禽流感病毒分离株NS基因同源性及等位基因类型分析

Homology and Allele Analysis of the NS Gene of Avian Influenza Virus Isolates

目的 克隆测定国内具有代表性的禽流感病毒 (AIV)的非结构 (NS)蛋白基因核苷酸序列 ,分析其同源性和等位基因类型 ,为进一步探索禽流感NS蛋白抗体监测方法奠定基础。方法 经RT PCR扩增了国内 3株H9N2、2株H5N1、2株H7N2亚型AIV分离株的NS蛋白基因 ,并把扩增的基因片段克隆到pGEM T载体中测序 ,将测序结果与GenBank中的核苷酸序列进行同源性比较 ,绘制基因进化树。结果 经测序获得了各AIV分离株NS基因的完整编码序列。同源性分析表明 ,3株H9亚型AIV的NS基因之间的同源性为 96 %～ 98% ;两株H5亚型AIVNS基因同源性为 91 6 % ;两株H7亚型AIV的NS基因同源性为 98 9%。H5和H9亚型分离株的NS基因之间的同源性均高于 90 % ;而H7N2亚型分离株与其它两种亚型分离株的NS基因同源性约为 6 0 %～ 70 %。在AIVNS基因系统发育进化树中 ,H5、H9亚型分离株都处于等位基因A群内 ;3株H9亚型分离株的进化关系较近 ,与香港、广东的部分H5N1病毒株起源相同 ,而 2株H5病毒的NS基因则处于不同分枝内 ;2株H7亚型分离株的NS基因都处于等位基因B群内 ,进化关系较近。结论 这 7株国内AIV分离株的NS基因之间的同源性差异较大 ,约为 6 0 %～ 99% ,且包括A。

Objective To clone and sequence the non-structure (NS) protein gene of avian influenza virus (AIV) isolates and analyze the homology and allele of the NS gene, and to set up a method for detection of antibodies against NS prote in. Method The NS gene of 7 AIV isolates was amplified by reve arse transcription polymerase chain reaction (RT-PCR). The DNA fragments were c loned into pGEM-T vector and sequenced subsequently. The nucleotide sequence wa s compared with GenBank data and the phylogenetic tree of NS gene was generated. Results The entire ORF sequences of NS1 and NS2 protein were obtained. Homology analysis showed that the NS gene of these 3 H9N2 strains exhi bited 96% ~ 98% identity with each other, as to 2 H5N1 strains, 91.6%, while the 2 H7N2 strains showed 98.9% ident ity. The identity between H5N1 and H9N2 isolates was more than 90%, and about 60 % ~ 70% between H7N2 and the other two subtypes. In NS gene phylogenetic tree, t he H5N1 and H9N2 isolates were situated in branch of allele A, while H7N2 isolat es in branch of allele B. The location of the 3 H9N2 isolates was very close, ha ving the same origin with some of the H5N1 isolated in Hong Kong and Guangdong. Conclusion There is a significant defference in the nucleotide sequence identity of NS gene of AIV isolated in China. The NS gene of these sev en AIV isolates belongs to different alleles, allele A and allele B.