Preparation, epitope identification and application of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) non-structural protein 12 antibody

Abstract

The invention provides preparation, epitope identification and application of a PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) non-structural protein 12 antibody. The preparation method comprises the following steps: constructing an eukaryotic expression vector pCDNA3.4-Nsp12-strep, transiently transfecting CHO cells, purifying by using a downstream strep tag to obtain PRRSV Nsp12 recombinant protein, immunizing a Balb / C mouse by using the purified Nsp12 as an antigen, separating splenocytes of the mouse, separating a single specific B cell by using an FACS method, obtaining an antibody variable region sequence of the single B cell through RT-PCR, respectively constructing heavy chain and light chain recombinant expression plasmids, and preparing the PRRSV recombinant protein. According to the present invention, two complete anti-PRRSV Nsp12 monoclonal antibodies 1N14 and 2S18 are obtained by using a recombinant plasmid as a template, transfecting CHO suspension cells, expressing and purifying by using Protein G, the application range of the 1N14 and 2S18 antibodies and the specific antigen epitope identified by the 1N14 antibody are further determined, and the sequence of the antigen epitope is 104YEFTGNGEDW113.

Description

Technical Field

[0001] The present invention relates to the field of biology, and in particular, to the preparation, epitope identification and application of an antibody against PRRSV nonstructural protein 12, and in particular, to the sequence, recognized antigenic epitope and application of an anti-PRRSV Nsp12 monoclonal antibody recombinantly expressed by CHO cells. Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the order Nidovirales, the genus Arterivirus, and the family Arteriviridae. It is a single-stranded positive-sense enveloped RNA virus. The virus has strict host specificity and tissue tropism, mainly infecting alveolar macrophages and dendritic cells, causing acute or persistent infection. Porcine alveolar macrophages are the main target cells for PRRSV replication in respiratory tract infection in vivo. The PRRSV genome is about 15 kb in length and can encode at least 10 open reading frames (ORFs). Among them, ORF1a and ORF1b account for three quarters of the viral genome, encoding the replicase polyprotein pp1a, pp1a-Nsp2N, pp1a-Nsp2TF, and pp1ab through ribosomal frameshifting, and are further hydrolyzed and processed into at least 16 functional non-structural proteins, including Nsp1α, Nsp1β, Nsp2TF, Nsp2N and Nsp2~6, Nsp7α, Nsp7β, and Nsp8~12, which are responsible for the replication and transcription of the viral genome.

[0003] Nsp12 is produced by the cleavage of the polyprotein pp1ab encoded by the ORF1b gene. It consists of 153 amino acids (aa) and is highly conserved in PRRSV strains of different lineages. Reverse genetics technology has shown that Nsp12 is involved in the synthesis of viral sgRNA and is indispensable in the replication of PRRSV-2. Studies have found that Nsp12 acts as the hub of a variety of PRRSV Nsp interaction networks, and its interaction with Nsp10 plays an important role in sgRNA synthesis and the coordination of the assembly of replication and transcription complexes. The functions of most proteins encoded by PRRSV have been resolved, but the functions of Nsp12 have been less reported, and its role in the viral immune pathogenesis is still unclear. Nsp12 can induce STAT1 phosphorylation and participate in maintaining the stability of KPNA6, which is conducive to viral replication. Previous studies in our laboratory have shown that Nsp12 interacts with host restriction factors PSMB1, RNF114 and Galectin-3. RNF114 can induce K27 polyubiquitination and proteasome degradation of Nsp12, while PSMB1 can activate the selective autophagy pathway to degrade Nsp12 by recruiting the iconic autophagy receptor protein NBR1, thereby inhibiting the replication of PRRSV in cells. The preparation of monoclonal antibodies against Nsp12 and the identification of the antigenic epitopes it recognizes will help to clarify the potential function of Nsp12 in the pathogenesis of PRRSV infection and provide valuable tools for future basic and applied research on PRRSV. Summary of the invention

[0004] In order to solve the above problems, the present invention uses single B cell amplification technology to obtain antibody variable region sequences against PRRSV Nsp12, and uses CHO cell recombinant expression and purification to obtain two highly specific and highly titer anti-PRRSV Nsp12 monoclonal antibodies 1N14 and 2S18, and identifies the specific antigen epitope sequence recognized by the 1N14 antibody, which lays a good foundation for further research on the biological functions of PRRSV Nsp12 and the design of new PRRS vaccines.

