广西鸡传染性支气管炎病毒地方分离株核蛋白基因的遗传变异分析

Genetic Variations of Nucleocapsid Protein Gene of Infectious Bronchitis Viruses Strains Isolated in Guangxi Province

应用RT-PCR方法扩增了广西1985～2008年的24株传染性支气管炎病毒(IBV)分离株的核(N)蛋白基因,并对此24株IBV的N蛋白基因进行了克隆、序列测定及分析。结果发现24株IBV分离株N蛋白均含有一个长1230bp的开放阅读框,编码由409个氨基酸残基组成的多肽,未发现碱基的缺失和插入,但存在较多点突变现象。与疫苗株H120核蛋白的3个保守碱性氨基酸区序列进行比较分析发现,大多数毒株在183～232位存在较多的氨基酸替代现象,部分毒株在334～363位也存在较多的氨基酸替代现象。与GenBank中的13个IBV参考毒株N蛋白基因序列进行比较分析,发现2004～2008年的21个分离株中有19个分布于第Ⅰ群中;1985～1988年的3个分离株之间N蛋白核苷酸及其推导氨基酸的同源性均达99%以上,均属于第Ⅲ群中,可见广西不同时期的IBV分离株在N基因进化关系上形成了自己独立的进化群。同时研究还发现GX-NN5分离株可能为基因重组病毒。以上结果表明了广西IBV毒株存在基因突变和基因重组现象。

Nucleocapsid （N） protein genes of 24 infectious bronchitis virus （IBV） strains isolated in Guangxi from 1985 to 2008 were amplified by reverse transcriptase polymerase chain reaction （RT-PCR）,and then were cloned, sequenced and compared with 13 IBV reference strains. The results showed that the N genes of 24 isolates were composed of 1 230 nucleotides,encoding a polypeptide of 409 amino acid residues. Deletion and insertion were not found in the N genes of 24 isolates,but it was found that point mutation inducing amino acidsubstitution was common in N genes of 24 isolates. Compared with the amino acids in three conserved basic regions of N genes among 24 Guangxi isolates and H120 strain,it was found that amino acid substitutions were common in 183 to 232 of N genes in the most isolates and in 334 to 363 of N genes in some isolates. Phylogenetic tree based on amino acid sequences of N genes from 24 IBV isolates and 13 reference IBV strains showed that 24 isolates were classified into four distinct clusters. 19 of 21 isolates from 2004 to 2008 could be classified into cluster Ⅰ. There were more than 99% identity of nucleotide sequence and deduced amino acid of in the N gene among the 3 Guangxi IBV isolates from 1985 to 1988,and these 3 isolates could be classified into cluster Ⅲ. Recombinations were also found in the genome of IBV isolate GX-NN5. These results suggested that gene mutations and recombinations had occured in the IBV strains isolated in Guangxi from 1985 to 2008.