草鱼呼肠孤病毒VP6蛋白在毕赤酵母中表达的初步研究

根据GeneBank中草鱼呼肠孤病毒(grass carp reovirus,GCRV)104株VP6蛋白的全基因序列(HM234682)设计一对特异引物,以提取的GCRV-104核酸为模板,采用RT-PCR技术扩增VP6蛋白编码基因,并将其克隆到含强启动子AOXⅠ的毕赤酵母(Pichia pastoris)表达载体pPICZB上。重组酵母质粒pPICZB-VP6经SacⅠ线性化,电击转化到毕赤酵母KM71菌株中,Zeocin抗性平板上筛选高拷贝转化子。阳性转化子在30℃,0.5%(V/V)的甲醇添加量的条件下,连续诱导3 d,表达产物经SDS-PAGE和Western-Blotting检测,结果表明成功实现了GCRV-104 VP6蛋白在毕赤酵母中的胞内表达,重组VP6蛋白相对分子质量约46 000,与天然VP6蛋白相对分子质量接近,并且可与鼠抗草鱼GCRV-104 VP6蛋白的多克隆抗体特异性结合。

Preliminary study on the expression of Grass carp reovirus VP6 protein in Pichia pastoris

The coding gene of grass carp reovirus(GCRV) VP6 protein was amplified by RT-PCR from the viral RNA genome extracted from virus infected Ctenopharyngodon idellus Kidney(CIK) cells with primers based on the gene sequence of GCRV 104 strain in GeneBank(HM234682).The amplification product of GCRV VP6 gene was oriently inserted into the Pichia expression vector pPICZB,which contained a strong promoter AOXⅠ.Thus,a Pichia-expression vector harboring the VP6 protein coding gene was constructed.The resultant construct was linearized with SacⅠand electroporated into P.pastoris KM71.Muti-copy transformants were selected on plates containing Zeocin.Positive transformants were induced on the condition of 30 ℃,0.5% methanol per day,for 3 days,the induced culture was assayed by SDS-PAGE and Western Blot.The result showed that relative molecular mas is 46 000,similar to the relative molecular mass of native GCRV-104 VP6 protein was detected,Western blotting analysis demonstrated this protein could be specifically hybridized with mouse polyclonal antibodies raised against GCRV VP6.