汉坦病毒A9株全长L片段cDNA克隆和核苷酸序列测定

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6533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2.46×105的蛋白,含有2151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21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.

Cloning and nucleotide sequencing of full length L segment cDNA of Hantavirus strain A9

Objective Cloning and nucleotide sequencing of full length cDNA sequence of L segment of Hantavirus strain A9.Methods The L segment cDNA of Hantavirus strain A9 was amplified by RT PCR.PCR products were cloned and sequenced .Full length cDNA clone of the L segment was obtained by subcloning.Results The L segment of strain A9 was 6 533 nucleotides in length and encoded a putative L polymerase that was 2 151 amino acids in length.Analysis of nucleotide and deduced amino acid sequences of the L segment of strain A9 showed a close relationship with the other Hantaviruses most notably the 76 118,Cl 1 and Cl 2 strains,with which they shared a 83 8% nucleotide identity. More distant similarity could also be seen with other Hantaviruses. Sequence comparison performed among the RNA dependent RNA polymerases of 27 negative stranded RNA viruses revealed that the same existence of six conserved regions and several strictly conserved residues located at the deduced amino acid sequence of A9 L segment. Conclusion The nucleotides sequence of L segment of strain A9 was similar to other Hantavirus L segments.Deduced amino acid sequence of A9 L segment shares 6 conserved regions with other RNA virus RNA polymerase.The conserved residues in these polymerases and their possible functions in light of the available structural information have been discussed.