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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 construct the infectious clone of enterovirus 71 (EV71) and establish a technological platform for the study of pathogenesis of EV71 and the development of drugs. Methods Stool samples of clinical hand-foot-mouth children were selected and stool samples were positive by fluorescence quantitative PCR. The whole genome of EV71 was amplified by RT-PCR and linked to TOPO-XL-PCR vector by TA cloning method. T7 polymerase system The linearized EV71 DNA sequence was transcribed into RNA in vitro and transfected into human rhabdomyosarcoma cell line RD-A. The virus was passaged and observed for pathological changes. Indirect immunofluorescence assay was used to further identify the infectious clones. The obtained infectious clones were subjected to whole genome sequencing and sequence alignment . Results The DNA fragment of about 7.5 kb in length of EV71 was obtained by RT-PCR. Typical enterovirus-induced cytopathic effect was observed 3 to 5 days after transfection in vitro. Specific immunofluorescence showed that the DNA sequence was C4a Subtype EV71 virus. Conclusion The full-length cDNA clone of infectious EV71 was constructed, which is conducive to further study on the pathogenesis and virulence genes of EV71 and the neutralization characteristics of the antibody in rabbits and the evaluation and development of the efficacy of vaccines and drugs.