Preparation, Epitope Identification and Application of an Antibody against Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 12

Abstract

The present invention provides the preparation, epitope identification and application of a PRRSV non-structural protein 12 antibody. By constructing a eukaryotic expression vector pCDNA3.4-Nsp12-strep, transiently transfecting CHO cells, and purifying the PRRSV Nsp12 recombinant protein using the downstream strep tag, the purified Nsp12 is used as an antigen to immunize Balb / C mice. The spleen cells of the mice are isolated, and single specific B cells are separated by the FACS method. The antibody variable region sequences of the single B cells are obtained by RT-PCR. Recombinant expression plasmids of the heavy chain and light chain are constructed respectively, transfected into CHO suspension cells, expressed, and 2 complete anti-PRRSV Nsp12 monoclonal antibodies 1N14 and 2S18 are obtained by Protein G purification. The application scopes of the 1N14 and 2S18 antibodies and the specific antigen epitope recognized by the 1N14 antibody are further determined, and its sequence is 104 YEFTGNGEDW 113 。

Description

Technical Field

[0001] The present invention relates to the field of biology. Specifically, it relates to the preparation, epitope identification and application of a PRRSV non-structural protein 12 antibody. Specifically, it relates to the sequence of a monoclonal antibody against PRRSV Nsp12 recombinantly expressed by CHO cells, the antigenic epitope recognized thereby, and its use. Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the order Nidovirales, family Arteriviridae, genus Arterivirus, and is a single-stranded positive-strand enveloped RNA virus. The virus has strict host specificity and tissue tropism, mainly infecting alveolar macrophages and dendritic cells, causing acute or persistent infections. Porcine alveolar macrophages are the main target cells for PRRSV replication in the in vivo respiratory tract infection. The PRRSV genome is approximately 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 and encode the replicase polyproteins 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 viral genome replication and transcription.

[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 novel antibodies against PRRSV Nsp12, as well as their specific preparation methods, antibody sequences, antigenic epitopes and uses. The technical solution of the present invention is as follows: The PRRSV Nsp12 gene sequence is cloned into the pcDNA3.4 vector to construct a eukaryotic expression vector. The 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. The spleen cells of the mice are isolated, and the specific B cells are isolated by the FACS method. The variable region sequences of the single B cell antibodies are obtained by RT-PCR. The heavy chain and light chain recombinant expression plasmids are respectively constructed and transiently transfected into CHO suspension cells. Two complete anti-PRRSV Nsp12 monoclonal antibodies 1N14 and 2S18 are expressed and purified by Protein G, and the usage ranges of the 1N14 and 2S18 antibodies and the specific antigenic epitope recognized by the 1N14 antibody are determined.

[0006] By the single B cell amplification technique, the heavy chain variable region and light chain variable region sequences (SEQ ID NO: 1-4) of two antibodies against PRRSV Nsp12 are obtained, and two monoclonal antibodies with the above variable region sequences are recombinantly expressed in 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 this antibody subtype is IgG2a, and the light chain is Kappa.

[0007] Another purpose of the present invention is to provide the sequence determination and preparation methods of the two recombinant PRRSV Nsp12 monoclonal antibodies.

[0008] Another purpose of the present invention is to provide a method for determining and preparing an antigenic epitope of PRRSV Nsp12. Brief Description of the Drawings

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

[0010] Figure 2 It is the Western blotting identification of the purified Nsp12 recombinant protein in Example 2. (A) Lane 1 is the protein molecular weight standard, and lane 2 is the purified Nsp12 recombinant protein. 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 with the correct size. (B) Lane 1 is the protein molecular weight standard, and lane 2 is the purified Nsp12 recombinant protein. The primary antibody is the positive serum of pigs infected with PRRSV, indicating that the purified protein can specifically react with the porcine positive serum, producing a band consistent with the expected size of PRRSV Nsp12, which can be used as an antigen protein for animal immunization. At the same time, it shows that after PRRSV infects pigs, it can induce the production of specific antibodies against Nsp12.

