草鱼呼肠孤病毒衣壳蛋白VP7基因真核表达载体pCI-VP7的构建及鉴定

将编码草鱼呼肠孤病毒(grass carp reovirus,GCRV)主要衣壳蛋白VP70.9kb的基因片段连接至克隆载体pMD19-T中,筛选阳性克隆并测序,经检测为正确序列后,再将目的片段克隆入真核表达载体pCI,筛选得到阳性重组质粒pCI-VP7。然后构建pCI-VP-GFP重组表达质粒(即GFP基因与VP7的一段上游基因融合表达),用PCR及酶切方法鉴定克隆的正确性。并用脂质体法将其转染入真核细胞COS-1和CIK进行瞬时表达,荧光显微镜观察及RT-PCR特异性检测。结果表明,GFP基因与VP7的一段上游基因被成功转染到COS-1和CIK细胞,并得到了很好的表达。进而证明pCI-VP7可以成功的表达,为GCRV基因疫苗的研制提供了实验资料。

Construction and identification of recombinant eukaryotic expression vector pCI-VP7 containing GCRV VP7 gene

A target sequence of 0. 9kb which codes the major capsid protein VP7 of grass carp reovirus (GCRV) was amplified by PCR. The target fragment was inserted into the pMD19-T vector. The positive clone was screened and sequenced. Sequencing result showed the nucleic acid sequence is right. Then the target gene was cloned into eukaryotic expression vector pCI and the positive clone was screened. The recombinant plasmid pCI-VP7 extracted from the positive clone were identified by PCR and digestion. Then the recom...