牛病毒性腹泻病毒长春分离株P125基因的克隆与序列测定

Cloning and sequencing of P125 gene of bovineviral diarrhea virus NCD strain

以MDBK细胞培养致细胞病变型牛病毒性腹泻病毒 (BVDV)NCD毒株 ,采用异硫氰酸胍一步法提取总RNA ,并根据BVDVNADL参考株序列设计合成的 1对引物 (W 1与W 2 ) ,进行反转录PCR (RT PCR) ,扩增出P12 5基因区约40 0bp的片段。经 pGEM T载体连接后克隆、测序 ,并与已发表的NADL、Osloss、SD 1、184、D、H、Yak等BVDV毒株序列进行比较。结果表明 :NCD株P12 5基因区无插入或缺失变异 ,但存在着核苷酸的替换 ;经聚类分析初步认为NCD株属于Ⅰa型。NCD株与长春 184、H株核苷酸序列差异较大 ,而亲缘关系较近 。

CytopathicbovineviraldiarrheavirusNCDstrainwaspropagatedintheMDBKcellandthecelltotalRNAisextractedby single stepmethodofacidguanidiniumthiocyanate phenol chloroformextraction .Afragmentofapproximately 40 0bpwasamplifiedbyre versetranscription polymerasechainreaction(RT PCR)withapairofprimersW 1/W 2 ,whichweredesignatedandsynthesizedbasedonthe publishedsequenceofreferencestrainNADL .ThefragmentwasclonedviapGEM Tvectorandsequenced ,thenthesequencewascompari sonwiththeBVDVNADL ,Osloss ,SD 1,184,H ,DandYakstrains .TheresultconfirmsthatNCDstrain ,likecivilstrains 184,H ,D , Yak ,doesnotpossessanyinsertionordeletionintheP12 5 generegion .Butnucleotideandaminoacidsubstitutionswerefoundinthese quenceanalysed ,whichdiffersfromNADL ,Osloss ,SD 1strains.ThesequenceandaminoacidhomologyofNCDwithgenotypeIaDstrains isthehighest ,98.1%and 97.7%respectively ;whilewithNADLare 82 .0 %and93 .0 % ,soitbelongstogenotypeIabydendrogram analysis .ButthereweremuchdifferencebetweenthesequenceofNCDand 184orHChangchunisolates ,andthethreestrainsshowhigh geneticrelation ,whichrevealedthattherewerestrainsfromdifferentgeneticorigininthesameregion .