MYCT1-TV真核表达载体的构建及鉴定

Construction and identification of MYCT1- TV eukaryotic expression vector

目的:构建MYCT1-TV(Myc target 1 transcript variant 1)真核表达载体,鉴定其在肝癌细胞系Bel7402中的表达。方法:以正常人外周血cDNA为模板,RT-PCR方法扩增该基因的开放阅读框,定向克隆至pEGFP-C1载体后送测序鉴定。将构建好的MYCT1-TV-GFP表达载体瞬时转染Bel7402细胞,RTPCR结合荧光显微镜观察绿色荧光蛋白表达情况并鉴定载体构建是否成功。结果:测序结果证实成功将MYCT1-TV的开放阅读框克隆至pEGFP-C1表达载体,且转染细胞后,MYCT1-TV mRNA的表达水平显著上调,荧光显微镜下可见细胞内重组绿色荧光蛋白的表达。结论:成功构建MYCT1-TV-GFP表达载体,为进一步研究MYCT1-TV在肝癌中的作用机制奠定了基础。

Objective：To construct MYCT1-TV（ Myc target 1 transcript variant 1）eukaryotic expression vector and detect its expression in Bel7402 cells. Methods：The entire open reading frame of MYCT1-TV complementary DNA（ cDNA）was obtained by RT-PCR from normal human blood and cloned into the pEGFP-C1 plasmid. The fi-delity of the constructs was then confirmed by sequencing. MYCT1-TV-GFP vector was determined by RT-PCR and fluorescence microscope observation after transiently transfected into Bel7402 cells. Results：Sequencing results verified that the entire open reading frame of MYCT1-TV was successfully cloned into the pEGFP-C1 vector. After transfected with MYCT1-TV-GFP vector,the mRNA level of MYCT1-TV was upregulated and recombined green fluorescent protein was expressed in Bel7402 cells. Conclusion：An eukaryotic expression vector of MYCT1-TV was successfully constructed which will lay a foundation for the further exploration on understanding the function of MY-CT1-TV in hepatocellular carcinoma.