深圳市手足口病重症感染性克隆的构建和分析

Construction and analysis of infectious clones of HFMD from serious cases in Shenzhen city

目的构建肠道病毒71型(EV71)的感染性克隆,为EV71致病机理的研究和药物的开发建立技术平台。方法选取临床手足口重症儿童粪便样品,经荧光定量PCR检测为阳性,RT-PCR方法扩增出EV71全基因组,通过TA克隆的方法连接到TOPO-XL-PCR载体中,应用T7聚合酶系统将线性化的EV71DNA序列体外转录成RNA,转染人横纹肌肉瘤细胞系RD-A细胞,病毒传代并观察病变,通过间接免疫荧光实验进一步鉴定,获得的感染性克隆进行全基因组测序和序列比对分析。结果 RT-PCR可以获得EV71全长约7.5kb的DNA片段,体外转录并转染后3～5d可观察到典型的肠道病毒致细胞病变,免疫荧光可看到特异标记,序列比对为C4a亚型的EV71病毒。结论构建出具有感染性的EV71全长cDNA克隆,在分子生物学水平上有利于深入研究EV71的致病机制和毒力基因、患者抗体的中和特性、疫苗和药物的效力评估与开发等。

Objective To find the virulence site and identify the anti-EV71 drugs, an infectious clone of HFMD from serious case was constructed and analyzed. Methods Two serious cases were determined of EV71 infection by fluorescence RT- PCR, and then the full length cDNA were obtained by RT-PCR and cloned into TOPO-XL-PCR vector. The in vitro transcripts of these clones were transfected into RD-A cells, and the successfully recovered viruses were determined by CPE （cytopathic effect） and IF （indirect immunofluorescence） observation. The whole genome of the infectious clone was sequenced and analyzed. Results We got an infectious clone from two constructions, the cytopathic efficiency of this clone was comparable to the isolated virus, and the whole genome sequence alignment showed it belonged to the C4a subtype. Conclusion The EVT1 infectious clone construction system has been set up in our lab, and will be used to identify the pathogenic factors and screening for anti-EV71 drugs.