gD糖蛋白基因表达质粒pcDNA3Kan-gD的构建及其在Hela细胞中的表达检测

Construction of gD glycoprotein gene expression plasmid pcDNA3Kan-gD and its expression in Hela cells

目的利用半定量RT-PCR技术及免疫荧光技术分析构建的单纯疱疹病毒gD糖蛋白基因真核表达质粒在Hela细胞中的瞬时表达情况。方法构建并提纯重组的真核表达质粒pcDNA3Kan-gD,转染培养的Hela细胞,采用半定量RT-PCR技术及免疫荧光技术检测外来转染的gD基因在Hela细胞中的瞬时表达。结果转染了pcDNA3Kan-gD重组质粒的Hela细胞RNA中能够扩增出gD基因的特异性条带,免疫荧光技术分析结果显示,转染了pcDNA3Kan-gD重组质粒的Hela细胞中能检测到特异性绿色荧光。结论真核表达质粒pcDNA3Kan-gD能够在Hela细胞中瞬时表达,有可能成为抗单纯疱疹病毒DNA疫苗候选物之一。

Objective To analyze the transient expression of herpes simplex virus glycoprotein D(g D)gene eukaryotic expression plasmid in Hela cells by using semi-quantitative RT-PCR and immunofluorescence.Methods The eukaryotic expression plasmid pc DNA3 Kan-g D was constructed and purified in large quantities.Hela cells were transfected and cultured.Semi-quantitative RT-PCR technique and immunofluorescence technique were used to detect the transient expression of externally transfected g D gene in Hela cells.Results Specific bands of g D gene could be amplified in the RNA of Hela cells transfected with the pc DNA3 Kan-g D recombinant plasmid,and the results of immunofluorescence analysis showed that specific green fluorescence could be detected in Hela cells transfected with pc DNA3 Kan-g D recombinant plasmid.Conclusions Eukaryotic expression plasmid pc DNA3 Kan-g D could be expressed transiently in Hela cells,these characteristics made it become one of the candidates for DNA vaccine against herpes simplex virus.