Antigenic epitope, monoclonal antibody and application of nsp12 protein of porcine reproductive and respiratory syndrome virus

Abstract

The present invention provides an antigenic epitope, monoclonal antibody and application of the nsp12 protein of porcine reproductive and respiratory syndrome virus. In this study, the expressed and purified nsp12 protein was used as an antigen to immunize mice, and five positive hybridoma cell lines that can stably secrete nsp12 mAb were successfully screened by immunological detection methods such as indirect ELISA and IFA. The results showed that the antigenic epitope recognized by monoclonal antibodies 3G11 and 9C2 is 93 TWGFESDTAY 102 , 2E3 recognizes the antigen epitope 115 DYNDAFRARQ 124 , 10G6 and 2A4 recognize the antigen epitope 142 PGPVIEPTL 150 Moreover, the three newly identified epitopes are all located on the surface of the nsp12 protein and are immunodominant epitopes. The three epitopes are highly conserved in the nsp12 protein between different strains of PRRSV-2 and can be used as targets for PRRSV-2 detection, which will help in the development of PRRSV detection methods.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an antigenic epitope of porcine reproductive and respiratory syndrome virus nsp12 protein, a monoclonal antibody and an application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an important infectious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV infection can damage the reproductive system of pregnant sows, leading to miscarriage, premature birth, stillbirth and mummified fetuses, while infection in nursery and fattening pigs mainly manifests respiratory diseases such as cough and wheezing, causing serious damage to the pig farming industry in countries around the world. PRRSV is an enveloped single-stranded positive-sense RNA virus belonging to the order Nidovirales and the genus Arterivirus of the family Arteriviridae. The full length of the genome is about 15kb. During the mRNA processing, a cap-like structure is formed at its 5' end, and there is a poly-A tail structure at the 3' end. The PRRSV genome contains 10 known open reading frames (ORF1a, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5a and ORF5-ORF7), of which ORF1a and ORF1b encode two large polyproteins pp1a and pp1ab, which are ultimately processed by four proteases encoded by ORF1a into at least 14 non-structural proteins related to genome replication and transcription. Among these non-structural proteins, nsp12 located at the C-terminus of the polyprotein encoded by ORF1b is a protein unique to arteriviruses, but the specific structure and function of nsp12 in the non-structural proteins encoded by PRRSV is still not very clear. Recent studies have found that nsp12 is a membrane-associated protein and a hub for the interaction of other nsps of PRRSV, speculating that nsp12 may be a major component of the replication and transcription complex (RTC). At the same time, through the reverse gene manipulation technology of PRRSV, the study confirmed that nsp12 is involved in the synthesis of positive and negative strand subgenomic mRNAs, but not in the synthesis of genomic RNA, indicating that nsp12 is essential for PRRSV replication. Since PRRSV infection can lead to a significant increase in the expression level of proinflammatory cytokines, studies have shown that nsp12 may be the cause of inducing increased pSTAT1-S727 and the expression of IL-1β and IL-8. It is speculated that nsp12 may participate in the pathogenesis of PRRSV by regulating the expression of cellular inflammatory factors. However, there are currently limited reports on the biological effects of PRRSVnsp12, and its protein characteristics and antigenicity are still unclear. More research is needed to analyze the exact function of nsp12 in PRRSV infection. Summary of the invention

[0003] To solve the above problems, this study used the expressed nsp12 protein as an antigen, prepared 5 specific monoclonal antibodies, and identified their antigenic epitopes, providing an effective tool for the development of diagnostic methods for PRRSV.

[0004] Specifically, the present invention provides three antigenic epitopes of nsp12 protein in a non-structural protein encoded by PRRSV, characterized in that the three antigenic epitopes are: 93 TWGFESDTAY 102 , 115 EDYNDAFRARQ 124 and 142 PGPVIEPTL 150 ;

[0005] Furthermore, the present invention provides a nucleic acid capable of encoding the above-mentioned antigenic epitopes, wherein the nucleic acid is a gene sequence capable of encoding three antigenic epitopes of the nsp12 protein in the non-structural protein encoded by the above-mentioned PRRSV;

[0006] Furthermore, the present invention provides a recombinant expression vector, which carries the above-mentioned nucleic acid.

[0007] Furthermore, the present invention provides a recombinant cell, which contains the above-mentioned recombinant expression vector, or has the above-mentioned nucleic acid integrated into its genome.

