不同禽源副黏病毒分离株F基因的克隆与序列分析  

Cloning and sequencing of F gene of avian paramyxovirus isolates

作　　者：王旋[1] 黄兵[2] 张训海[1] 于可响[2] 钟登科[1] 宋敏训[2] 龚争[1] 赵磊[1] 

机构地区：[1]家禽疫病防控监测安徽省重点实验室,安徽凤阳233100 [2]山东省农业科学院家禽研究所,山东济南250023

出　　处：《中国兽医学报》2010年第8期1043-1048,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30671556)

摘　　要：分别对鸡源、鸭源、鹅源新城疫病毒（NDV）强毒分离株（WF00C、WF00D、WF00G）的F基因进行RT-PCR扩增、克隆和序列测定,并对其核苷酸和推导氨基酸序列进行分析。结果表明,各分离株F基因均含1个完整的开放阅读框（ORF）,全长为1 662 bp,编码553个氨基酸,有6个潜在的糖基化位点,裂解位点区的氨基酸序列均为112R-R-Q-K-R-F117,与强毒株特征相符,具有第101位的K（赖氨酸）和121位的V（缬氨酸）,符合基因Ⅶ型NDV特征;WF00C株有13个半胱氨酸（Cys）残基,WF00D株和WF00G株各有12个Cys残基;在限制性内切酶（RE）位点（nt334~1 682）的分布上,3个毒株均存在多数鹅源分离株特有的973 nt和1 249 nt RsaⅠ位点;WF00D株与WF00C株、WF00G株以及GenBank的15株各基因型代表株的F基因编码区同源性进行比较,其核苷酸序列同源性为84.7%~99.3%,推导的氨基酸序列同源性为88.4%~98.9%;3个分离株间的同源性较高,其与ZJ1、SF02和NA_1等鹅源毒株之间差异较小,而与LaSota、F48E9等毒株之间差异明显。根据NDV系统发育进化树、酶切位点分析以及基因分型结果,证明它们的基因型均为Ⅶd亚型。The whole sequence of F gene of avian paramyxovirus isolates was amplified by reverse transcription-polymerase chain reaction（RT-PCR）,and further cloned into pMD18-T vector,and positive recombinant plasmids were obtained by polymerase chain reaction（PCR）.The sequence analysis showed that all the nucleotide sequence of the F gene were 1 662 bp in length and had a single opening read frame（ORF）,which encoded polypeptide of 533 animo acid,contained 6 potential asparagine-linked glycosylation sites,their amino acid sequence of cleavage site region residues were 112R-R-Q-K-R-F117,matched to the characteristics of virulent strain,the isolates possessed K101and V121,which were similar to the characteristic of genotype Ⅶ;WF00C strain contained 13 cysteine（Cys） residues,but WF00D and WF00G strains only had 12Cys residues,all isolates contained 973 nt and 1 249 nt RsaⅠrestriction endonuclease（RE）sites（nt334-1 682）.Comparison of F gene nucleotide sequences of WF00D strain with 15 other published sequences from GenBank showed that the homology of the nucleotide was 85.0%-99.3%,the homology of the deduced amino acid sequences was 88.8%-98.9%.Three isolates from Anhui shared higher homonology each other,and higher homonology compared with ZJ1、SF02、NA_1 too,but lower homonology compared with F48E9、LaSota.The results of phylogenetic tree,restriction site analysis and genotype characterization showed that three strains WF00C,WF00D,WF00G belonged to novel subgenotype Ⅶd.

关 键 词：禽副黏病毒 新城疫病毒 F基因 克隆 序列分析 

分 类 号：S852.65[农业科学—基础兽医学]