 and for the early diagnosis of PRRSV.

[0005] Specifically, this study screened and obtained a monoclonal antibody 1C11 that targets the PRRSV Nsp4 protein and has neutralizing activity; Furthermore, the present invention provides a monoclonal antibody that can simultaneously and specifically recognize the Nsp4 protein of all PRRSV-2 representative strains and also recognize the Nsp4 protein of PRRSV-1 representative strains.

[0006] Furthermore, the present invention provides a monoclonal antibody that can inhibit viral replication to a certain extent.

[0007] Preferably, the antibody is a monoclonal antibody 1C11, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as follows: MNFGLSLIFLALILKGWEFEVQLQESGGGLVKPGGSLKLSCAASGFTFS TYVMS WVRQIPEKRLEWVA SISSGGSYTYYPDSVKG RFTISRDNARNILYLQMSSLRSEDTAMYYCAR GDPPYGDFAY WGQGTTLTVSS(SEQ IDNO.3); The amino acid sequence of the light chain variable region is as follows: MGIKMESQTQVQLPANQCFSHNVQRTNCSLQSPAILSASPGEKVTMTC RASSSVNYMH WYQQKPGSSPKPWIY DTSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYC QQWSRNPWT FGGGTKLEIKRADAAPTVS (SEQID NO.4), where the underlined part is CDR1-3.

[0008] Another aspect of this invention elucidates the mechanism of action of the neutralizing antibody and identifies its key binding site with the antigen. This invention provides a novel antigenic epitope in the PRRSV Nsp4 protein, epitope 123ITEAGELV130, which is a novel antigenic epitope first identified in the Nsp4 protein; this epitope is located in its domain II (89-153aa). Structural analysis of different PRRSV Nsp4 protein lineages revealed that this epitope exhibits good conservation among different strains, demonstrating its potential for detecting different PRRSV-2 lineages.

[0009] Beneficial effects In this study, we prepared a monoclonal antibody that recognizes different epitopes of the Nsp4 protein, using the expressed Nsp4 protein as the antigen. Epitope 123ITEAGELV130 is a novel antigenic epitope identified for the first time in the Nsp4 protein. Notably, this epitope is located in its domain II (89-153aa). Structural analysis of the PRRSV Nsp4 protein from different lineages revealed that this epitope is well conserved among different strains, demonstrating its potential for detecting different lineages of PRRSV-2. Particularly noteworthy is that, despite the low homology of the Nsp4 amino acid sequences between PRRSV-2 and PRRSV-1, the monoclonal antibody 1C11 specifically recognizes both all representative PRRSV-2 strains and representative PRRSV-1 strains. Furthermore, we found that the monoclonal antibody 1C11 has a certain inhibitory effect on viral replication. Although the specific mechanism of its inhibitory effect on viral proliferation is unclear, this result suggests the potential for the development and application of monoclonal antibodies targeting the Nsp4 protein in antiviral formulations. In summary, this study enhances our understanding of the antigenic properties of the PRRSV Nsp4 protein, providing new evidence for understanding the Nsp4 immune recognition mechanism and developing diagnostic tools based on antigenic epitopes. Attached Figure Description

[0010] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0011] Figure 1 The expression and identification of Nsp4 protein includes: (A) PCR amplification of the Nsp4 gene; (B) SDS electrophoresis analysis of Nsp4 expressed protein; (C) Western spectroscopy detection of Nsp4 expressed protein with tag antibody; and (D) Western spectroscopy detection of positive serum antibody against Nsp4 expressed protein. Figure 2 Screening and identification of anti-PRRSV Nsp4 monoclonal antibodies: (A) Indirect immunofluorescence detection of monoclonal antibodies; (B) Monoclonal antibodies can recognize Nsp4 eukaryotic transfected protein; (C) Monoclonal antibodies can recognize PRRSV-infected cells expressed protein.

[0012] Figure 3 Identification of monoclonal antibody epitopes: (A) Western spectral analysis of Nsp4 truncated protein expressed by monoclonal antibody; (B) Western spectral analysis and ELISA analysis of Nsp4 (115-130 aa) truncated protein expressed by monoclonal antibody; (C) Western spectral analysis and ELISA analysis of Nsp4 (121-130 aa) truncated protein expressed by monoclonal antibody.

