人Dact2基因真核表达载体的构建及其在结肠癌细胞中的表达

Construction and identification of eukaryotic expression vector of Dact2

目的构建人Dact2基因真核表达载体。方法以人胎肝cDNA文库为模板,利用RT-PCR方法获得Dact2编码序列,构建并鉴定表达Dact2的pCMV6-Entry-GFP真核表达载体。Lipofectamine 2000转染结肠癌细胞株HT29,荧光显微镜和流式细胞仪观察和分析转染效率,Western blot检测Dact2蛋白表达。结果通过PCR扩增获得了2 322 bp的Dact2编码序列,通过酶切及测序证实Dact2序列正确插入真核表达载体pCMV6-Entry-GFP;转染空载体和Dact2表达质粒48 h后,荧光显微镜下观察,可见两组细胞均表达绿色荧光蛋白,流式细胞仪分析结果显示瞬时转染效率分别为41.36%和39.88%。Western blot检测证实转染表达质粒组的细胞Dact2蛋白呈阳性表达。结论成功构建了人Dact2真核表达载体,为其功能研究奠定了基础。

Objective To construct the eukaryotic expression vector of human Dact2 gene. Methods The coding sequence of Dact2 was amplified from human fetal liver cDNA library and cloned into pCMV6-Entry-GFP vector. The pCMV6-Entry-GFP-Dact2 was identified by restriction endonucleases and sequencing assay. After transfection of pC- MV6-Entry-GFP-Dact2 into a colon carcinoma cell line-HT29, the transfection efficiency was analyzed by fluorescence microscope and flow cytometry, and Dact2 expression was detected by Western blot. Results A 2 322 bp coded sequence was obtained and cloned into pCMV6-Entry-GFP vector correctly. The efficiency of transfection were 41.36% and 39.88% in the control and Dact2 expression vector transfection group after 48 hours of transfection. Western blot showed Daet2 expression was positive in pCMV6-Entry-GFP-Dact2 transfection group. Conclusion Human Dact2 eukaryotic expression vector was successfully constructed.