Antibody aiming at Nsp7 protein of porcine reproductive and respiratory syndrome virus or antigen binding fragment thereof and application of antibody or antigen binding fragment

Abstract

The invention discloses an antibody aiming at porcine reproductive and respiratory syndrome virus (PRRSV) Nsp7 protein or an antigen binding fragment thereof, and the antibody comprises a heavy chain and a light chain, and can specifically recognize and detect the Nsp7 protein. The Nsp7 is only expressed during virus infection, and vaccine immunization does not induce the generation of the antibody, so that infection and immune identification (DIVA) can be realized by detecting the anti-Nsp7 antibody. Meanwhile, Nsp7 is highly conservative in different PRRSV strains, the broad spectrum and stability of detection can be improved, and the Nsp7 is suitable for infection monitoring and immune identification of PRRSV.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to an antibody or antigen-binding fragment thereof targeting the Nsp7 protein of porcine reproductive and respiratory syndrome virus and its applications. Background Technology

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as blue ear disease in pigs, is a highly contagious disease caused by the PRRS virus (PRRSV). This virus can infect pigs of all ages through multiple routes, including contact and airborne transmission, but it is particularly harmful to pregnant sows and piglets. Clinical manifestations are mainly reproductive disorders in sows (such as abortion, stillbirth, mummified fetuses, and weak piglets) and respiratory symptoms in piglets, significantly increasing piglet mortality. PRRSV has significant immunosuppressive properties, targeting and destroying alveolar macrophages, leading to decreased immunity and susceptibility to secondary bacterial or viral infections, creating a vicious cycle of "immunosuppression—multiple infections." This causes significant economic losses to the global pig industry and severely restricts the sustainable development of intensive pig farming. Currently, PRRS prevention and control mainly relies on comprehensive measures centered on "vaccination + monitoring and eradication," with accurate and rapid diagnostic technologies being key to effective monitoring and epidemic tracing.

[0003] While various diagnostic methods exist for porcine reproductive and respiratory syndrome (PRRS), each has its limitations. Virus isolation, considered the "gold standard" for etiological detection, yields accurate results, but its cumbersome and time-consuming process makes it difficult to meet the needs of rapid screening at the grassroots level. In molecular biology testing, techniques such as RT-PCR, real-time quantitative PCR, and loop-mediated isothermal amplification (LAMP), while possessing high sensitivity and specificity, rely on sophisticated instruments and specialized operation, making them unsuitable for on-site application in pig farms. Among serological tests, indirect immunofluorescence assays and virus neutralization assays, while specific, are limited by low throughput, high subjectivity, or the delayed production of neutralizing antibodies (1-2 months post-infection), hindering early diagnosis. In contrast, enzyme-linked immunosorbent assay (ELISA) has become the mainstream technology for PRRSV serological detection due to its high throughput, low cost, and ease of operation. However, current ELISA kits still face challenges such as single-target targeting, difficulty in distinguishing between wild-type virus infection and vaccine immunization, and reliance on imported core reagents, restricting their accurate application at the grassroots level. Therefore, developing PRRSV monoclonal antibodies with independent intellectual property rights can not only provide core raw materials for establishing detection methods such as sandwich ELISA and blocking ELISA with high sensitivity and specificity, but also is expected to break through existing technical bottlenecks in distinguishing between wild-type and vaccine strains and covering multiple genotype strains, providing key support for the development of domestically produced and precise rapid on-site detection technology for PRRSV.