[0008] Furthermore, the present invention provides a fusion protein, which is formed by the repetition or combination of any one or more of the three antigenic epitopes of the above-mentioned nsp12 protein, or the fusion of the above-mentioned repetition or combination with a carrier protein or other antigenic epitope protein.

[0009] Furthermore, the present invention provides a monoclonal antibody that specifically recognizes the antigenic epitope of the nsp12 protein.

[0010] Preferably, the monoclonal antibodies are: monoclonal antibody 3G11, whose heavy chain variable region amino acid sequence is shown in SEQ ID NO.3 (EVQLQESGAELVKPGASVKLSCKASGYTFTSYYMFWVKQRPGQGLEWIGEINPSNGGTNFNERFKSKATLTVDKSSST ACMQVSGVASEDCAVYYCTRV), and whose light chain variable region amino acid sequence is shown in SEQ ID NO.4 (SLGERVTITCKASQDINNYLGWFQQNPGKSPKTLIYRSNRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQY DEFPPTFGAGTKLGLKR);

[0011] Monoclonal antibody 2E3, whose heavy chain variable region amino acid sequence is shown in SEQ ID NO.5 (SLSSPSTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAREISLLRLYYFDYWGQGTTVTVSS), and whose light chain variable region amino acid sequence is shown in SEQ ID NO.6 (WNQQKPGQPPRLLIYLVSNLQSGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWS);

[0012] The amino acid sequence of the heavy chain variable region of monoclonal antibody 10G6 is shown in SEQ ID NO.7 (EVQLKESGAELVKPGASVKLSCKASGYTFTSYYMFWVKQRPGQGLEWIGEINPSNGGTNFNERFKSKATLTVDKSSSTAYMQLSGLTSEDSAVYYCTRDLFAYWGQGTMVTVSS), and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8 (WNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQH).

[0013] Furthermore, the present invention provides the use of the three antigenic epitopes of the nsp12 protein and / or monoclonal antibodies in the preparation of reagents or kits for detecting PRRSV infection or detecting PRRSV antibodies.

[0014] Furthermore, the present invention provides the use of the three antigenic epitopes of the nsp12 protein and / or monoclonal antibodies in the preparation of drugs or vaccines for preventing and treating PRRSV infection.

[0015] Furthermore, the present invention provides the use of the three antigenic epitopes of the nsp12 protein in the preparation of PRRSV antibodies.

[0016] Beneficial Effects

[0017] In this study, we prepared five monoclonal antibodies against nsp12 protein, all of which had good reactivity with nsp12 protein. Subsequently, we identified five monoclonal antibodies that could recognize three antigenic epitopes in nsp12 protein ( 93 TWGFESDTAY 102 , 114 EDYNDAFRAR 123 and 142 PGPVIEPTL150 The results verified that these are conserved antigenic epitopes, and all monoclonal antibodies targeting these antigenic epitopes can recognize the nsp12 protein of PRRSV-2 strains, indicating that monoclonal antibodies targeting the above three antigenic epitopes have the potential to be used as differential diagnosis to distinguish between PRRSV-1 and PRRSV-2 strains. In short, this study provides new selection targets for PRRSV detection based on antigenic epitopes.

[0018] Furthermore, the present invention provides a specific sequence of a novel monoclonal antibody against nsp12 protein, wherein, in the variable region sequence of the monoclonal antibody 3G11, the amino acid sequence of the heavy chain variable region is: the amino acid sequence shown in the sequence SEQ ID NO.3, or a conservative variant obtained by adding, deleting, replacing or modifying the sequence SEQ ID NO.3 with one or more amino acids; the amino acid sequence of the light chain variable region is: the amino acid sequence shown in the sequence SEQ ID NO.4, or a conservative variant obtained by adding, deleting, replacing or modifying the sequence SEQ ID NO.4 with one or more amino acids;

[0019] Among them, in the variable region sequence of the monoclonal antibody 2E3, the amino acid sequence of the heavy chain variable region is: the amino acid sequence shown in the sequence SEQ ID NO.5, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the sequence SEQ ID NO.5; the amino acid sequence of the light chain variable region is: the amino acid sequence shown in the sequence SEQ ID NO.6, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the sequence SEQ ID NO.6;

