可调控CYP4A1的逆转录病毒重组质粒的构建与鉴定  

Construction and Identification of a Recombinant Retroviral Vector of Full CYP 4A1 Gene Regulated by Tetracycline

作　　者：秦欢[1,2] 汪炳华[1] 周赤燕[1] 黄丽丽[1] 

机构地区：[1]武汉大学医学院生物化学与分子生物学系,湖北武汉430071 [2]武汉市妇女儿童医疗保健中心检验科,湖北武汉430016

出　　处：《武汉大学学报（医学版）》2009年第1期1-5,37,共6页Medical Journal of Wuhan University

基　　金：湖北省卫生厅资助课题(编号:JX2B24)

摘　　要：目的:运用分子生物学技术克隆CYP 4A1全长cDNA基因,进一步构建可由强力霉素诱导表达CYP4A1的逆转录病毒重组质粒。方法:运用逆转录-聚合酶链反应(RT-PCR)从大鼠肝组织中扩增出CYP 4A1全长基因,克隆至pMD18-T载体,将构建正确的pMD18-CYP 4A1质粒采用双酶切亚克隆到逆转录病毒载体载体pRe-vTRE中,采用酶切反应、PCR鉴定和序列测定鉴定重组逆转录病毒载体pRevTRE/CYP 4A1;在脂质体介导下分别将质粒pRevTRE/CYP 4A1与逆转录病毒四环素调节质粒pRevTet-on转染至包装细胞PT67,分别经抗性筛选获得稳定产生病毒的包装细胞系后收集转染后的细胞上清。用荧光定量PCR测定病毒滴度。结果:经酶切鉴定、PCR鉴定和序列测定证实成功构建了pRevTRE/CYP 4A1逆转录病毒重组质粒;转染pRevTet-on的PT67细胞病毒滴度为7.2×1010copies/L,转染pRevTRE/CYP 4A1的PT67细胞病毒滴度为5.46×109copies/L。结论:成功构建了含四环素调控CYP 4A1基因的逆转录病毒重组质粒,建立了能产较高滴度逆转录病毒的包装细胞系,为CYP 4A1基因功能及其相关疾病的研究打下实验基础。Objective： To clone full CYP 4A1 gene and construct a recombinant retroviral vector of full CYP 4A1 gene regulated by tetracycline. Methods： The full CYP 4A1 gene was amplified from normal liver by reverse transcriptase-polymerase chain reaction （RT-PCR） and then cloned into pMD18-T vector. The CYP 4A1 gene was subcloned into retroviral vector pRevTRE digested by restriction enzymes BamH l and Hind Ⅲ; pRevTRE/CYP 4A1 and pRevTet-On were separately transfected into packaging cell PT67 by lipofectamine 2000. The transfected PT67/Tet-On and PT67/TRE CYP 4A1 cells were selected by the corresponding antibiotics and identified by RTPCR. The recombinant retrovirus supernants were harvested and the retrovirus titer was determined by fluorescent quantitative PCR. Results： The restriction endonuclease digestion, PCR analysis and DNA sequencing confirmed that the recombinant pRevTRE/CYP 4A1 vector was constructed successful. The virus titer of PT67/Tet on was 7.2 × 10^10 copies/L, and the virus titer of PT67/TRECYP 4A1 was 5. 47 × 10^9 copies/L. Conclusion. A recombinant retroviral vector with CYP 4A1 regulated by tetracycline and stable virus producing lines have been successfully constructed, which provides a good basis for further research on the function of CYP 4A1 gene.

关 键 词：细胞色素P450 CYP 4A1 TA克隆 荧光定量检测 Tet—on诱导表达系统 

分 类 号：R543.1[医药卫生—心血管疾病]