HBV-S基因四环素调控双表达载体的构建及其调控表达研究  

Construction of the tetracycline regulated expression vector of HBV-S gene and its expression in eukaryotic cells

作　　者：梁丽[1] 雷宇[2] 倪艳[2] 王蜀强[2] 彭凤英[3] 周智[1] 陈压西[2] 任红[2] 

机构地区：[1]重庆医科大学附属第二医院感染病科 [2]重庆医科大学病毒性肝炎研究所,重庆400010 [3]重庆医科大学附属第一医院传染科,重庆400016

出　　处：《重庆医科大学学报》2011年第7期833-836,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:30100158);重庆市渝中区科委基金资助项目(编号:2007-2009);重庆市科委自然科学基金计划资助项目(编号:CATC.2009BB5063);重庆市教委基金资助项目(编号:KJ070317)

摘　　要：目的:构建在同一载体中含乙肝病毒表面抗原基因(Hepatitis B virus surface gene,HBV-S)和四环素阻抑蛋白基因的双表达载体,研究在真核细胞中四环素对S基因表达的调控。方法:在双表达载体pVITRO3一个启动子后的多克隆位点插入四环素阻抑蛋白基因,用含四环素操纵子的启动子取代另一个启动子,并在其多克隆位点插入S基因,在同一载体中构建成S基因的四环素调控表达系统。用脂质体将重组质粒转染HepG2细胞,潮霉素400μg/ml进行抗性筛选,20 d形成抗性克隆,建立在四环素诱导下能稳定表达S基因的细胞系。以不同浓度的四环素(0.5、1.0、2.0、3.0μg/ml)进行诱导,用逆转录PCR和微粒子酶免疫分析法检测S抗原的表达,以确定四环素的最佳浓度。结果:转染了重组质粒的HepG2细胞,在无四环素环境下,表达HBsAg水平极低,72 h时S/N值平均为1.57,在不同浓度的四环素诱导下,HBsAg表达量明显增加,72 h时S/N平均值分别为4.24、4.23、4.18、4.20,未发现与四环素的浓度之间有剂量关系。结论:成功构建了在同一质粒中表达HBV-S基因和四环素阻抑蛋白基因的双表达调控载体,在不同浓度四环素诱导下均有HBsAg表达,并随时间延长而表达量增高,但在同一时间点无明显差异。Objective： To construct a co-expression vector which expresses both HBsAg and Tet Repressor protein and in which the expression of HBsAg can be regulated by tetracycline,and to study the regulating role of tetracycline in the HBV-S expression in HepG 2 cell.Methods：Tetracycline repressor（TetR）gene was insert into the first multiple cloning site,which was just after the first promotor in pVITRO3,a co-expression vector,then the second promotor was partly replaced by another one which contained tetracycline operator.Then,HBs gene was inserted into the second multiple cloning site.The constructed vector was transfected into HepG2 by means of lipofectamine.And 400 μg/ml Hygromycin B was used to kill the untransfected host cell line.After 20 days selection,the cell line which expressed HBsAg stably after being induced by tetracycline was generated.Then the HBsAg expression was induced by various concentrations of tetracycline（0.5,1.0,2.0,3.0 μg/ml）.And HBsAg was detected by means of RT-PCR and MEIA（Microparticle Enzyme Immunoassay）to determine the appropriate concentration of tetracycline.Results：HBsAg was expressed little without tetracycline in transfected HepG2.The average S/N in 72 hour was 1.57.And the S/N of HBsAg was increased remarkably but had no difference after being induced by various concentrations of tetracycline.The average S/N in 72 hour was 4.24,4.23,4.18 and 4.20 respectively.Conclusion：The basal expression was very low in the tetracycline regulated expression vector of HBV-S gene without inducer.After being induced by various concentrations of tetracycline,HBsAg expression was sharply increased with the incubation time prolongation,but the expression quantity showed no difference at the same time point.

关 键 词：乙肝病毒 四环素 调控表达 

分 类 号：R512.6[医药卫生—内科学]