鸡新城疫病毒分离株F基因的分子特性分析

Molecular characteristic analysis of Newcastle Disease Virus Chicken isolates' F gene

【目的】对新城疫病毒（NDV）分离毒株进行分子特征分析,了解免疫鸡群发生新城疫的原因。【方法】用非免疫鸡胚从病鸡肺脏中分离新城疫病毒;参考GenBank上发表的NDV的F基因序列设计引物,应用RT-PCR方法对新城疫病毒分离株进行扩增,并构建克隆载体,对阳性质粒测序后进行F基因核苷酸及推导氨基酸序列分析。【结果】从发病鸡群中分离到6株NDV,分离毒株间的F基因核苷酸和F蛋白氨基酸同源性分别在99.6%-99.9%和99.1%-99.8%;分离毒株与参考毒株的F基因核苷酸和F蛋白氨基酸序列同源性分别在83.2%-87.3%和90.2%-93.8%;分离毒株F蛋白裂解位点的氨基酸顺序为^112R-R-Q-K-R-F^117,符合强毒株的分子特征;6个分离株均属NDV基因Ⅶ型。【结论】NDV分离株的F基因已经发生明显变异,与La Sota、Clone-30、V4等常用疫苗株在遗传进化上的亲缘关系较远,这可能是免疫鸡群发生新城疫的重要原因之一。

【Objective】 The reseaon for the outbreak of Newcalstle Disease in vaccinated chickens was investigated by analysing molecular characteristics of NDV isolates.【Method】 Six isolates of Newcastle Disease Virus（NDV） were separated from suspected diseased chickens.According to the sequence published in GenBank,a couple of primers were designed,and the six isolates of NDV were amplified by RT-PCR.The target gene was obtained,and was cloned to the pMD18-T vector,thus the cloning vector was constructed.The sequences of positive plasmids,nucleotide sequences and amino acid sequences were analyzed.【Result】 Results demonstrated that the nucleotide homology and amino acid homology of F fragment among the six NDV isolates were 99.6%-99.9% and 99.1%-99.8% respectively.The nucleotide homology and amino acid homology of F fragment between the isolated strain and the reference strain were 83.2%-87.3% and 90.2%-93.8% respectively.The amino acid sequence of F protein cleavage site was 112R-R-Q-K-R-F117,consistent with the molecular characteristics of virulent strain.The molecular characteristics of F gene indicated that the six isolates belonged to Ⅶ genotype.【Conclusion】 F gene of the six NDV isolates had a far genetic relationship with the vaccine strain e.g.La Sota,Clone-30,V4,which was the probable reason that vaccinated chickens still catch Newcastle Disease.