Prokaryotic Expression Of PRRSV Nsp1α Gene In E.coli And Preparation Of Polyclonal Anti-nsp1α Antibody And Monoclonal Anti-nsp1α Antibody

Porcine reproductive and respiratory syndrome (PRRS) is caused by porcine reproductive and respiratory syndrome virus (PRRSV), which leads to reproductive failure in sows and respiratory problems in piglets. This infectious disease is first discovered in the United States. Now it is widely popular in the world’s major countries. In 1996, Guo et al first isolated PRRSV from aborted fetuses pig, which confirm the presence of PRRS in China. In the following years, the epidemic spread in most of the country and rises in some areas. It has caused great economic losses the pig industry.PRRSV is an enveloped single-strand RNA virus. Studies have shown that the virus can cause inhibition of the body immune suppression which form persistent infection and has antibody-dependent enhancement. The virus genetic sequence has significantly variation. PRRSV genome encodes proteins that can be divided into structural proteins and non-structural proteins. The main function of the non-structural protein is in the synthesis phase of replication of the virus and the regulation of host immune responses in viral RNA. The non-structural protein la (nspla) can suppress the Ⅰ-interferon, which play an important role in PRRSV escaping immune process.In this study, the nsp1α gene fragment was amplified and was inserted into prokaryotic expression vector. The recombinant plasmids express the recombinant proteins which are immunogen. Then polyclonal antibodies and monoclonal antibodies were prepared. This study includes two sections.1. Prokaryotic expression of PRRSV nspla gene in E.coli and preparation of polyclonal anti-nsp1α antibodyThe nspla gene was amplified from the eukaryotic recombinant plasmid pcDNA3.1-nsp1α. Then the nsp1α was inserted into prokaryotic expression vector pET32a. The recombinant plasmid pET32a-nsp1α was transformed into E. coli BL21 (DE3). The E.coli BL21 (DE3) was train at 37 ℃ and induced 4h with 1mM isopropyl thiogalactoside (IPTG). The recombinant protein was abundantly expressed in the form of inclusion bodies. The bacteria liquid was lysed with ultrasound. The inclusion bodies were washing with a series of buffer and were collected with 8M urea buffer. The refolding inclusion body protein immunized rabbits with three immunizations. The refolding protein was used as coating antigen. The antibody titer for indirect ELISA is 1:2048000 or more.2. Prokaryotic expression of PRRSV nsp1α gene in E.coli and preparation of monoclonal anti-nsp1α antibodyThe nspla gene was amplified from the eukaryotic recombinant plasmid pcDNA3.1-nsp1α and inserted into prokaryotic expression vector pET28a. The recombinant plasmid pET28a-nsp1α was transformed into E.coli BL21 (DE3). The E.coli BL21 (DE3) was trained at 37℃ and induced 6h with 1mM isopropyl thiogalactoside (IPTG). The recombinant protein was abundantly expressed in the form of inclusion bodies; Indirect ELISA showed that the recombinant protein could react swine PRRSV positive serum, which has good immunological reactivity. The recombinant protein immunizes BALA/c mice which include three times immunization and a booster immunization. Then spleen cells of mice were fused with SP2/0 tumor cells. The pET28a-nsp1α recombinant protein as coating antigen for the initial screening which use indirect ELISA.The indirect immunofluorescence assay was further screening. And hybridomas cells which secreting anti-nsp1α antibody was obtained finally. Then mouse ascites was produced which the indirect ELISA titer is 1:512000.Anti-nsp1α monoclonal and polyclonal antibodies were detected through western blot and indirect immunofluorescence and immunohistochemistry assay. The results show that anti-nsp1α polyclonal and monoclonal antibodies are capable of specifically recognizing the eukaryotic recombinant proteins pcDNA3.1-nspla and natural PRRSV nspla. This study provides a basis for the development of PRRSV diagnostic methods based on the nsp1α detection and a further study of nsp1α function.