共表达丙性肝炎病毒结构蛋白及分泌型荧光素酶Gluc的慢病毒制备与鉴定  

Development and Identification of the Recombinant Lentivirus Co-expressing HCV Structural Protein and Secreted Gaussia Luciferase(Gluc)

作　　者：张玲[1] 刘晓明[1,2] 宋敬东[1] 新燕[2] 邓瑶[1] 谭文杰[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京102206 [2]内蒙古医科大学,微生物教研室,呼和浩特010059

出　　处：《病毒学报》2015年第2期174-179,共6页Chinese Journal of Virology

基　　金：国家自然科学基金(81373229);传染病科技重大专项(2009ZX10004-705;-715)

摘　　要：旨在制备共表达丙型肝炎病毒(Hepatitis C virus,HCV)结构蛋白及分泌型荧光素酶Gluc慢病毒VSVppHCV。通过构建共表达HCV结构蛋白及分泌型荧光素酶Gluc的慢病毒表达载体pCSGluc2aCE1E2,与包膜质粒pVSVG,包装质粒pHR′CMVΔ8.2共转染HEK 293T的方式获取慢病毒,将慢病毒感染细胞后,对细胞上清中Gluc荧光素酶活性、细胞中HCV特异蛋白的表达等进行表达鉴定。结果表明:浓缩后获得的慢病毒浓度为1×108 TU/mL,电镜负染观察到直径约为100nm带包膜的浓缩慢病毒颗粒;慢病毒VSVpp-HCV感染细胞后,细胞内HCV特异蛋白及上清中Gluc荧光素酶的表达呈平行关系。因此通过检测Gluc荧光素酶的表达水平可反映HCV结构蛋白表达水平,这为建立基于慢病毒感染的带有报告基因的HCV转基因小鼠模型提供了基础。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 293Tcells with pHR′CMVΔ8.2and 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.

关 键 词：慢病毒 HCV 结构蛋白 分泌型荧光素酶 

分 类 号：R373.2[医药卫生—病原生物学]