[0004] PRRSV belongs to the genus Arteritisvirus of the family Arteritisviridae. It is a single-stranded positive-sense RNA virus with a genome length of approximately 15 kb. It is mainly divided into two genotypes: PRRSV-1 (European type) and PRRSV-2 (American type). Currently, the predominant strain in my country is PRRSV-2, with lineage 1 NADC30-like strains becoming the dominant circulating strain. The Nsp7 protein, encoded by ORF1b, has a molecular weight of approximately 28.65 kDa and is a key component of the PRRSV non-structural protein family. It participates in the formation of the viral RNA polymerase complex and plays a central role in viral genome replication. Unlike structural proteins such as GP5 and N, Nsp7 is only expressed intracellularly after viral infection of host cells. Currently available commercial PRRS vaccines (including inactivated and attenuated vaccines) do not contain the complete ORF1b coding region and therefore cannot induce the body to produce specific antibodies against Nsp7. Therefore, Nsp7 can serve as an ideal target for infection-immunity differentiation (DIVA): naturally infected pigs produce both anti-Nsp7 antibodies and antibodies against structural proteins, while vaccine-immunized pigs only produce antibodies against structural proteins. Accurate differentiation between these two types of antibodies can be achieved by detecting anti-Nsp7 antibodies, thus overcoming the technical limitations of existing detection methods in effectively differentiating infection and immune status. Furthermore, Nsp7 exhibits high sequence conservation (homology typically exceeding 85%) among different PRRSV strains. Compared to highly variable structural protein targets, it is less prone to false negatives due to viral mutations, significantly improving the broadness and stability of the detection. Therefore, establishing a detection system based on the Nsp7 protein has significant application value for PRRSV infection monitoring, immune differentiation, and multi-strain coverage. Summary of the Invention

[0005] This invention aims to provide an antibody or antigen-binding fragment thereof targeting the Nsp7 protein of porcine reproductive and respiratory syndrome virus (PRRSV). The antibody or antigen-binding fragment comprises a heavy chain and a light chain, enabling it to specifically recognize the PRRSV Nsp7 protein and can be used to detect the presence or content of Nsp7 protein in a sample. Since the Nsp7 protein is expressed only after viral infection of host cells, and existing vaccine immunization does not induce the production of anti-Nsp7 antibodies, detecting anti-Nsp7 antibodies can effectively differentiate naturally infected pigs from vaccine-immunized pigs, making it suitable for infection-immune differential diagnosis (DIVA) detection. Furthermore, Nsp7 exhibits high sequence conservation among different PRRSV strains, making it less prone to false negatives compared to structural protein targets that are more susceptible to mutation, thus improving the broadness and stability of detection. The antibody or antigen-binding fragment provided by this invention is suitable for establishing detection methods for PRRSV infection monitoring, immunodiagnosis, and multi-strain coverage, and has significant application value.

[0006] To achieve the above objectives, in a first aspect, the present invention provides an antibody against the Nsp7 protein of porcine reproductive and respiratory syndrome virus, wherein the amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO:2.

[0007] In some embodiments, the antibody type is IgG1 and the light chain is a kappa light chain.

[0008] In a second aspect, the present invention provides an antigen-binding fragment that specifically binds to an antibody as described in the first aspect.

[0009] In a third aspect, the present invention provides a nucleic acid molecule encoding the antibody described in the first aspect, the nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO:3, and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO:4.

[0010] In a fourth aspect, the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the third aspect.

[0011] The optimized vector is the pGEX-4T-1 expression vector.

[0012] In a fifth aspect, the present invention provides a host cell comprising the recombinant expression vector described in the fourth aspect.

[0013] In a sixth aspect, the present invention provides a GST-Nsp7 fusion protein, which is obtained by inducing expression of the recombinant expression vector described in the fourth aspect in the host cells described in the fifth aspect.

[0014] In a seventh aspect, the present invention provides the use of the fusion protein as described in the sixth aspect in the immunization preparation of antibodies against porcine reproductive and respiratory syndrome virus Nsp7 protein.

[0015] In an eighth aspect, the present invention provides the use of the antibody or its antigen-binding fragment thereof in the preparation of a reagent or kit for detecting porcine reproductive and respiratory syndrome virus Nsp7 protein.

[0016] Working principle and beneficial effects of the present invention: The antibody in this invention contains a specific sequence of heavy chain variable regions and light chain variable regions, enabling it to specifically recognize PRRSV. The Nsp7 protein, expressed only during PRRSV infection, cannot be induced by vaccine immunization to produce anti-Nsp7 antibodies, which can differentiate between natural infection and vaccine immunization. Furthermore, the Nsp7 protein is highly conserved across different PRRSV strains, improving the broadness and stability of detection. This invention solves the problems of existing PRRSV detection technologies, such as the inability to distinguish between infection and immunity, single target, and reliance on imported core reagents. It provides key biological reagents for PRRSV infection monitoring and immunodiagnostic methods, and has significant practical application value. Attached Figure Description

