柯萨奇病毒A组16型cDNA感染性克隆的构建  

Construction of infectious DNA clones of coxsackievirus A16

作　　者：李嘉祺[1] 李洪哲[1] 郑惠文[1] 宁若彤 范海涛 杨泽宁[1] 胡雅洁[1] 刘龙丁[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《中国生物制品学杂志》2018年第1期14-17,23,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31300143);云南省应用基础研究计划项目(2015FB193)

摘　　要：目的构建柯萨奇病毒A组16型(coxsackievirus A16,CA16)c DNA感染性克隆。方法提取CA16基因组RNA,经RT-PCR得到基因组全长c DNA,插入至TOPO-XL-PCR载体,获得CA16全长c DNA克隆。采用T7聚合酶系统将线性基因组c DNA体外转录出病毒基因组RNA,纯化后转染Vero细胞,获得拯救病毒。通过RT-PCR、基因测序及免疫荧光法对拯救病毒进行鉴定,并对拯救病毒与母本病毒的增殖特性进行比较。结果病毒基因组RNA转染Vero细胞后48 h,可见典型的肠道病毒致细胞病变。拯救病毒经RT-PCR、基因测序和免疫荧光鉴定为CA16,且与母本野生型病毒增殖特性基本一致。结论成功构建了具有感染性的CA16全长c DNA克隆,为研究CA16基因功能和研发CA16新型疫苗奠定了基础。Objective To construct the infectious c DNA clones of coxsackievirus A16（CA16）. Methods The genomic RNA of CA16 was extracted, and full-length genomic c DNA was amplified by RT-PCR and inserted into vector TOPO-XLPCR to obtain full-length CA16 c DNA clone. Linear genomic c DNA was transcribed to viral genomic RNA in vitro by using T7 polymerase system, purified and transfected to Vero cells, and the produced rescued virus was identified by RTPCR, gene sequencing and immunofluorescence assay（IFA）. The proliferation characteristics of rescued virus and its parental virus were compared. Results Typical cytopathic effect（CPE）caused by enterovirus was observed in Vero cells48 h after transfection with viral genomic RNA. The rescued virus was identified as CA16 by RT-PCR, gene sequencing and IFA, of which the proliferation characteristic was consistent with that of wild CA16. Conclusion The full-length infectious c DNA clone of CA16 was successfully constructed, which laid a foundation of study on gene function of CA16 and development of a novel CA16 vaccine.

关 键 词：柯萨奇病毒A组16型 感染性克隆 病毒拯救 

分 类 号：R373.23[医药卫生—病原生物学] Q789[医药卫生—基础医学]