EV71济南分离株感染性克隆的构建及其生物学特征分析

Construction of a DNA-launched infectious clone of an EV71 strain based on isolates from Ji'nan and identification of the virus' biological characteristics

目的构建以济南本土分离株为模板的EV71感染性克隆,转染细胞以获得拯救病毒并观察其生物学特性,为研究EV71病毒基因结构和功能奠定基础。方法分段扩增EV71基因组cDNA并顺序连接至pCDNA3.1载体,同时在序列两端引入自剪切核酶序列,构建能直接转染细胞的病毒基因组全长cDNA克隆,转染RD细胞后获得拯救病毒。通过噬斑形成试验、间接免疫荧光试验和感染性滴度测定观察拯救病毒的生物学特征。结果成功构建了病毒基因组全长cDNA克隆,转染RD细胞后观察到典型细胞病变,收获的病毒经RT-PCR扩增出特异性目的片段,全序列测定结果表明拯救病毒与父本株相比具有3个同义突变,未导致推导氨基酸的变异;拯救的子代病毒感染RD细胞后形成典型的空斑;间接免疫荧光试验检测拯救病毒感染细胞可见特异性荧光,荧光密度较父本株病毒稍弱;拯救病毒传代稳定,其感染性滴度(10-3.48)较父本株(10-5.0)稍低。结论成功构建了真核载体的病毒全长cDNA感染性克隆,拯救病毒与野生株具有相似的生物学性状和感染性。

Objectives To construct a full-length cDNA clone of infectious EV71 based on isolates from Ji＇nan and to observe the biological characteristics of the rescued virus in order to lay the foundation for further research on the structure and function of the genes of EV71. Methods The genomic sequence of the JN200804 strain of EV71 was amplified and cloned into a pCDNA3.1 vector,and ribozyme elements were introduced at both termini of the viral genomic cDNA sequence to construct a full-length cDNA clone that could be directly transfected into cells to yield a rescued virus.The biological characteristics of the virus were preliminarily observed using aplaque formation assay,indirect immunofluorescence assay（IFA）,and determination of the infectious titer of the virus. Results The full-length cDNA clone was transfected into RD cells,causing typical CPE.The rescued virus was identified using RT-PCR and sequenced.Sequencing indicated that the rescued virus has three synonymous mutations compared to the wild-type strain,without no variation in amino acids.The rescued virus was found to typically form plaque.An IFA of infected cells resulted in specific fluorescence that was less intense than that of cells infected with the wild-type virus.The rescued virus propagates stably,and the infectious titer（10-3.48）was slightly lower than that of the mild strain（10-5.0）. Conclusion A full-length cDNA clone of EV71 was successfully constructed with eukaryotic vector,and the rescued virus had similar biological characteristics and infectivity to the wild-type strain.