摘要
以传染性法氏囊病病毒 (IBDV )浙江分离株 (ZJ2 0 0 0 )基因组 RNA为模板 ,采用 L ong- accurate RT- PCR(L A-PCR)一步法扩增并克隆了 IBDV ZJ2 0 0 0株基因组 A节段全长 c DNA。序列测定结果表明 ,克隆的 A节段全长共32 5 9个核苷酸 ,包括 5′、3′端的非编码区 (NCRs)和 2个部分重叠的开放阅读框 (ORF1和 ORF2 ) ,与参比的血清 型毒株核苷酸序列的同源性高达 95 .2 %~ 99.2 %。二级结构预测表明 ,在 5′- NCRs和 3′- NCRs存在 1个大型的茎环发夹结构。 ORF2编码 14 5个氨基酸的 VP5 ,与参比毒株的同源性高达 98.6 %~ 10 0 %。 ORF1编码 10 12个氨基酸的VP2 / VP4 / VP3,在氨基酸水平上 VP2、VP3、VP4与参比毒株的同源性分别达 94 .9%~ 98.8%、96 .1%~ 98.5 %、97.1%~ 99.2 %。ZJ2 0 0 0共有 14~ 38个氨基酸的替代 ,其中特有氨基酸 6个 ,变异大多数集中在 VP2高变区 ,突变率达 2 .9%~ 11%。第 2个小亲水区内 2 80位氨基酸由 S替代了 N、2 90位 M替代了 L。这 2个突变可能与 IBDV的抗原性有关。VP2 - VP4剪切位点附近 5 11和 5 4 0位氨基酸的变异可能使 ZJ2 0 0 0株的毒力增强。分子系统进化树分析表明 ,ZJ2 0 0 0与欧洲 Cu- 1株、P2株、CEF94株和中国 Harbin株的关系最近 ,而与欧洲、香港、?
The full length segment A of infectious bursal disease virus(IBDV ZJ2000) isolated in Zhejiang province was amplified by long and accurate PCR in a single step,cloned into T vector and sequenced by prime walking.The result showed that the segment A contains 3 259 nucleotides including 5′ and 3′ end noncoding region and two partially overlapped Open Reading frames ORF1 and ORF2.Compared with other IBDV strains,ZJ2000 segment A shared 95 2% 99 2% homology at nucleotide level.A hairpin structure was predicted in 5′ NCRs and 3′ NCRs of the segment.VP5 was composed of 145 amino acid residues shared 98 6% 100% homology with other strains.The sequence of the region coding for polyprotein(VP2,VP4,VP3) was located from 132 to 3 146 ,the deduced amino acids sequence of VP2,VP3,VP4 shared 94 9% 98 8%,96 1% 98 5%,97 1% 99 2% homology with other strains respectively.14 38 amino acid substitutions were observed in polyprotein,and six amino acids were unique for ZJ2000,most amino acid substitutions were in the VP2 variable region with a mutation rate of 2 9% 11%.Two amino acids were substituted at 280(N to S) and 290(L to M) in the second minor hydrophic peak of ZJ2000 which may involve in antigenic variation.The two key amino acid exchanges near the cleavage site of VP2 VP4 may play an important role in increasing the virulence of ZJ2000.Phylogenetic tree analysis indicated ZJ2000 is more related to the European classical strains than those of vvIBDV and variant strains.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2002年第1期10-14,共5页
Chinese Journal of Veterinary Science
基金
国家"8 63"高科技资助项目 (No.10 1-j99-0 2 )