人CYP39A1基因过表达慢病毒载体构建及其在HepG2中表达

Construction of lentiviral vector with CYP39A1 gene overexpression and its expression in human HepG2 cells

[目的]构建人CYP39A1基因过表达慢病毒载体,并建立CYP39A1基因过表达的HepG2细胞株,为研究CYP39A1在肝细胞肝癌中的作用机制奠定基础。[方法]构建具有嘌呤霉素抗性的CYP39A1慢病毒载体,经酶切鉴定及DNA测序鉴定后,包装成重组慢病毒颗粒;用嘌呤霉素筛选获得CYP39A1过表达的HepG2稳转细胞,采用qPCR和Western Blot检测CYP39A1的mRNA和蛋白相对表达量。[结果]成功扩增CYP39A1基因片段,大小为1451 bp。确定嘌呤霉素在HepG2中的最佳筛选浓度为2.0μg/mL,目的基因CYP39A1被慢病毒成功导入HepG2细胞中,荧光显微镜下能直接观察到EGFP。成功构建CYP39A1过表达及其阴性对照病毒,CYP39A1过表达病毒滴度为1.0×10^(9) TU/mL,阴性对照病毒的滴度为3.0×10^(8) TU/mL。转染CYP39A1过表达组和阴性对照组的qPCR实验结果显示:在HepG2细胞中,过表达组的CYP39A1的mRNA表达量为阴性对照组的516.55±85.24倍(P<0.001)。Western Blot也显示有Flag-CYP39A1的蛋白过表达。[结论]成功构建CYP39A1过表达的HepG2稳转细胞株,且CYP39A1的过表达组mRNA和蛋白均有上调。可为进一步探讨CYP39A1基因在肝细胞肝癌发生发展中的作用提供工具。

[Objective]To construct the lentiviral vector overexpressing human CYP39 A1 and establish HepG2 cell line overexpressing CYP39 A1 through stably infecting human hepatocellular carcinoma(HepG2)cells,and lay the foundation for the study of the mechanism of CYP39 A1 in hepatocellular carcinoma.[Method]CYP39 A1 lentivirus vector with purinomycin resistance was constructed,and then it was packaged into recombinant lentivirus particles after enzymatic digestion and DNA sequencing.HepG2 cells stably overexpressed CYP39 A1 were screened by purinomycin.The mRNA and protein relative expression levels of CYP39 A1 were detected by real-time fluorescence quantitative polymerase chain reaction and Western Blot.[Result]The CYP39 A1 gene fragment was successfully amplified,about 1451 bp in size.The optimal screening concentration of purinomycin in HepG2 was determined to be 2.0μg/mL.The target gene CYP39 A1 was successfully transfected into HepG2 cells by lentivirus,and EGFP could be directly observed under fluorescence microscope.CYP39 A1 overexpression lentivirus and its negative control virus were successfully constructed.The titer of CYP39 A1 overexpression virus was 1.0×10^(9) TU/mL,and the titer of negative virus was 3.0×10^(8) TU/mL.In HepG2 cells,the relative mRNA expression of CYP39 A1 in the overexpression group was 516.55±85.24 folds than that in the negative control group(P<0.001).Western Blot also indicated the Flag-CYP39 A1 was determined in the overexpression group.[Conclusion]The CYP39 A1 gene overexpression lentiviral vector were constructed and packaged successfully.HepG2 cell lines overexpressing CYP39 A1 were successfully constructed,which can provide a tool for further exploration of the role of CYP39 A1 gene in the occurrence and development of hepatocellular carcinoma.