携带eGFP基因慢病毒颗粒的制备及鉴定  

Preparation and Appraisal of the Recombinant eGFP-containing Lentiviral Particles

作　　者：陈瑜[1,2] 杨耀云[1,2] 鲁帅尧[1,2] 王晓囡[1,2] 和占龙[1,2] 乞素冬[1,2] 李鸿钧[1,2] 

机构地区：[1]中国医学科学院,北京协和医学院医学生物研究所 [2]云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《生物技术》2013年第6期76-80,共5页Biotechnology

基　　金：协和青年科研基金项目("hFGF21对Ⅱ型糖尿病的靶向基因治疗研究";2012D14)资助;云南省应用基础研究面上项目("抵抗素与饮食诱导的Ⅱ型糖尿病猕猴模型的关联性研究";2009ZC185M)资助

摘　　要：目的:通过eGFP基因慢病毒颗粒的包装和感染,明确重组慢病毒对外源基因在细胞中的转导能力。方法:把慢病毒质粒Lv105-eGFP转染到293 Ta细胞中并包装为慢病毒颗粒,经电镜检测病毒颗粒和实时定量PCR(qPCR)测定病毒滴度,之后感染H1299细胞,通过荧光显微镜观察比较eGFP在靶细胞中的表达效率。结果:eGFP在包装细胞中转染和表达效率在48h时达90%以上,到72h时细胞病变效应(CPE)逐渐显著;电镜检测可看到典型的慢病毒颗粒形态特征;病毒原液的滴度是4.4×107VG/mL、浓缩液的是2.68×1010VG/mL;1μL病毒原液感染H1299细胞时,eGFP表达水平可达10%、增加到100μL感染时表达水平达到99%以上,证明eGFP基因得到有效转导并可在细胞中高水平表达绿色荧光蛋白。结论:成功包装了eGFP慢病毒颗粒并可在靶细胞中获得高效表达。Objective： The transduction capacity for the target gene carried in lentiviral particles in cells was determined by the package and infection of lentiviral particles with the enhanced green fluorescent protein （eGFP） . Method： First, the lentiviral recombinant plasmidLvl05 -eGFP was transfected and packaged into 293Ta cells. Second, the lentiviral particles were identified with the electron microscopy and use real -time quantitative PCR （qPCR） to gauge the titer recombinant lentivirus and then infect H1299 cells. After that,the expres- sion of eGFP was detected by fluorescence microscopy in H1299 cells. Result：First,the effiency of the transduction and expression of the eGFP in the cells was up to 90% in 48hs,and after 72hs, the cytopathic effect was increasingly significant. Typical morphological charac- teristics of lentiviral particles was found through the election microscope and the titer of the virus changed from 4. 77 × 107VG/mL to 2. 68 × 10-10VG/mL after concentration. The H1299 cells was infected with 1 μL virus stock,the expression of eGFP was up to 10%, but the ex- pression was above 99% when the volumn of the virus became lO0μL,which suggested that the eGFP had been effectively transducted and expressed ~een fluorescent protein in cells. Conclusion：The eGFP lentiviral particles had been successfully packaged and made the parti- cle high -level express in the target cell, which would be useful to a follow- up research.

关 键 词：增强绿色荧光蛋白 慢病毒颗粒 包装 细胞 表达 

分 类 号：Q812[生物学—生物工程]