[0011] Figure 3 It is the result of FACS sorting of mouse single B cells in Example 4. Using the surface marker molecule CD138 of B cells as the FACS sorting antibody to screen positive cells with fluorescence signals. (A) The blank control of the cells of the immunized mouse shows that there is no fluorescence signal in the cells. (B) The CD138-labeled fluorescence signal of the cells of the immunized mouse. The abscissa 10 2 On the right is the cell population of memory B cells positive for CD138. Approximately 7.76% of the positive cells are marked from it for the collection of single specific B cells.

[0012] Figure 4It is the Coomassie brilliant blue staining identification and titer determination of the purified Nsp12 recombinant antibody in Example 4. (A) Lane 1 is the protein molecular weight 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 correctly sized and clear heavy and light chain bands. (B) The antibody titers of 1N14 and 2S18 were detected by ELISA, indicating that the titers of these two antibodies can both reach 1:256000.

[0013] Figure 5 It is the identification result of antibody subtypes 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 It is the immunofluorescence test and Western blotting identification results of Nsp12 overexpressed in 293T cells using the 1N14 and 2S18 antibodies in Example 5. (A) The coding sequences of Nsp12 and the Myc tag were cloned into the pCAGGS vector to construct a eukaryotic expression plasmid of Nsp12-Myc and transfected into 293T cells. The results show that the Myc tag antibody can specifically recognize the protein expressed by the cells transfected with the Nsp12-Myc plasmid, producing red fluorescence; both the 1N14 and 2S18 antibodies can specifically recognize the protein expressed by the cells transfected with the Nsp12-Myc plasmid, producing green fluorescence, indicating that the above two antibodies can both recognize the non-denatured Nsp12 in the immunofluorescence test. (B) Protein samples were collected after transfection of Nsp12-Myc cells, added with SDS protein loading buffer and boiled for denaturation, and then detected by Western blotting. The results show that the 1N14 antibody can specifically recognize the cell protein sample transfected with the Nsp12-Myc plasmid, while 2S18 cannot, indicating that the 1N14 antibody can recognize the denatured and reduced Nsp12 in the Western blotting test. 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 7It is a schematic diagram of the specific segmented positions for initially screening the antigen epitopes recognized by the 1N14 antibody in Example 6 and the Western blotting identification diagram. (A) Five prokaryotic expression plasmids of truncated Nsp12 fragments were constructed and are shown as T1, T2, T3, T4, and T5 respectively as depicted. (B - C) The plasmids were transformed into BL21(DE3) Escherichia coli competent cells to induce the expression of the five truncated Nsp12 fragments, and protein samples were prepared and subjected to Western blotting identification with His-tag antibody and 1N14 antibody respectively. The results showed that the 1N14 antibody could recognize the T2 and T5 fragments, both of which contain 104 YEFTGNGEDW 113 sequences.

[0016] Figure 8 It is the ELISA identification diagram of the minimum antigen epitope in Example 6. (A) Four polypeptides were truncated and synthesized from the amino acid sequence of the T5 fragment and are denoted as P1 - P4. The results showed that the 1N14 antibody could only recognize the P1 peptide segment, i.e., the 101 - 120 peptide segment. (B) Eleven polypeptides were truncated and synthesized from the P1 peptide segment and are denoted as 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 could not recognize P7 and P9, it indicates that the polypeptide recognized by the 1N14 antibody cannot be further truncated, and the shortest epitope peptide sequence recognized by the 1N14 antibody is 104 YEFTGNGEDW 113 .

[0017] Figure 9 It is the antigen epitope and three-dimensional structure prediction analysis of PRRSV Nsp12 in Example 7. (A) The immune epitope database (IEDB) was used to predict the potential B cell epitopes in the full-length amino acid sequence of Nsp12. The regions above the red dashed line may have antigen epitopes. The results showed that the 104 YEFTGNGEDW 113 region where the antigen epitope recognized by the 1N14 antibody is located was predicted to have potential B cell epitopes. (B) The three-dimensional spatial structure of Nsp12 was constructed using the SWISS-MODEL tool, and the spatial structure position of the 104 YEFTGNGEDW 113 antigen epitope (blue) was analyzed and visualized. The results showed that the identified antigen epitope was displayed on the surface of the Nsp12 three-dimensional spatial structure.

