新型高致病性猪繁殖与呼吸综合征病毒弱毒株全长cDNA克隆的构建与分析  

Construction and analysis of a novel full cDNA clone of live-attenuated highly pathogenic porcine reproductive and respiratory syndrome virus strain

作　　者：王松豪[1] 郑海学[1] 杨帆[1] 曹伟军[1] 刘芳[1] 张艳[1] 刘华南[1] 郭建宏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室国家口蹄疫参考实验室,甘肃兰州730046

出　　处：《中国兽医科学》2013年第5期458-465,共8页Chinese Veterinary Science

基　　金：甘肃省高层次人才科技创新创业扶持行动项目(1013JHTA008);公益行业专项(200903055-04;2008FY130100)

摘　　要：为了获得高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)弱毒株的全基因组序列和病毒基因组全长cDNA克隆,根据高致病性猪繁殖与呼吸综合征病毒株(EF635006)和经典CH-1a株(AY032626)全基因组序列设计并合成特异引物,进而用RT-PCR分6段扩增了HP-PRRSV弱毒株的全基因组。将扩增的各个cDNA重叠片段分别克隆到pMD20-T载体中,建立了PRRSV弱毒株的初级cDNA克隆,并对基因组cDNA序列进行了测定。根据测序结果,选择特异的酶切位点,将各个亚克隆产物逐段亚克隆入经过改造的低拷贝pOK12载体中,通过引入MluⅠ和EcoRⅠ酶切位点将聚合酶Ⅱ、Ⅰ和T7启动子序列及榔头锤酶基因置于病毒基因组的5′端,引入NotⅠ和FseⅠ将聚合酶Ⅱ、Ⅰ终止子序列及戊型肝炎病毒核酶基因置于病毒基因组的3′端,结果得到了病毒基因组全长cDNA克隆ppAPRRSVOK12。测序结果表明,该毒株基因组全长15 319bp[不包括poly(A)尾];全基因序列比对结果显示,该毒株属于北美洲型毒株,与HuN4的同源性高达99.5%。测序及酶切鉴定结果证实,HP-PRRSV弱毒株基因组全长cDNA克隆已构建成功。To obtain the genome sequence and full cDNA clone of a live-attenuated highly patho porcine reproductive and respiratory syndrome virus（HP-PRRSV） strain, PCR primers were designe g d enlc ac cording to the sequences of HP-PRRSV HuN4 and classical CH-1a. Six overlapped fragments spanning the full genome were subsequently amplified by RT-PCR. Each PCR product was cloned into pMD20-T vector and then sequenced. The subclone products were cloned into modified pOK12 vector in a proper order by suitable enzyme sites to obtain viral full genomic cDNA clone, named ppAPRRSVOK12. In addition, polymerases Ⅱ , Ⅰ and the T7 RNA polymerase promoter and hammerhead ribozyme fragments were inserted into the upstream of the viral genomie 5r-terminal sequences by introduced restriction enzymes Mlu Ⅰ and EcoR Ⅰ ,and the terminator fragments of polymerases Ⅱ , Ⅰ and hepatitis E virus ribozyme were inserted into the downstream of the viral genomic 3＇-terminal sequences by introduced restriction enzymes Not I and Fse I. The results showed that the viral genome was 15 319 bp in length excluding a poly（A） tail. The whole viral genomic sequence shared 99.5% nucleotide identity with HP-PRRSV HUN4, which belonged to North-American-type PRRSV strain. Sequencing and enzymatic digestion results showed that the viral full genomic eDNA clone was constructed successfully.

关 键 词：高致病性猪繁殖与呼吸综合征病毒 基因组全长cDNA克隆 感染性分子克隆 序列分析 

分 类 号：S852.659.6[农业科学—基础兽医学]