Construction Of The Double Genes Prokaryotic Expression Vector And P.pastoris Expression Vectors Of PRRSV N Gene And Study On Their Expression

The N genes of both North American(N_A) and European(N_E) genotype PRRSV were amplified by RT-PCR and two hands of study were carried out as follow in this study:(1) The double genes fusion expression vector was constructed and relative research on expression was then carried out.(2) The Pichia pastoris(P.pastoris) expression vectors pPIC9K-N_A and pPIC9K-N_E were constructed respectively and relative researches on expression were carried out.It was of great significance to prevention and control of PRRS and established foundation for further study on structure and function of N protein,preparation of diagnostic reagent and differential diagnosis and so on.1.The Construction of the double genes fusion expression vector pET-32a(+)-N_A-N_E and study on its expression.According to sequences of the North American and European genotype PRRSV N gene,two specific primers were designed,and the N_A and N_E gene were obtained by RT-PCR.The cloning vectors pMD19-T-N_A and pMD19-T-N_E were then constructed by inserting the N_A and N_E gene into pMD19-T simple vector,and sequencing identifications were then carried out.The recombinant plasmid pET-32a(+)-N_A was constructed by connecting the digested products of pMD19-T-N_A and pET-32a(+) with Ncoâ… & EcoRâ… ,while the double genes fusion expression vector pET-32a(+)-N_A-N_E was then constructed by connecting the digested products of pET-32a(+)-N_A and pMD19-T-N_E with EcoRâ… & Notâ… .The molecular weight of the identification products by PCR and different digestions of fusion expression vector pET-32a(+)-N_A-N_E were coincident with expectation.So it displayed that the double genes fusion expression vector pET-32a(+)-N_A-N_E was successfully constructed by the identification results.The vector pET-32a(+)-NA-EU-N was then transformed into E.coli BL21 which was then induced by IPTG with a optimal concentration-1.0mM/L and a optimal time-3h.The result was coincident with expectation that the products were 49kD while analyzed by SDS-PAGE and the fusion protein N possesed the reactionogenicity to PRRSV positive serum antibody while analyzed by Western blotting.2.The construction of the P.pastoris expression vectors pPIC9K-N_A and pPIC9K-N_E and study on their expression. The N_A gene was obtained again by PCR with a pair of enzyme digest site changed primer and cloned it into pMD19-T simple vector to construct a new vector called pMD19-T-N_Al.The N_A and N_E gene obtained from the double digestion products of the vectors pMD19-T-N_Al and pMD19-T-N_E with EcoRâ… & Notâ… were inserted into the P.pastoris expression vector pPIC9K linearized with the same enzyme to construct the expression vectors pPIC9K-N_A and pPIC9K-N_E and identifications of the double expression vectors by PCR and double digestion were the carried out respectively.It displayed that the expression vectors pPIC9K-N_A and pPIC9K-N_E were successfully constructed by the results that the products of PCR and double digestion were as the same molecular weight as that we expected. The P.pastoris expression vectors pPIC9K-N_A and pPICgK-N_E were electro transformed into GS115 after linearizing with Sacâ… respectively and the multi-copy transformants obtained by sequentially screening on G-418/YPD medium plates were then identified and the results of identification showed that the transformants were Mut⁺ and positive by PCR.The recombinants of P.pastoris were then induced by methanol and the maximum products were emerged when they were induced for 96h.The double genotypes N protein expressed in P.pastoris were both 15kD approximately and had the reactionogenicity to propotional positive antibody based on the Dot blotting.