同时针对VEGF和p53腺病毒干扰载体的构建及干扰作用

目的:构建能同时干扰VEGF和p53基因表达的RNAi腺病毒干扰载体,观察其对肿瘤细胞VEGF和p53的干扰作用.方法:先分别构建能干扰p53和VEGF的质粒载体,依次将构建干扰载体的干扰序列和H1启动子序列切下并连接到pAdTrack上,构建成pAdTrack/VEGF/p53,将构建的质粒转染含有pAdEasy-1的BJ5183工程菌中,回收并酶切鉴定重组腺病毒载体,将阳性腺病毒载体感染293细胞并收集病毒,采用荧光定量PCR检测腺病毒干扰载体同时降低VEGF和p53表达的作用.结果:经过酶切鉴定,pAd/VEGF/p53载体中含有插入的2个启动子和干扰序列.转染腺病毒干扰载体后,肿瘤细胞中的VEGF和p53 mRNA表达量显著降低.结论:构建能同时干扰VEGF和p53基因的腺病毒干扰载体可以显著降低MCF-7细胞中VEGF和p53 mRNA的表达.

Construction of RNAi Adenovirus Targeting both p53 and VEGF Synchronously and Its Gene Silencing Function

Objective: To construct RNAi adenovirus targeting both VEGF and p53 in the same time. Methods: The interfering plasmids targeting VEGF and p53 were constructed respectively. Then the H1 promoter and RNAi sequence were cut and ligated to pAdTrack successively to generate pAdTrack/VEGF/p53, which was transfected into BJ5183 cells containing pAdEasy-1 to generate pAd/VEGF/p53 after homologous recombination, harvesting the adenovirus after infecting 293 cells. The knockdown of VEGF and p53 by pAd/VEGF/p53 was detected through real-time PCR after infecting MCF-7 cells. Results: The constructed pAd/VEGF/p53 containing being inserted two H1 promoters and RNAi sequences was identified by BamH digesting. The VEGF and p53 mRNA level of MCF-7 cells decreased obviously after being infected by pAd/VEGF/p53. Conclusions: The RNAi adenovirus targeting both VEGF and p53 in the same time was successfully constructed and it can silence both genes obviously.