手足口病病原微生物EV71病毒株的分离与鉴定

目的：探讨手足口病病原体EV71病毒的分离方法。方法：取手足口病患儿的粪便,经磷酸盐缓冲液（PBS）稀释后用0.22μm滤膜过滤,将悬液接种于Vero细胞。通过EV71病毒致细胞病变效应（CPE）分析、电镜分析、EV71病毒特异性核酸及蛋白的聚合酶链反应（PCR）及Western Blotting分析来鉴定病毒分离和致病效果。检测病毒悬液对于宿主细胞Vero增殖抑制的效果。结果：标本悬液接种Vero细胞48h后出现CPE。收集感染细胞的培养基上清液,连续感染2次,均出现同样的CPE。电镜检测发现在感染组Vero细胞的细胞质和细胞核周围均有大量聚集的病毒颗粒。EV71特异性核酸RT-PCR检测证实,感染组Vero细胞中可以扩增出EV71病毒特异性的核酸条带。Western Blotting检测发现,在感染组细胞中有高表达EV71特异性抗原蛋白,而未感染组细胞不表达该病毒抗原。MTT检测结果显示,EV71病毒显著抑制宿主细胞Vero的体外增殖和生长,并且呈时间依赖性。结论：成功分离出人手足口病致微生物EV71病毒株（该EV71病毒株编号为EV71-Tj-100623）。

The Pathogen of Enterovirus 71 on Hand-Foot-Mouth Disease Isolated and Identified

Objective：To study the pathogenic molecular mechanisms of enterovirus 71 and screen the anti-virus drug for it.Methods：The stool from a patient of hand-foot-mouth disease was diluted by phosphate buffer solution（PBS）.After filtrated by 0.22μm filter,the suspends of stool water were inoculated into cell medium of Vero cell lines.The scanning electron microscope assay,PCR and western blotting assay were used to analyze the cytopathic effect（CPE）,which was induced by enterovirus 71.Besides,the cell proliferation inhibitor rates on Vero,which was induced by enterovirus 71 were tested.Results：The cytopathic effect on Vero cells was found on 48 hours,which were inoculated stool solution.The same CPE on Vero cells could also found in different three cell phases.The results of electron microscope assay indicated that a large number of virus were found congregated on the cytoplasm and nucleolus in infected group.Furthermore,the results of polymerase chain reaction（PCR） and western blotting revealed that the nucleic acid and protein of enterovirus 71 were enriched by these assay in the infected group,however,the positive antigens of enterovirus 71 were not found in the blank control Vero cell group.On the other hand,the result shown that enterovirus 71 could inhibit the proliferation and growth of Vero cells in vitro.Conclusions：We have isolated the human entrovirus 71 and signed it for EV71-Tj-100623.