血管内皮细胞生长因子RNA干扰真核表达载体的构建

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（RNM）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Keynolds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 interferingRNA，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、PCK和DNA测序进行鉴定。结果构建针对人VEGF的RNM真核细胞表达载体经限制性内切酶酶切、PCK和DNA测序证实与设计完全一致。结论针对人VEGF的RNM真核细胞表达载体构建成功。

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

[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.