[0020] Among them, in the variable region sequence of the monoclonal antibody 10G6, the amino acid sequence of the heavy chain variable region is: the amino acid sequence shown in the sequence SEQ ID NO.7, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the sequence SEQ ID NO.7; the amino acid sequence of the light chain variable region is: the amino acid sequence shown in the sequence SEQ ID NO.8, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the sequence SEQ ID NO.8;

[0021] The use of the above antibodies can solve the technical problems of the inability to accurately quantify the existing PRRSV virus and the inability to verify the expected expression effect, and solve the key problem of vaccine preparation. At the same time, the pharmaceutical composition containing the antibody can prevent and / or treat PRRSV diseases caused by pigs regardless of species, and can not only solve the porcine reproductive and respiratory syndrome caused by the classical strain of PRRSV, but also solve the porcine reproductive and respiratory syndrome caused by the variant strain of the porcine reproductive and respiratory syndrome virus, with a broad spectrum; make up for the lack of commercial vaccines, can be used for emergency prevention and / or treatment, reduce the incidence rate, and reduce the mortality rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 SDS-PAGE and Western blot detection of fusion expression of nsp12 protein, where A: M is the protein molecular weight standard; 1: pCold-nsp12 induced expression of the whole bacteria; 2: pCold-nsp12 induced expression of the supernatant after ultrasonic lysis; 3:

[0023] Precipitation after ultrasonic lysis of pCold-nsp12 induced expression; B: Western blot detection using His tag antibody; C: Western blot detection using positive serum from pigs infected with HuN4 strain.

[0024] Figure 2 ELISA screening and identification of monoclonal antibodies against nsp12 protein, including: A: using the expressed His-nsp12 protein as the coating antigen, positive monoclonal antibodies against nsp12 were screened by indirect ELISA; B: Western blot identification of 5 monoclonal antibodies using the expressed His-nsp12 protein; C: IFA identification of 5 monoclonal antibodies using PRRSV-infected Marc-145 cells.

[0025] Figure 3 Construction strategy of PRRSV nsp12 protein truncations and identification of antigenic epitopes by Western blot and ELISA, including: A: Construction strategy of PRRSV NSP12 protein truncations; B: Identification of mAb antigenic epitopes by indirect ELISA; DF: Identification of mAb antigenic epitopes by Western blot;

[0026] Figure 4 Conservative analysis of PRRSV nsp12 protein antigenic epitopes and three-dimensional structural model of nsp12 protein, where A: Conservative analysis of PRRSV nsp12 protein antigenic epitopes; B: Spatial structure and position of the identified epitopes, in the predicted 3D structure of PRRSVnsp12, the identified epitopes 93 TWGFESDTAY 102In the highlighted in red, the epitope 115 EDYNDAFRARQ 124 Highlighted in blue, the epitope 142 PGPVIEPTL 150 Highlighted in orange.

[0027] Figure 5 Characterization of IFA reactivity of nsp12 monoclonal antibody with different PRRSV strains. DETAILED DESCRIPTION

[0028] In order to make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples, which are merely illustrative and are not intended to limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained by commercial routes or conventional methods unless otherwise specified.

[0029] Cells, viruses and experimental animals used in this application

[0030] SP2 / 0 cells, HEK293T cells, and Marc-145 cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO2. PRRSV-2 strains including HuN4, CH-1a, and ZJhz021, as well as positive porcine sera against PRRSV-2 strains, were prepared and stored in our laboratory.

[12] The pCAGGS-Nsp12-HA plasmid was constructed and maintained by our laboratory; 6-8 week-old BALB / c mice were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.

[0031] Example 1 Construction of nsp12 protein expression plasmid

[0032] According to the nsp12 sequence in the genome of PRRSV HuN4 strain (GenBank: EF635006), the forward primer pColdI-nsp12-F: 5'-ACCCTCGAGGGATCC GAATTC ATGGGCCGCCATTTTACC-3' (SEQ ID NO.1, the underline is the EcoRI restriction site) and reverse primer pColdI-nsp12-R: 5'-GGTAAAATGGCGGCCCAT GAATTCGGATCCCTCGAGG GT-3' (SEQ ID NO.2, the underline is the EcoRI restriction site), and then the nsp12 gene was amplified by RT-PCR using the cDNA of PRRSV HuN4 strain as a template. The reaction system was 50μl: including 25μl of Q5 ultra-fidelity premixed enzyme (2×), 1μl of upstream and downstream primers, 50ng of cDNA template, and ddH2O to 50μl. PCR reaction conditions: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 58℃ annealing for 20s, 72℃ extension for 1min30s, a total of 35 cycles; finally, 72℃ extension for 10min; at the same time, the pColdI plasmid was treated with EcoRI restriction enzyme digestion, and then the PCR product and the enzyme digestion product were purified by agarose gel electrophoresis and gel recovery kit, and then cloned into the pColdI plasmid by homologous recombination, and positive clones were identified by PCR amplification and sequencing.

