逆转录病毒载体介导的HSVtk基因表达及提高重组逆转录病毒滴度的研究  

Retroviral vector-mediated HSVtk gene expression and acquisition of high titer recombinant virus

作　　者：曾赵军[1] 胡维新[1] 罗赛群[1] 陈迁[1] 

机构地区：[1]中南大学湘雅医学院分子生物学研究中心,长沙410078

出　　处：《中华实验和临床病毒学杂志》2004年第4期332-336,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：CMB(美国中华医学会)基金资助(99 698)

摘　　要：目的　探讨逆转录病毒载体介导的单纯疱疹病毒胸苷激酶 (HSVtk)基因表达及提高逆转录病毒滴度的措施。方法　将HSVtk基因插入逆转录病毒载体pRevTRE中 ,构建重组逆转录病毒载体。通过微乒乓感染法导入包装细胞PA317中 ,以潮霉素B筛选克隆细胞 ,浓缩克隆细胞上清制备重组逆转录病毒。在不同时间及不同丁酸钠浓度下 ,检测分析经筛选获得阳性克隆靶细胞中有无HSVtk基因的表达及如何获得高滴度的重组病毒液。结果　成功构建了重组逆转录病毒载体RevTRE tk ,制备的重组病毒感染靶细胞后有HSVtk基因的表达。结论　通过微乒乓感染法在包装细胞PA317培养 30h和 10mmol L丁酸钠浓度下 ,经冷冻超速离心能获得携HSVtk基因的高滴度逆转录病毒颗粒 ,为其基因治疗的应用及研究奠定了基础。Objective To explore the HSVtk gene expression mediated by the retroviral vector and to obtain high titer recombinant retroviral virus. Methods The recombinant vector pRevTRE/HSVtk was constructed by inserting HSVtk gene into pRevTRE. The recombinant retrovirus,which was produced from cloned PA317 cells screened by hygromycin B after“micro-pingpong” technique transferring with pRevTRE/HSVtk plasmids DNA by using modified calcium phosphate precipitation method. HSVtk gene expression was performed on target cells and virus titers were detected in different cultured temper, time and sodium butyrate concentration. Results The recombinant retroviral vector pRevTRE/HSVtk was constructed and HSVtk gene expression was detected on target cells after they were infected with the recombinant retrovirus. Conclusion High titer of retroviruses could be obtained in the culture medium of PA317 cell line through “micro-pingpong” technique at 30 hours and 10 mmol/L sodium butyrate concentration followed by frozen ultrafiltration.

关 键 词：HSV TK基因 逆转录病毒载体 高滴度 表达 介导 包装细胞 重组病毒 潮霉素 筛选 

分 类 号：R346[医药卫生—基础医学]