[0013] Figure 4 The results are: (A) IFA identification of the monoclonal antibody 1C11 against different PRRSV strains; (B) Western spectroscopy identification of the monoclonal antibody against cells infected with different PRRSV strains.

[0014] Figure 5 The results showed that monoclonal antibody 1C11 inhibited PRRSV replication. (A) IFA detection of monoclonal antibody 1C11 incubated with rHuN4-F112 virus; (B) detection of viral nucleic acid copy number; (C) determination of viral proliferation titer. Detailed Implementation

[0015] In the following examples, experimental methods without specific conditions are generally performed under conventional conditions, such as those described in "A Concise Guide to Molecular Biology Experiments" (edited by FM Osber, RE Kingston, JG Seidman, et al., translated by Ma Xuejun and Shu Yuelong. Beijing: Science Press, 2004).

[0016] Cells, plasmids, virus strains and laboratory animals Marc-145 cell line (African green monkey kidney epithelial cell line), human embryonic kidney HEK293T cell line, and myeloma cell line SP2 / 0 cell line were maintained in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum at 37°C and 5% CO2. E. coli BL21(DE3) was purchased from Shanghai Sangon Biotech Co., Ltd. pcoldI-TF and pCAGGS-Nsp4-Myc were preserved in this laboratory. PRRSV... The HuN4 strain (GenBank: EF635006), CH-1a strain (GenBank: AY032626.1), and PRRSV-1 are stored in our laboratory. The HuN4-F112 strain is an attenuated vaccine strain of HuN4 after 112 consecutive passages in Marc-145 cells, prepared and stored in our laboratory. The NADC30-like strains ZJhz2021 and HBap4-2018 (GenBank: MZ579701.1) were also used. 4-6 week old BALB / c mice were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. All mouse experiments were approved by the Animal Ethics Committee of the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, and were conducted in accordance with the approved animal ethics guidelines and procedures in the biosafety level 2 laboratory of the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

[0017] Antibodies and serum The monoclonal antibodies mouse anti-His antibody (66005-1), mouse anti-β-actin antibody (60008–1), horseradish peroxidase (HRP) conjugated anti-mouse IgG antibody (SA00001-1), and HRP conjugated anti-rabbit IgG antibody (SA00001-2) were purchased from Peoteintech Group; the rabbit anti-Myc tag antibody (AE070), mouse anti-GST antibody (AE001), and rabbit anti-CALR antibody (A1006) were purchased from ABclonal Technology; the goat anti-mouse IgG fluorescent secondary antibody conjugated with Alexa Fluor 488 (A21207) and the goat anti-rabbit IgG fluorescent secondary antibody conjugated with Alexa Fluor 594 (A11029) were purchased from Thermofisher; the monoclonal antibody PRRSV N was prepared and stored in our laboratory; and the positive sera from the PRRSV challenge group and the negative group at 0, 7, 14, 21, and 28 days were stored in our laboratory.

[0018] Construction of Nsp4 recombinant protein expression plasmid Based on the Nsp4 gene sequence of the PRRSV HuN4 isolate, a forward primer pCold-TF-HuN4-Nsp4-F: 5'-ATGGAGCTCGGTACC was designed. CTCGAG GGCGCTTTCAGAAC-3' (SEQ ID NO.1, the underlined part is the XhoI restriction site) and reverse primer pCold-TF-HuN4-Nsp4-R:5'-ACTGCAGGTCGAC AAGCTT The gene fragment CTATTCCAGTTCGG-3' (SEQ ID NO.2, underlined area indicates HindIII restriction site) was cloned into the pcoldI-TF vector (double restriction sites are XhoI and HindIII) to generate the recombinant plasmid pcoldI-TF-Hu-Nsp4. The corresponding gene fragments were then cloned into the eukaryotic vector pCAGGS and the prokaryotic expression vector PGEX-6P-1, respectively, to prepare eukaryotic expression plasmids and recombinant E. coli proteins. All recombinant plasmids used in this study were validated by DNA sequencing.

