鸡传染性支气管炎病毒中国地方分离株膜蛋白基因的克隆及序列分析

Cloning and sequence analyzing of M genes of 9 infectious bronchitis virus isolated in China

本研究选取有代表意义的9株鸡传染性支气管炎病毒（IBV）国内分离株．利用所设计的一对特异引物，通过RT—PCR的方法成功地扩增了膜蛋白基因即M基因全长片段．通过克隆、序列测定获得了9个分离毒株M基因的全长核苷酸序列。并将各IBV国内分离株与GenBank中注册的一些毒株的M基因核苷酸序列及推导的氨基酸序列进行比较、系统进化关系分析发现：IBV中国地方分离毒株大部分属于Mass型；不同毒株间存着重组、缺失、插入及点突变等变异，从ATG至第150bp区段的核苷酸序列变异频率最高；IBV中国地方分离株M基因的变异属于高频率的同义突变；国内的IBV分离株分为两个基因群，1群分离毒株与GenBank中Mass型的毒株亲缘关系较近．毒株间核苷酸序列同源性为95．7%～100％，其相应的氨基酸序列同源性为98．2%～100%；Ⅱ群分离毒株与众多参考毒株的亲缘关系都比较远，毒株间彼此核苷酸序列同源性为89．7％～91．9%，氮基酸序列同源性为92．0-96．0%。

A pair of primers were designed and synthesized according to the published matrix （M） protein gene sequences of IBV. The full length cDNA of M gene of 9 IBV strains isolated in China were amplified by RT-PCR. The amplified products were sequenced. We analyzed and compared the nucleotide sequences and deduced amino acid sequences of the M protein genes of all IBV isolates in China with the published sequences of reference strains in GenBank, and then constructed the phylogenetic tree based on the nucleotide sequences. The results showed that： （1） Though the nucleotide sequences of the M gene of the isolated IBV strains in China were conserved, there were recombinations, deletions, insertions or mutations within the IBV isolates in China, Furthermore, a fragment of 150bp from start codon ATG to 150nt showed the most variation; （2） The variations of nucleotide sequence of M protein mostly resulted from silent mutation; （3） On the basis of the phylogenetic tree of M gene, it appeared that the IBV isolates in China fall into two genetic groups, the strains of group Ⅰ closely related to Beaudette strain and KB8523 strain, with 95.7% to 100% nucleotide sequence homology and 98.2% to 100% deduced amino acid sequence homology and the strains of group Ⅱ having no close relation ship with all the published M gene sequences of IBV, with 87. 7% to 94. 9%. nucleotide sequence homology and 92. 0% to 96. 0% deduced amino acid sequence homology within the strains of this group,