[0018] Figure 10Indirect ELISA analysis of the antigenic epitope peptide recognized by 1N14 antibody in Example 8 reacting with porcine PRRSV positive serum. The results showed that porcine PRRSV positive serum could specifically recognize the antigenic epitope peptide, while porcine negative serum could not.

[0019] Figure 11 Reactivity analysis of 1N14 and 2S18 antibodies with different lineages of PRRSV strains and Nsp12 sequence conservation analysis in Example 9. (A) Immunofluorescence analysis of 1N14, 2S18 antibodies and the N protein antibody stored in the laboratory with 4 lineages (L1, L3, L5, L8) of PRRSV strains. The results showed that 1N14 antibody could recognize L1, L5, L8 strains, and 2S18 antibody could recognize strains of all lineages. (B) Amino acid sequence alignment of Nsp12 of different lineages of PRRSV strains. The results showed that the 104 YEFTGNGEDW 113 antigenic epitope recognized by 1N14 antibody was conservatively present in L1, L5, L8 lineages.

[0020] Figure 12 Subcellular localization analysis of Nsp12 in cells after PRRSV infected permissive cells and target cells at different times in Example 9. PRRSV was infected into (A) MARC-145 cells and (B) porcine alveolar macrophages for 8, 16, 24 and 32 hours respectively, and then laser confocal analysis was carried out. The primary antibodies were 1N14 antibody and the N protein antibody stored in the laboratory, and the secondary antibody was a green fluorescence secondary antibody. Detailed implementation mode

[0021] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further illustrates the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0022] Example 1 Expression and purification of PRRSV Nsp12 recombinant protein in CHO cells

[0023] The inventor first constructed the eukaryotic expression plasmid pCDNA3.4-Nsp12-strep, and the 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 analyzed by BLAST, 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 the NCBI reference. The Nsp12 fragment was obtained by PCR amplification and gel recovery, and was homologously recombined into the pcDNA3.4 eukaryotic expression vector. After sequencing analysis and comparison, the inserted gene fragment was completely identical to the PRRSV Nsp12 gene sequence, and this recombinant plasmid was named pcDNA3.4-Nsp12-strep. The specific PCR reaction conditions were: 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, and 30 cycles were set for the denaturation to extension process; final extension at 72°C for 10 min.

[0024] The CHO cells stored in liquid nitrogen were resuscitated and cultured in suspension. One day before transfection, the CHO cells were counted and the cell density was adjusted to 3×10 6 cells / mL, and their suspension growth was maintained for 24 h. On the day of transfection, the final density of CHO cells was adjusted to 6×10 6 cells / mL with freshly preheated medium at 37°C. Taking 100 mL of cell volume as a unit to configure the transfection system, 100 μg of plasmid DNA was diluted with 4 mL of Opti PRO TM SFM and slowly mixed; 100 mg of PEI transfection reagent was diluted with 4 mL of Opti PRO TM SFM and slowly mixed. The 4 mL of diluted PEI was added to the 4 mL of diluted plasmid DNA. Slowly invert and mix, and incubate the complex at room temperature for 20 minutes. The incubated complex was slowly added dropwise to 100 ml of cell suspension, and placed in a shaking incubator at 37°C, 8% CO2, and 100 r / min for suspension culture. After 18 - 22 hours, 600 μL of enhancer and 24 mL of adjuvant were added, and the culture was continued in the shaking incubator for 5 days. On the 3rd and 5th days after transfection, the supernatant and cell samples were collected respectively for SDS-PAGE electrophoresis and Coomassie brilliant blue staining identification to analyze the expression of recombinant protein ( Figure 1A). Subsequently, the target protein was purified using strep beads. The general process was as follows: The collected sample was incubated with pre-treated strep beads by shaking for 3 h. The medium was washed with PBST washing solution containing 8.8 g NaCl and 20 mL 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 for identification to analyze the purification effect of the recombinant protein. Figure 1 B).

