广西巴马小型猪PERV-A亚型毒株全基因cDNA克隆的构建与分析  

Construction and analysis of the PERV-A strain full-length complementary dna clone from Guangxi Bama minipig

作　　者：钟雅婷[1] 黄红梅[1] 钟华[2] 欧阳康[1] 马玲[1] 廖艳娟[2] 白安斌[1] 吴健敏[1] 

机构地区：[1]广西兽医研究所广西畜禽疫苗新技术重点实验室,广西南宁530001 [2]广西民族大学化学与生态工程学院,广西南宁530006

出　　处：《中国兽医学报》2015年第11期1740-1747,共8页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31260613);广西自然科学基金资助项目(2013GXNSFBA019123);广西重点实验室开放基金资助项目(12-071-28-B-3)

摘　　要：参照GenBank已发表的猪内源性逆转录病毒(PERV-A亚型)全基因序列,用软件DNAman分析PERV-A亚型全基因序列单一酶切位点,设计3对特异性引物及1对套式引物,分4段从仅携带PERV-A亚型毒株的广西巴马小型猪外周血淋巴细胞中扩增巴马小型猪PERV(PERV-A-BM)全基因组序列,并将4个覆盖全基因组序列的重叠基因片段,通过pMD 18-T中间载体依次连接并克隆到pBluescript II SK(-)载体上,构建全基因cDNA克隆。同时通过定点突变和化学合成的方法成功恢复PERV-A-BM全基因cDNA克隆中存在的15个保守突变碱基,将突变后的PERV-A-BM全长序列上传GenBank,登录号为HM159246。为便于鉴定将PERV-A-BM全基因cDNA克隆第8408位碱基由A沉默突变为C,引入新的单一酶切位点Aat II,获得cDNA克隆的突变株。酶切鉴定及测序结果表明,PERV-A-BM前病毒基因组全长8 774bp,分为两端的5′、3′非编码区(UTR)及中间的开放阅读框(ORF),编码gag、pol和env基因。与不同亚型标准株gag和pol基因的氨基酸序列具有较高同源性,而env基因的同源性则较低。本研究为下一步拯救出该病毒提供了可直接使用的全长cDNA真核表达质粒。Based on the whole genome of porcine endogenous retrovirus（PERV-A）in GenBank,we analyzed the restriction sites of PERV-A genome with software DNAman,and designed 3pairs of specific primers and 1pair of nested primers to amplify the whole genome of PERV-A strain from the peripheral blood lymphocytes of a Guangxi Bama minipig that only carried PERV-A.Then,the 4resulting overlapping fragments were joined in turn and cloned into the binary vector pBluescript II SK（-）with pMD18-T as the intermediate vector to construct the full-length cDNA clone.Meanwhile,15 mutations of PERV-A-BM cDNA clone were restored by gene site-directed mutagenesis and chemosynthesis.The modified PERV-A-BM was then registered at GenBank,getting an accession number：HM159246.For easy identification,we induced site mutation during the cDNA clone construction by replacing the base adenine（A）with cytosine（C）at the 8408 bps site of PERV-A-BM through site-specific mutagenesis.The single site of restricting enzymes Aat II was also induced into the mutant virus.The enzyme digestion and sequencing results show that the full length of the proviral genome sequence of PERV-A-BM was 8774 bp,with 5′UTR,3′UTR at both ends and ORF in the middle,containing the encoded gen gag,pol and env.The amino acid sequences of the obtained gag and pol have a high homology with those in other PERV subtypes,while the homology in env genes was low.This research has provided a ready eukaryotic expression plasmid of a full-length cDNA for further rescuing PERV.

关 键 词：巴马小型猪 PERV 全基因克隆 序列分析 

分 类 号：S852.65[农业科学—基础兽医学]