增强型绿色荧光蛋白与单纯疱疹病毒Ⅰ型胸苷激酶共表达质粒载体构建及在肝癌细胞株HepG2表达的研究

Construction and expression of enhanced green fluorescent protein and HSV1-TK co-expression vector

目的构建增强型绿色荧光蛋白与单纯疱疹病毒Ⅰ型胸苷激酶基因的真核共表达质粒载体pIRES2-EGFP-TK,并鉴定其在肝癌细胞株HepG2中转染和表达。为肝癌靶向基因治疗提供实验依据。方法2008年6月重庆医科大学附属第二医院从含有目的基因的质粒PORF-HSV1-TK中获得HSV1-TK基因片段,连接到增强型绿色荧光蛋白共表达载体pIRES2-EGFP,得到重组质粒pIRES2-EGFP-TK并进行鉴定、测序。将重组质粒用脂质体法转染肝癌细胞株HepG2,荧光显微镜下观察增强型绿色荧光蛋白基因(EGFP)在细胞内的表达,逆转录聚合酶链反应RT-PCR检测胸苷激酶TK的mRNA表达水平,免疫印迹Western blotting法检测TK蛋白表达情况,新霉素类似物G418筛选出阳性克隆。结果重组克隆载体内的TK序列与GeneBank报告的序列完全一致。肿瘤细胞转染后在荧光显微镜下可观察到有绿色荧光出现,G418筛选的最佳浓度为600mg/L,TK的mRNA表达水平高,TK蛋白有明显表达。结论实验成功构建EGFP和TK真核共表达载体,在肝癌细胞能有效的表达。

Objective To construct EGFP and HSV1-TK co-expression vector and to detect its expression level in hepatoma carcinoma cell line HepG2, and thus to provide experimented bases for the target gene therapy of liver cancer. Methods HSVI-TK gene was isolated from pORF-HSV1TK plasmids and cloned into eukaryotic expression plasmid pIRES2-EGFP. The recombinant plasmid plRES2-EGFP-TK was identified by restriction endonuclease digestion and DNA sequencing. Then recombinant plasmid was transfected into hepatoma carcinoma cell line HepG2 by liposome reagent, and the expression of EFGP in cell were observed by fluorescence microscopy , TK protein and mRNA was analyzed by Western blotting and RT-PCR respectively. Results The sequence of the cloned DNA fragment was identical to HSV1-TK that was reported on Gene bank. The recombinant expression plasmid was successfully transfected into HepG2 cell line, and green fluorescent was observed under fluorescent microscope, positive clones were picked out by G418 screening. The optimal G418 screening concentration was 600g/L.The mRNA expression level of TK was high and its protein expression was found. Conclusion The recombinant eukaryotic co-expression vector of EGFP and HSV 1-TK was successfully constructed and effectively expressed in HepG2 cell line.