乙型肝炎病毒启动子调控的增强型绿色荧光蛋白真核表达载体的构建与表达

Construction and expression of EGFP controlled HBV promoters in eukaryotic cells

目的研究乙型肝炎病毒(HBV)启动子(含增强子)调控下的增强型绿色荧光蛋白(EGFP) 报告基因在肝癌细胞中的表达。方法用聚合酶链反应(PCR)技术分别扩增出HBV的4个启动子,载入 T载体,测序后插入到含EGFP报告基因的质粒pEGFP-1,构建出HBV不同启动子调控的EGFP基因表达载体,经酶切、测序鉴定各重组体。采用脂质体介导法将4种构建好的载体转染肝癌细胞株H e p G 2,用倒置荧光显微镜观察各重组体转染细胞中EGFP的表达。结果各目的片段测序正确并成功插入表达载体中, 转染了各质粒的阳性细胞克隆均可检测出E G F P的表达,不同启动子调控下的蛋白表达量有所不同。结论 H B V启动子调控下的E G F P报告基因能够在肝癌细胞中特异表达,不同启动子表达效率之间存在一定的差异性,从而为肝脏疾病的基因治疗提供了一个新的选择。

Objectives To construct four expression vectors carrying enhanced green fluorescent protein （EGFP） gene under the control of different HBV promoters, and to detect and analyze their expressions in hepatoma cell lines. Methods Four HBV promoters were amplified using PCR, and they were inserted into the T-vector and identified using restriction enzymes and sequencing, then cloned into the expression vector pEGFP-1. The four recombinant plasmids were transfected into human hepatoma cell line HepG2 by lipofectamine2000, and the positive cell clones were detected using fluorescence microscopy. Results All target fragments were separately obtained and successfully cloned into the expression vector. The expressions of EGFP under the control of the four promoters were detected. The expressions of EGFP controlled by different promoters had some differences. Conclusions Reporter gene EGFP under the control of four HBV promoters can be specifically expressed in hepatoma cell line HepG2, and different promoters give different results; this may provide another option in gene therapy of liver diseases.