肠道病毒71型（EV71）基因组全长cDNA感染性克隆的构建及鉴定  

Construction and identification of infectious full-length cDNA clone of enterovirus 71

作　　者：郭会杰[1] 李秀玲[1] 

机构地区：[1]北京生物制品研究所病毒性疫苗研究二室,100024

出　　处：《中华微生物学和免疫学杂志》2010年第9期809-815,共7页Chinese Journal of Microbiology and Immunology

基　　金：国家科技支撑计划（2008BA169D03）

摘　　要：目的 构建肠道病毒71型（enterovirus 71,EV71）基因组全长cDNA感染性克隆,为EV71分子遗传学、致病机制及疫苗研究等建立技术平台.方法 根据EV71 085株病毒全基因组序列,分4个片段对基因组cDNA进行扩增后,将扩增片段定向、顺序克隆至pBluescript SK（＋）载体,构建病毒基因组全长cDNA克隆,经体外转录为RNA后,转染Vero细胞获得拯救病毒.经RT-PCR、间接免疫荧光（IFA）以及电镜检测等方法鉴定拯救子代病毒.结果 成功构建Ev71基因组全长cDNA克隆,其转染Vero细胞后,可观察到典型的细胞病变;所获得的拯救子代病毒,可被EV71特异性抗血清中和;采用EV71特异性引物进行RT-PCR扩增,可出现长约226 bp的特异性条带;IFA检测可见感染细胞出现黄绿色荧光;透射电镜检测可见20～30 nm球形病毒颗粒;子代病毒在Vero细胞上连续传8代,滴度维持在4.5～6.0 lgCCID50/ml,略低于母本株（7.5 lgCCID50/ml）;噬斑检测发现,空斑形态、大小均一,但比母本株小.结论 成功构建EV71基因组全长cDNA感染性克隆,为深入探讨EV71的致病机制和疫苗研制奠定了基础.Objective To construct an infectious full-length cDNA clone of enterovirus 71（EV71）and develop a technological platform for study on vaccine development as well as molecular virology of EV71.Methods According to the nucleotide sequence of EV71 strain 085 isolated in China,four pairs of primers were designed for amplification of four end to end overlapping subgenomic cDNA fragments,the cDNA fragments were directional cloned into pBluescript SK（＋）vector,and the virus genome cDNA clone was obtained by ligation orderly.The rescued virus of parental strain 085 from RNA transfected host cells was identified by RT-PCR,IFA,titration as well as transmission electron microscope（TEM）after the transcription of the full-length cDNA clone in vitro.Results The full-length cDNA clone was constructed successfully,and the typical CPE was observed after its transcription into Vero cells.The rescued virus with 20-30 nm in diameter can not only be neutralized by EV71 special anti-serum but also react with anti-EV71 monoclonal antibody that virus infected cells stained with FITC can be detected by IFA.After amplification from the total RNA extraction of virus infected cells by RT-PCR with EV71 special primers,the 226 bp products can be detected.The growth curve showed that the rescued virus can propagate in Vero cells stably with a titer of 4.5 ～6.0 lgCCID50/ml during 8 passages.The plaque formed by rescued virus is identical as parental virus in morphology but smaller in size.Conclusion An infectious full-length clone of EV71 was developed successfully,which will be used for further study on pathogenesis and vaccine development of EV71.

关 键 词：肠道病毒71型 全长CDNA克隆 体外转录 病毒拯救 

分 类 号：R3[医药卫生—基础医学]