[0019] Prokaryotic expression, identification and purification of Nsp4 recombinant protein Take 200 μL of correctly sequenced bacterial culture and inoculate it into a conical flask containing 200 mL of ampicillin-resistant LB broth. Incubate at 37°C and 200 rpm for OD. 600When the β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mol / L, the culture was carried out at 16℃ and 180 r / min for 16 h, centrifuged, and the bacterial pellet was collected. The pellet was washed with PBS and resuspended, and then sonicated on ice. The supernatant was collected and incubated with QIAGEN Ni-NTA affinity chromatography medium overnight at 4℃. The target protein was eluted with imidazole buffer of different concentrations. The protein was then analyzed by SDS-PAGE and identified by Western blot using HuN4 positive serum and His-tagged antibody. After successful identification, the protein was quantified, the concentration was determined, and the protein was stored at -80℃. The results showed that the Nsp4 gene of PRRSV HuN4 strain was cloned into the pcold-TF expression vector, and the target fragment of approximately 612 bp was obtained by PCR amplification, which was consistent with the expectation. Figure 1 A). Positive strains were induced with IPTG at 16℃ for 16 h. The supernatant from cell lysis was purified by nickel column chromatography and then detected by SDS-PAGE electrophoresis as a soluble TF-Nsp4 fusion protein of approximately 73 kDa. Figure 1 B). The protein was detected by Western blot using His-tagged protein antibody and PRRSV-positive porcine serum as primary antibodies. The results showed that both antibodies could specifically react with the recombinant protein (Figure 1C, D), indicating that soluble PRRSV Nsp4 fusion protein was obtained.

[0020] Mouse immunization and hybridoma cell preparation The concentration of purified Nsp4 protein was determined. 100 μg of recombinant protein was mixed with an equal volume of Freund's complete adjuvant (for initial immunization) or incomplete adjuvant and emulsified. BALB / c mice were injected subcutaneously at multiple sites. Starting 14 days after immunization, tail vein blood was collected every 7 days. Serum was separated by centrifugation, and serum antibody levels were detected using indirect ELISA and IFA. Once the ELISA titer reached 1:100,000, mice were boosted with tail vein injection of TF-Nsp4 recombinant protein. Three days later, spleens from these mice were harvested for cell fusion. The boosted mouse spleen cells were fused with SP2 / 0 cells. Positive hybridoma cells were selected using a preferential dilution method and subjected to at least three subclonal cultures. Indirect ELISA was used to screen for monoclonal hybridoma cell lines that stably secreted Nsp4 antibodies. Results showed that one monoclonal hybridoma cell line stably secreted specific antibodies against Nsp4 protein, named 1C11 (…). Figure 2A). Further analysis using eukaryotically expressed Nsp4 protein in HEK293T cells and protein samples from PRRSV HuN4-infected Marc-145 cells was conducted via Western blot analysis to identify the reactivity of the mAb to Nsp4 protein. The results showed that the monoclonal antibody 1C11 specifically recognized both eukaryotically expressed Nsp4 and naturally occurring Nsp4 from viral infection. Figure 2 (B, 2C). This indicates that the mAb obtained is a specific monoclonal antibody against the PRRSV Nsp4 protein. The selected monoclonal cells were expanded and cryopreserved for seed preservation.

[0021] Sequencing was performed on the expanded hybridoma cell line 1C11, and the results showed... The amino acid sequence of the heavy chain variable region of the monoclonal antibody is as follows: MNFGLSLIFLALILKGWEFEVQLQESGGGLVKPGGSLKLSCAASGFTFS TYVMS WVRQIPEKRLEWVA SISSGGSYTYYPDSVKG RFTISRDNARNILYLQMSSLRSEDTAMYYCAR GDPPYGDFAY WGQGTTLTVSS(SEQ IDNO.3); The amino acid sequence of the light chain variable region is as follows: MGIKMESQTQVQLPANQCFSHNVQRTNCSLQSPAILSASPGEKVTMTC RASSSVNYMH WYQQKPGSSPKPWIY DTSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYC QQWSRNPWT FGGGTKLEIKRADAAPTVS (SEQID NO.4), where the underlined part is CDR1-3.

