血管生成抑制因子METH-1腺病毒载体的构建

Construction of antiangiogenesis METH1 gene recombinant adenovirus vector

目的:利用大肠杆菌同源重组构建重组腺病毒载体并在293细胞制备高滴度的病毒。方法:实验于2004-08/2005-08在中山大学眼科中心国家重点实验室完成。自真核表达载体pMD18-T-METH1中酶切出METH1基因,亚克隆至带有增强型绿色荧光蛋白表达盒的腺病毒穿梭质粒pAdTrack-CMV中,形成转移质粒pAdTrack-CMV-METH1,在大肠杆菌BJ5183内与腺病毒骨架质粒pAdEasy-1同源重组,得到重组腺病毒载体pAd-METH1。以pAd-METH1为模板,经DNA测序正确后,用PacI酶切线性化pAd-METH1,转染293细胞,包装成重组病毒颗粒,荧光显微镜观察转染细胞增强型绿色荧光蛋白的表达,采用聚合酶链反应方法对重组腺病毒进行鉴定。结果:①构建了携带外源基因人血管生成抑制因子的穿梭载体pAdTrack-CMV-METH1。②制备了METH1重组腺病毒载体pAdEasy-METH1。③METH1重组腺病毒可感染293细胞并在293细胞中进行有效的复制。结论:应用细菌内同源重组能够快速构建腺病毒载体,制备高滴度重组病毒,为METH1基因功能研究奠定了基础。

AIM： To construct a recombinant adenovirus vector with Bacterium coli and prepare adenoviruses with high titer and purity by amplifying in 293 cells. METHODS： The experiment was conducted in the national key laboratory of Ophthalmic Center of Sun Yat-sen University between August 2004 and August 2005. METH1 gene was collected from eukaryotic vector pMD18-T- METH1 and subcloned into adenovirus shuttle plasmid pAdTrack-CMV containing enhanced green fluorescent protein gene （EGFP） expression cassette to form transfer vector of pAdTrack-CMV-METH1, which was co- transformed into BJ5183 bacterial cells with adenovirus backbone vector pAdEasy-1. After the recombinant adenovirus vector pAd-METH1 was obtained, DNA sequencing was performed with pAd-METH1 as template. The identified recombinant adenovirus plasmid DNA was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. The expression of EGFP was detected by fluorescent microscope, and the recombinant adenoviruses in 293 cells were identified by PCR. RESULTS： ①The shuttle plasmid pAdTrack-CMV-METH1 with exogenous gene of human antiangiogenesis was constructed. ② The recombinant adenoviruses pAdEasy-METH1 was prepared. ③ The recombinant adenovirus had infected 293 cells and replicated in .293 cells. CONCLUSION： The adenovirus vector can be fast constructed by homologous recombination in bacterium and the adenoviruses with high titer and purity have been achieved, which lays a foundation for the research on the function of METH1 gene.