[0017] Figure 1 The image shows the SDS-PAGE results of imidazole eluted samples collected at different times in Example 1; where M is the protein molecular weight standard (Marker), 1 is the uninduced bacterial protein, 2 is the induced bacterial protein, and 3-14 are the eluted samples collected at different times with a concentration of 250 mM imidazole. Figure 2 This is a graph showing the results of serum titer detection in mice after immunization in Example 2; Figure 3 This is a graph showing the results of ascites titer detection in mice after injection of cell line Nsp7-4 in Example 3; Figure 4 This is the result of SDS-PAGE analysis of the purified Nsp7-4 monoclonal antibody in Example 3; where M is the protein molecular weight standard (Marker) and 1-3 are the purified Nsp7-4 monoclonal antibody. Figure 5 This is a graph showing the results of the Nsp7-4 monoclonal antibody specificity detection in Example 3. Detailed Implementation

[0018] The following detailed description illustrates the specific implementation method: Example 1: Obtaining the GST-Nsp7 fusion protein of porcine reproductive and respiratory syndrome virus (PRRSV) In this embodiment, the GST-Nsp7 fusion protein was prepared. The specific preparation method includes the following steps: 1. Construction of recombinant expression vectors The Nsp7 protein (NCBI reference sequence number: NP_740601.1) of the porcine reproductive and respiratory syndrome virus (PRRSV) NADC30-like strain was selected as the design target for the recombinant antigen. Bioinformatics analysis showed that the Nsp7 protein is 259 amino acids in length, lacks a signal peptide and transmembrane structure, exhibits overall weak hydrophilicity with localized hydrophobic regions, and is suitable for soluble expression in Escherichia coli.

[0019] This invention uses pGEX-4T-1 as the expression vector, which carries a glutathione S-transferase (GST) tag. GST can bind highly specifically to immobilized glutathione (GSH) resin, exhibiting strong binding and low non-specific adsorption, making it suitable for affinity purification of fusion proteins. Furthermore, GST itself is a highly soluble protein (molecular weight approximately 26 kDa), and its fusion with the target protein helps improve the solubility and stability of the recombinant protein in prokaryotic systems.

[0020] Based on the amino acid sequence of the PRRSV Nsp7 protein, the corresponding nucleotide sequence was codon-optimized for a prokaryotic expression system, and gene synthesis was commissioned to Anshengda Biotechnology Co., Ltd. The synthesized Nsp7 gene fragment was digested, ligated, and cloned into the multiple cloning site of the pGEX-4T-1 vector to construct the recombinant expression plasmid pGEX-4T-1-Nsp7. This recombinant plasmid can be used to express the GST-Nsp7 fusion protein in host cells, thus providing a foundation for the subsequent preparation of recombinant antigens and their immunological applications.

[0021] 2. Expression and purification of GST-Nsp7 fusion protein After confirming the correct BL21(DE3) clone containing the recombinant expression vector pGEX-4T-1-Nsp7, plasmid was extracted and transformed using standard methods. Single colonies were then picked and cultured in LB medium containing ampicillin resistance until the bacterial concentration reached A... 600 When the concentration was 0.5, expression was induced for 18 h with 0.1 mM IPTG at 200 rpm and 16℃. The cells were collected by centrifugation at 4℃ and 4000 rpm for 20 min, resuspended and washed with 20 mL PBS, and then sonicated on ice for 3 min with Binding buffer at 30 W power. The sonication was repeated for 2 s with a 2 s interval until the bacterial solution was relatively clear. The supernatant was collected by centrifugation at 4℃ and 12000 rpm for 20 min for purification.

[0022] The target protein (pGEX-4T-1-Nsp7) was purified by nickel column affinity chromatography: 50% NI-NTA column was packed, washed with 4 mL of deionized water, and then equilibrated with 5 mL of binding buffer. The lysate containing the recombinant Nsp7 protein was added to the column, and the permeate was collected and loaded again. Unbound proteins were washed away with binding buffer, followed by 20 mL of washing buffer. Proteins were then eluted with 250 mM imidazole elution buffer. The purity of the eluted sample was determined by SDS-PAGE electrophoresis. The results are shown below. Figure 1As shown, the GST-Nsp7 fusion protein obtained by affinity chromatography had high purity. The concentration of the purified GST-Nsp7 fusion protein was determined by BCA method.

