16株猪2型圆环病毒全基因组克隆及序列分析

Complete Genomic Cloning and Sequence Analysis of PCV2 of Sixteen Strains

采用猪2型圆环病毒(PCV2)型特异性引物,通过PCR方法直接从疑似PMWS病猪肺、淋巴结病料中扩增出16株PCV2的全基因组序列,将扩增产物回收连接到pMD19-T,转化到DH5α后进行克隆测序。结果显示:16株PCV2中,有12株基因组全长为1767bp,1株全长为1766bp,3株PCV2的基因组全长为1778bp。同源性分析结果表明,16株PCV2之间的核苷酸同源性为95.7%～100%,与GenBank已发表的9株PCV2分离株基因组同源性为94.3%～99.9%,与2株PCV1分离株基因组的同源性为76.6%～77.3%;遗传进化树显示16株PCV2主要分为2种基因型,其中134-ZF1、134-ZF2、134-ZF3、141-ZF1、141-ZF2、141-ZF3、142-TL1、142-TL2、142-TL3、142-TL4、142-TL5和148-ZF3这12株与AF055394(法国株)同属于PCV2b亚型,亲缘关系较近;133-ZF1、143-2-15、148-ZF1和WJQ001与AY181946(中国株)同属于PCV2d亚型,组成一个分支。所得的16株PCV2的ORF2核苷酸及其所推导的氨基酸序列与参考株同源性分别为90.2%～99.9%和92.1%～100.0%,存在较大的变异。

According to the sequences of porcine circovirus type 2（PCV2） published in GenBank,the specific primers were designed to amplify the complete genome of sixteen PCV2strains from lung,and lymph nodes of suspected PMWS pigs by PCR,and the amplified products were recovered and connected to pMD19-T,transformed to DH5αand cloned and sequencing.The results showed that the complete genome of twelve out of the sixteen strain all 1767bp in length,one strains was 1766bp,and three strains were 1778bp.The homology of nucleotide sequences of the sixteen strains was 95.7%～100%.Compared with nine PCV2in Genbank,the homology was 94.8%～99.9%,and compared with two PCV1in Genbank,the homology was 76.6%～77.3%.Phylogenetic tree analysis showed that the sixteen strains could be divided into two genotypes,12strains（134ZF1,134-ZF2,134-ZF3,141-ZF1,141-ZF2,141-ZF3,142-TL1,142-TL2,142-TL3,142-TL4,142-TL5and 148-ZF3） were close to the representative strain of France（AF055394） and belong to PCV2b;4strains（133-ZF1 、 143-2-15 、 148-ZF1,WJQ001） were close to the representative strains of China（AY181946）,forming a branch PCV2.The ORF2of the sixteen strain had 90.2%～ 99.9% nucleotide sequence identity and 92.1%～100.0%amino acid identity.