细胞水平狂犬病病毒感染的RNA干扰  

RNA Interference against Rabies Virus Infection in Susceptible Cells

作　　者：张守峰[1] 李清竹[2] 李忠[1] 刘晔[3] 张菲[3] 卢彦欣[1] 夏咸柱[1] 扈荣良[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]沈阳农业大学畜牧兽医学院,辽宁沈阳110161 [3]军事医学科学院军事兽医研究所,吉林长春130062

出　　处：《病毒学报》2006年第6期462-465,共4页Chinese Journal of Virology

基　　金：国家自然科学基金项目(30300255);军队医学杰出中青年基金项目(04J016)

摘　　要：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。Nine siRNA expression plasmids against Rabies virus （RV） glycoprotein and nucieoprotein 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 TCID50 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.

关 键 词：狂犬病病毒 RNAI 感染 糖蛋白 核蛋白 

分 类 号：R512.99[医药卫生—内科学]