VEGF基因RNA干扰质粒的构建与鉴定

Construction and Identification of VEGF Gene RNA Interference Recombinant Plasmid

目的构建干扰血管内皮生长因子(vascular endothelial growth factor,VEGF)基因RNA表达的重组质粒。方法设计3组针对VEGF基因的核糖核酸干扰(RNA interference,RNAi)序列,应用基因重组技术克隆入载体pcDNA3.1中,重组质粒分别命名为pcDNA3.1-VEGF-LH1、pcDNA3.1-VEGF-LH2和pcDNA3.1-VEGF-LH3,用DNA直接测序法和双酶切法鉴定构建的重组质粒。结果 DNA测序证实克隆入载体pcDNA3.1(-)中的序列正确,双酶切证实RNA干扰序列正确插入载体pcDNA3.1(-)。结论成功构建针对VEGF基因的RNAi质粒,为进一步研究该RNAi质粒在脉络膜新生血管形成中的作用奠定了基础。

Objective To construct the recombinant plasmid which interferes the RNA expression of vascular endothelial growth factor（VEGF） gene.Methods Three groups of RNA interference（RNAi） sequences were designed according to VEGF gene of pigment rats.The recombination technology was applied to insert all RNA interference sequences into the plasmid carrier pcDNA3.1（-）.The recombinant plasmids were named pcDNA3.1-VEGF-LH1,pcDNA3.1-VEGF-LH2 and pcDNA3.1-VEGF-LH3.These recombinant plasmids were identified by DNA direct sequencing method and double enzyme appraisal method.Results DNA sequencing confirmed that the sequences cloning into the plasmid carrier pcDNA3.1（-） were correct.Double enzyme appraisal affirmed that the RNA interference sequences were inserted into carrier pcDNA3.1（-） correctly.Conclusion The VEGF gene RNA interference recombination plasmids are successfully constructed,which lays the foundation to further study the function of the RNAi plasmid in the whole process of choroidal neovascularization（CNV） generation.