高效包装重组慢病毒及其定量方法的建立  

Packaging recombinant lentivirus efficiently and establishing quantifying method for lentivirus

作　　者：张玲[1] 翁云层 张云 帅丽芳[3] 李婷婷[1] 王文敬[1] 李红卫[1] 赵卫[4] 黎诚耀[1] 

机构地区：[1]南方医科大学生物技术学院输血医学系,广州510515 [2]山东省潍坊市临朐县中医院骨外科,潍坊262600 [3]广州军区疾病预防控制中心,广州510515 [4]南方医科大学公共卫生与热带医学学院三级生物安全实验室,广州510515

出　　处：《白求恩医学杂志》2015年第1期3-5,F0004,共4页Journal of Bethune Medical Science

基　　金：国家自然科学基金资助项目(编号:81301433)

摘　　要：目的高效包装不同启动子的重组慢病毒(LV),建立LV的定量方法。方法采用分子克隆方法获得p FUGW、p TY-EF1α-EGFP及p TY-CMV-EGFP 3种转移质粒,然后采用脂质体法与另两种包装质粒p SPAX2及p MD2.G共同转染293T细胞,制备含有不同启动子的LV。设计针对3’-LTR的探针和引物,建立LV的荧光定量PCR定量方法。结果获得了不同启动子的转移质粒,制备了3种不同启动子的LV,应用荧光定量PCR方法测定制备的病毒,载量能达到109copies/ml。结论成功构建了不同启动子的转移质粒,获得了三种LV,建立了针对LV的荧光定量PCR方法,为后续研究不同启动子在多种细胞系中驱动目的蛋白的表达奠定了基础。Objective To package recombinant lentivirus（ LV） efficiently and establish quantitative method for LV. Methods Firstly,gene cloning method was used to construct three transfer plasmids p FUGW,p TY-EF1α-EGFP and p TY-CMV-EGFP and then 293 T cells were co-transfected with packaging plasmids p SPAX2 and p MD2. G by liposome,lentivirus with different promoters were obtained. The probe and primers aimed at 3＇-LTR were designed and the fluorescent quantitative method for LV was set up. Results Transfer plasmids with different promoters were obtained,LV with three different promoters was manufactured,the QPCR method worked well and lentivirus load could achieve 109 copies / ml by it. Conclusion Transfer plasmids with different promoters have been successfully constructed,three kinds of lentivirus have been acquired and the QPCR method for LV has been established,which has laid the foundation for the follow-up study of different promoters driven protein expression among various cells.

关 键 词：重组慢病毒 绿色荧光蛋白 荧光定量PCR 

分 类 号：R346[医药卫生—基础医学]