[0005] Specifically, the purpose of the present invention is to provide two new antibodies against PRRSV Nsp12 and their specific preparation methods, antibody sequences, antigenic epitopes and uses. The technical scheme of the present invention is: the PRRSV Nsp12 gene sequence is cloned into the pcDNA3.4 vector to construct a eukaryotic expression vector, CHO cells are transiently transfected, and the protein is purified through the downstream strep tag, the purified PRRSV Nsp12 is used as an antigen to immunize Balb / C mice, mouse spleen cells are separated, specific B cells are separated by FACS, and single B cell antibody variable region sequences are obtained by RT-PCR, heavy chain and light chain recombinant expression plasmids are constructed respectively, CHO suspension cells are transiently transfected, and two complete anti-PRRSVNsp12 monoclonal antibodies 1N14 and 2S18 are expressed and purified by Protein G, and the use range of 1N14 and 2S18 antibodies and the specific antigenic epitopes recognized by 1N14 antibodies are determined.

[0006] Through single B cell amplification technology, two antibody heavy chain variable region and light chain variable region sequences (SEQ ID NO: 1-4) against PRRSV Nsp12 were obtained, and two monoclonal antibodies with the above variable region sequences were recombinantly expressed using CHO cells. Among them, the 1N14 antibody can specifically recognize PRRSV Nsp12 and can be used for immunofluorescence and Western blotting detection. The heavy chain of this antibody subtype is IgG1 and the light chain is Kappa. This antibody can specifically recognize the 104-113 peptide segment of PRRSV Nsp12, and the amino acid sequence it recognizes is 104 YEFTGNGEDW 113 (SEQ ID NO: 5). The 2S18 antibody can specifically recognize PRRSV Nsp12 and can be used for immunofluorescence detection. The heavy chain of the antibody subtype is IgG2a and the light chain is Kappa.

[0007] Another object of the present invention is to provide a method for determining the sequence and preparing the two recombinant PRRSV Nsp12 monoclonal antibodies.

[0008] Another object of the present invention is to provide a method for determining and preparing a PRRSV Nsp12 antigenic epitope. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1The results of the expression and purification of the recombinant Nsp12 protein after transient transfection of the pcDNA3.4-Nsp12-strep eukaryotic expression plasmid into the suspended cultured CHO cells in Example 1 were identified by Coomassie brilliant blue staining. (A) Lane 1 is the protein molecular mass standard, lane 2 is the untransfected CHO cell control, lanes 3-4 are cells collected on the 3rd and 5th days after transfection, and lanes 5-6 are supernatants collected on the 3rd and 5th days after transfection. The results show that the recombinant Nsp12 protein is mainly expressed in CHO cells. On the 3rd day after transfection, the expected band can be seen. On the 5th day after transfection, the band is clear and obvious, with a size of about 17 kDa, which is consistent with the expected size of PRRSV Nsp12. (B) Lane 1 is the protein molecular mass standard, and lane 2 is the recombinant Nsp12 protein after strep beads purification. The band is clear and single, with a size of about 17 kDa, which is consistent with the expected size of PRRSV Nsp12.

[0010] Figure 2 This is the Western blotting identification of the purified Nsp12 recombinant protein in Example 2. (A) Lane 1 is the protein molecular mass standard, lane 2 is the purified Nsp12 recombinant protein, and the primary antibody is the strep tag antibody, indicating that the purified protein can specifically react with the strep tag antibody, and the band is clear, which is the target protein of the correct size. (B) Lane 1 is the protein molecular mass standard, lane 2 is the purified Nsp12 recombinant protein, and the primary antibody is the positive serum of PRRSV-infected pigs, indicating that the purified protein can specifically react with the pig positive serum to produce a band consistent with the expected size of PRRSV Nsp12, which can be used as an antigen protein for animal immunization, and at the same time indicates that PRRSV can induce the production of specific antibodies against Nsp12 after infecting pigs.