[0022] Expression of Nsp4 overlapping recombination truncated protein The antigenic epitopes of monoclonal antibodies were identified using a segmented truncated expression method for Nsp4 protein. Nsp4 was divided into truncated segments of different lengths. Using PRRSV HuN4 cDNA as a template, gene fragments encoding the truncated segments were amplified, and each fragment was cloned into the prokaryotic expression vectors pColdI or pGEX-4T-1, and then transformed into BL21(DE3) competent cells for induced expression. The results showed that the monoclonal antibody 1C11 could simultaneously recognize Nsp4-A (1-153 aa) and Nsp4-B (77-204 aa). Nsp4-B was further truncated into Nsp4-B1 (77-100 aa), B2 (89-130 aa), and B3 (115-153 aa). Western spectroscopy of the fusion-expressed proteins showed that the monoclonal antibody 1C11 could simultaneously recognize Nsp4-B2 (89-130 aa) and Nsp4-B3 (115-153 aa) (Figure 3A), indicating that the epitope recognized by 1C11 is located at 115-130 aa. The 115-130 aa epitope recognized by 1C11 was further divided into four truncated proteins, and the reactivity of 1C11 was tested. The results showed that the monoclonal antibody 1C11 could react with HE3 (121-130 aa) and HE4 (118-130 aa), but not with HE1 (115-127 aa) and HE2 (115-124 aa). Figure 3 B) indicates that 1C11 recognizes the antigenic epitope located at HE3 (121-130 aa). Further, amino acid deletion was performed sequentially from the N-terminus or C-terminus of epitopes 121-130 aa to construct a truncated expression protein. The reactivity of the monoclonal antibody was validated using the truncated protein. The results showed that the monoclonal antibody 1C11 only recognized MC5 (122-130 aa) and MC4 (123-130 aa), and showed no reactivity to MC1 (121-129 aa), MC2 (121-128 aa), and MC3 (124-130 aa). Figure 3 C). This indicates that the antigenic epitope recognized by 1C11 is located at... 123 ITEAGELV 130 .

[0023] To analyze the conservation of the newly identified antigenic epitopes, we selected four lineages of PRRSV-2 (L1, L3, L5, and L8) and the LV strain of PRRSV-1 as reference strains. We analyzed the amino acid sequence encoded by Nsp4. The results showed that the epitope... 123 ITEAGELV 130The Nsp4 amino acid composition of PRRSV-2 is highly conserved. To further identify whether differences in Nsp4 amino acids among different strains affect the recognition ability of the monoclonal antibody, the monoclonal antibody 1C11 was subjected to Western blot analysis with Marc-145 cells infected with highly pathogenic PRRSV strains HuN4, HuN4-F112, CH-1a, ZJhz021 (a NADC30-like strain), and HBap4-2018. The results showed that 1C11 could specifically recognize the Nsp4 amino acid composition of the above five PRRSV strains. Figure 4 A). Simultaneously, the IFA response of cells infected with different representative strains of PRRSV-2 and PRRSV-1 was detected using the clonal antibody 1C11. The results showed that the responses to HuN4, CH-1a, and ZJhz021 were consistent with the results of Western blot analysis. The monoclonal antibody 1C11 could recognize all representative strains of PRRSV-2, and also recognized representative strains of PRRSV-1. Figure 4 B).

[0024] Enzyme-linked immunosorbent assay (ELISA) Using the expressed Nsp4 protein as the antigen, the antigen was coated onto the adsorption plate with carbonate coating buffer (50 mM, pH 9.6) at a final concentration of 10 μg / mL. After overnight coating at 4°C, the liquid was discarded, and the plate was washed three times with PBS solution containing 0.5% Tween-20 (PBST). Then, it was blocked with PBST containing 5% skim milk powder at 37°C for 2 h. After three washes, the plate was incubated at 37°C for 1 h with the prepared monoclonal antibody or porcine serum (1:100 dilution) as the primary antibody. After washing, HRP-labeled mouse or porcine secondary antibody was added and incubated for 1 h. After washing the secondary antibody, TMB chromogenic solution (PO206, Beyotime Biotechnology) was added and incubated at room temperature for 15 min. Finally, stop solution was added to terminate the chromogenic reaction, and the OD was read using a microplate reader. 450nm The absorbance at the specified location. For PRRSV N protein ELISA detection, the procedure was performed according to the instructions of the IDEXX Porcine Reproductive and Respiratory Syndrome Indirect ELISA Kit, and the final result is expressed as the S / P value.

[0025] Western blot for proteins The collected protein expression samples were mixed with 5× loading buffer (LT101, Epizyme Biotech), boiled for 10 min, and the proteins in the samples were separated by 12% SDS-PAGE electrophoresis. After electrophoresis, the samples were transferred to NC membranes and then blocked at room temperature for 2 h with TBST solution containing 5% skim milk powder (TBS solution containing 1% Tween-20). After blocking, the samples were washed 3 times with washing buffer, and the selected monoclonal antibodies or tag antibodies, positive serum, etc. were added as primary antibodies and incubated for another 1 h. After washing with washing buffer, the samples were incubated at room temperature for 1 h with the corresponding HRP-labeled unlabeled secondary antibody. After washing, the samples were developed using ECL developing solution (SB-WB004, share-bio) under a Tianneng developer.

