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目的:应用逆转录-聚合酶链反应(RT-PCR)、核苷酸序列分析等技术,尝试建立一种可直接从鼠肺标本中进行汉坦病毒基因分析的方法。方法:提取经免疫荧光法检测汉坦病毒抗原阳性的鼠肺细胞总RNA,经逆转录获取病毒cDNA,纯化,克隆于pMD-18T载体,测定S基因片段核苷酸序列。结果:采用RT-PCRT/A克隆技术成功地从鼠肺组织中扩增出汉坦病毒S基因全序列,序列长为1701nt。只有一个编码N蛋白的开放读码框架(ORF),起始密码子为第37nt,终止于1326nt,推导其编码的蛋白长为429个氨基酸。与该标本分离株(简称TJJ16毒株)S基因的全序列相一致。结论:该方法可对汉坦病毒抗原检测阳性而病毒分离阴性的鼠肺标本进行分子生物学特征性分析,从而建立一种对汉坦病毒在宿主间的流行情况分析的方法。
OBJECTIVE: To establish a method for gene analysis of Hantaan virus directly from mouse lung specimens by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequence analysis. Methods: The total RNA of rat lung cells positive for Hantavirus antigen was detected by immunofluorescence method. The cDNA was purified by RT-PCR and cloned into pMD-18T vector. The nucleotide sequence of the S gene was determined. Results: The complete sequence of Hantavirus S gene was successfully amplified from murine lung tissue by RT-PCRT / A cloning technique with a sequence length of 1701nt. There is only one open reading frame (ORF) encoding the N protein with the start codon of 37nt and the end of 1326nt. The deduced protein length is 429 amino acids. Consistent with the full sequence of the S gene of the specimen isolate (TJJ16 strain for short). Conclusion: This method can be used to analyze the molecular characteristics of Hantavirus antigen-positive and virus-negative mouse lung specimens, so as to establish a method to analyze the prevalence of Hantavirus among hosts.