[0011] Figure 3 The results of FACS sorting of single B cells in mice in Example 4. CD138, a marker molecule on the surface of B cells, was used as the FACS sorting antibody to screen positive cells with fluorescent signals. (A) Blank control of immunized mouse cells, where no fluorescent signals were observed. (B) Fluorescent signals of CD138-labeled mouse cells after immunization, with the horizontal axis 10 2 The right side shows a population of CD138-positive memory B cells, from which approximately 7.76% of positive cells were marked for collection of single specific B cells.

[0012] Figure 4The Coomassie Brilliant Blue staining and titer determination of the purified Nsp12 recombinant antibody in Example 4. (A) Lane 1 is the protein molecular mass standard, lane 2 is the eluted sample of the 1N14 antibody, and lane 3 is the eluted sample of the 2S18 antibody. The results show that the purified 1N14 and 2S18 antibodies have clear heavy and light chain bands of the correct size. (B) The ELISA method was used to detect the antibody titers of 1N14 and 2S18, indicating that the titers of the two antibodies can reach 1:256000.

[0013] Figure 5 This is the result of the identification of the antibody subtype in Example 5. Among them, the heavy chain of the 1N14 antibody subtype is IgG1, and the light chain is Kappa; the heavy chain of the 2S18 antibody subtype is IgG2a, and the light chain is Kappa.

[0014] Figure 6 The results of immunofluorescence test and Western blotting identification of Nsp12 overexpressed in 293T cells using 1N14 and 2S18 antibodies in Example 5. (A) The coding sequences of Nsp12 and Myc tags were cloned into the pCAGGS vector to construct the eukaryotic expression plasmid of Nsp12-Myc, and transfected into 293T cells. The results showed that the Myc tag antibody can specifically recognize the protein expressed by the cells after transfection with the Nsp12-Myc plasmid, producing red fluorescence; 1N14 and 2S18 antibodies can specifically recognize the protein expressed by the cells after transfection with the Nsp12-Myc plasmid, producing green fluorescence, indicating that the above two antibodies can recognize the undenatured Nsp12 in the immunofluorescence test. (B) The protein samples after Nsp12-Myc transfection cells were collected, added with SDS protein loading buffer, boiled and denatured, and Western blotting was performed. The results showed that the 1N14 antibody could specifically recognize the cell protein sample after transfection of Nsp12-Myc plasmid, while 2S18 could not, indicating that the 1N14 antibody could recognize Nsp12 after denaturation and reduction in the Western blotting experiment. In summary, the antigen epitope recognized by the 1N14 antibody is a linear epitope, and the antigen epitope recognized by the 2S18 antibody is a conformational epitope.

[0015] Figure 7The following is a schematic diagram of the specific segmented positions and Western blotting identification diagrams of the preliminary segmented screening of the 1N14 antibody recognition antigen epitope in Example 6. (A) Five prokaryotic expression plasmids of truncated Nsp12 fragments were constructed, as shown in the figure, represented by T1, T2, T3, T4, and T5, respectively. (B-C) The plasmids were transformed into BL21 (DE3) Escherichia coli competent cells to induce the expression of five truncated Nsp12 fragments, and protein samples were prepared and Western blotting was performed with His tag antibodies and 1N14 antibodies, respectively. The results showed that the 1N14 antibody could recognize T2 and T5 fragments, both of which contained 104 YEFTGNGEDW 113 sequence.

[0016] Figure 8 This is the ELISA identification diagram of the minimum antigen epitope in Example 6. (A) The amino acid sequence of the T5 fragment was truncated and synthesized into 4 polypeptides, represented by P1-P4. The results showed that the 1N14 antibody could only recognize the P1 peptide segment, that is, the 101-120 peptide segment. (B) The P1 peptide segment was truncated and synthesized into 11 polypeptides, represented by P5-P15. The results showed that the 1N14 antibody could only recognize the P8 peptide segment and had no reaction with other peptide segments. Since the P7 and P9 peptide segments contain sequences that are only 1 aa shorter than the P8 peptide segment, and the 1N14 antibody cannot recognize P7 and P9, it means that the polypeptide recognized by the 1N14 antibody cannot be further truncated. The shortest epitope peptide sequence recognized by the 1N14 antibody is 104 YEFTGNGEDW 113 .

