马传染性贫血病病毒驴白细胞弱毒疫苗株前病毒全基因组核苷酸序列分析

Complete Sequence of Proviral DNA of EIAV Chinese Donkey Leukocyte Attenuated Strain

马传染性贫血病病毒 (EIAV)是马传染性贫血病 (简称马传贫 ,EIA)的病原。我国的EIAV弱毒疫苗是迄今为止世界上唯一投入应用的慢病毒疫苗 ,成功地控制了我国EIA的流行。本研究从EIAV弱毒疫苗株 (DLA)感染的驴白细胞中提取前病毒DNA ,以提取的前病毒DNA为模板 ,利用PCR技术分 3个片段扩增EIAV前病毒 ,这 3个片段覆盖全部前病毒DNA。将这 3个片段分别克隆到载体质粒pBluescrtiptsK中 ,得到 3个重组质粒P2 .8,P2 .4,P4.1,经酶切鉴定后 ,测序。对测序结果分析、拼接 ,得到EIAV驴白细胞弱毒疫苗株前病毒基因组全序列。EIAVDLA株前病毒基因组全长 82 6 6bp。基因组两端是长为334bp的LTR ,其中U3为 2 14bp ,R为 81bp ,U5为 39bp。前病毒基因组有 3个较大的开放阅读框架 (ORF) ,分别编码gag、pol和env基因。gag基因位于 732～ 1931位碱基之间 ,全长 12 0 0bp ;pol基因位于 172 7～ 5 12 8位碱基之间 ,全长 34 0 2bp ;env基因位于5 32 4～ 7912之间 ,长为 2 5 89bp。此外前病毒还有 3个小的ORF ,S1位于 5 132～ 5 2 84位碱基之间 ,长为 15 3bp ;S2位于 5 2 98～ 5 5 0 1位碱基之间 ,长为 2 0 4bp ;S3位于 72 5 8～ 76 5 9位碱基之间 ,长为 40 2bp。本研究结果为进一步确定EIAV的基因功能、揭示马传贫疫苗毒的致弱?

Equine infectious anemia virus (EIAV)is the etiological agent of equine infectious anemia (EIA). Donkey leukocyte attenuated equine infectious anemia virus (DLA EIAV)vaccine was the only successful lentivirus vaccine prepared in China,which has been applied extensively in China against EIA for twenty years. In this study,provirl DNA was extracted from donkey leukocyte by EIAV DLA strain.Three fragments covering the entire EIAV genome were amplified by polymerase chain reaction (PCR) using the proviral DNA as template.These PCR products were cloned into plasmid pBluescrip sK,and obtained the three recombinant plasmid P2.8,p2.4 and p4.1,respectively.Following recombinant plasmids were confirmed by restriction enzyme digesting,they were sequenced.The complete nucleotide sequence of EIAV DLA strain proviral DNA was determined by analyzing each of the three parts and connecting them as a whole.The genome of EIAV DLA was 8266 bp in length.Both ends of EIAV DLA provirus were identical long terminal repeat (LTR)sequences of 334bp in length.The LTRs include U3,R and U5 regions in turn,and the sizes of them were 214bp,81bp and 39bp,respectively.The genome of EIAV DLA provirus contains three long open reading frames (ORF)corresponding to gag,pol and env genes,respectively.Gag gene is 1200bp located at 732__1931.pol gene is 3402bp at position 1727__5128.Env gene is 2589bp at position 5324__7912.The provirus has three small ORFs,ie S1,S2 and S3,with sizes of 153 bp (nucleotides 5132__5284),204bp(nucleotides 5298__5501)and 402bp (nucleotides 7258__7659),respectively.