[0026] Indirect immunofluorescence assay (IFA) Marc-145 cells were infected with HuN4 or different strains. Cell samples were collected at 24 h or at 3, 6, 9, 12, 15, 18, 21, 24, and 36 h. The collected Marc-145 cells were fixed with 4% paraformaldehyde at room temperature for 30 min, incubated with 0.5% Triton X-100 at room temperature for 15 min to permeate the cell membrane, blocked with 5% BSA solution for 2 h, and incubated overnight at 4 °C with monoclonal antibody or anti-PRRSV N protein antibody prepared in this study as primary antibody. The cells were incubated at 37 °C in the dark for 1 h with Alexa Fluor 488-bound goat anti-mouse IgG fluorescent secondary antibody or Alexa Fluor 594-bound goat anti-rabbit IgG fluorescent secondary antibody. After washing, the cells were observed under a fluorescence microscope.

[0027] In vitro inhibition experiment Antibody or purified ascites fluid was inactivated and mixed with 0.1 MOI rHuN4-F112. After vortexing and incubation at 37°C for 1 h, the mixture was added to wells of Marc-145 cells. After 36 hpi, the supernatant was discarded. Cells were stained with anti-PRRSV N antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG fluorescent secondary antibody, and the fluorescence intensity of infected cells was observed under a fluorescence microscope. Simultaneously, cell supernatants were collected at different time points post-infection for virus titration, and viral nucleic acid was extracted and the viral copy number in the supernatant was detected by qPCR. The results showed that with increasing concentration of monoclonal antibody 1C11, the fluorescence intensity of virus-infected cells significantly decreased. Figure 5 A) After incubating monoclonal antibody 1C11 with PRRSV, cell samples infected at different time points were tested. The results showed that within 48 hours after viral infection, the viral nucleic acid copy number and viral proliferation titer were both lower than those in the control group. Figure 5 (B and 5C). This indicates that the monoclonal antibody 1C11 can inhibit the proliferation of PRRSV on cells to a certain extent.

[0028] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A monoclonal antibody 1C11 targeting the PRRSV Nsp4 protein, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as follows: MNFGLSLIFLALILKGWEFEVQLQESGGGLVKPGGSLKLSCAASGFTFS TYVMS WVRQIPEKRLEWVA SISS GGSYTYYPDSVKG RFTISRDNARNILYLQMSSLRSEDTAMYYCAR GDPPYGDFAY WGQGTTLTVSS (SEQ ID NO.3); The amino acid sequence of the light chain variable region is as follows: MGIKMESQTQVQLPANQCFSHNVQRTNCSLQSPAILSASPGEKVTMTC RASSSVNYMH WYQQKPGSSPKPWIY DTSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYC QQWSRNPWT FGGGTKLEIKRADAAPTVS (SEQ ID NO.4), where the underlined part is CDR1-3.

2. A novel antigenic epitope peptide in the PRRSV Nsp4 protein, characterized in that... The amino acid sequence of the antigenic epitope peptide is 123ITEAGELV130 (SEQ ID NO.5). This epitope is a novel antigenic epitope first identified in the Nsp4 protein; the antigenic epitope is located in its domain II (89-153aa).

3. The use of the monoclonal antibody of claim 1 and / or the antigenic epitope peptide of claim 2 in the preparation of medicaments for the treatment and / or prevention of PRRSV.

4. A pharmaceutical composition for treating and / or preventing PRRSV, said pharmaceutical composition comprising an effective amount of the monoclonal antibody of claim 1, said pharmaceutical composition being capable of inhibiting the proliferation of PRRSV.

5. The use of a monoclonal antibody as described in claim 1, and / or the antigenic epitope peptide of claim 2, in the preparation of a reagent for detecting and recognizing PRRSV.

6. A reagent for detecting PRRSV, characterized in that... The reagent is a kit / or test strip.

7. A test strip for detecting PRRSV, characterized in that, The test strip contains the monoclonal antibody as described in claim 1.

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