不同启动子指导的HSV1-sr39TK/GCV系统对小鼠淋巴细胞的敏感性研究  

Killing Effects on Mouse Lymphocytes Mediated by HSV1-sr39TK/GCV System Directed by Different Promoters

作　　者：高飞[1] 徐开林[1] 潘秀英[1] 鹿群先[1] 杜冰[1] 

机构地区：[1]徐州医学院附属医院血液科,江苏省徐州市221002

出　　处：《医学分子生物学杂志》2008年第2期124-128,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金(No.30170389);江苏省高校自然科学研究计划(No.02KJB320013)~~

摘　　要：目的观察慢病毒载体中不同启动子指导的突变型单纯疱疹病毒1型胸苷激酶(HSV1-sr39TK)基因在小鼠T淋巴细胞内的表达差异,并进一步比较对丙氧鸟苷(ganciclovir,GCV)的敏感性。方法用亚克隆技术将PCR扩增的HSV1-sr39TK基因分别连接至慢病毒载体不同启动子后,然后在HSV1-sr39TK基因后以内部核糖体进入位点(internal ribosome entry sites,IRES)连接绿色荧光蛋白(green fluorescent pro-tein,GFP)报告基因;采用三质粒包装系统包装病毒,将病毒感染刀豆蛋白A(concanavalin,ConA)激活的小鼠淋巴细胞,分别用流式细胞术、RT-PCR法鉴定HSV1-sr39TK和GFP基因在小鼠淋巴细胞的表达,用Cell Counting Kit-8(CCK-8)法观察感染不同病毒的淋巴细胞对前体药物GCV的敏感性。结果不同启动子指导的HSV1-sr39TK及GFP基因均可在小鼠淋巴细胞表达,巨细胞病毒(cytomegalovirus,CMV)启动子驱动表达能力最强,磷酸甘油激酶(phosphoglycerokinase,PGK)启动子最弱。感染含CMV启动子病毒的淋巴细胞对GCV的IC50值最小。结论在小鼠淋巴细胞内CMV启动子驱动的慢病毒载体HSV1-sr39TK基因表达最强,对前体药物GCV敏感性最高。Objective To investigate the expression variance of mutant herpes simplex virus type 1 thymidine kinase （HSV1-sr39TK） gene in mouse T lymphocytes directed by different internal promotors, and the killing effects on mouse T lymphocytes mediated by HSV1-sr39TK/Ganciclovlr （GCV） system. Methods The HSV1-sr39TK gene was subcloned into lentivectors and linked to different promoters. Then GFP reporter gene was linked by internal ribosome entry sites （IRES）. Lentivirus generated by transient three-plasmid transfection was used to infect the concanavalin A （Con A） stimulant T lymphocytes. The expressions of HSV1-sr39TK and GFP genes in mouse T lymphocytes were estimated by fluorescence activated cell sorter （FACS） and reverse transcriptase polymerase chain reaction （RT-PCR）. The GCV killing effects on mouse lymphocytes were meas- ured by using cell count kit-8 （CCK-8）. Results Both HSV1-sr39TK and GFP genes were ex- pressed effectively in mouse T lymphocytes, the initial expression ability of cytomegalovirus （CMV） promoter was strongest and that of phosphoglycerokinase （PGK） promoter was weakest. The IC50 value of CMV promoter was lowest. Conclusion Compared with other promoters, the CMV promoter is best in directing HSV1-sr39TK gene expression in mouse lymphocytes and in inducing T cell sensitivity to GCV.

关 键 词：慢病毒载体 丙氧鸟苷 单纯疱疹病毒Ⅰ型胸苷激酶 淋巴细胞 

分 类 号：Q782[生物学—分子生物学]