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目的构建含增强型绿色荧光蛋白(EGFP)报告基因的HCV复制子表达载体,并实现其在细胞中的复制表达。方法用分子生物学基因克隆技术对HCV 2a型复制子的基因进行改造,用EGFP基因替代HCV基因组中的包膜基因(E1和E2)体外构建重组单顺反子HCV亚基因组复制子真核表达质粒pcDNA-JFH1-EGFP,经限制性内切酶酶切分析和测序鉴定;脂质体介导转染人肝癌细胞系Huh-7细胞,用荧光显微镜观察EGFP表达,采用半定量RT-PCR方法检测重组复制子的HCV RNA负链,采用Western blot检测HCV NS3蛋白的复制表达,并观察IFN-α对重组质粒表达的HCV RNA复制的抑制作用。结果构建的4个重组质粒酶切分析与预期相符,HCV亚基因复制子表达载体中未发生EGFP和HCV编码区读码框架改变,转染重组载体Huh-7细胞检测到HCV负链及EGFP和HCV NS3蛋白表达。转染后48h,1 000IU/ml和2 000IU/ml IFN-α处理的细胞HCV RNA表达水平分别为未处理组的20.0%和7.6%。结论含EGFP报告基因的单顺反子HCV亚基因组复制子表达载体pcDNA-JFH1-EGFP构建成功,在Huh-7细胞中能有效复制表达,为进一步研究HCV提供了实验平台。
Objective To construct HCV replicon expression vector containing enhanced green fluorescent protein (EGFP) reporter gene and to express it in cells. Methods The gene of HCV 2a replicon was modified by molecular biological gene cloning, and the eukaryotic expression of recombinant monocistronic HCV subgenomic replicon was constructed by using EGFP gene instead of envelope gene (E1 and E2) in HCV genome The plasmid pcDNA-JFH1-EGFP was digested with restriction endonuclease and sequenced. Lipofectamine was transfected into human hepatocellular carcinoma cell line Huh-7. The expression of EGFP was observed by fluorescence microscopy. Semi-quantitative RT-PCR HCV RNA negative strand was detected. Western blot was used to detect the expression of HCV NS3 protein. The inhibitory effect of IFN-α on HCV RNA replication was also observed. Results The constructed recombinant plasmids were in accordance with the expectation. The HCV subgenomic replicon expression vector did not change the reading frame of EGFP and HCV coding region. The HCV negative strand and EGFP were transfected into Huh-7 cells HCV NS3 protein expression. 48h after transfection, HCV RNA expression levels of cells treated with 1 000 IU / ml and 2 000 IU / ml IFN-α were 20.0% and 7.6% respectively of untreated cells. CONCLUSION: The monocistronic HCV subgenomic replicon expression vector pcDNA-JFH1-EGFP containing EGFP reporter gene was successfully constructed and could be efficiently replicated in Huh-7 cells, providing an experimental platform for further study on HCV.