[0017] Fig. 9 The following is the predicted analysis of the antigenic epitope and three-dimensional structure of PRRSV Nsp12 in Example 7. (A) The potential B cell epitopes in the full-length amino acid sequence of Nsp12 were predicted using the immune epitope database (IEDB). The regions with scores above the red dotted line may contain antigenic epitopes. The results show that the 1N14 antibody recognizes 104 YEFTGNGEDW 113 The region where the antigen epitope is located is predicted to have potential B cell epitopes. (B) The three-dimensional structure of Nsp12 was constructed using the SWISS-MODEL tool and analyzed and visualized using the PyMOL software 104 YEFTGNGEDW 113 Spatial structural location of the antigen epitope (blue). The results show that the identified antigen epitope is displayed on the surface of the three-dimensional structure of Nsp12.

[0018] Fig.10This is an indirect ELISA analysis of the reaction between the antigenic epitope peptide recognized by the 1N14 antibody and the porcine PRRSV positive serum in Example 8. The results show that the porcine PRRSV positive serum can specifically recognize the antigenic epitope peptide, while the porcine negative serum cannot recognize the antigenic epitope peptide.

[0019] Fig.11 This is the reactivity of 1N14 and 2S18 antibodies with different lineages of PRRSV strains and the conservation analysis of Nsp12 sequences in Example 9. (A) Immunofluorescence analysis of 1N14, 2S18 antibodies and laboratory-preserved N protein antibodies with four lineages (L1, L3, L5, L8) of PRRSV strains. The results showed that the 1N14 antibody can recognize L1, L5, and L8 strains, and the 2S18 antibody can recognize strains of all lineages. (B) Comparison of the amino acid sequences of Nsp12 of different lineages of PRRSV strains. The results showed that the 1N14 antibody recognized 104 YEFTGNGEDW 113 The antigenic epitope is conserved in the L1, L5, and L8 lineages.

[0020] Fig.12 This is the localization analysis of Nsp12 in cells after PRRSV infection of permissive cells and target cells for different time periods in Example 9. PRRSV was infected with (A) MARC-145 cells and (B) porcine alveolar macrophages for 8, 16, 24 and 32 hours, respectively, and laser confocal analysis was performed. The primary antibodies were 1N14 antibody and laboratory-preserved N protein antibody, and the secondary antibody was a green fluorescent secondary antibody. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0022] Example 1 CHO cell expression and purification of PRRSV Nsp12 recombinant protein

[0023] The inventors first constructed the pCDNA3.4-Nsp12-strep eukaryotic expression plasmid, and the designed primer sequences are shown in Table 1 (SEQ ID NO: 6 and SEQ ID NO: 7). The above primers were all PRRSV Nsp12-specific sequences after BLAST analysis, and the primers were synthesized by Shanghai Qingke Biotechnology Co., Ltd. The template was the Nsp12 gene sequence of the PRRSV HuN4 strain (GenBank ID: EF635006.1) in NCBI, and the Nsp12 fragment was obtained by PCR amplification and gel recovery, and it was homologously recombined into the pcDNA3.4 eukaryotic expression vector. After sequencing analysis and comparison, the inserted gene fragment was completely consistent with the PRRSV Nsp12 gene sequence, and the recombinant plasmid was named pcDNA3.4-Nsp12-strep. The specific PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s; annealing at 56°C for 30 s; extension at 72°C for 1 min, with 30 cycles from denaturation to extension; and final extension at 72°C for 10 min.

[0024] The CHO cells stored in liquid nitrogen were revived and cultured in suspension. One day before transfection, the CHO cells were counted and the cell density was adjusted to 3×10 6 On the day of transfection, the final density of CHO cells was adjusted to 6×10 cells / mL with fresh culture medium preheated at 37°C. 6 cells / mL. The transfection system was prepared based on a cell volume of 100 mL, and 4 mL of Opti PRO TM Dilute 100 μg of plasmid DNA in SFM and mix slowly; use 4 mL of Opti PRO TM Dilute 100mg PEI transfection reagent with SFM and mix slowly. Add 4mL of diluted PEI to 4mL of diluted plasmid DNA. Slowly turn over and mix, and incubate the complex at room temperature for 20 minutes. Slowly add the incubated complex to 100ml of cell suspension, place it in a 37℃, 8% CO2, 100r / min shaking incubator for suspension culture, add 600μL enhancer and 24mL auxiliary material after 18-22 hours, and continue to culture in the shaking incubator for 5 days. On the 3rd and 5th days after transfection, collect the supernatant and cell samples, respectively, perform SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining to analyze the expression of recombinant protein ( Figure 1A). Then, the target protein was purified using strep beads. The general process is as follows: the collected sample was incubated with pre-treated strep beads for 3 hours by shaking. The medium was washed with PBST containing 8.8g NaCl and 20mL 1M Tris-HCl, and finally eluted with biotin. The eluate was collected, which was the purified Nsp12 recombinant protein. Coomassie brilliant blue staining was performed to identify the purification effect of the recombinant protein ( Figure 1 B).

