携带人生存素基因短发夹RNA表达载体重组腺病毒的构建及其生物学作用

Construction of recombinant adenovirus with short hairpin RNA expression vector of survivin and its biological effect

目的构建携带人生存素(survivin)基因短发夹RNA(shRNA)表达载体的重组腺病毒。方法构建并筛选重组质粒pShuttles-urvivin,将其与腺病毒骨架质粒共转染HEK-293细胞,经同源重组产生腺病毒;PCR鉴定并测定病毒滴度;β-半乳糖染色检测病毒对人乳腺癌细胞MCF-7的感染效率;流式细胞术、RT-PCR和W estern免疫印迹法检测其生物学功能。结果经PCR鉴定重组腺病毒构建成功;48 h后感染率达75%以上;腺病毒感染MCF-7细胞后48 h,生存素mRNA和蛋白的表达均受到抑制;腺病毒感染MCF-7细胞后,细胞分裂受阻于G2/M期,在48和72 h有凋亡峰出现。结论成功构建人生存素基因shRNA表达载体的重组腺病毒。

AIM To construct recombinant adenovirus with short hairpin RNA （shRNA） expression vector of survivin. METHODS After pShuttle-survivin was constructed and screened, cotransfected it with bone plasmid of adenovirus into HEK-293 cells to get recombinant adenovirus. Virus titer was determined after verification by PCR. Efficiency of adenovims infecting MCF-7 cells was detected by β- galactose staining. Its biological effects were observed by means of flow cytometric analysis, RT-PCR and Western blot. RESULTS Adenovirus was constructed successfully. Total of 75% of MCF-7 cells were infected after treated with adenovirus. After infected with adenovirus, RT-PCR and Western blot analysis showedthat survivin was suppressed at 48 h on the level of mRNA and protein. Flow cytometric analysis indicated that survivin shRNA induced the cell cycle arrest at G2/M phase, and induced apoptosis at 48 and 72 h. CONCLUSION Recombinant adenovirus with shRNA targeting survivin may be an effective way of gene therapy on human breast cancer.