[0025] Example 2 Analysis of antigen 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 for denaturation, and Western blotting analysis was performed. Using the strep-tag antibody as the primary antibody, the purified Nsp12 recombinant protein could be specifically recognized, generating clear and correctly sized target bands. 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 specifically react with the pig positive serum, generating a band consistent with the expected size of PRRSV Nsp12. Figure 2 B), indicating that after PRRSV infects pigs, it can induce the production of specific antibodies against Nsp12, and this recombinant protein can be used as an antigen for the next animal immunization.

[0027] Example 3 Animal immunization

[0028] Five 4-week-old female Balb / C mice were immunized with the purified Nsp12 recombinant protein. Another group of five mice was used as a negative control. Before immunization, the concentration of the purified protein was measured. 50 μg of the purified protein was mixed with MnJ(β) colloidal manganese adjuvant, and immunization was performed by multi-point injection into muscle, subcutaneous, and peritoneal cavities. The second immunization was carried out 10 days after the first immunization, and the third and booster immunizations were carried out 7 days later. The methods and doses refer to the first immunization.

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

[0030] (1) Memory B cells were sorted by FACS.

[0031] Blood was collected from the eyes of immunized mice, and the serum was taken as a positive control, and the mice were sacrificed. The sacrificed mice were soaked in 75% alcohol for 5 min, the mice were fixed, the abdominal skin of the mice was gently cut open with a sterilized scissors, the spleens of the immunized mice were aseptically removed, placed in a culture dish, a 40 μm cell sieve was placed on a 50 mL centrifuge tube, the spleen was ground with a disposable grinding rod, DMEM was added dropwise to rinse the spleen, and the grinding fluid was collected and centrifuged at 1000 rpm for 5 min. Using the marker CD138 of mouse memory B cells, the cell population of memory B cells was sorted and labeled by FACS ( Figure 3 ).

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

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

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

[0035] The heavy chain plasmid DNA and the 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 centrifuging and filtering the cell supernatant, it was combined with Protein G equilibrated with the column, the impurities were washed with buffer, and eluted with glycine with pH = 2.5 to obtain two recombinant antibodies, named 1N14 and 2S18 respectively. The purified recombinant antibodies were identified by SDS-PAGE electrophoresis and Coomassie brilliant blue staining to analyze the antibody expression. The results showed that clear heavy chain and light chain bands were produced after purification of 1N14 and 2S18 antibodies( Figure 4 A). Subsequently, the titers of the purified 1N14 and 2S18 antibodies were detected by ELISA method, and the titers of both could reach 1:256000( Figure 4 B).

[0036] Example 5 Subtype identification and reactivity specificity analysis of PRRSV Nsp12 antibody

[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 heavy chain of the 1N14 antibody subtype was IgG1 and the light chain was Kappa; the heavy chain of the 2S18 antibody subtype was IgG2a and the light chain was Kappa( Figure 5 ). To verify the reaction specificity of 1N14 and 2S18 antibodies, immunofluorescence and Western blotting analyses were performed on Nsp12 overexpressed in 293T cells using the above two antibodies respectively. The coding sequences of Nsp12 and Myc tag were cloned into the pCAGGS vector to construct a eukaryotic expression plasmid of Nsp12-Myc, and 293T cells were transfected. The results showed that the Myc tag antibody could specifically recognize the protein expressed by the cells transfected with the Nsp12-Myc plasmid, producing red fluorescence( Figure 6 A); both 1N14 and 2S18 antibodies could specifically recognize the protein expressed by the cells transfected with the Nsp12-Myc plasmid, producing green fluorescence( Figure 6 A), indicating that the above 1N14 and 2S18 antibodies could specifically recognize the native Nsp12 in the immunofluorescence assay. Protein samples after Nsp12-Myc transfection of cells were collected, added with SDS loading buffer, boiled for denaturation, and Western blotting analysis was performed. The results showed that the 1N14 antibody could specifically recognize the cell protein sample transfected with the Nsp12-Myc plasmid, while 2S18 could not recognize( Figure 6 B), indicating that the 1N14 antibody could recognize the denatured and reduced Nsp12 in the Western blotting assay. In summary, both 1N14 and 2S18 antibodies had good reaction specificity, and the antigen epitope recognized by the 1N14 antibody was a linear epitope, while the antigen epitope recognized by the 2S18 antibody was a conformational epitope.

