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目的:建立汉坦病毒(HV)包膜糖蛋白M的克隆载体;研究M基因的变异情况,并对其序列进行系统发生树分析。方法:应用逆转录聚合酶链反应(RT-PCR)扩增GM04-38M片段的基因,产物纯化后克隆于PMD-18T载体,经氨苄青霉素筛选,酶切鉴定,并进行序列测定,应用DNASTAR软件将它与世界范围内分离的病毒株同一基因序列分析比较。结果:筛选出含有HVM蛋白基因的克隆。GM04-38株M片段的全基因序列共3651个核苷酸,4种核苷酸的比例分别为:A30.46%,T30.13%,G20.84%,C18.57%。序列同源分析表明,GM04-38株与Z37株核苷酸同源性最高(97.3%),属于SEO型HV。与其他SEO各株的差别均小于18.0%;绘出了核苷酸系统发生树。结论:成功地建立汉坦病毒M蛋白基因克隆载体;中国不同地区汉坦病毒流行株基因序列存在明显差异。这为研究HV遗传与变异以及制备有效的亚单位疫苗奠定了基础。
OBJECTIVE: To establish a cloning vector of Hantavirus (HV) glycoprotein M, to study the variation of M gene, and to analyze its sequence by phylogenetic tree. Methods: The gene of GM04-38M was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The purified product was cloned into PMD-18T vector, screened by ampicillin, identified by restriction enzyme digestion and sequenced. DNASTAR software It is compared with the sequence analysis of the same gene of virus strains isolated worldwide. Results: Clones containing the HVM protein gene were screened out. The total gene sequence of GM04-38 strain M fragment was 3651 nucleotides in total. The proportions of the four nucleotides were A30.46%, T30.13%, G20.84% and C18.57%, respectively. Sequence homology analysis showed that the nucleotide homology between GM04-38 and Z37 strains was the highest (97.3%), belonging to SEO type HV. Differences from all other SEO strains were less than 18.0%; nucleotide phylogenetic trees were mapped. Conclusion: The Hantavirus M protein gene cloning vector was successfully established. There were significant differences in the gene sequences of Hantavirus epidemic strains in different regions of China. This laid the foundation for the study of HV inheritance and mutation as well as preparation of effective subunit vaccines.