血管内皮细胞生长因子RNA干扰真核表达载体的构建

Construction of eukaryotic expression vector of RNA interference specific for vascular endothelial growth factor

目的构建特异性的人血管内皮细胞生长因子(VEGF)基因siRNA真核细胞表达载体,为探索RNA干扰(RNAi)分子靶向治疗非小细胞肺癌(NSCLC)的作用奠定基础。方法根据GenBank数据库提供的VEGF基因核苷酸序列,按照Reynolds设计原则,针对VEGF的序列及二级结构选择靶序列,设计双链小干扰RNA(small interfering RNA,siRNA),再转化为能表达其小发卡结构RNA(Small hairpin RNAs,shRNA)的DNA序列,并与pRNAi-H1质粒定向连接,构建受控于人RNA聚合酶Ⅲ启动子Hl的真核表达载体pRNAi-H1VEGF siRNA,经限制性内切酶酶切、PCR和DNA测序进行鉴定。结果构建针对人VEGF的RNAi真核细胞表达载体经限制性内切酶酶切、PCR和DNA测序证实与设计完全一致。结论针对人VEGF的RNAi真核细胞表达载体构建成功。

[Objective] To construct eukaryotic expression vector of siRNA specific for VEGF as a molecular target tool to cleave VEGF mRNA in NSCLC cell, and to expore the feasibility of it. [Methods] Genome sequences of VEGF fusion gene was retrieved from Genbank, siRNA （small interfering RNA） was designed according to the Reynolds＇s principle of RNAi-based medicine, and was converted into cDNA coding expression of shRNA （small hairpin RNAs） of siRNA for VEGF fusion gene. The cDNA was synthesized and inserted into plasmid pRNAi-H1. The pRNAi-H1 VEGF siRNA of recombinant plasmid being eukaryotic expression vector was controlled by the H1 promoter of RNA polymeraseⅢ, and identified by the restriction map, PCR and the sequence analysis. [Results] The pRNAi-H1 VEGF siRNA of recombinant plasmid identificated by the restriction map and the sequence analysis completely coincided with the designs. [Conclusion] The siRNA eukaryotic expression vector against VEGF mRNA has been successfully conctructed.