肠道病毒71型(EV71)感染性克隆的构建  

Construction of a full-length infectious cDNA clone of enterovirus 71(EV71)

作　　者：杨秀惠[1,2] 严延生[1,2] 何爱华[2] 周勇[2] 张红榕[2] 潘伟毅[2] 许江阳[2] 林其财[2] 

机构地区：[1]福建医科大学,福州350004 [2]福建省疾病预防控制中心,福州350001

出　　处：《中国人兽共患病学报》2010年第5期413-416,共4页Chinese Journal of Zoonoses

基　　金：福建省自然科学基金计划资助项目(2006J0081)

摘　　要：目的构建肠道病毒71(enterovrius 71,EV71)的感染性克隆,为研究EV71毒力基因及新型疫苗设计等建立一个技术平台。方法用RT-PCR方法扩增出EV71FJ08149株全基因组,通过TA克隆组装进TOPO-XL-PCR载体中,获得全长cDNA克隆pFJ08149T5和pFJ08149T25。应用T7聚合酶系统在体外将线性化后全长cDNA克隆转录出RNA并转染RD细胞。通过间接免疫荧光试验、RT-PCR、序列测定及免疫电镜等对拯救病毒进行鉴定。结果 RNA转染后72h观察到典型的肠道病毒致细胞病变,拯救病毒经RT-PCR、间接免疫荧光和免疫电镜检测鉴定为EV71型,表明已成功构建了EV71感染性克隆。结论本研究成功构建出具有感染性的EV71全长cDNA克隆,为深入研究EV71的致病机制等提供了有利的工具。In order to explore virulence determinants of enterovirus type 71（EV71） and develop novel vaccines,a full-length genomic cDNA clone was constructed in this study. Full-length cDNA of EV71 strain FJ08149 was obtained by RT-PCR and subsequently cloned into vector TOPO-XL-PCR. Two positive cDNA clones,pFJ08149T5 and pFJ08149T25,respectively,were obtained and linearized for RNA synthesis in vitro using T7 polymerase. The synthesized RNA was transfected into RD cells and typical CPE of enterovirus was observed on 72 hours post-transfection,the rescued virus was identified by indirect immunofluorescence（IFA）,RT-PCR,sequencing and immunoelectron microscopy,demonstrated successful recovery of infectious EV71 viruses from transfected cells. The construction of infectious EV71 cDNA clone would facilitate ongoing research on pathogenesis of EV71.

关 键 词：肠道病毒71型 感染性克隆 病毒拯救 体外转录 转染 

分 类 号：R373.2[医药卫生—病原生物学]