Generation Of Monoclonal Antibodies Against Nsp7 Protein Of Porcine Reproductive And Respiratory Syndrome Virus And Identification Of Antigen Epitopes On Nsp7 Protein Of PRRSV

Porcine reproductive and respiratory syndrome is caused by infection of PRRSV. The disease was first discovered in the Unites States and the virus was isolated in the Netherlands in 1991. In China, the first strain, named CH-la, was isolated in 1996. At present, the disease is prevalent in many pig-breeding countries all over the world, resulting in a huge economic loss for global pig industry. Nsp7 protein is a major non-structural protein of PRRSV with high immunogenicity. It was verified that the specific antibody against Nsp7 protein was present in pigs infected with PRRSV and the antibody can remain for a long time. Besides, owing to its high conservation among different strains, the protein is an ideal target protein for developing a diagnostic method. However, little is known about the epitopes of Nsp7 by now. In this study, monoclonal antibody and synthesized polypeptides were used to confirm the epitopes of Nsp7, laying a foundation for the development of new diagnostic reagents. The main contents of the study are as following:1. The cloning and expression of Nsp7 gene of PRRSV WUHl strainTotal RNA of PRRSV WUH1 strain was extracted and Nsp7 gene was amplified by RT-PCR using specific primers, and was cloned into prokaryotic expression vector pET-28a, generating prokaryotic expression plasmid pET-28a-Nsp7. High efficient expression of 6His-Nsp7 was achieved by IPTG induction and the fusion protein has a molecular weight of 37kDa, mainly in a soluble form. Western Blotting verified that expression product possessed a nice reactivity.2. The preparation of monoclonal antibody against Nsp7 protein of PRRSV WUHl strainThe prokaryotic expression product of PRRSV NSp7 protein was purified via Ni-NTA column and used as immunogen to immunize 6-week female BALB/c mice. By fusion and indirect ELISA screening,19 strains of monoclonal antibody against Nsp7 protein was obtained, namely D1F9,D3G10,D1E9,B2B3,B3D6,BIF,B2E11,B1F5,D1C11,C4G7,C2A8,C2D11,B2G3,C3C7,B1F8,C4B2,B4H10,B4E2 and D1C10. Chromosome counting of hybridoma was performed by colchicine lysis and the result showed that the numbers of chromosome were 90 to 105. Subtyping analysis revealed that all strains were IgG1 except that B1F7 was IgM subtype. Mice were injected with 19 strains of hybridoma for production of ascites in large quantity. The constructed eukaryotic expression plasmid PCAGGS-HA-Nsp7 was transinfected into Hela cells and the expressed protein was collected. Western blotting and IFA confirmed that all these strains of monoclonal antibody reacted specifically with eukaryotically expressed Nsp7 protein except D1F9.3. The identification of the epitopes of PRRSV Nsp7 proteinAccording to the results of potential epitopes prediction by bioinformatic software, Nsp7 protein was truncated into 4 fragments (pN71(1～116aa), pN72(56～163aa), pN73(112～213aa) and pN74(42～252aa)). The prokaryotic expression vectors for the 4 fragments, pET-28a-pN71, pET-28a-pN72, pET-28a-pN73 and pET-28a-pN74 were transformed into E. coli BL21 (DE3) and four proteins were efficiently expressed after IPTG induction. We found by Western Blotting and indirect ELISA that 13 of the 19 strains of monoclonal antibody against Nsp7 protein could react with pN72 directly or indirectly.Based on this, a series of 16 short peptides containing complete pN72 fragment and other potential epitopes were designed. Each polypeptide was 15 aa in length and there is a repeat of 5aa between each two neighboring short peptides. By indirect ELISA with 19 strains of anti-Nsp7 monoclonal antibody, we found that D1E9, D1C10, D3G10 had specific reaction with SP9 and SPIO. B1F5 had specific reaction with SP10 and other antibodies had no reaction with the polypeptides. Furthermore, the reaction of 16 polypeptides with the sera of pigs artificially infected with PRRSV showed that only SP10 could be recognized by positive sera, indicating that SP10 was a specific epitope of PRRSV.In this study, we identified a specific epitope on PRRSV Nsp7 protein (SP10) for the first time, providing useful information for the establishment of new diagnostic method and the development of gene engineering vaccines, and laying the basis for the study on interaction between PRRSV Nsp7 protein and host immune system.