汉坦病毒GM04-38株包膜糖蛋白M的基因克隆与序列分析

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。

Cloning and sequence analysis of envelope glycoprotein M gene of Hantavirus, GM04-38 strain

Objective:To construct the cloning vector of glycoprotein M gene of Hantavirus,to study the differences of glycoprotein M genes from the world around,and to analyze the sequence of M gene by the phylogenetic tree.Methods:Hantavirus GM04-38 strain envelope glycoprotein M gene was amplified by RT-PCR and the product was recombined with the PMD-18T vector.The clones that carried the M gene were identified.After sequenced,the gene sequence was handled with the software DNAStar compared with 25 strains worldwide,and the phylogenetic tree was drawn.Results:The GM04-38 complete M segment had3 651 nucleotides in length,of which,the ratio of type A,T,G and C was 30.46%,30.13%,20.84%,and 18.57%,respectively.The map of the phylogenetic tree showed that Hantavirus had several gene types and GM04-38 strain belonged to SEO-type Hantavirus.The analysis of the sequence showed that GM04-38 strain had the least difference in sequence from Z37 strain with a value of 2.7%,and the largest difference from Gou3 strain with a value of 18.0%.The sequence differences between GM04-38 strain and the other SEO-type Hantavirus strains ranged from 4.2% to 5.7%.Conclusion:The cloning vector containing GM04-38 glycoprotein M gene is successfully constructed.The phylogenetic tree shows that there are significant differences in the sequences of the Hantaviruses isolated from China.This will contribute to the researches on molecular genetics and epidemiology of the virus and effective subunit vaccine.