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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 eukaryotic co-expression vector pIRES2-EGFP-TK of enhanced green fluorescent protein and herpes simplex virus type 1 thymidine kinase gene and identify its transfection and expression in hepatoma cell line HepG2. Provide experimental evidence for liver cancer gene therapy. METHODS: The HSV1-TK gene fragment was obtained from the PORF-HSV1-TK plasmid containing the gene of interest at the Second Affiliated Hospital of Chongqing Medical University in June 2008 and linked to the enhanced green fluorescent protein co-expression vector pIRES2-EGFP. The recombinant plasmid pIRES2- EGFP-TK and identified, sequenced. The recombinant plasmids were transfected into HepG2 cells by lipofectamine. The expression of enhanced green fluorescent protein (EGFP) in cells was observed under a fluorescence microscope. The expression of thymidine kinase TK mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) The expression of TK protein was detected by Western blotting. The positive clone was screened by the neomycin analogue G418. Results The sequence of TK in the recombinant cloning vector was exactly the same as that reported in GeneBank. The green fluorescence appeared under the fluorescence microscope after transfection of tumor cells. The optimum concentration of G418 for screening was 600 mg / L, the expression of TK mRNA was high, and the expression of TK protein was obvious. Conclusion The eukaryotic co-expression vector of EGFP and TK was constructed successfully and could be efficiently expressed in hepatoma cells.