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目的对一种肠道病毒71型(enterovirus 71,EV71)毒株进行一般生物学特性检测及分子鉴定。方法将EV71毒株接种于人横纹肌瘤(rhabdomyosarcoma,RD)细胞进行病毒培养,通过EV71致细胞病变效应(CPE)分析、CCID50法检测子代病毒产量、实时荧光定量PCR(qRT-PCR)检测EV71核酸合成情况以及免疫荧光印迹检测EV71蛋白表达水平,来确认其生物学特性;RT-PCR扩增EV71保守区的基因片段后进行基因测序,并将测序结果经BLAST程序进行同源性比对。结果该毒株接种RD细胞48 h后出现明显的CPE,其可在RD细胞中增殖,最终导致细胞死亡;该毒株在RD细胞中处于快速复制状态,其核酸合成与蛋白表达随时间的延长大量增加;该毒株与EV71/wuhan/3018/2010(KF501389)的核苷酸序列同源性为100%。结论该EV71毒株为EV71/wuhan/3018/2010,在RD细胞中可快速增殖,毒力较强,适合作为抗EV71药物筛选毒株。
Objective To test the general biological characteristics and molecular identification of a enterovirus 71 (EV71) strain. Methods EV71 strains were inoculated into human rhabdomyosarcoma (RD) cells for virus culture. EV71 induced cytopathic effect (CPE) analysis was performed. CCID50 method was used to detect the progeny virus yield. Real-time quantitative PCR (qRT-PCR) Nucleic acid synthesis and immunofluorescence staining were used to detect the expression of EV71 protein to confirm its biological characteristics. RT-PCR was used to amplify the gene fragment of EV71 conserved region and then sequenced. The results of BLAST program were used for homology comparison. Results The strain showed obvious CPE after being inoculated on RD cells for 48 h, which could proliferate in RD cells and eventually lead to cell death. The strain was rapidly replicated in RD cells, and the expression of nucleic acid and protein expression were prolonged with time The nucleotide sequence homology between this strain and EV71 / wuhan / 3018/2010 (KF501389) was 100%. Conclusion The EV71 strain is EV71 / wuhan / 3018/2010, which can proliferate rapidly in RD cells with strong virulence and is suitable as an anti-EV71 drug screening strain.