B组轮状病毒WH-1株NSP2基因序列和蛋白质结构分析

Analysis of non-structural protein NSP2 gene sequence and protein structure of human group B rotavirus strain WH-1

目的 克隆成人腹泻标本WH 1中B组轮状病毒组 (groupBrotavirus ,GBRV)非结构蛋白NSP2基因 ,分析其核苷酸序列 ,比较与其它GBRV基因同源性 ,预测其mRNA二级结构、蛋白质二级结构。方法 利用逆转录 聚合酶链反应 (RT PCR)技术 ,从成人腹泻标本WH 1中扩增GBRVNSP2基因 ,克隆载体pUCmT ,对NSP2基因进行核苷酸序列分析。利用GeneBee软件比较与其它GBRV毒株NSP2基因同源性 ,Rnaviz2 .0软件绘制NSP2基因的二级结构 ,PredictProtein软件分析NSP2蛋白结构。结果 成人腹泻标本WH 1中GBRV非结构蛋白NSP2基因全长 10 0 7bp ,与ADRV核苷酸序列的同源性达 98% ,与印度加尔各达分离株CAL 1达 82 % ,与IDIR(鼠 )同源性仅为 79%。氨基酸序列与ADRV的同源性达 98.4 % ,与CAL 1达 97.7% ,而与IDIR仅为 89.0 %。其mRNA折叠形成多达 2 6个发卡环状结构。NSP2蛋白是由 30 1个氨基酸残基组成的多肽 ,含有 2个潜在的N 糖基化位点和多个磷酰化位点。结论 成人腹泻标本WH 1中GBRV非结构蛋白NSP2基因和氨基酸序列与人GBRV有较高的同源性 ,而与动物中GBRV同源性相对较低 ,其中与ADRV的同源性最高 ,推测GBRVWH 1株与ADRV具有相同起源。

Objective To clone and sequece the non-structural protein NSP2 gene of human group B rotavirus (GBRV) strain WH-1, compare identity to other GBRV and predict the secondary structure of its mRNA and protein. Methods The NSP2 gene of GBRV strain WH-1 was amplified by reverse trarnscription polymerase chain reaction (RT-PCR) and ligated to plasmid pUCm-T and transformed into E.coli competent cells One-shot Top-10F′, the NSP2 gene of GBRV strain WH-1 on the cloned plasmid pT-NSP2 was sequenced, the identity to other GBRV was analyzed by software GeneBee, the secondary structure of WH-1 NSP2 mRNA was predicted by software RNA structure 3.7 and Rnaviz 2.0, and the protein of NSP2 was analyzed by PredictProtein. Results The nucleotide sequence of WH-1 NSP2 showed high identities (more than 90%) to that of other human GBRV such as ADRV in China, while identities tothat of animal GBRV such as IDIR(Rat) was 79%. The secondary structure of WH-1 NSP2 mRNA was constructed by 28 stem-loop structures. The polypeptide encoded by WH-1 NSP2 contains 301 amino acids. The protein contains two potential N-linked glycosylation sites and some phosphorylation sites. Comparied with other GBRV WH-1 NSP2 demonstrated 98.4% and 97.7% of amino acids similarity with ADRV and CAL-1 respectively, but only 89.0% with IDIR. Conclusin GBRV WH-1 and ADRV might originate from a common ancestral virus.