猪流行性腹泻病毒地方株LJB/03分离及培养特性  

Isolation and Characteristics of Virus Culture of Porcine Epidemic Diarrhea Virus LJB/03

作　　者：毛雅元[1] 张桂红[2] 葛俊伟[1] 姜艳平[1] 乔薪瑗[1] 崔文[1] 李一经[1] 

机构地区：[1]东北农业大学动物医学院微生物与免疫学实验室,哈尔滨150030 [2]瑞普(保定)生物药业有限公司,保定071000

出　　处：《病毒学报》2010年第6期483-489,共7页Chinese Journal of Virology

基　　金：黑龙江省十一五重大科技攻关项目(GA06B202-4)

摘　　要：从黑龙江省某猪场疑为病毒性腹泻的发病猪采集腹泻粪便样品,以RT-PCR法扩增出猪流行性腹泻病毒M基因后,采用细胞培养法进行病毒分离。对细胞培养分离物进行间接免疫荧光、免疫电镜观察、RT-PCR及ELISA法检验,其中间接免疫荧光试验可见培养细胞中存在明显的特异性绿色荧光;免疫电镜下可见大小符合预期、有囊膜、花瓣状的典型冠状病毒结构特征;RT-PCR检测证实存在PEDVM基因;间接ELISA检测中平均P/N比值为7.6;从而确认为分离到一株猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV),命名为PEDVLJB/03株。随后,对该分离毒株的培养特性及如何提高病毒滴度进行探索。通过摸索该分离毒株的蚀斑形成条件,建立了PEDV蚀斑形成方法,并采用该方法进行病毒的蚀斑纯化,纯化得到PEDV大蚀斑克隆株和小蚀斑克隆株。对大、小两种蚀斑克隆株的病毒滴度测定结果表明,大小蚀斑克隆株细胞感染滴度相差明显。Porcine epidemic diarrhea virus（PEDV） LJB/03 strain was isolated from the feces of piglets suspected to be suffering from a severe diarrhea in Heilongjiang Province,and was identified by immunofluorescence test,immunelectronmicroscopy,RT-PCR and indirect ELISA assay.Characteristics of the virus culture and the methods of improvement of virus titer were explored.The results showed that the virus had the typical appearance of the coronavirus.Analysis of the nucleotide sequences of RT-PCR products revealed 98% homology with the reference strains.Indirect immunofluorescence assay showed a significant presence of green fluorescence,and an average P/N ratio of 7.6 by indirect ELISA assay.Taken together,these tests showed positive isolation of PEDV.Using the virus plaque purification cloning methods established in the test,the purified PEDV large plaque and small plaque were obtained,and the large plaque and small plaque titers were measured with significant difference.These results provide potential for the application of PEDV on the basis of the biological features of isolated virus.

关 键 词：猪流行性腹泻病毒 分离 培养特性 

分 类 号：S852.65[农业科学—基础兽医学]