牛病毒性腹泻病毒E2基因在毕赤酵母GS115中的表达与鉴定

将牛病毒性腹泻病毒(BVDV)河北分离株HB-bd毒株E2基因去除跨膜区获得sE2基因,将sE2基因克隆入巴斯德毕赤酵母(P.Pastoris)分泌型表达载体pPIC9K中,筛选培养后提取阳性重组质粒,经酶切和PCR鉴定,命名为pPIC9K-sE2.重组质粒pPIC9K-sE2经Sal Ⅰ酶切线性化,电穿孔导入P.Pastoris GS115基因进行整合,使外源基因sE2稳定地整合到P.Pastoris染色体中,G418筛选得到阳性高拷贝转化子GS115-pPIC9K-sE2.经甲醇诱导表达后,sE2融合蛋白获得了表达,表达产物经SDS-PAGE、Western-blot和Dot-ELISA分析,确定其表达的sE2融合蛋白相对分子质量大小为37 000,且具有天然蛋白的抗原特异性.免疫原性研究证明P.Pastoris表达的sE2蛋白能刺激动物产生特异抗体.

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

The sE2 gene removed C-terminal transmernbrane domain was obtained from isolated HB-bd strain of BVDV by PCR.The sE2 gene was subcloned into the expression vector pPIC9K and then transfected into DH5a.The recombinant plasmids were extracted and identificated by enzyme digestion and PCR.The positive recombinant plasmids were designated by 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 its immunological activity indicated that the sE2 protein expressed in P.Pastoris could induce animals to produce BVDV specific antibodies against E2 protein.