靶向血管内皮细胞因子受体3小干扰RNA腺病毒表达载体的构建

Construction of the recombinant adenovirus expressing the short interfering RNA targeting vascular endothelia growth factor receptor 3 gene

目的构建靶向血管内皮细胞因子受体3(VEGFR-3)的小干扰RNA重组腺病毒表达载体。方法将构建的靶向VEGFR-3特异性小干扰RNA真核表达载体pGenesil-siRNA的启动子及siRNA序列克隆入穿梭质粒pAdTrack,将质粒线性化处理后转化入pAdEasy。重组腺病毒载体线性化处理后转染HEK293细胞,包装重组腺病毒,并进行PCR鉴定、病毒滴度测定;扩增后感染结肠癌LoVo细胞并进行Western blotting检测VEGFR-3蛋白的表达。结果双酶切鉴定及测序证实pAdTrack-pG-siRNA及pAd-VEGFR3-siRNA质粒构建正确,扩增纯化测定重组病毒pAd-VEGFR3-siRNA的滴度为5.6×109pfu/mL。病毒感染结肠癌LoVo细胞后测定VEGFR-3蛋白表达明显降低。结论靶向VEGFR-3小干扰RNA的腺病毒载体构建成功。

Objective To construct the recombinant adenovirus expression vector of a short interfering RNA （siRNA） tar- geting Vascular endnthelia growth factor receptor 3 （ VEGFR - 3） gene. Methods The pGenesil - siRNA expression vector promoter and siRNA, which was constructed, were subcloned to pAdTrack shuttle plasmid. The product was iinearized anti recombinated with pAdEasy. After linearization by Pael, the recombinant adenovirus piasmid was transfected into 293 cells for packaging and amplification, then was further identified by PCR analysis. Western blotting was used to detec! the expression of VEGFR -3 protein in the eolorectal cancer cell lines LoVo infected with the adenovirus. Results The pAdTraek-pG - shRNA andpAd - VEGFR3 - siRNA plasmids had been successfully constructed. File titer of the recombinant virus pad -VEGFR3 -siRNA was 5.6 x 10^9pfu/mL after ampiificated and purified. VEGFR -3 protein expression decreased significantly in the eoloreetal cancer LoVo cells lines after infection by the recombinant virus. Conclusion We have suceessfully constructed the recombinant adenovirus pad -VEGFR3 -siRNA targeting VEGFR- 3 gene.