猪圆环病毒Ⅱ型新疆分离株全基因组及ORF2基因的克隆与测序

根据GenBank中已发表的猪Ⅱ型圆环病毒(PCV2)全基因组序列,设计合成了2对特异性引物。将PCV2新疆分离株(PCV2-XJ)用PK15细胞培养数代,从细胞培养物中提取病毒总DNA,并取其作为模板,PCR扩增出病毒全基因和结构基因(ORF2)。将PCR回收产物克隆到pMD18-T载体,成功构建了重组质粒pMD18-TPCV2和pMD18-T-ORF2,并对筛选出的阳性质粒进行测序。应用序列分析软件对测序结果分析可知,克隆得到的PCV2-XJ基因组全长1 768 bp。通过序列分析结果显示,PCV2-XJ与国内外PCV1、PCV2参考毒株的核苷酸同源性分别为99.5%～99.7%和68.7%;与HuB08(FJ041151)参考株核苷酸同源性最高(99.7%)。PCV2-XJ株ORF2基因与参考株核苷酸和氨基酸同源性分别高达99.3%和98.8%。

Cloning and Sequence Analysis of Complete Genome and ORF2 Gene of Xinjiang Isolates of Porcine Circovirus Type2

According to the published genome sequences of PCVg,two pairs of specific primer were designed to amplify the full length of PCV2 DNA and ORF2 gene from PK-15 cell line infected with Xinjiang isolate（PCV2-XJ）. The amplified fragments were cloned into pMD18-T vector to obtain the recombinant plasmid pMD18-T-PCV2 and pMD18-T-ORF2,and the positive recombinant plasmids were sequenced. Se- quence analysis showed that the genome of PCV2-XJ consisted of 1 768 bp. The result of sequence analysis showed that the nucleotide homology of reference isolates from PCV2-XJ and PCV1,PCV2 were 99.5 %- 99.7% and 68.7%. The reference isolates nucleotido homology HUB08 strain （FJ041151） were the highest （99.7 %）. The reference isolates nucleotide and amino acid homology of PCV2-XJ strains ORF2 gene were 99.3 % and 98.8 % respectively.