[0025] Example 2 Analysis of antigenic specificity of purified PRRSV Nsp12 recombinant protein

[0026] To determine the specificity of the purified protein, SDS loading buffer was added to the purified PRRSV Nsp12 recombinant protein, boiled and denatured, and Western blotting analysis was performed. Using the strep tag antibody as the primary antibody, it can specifically recognize the purified Nsp12 recombinant protein and produce a clear, correct-sized target band ( Figure 2 A), indicating that the recombinant protein was correctly expressed and purified. Using the positive serum of pigs infected with PRRSV as the primary antibody, the results showed that the protein could react specifically with the positive serum of pigs, producing a band consistent with the expected size of PRRSV Nsp12 ( Figure 2 B), indicating that PRRSV infection in pigs can induce the production of specific antibodies against Nsp12, and the recombinant protein can be used as an antigen for the next step of animal immunization.

[0027] Example 3 Animal Immunization

[0028] The purified Nsp12 recombinant protein was used to immunize 5 4-week-old female Balb / C mice. Another group of 5 mice was used as a negative control. Before immunization, the concentration of the purified protein was determined. 50 μg of purified protein was mixed with MnJ (β) colloidal manganese adjuvant and immunized by intramuscular, subcutaneous, and intraperitoneal multi-point injection. The second immunization was performed 10 days after the first immunization, and the third and booster immunizations were performed 7 days later. The method and dosage refer to the first immunization.

[0029] Example 4 Sequence cloning and recombinant expression of PRRSV Nsp12 antibody

[0030] (1) FACS was used to separate memory B cells.

[0031] Blood was collected from the eyeballs of the immunized mice, serum was taken as a positive control, and the mice were killed. The killed mice were soaked in 75% alcohol for 5 minutes, the mice were fixed, and the abdominal skin of the mice was gently cut with sterilized scissors. The spleen of the immunized mice was removed under sterile conditions and placed in a culture dish. A 40μm cell sieve was placed on a 50mL centrifuge tube, and the spleen was ground with a disposable grinding rod. DMEM was added to rinse the spleen, and the grinding solution was collected and centrifuged at 1000rpm for 5 minutes. Using the mouse memory B cell marker CD138, the cell population of memory B cells was marked by FACS sorting ( Figure 3 ).

[0032] (2) Amplification and cloning of antibody variable region sequences

[0033] After the memory B cell population was isolated by FACS, it was sorted into a 96-well plate containing a lysis solution, and the B cells were lysed to release intracellular RNA. The antigen-specific single B cell antibody variable region gene sequence was obtained by reverse transcription, PCR and sequencing. After comparison with the NCBI gene library, the antibody variable region sequence that was sequenced correctly was obtained, of which the heavy chain variable region nucleotide sequence of one antibody was SEQ ID NO: 8 and the light chain variable region nucleotide sequence was SEQ ID NO: 9, and the heavy chain variable region nucleotide sequence of another antibody was SEQ ID NO: 10 and the light chain variable region nucleotide sequence was SEQ ID NO: 11. The above sequences were cloned into the pcDNA3.4 vector containing the corresponding constant region.