[0033] Expression and purification of nsp12 recombinant protein

[0034] The pColdI-His-nsp12 positive strain transformed into BL21(DE3) competent cells was inoculated into 200 ml LB liquid medium containing ampicillin resistance and cultured at 37°C with shaking at 200 r / min. 600 When nm reached 0.6, IPTG was added at a final concentration of 1 mol / L, and protein expression was induced at 16°C, 200 r / min for 16 h. The cells were collected by centrifugation at 8000 g for 5 min at 4°C, resuspended in 20 mL of buffer (50 mM Tris-HCl, 250 mM NaCl, pH 8.0), and ultrasonically lysed for 15 min under ice bath conditions. The lysate was then centrifuged at 12000 g for 30 min at 4°C, and the nsp12 protein with His tag was purified using QIAGEN Ni-NTA affinity chromatography resin and analyzed by SDS-PAGE. At the same time, Western blot identification was performed using PRRSV HuN4 positive serum and His tag antibody, respectively.

[0035] The results showed that the protein band corresponding to His-nsp12 was observed at about 19 KDa, and the expressed protein was mainly present in the precipitate of the ultrasonic lysis product. TM After purification with excel affinity column, the purity of nsp12 protein can reach more than 95% ( Figure 1 A). In order to further identify the target protein, anti-His monoclonal antibody and anti-PRRSV positive pig serum were used as primary antibodies, respectively. Western blot was used for identification. The results showed that the detection results of the two antibodies were consistent, and the nsp12 protein-specific band was detected at 19KDa ( Figure 1 B) indicates that the expressed and purified protein can be used as an antigen for immunizing mice.

[0036] Example 2 Screening and preparation of monoclonal antibodies against nsp12

[0037] Mouse immunization and hybridoma cell preparation

[0038] 100 μg of purified nsp12 recombinant protein was mixed with an equal amount of Freund's complete adjuvant (first immunization) or incomplete adjuvant for emulsification, and three female BALB / c mice aged 6 to 8 weeks were immunized. The same dose was used for booster immunization every 14 days, for a total of four immunizations. Three days after the last immunization, the spleen cells of the immunized mice were taken and fused with SP2 / 0 cells using PEG-1450. The fused cells were cultured in a selective medium containing DMEM containing HAT and 20% FBS. After 10 days of cell fusion, the supernatant of the fused cells was collected, and antibodies against the nsp12 protein were detected by indirect ELISA, positive hybridoma cells were screened, and the obtained positive hybridoma cell lines were subcloned three times by limiting dilution. In order to obtain monoclonal antibodies against Nsp12 protein, mice were immunized with purified fusion protein His-nsp12, and cell fusion was performed 3 days after booster immunization. After screening by indirect ELISA, it was found that the antibodies secreted by 5 hybridoma cells were highly reactive to the fused His-nsp12 protein, and their OD 450 The values ​​were all greater than 1.5, and the five positive hybridoma cell lines secreting anti-PRRSV Nsp12 protein antibodies were named 3G11, 9C2, 2E3, 10G6 and 2A4 ( Figure 2 A). At the same time, 5 monoclonal antibodies were used to detect His-nsp12 protein by Western blot, and the results showed that they specifically recognized the nsp12 protein band ( Figure 2 B). IFA detection was performed using Marc-145 cells infected with PRRSV strains. The results showed that all five mAbs could specifically recognize PRRSV-infected cells ( Figure 2 C). In order to identify the specificity of the five mAbs in recognizing the PRRSV nsp12 protein, the five mAbs were identified by Western blot using cell samples harvested 48 hours after HEK293T cells were transfected with the eukaryotic expression plasmid pCAGGS-Nsp12-HA, and cell samples harvested 48 hours after Marc-145 cells were infected with the PRRSV HuN4 strain. The results showed that the five mAbs could specifically recognize not only the eukaryotic nsp12 protein, but also the nsp12 protein expressed by the HuN4 strain in Marc-145 cells.