[0023] Example 2: Establishment of a monoclonal antibody cell line for porcine reproductive and respiratory syndrome virus Nsp7 protein This embodiment prepared a monoclonal antibody cell line for PRRSV Nsp7 protein. The specific preparation method includes the following steps: 1. Mouse immunization The fusion protein obtained in Example 1 contains the PRRSV Nsp7 protein antigenic epitope and can be used as an Nsp7 antigen to immunize Balb / c mice. For the first immunization (day 1 of the first immunization), 100 μg of the immunogen was emulsified with Freund's complete adjuvant at a 1:1 volume ratio, administered subcutaneously at five sites on the back and abdomen of each mouse. A second immunization was performed on day 15, using Freund's incomplete adjuvant emulsified with the immunogen at a 1:1 volume ratio, at a dose of 50 μg per mouse, following the same method. A third immunization was performed on day 29, following the same method as the second immunization. On day 36, a small amount of tail blood was collected for ELISA testing. If the antibody titer was greater than 1:10000, antigen pulse immunization and spleen cell fusion could then be performed. If the antibody titer was less than 1:10000, a fourth immunization was performed on day 43, following the same method. Three days before cell fusion, an intraperitoneal pulse immunization was performed, with 100 μg of the immunogen injected directly into the peritoneum without adjuvant.

[0024] 2. Mouse serum titer detection (1) Antigen coating: The PRRSV Nsp7 antigen obtained in Example 1 was prepared into a coating solution of 1 μg / mL. 100 μL was added to each well and the solution was coated overnight at 4°C.

[0025] (2) Washing: On the second day, discard the liquid in the well, pat dry, and wash twice with PBST in a plate washer.

[0026] (3) Blocking: Add 200 μL of blocking solution to each well and incubate at 37℃ for 1 h.

[0027] (4) Preparation of serum (primary antibody): Blood was collected from the tail of mice, and serum from unimmunized mice was used as a negative control.

[0028] (5) Add primary antibody: Use blocking solution to serially dilute the serum samples to be tested. Use non-immunized mouse serum diluted 1:2500 as a negative control and blocking solution as a blank control. Incubate at 37℃ for 1 h, discard the liquid in the well, pat dry, wash twice with PBST, and pat dry.

[0029] (6) Add enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody (HRP-goat anti-mouse) 5000 times with blocking buffer, 100 μL / well, incubate at 37℃ for 1 h, discard the liquid in the well, and pat dry.

[0030] (7) Color development and measurement: Add 50 μL of TMB chromogenic solution to each well, incubate at 37℃ in the dark for 15 min, then add 100 μL of 2 M sulfuric acid to terminate the reaction. Measure the A value at 450 nm using a microplate reader. Calculation: The highest antiserum dilution factor when the ratio of the A value of the test well to the A value of the negative control well (P / N) is ≥2.1 is taken as the serum titer. Mice with a dilution greater than 1:100,000 are prepared for the next fusion step. The results are as follows: Figure 2 As shown, the reaction of Nsp7 protein with immune serum was identified by indirect ELISA, and the titer of mouse serum was 1:64000.

[0031] 3. Cell fusion Aseptically, spleen cells from immunized mice were mixed with myeloma cells from SP2 / 0 mice at a ratio of approximately 5:1 in a 50 mL centrifuge tube. After washing twice with culture medium, the supernatant was discarded. Over 50 seconds, 0.9 mL of preheated PEG-1500 was slowly added to disperse the cells as evenly as possible. The mixture was allowed to stand for 1 min. 20 mL of preheated serum-free DMEM culture medium at 37°C was slowly added dropwise, 2 mL over the first two minutes and 18 mL over the next two minutes, all within 4 minutes. The mixture was allowed to stand for 3 minutes, then centrifuged at 800 rpm for 5 minutes and the supernatant was discarded. Preheated FBS and HAT culture medium were added, and the mixture was gently pipetted to mix. The mixture was then transferred to 96-well plates at a density of 200 μL per well and incubated.