[0034] (3) Recombinant expression of antigen-specific antibodies

[0035] The heavy chain plasmid DNA and light chain plasmid DNA were transiently transfected into suspended CHO cells at a ratio of 1:1 using PEI to recombinantly express antigen-specific antibodies. Five days after transfection, the cell supernatant was collected and the recombinant antibodies secreted and expressed in the supernatant were purified using Protein G beads. After centrifugation and filtration of the cell supernatant, it was combined with Protein G equilibrated in the column, impurities were washed with buffer, and two recombinant antibodies were eluted with glycine at pH = 2.5, named 1N14 and 2S18. The purified recombinant antibodies were identified by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining to analyze the antibody expression. The results showed that after purification, both 1N14 and 2S18 antibodies produced clear heavy chain and light chain bands ( Figure 4 A). Subsequently, the titers of the purified 1N14 and 2S18 antibodies were detected by ELISA, and the titers of both antibodies were as high as 1:256000 ( Figure 4 B).

[0036] Example 5 Identification of subtypes and analysis of reaction specificity of PRRSV Nsp12 antibodies

[0037] According to the instructions of the monoclonal antibody subtype identification kit, the subtypes of 1N14 and 2S18 antibodies were identified. The results showed that the subtype of 1N14 antibody was IgG1 heavy chain and Kappa light chain; the subtype of 2S18 antibody was IgG2a heavy chain and Kappa light chain. Figure 5 ). To verify the specificity of the reaction of 1N14 and 2S18 antibodies, immunofluorescence and Western blotting analysis were performed on Nsp12 overexpressed in 293T cells using the above two antibodies. The coding sequences of Nsp12 and Myc tags were cloned into the pCAGGS vector, and the eukaryotic expression plasmid of Nsp12-Myc was constructed and transfected into 293T cells. The results showed that the Myc tag antibody can specifically recognize the protein expressed by cells after transfection with the Nsp12-Myc plasmid, producing red fluorescence ( Figure 6 A); 1N14 and 2S18 antibodies can specifically recognize the protein expressed by cells after transfection of Nsp12-Myc plasmid, producing green fluorescence ( Figure 6 A), indicating that the above 1N14 and 2S18 antibodies can specifically recognize the undenatured Nsp12 in the immunofluorescence test. The protein samples of Nsp12-Myc transfected cells were collected, added with SDS loading buffer and boiled for denaturation, and then subjected to Western blotting analysis. The results showed that the 1N14 antibody can specifically recognize the cell protein samples transfected with Nsp12-Myc plasmid, while 2S18 cannot ( Figure 6 B), indicating that the 1N14 antibody can recognize Nsp12 after denaturation and reduction in the Western blotting experiment. In summary, both the 1N14 and 2S18 antibodies have good reaction specificity, and the antigenic epitope recognized by the 1N14 antibody is a linear epitope, while the antigenic epitope recognized by the 2S18 antibody is a conformational epitope.

[0038] Example 6 Identification of antigenic epitopes recognized by PRRSV Nsp12 antibodies

[0039] First, PRRSV Nsp12 (1-153aa) was divided into two segments (T1: 1-100aa; T2: 50-153aa), and expression plasmids were constructed according to the primers in Table 1 (SEQ ID NO: 8-11). The segmentation schematic is shown in Figure 7A. The PCR reaction conditions are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s; annealing at 56°C for 30 s; extension at 72°C for 45 s, with 35 cycles from denaturation to extension; final extension at 72°C for 10 min. The fragments were connected to the pCold-TF vector and transformed into BL21 (DE3) Escherichia coli competent cells. Positive clones were selected, and single colonies were selected for verification. After verification, expression was induced. The bacteria were cultured in a 37°C shaker until the OD value reached 0.6-0.8, and 1 mM IPTG was added to induce expression. After induction at 16°C for 24 h, the bacterial solution was collected for ultrasound. The results showed that the 1N14 antibody can recognize the T2 fragment ( Figure 7 B). Subsequently, PRRSV Nsp12 (1-153aa) was divided into three segments (T3: 1-50aa; T4: 51-100aa; T5: 101-153aa), and expression plasmids were constructed according to the primers in Table 1 (SEQ ID NO: 12-17), and sequencing, transformation, induction, and protein samples were collected. The results showed that the 1N14 antibody could recognize the T5 segment ( Figure 7 C). In summary, the antigenic epitope recognized by the 1N14 antibody is located in the T5 region, i.e., the 101-153aa region of PRRSV Nsp12.

