摘要
目的研究小分子干扰RNA(siRNA)对狂犬病病毒(RV)不同毒株复制的交叉抑制效果。方法采用体外转录和RNA酶III消化长片段双链RNA的方法制备8条以RV的PV株核蛋白(N)基因为靶基因的21ntsiRNA,用以转染已经感染了不同滴度PV、CTN或CVS株RV的BSR细胞,采用直接免疫荧光法观察转染的siRNA对已感染BSR细胞的不同毒株RV复制的抑制效果,并分析这种抑制效果与靶基因序列的相关性。结果不同21ntsiRNA均对PV株的复制产生了较强的抑制作用;对CTN株和CVS株的交叉抑制作用观察结果表明,21ntsiRNA与靶基因在碱基错配高达5个的情况下仍对病毒复制保持抑制效应。然而,siRNA与靶基因碱基错配的位置与抑制作用的丧失高度相关。3’端第2个碱基的错配将使抑制作用表失,随后的碱基错配对抑制作用的影响依次降低;中部碱基错配影响较小;而5’端碱基错配对抑制作用几乎没有影响。结论siRNA对靶基因的抑制作用的丧失与其同靶基因序列碱基错配的位置相关,3’端碱基错配可降低其抑制作用的特异性,产生偏靶效应的范围和概率可能增大,这为设计独特的siRNA序列提供了新的思路。
Objective To explore the cross-inhibitory effect of small interfering RNA （siRNA） to rabies virus （RV） PV strain N gene on replication of different strains of RV. Methods Eight 21nt siRNAs complementary to different positions at N gene of PV strain of RV and PV strain N gene siRNA mixtures （siRNA Cocktails ） were prepared by transcription in vitro and digestion of long double-stranded RNA with RNase Ⅲ respectively. These 2 lnt siRNAs were transfected into BSR cell monolayer infected with PV, CTN, or CVS strains of RV at different titers and the direct immunofluorescence assay （FA） was used to measure cross-inhibitory effects of 21nt siRNAs on replication of RV in BSR cells. The relationship between the cross-inhibitory effect of siRNAs and the sequence of the target gene was analyzed. Results Strong inhibitory effects to replication of PV strain were induced by siRNAs. The further cross inhibitory experiments also showed that all of the eight 21nt siRNAs could inhibit replication of heterogenous RV strains infection, including CVS and CTN strains, even when siRNA mismatching to target mRNA with five nucleotides., Nevertheless, the level of the inhibitory effect was related to the site of the mismatched base pairs. Mismatching nucleotides at the 3＇ end of siRNA chain with target mRNA played a crucial role in losing the inhibitory ability, while that at the middle part of the siRNA chain could only decrease the inhibitory effect. A mismatched base pair at 5＇ end did not influence the inhibition. Conclusions The site of mismatched base pair between siRNA chain and target mRNA play a role in the level of cross-inhibitory effect of the siRNA. The specificity of the inhibition can be decreased by a mismatched base pair at 3＇ end, leading to a higher incidence of extended off-target effect. A specific siRNA sequence can be set up basing on the results.
出处
《中国医药生物技术》
CSCD
2008年第3期204-209,共6页
Chinese Medicinal Biotechnology
