青海牦牛病毒性腹泻病毒的分离与鉴定  

Isolation and identification of a bovine viral diarrhea virus isolate from yak in Qinghai Province

作　　者：张芳芳[1,2] 王光华[1] 郑福英[1] 宫晓炜[1] 周继章[1] 吴润[2] 邱昌庆[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部兽医公共卫生重点开放实验室,甘肃兰州730046 [2]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《中国兽医科学》2011年第11期1101-1105,共5页Chinese Veterinary Science

基　　金：科技部基础工作专项(2008FY210200)

摘　　要：采集青海省泽库县某牧户疑似牦牛病毒性腹泻病牛的样品,将RT-PCR检测为阳性的样品处理后接种MDBK细胞,并盲传至第10代,检测每一代细胞中牛病毒性腹泻病毒的E0基因。通过分离株接种细胞后产生的病变效应观察、免疫荧光试验、电镜观察、RT-PCR扩增以及序列分析鉴定了该病毒。结果表明,盲传的每代细胞中均可检测到E0基因;接毒后的细胞在盲传至第7代时出现明显的细胞病变;免疫荧光试验中能观察到细胞内发出的特异性荧光;经浓缩、纯化的病毒在电镜下呈直径为40～60nm的病毒粒子,将其命名为QHZK株。对克隆的E0基因测序后提交至GenBank(登录号:JF927789),并将获得的序列与其他国内外分离株的E0序列比对,同源性为73.6%～98.2%;系统进化分析表明该分离株属于牛病毒性腹泻病毒1b亚型。Samples were collected from yak with suspected bovine viral diarrhea on a yak farm in Zeku County,Qinghai Province.MDBK cell lines were inoculated with RT-PCR positive specimens,followed by blind passage culture for 10 generations,and the E0 gene of bovine viral diarrhea virus（BVDV） was detected from the cell cultures of each generation.The isolates were cultured in MDBK cell lines,and identified by cytopathic effect（CPE）,direct immunofluorescence assay（DIFA）,electron microscopy（EM）,RT-PCR and sequence analysis. The results indicated that the E0 gene was detected from the cell cultures of each generation,and typical CPE was observed from cell cultures of the seventh generation.Specific fluorescence in cell cultures can be see in DIFA.Virions of 40-60nm were observed under EM,and was named QHZK strain.The obtained E0 gene sequence was submitted to GenBank with the accession number：JF927789.Alignment with other 9 strains of BVDV available in GenBank showed a homology of 73.6%-98.2% for nucleotide sequence.The phylogenetic analysis indicated that the isolated strain belonged to BVDV 1b.

关 键 词：牛病毒性腹泻病毒 E0基因 分离 鉴定 

分 类 号：S852.659.6[农业科学—基础兽医学]