[0040] In order to accurately identify the antigen epitope recognized by the 1N14 antibody, the amino acid sequence of the T5 fragment was truncated and synthesized into four peptides, represented by P1-P4. The results showed that the 1N14 antibody could only recognize the P1 peptide segment, that is, the 101-120 peptide segment ( Figure 8 A). Furthermore, the P1 peptide was truncated to synthesize 11 peptides, represented by P5-P15. The results showed that the 1N14 antibody could only recognize the P8 peptide and had no reaction with other peptides ( Figure 8 B). Since the P7 and P9 peptides are only 1aa shorter than the P8 peptide, and the 1N14 antibody cannot recognize the P7 and P9 peptides, it means that the polypeptide recognized by the 1N14 antibody cannot be further shortened, and the P8 peptide is the shortest epitope peptide that the 1N14 antibody can recognize. In summary, the specific antigen epitope sequence recognized by the 1N14 antibody is 104 YEFTGNGEDW 113 .

[0041] Example 7 PRRSV Nsp12 antigen epitope and spatial structure analysis

[0042] The full-length amino acid sequence of PRRSV Nsp12 was input into the Immune Epitope Database (IEDB) for B cell epitope prediction. The white area above the red dotted line is the potential B cell epitope in the amino acid sequence of PRRSV Nsp12. The horizontal axis is the corresponding PRRSV Nsp12 amino acid position. 104YEFTGNGEDW 113 The peptide region is predicted to contain B cell linear antigen epitopes ( Fig. 9 A). The three-dimensional structure of PRRSV Nsp12 was constructed using the SWISS-MODEL tool and analyzed and visualized using the PyMOL software. 104 YEFTGNGEDW 113 Spatial structural location of the antigenic epitope (blue) in PRRSV Nsp12 ( Fig. 9 B) The results showed that the identified antigen epitopes 104 YEFTGNGEDW 113 Displayed on the surface of the three-dimensional structure of PRRSV Nsp12.

[0043] Example 8 ELISA analysis of PRRSV Nsp12 antigen epitope peptide and pig positive serum

[0044] To analyze the identified antigen epitope peptides ( 104 YEFTGNGEDW 113 )The immunogenicity of the virus under the conditions of animal infection was further tested to see whether the positive antiserum induced by PRRSV infection in pigs could stimulate specific antibodies against the antigenic epitope peptide segment. The synthesized Nsp12 antigenic epitope peptide was used as an antigen to coat the ELISA reaction plate, and the pig PRRSV positive serum and pig negative serum preserved in this laboratory were diluted in a gradient of 1:10, 1:20, 1:40 and 1:80, respectively, and used as the primary antibody for indirect ELISA analysis. The results showed that the PRRSV positive serum could specifically recognize the antigenic epitope peptide, while the negative serum could not recognize the antigenic epitope peptide, indicating that the antiserum produced after PRRSV infection in pigs contained specific antibodies against the antigenic epitope peptide sequence ( Fig.10 ). Since Nsp12 encoded by PRRSV in pigs contains this peptide, and the indirect ELISA reaction using this peptide as an antigen can react with PRRSV positive serum, that is, PRRSV induces pigs to produce a specific immune response to this peptide and produce antibodies against this peptide, indicating that this antigen epitope peptide is immunogenic.

[0045] Example 9 Determination of the application scope of PRRSV Nsp12 monoclonal antibody

[0046] The main prevalent strain in my country is PRRSV-2. According to the ORF5 gene, PRRSV-2 can be further divided into 9 lineages. The main strains isolated in my country in recent years are lineage 1 (L1), lineage 8 (L8), lineage 3 (L3), and lineage 5 (L5) strains. MARC-145 cells were infected with the above four lineage strains preserved in the laboratory, and immunofluorescence analysis was performed using 1N14 antibody, 2S18 antibody and N protein antibody preserved in the laboratory as primary antibodies. The results showed that the 1N14 antibody can recognize L1, L5, and L8 strains, but not the L3 strain; the 2S18 antibody can recognize all lineage strains and produce specific red fluorescence ( Fig.11 A), indicating that the spatial conformational epitope recognized by the 2S18 antibody is conserved in the four lineages, so it can react with strains of the four lineages. The Nsp12 amino acid sequences of representative strains of different lineages were compared and analyzed, and the results showed that the 1N14 antibody recognized 104 YEFTGNGEDW 113 The antigenic epitope is conserved in the L1, L5, and L8 lineages and is a conserved linear epitope ( Fig.11 B). Furthermore, the 1N14 antibody was used to perform laser confocal microscopy to analyze the subcellular distribution and accumulation of Nsp12 in cells after PRRSV infection of permissive cell lines (MARC-145 cells) and primary target cells (PAM cells) at different times. The results showed that PRRSV infected MARC-145 cells ( Fig.12 A) and PAM cells ( Fig.12 B), the viral N protein is distributed in both the cytoplasm and the nucleus, producing specific green fluorescence; however, in the two different cells, Nsp12 is distributed in the cytoplasm after viral infection. At 8 hpi, Nsp12 can be expressed in the cytoplasm, producing specific green fluorescence, which gradually accumulates and increases with the extension of infection time, and is distributed in the cytoplasm.

