猪圆环病毒2型的分离鉴定及全基因组克隆和序列分析

通过PCR方法对采自四川省某规模化猪场一批疑似断奶仔猪多系统衰弱综合征病例的肺脏、淋巴结进行PCV-2的抗原检测,并将检测结果为阳性的对应组织研磨、过滤除菌后接种无PCV-1污染的PK-15细胞,盲传15代后,IFA方法检测出有明显的特异性荧光,将该毒株命名为PCV-2-SC株,并对PCV-2-SC病毒基因组全长进行扩增及克隆、测序与比对分析.测序结果显示该病毒基因组全长1 767 bp,分离株与国内外参考毒株核苷酸序列相似性在95.2％～99.5％之间,进化树分析表明,PCV-2分离毒株在进化上存在地域相关性.PCV-2四川株的分离鉴定为其诊断试剂的研制及检测方法的建立奠定了基础.

Isolation, Identification, Genetic Cloning and Sequence Analysis of PCV-2

The lungs and lymph nodes collected from the pigs with typical PMWS in a commercial pig-farm of Sichuan were detected by PCR assay.The positive lungs and lymph nodes were homogenized and filtered,and the suspension was inoculated in a layer of PK-l5cells in absence of PCV-l through 15passages.Specific fluorescence appared clearly in the infected cells via IFA assay,and the isolated strain was named as PCV-2-SC.Meanwhile,the full length of viral genome was amplified with a pair of specific primers by PCR and cloned into DH5α,and the recombinant plasmid was sequenced and analyzed.Sequence analysis showed that its complete genome was composed of 1 767nucleotides.The similarity of nucleotide sequence in PCV-2-SC strain shared 95.2%-99.5%in comparison with that of others.And the phylogenetic tree analysis showed that,the geographic factor has an influence on evolution of PCV-2strains.This work will lay a foundation to further research for diagnostic agent and detective methods of PCV-2.