[0039] Expression of nsp12 overlapping peptides

[0040] The antigenic epitopes recognized by the monoclonal antibody were identified by using the segmented truncated expression of nsp12 protein. nsp12 was designed to be divided into 17 overlapping peptides. PRRSV HuN4 cDNA was used as a template and the primers in Table 1 were used to amplify the gene fragments encoding the 17 peptides. Each gene fragment was cloned into a prokaryotic expression vector and transformed into BL21 (DE3) competent cells for induced expression.

[0041] Table 1 Primers used to amplify nsp12 overlapping peptide P1-P17 gene fragments

[0042]

[0043] Western blot detection

[0044] The collected protein expression samples were mixed with 5× protein loading buffer, boiled for 10 minutes, and separated by 10% SDS-PAGE electrophoresis, and stained with Coomassie Brilliant Blue R-250. At the same time, the electrophoresed protein samples were transferred to the NC membrane, and the NC membrane was blocked with TBS solution containing 5% skim milk at room temperature for 2 hours. The NC membrane was then incubated with the screened monoclonal antibodies or label antibodies, positive serum, etc. at room temperature for 1 hour, washed 3 times with 0.5% Tween-20 (TBST), and incubated with HRP-labeled goat anti-mouse IgG (H+L) as the secondary antibody (1:10000) at room temperature for 1 hour, washed 3 times with TBST, and developed with ECL. The immunoblot results were detected by the Tianneng imaging system. The results showed that the 5 mAbs could not only specifically recognize the eukaryotic expressed nsp12 protein, but also the nsp12 protein expressed by HuN4 strain infected Marc-145 cells.

[0045] Indirect immunofluorescence (IFA) assay

[0046] Marc-145 cells were infected with 0.1 MOI of PRRSV HuN4, CH-1a, and ZJhz021 strains, respectively. After 24 hours of infection, the infected Marc-145 cells were fixed with pre-cooled 80% ethanol. After washing the cells three times with PBS, the monoclonal antibodies obtained by screening were added, and the cells were exposed to 37°C for 1 hour. After washing with PBS three times, 1:1000 diluted FITC-labeled goat anti-mouse IgG (H+L) was added, and the cells were incubated at 37°C in the dark for 30 minutes. After washing with PBS, the detection results were recorded under an inverted fluorescence microscope.

[0047] Amino acid sequence analysis and structure prediction

[0048] The full-length genes of 26 PRRSV-2 isolates were obtained from the GenBank database, and the nsp12 gene of each strain was selected to compare and analyze the conservation of the amino acid sequence using DNASTAR MegAlign software. The spatial structure of the PRRSV nsp12 protein was constructed using SWISS-MODEL, and the spatial structural position of the visualized antigen epitope in the NSP12 protein was analyzed using PyMOL software.

