HIV－1载体中存在着提高转录和翻译基因的元件  

Elements in HIV-1 Vector Enhancing Transcription and Translation

作　　者：孙凤强[1] 方彧聃[1] 王娟[1] 张帆[1] 马海燕[1] 张敬之[1] 

机构地区：[1]上海交通大学医学遗传研究所,卫生部医学胚胎分子生物学重点实验室,上海市200040

出　　处：《医学分子生物学杂志》2010年第5期377-381,共5页Journal of Medical Molecular Biology

基　　金：国家高技术研究发展计划（863计划）（No.2007AA021206）,国家自然科学基金（No.30870943）,上海市自然科学基金（No.08ZRl412100）

摘　　要：目的研究HIV-1载体中的一些元件如Rev和Tat蛋白对其骨架的转录及外源基因表达水平的影响。方法将HIV-1表达GFP载体（FUGW）单独或分别与Rev蛋白表达质粒（pLP2）、Tat蛋白表达质粒（pcDNA3.1-Tat），及表达Rev和Tat蛋白的质粒（△8．9）等摩尔共转染人293T细胞后，经实时定量RT-PCR、FACS、荧光显微镜镜检等方法检测，比较其表达量。结果Rev与RRE结合后，载体骨架及外源基因的转录是单独转染FUGW时的3倍，Tat与TAR结合后，则提高其骨架及外源基因的转录近4倍，而Rev和Tat蛋白的协同作用，其转录本则可提高至6倍。FACS和荧光显微镜镜检也显示GFP蛋白表达量明显提高。F-TPO载体（HIV-1载体乳腺特异表达促血小板生成素）与△8．9在小鼠乳腺上皮细胞HC-11共转染和表达，则TPO蛋白的表达量接近pcDNA3．1-TPO载体的8倍。结论HIV-1载体中存在着提高转录和翻译基因的元件，可提高其骨架的转录和外源基因的表达，且该现象并不依赖于细胞类型和外源基因的种类。Objective To study the impact of some HIV-1 vector elements, like Rev and Tat protein, on its backbone transcription and transgene expression. Methods We transfected HIV-1 vector containing GFP expression cassette, either alone, or together with rev plasmid （pLP2）, tat plasmid （pcDNA3.1-Tat）, or rev and tat plasmid （△8.9）, into 293T cells, and detected the backbone transcription and GFP expression by real time RT-PCR, FACS and fluorescence micro- scope respectively. Results The transcription was enhanced to 3-fold in the case of co-transfection with pLP2, 4-fold with pcDNA3.1-Tat, and 6-fold with A8.9. GFP expression was also significantly enhanced as assessed by FACS and fluorescence microscopic observation. Moreover, the con- tent of thrombopoietin （TPO） protein in HC-11 cell culture, a murine mammary epithelial cell line, after co-transfection of HIV-1 expressing TPO vector （F-TPO） with △8.9, was nearly seven times higher than its negative control, as determined by ELISA. Conclusion Therefore, we pro- pose that some elements in HIV-1 vector as described here are able to enhance transcription and translation, which is independent of cell types and transgenes.

关 键 词：HIV-1载体 Rev蛋白 TAT蛋白 Rev应答元件 转录启动反应因子 

分 类 号：Q752[生物学—分子生物学]