含tk和EGFP双顺反子重组腺病毒载体的构建及其在鼠内皮细胞中的表达

Construction and Expression of Bicistronic Adenovirus Vector Carrying Thymidine Kinase and Enhanced Green Fluorescent Protein Gene.

目的:构建含有单纯疱疹病毒胸腺嘧啶核苷激酶(HSV-tk)和增强绿色荧光蛋白(EGFP)的重组腺病毒载体,体外转染鼠内皮细胞,并检测其表达。方法:利用脑炎心肌炎病毒的内部核糖体进入位点(IRES)将HSV-tk与EGFP连接,构建携带HSV-tk和EGFP双顺反子的穿梭质粒,经细菌内同源重组获得复制缺陷腺病毒载体。转染鼠内皮细胞,并检测腺病毒表达情况。结果:成功构建了含有HSV-tk和EGFP双顺反子重组腺病毒载体,荧光显微镜下可观察到绿色荧光的转基因细胞;体外检测结果表明丙氧鸟苷(GCV)对转染有tk-EGFP的鼠内皮细胞有明显杀伤作用,而对未转染的鼠内皮细胞无明显的毒性。结论:含HSV-tk和EGFP基因双顺反子腺病毒载体的构建可以为肿瘤抗血管形成基因治疗提供了新的手段。

Objective： To construct a recombinant adenovirus vector carrying gene of herps simplex virus thymidine kinase （ HSV - tk） and enhanced green fluorescent protein （ EGFP）, and detect its expression in rat endothelial cells. Methods： Molecular cloning techniques were used to construct an adenovirus vector. The internal ribosome entry site （IRES） of encephalomyocarditis virus （EMCV）, which could coordinate expression of two genes in a single vector, was optioned. The recombinant advenovirus vector was obtained by cotransforming pShuttleCMV - tk - EGFP and adenovirus genomic DNA plasmid of pAdeasy - 1 vector in bacterial cells. Then the vector containing tk - IRES - EGFP was transfected into rat endothelial cells （RECs）. Results： An adenovirus vector carrying tk - IRES - EGFP was constructed. The fluorescence microscope findings showed that the tk - IRES - EGFP gene was successfully transferred into RECs. There were no differences in the growth pattern or the morphology between RECs and REC/tk - IRES - EGFP cells. In vitro experiments demonstrated dose - dependent cell killing by transduction of tk - IRES - EGFP gene followed by Ganciclovir （ GCV ） treatment. Conclusion： These results suggest that this new kind of adenovirus vector can serves as a new tool and method for tumor anti- angiogenic gene therapy.