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.
出处
《中国兽医科学》
CAS
CSCD
北大核心
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)