[0032] 4. Screening of positive hybridoma cells Ten days after cell fusion, when the fused cells filled more than 50% of the wells, hybridoma cells were screened using an indirect ELISA method.

[0033] 5. Subcloning of positive hybridoma cells Subcloning was performed on the positive wells using a limiting dilution method. The number and location of cell clusters in the positive wells were observed under an inverted microscope. Cell clusters were then aspirated using a 200 μL pipette tip in a clean bench, and the cell count was diluted to 1-2 cells per 100 μL. Prepared feeder cells were then added to 100 μL of the diluted cells in 96-well plates, labeled, and incubated at 37°C for 9 days using a 5% CO2 incubator. After three subcloning cycles until one cell was found per well, the titer of the cell supernatant was measured using an indirect ELISA method. The positive rate reached 100%. The culture was expanded, and the cell line was preserved and labeled as Nsp7-4.

[0034] Example 3: Preparation and Identification of Monoclonal Antibodies Against Porcine Reproductive and Respiratory Syndrome Virus Nsp7 Protein In this embodiment, a monoclonal antibody against the Nsp7 protein was prepared and its subclass was identified. The specific steps are as follows: 1. Preparation and titer determination of ascites fluid containing Nsp7 protein monoclonal antibody 12-16 week old female BALB / c mice were intraperitoneally injected with 0.5 mL of sterile liquid paraffin. Ten days later, each mouse was injected with 0.5 mL of a suspension of Nsp7-4 cell line preserved in Example 2 (5 × 10⁻⁶ cells). 5 (Each mouse was injected with the cell suspension, and the day of injection was counted as day 1). On day 7, after significant abdominal distension, ascites fluid was collected, centrifuged at 3000 rpm for 20 min, adipose tissue was removed, and the supernatant was collected and stored at -20℃ for later use. The titer of the ascites fluid was determined by indirect ELISA, and the results are as follows: Figure 3 As shown: Ascites antibody titer 1:1280000.

[0035] 2. Purification of monoclonal antibodies Ascites fluid was collected, and Nsp7-4 monoclonal antibody was purified using the caprylic acid-ammonium sulfate precipitation method, as follows: Ascites fluid was collected and centrifuged at 12000 rpm for 5 min at 4°C. The supernatant was collected, and 2 volumes of 0.06 M acetate buffer (pH 4.0) were added to adjust the pH to 4.5. 33 μL of caprylic acid was added per mL of ascites fluid, and the mixture was stirred at room temperature for 30 min. The mixture was then incubated at 4°C for 1 h to allow for complete precipitation of contaminating proteins. The mixture was centrifuged at 1000 g for 30 min at 4°C. 0.277 g of ammonium sulfate powder was added per mL of the supernatant. The beaker was placed on a magnetic stirrer and stirred for 1 h. The mixture was then centrifuged at 10000 g for 20 min at 4°C. The supernatant was discarded, and the precipitate was dissolved in PBS. SDS-PAGE was used to identify the purity of the monoclonal antibody. The results are shown below. Figure 4 As shown: High-purity Nsp7-4 monoclonal antibody was obtained.

[0036] 3. Identification of Monoclonal Antibody Types and Subclasses The mouse subtype identification kit was used to perform the experimental procedures. The Nsp7-4 monoclonal antibody was identified as IgG1 and kappa light chain.

