含HIV-1 Tat基因重组反转录病毒表达载体构建与表达Tat蛋白功能检测  

Construction of a recombinant retroviral vector containing HIV-1 Tat gene and functional detection of the expressed Tat in target cells

作　　者：卢春[1] 黄丽[1] 曾怡[1] 

机构地区：[1]南京医科大学微生物学与免疫学系,210029

出　　处：《中华微生物学和免疫学杂志》2003年第9期691-695,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 (3 0 10 0 160 ;3 0 2 71179)

摘　　要：目的　构建含HIV 1Tat基因重组反转录病毒表达载体及评价表达Tat蛋白的功能。方法　使用HindⅢ将HIV 1Tat1 0 1 蛋白编码基因从pEV质粒中切出 ,插入到表达质粒LZRSpBMN Z中 ,构建成重组反转录病毒表达质粒LZRS Tat1 0 1 。采用磷酸钙转染法将重组质粒LZRS Tat1 0 1 转染进含反转录病毒env、gal和pol编码基因的包装细胞phoenix(ФNX)中 ,嘌呤霉素筛选获得稳定细胞系。免疫组化 (IHC)染色检查Tat在临时转染和稳定转染ФNX细胞中的表达水平。收集临时转染和稳定转染包装细胞分泌的病毒上清 ,并分别感染 2 93细胞 ,Westernblot检测Tat在 2 93中表达。与此同时 ,收集感染 2 93细胞培养上清 ,加入到HL3T1细胞 (HeLa HIV 1 LTR CAT报告基因 )中 ,共培养 72h后收集细胞 ,提取蛋白作CAT活性检测。结果　①含Tat1 0 1 基因重组反转录病毒表达质粒转染包装细胞后 ,Tat在临时转染ФNX中表达水平显著高于在稳定转染中的水平 ;②临时转染病毒感染 2 93细胞 ,Tat在感染细胞中得到了表达 ,且分泌至上清中的Tat蛋白能够激活靶细胞HL3T1中HIV 1的LTR启动子 ,使得其下游的CAT基因得到表达。结论　重组LZRS Tat1 0 1 反转录病毒能够在其感染靶细胞中表达Tat蛋白 ,且表达蛋白具有转录激活功能。Objective To construct a recombinant retroviral vector containing HIV-1 Tat gene and evaluate the function of the expressed Tat in target cells. Methods HIV-1 Tat_ 101 gene was isolated from pEV plasmid by HindⅢ digestion and then cloned into expression plasmid LZRSpBMN-Z for constructing a recombinant retroviral expression plasmid named LZRS-Tat_ 101 . The construct of LZRS-Tat_ 101 was then transfected into packaging cell lines Phoenix (ФNX)using calcium phosphate which contained env and gal encoding for structural proteins while pol encoding for 3 enzymes (reverse transcriptase, protease and integrase) essential for retroviral integration and replication. The stable transfected cell lines was obtained by using puromycin to screen for more than 3 days. Then immunohistochemical (IHC) staining was carried out to detect the expression level of Tat_ 101 protein in above transiently and stably transfected ФNX respectively. Supernatants containing recombinant virus collected from transient and stable transfected cells were employed to infect 293 cells respectively and expressed Tat in 293 cells was tested by Western blot. Meantime, supernatants of infected 293 cells was added to HL3T1 cell (HeLa cell lines has contained a HIV-1-LTR/CAT reporter construct) to establish a co-culture system. After incubation for 72?h, protein was extracted from HL3T1 cells and used for CAT activity assay. Results ① After LZRS-Tat_ 101 was transfected into ФNX, the amount of expressed Tat in transient transfection cells was significantly more than that in stable transfection cells; ② Tat could be detected not only in 293 cells but also in the supernatants from 293 cells culture and the Tat in the supernatanst activated HIV-1 LTR promoter in HL3T1, resulting in the downstream high expression of CAT. Conclusion We conclude that construction of recombinant retrovirus LZRS-Tat_ 101 can express Tat protein in target cells and the expressed Tat is functionally active and shows a trans-activation activity.

关 键 词：HIV-1Tat 基因重组 反转录病毒表达载体 表达 TAT蛋白 检测 转录激活 

分 类 号：R392[医药卫生—免疫学]