南京医科大学学报（英文版）2003，Vol.17Issue(6)：261-269,9.
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 Recombinant Retroviral Vector Containing HIV-1 Tat Gene and Functional Detection of Expressed Tat in Target Cells
摘要
Abstract
Objective: To construct 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 recovcred from pEV plasmid by Hind Ⅲ digestion and cloned into expression plasmid LZRSpBMN-Z to construct recombinant retroviral expression plasmid named LZRS- Tat 101. Using the method of calcium phosphate, the construct of LZRS-Tat1o1 was then transfected into packaging cell lines Phoenix (ФNX) which contained env and gal geres encoding structural proteins and pol gene coding for 3 enzymes (reverse transcriptase , protease and integrase ) essential for retroviral integration and replication. The stable transfected cell lines was obtained 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 both transiently and stably trancfected ФNX, respectively. The supematants containing recombinant virus collected from transient and stable transsfected cells were employed to infect 293 cells, respectively, and the expressed Tat in 293 cells was tested by Western blot. Meantime, the supematants of infected 293 cells was further added to HL3 T1 cells which were Hela cell lines containing an HIV- 1-LTR/ CAT reporter construct to establish a co-culture system. After co-culture for 72 hours, the protein was extracted from HL3 T1 cells and used for CAT activity assay. Results: After LZRS- Tat 1o1 was transfected into ФNX, the amount of expressed Tat in transient transfection cells was significantly higher than that in stable transfection cells; Tat could be detected not only in 293 cells but also in the supematatats from 293 cells culture, and Tat in the supematants could activate HIV- 1 LTR promoter in HL3 T1, resulting in high expression of CAT located at the downstream of LTR. Concluusion: The construct of recombinant retravirus LZRS- Tat 101 could express Tat protein in target cells and the expressd Tat was functionally active and can really exhibit the ability to activate transcription.关键词
HIV-1 Tat/反转录病毒表达载体/转录激活Key words
HIV-1 Tat/retroviral expressionvector/the ability to activate transcription分类
医药卫生引用本文复制引用
卢春,钱超,唐桂霞,黄丽,曾怡..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[J].南京医科大学学报（英文版）,2003,17(6):261-269,9.基金项目
Supported by the NationalNatural Science Found ation of China(30100160,30271179) （30100160,30271179）
