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根据GenBank发表的传染性支气管炎病毒 (infectiousbronchitisvirus,IBV)Beaudette毒株核衣壳 (nucleocap sid ,N)基因序列设计一对特异性引物 ,利用RT -PCR方法分别扩增了疫苗毒株IBV -H5 2、嗜腺胃ZJ971株、嗜肾IBV -X和N株的N基因ORF ,并克隆到质粒载体 pBluescriptSK( +) ,测序后用PCgene和DNAStar软件分析。结果显示 :4株IBV的N基因均含有一个长 12 30bp的ORF ,编码一由 40 9个氨基酸残基组成的N蛋白。嗜肾IBV -X和N毒株的N基因存在HindⅢ酶切位点。与来自GenBank的另外 8株IBV比较 ,ZJ971株与它们的N蛋白的核苷酸及氨基酸序列同源性分别在 88 2 %～ 98 6 %和 91 2 %～ 97 8% ;N株则分别为 86 6 %～ 87 4%和90 5 %～ 91 5 % ;X株分别为 86 3%～ 87 4%和 90 0 %～ 91 5 % ;H5 2分别为 90 4%～ 98 3 %和 92 0 %～98 0 %。ZJ971株、N和X株的N基因的突变特点是散在的点突变。嗜腺胃ZJ971株核衣壳蛋白的氨基酸序列的最显著的遗传变异特征是H111D、S117A、V14 2 L、P171A和E4 0 1D ;嗜肾IBV -X和N株核衣壳蛋白的氨基酸序列的最显著的遗传变异特征是A4 6S、A4 8P、L189R、N190 S、E2 2 0 D、I2 2 3A、V2 3 6Y、S2 37K、K2 4 0 Q、Q2 86R、T2 99K、R30 1E、E33 4 D、V33 5E、V336P和T351S。
Abstract：Two specific primers were designed according to the published sequence of nucleocapsid(N)protein gene of strain Beaudette of infectious bronchitis virus(IBV)from GenBank.cDNA of N gene was amplified by reverse transcription polymerase chain reaction(RT-PCR)from viral RNAs of proventriculopathogenic IBV strain ZJ971,nephropathogenic IBV strains N,X and IBV vaccine strain H52 respectively.They were cloned into vector pBluescript SK(+) and sequenced by the dideoxy-mediated chain termination method.The results indicated that the complete open reading frame(ORF)of N gene is 1230bp in length,which encodes a protein of 409 amino acids.Compared with N gene of the other 8 IBV strains from GenBank,the homology of nucleotide and deduced amino acid sequences was 88.2%-98.6% and 91.2%-97.8% for ZJ971,86.6%-87.4% and 90.5%-91.5% for N,86.3%-87.4% and 90.0%-91.5% for X,90.4%-98.3% and 92.0%-98.0% for H52 respectively.The characteristics of variation of N gene for ZJ971,X and N strains was scattered point mutation without obvious HVR, while those of X and N tend to vary in middle and rear regions relatively.These findings showed that variation of nucleocapsid protein gene of IBV with different tissue tropism was different.Variation of amino acid sequence of nucleocapsid protein of proventriculopathogenic IBV strain ZJ971 was characterized with H 111D,S 117A,V 142L,P 171A,E 401D;and that of nephropathogenic IBV strains N and X was characterized with A 46S?A 48P?L 189R?N 190S?E 220D?I 223A?V 236Y?S 237K?K 240Q?Q 286R?T 299K?R 301E?E 334D?V 335E?V 336P and T 351S.
[1]JiaW ,KaracaK ,ParrishCR ,etal.Anovelvariantofavianinfec tiousbronchitisvirusresultingfromrecombinationamongthreedif ferentstrains[J].ArchVirol,1995,140:259～271.
[2]CavanaghD ,NaqiSA .Infectiousbronchitis[A].CalnekBN ,BarnesCW ,McDougaldLRetal.DiseasesofPoultry[M].TentheditionIowaUniversityPress,AmesIowa,1997511-526.
[3]BoursnellMEG ,BrownTDK ,FouldsIJ,etal.Completionofthesequenceofthegenomeofcoronavirusavianinfectiousbronchitisvirus[J].JGenVirol,1987,68:57-77.
[4]SternDF ,KennedySIT .Coronavirusmultiplicationstrategy.I .I dentificationandcharacterizationofvirus-specifiedRNA[J].JVi rol,1980,34:665-674.
