NF-κB/p65特异的RNAi腺病毒载体的构建及其对p65表达的抑制效应

Construction of NF-κB/p65-targeting RNAi combinant adenovirus vector and its suppressive effect on the expression of p65 in ECV304 cells

目的构建NF-κB p65亚基特异的RNA干扰(RNAi)腺病毒表达载体,并验证其对p65亚基的基因沉默效应。方法设计合成三对针对p65 mRNA不同位点的短发夹RNA(shRNA)编码序列,克隆到穿梭载体中,通过体外同源重组将短干扰RNA(siRNA)表达盒转移到腺病毒骨架质粒,构建RNAi腺病毒表达载体;在HEK293A细胞中包装并扩增病毒、空斑实验法进行病毒滴度测定;腺病毒感染人脐静脉内皮细胞株ECV304细胞,Western印迹法和免疫细胞化学法验证构建的RNAi腺病毒对p65蛋白表达的抑制效应。结果成功构建了NF-κB p65亚基特异的RNAi腺病毒表达载体,获得高滴度的腺病毒液;RNAi腺病毒感染ECV304细胞后可以高效抑制p65蛋白的表达,且对p65蛋白表达的抑制作用可持续6 d以上。结论应用RNAi腺病毒表达载体能有效阻断目的基因的表达。

Objective To construct RNAi combinant adenoviral expressive vectors specific to p65 subunit of NF-κB and to observe their gene silencing effect on p65 subunit. Methods Three pairs of complementary single-strand DNA oligos targeting three various sites of p65 mRNA were designed and synthesized. Annealling was used to generate double-strand oligos （ds-oligos）, and then the ds-oligos were cloned into pENTR^TM/U6 to generate the entry clone named pENTR. Recombination reaction in vitro with the pENTR and pAd/BLOCK-iT^TM-DEST was used to creat the adenovirus plasmid which contains the RNAi cassette. Then, the adenovirus plasmids digested with PacI were transfected into HEK293A cells to product adenovirus, and latter infected the HEK293A cells to amplify the adenoviral stock. Plaque forming assay was used to titer the adenoviral stock. The p65 gene silencing effect induced by the RNAi adenovirus was detected by Western blot and immunocytochemistry assay in ECV304 cells. Results The RNAi adenovirns specific to p65 subunit of NF-κB were produced with titer of 3.0 × 10^9pfu/ml to 2.5 × 10^10pfu/ml. The expression of p65 protein in ECV304 cells could be down-regulated efficiently by the RNAi adenovirus 48-72 h after infection, which would last for more than 6 days after infection. Conclusion RNAi adenovirus is an important tool inhibiting the expression of target gene efficiently.