[0047] Table 1 Primer sequences used in the present invention

[0048]

[0049] In summary, the present invention provides sequences of two recombinant PRRSV Nsp12 monoclonal antibodies expressed by CHO cells, recognized antigenic epitopes and uses thereof. The PRRSV Nsp12 coding sequence is cloned into the pCDNA3.4 vector to construct a eukaryotic expression vector pCDNA3.4-Nsp12-strep, which is transfected into suspended CHO cells to transiently express PRRSV Nsp12 recombinant protein, and the protein is purified using the downstream strep tag. The purified Nsp12 is used as an antigen to immunize Balb / C mice, and mouse spleen cells are isolated. Single specific B cells are isolated by FACS, and the antibody variable region sequence of a single B cell is obtained by RT-PCR. The heavy chain and light chain recombinant expression plasmids are constructed respectively, transfected into CHO suspension cells, and expressed and purified using Protein G to obtain two complete anti-PRRSV Nsp12 monoclonal antibodies 1N14 and 2S18. Among them, the 1N14 antibody can specifically recognize PRRSV Nsp12 and can be used for immunofluorescence and Western blotting detection. The heavy chain of this antibody subtype is IgG1 and the light chain is Kappa. This antibody can specifically recognize the 104-113 peptide segment of PRRSV Nsp12. The amino acid sequence it recognizes is 104 YEFTGNGEDW 113 The 2S18 antibody can specifically recognize PRRSV Nsp12 and can be used for immunofluorescence detection. The heavy chain of this antibody is IgG2a and the light chain is Kappa.

[0050] It should be understood that although the present invention has been described in detail above with general descriptions and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. Two monoclonal antibodies that specifically recognize PRRSV nonstructural protein 12, characterized in that The monoclonal antibodies are 1N14 and 2S18, wherein the amino acid sequence of the heavy chain variable region of monoclonal antibody 1N14 is shown in SEQ ID NO: 1, and the sequence of the light chain variable region is shown in SEQ ID NO: 2; The monoclonal antibody 2S18 is characterized in that the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:3, and the sequence of its light chain variable region is shown in SEQ ID NO:

4.

2. A polynucleotide, characterized in that The polynucleotides respectively encode the monoclonal antibody 1N14 and / or the monoclonal antibody 2S18 according to claim 1.

3. The polynucleotide according to claim 2, wherein The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1N14 is shown in SEQ ID NO:8, and the sequence of the light chain variable region is shown in SEQ ID NO:9; The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 2S18 is shown in SEQ ID NO:10, and the sequence of the light chain variable region is shown in SEQ ID NO:

11.

4. An antigenic epitope of PRRSV Nsp12, characterized in that: The antigenic epitope is the 104-113 peptide segment of PRRSV Nsp12, and its amino acid sequence is shown in SEQ ID NO:5 ( 104 YEFTGNGEDW 113 ).

5. Use of the monoclonal antibody according to claim 1 or the polynucleotide according to claim 2 or 3 in the detection of PRRSV for non-diagnostic purposes.

6. The use according to claim 5, characterized in that The antibody is specific for the antigenic epitope of PRRSV Nsp12.

7. A kit comprising the monoclonal antibody of claim 1 or the polynucleotide of claim 2 or 3.

8. A method for detecting the presence or level of PRRSV or its Nsp12 protein in a sample for non-diagnostic purposes, comprising using the monoclonal antibody of claim 1 or the polynucleotide of claim 2 or 3.

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