[0049] The results showed that in order to determine the antigenic epitopes recognized by the five mAbs in nsp12 protein, we used 17 overlapping expressed nsp12 and its truncated peptides as antigens ( Figure 3 A), 5 monoclonal antibodies were tested by Western blot and ELISA. Among them, for the three overlapping truncated GST fusion proteins A (1-68aa), B (52-102aa) and C (95-154aa) cloned in the pGEX-6p-1 vector, the 5 monoclonal antibodies could not recognize the truncated A (1-68aa), while the monoclonal antibodies 9C2 and 3G11 only recognized the truncated B (52-102aa) but not the truncated C (95-154aa); the other three monoclonal antibodies 2E3, 2A4 and 10G6 could only recognize the truncated C (95-154aa) but not B (52-102aa) ( Figure 3 B and 3C). Based on this, truncation B (52-102aa) was further divided into 4 overlapping truncated peptides, and truncation C (95-154aa) was further divided into 10 overlapping truncated peptides. After fusion expression with GST, Western blot and ELISA were performed. The results showed that 3G11 and 9C2 did not recognize the B1 (52-82) antigen region, but could simultaneously recognize B2 (83-102), B3 (88-102) and B4 (93-102) ( Figure 3 D), based on this, the antigen epitopes recognized by the three monoclonal antibodies 3G11 and 9C2 were preliminarily determined to be 93 TWGFESDTAY 102. Monoclonal antibody 2E3 can recognize C1 (95-132aa) and C3 (95-127aa) at the same time, but not C2 (125-154aa), C4 (95-122aa), C5 (95-117aa) and C6 (95-112aa). Therefore, it is speculated that the antigen region it recognizes exists in the C-terminus of C3. On this basis, the C-terminus of C3 was further divided into 5 peptide segments for truncated expression and identification. The results showed that monoclonal antibody 2E3 only recognized C3.4 (115-124aa), but not C3.1 (118-127), C3.2 (117-126aa), C3.3 (116-125aa) and C3.5 (114-123aa) ( Figure 3 E), so it is speculated that the antigen epitope recognized by the three monoclonal antibodies of 2E3 is 115 DYNDAFRARQ 124 The results of the monoclonal antibodies 2A4 and 10G6 showed that they could simultaneously recognize C2 (125-154), C7 (130-154), C8 (135-154) and C9 (140-154), but not C10 (145-154). Therefore, the above-mentioned minimum recognition region C9 was truncated into 5 peptide segments for expression and identification. The results showed that 2A4 and 10G6 could simultaneously recognize C9.2 (141-150) and C9.3 (142-151), but not C9.1 (140-149), C9.4 (143-152) and C9.5 (144-153) ( Figure 3 F), based on which it is inferred that the antigenic epitopes recognized by 10G6 and 2A4 are 142 PGPVIEPTL 150 .

[0050] Three monoclonal antibodies with better effects were selected for sequencing analysis, among which,

[0051] Monoclonal antibody 3G11, whose heavy chain variable region amino acid sequence is shown in SEQ ID NO.3 (EVQLQESGAELVKPGASVKLSCKASGYTFTSYYMFWVKQRPGQGLEWIGEINPSNGGTNFNERFKSKATLTVDKSSSTACMQVSGVASEDCAVYYCTRV), and whose light chain variable region amino acid sequence is shown in SEQ ID NO.4 (SLGERVTITCKASQDINNYLGWFQQNPGKSPKTLIYRSNRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPPTFGAGTKLGLKR);

[0052] Monoclonal antibody 2E3, whose heavy chain variable region amino acid sequence is shown in SEQ ID NO.5 (SLSSPSTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAREISLLRLYYFDYWGQGTTVTVSS), and whose light chain variable region amino acid sequence is shown in SEQ ID NO.6 (WNQQKPGQPPRLLIYLVSNLQSGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWS);

[0053] The amino acid sequence of the heavy chain variable region of monoclonal antibody 10G6 is shown in SEQ ID NO.7 (EVQLKESGAELVKPGASVKLSCKASGYTFTSYYMFWVKQRPGQGLEWIGEINPSNGGTNFNERFKSKATLTVDKSSSTAYMQLSGLTSEDSAVYYCTRDLFAYWGQGTMVTVSS), and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8 (WNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQH).

[0054] In order to analyze the conservation of the antigenic epitopes recognized by the monoclonal antibodies obtained in this study, four lineage reference strains of L1, L3, L5 and L8 were collected from 26 strains of type 2 PRRSV. The amino acid sequences encoded by nsp12 of the four major epidemic lineage reference strains were compared and analyzed using DNASTAR MegAlign software. The results showed that the epitopes 93 TWGFESDTAY 102 Highly conserved among the four lineages; epitope 115 DYNDAFRARQ 124 It is highly conserved among L8, L5, and L3, with only 1 amino acid difference in L1 ( 118 D- 118 E); epitope 142 PGPVIEPTLQ 150 Highly conserved in L8, L5, and L3, with two amino acid differences in L1 ( 144 PV 145 - 144 HT 145 )( Figure 4The three-dimensional structure of PRRSV nsp12 was constructed using the SWISS-MODEL server, and the positions of the three newly identified linear epitopes in the spatial structure were visualized using PyMOL software. The results showed that the epitopes 93 TWGFESDTAY 102 Can form a stable β-fold, epitope 115 DYNDAFRARQ 124 Exists in α-helical form, epitope 142 PGPVIEPTL 150 It exists in the form of random coils, and all three are exposed on the surface of the structure, which is conducive to the recognition of antigenic epitopes.

