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目的 测定我国疫苗株Z10的M片段全基因序列 ,了解其分子基础 ,并分析其与其它汉坦病毒株的遗传学差异。方法 从Z10株病毒感染的细胞提取总RNA ,将逆转录PCR扩增的产物纯化后克隆于T载体并进行序列测定 ,应用DNASTAR软件分析比较。结果 Z10株M片段的全基因序列共 36 15个核苷酸 ,四种核苷酸的比例分别为A 30 15 % ,T 2 9 41% ,G 2 1 72 % ,C 18 73%。以第二种读码方式推导出其最大读码框架从 41到 34 48,共编码 1135个氨基酸。序列同源分析表明 ,Z10株与HTN型核苷酸同源性为 83 6 %～ 87 4% ,与SEO ,DOB和Thai型为 6 9 5 %～ 70 8% ,而与PUU和HPS各型病毒同源性仅为 5 7 6 %～ 5 9 9%。氨基酸同源分析Z10株与HTN型同源性高达 94 2 %～96 0 % ,与SEO ,DOB和Thai型为 73 3%～ 76 6 % ,而与PUU和HPS各型病毒同源仅为 5 3 2 %～5 6 3%。绘出了核苷酸和氨基酸的系统发生树。结论 Z10株为不同于已报告M片段全基因序列的HTN型国内外毒株的另一亚型 ,并具有 16个独特的氨基酸。
Objective To determine the M gene sequence of vaccine strain Z10 in China, understand its molecular basis and analyze its genetic difference with other Hantavirus strains. Methods Total RNA was extracted from cells infected with Z10 virus. The products amplified by RT - PCR were purified and cloned into T vector and sequenced. DNASTAR software was used to analyze and compare the results. Results The sequence of the full-length M segment of Z10 strain was 36 15 nucleotides. The proportions of the four nucleotides were A 30 15%, T 2 9 41%, G 2 1 72% and C 18 73%, respectively. The second reading frame deduced the largest reading frame from 41 to 3448, encoding a total of 1135 amino acids. Sequence homology analysis showed that the homology between Z10 strain and HTN nucleotide was 83 6% -87 4%, while it was 65% -70 8% with SEO, DOB and Thai, but not with PUH and HPS Virus homology was only 576% ~ 599%. Amino acid homology analysis showed that the homology between Z10 and HTN was 94 2% ~ 96 0%, which was 73 3% ~ 76 6% with SEO, DOB and Thai, but only 5 with PUH and HPS 3 2% ~ 5 6 3%. The phylogenetic tree of nucleotides and amino acids is plotted. Conclusion Z10 strain is a different subtype of domestic and foreign HTN strains which has different sequence from the reported M fragment and has 16 unique amino acids.