[0038] Example 6 Identification of Antigen 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 schematic diagram of the segmentation is shown in Figure 7A. The 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 45 s, with 35 cycles set from denaturation to extension; final extension at 72°C for 10 min. The fragments were respectively ligated to the pCold-TF vector, transformed into BL21(DE3) Escherichia coli competent cells, positive clones were selected, and single colonies were picked for verification. After correct verification, induction expression was carried out. The bacteria were cultured in a shaker at 37°C until the OD value reached 0.6 - 0.8, then 1 mM IPTG was added for induction expression. After induction at 16°C for 24 h, the bacterial solution was collected for sonication. The results showed that the 1N14 antibody could 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). Expression plasmids were constructed according to the primers (SEQ ID NO:12 - 17) in Table 1, and sequencing, transformation, induction, and protein sample collection were carried out. The results showed that the 1N14 antibody could recognize the T5 fragment ( Figure 7 C). In summary, the antigenic epitope recognized by the 1N14 antibody was located in the T5 region, that is, the 101 - 153aa region of PRRSV Nsp12.

[0040] To precisely identify the antigenic epitope recognized by the 1N14 antibody, the amino acid sequence of the T5 fragment was truncated and synthesized into 4 polypeptides, denoted as 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). Further, the P1 peptide segment was truncated and synthesized into 11 polypeptides, denoted as P5 - P15. The results showed that the 1N14 antibody could only recognize the P8 peptide segment and did not react with other peptide segments ( Figure 8 B). Since the P7 and P9 peptide segments were only 1 aa shorter than the P8 peptide segment, and the 1N14 antibody could not recognize the P7 and P9 peptide segments, it indicated that the polypeptide recognized by the 1N14 antibody could not be truncated further, and the P8 peptide segment was the shortest epitope peptide that the 1N14 antibody could recognize. In summary, the specific antigenic epitope sequence recognized by the 1N14 antibody was 104 YEFTGNGEDW 113 .

[0041] Example 7 Analysis of the Antigenic Epitope and Spatial Structure of PRRSV Nsp12

[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 was the potential B cell epitope in the amino acid sequence of PRRSV Nsp12. The abscissa was the corresponding amino acid position of PRRSV Nsp12, and the 104YEFTGNGEDW 113 The peptide region was predicted to have B-cell linear epitopes ( Figure 9 A). The three-dimensional structure of PRRSV Nsp12 was constructed using the SWISS-MODEL tool and visualized by analysis with the PyMOL software 104 YEFTGNGEDW 113 The spatial structural position of the epitope (blue) in PRRSV Nsp12 Figure 9 B). The results showed that the identified epitopes 104 YEFTGNGEDW 113 were displayed on the surface of the three-dimensional structure of PRRSV Nsp12.

[0043] Example 8 ELISA analysis of the PRRSV Nsp12 epitope peptide and porcine positive serum

[0044] To analyze the immunogenicity of the identified epitope peptide ( 104 YEFTGNGEDW 113 ) under virus-infected animal conditions, it was further detected whether specific antibodies against this epitope peptide could be elicited in the positive antiserum induced after PRRSV-infected pigs. The synthesized Nsp12 epitope peptide was used as an antigen to coat the ELISA reaction plate. The porcine PRRSV positive serum and porcine negative serum stored in this laboratory were respectively serially diluted at 1:10, 1:20, 1:40, and 1:80 as primary antibodies for indirect ELISA analysis. The results showed that the PRRSV positive serum could specifically recognize this epitope peptide, while the negative serum could not recognize this epitope peptide, indicating that the antiserum produced after PRRSV-infected pigs contained specific antibodies against this epitope peptide sequence ( Figure 10 ). Since the Nsp12 encoded by PRRSV in pigs contains this peptide segment, and the indirect ELISA reaction using this peptide segment as an antigen can react with the PRRSV positive serum, that is, PRRSV induced a specific immune response in pigs against this peptide segment and produced antibodies against this peptide segment, indicating that this epitope peptide has immunogenicity.

