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

关键词:  VEGFR-3 基因  RNA 干扰  腺病毒  表达载体

Key words:  VEGFR-3  RNA interference  adenovirus  expression vector

摘要:

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5.6×109 pfu/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。

Abstract:

Objective 　To construct the recombinant adenovirus expression vector of a short interfering RNA (siRNA) targeting Vascular endothelia 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 linearized and recombinated with pAdEasy. After linearization by PacI, the recombinant adenovirus plasmid was transfected into 293 cells for packaging and amplification, then was further identified by PCR analysis. Western blotting was used to detect the expression of VEGFR-3 protein in the colorectal cancer cell lines LoVo infected with the adenovirus. 　Results　The pAdTrack pG-shRNA andpAd-VEGFR3-siRNA plasmids had been successfully constructed. The titer of the recombinant virus pAd-VEGFR3 -siRNA was 5.6×109 pfu/mL after amplificated and purified.VEGFR-3 protein expression decreased significantly in the colorectal cancer LoVo cells lines after infection by the recombinant virus. 　Conclusion　We have successfully constructed the recombinant adenovirus pAd-VEGFR3-siRNA targeting VEGFR-3 gene.