1株汉坦病毒的分离及其S基因片段的克隆和序列分析  

Molecular cloning and nucleotide sequence analysis of the S-segment in Hantavirus isolated in Zhejiang province

作　　者：徐芳[1] 姚苹苹[1] 朱函坪[1] 谢荣辉[1] 

机构地区：[1]浙江省疾病预防控制中心,杭州310051

出　　处：《中国人兽共患病学报》2009年第7期665-668,共4页Chinese Journal of Zoonoses

摘　　要：目的对浙江省肾综合征出血热（Hemorrhagic Fever with Renal Syndrome，HFRS）疫区-龙泉，新分离到的毒株的S基因片段进行克隆并进行序列测定及分析，以确定毒株的型别和基因变异程度，为进一步研究病毒进化和变异提供了有利条件。方法采用直接免疫荧光检测疫区鼠肺HV抗原。将HV抗原阳性的鼠肺标本接种Vero-E6细胞，分离病毒。参考GenBank发表的汉坦病毒核蛋白基因序列，设计合成一对引物，提取分离株细胞培养物的总RNA，应用逆转录聚合酶链反应（RT-PCR）方法扩增新毒株S片段基因，并克隆入载体，纯化后进行核苷酸序列测定及分析。结果成功分离到的新毒株S片段的全基因序列共1 700个核苷酸，只有一个开放读码框架，共编码429个氨基酸。新毒株与HTN型毒株比较，核苷酸同源率为85.7%-91.9%。与SEO型毒株比较，核苷酸同源率为71.2%-75%，表明该毒株为HTN型毒株。结论浙江HFRS疫区新分离汉坦病毒株可以定型为HTN型毒株，可能为新的亚型。One strain of Hantavirus（HV） was isolated from patients with hemorrhagic fever with renal syndrome （HFRS） in Zhejiang province and its S-segment was cloned and submitted to nucleotide sequence analysis in order to determine the type of strain and extent of genetic variation for further study on its evolution and mutation. The HV antigen was detected from mouse lungs in endemic area by direct immunofluorescene test and the HV-positive sample of mouse lung was inoculated to Veto-E6 cells to isolate virus. The total cellular RNA was extracted from infected cell culture and the S segment gene was amplicated by RT-PCR. Then, the purified PCR product was cloned into T vector for sequencing. The result showed that the full-length sequence of the S segment was 1 700 bp with one open reading frame （ORF） encoding 429 amino acids. Comparison with Hantavirus HTN type showed 85.7%-91.9% homology at the nucleotide level. In comparison with the SEO type of viruses, the homology at nucleotide level was shown to be 71.2%-75%. This new HV strain may be typed as to HTN virus and may be a new subtype of this virus.

关 键 词：汉坦病毒 病毒分离 S片段 序列分析 

分 类 号：R373[医药卫生—病原生物学]