不同来源的5株新城疫病毒的分离·鉴定与基因型分析

Isolation and Identification of Newcastle Disease Viruses from Different Species and Analysis of Their Genotype

[目的]分离鉴定不同来源的5株新城疫病毒(NDV),并对其基因型进行分析。[方法]从江苏和广西发病孔雀、鸡、鹅群分离到5株NDV。参照文献合成1对引物P1和P2,用于扩增基质蛋白基因(M)1161nt至融合蛋白基因(F)889nt之间长约970nt的片段。用TrizolRNA试剂盒抽提5株NDV基因组,再按照Promega说明书,用上游引物P1合成F基因cDNA,合成的cDNA作为聚合酶链式反应(PCR)的扩增模板,用PCR反应扩增F基因目的片段。将所得产物进行序列测定,获得这5株NDVF基因5′端约890nt序列。采用DNAMAN软件对F基因47～420nt间的片段进行序列分析,并推导出相应的氨基酸序列。在此基础上,从GenBank中选取32个参考毒株与这5个分离株进行遗传变异分析,并绘制系统发育进化树。[结果]同源性分析比较表明,5株NDV分离株之间的差异性很小,它们之间的同源性为95.00%～97.00%,其中2株广西分离株之间的同源性为97.07%,3株鹅源分离株之间的同源性为96.43%。所有毒株F蛋白裂解区氨基酸序列均为112R-R-Q-K-R-F117,符合强毒株裂解区位点特征。这5株NDV分离株均属于基因Ⅶ型和Ⅶd亚型。[结论]近几年流行的NDV的主导基因型为基因Ⅶ型,且来自不同宿主的毒株之间的差异性较小。

[Objective] The research aimed to isolate and identify the Newcastle disease viruses（NDV）from different species and analyze their genotype.[Method] Five NDV isolates were isolated from visceral organs of diseased birds during outbreaks of chickens,geese and peafowl in recent years in Jiangsu and Guangxi Provinces.One pair of primer,P1 and P2,composed by according to reference were used to amplify about 970 nt fragment from matrix protein（M）gene 1161 nt to fusion protein（F）gene 889 nt.Five NDV isolates genome were extracted by Trizol RNA kit,cDNA of F gene composed by using P1 was used as template of PCR to amplify the target fragment of F gene.The product sequencing was determined and obtained about 890 nt sequence at the 5＇terminal of 5 NDV isolates F gene.374 nt sequence analysis between 47 to 420 nt was made by DNAMAN soft,and deduced corresponding amino acid sequence.A phylogenic tree was constructed based on the 374 nt sequence data of 5 NDV isolates and 32 NDV reference strains from the Genbank.[Result] The results showed that 5 NDV isolates had a little difference with the homology of 95.00%-97.00%.Thereinto,the homology of 2 isolates from the Guangxi Zhuang Autonomous Region was 97.07%,and the homology of 3 isolates form gooses was 96.43%.The amino acid sequences of F protein cracking of 5 isolates were all 112R-R-Q-K-R-F117,meeting the characters of location point of cleavage of virulent NDV.All 5 NDV isolates belonged to genotype Ⅶ,subgenotype Ⅶd.[Conclusion] The study indicates that the main genotype of popular NDV these years is genotype Ⅶ,and the isolates from different host had a little difference.