Zhang Ling, Liu Xiaoming, Song Jingdong, Xin Yan, Deng Yao, Tan Wenjie
Bing Du Xue Bao. 2015 Mar;31(2):174-9.
To develop a recombinant lentivirus co-expressing structural protein of hepatitis C virus (HCV) and secreted Gaussia Luciferase (Gluc), we first constructed an expression vector that encoded HCV structural protein (C, E1, E2) and GLuc named pCSGluc2aCE1E2. The expression of HCV proteins and Gluc was confirmed by an immunofluorescence assay (IFA) and the detection of luciferase activity. Recombinant lentivirus (VSVpp-HCV) was developed by the co-transfection of pCSGluc2aCE1E2 into 293T cells with pHR'CMVA8.2 and pVSVG. The infectivity of VSVpp-HCV was confirmed by luciferase activity detection, IFA and western blotting. Virus-like particles were identified using electron microscopy after concentration. The results showed that the level of luciferase activity correlated with the expression of HCV protein after the infection of cells with lentivirus VSVpp-HCV. Therefore, the expression level of HCV proteins could be evaluated by detecting the luciferase activity of Gluc. In conclusion, this research pave a way for the development of transgenic mice that express HCV proteins and Gluc, which enable the evaluation of anti-HCV therapy and vaccine in vivo.
为构建共表达丙型肝炎病毒(HCV)结构蛋白和分泌型高斯荧光素酶(Gluc)的重组慢病毒,我们首先构建了一个编码HCV结构蛋白(C、E1、E2)和GLuc的表达载体,命名为pCSGluc2aCE1E2。通过免疫荧光分析(IFA)和荧光素酶活性检测证实了HCV蛋白和Gluc的表达。通过将pCSGluc2aCE1E2与pHR'CMVA8.2和pVSVG共转染到293T细胞中,构建了重组慢病毒(VSVpp-HCV)。通过荧光素酶活性检测、IFA和蛋白质印迹法证实了VSVpp-HCV的感染性。浓缩后使用电子显微镜鉴定病毒样颗粒。结果表明,用慢病毒VSVpp-HCV感染细胞后,荧光素酶活性水平与HCV蛋白的表达相关。因此,可通过检测Gluc的荧光素酶活性来评估HCV蛋白的表达水平。总之,本研究为开发表达HCV蛋白和Gluc的转基因小鼠铺平了道路,这使得能够在体内评估抗HCV治疗和疫苗。