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

[0046] The main prevalent strain in China is PRRSV-2. PRRSV-2 can be further divided into 9 lineages based on the ORF5 gene. In recent years, the main isolated strains in China are those of lineage 1 (L1), lineage 8 (L8), lineage 3 (L3), and lineage 5 (L5). The above four lineage strains preserved in the laboratory were used to infect MARC-145 cells. Immunofluorescence analysis was performed using 1N14 antibody, 2S18 antibody, and the N protein antibody preserved in the laboratory as primary antibodies. The results showed that the 1N14 antibody could recognize L1, L5, and L8 strains, but not the L3 strain; the 2S18 antibody could recognize all lineage strains, producing specific red fluorescence ( Figure 11 A), indicating that the conformational epitopes recognized by the 2S18 antibody are conserved among the four lineages, so it can react with the strains of the four lineages. The amino acid sequences of Nsp12 of representative strains of different lineages were aligned and analyzed. The results showed that the 104 YEFTGNGEDW 113 antigenic epitopes recognized by the 1N14 antibody are conservatively present in lineages L1, L5, and L8, and are conservative linear epitopes ( Figure 11 B). Further, a laser confocal experiment was performed on the localization of Nsp12 in cells at different times after PRRSV infection of the permissive cell line (MARC-145 cells) and primary target cells (PAM cells) using the 1N14 antibody, to analyze the subcellular distribution and accumulation of Nsp12 in cells after virus infection. The results showed that after PRRSV infection of MARC-145 cells ( Figure 12 A) and PAM cells ( Figure 12 B), the N protein of the virus was distributed in both the cytoplasm and the nucleus, producing specific green fluorescence; however, in the two different cells, Nsp12 was distributed in the cytoplasm after virus infection. At 8 hpi, Nsp12 could be expressed in the cytoplasm, producing specific green fluorescence, and gradually accumulated and increased with the prolongation of the infection time, and was all distributed in the cytoplasm.

[0047] Table 1 Primer sequences used in this invention patent

[0048]

[0049] In summary, the present invention provides the sequences of two recombinant PRRSV Nsp12 monoclonal antibodies expressed by CHO cells, the antigenic epitopes recognized thereby, and their uses. By cloning the PRRSV Nsp12 coding sequence into the pCDNA3.4 vector, the eukaryotic expression vector pCDNA3.4-Nsp12-strep was constructed, and it was transfected into suspension CHO cells for transient expression of the PRRSV Nsp12 recombinant protein. The protein was purified using the downstream strep tag, and the purified Nsp12 was used as an antigen to immunize Balb / C mice. The spleen cells of the mice were isolated, and single specific B cells were separated by the FACS method. The antibody variable region sequences of the single B cells were obtained by RT-PCR. Recombinant expression plasmids of the heavy chain and light chain were constructed respectively, transfected into CHO suspension cells, and two complete anti-PRRSV Nsp12 monoclonal antibodies 1N14 and 2S18 were expressed and purified using Protein G. 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 . The 2S18 antibody can specifically recognize PRRSV Nsp12 and can be used for immunofluorescence detection. The heavy chain of this antibody subtype is IgG2a, and the light chain is Kappa.

[0050] It should be understood that although the content of the present invention has been described in detail above with general descriptions and specific implementation examples, some modifications or improvements can be made on the basis of the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope claimed by the present invention.

Claims

1. Two monoclonal antibodies that specifically recognize porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 12, characterized in that The monoclonal antibodies are 1N14 and 2S18 respectively. Among them, 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; 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 the light chain variable region is shown in SEQ ID NO:

4.

2. A polynucleotide, characterized in that, The polynucleotides respectively encode monoclonal antibody 1N14 and / or monoclonal antibody 2S18 as claimed in 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 peptide of PRRSV Nsp12, characterized in that, The epitope peptide is the 104-113 peptide segment of PRRSV Nsp12, and its amino acid sequence is shown in SEQ ID NO:

5.

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

6. A kit, which comprises the monoclonal antibody of claim 1 or the polynucleotide as claimed in claim 2 or 3.

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

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