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目的 克隆我国分离的汉坦病毒A9株L片段全长cDNA ,并测定其核苷酸序列。方法 用逆转录 聚合酶链反应 (RT PCR)技术分段扩增汉坦病毒A9株全部L片段 ,用T A克隆方法进行PCR产物克隆 ,测定PCR产物的核苷酸序列。通过亚克隆将分段的L片段连接成全长cDNA克隆。结果 A9株的基因组L片段长度为 6 5 33个核苷酸 ,腺嘌呤核苷酸和尿嘧啶核苷酸丰富 (%A +U =62 47)。包含有一个单一的开放读码框架 (ORF) ,编码一个标准的质量为 2 46× 10 5 的蛋白 ,含有 215 1个氨基酸。A9株与 76 118、C1 1和C1 2株的同源性最高 ,达到 83 8%。与TULA病毒的关系较远 ,其核酸序列的同源性为 65 8%。将推导的A9株编码的氨基酸序列与其他 2 1种负链RNA病毒的依赖RNA的RNA聚合酶的氨基酸序列以及汉坦病毒几个代表株的L片段氨基酸序列进行比较 ,显示A9编码的RNA聚合酶也有 6个比较保守的区域以及几个极端保守的氨基酸残基。结论 汉坦病毒A9株L片段具有和其他汉坦病毒RNA聚合酶相似的核苷酸一级结构 ,通过对推导的氨基酸分析 ,该片段具有一些在RNA病毒聚合酶中都存在的保守区域
Objective To clone the full-length cDNA of L segment of Hantavirus A9 isolated in China and determine its nucleotide sequence. Methods All the L fragments of Hantavirus A9 strain were amplified by reverse transcription polymerase chain reaction (RT PCR). The PCR products were cloned by T A cloning method. The nucleotide sequence of PCR products was determined. The segmented L fragments were ligated into full-length cDNA clones by subcloning. Results The length of the genomic L fragment of A9 strain was 6533 nucleotides, and adenine nucleotide and uracil nucleotide were abundant (% A + U = 62 47). Contains a single open reading frame (ORF) encoding a standard protein of 2 46 × 10 5 with 215 1 amino acids. The highest homology between strains A11 and 76 118, C1 1 and C1 2 was 83.8%. Its relationship with TULA virus is far, and its nucleotide sequence homology is 65.8%. The amino acid sequence encoded by the A9 strain was compared with the amino acid sequences of the RNA-dependent RNA polymerase of the other 21 negative-strand RNA viruses and the L-segment amino acid sequence of several representative strains of Hantavirus and showed that the A9-encoded RNA was polymerized The enzyme also has six more conserved regions and several extremely conserved amino acid residues. Conclusion The H segment of A9 strain of Hantaan virus has similar nucleotide structure to other Hantavirus RNA polymerase. Based on deduced amino acid analysis, this fragment has some conserved regions in RNA virus polymerase