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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/VEGE/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。
OBJECTIVE: To construct an RNA interference vector that can simultaneously interfere with the expression of VEGF and p53, and to observe its interference effect on VEGF and p53 in tumor cells. METHODS: Plasmid vectors capable of interfering with p53 and VEGF were constructed. The interference vector and H1 promoter sequence were cut out and ligated into pAdTrack to construct pAdTrack / VEGF / p53. The constructed plasmids were transfected into pAdEasy-1 BJ5183 engineered bacteria, the recombinant adenoviral vector was recovered and digested with restriction endonucleases. 293 cells were infected with positive adenovirus vector and virus was collected. Fluorescent quantitative PCR was used to detect adenovirus interference vector and reduce the expression of VEGF and p53. Results: After digestion, pad / VEGE / p53 vector contained two inserted promoters and interference sequences. Transfection of adenovirus interference vector, tumor cells in the expression of VEGF and p53 mRNA was significantly reduced. CONCLUSION: Construction of adenovirus vector that can simultaneously interfere with VEGF and p53 gene expression can significantly reduce the expression of VEGF and p53 mRNA in MCF-7 cells.