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目的建立能表达丙型肝炎病毒核心蛋白(HCV core)的人肝癌细胞SMMC-7721的稳定转染细胞株。方法构建含目的基因HCV core的重组质粒,转染HEK293T细胞,包装获得含ZsGreen和HCV core基因的慢病毒后,感染SMMC-7721人肝癌细胞,采用实时荧光定量PCR检测HCV core mRNA表达,采用免疫荧光细胞化学染色和Western blot法检测HCV core蛋白表达,筛选稳定表达HCV core的细胞株。结果质粒酶切和序列测定证实重组载体构建正确;慢病毒包装48 h后可见清晰ZsGreen表达,感染SMMC-7721细胞后筛选获得稳定转染的细胞株,实时荧光定量PCR检测到HCV core mRNA,免疫荧光细胞化学染色和Western blot法均检测出HCV core蛋白表达。结论成功构建了表达HCV core蛋白的SMMC-7721人肝癌细胞的稳定转染细胞株。
Objective To establish a stable transfected human hepatocellular carcinoma cell line SMMC-7721 expressing hepatitis C virus core protein. Methods Recombinant plasmids containing target gene HCV core were constructed and transfected into HEK293T cells. The lentivirus containing ZsGreen and HCV core genes were packaged and transfected into SMMC-7721 human hepatocellular carcinoma cells. HCV core mRNA expression was detected by real-time fluorescence quantitative PCR. HCV core protein expression was detected by fluorescent cytochemistry and Western blot, and the cell lines stably expressing HCV core were screened. Results The recombinant vector was confirmed by restriction enzyme digestion and sequencing. The expression of ZsGreen was clearly seen after 48 h of packaging with lentivirus. The stable transfected cell lines were obtained after SMMC-7721 cells were infected, and HCV core mRNA was detected by real-time fluorescence quantitative PCR. HCV core protein expression was detected by fluorescence cytochemistry and Western blot. Conclusion The stable transfected cell line of SMMC-7721 human hepatoma cells expressing HCV core protein was successfully constructed.