以HIV-1 5'LTR为靶点的药物筛选细胞模型的构建  

Construction of an Drug-Screening Cell Model based on HIV-1 5′LTR

作　　者：李建彬[1,2] 陈斌[2] 米志强[2] 安小平[2] 李存[2] 刘大斌[2] 王晓娜[2] 范华昊[2] 方祥[1] 童贻刚[2] 

机构地区：[1]华南农业大学食品学院,广东广州510642 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《微生物学杂志》2011年第6期96-100,共5页Journal of Microbiology

摘　　要：构建以人类免疫缺陷病毒(HIV-1)5'LTR为靶点的药物筛选细胞模型,用于体外筛选潜在的对HIV-1启动子具有抑制作用的药物,或用于筛选与HIV-1复制相关的宿主因子。通过PCR扩增HIV-1 5'LTR片段和胸苷激酶基因(thymidine kinase gene,TK基因),以这2片段为模板进行重叠PCR将两者连接起来,连接产物经酶切后与pcDNA3.1载体连接;将连接正确的质粒转染HEK293细胞同时用G418加压筛选获得稳定细胞系;加入药物更昔洛韦(GCV)检测TK基因的表达。成功构建了HIV-1 5'LTR调控TK基因表达的稳定细胞系,在其培养过程中加入药物更昔洛韦时,细胞逐渐死亡。构建了以HIV-1 5'LTR为靶点的药物筛选细胞模型,该模型利用TK基因作为报告基因方便灵敏,可用于筛选针对HIV-1 5'LTR的潜在抗HIV药物。A drug-screening cell model based on HIV-1 5′LTR was constructed for in vitro screening the latent drugs targeting HIV-1 promoter and inhibit it or host factor used for screening interrelated to HIV-1 replication.HIV-1 5′LTR and HSV-TK（thymidine kinase） gene were amplified with PCR and then joined them with overlapping PCR using these two fragments as templates.The joined product was cloned into pcDNA3.1 expression vector after digested with enzyme.The correctly joined recombinant plasmid was transfected into HEK293 cells,and at the same time stable cell lines were selected with 0.8 mg/mL G418 under pressure and tested with GCV for the expression of TK gene.The results showed that stable cell lines expressing TK gene promoter of HIV-1 5′LTR were successfully constructed,and during the cultivation the cells gradually to die when adding GCV.Therefore,the cell model for screening drug targeting HIV-1 5′LTR was constructed.The model used TK gene as a reporter gene was convenient and sensitive,and could be used for large-scale screening for latent drug targeting HIV-1 5′LTR.

关 键 词：HIV LTR TK自杀基因 药物筛选 

分 类 号：R392.12[医药卫生—免疫学]