鲤春病毒血症病毒糖蛋白基因在毕赤酵母中的分泌表达

Secreting expression of glycoprotein gene of spring viremia of carp virus in Pichia pastoris

根据Gen Bank中鲤春病毒血症病毒(SVCV)糖蛋白全基因序列设计特异性引物,以SVCV欧洲株病毒核酸为模板,通过RT-PCR方法获得欧洲株SVCV-G基因,克隆至p PICZαA,构建p PICZαASVCV1540表达质粒。以限制性内切酶Sac I线性化p PICZαASVCV1540后通过化学法转化毕赤酵母感受态细胞X33和GS115,添加1%甲醇进行诱导表达,SDS-PAGE电泳分析表达产物显示其分子质量为70 ku。Western Blot分析显示该蛋白可以被SVCV山羊多抗识别,表明目的蛋白具有反应原性。

A DNA fragment, which encoded the glycoprotein of spring viremia of carp virus （ SVCV） European strain, was amplified using designed specific primers by RT-PCR.The complete glycoprotein gene then was cloned into pPICZαA. The linearized recombinant plasmid pPICZαASVCV1540 digested by SacI was integrated into the Pichia pastoris comptent cells X33and GS115 by chemical approaches respectively.The 70 ku secreting protein were found by SDS-PAGE analysis when the recombinant yeast colonies were induced with 1% methanol.The reactinogenicity of induced protein was deter-mined with goat polyclonal antibodies against SVCV by Western Blot analysis.