含EGFP报告基因单顺反子HCV亚基因组复制子载体的构建和表达

Development of a monocistronic HCV subgenomic replicon with the EGFP reporter gene and its expression in cultured cells

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 develop an HCV subgenomic replicon containing the EGFP reporter gene that replicates efficiently in cultured cells. Methods Molecular cloning was used to substitute a DNA fragment encoding EGFP for HCV envelope genes E1 and E2 in a eukaryotic expression vector of the full-length HCV replicon in order to generate a monocistronic HCV subgenomic replicon with the EGFP reporter, designated pcDNA-JFH1-EGFP. This substitution was verified using enzymatic digestion and DNA sequencing. The pcDNA-JFH1-EGFP was transfected into Huh-7 human hepatoma cells using liposome mediation, and EGFP expression in transfected cells was examined using fluorescence microscopy. Minus-strand RNA and the NS3 protein of HCV were respectively detected using semi-quantitative RT-PCR and Western blot analysis. Inhibition of recombinant plasmid DNA-based HCV RNA replication by IFN-α was also investigated. Re- sults Enzymatic digestion of four recombinant plasmids was in line with expectations, and DNA sequencing indicated that there were no nucleotide mutations or open reading frame changes in the HCV and EGFP genes. EGFP green fluorescence was observed in transfeeted cells under a fluorescence microscope, and HCV minus RNA of both upstream and down stream fragments was detected using RT-PCR. Western blot analysis indicated HCV NS3 protein expression. Expression of HCV RNA was 200/oo in transfected cells treated with 1 000 IU/ml or 2 000 IU/ml of IFN-α and 7. 6% in untreated cells. Conclusion A monocistronic HCV subgenomic replicon with the EGFP reporter was successfully constructed. This replicon can correctly express EGFP and HCV proteins and replicate efficiently in Huh-7 cells. This provides an effective platform and basis for further study of HCV.