[0055] Application of nsp12 monoclonal antibody in the detection of different PRRSV

[0056] In order to analyze the application of the above mAbs in different PRRSV detection, the five mAbs were respectively used for IFA identification with Marc-145 cells infected with three different PRRSV-2 isolates, namely the highly pathogenic PRRSV virulent strain HuN4, the classical strain CH-1a and the NADC30 strain ZJhz021. The results showed that the five mAbs could produce specific fluorescence with the three PRRSV strains mentioned above ( Figure 5 ), which is present in the epitope 115 DYNDAFRARQ 124 middle 118 D / 118 The amino acid mutation of E did not affect the recognition of the antigen epitope by the monoclonal antibody. The results showed that the five monoclonal antibodies obtained had good reactivity to the HP-PRRSV-like strain, CH-1a-like strain and NADC30-like strain in PRRSV-2 and could be used for the detection of the above strains.

[0057] In summary, from the above results we can see that we identified 5 monoclonal antibodies that can recognize three antigenic epitopes in the nsp12 protein ( 93 TWGFESDTAY 102 , 115 DYNDAFRARQ 124 and 142 PGPVIEPTL 150 ), the nsp12 amino acid sequence of 26 PRRSV-2 reference strains of different lineages was compared and analyzed, indicating that the antigenic epitope 93 TWGFESDTAY 102 It is completely conserved among the four lineages of PRRSV-2 isolates currently prevalent in China; and the antigenic epitope 114-123aa ( 115 DYNDAFRARQ 124) and epitope 142-150aa ( 142 PGPVIEPTL 150 ) is completely conserved between L8, 5, and 3, while only 1 or 2 amino acid differences exist in Lineage 1 ( 118 D / E or 144 PV 145 / 144 HT 145 ). The IFA test results based on different strains confirmed that the three monoclonal antibodies could specifically recognize the ZJhz021 strain of the NADC30-like strain belonging to Lineage1, indicating that although there are mutant amino acids in Lineage1, 118 The amino acids of the D / E mutation cannot affect the antigenic changes of the nsp12 protein and belong to conserved antigenic epitopes. All monoclonal antibodies targeting these antigenic epitopes can recognize the nsp12 protein of the PRRSV-2 strain.

[0058] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. An antigenic epitope polypeptide of nsp12 protein in a nonstructural protein encoded by PRRSV, characterized in that The antigen epitopes are respectively: TWGFESDTAY, EDYNDAFRARQ and PGPVIEPTL.

2. A nucleic acid capable of encoding the above-mentioned antigenic epitope, wherein the nucleic acid is a gene sequence capable of encoding an antigenic epitope polypeptide of the nsp12 protein in the non-structural protein encoded by PRRSV according to claim 1.

3. A recombinant expression vector carrying the nucleic acid according to claim 2.

4. A recombinant cell, comprising the recombinant expression vector of claim 3, or the nucleic acid of claim 2 integrated into its genome.

5. A fusion protein, which is formed by the repetition or combination of one or more of the antigenic epitope polypeptides of the nsp12 protein in claim 1, or the fusion of the above repetition or combination with a carrier protein or other antigenic epitope proteins.

6. A monoclonal antibody that specifically recognizes the antigenic epitope polypeptide of the nsp12 protein according to claim 1, wherein the monoclonal antibodies are: monoclonal antibody 3G11, whose heavy chain variable region amino acid sequence is shown in SEQ ID NO.3, and whose light chain variable region amino acid sequence is shown in SEQ ID NO.4; monoclonal antibody 2E3, whose heavy chain variable region amino acid sequence is shown in SEQ ID NO.5, and whose light chain variable region amino acid sequence is shown in SEQ ID NO.6; monoclonal antibody 10G6, whose heavy chain variable region amino acid sequence is shown in SEQ ID NO.7, and whose light chain variable region amino acid sequence is shown in SEQ ID NO.

8.

7. Use of the antigenic epitope polypeptide of the nsp12 protein according to claim 1 and / or the monoclonal antibody according to claim 6 in the preparation of a reagent or kit for detecting PRRSV infection or detecting PRRSV antibodies.

8. Use of the antigenic epitope polypeptide of the nsp12 protein according to claim 1 and / or the monoclonal antibody according to claim 6 in the preparation of a drug or vaccine for preventing and treating PRRSV infection.

9. Use of the antigenic epitope polypeptide of the nsp12 protein in claim 1 in preparing PRRSV antibodies.

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