野生型PTEN基因重组腺病毒表达载体构建的改进及其鉴定

Improvement of construction of wild-type PTEN gene recombinant adenovirus expression vector and its identification

目的为改进构建野生型抑癌基因PTEN重组腺病毒表达载体的方法。方法用抑癌基因PTEN与带有绿色荧光蛋白基因的穿梭载体pAdTrack-CMV重组后转化含腺病毒骨架载体pAdEasy1的E.coliBJ5183（Ad-1 cells）,以获得重组腺病毒质粒,经限制酶PacⅠ线性化后,转染293细胞,检测PTEN蛋白的表达,并以Ad-PTEN感染人乳腺癌MCF-7细胞后,测其感染率。结果获得PTEN基因的重组腺病毒表达载体（Ad-PTEN）,受Ad-PTEN转染的293细胞发生肿胀或圆缩的形态改变,经PCR对传代的Ad-PTEN分析证实得到目的基因PTEN,荧光显微镜下能观察到293细胞内有绿色荧光。通过Western blot可检测到293细胞中PTEN蛋白高效表达。Ad-PTEN感染的人乳腺癌MCF-7细胞,感染率达80%-90%。结论PTEN重组腺病毒表达载体可在细菌体内进行同源重组而构建,方法较简便、快速,且生成的病毒滴度高、感染率高。

Objective To improve the construction of adenovirus expression vector harbouring wide-type tumor suppressor gene PTEN. Methods PTEN gene was inserted into the shuttle vector pAdTrack-CMV containing green fluorescence protein （GFP） gene, the recombinant plasmid containing adenovirus backbone vector pAdEasy-1 was then transformed into E. coli （BJ5183）. PTEN protein expression in the recombinant was determined after Pac I linearization and 293 cell transfection, and its infection rate was detected by human breast cancer cell line MCF-7 infection. Results Recombinant adenovirus expression vector Ad-PTEN was obtained. The transfected 293 cells showed cytopathic effect. PTEN gene and protein expression and green fluorescence were observed in passage 293 cells. The infection rate of MCF-7 cells was 80 % --90 %. Conclusion Recombinant adenovirus expression vector Ad-PTEN can be constructed by homologous recombination with bacteria. The construction method is simple and rapid, and Ad-PTEN infection test exhibits high virus titer and infection rate.