人巨细胞病毒融合表达载体的构建及鉴定

Construction of Recombinant Plasmid for Fusion Expression of Human Cytomegalovirus and Identification of Expressed Product

目的构建含人巨细胞病毒(HCMV)gB680糖蛋白基因和突变的pp65蛋白基因(pp65m)的融合表达载体,并检测其在COS-7细胞中的表达。方法用PCR法从HCMV基因组中钓取gB680和pp65m蛋白基因,分别亚克隆入pMD18-T载体中,经酶切鉴定并测序后,构建真核表达载体pVAX1/gB680+pp65m,以ELISA法检测其在COS-7细胞中的瞬时表达。结果重组质粒含有gB680和pp65m融合基因,并能在COS-7细胞中表达。结论已成功构建了人巨细胞病毒融合表达载体,并可在真核细胞中表达。

Objective To construct the fusion expression vector containing the genes encoding gB680 glycoprotein and mutant pp65 （pp65m） protein of human cytomegalovirns （HCMV） and determine the expressed product in COS-7 cells. Methods Amplify gB680 and pp65m genes from genome of HCMV by PCR and subclone to vector pMD18-T respectively. After identification of the constructed recombinant plasmids by restriction analysis and sequencing, clone gB680 and pp65m genes into plasmid pVAX1. Transfect COS-7cells with the constructed eukaryofic expression vector pVAX1/gB680 ＋ pp65m, and identify the expressed product by ELISA. Results The constructed eukaryotic expression vector contained gB680 and pp65m fusion gene and expressed in COS-7cells. Conclusion The recombinant plasmid for fusion expression of HCMV gene was successfully constructed and expressed in eukaryotic cells.