[0037] Meanwhile, GENEWIZ (Suzhou Genewiz Biotechnology Co., Ltd.) was commissioned to sequence the monoclonal antibody produced by the Nsp7-4 cell line preserved in Example 2 (i.e., the Nsp7-4 monoclonal antibody purified in step 2 of Example 3). The results are as follows: The nucleotide sequence of the heavy chain variable region of the Nsp7-4 monoclonal antibody is: SEQ ID NO:3 5’-CAGGTCCAGCTGCAGCAGTCTGGGCCTGAGGTGGTGAGGCCTGGGGTCTCAGTGAAGATTTCCTGCAAGGGTTCCGGCTACACATTCACTGATTATGCTATACACTGGGTGAAGCAGAGTCATGCAAAGAGTCTAGAGTGGATTGGACTTATTAGTACTTACAATGGTATTACAAACTACAACCAGAAGTTTAACGGCAAGGCCACAATGACTGTAGACATATCCTCCAGCACAGCCTATATGGAACTTGCCAGATTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGATCCCGCGCCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA-3’; The corresponding amino acid sequence is: SEQ ID NO:1 QVQLQQSGPEVVRPGVSVKISCKGSGYTFTDYAIHWVKQSHAKSLEWIGLISTYNGITNYNQKFNGKATMTVDISSSTAYMELARLTSEDSAIYYCARSRAYFDYWGQGTTLTVSS; The nucleotide sequence of the light chain variable region is: SEQ ID NO:4 5’-GACATCCAGATGACACAGTCTCCATCCTCACTGTCTGCATCTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCCAGCCAAGACATTAACAAGCATATTGCTTGGTACCAACATAAGCCTGGAAAAGGTCCTAGGCTGCTCATACAATACACATCTACATTACAGCCAGGCATCCCATCAAGGTTCAGTGGAGGTGGGTCTGGGAGAGATTTTTCCTTCAGCGTCAGCAACCTGGAGCCTGAAGATATTGCGACTTATTATTGTCTACAGTATGATAATCTTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA-3’; The corresponding amino acid sequence is: SEQ ID NO:2 DIQMTQSPSSLSASLGGKVTITCKASQDINKHIAWYQHKPGKGPRLLIQYTSTLQPGIPSRFSGGGSGRDFSFSVSNLEPEDIATYYCLQYDNLYTFGGGTKLEIK .

[0038] 4. Specificity detection of monoclonal antibodies The following proteins were used as antigens: porcine reproductive and respiratory syndrome virus (PRRSV) Nsp7 protein (NSP), PRRSV N (2–87 aa) protein (N), PRRSV GP5 protein (GP5), porcine pseudorabies virus gE (52–260 aa) protein (gE-52–260), porcine pseudorabies virus gB (59–337 aa) protein (gB-59–337), porcine pseudorabies virus gB (507–734 aa) protein (gB-507–734), porcine foot-and-mouth disease virus 3ABC protein (3ABC), African swine fever virus I226R protein (I226R), African swine fever virus CD2v protein (CD2v), African swine fever virus p30 protein (p30), and African swine fever virus p72 protein (p72). All the antigens used in the tests were obtained by inducing prokaryotic expression in the laboratory.

[0039] The ELISA plate was coated under the same conditions, using the Nsp7-4 monoclonal antibody purified in step 2 of Example 3 as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody. A values ​​were read on the ELISA reader. 450 The values ​​were calculated by taking the average of three replicates for each sample. The specificity of the monoclonal antibody was detected using an indirect ELISA method (refer to Example 2). The results are as follows: Figure 5 As shown, the monoclonal antibody produced by the cell line Nsp7-4 preserved in Example 2 (i.e., the Nsp7-4 monoclonal antibody purified in step 2 of Example 3) has good specificity and can be used for qualitative or quantitative detection of porcine reproductive and respiratory syndrome virus Nsp7 protein in the sample to be tested.

[0040] It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this invention. These modifications and improvements should also be considered within the scope of protection of this invention, and will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An antibody against the Nsp7 protein of porcine reproductive and respiratory syndrome virus, characterized in that, The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO:

2.

2. The antibody according to claim 1, characterized in that, The antibody type is IgG1, and the light chain is the kappa light chain.

3. An antigen-binding fragment that specifically binds to the antibody as described in claim 1 or 2.

4. A nucleic acid molecule encoding the antibody of claim 1 or 2, characterized in that, The nucleic acid molecule contains a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO:3, and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO:

4.

5. A recombinant expression vector, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 4.

6. The recombinant expression vector according to claim 5, characterized in that, The vector is the pGEX-4T-1 expression vector.

7. A host cell, characterized in that, The host cell comprises the recombinant expression vector as described in claim 5 or 6.

8. A GST-Nsp7 fusion protein, characterized in that, The fusion protein is obtained by inducing expression of the recombinant expression vector of claim 5 or 6 in the host cells of claim 7.

9. The use of the fusion protein according to claim 8 in the immunization preparation of antibodies against porcine reproductive and respiratory syndrome virus Nsp7 protein.

10. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3 in the preparation of reagents or kits for detecting porcine reproductive and respiratory syndrome virus Nsp7 protein.

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