[5]CanvanaghD ,DavisPJ,PappinDJC ,etal.CoronavirusIBV :par tialaminoterminalsequencingofspikepolypeptideS2identifiesthesequenceArg-Arg-Phe-Arg-ArgatthecleavagesiteofthespikeprecursorpolypeptideofIBVstrainsBeaudetteandM41[J].VirusRes,1986,4:133-143.
[6]CavanaghD ,DavisPJ ,DarbyshireJH ,etal.CoronavirusIBV :virusretainingspikeglycopolypeptideS2butnotS1isunabletoinducevirus-neutralizingorhemaglutinationinhibitingantibody,orinducechickentrachealprotection[J].JGenVirol,1986,67:1435-1442.
[7]BootsAMH ,Benaissa-TrouwBJ ,HesselinkW ,etal.Inductionofanti-viralimmuneresponsesbyimmunizationwithrecombination-DNAencodedaviancoronavirusnucleocapsidprotein[J].Vaccine,1992,10:119-124.
[8]WilliamsAK ,WangL ,SneedLW ,etal.Comparativeanalysisofthenucleocapsidgenesofseveralstrainsofinfectiousbronchitisvirusandothercoronavirus[J].VirusRes,1992,25:213-222.
[9]SapatsSI,AshtonF ,WrightPJ,etal.NovelvariationintheNpro teinofavianinfectiousbronchitisvirus[J].Virol,1996,226:412-417.
[10]VanderMeerY ,SnijderEJ,DobbeJC ,etal.LocalizationofmousehepatitisvirusnonstructuralproteinsandRNAsynthesisin dicatesaroleforlateendosomesinviralreplication[J].JVirol,1999,73:7641-7657.
[11]DenisonMR ,SpaanWJ,VanderMeerY ,etal.Theputativeheli caseofthecoronavirusmousehepatitisvirusisprocessedfromthereplicasegenepolyproteinandlocalizesincomplexesthatareactiveinviralRNAsynthesis[J].JVirol,1999,73:6862-6871.
[12]NdifunaA ,WatersAK ,ZhouML ,etal.Recombinantnucleocapsidproteinispotentiallyaninexpensive,effectiveserodiagnosticreagentforinfectiousbronchitisvirus[J].JVirolMethods,1998,70(1):37-44.
[13]SeahJN ,YuL ,KwangJ .LocalicationoflinearB -cellepitopesoninfectiousbronchitisvirusnucleocapsidprotein[J].VeterinaryMi crobiology.2000,75:11-16.
[14]周继勇.传染性腺胃炎病毒(暂定名)ZJ971株的一些生物学特性[J].畜牧兽医学报,2000,31(3):227-234.
[15]荣骏弓,吴东来,谷守林,等.腺胃病变型传染性支气管炎病毒强毒株的培育[J].中国兽医学报,2001,21(4):327-330.
[16]周继勇,于涟,马有智,等.鸡肾型传染性支气管炎病毒分离及鉴定[J].浙江农业大学学报,1998,24(3):303-307.
[17]周继勇.鸡传染性腺胃炎病毒(暂定名)的致病性[J].中国兽医学报,2000,20(2):124-127.
[18]周继勇,沈行燕,程丽琴,等.新变异的禽传染性支气管炎病毒ZJ971毒株S基因克隆及序列分析[J].中国农业科学,2001,34(4):445-450.
[19]ZhouML ,CollissonEW .Theaminoandcarboxyldomainsoftheinfectiousbronchitisvirusnucleocapsidproteininteractwith3’ge nomicRNA[J].VirusRes,2000,67:31-39.
[20]ZhouML ,WilliamsAK ,ChungS ,etal.TheinfectiousbronchitisvirusmucleocapsidproteinbindsRNAsequencesinthe3’terminusofthegenome[J].Virology,1996,217:191-199.
[21]NelsonGW ,StohlmanSA ,TaharaSM .Interactionbetweennu cleocapsidproteinandleader/intergenicsequenceofmousehepatitisvirusRNA[J].JGenVirol,2000,81:181-188.
[22]ColognaR ,SpagnoloJF ,HogueBG .Identificationofnucleocapsidbindingsiteswithincoronavirus-defectivegenomes[J].Virology,2000,277:235-249.
基本信息:
DOI：
中图分类号:S852.65
引用信息:
[1]周继勇,张德勇,丁红梅,程丽琴,陈吉刚.不同组织嗜性传染性支气管炎病毒核衣壳基因的遗传变异性分析[J].病毒学报,2003(01):59-63.
基金信息:
国家自然科学基金 ( 39970 0 30 );; 浙江省自然科学基金( 399411);; 浙江省重点科研项目 ( 99110 2 0 30 )资助