STAT1过表达慢病毒载体的构建及其在MHCC97L细胞中的表达

Construction of STAT1 gene over-expression lentivirus vec- tor and expression in MHCC97L cells

目的:构建STAT1基因过表达慢病毒载体并实现其在人肝癌细胞MHCC97L中的表达。方法:根据人STAT1基因mRNA序列,全基因合成STAT1基因,构建至过表达载体并转化至感受态细胞DH5α,进行测序验证。在脂质体介导下转染293T细胞,包装生产慢病毒。慢病毒载体转染人肝癌细胞MHCC97L,用荧光定量PCR和Western blot检测原始细胞株(MHCC97L),稳转细胞株(MHCC97-stat1)中STAT1的表达情况,确定STAT1过表达的有效性。结果:测序证实STAT1基因过表达载体构建成功。过表达慢病毒包装成功。慢病毒转染人肝癌细胞MHCC97L 48 h后,STAT1基因在mRNA水平和蛋白质水平上表达显著增加。结论:STAT1基因过表达慢病毒载体构建成功,并实现其在人肝癌细胞MHCC97L中的表达。

Objective： To construct lentiviral vectors of STAT1 gene overexpression and achieve their expression in MHCC97L of human hepatoma cells. Methods： Based on the mRNA sequence of human STAT1 gene, we made the total synthesis of STAT1 gene, to construct overexpression vectors and transformed them into competent cells DH5 a which we verified by sequencing. Under the liposome mediation 293T cells were transfected to achieve the lentivirus packaging production. Then MHCC97L of human hepatoma cells were transfected by tentiviral vectors. The ex- pression of STAT1 gene in both original cell lines （MHCC97L）and stable transfected cell lines （MHCC97-stat1）were detected by fluorescence quantitative PCR and Western blot in order to determine the validity of STAT1 gene overexpression. Results： It is confirmed that the overexpression vectors of STAT1 gene were constructed successfully by sequencing. The packaging of overex- pressing lentivirus was successfu1.48h after the lentivirus transfected MHCC97L of human hepatoma cells, the expression of STAT1 gene increased significantly at both the mRNAlevel and protein level. Conclusion： The lentiviral vectors of STAT1 gene overexpression were constructed successfully and we achieved their expression in MHCC97L of human hepatoma cells.