携带CBP短发夹RNA基因腺病毒表达载体的构建

目的:构建编码4条大鼠CREB结合蛋白(rCBP)短发夹RNA(shRNA)的腺病毒,检测RNA干扰技术(RNAi)下调rCBP mRNA的能力.方法:根据RNAi原理,设计4条针对rCBP的shRNA序列.PCR技术和酶切连接技术构建重组穿梭质粒Pgenesil4-rCBP-EGFP;体外同源重组技术构建可同时编码4...

Construction of the adenovirus coding CBP short hairp in RNA gene

Objective： To construct adenovirus of CREB-binding protein（rCBP） that encoding short hairpin RNAs（shRNAs）,and to evaluate the down regulation effects of RNA interference（RNAi） on rCBP mRNA.Methods： On the basis of RNAi principles,4 rCBPs encoding shRNAs were designed first.Then the Pgenesil4-rCBP-EGFP recombinated shuttle vector was constructed by PCR 、enzyme digestion and connection methods.In vitro homologous recombination was applied to construct the adenovirus of 4 rCBPs encoding shRNAs（CBP-shRNA/Ad）,which was verified and detected with titre method.The transfection rate of CBP-shRNA/Ad was observed by fluorescence microscope,and the down regulation ability of CBP-shRNA/Ad was assessed with RT-PCR and real time PCR.Results： The success of CBP-shRNA/Ad construction was confirmed by enzyme digestion and vector sequencing.The transfection rate of CBP-shRNA/Ad with multiplicity of infection 25 was up to 80%.After transfection of the same dose of CBP-shRNA/Ad,rCBP mRNA content in the transfected VSMCs decreased persistently at 24th h,48th h and 72th h（P0.05）At the 72th h,rCBP mRNA content was less than that of the negative control group（P0.05）.Conclusions： CBP-shRNA/Ad could down regulate rCBP mRNA content without inducing obvious adverse effects.This study provides evidence for possibility of treating vascular hyperplasia by regulating gene.