Ⅱ型圆环病毒山东泰安株的分离鉴定及系统进化分析  

ISOLATION AND IDENTIFICATION OF PCV2SDT AND PHYLOGENETIC ANALYSIS

作　　者：娄忠子[1] 王天户[1] 董李民 刘思当[1] 

机构地区：[1]山东农业大学动物科技学院基础兽医系,山东泰安271018 [2]莱阳市河洛镇兽医站,山东莱阳265200

出　　处：《山东农业大学学报（自然科学版）》2007年第4期509-514,共6页Journal of Shandong Agricultural University：Natural Science Edition

摘　　要：采用双抗体夹心ELISA方法和PCR技术对来自山东泰安的病猪组织病料进行猪Ⅱ型圆环病毒（PCV2）检测。将检测为阳性的病料悬液接种无PCV1污染的PK-15细胞进行病毒分离,获得一株PCV2,命名为猪Ⅱ型圆环病毒山东泰安株（PCV2 sdt）。间接免疫荧光试验（IFA）检测可见接种病毒细胞中散在特异性荧光。提取全基因组为模板,进行病毒基因组1号开放阅读框（ORF1）、2号开放阅读框（ORF2）和全基因组序列扩增,得到长度分别约为999bp、749bp和1767bp左右的产物条带。将产物克隆到pMD18-T载体上,构建重组质粒pMD18-T-ORF1、pMD18-T-ORF2和pMD18-T-PCV2。测序结果显示,所分离病毒的ORF1、ORF2和全基因组序列长度分别为945bp、702bp和1767bp。DNAStar生物学软件分析发现,PCV2分离毒株与参考株的全基因组同源性介于95.4%-98.8%之间,ORF1的同源性介于97.0%-98.8%,ORF2的同源性略低,介于91.9%-98.4%之间。With the help of antigen-capture enzyme-linked immunosorbent assay and polymerase chain reaction,different tissues of ailing piglets, which were from Taian Shandong province,were detected for porcine circovirus type 2（PCV2）.The positive samples were treated and then inoculated PK-15 which was not polluted by PCV1 to isolate PCV2.Successfully a strain of PCV2 named PCV2sdt was obtained.After the inoculation of virus sample,indirect fluorescent antibody（IFA） method was used to find the effect,and fluorescence in the cells was detected.DNA templates from the cells were extracted and used to amplify ORF1,ORF2 and the whole genomic sequence,DNA bands of amplification products about 999bp,749bp and 1767bp were obtained.The products were cloned to pMD18-T vector to construct recombinant plasmid pMD18-T-ORF1,pMD18-T-ORF2 and pMD18-T-PCV2.Sequencing results showed the sequence length of ORF1,ORF2 and the whole genomic length of the virus isolated were 945bp,702bp and 1767bp respectively.Compared with the reference strains of PCV2,the homology of genome is about 95.4%～98.8%,ORF1 is about 97.0%～98.8% and ORF2 is about 91.9%～98.4%.

关 键 词：猪Ⅱ型圆环病毒 双抗体夹心ELISA PCR 病毒分离 间接免疫荧光试验 

分 类 号：S852.651[农业科学—基础兽医学]