Expression of E2 gene of bovine viral diarrhoea-mucosal disease virus in Pichia Pastoris and identification of its expression product.

The sE2 gene of HB-bd strain isolated of BVDV was obtained from E2 gene removed C-terminal transmernbrane domain by PCR.The sE2 gene was subcloned into the expression vector pPIC9K and then transfected into DH5α.The recombinant plasmids were extracted and identificated by enzyme digestion and PCR.The positive recombinant plasmids was designated pPIC9K-sE2.The recombination expression vector,pPIC9K-sE2 was linearized by Sal Ⅰ and stransformed into P.pastoris GS115 by electroporation.The sE2 genes were integrated stably into chromosome of P.pastoris.High-copied transformants GS115-pPIC9K-sE2 were obtained by G418 screening.Expression of sE2 fusion protein was induced in recombinant P.pastoris GS115 by the addition of 1% methanol at 30℃,shaking at 200 r/min.The results of SDS-PAGE and Western-blot showed that expressed fusion proteins were provided with antigenic characteristic of BVDV.The results of studies on immunological activity indicated that the sE2 protein expressed in P.pastoris could induce animals to produce BVDV specific antibodies against E2 protein.