【摘要】 目的:构建乙型肝炎病毒S基因的四环素调控表达载体,并研究在真核细胞中四环素对S基因的调控表达,以期用于基因治疗实验。方法:将乙型肝炎病毒S基因插入启动子内含有2个四环素超纵子(TO)的质粒pcDNA4,并用脂质体将重组质粒pcDNA4-S和含有四环素抑制基因的质粒pcDNA6共转染入真核细胞HepG2中,用Zeocin(400μg/ml)及Blasticidin(10μg/ml)进行抗性筛选,约20天形成抗性克隆,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(0.5μg/ml,1.0μg/ml,2.0μg/ml,3.0μg/ml)进行诱导,用微粒子酶免疫分析法(MEIA)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HBsAg表达量很低,当加入不同浓度的四环素诱导后,HBsAg表达量明显增加,但所测HBsAg的S/N值无明显差异。结论:成功构建了乙型肝炎病毒S基因四环素调控表达载体,并在真核细胞中进行了四环素的调控表达。更多还原

【Abstract】 Objective:To construct tetracycline regulating expression system of HBV-S gene,and study its regulating expression of HBVAg by tetracycline in HepG 2 cells. Methods:Insert HBs gene into the multiple cloning site of pcDNA4,which contain tetracycline operator in the promoter,and the constructed vector with pcDNA6 was co-transferred into HepG2 by means of lipofectamine,and use 400μg/ml Zeocin and 10μg/ml Blasticidin to kill the untransfected host cell line. After 20 days’select,the cell line which express HBsAg stablely after induced by tetracycline was generated. Then the HBsAg expressing was induced by varying concentration of tetracycline(0.5μg/ml,1.0μg/ml,2.0μg/ml,3.0μg/ml).Amount of HBsAg was detected by means of MEIA (Microparticle Enzyme Immunoassay) to determine the appropriate concentration of tetracycline. Result:HBsAg had been expressed at low quantity without tetracycline in transfected HepG2. However the S/N of HBsAg were high but not different after induced by varying concentration of tetracycline. Conclusion:Tetracycline regulating expression system of HBV-S gene was constructed successfully and HBsAg was highly expressed in the vector induced by tetracycline.更多还原