细胞水平狂犬病病毒感染的RNA干扰

以质粒pSilencer2.1-U6 Hygro为基础,设计并构建了针对狂犬病病毒(RV)糖蛋白和核蛋白mRNA的共9个siRNA表达载体,转染BHK-21细胞系后,在潮霉素-B的筛选压力下,获得9个稳定转录相关siRNA的BHK-21细胞株。1000TCID50的RV分别感染24孔板内的上述9株细胞,48h后以直接免疫荧光法检测各株细胞上RV的增殖,结果显示,在经不同siRNA表达载体转染的BHK-21细胞中,RV增殖水平有不同程度下降,RV增殖水平最低者为对照细胞的1%,即RNA干扰效应最高可阻断99%RV的感染。针对其中阻断水平超过90%的靶序列G69和N19,人工合成其双链siRNA,瞬时转染BHK-21细胞后,仍可达到80%以上的感染阻断率。本试验为有效阻断RV早期感染提供了新选择,为通过RNAi研究RV的基因组功能提供了新的依据。

RNA Interference against Rabies Virus Infection in Susceptible Cells

Nine siRNA expression plasmids against Rabies virus(RV) glycoprotein and nucleoprotein mRNAs were designed and constructed based on the vector pSilencer 2.1-U6 Hygro(Ambion).Baby hamster kidney cell line BHK-21 was transfected by above-mentioned plasmids respectively and screened by Hygromycin-B,and subsequently,nine cloned strains were gained in which the corresponding siRNAs could be transcribed stably.Every cloned cell strain in a 24-well culture plate was infected by 1000 TCID_(50) of RV and the replication of the virus was detected by direct immunofluorescence assay(DIF) and RT-PCR after 48h.The result of detection discovered that the inhibition ratios of RV replication were from 10% to 99%,and there were two exceeding 90% among them.Consequently,two double-stranded siRNA molecules(G69 and N19) aimed at the two RNAi target sequences were synthesized and transfected into BHK-21 transiently.The DIF displayed that about 80% of the virus replication was inhibited after the transfection.This study provided an alternative to block the infection of RV before immune response and to research the functions of RV genome.