Tet-on基因表达系统反应质粒P^(TRE-HIF-1α)的构建和表达鉴定  

Construction and Expression Analysis of Recombinant Vector P^(TRE-HIF-1α) of Tet-on Gene Expression System

作　　者：徐宗全[1] 陈孝平[1] 张万广[1] 王其[1] 关剑[1] 李高鹏[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院肝脏外科中心,武汉430030

出　　处：《中国普外基础与临床杂志》2008年第12期887-891,共5页Chinese Journal of Bases and Clinics In General Surgery

基　　金：国家自然科学基金项目(编号:30371395)~~

摘　　要：目的克隆和构建携带人低氧诱导因子-1α(HIF-1α)的Tet-on基因表达系统反应质粒PTRE-HIF-1α。方法以缺氧的肝癌细胞株HepG2总RNA为模板,进行RT-巢式PCR,获得HIF-1α的cDNA,克隆入Tet-on基因表达系统反应质粒PTRE2hyg,酶切重组子鉴定。将构建好的PTRE-HIF-1α用脂质体法转入HepG2Tet-on细胞,在强力霉素的作用下,用RT-PCR及Westernblot法鉴定重组质粒的表达。结果扩增出HIF-1α的cDNA测序结果与Genbank记载完全一致,并成功克隆入PTRE2hyg,将反应质粒PTRE-HIF-1α转入HepG2Tet-on细胞,可以完整有效表达HIF-1α且受强力霉素的调控。结论成功克隆和构建携带HIF-1α基因的Tet-on基因表达系统反应质粒PTRE-HIF-1α,并证明其表达能在HepG2Tet-on细胞中受强力霉素调控。Objective To construct the responsive plasmid P^TRE-HIF-1α of Tet-on gene expression system and examine its expression. Methods RT-nested PCR was performed on the total RNA extracted from hypoxia HepG2 cells to obtain the eDNA of HIF-1α, which was inserted into the responsive plasmid P^TRE2hyg. DNA sequencing was performed after the recombinant of responsive plasmid P^TRE-HIF-1α was identified by endonuclease digestion. This recombinant vector was transfected into HepG2^Tet-on cells by means of liposome and its expression was examined by RT-PCR and Western blot under the control of deoxycycline. Results The amplified products were confirmed as the eDNA of HIF-1α by DNA sequencing. The responsive plasmid P^TRE-HIF-1α verified by edonuclease digestion, was capable of expression in HepG2^Tet-on cells and could be controlled by deoxycycline. Conclusion The responsive plasmid P^TRE-HIF-1α of Tet-on expression system is constructed successfully, and it can express under the regulation of deoxycycline in the HepG2^Tet-on cells.

关 键 词：低氧诱导因子-1Α 巢式PCR Tet-on基因表达系统 反应质粒 克隆 

分 类 号：R735.7[医药卫生—肿瘤] R730.231[医药卫生—临床医学]