水貂源犬瘟热病毒的分离及鉴定  

Isolation and identification of canine distemper virus from mink

作　　者：王小龙 王承宝[2] 王雅文[1] 陈杰[1] 闫喜军 赵建军[1] WANG Xiaolong;WANG Chengbao;WANG Yawen;CHENG Ji;YAN Xijun;ZHAO Jianjun(Research Institute of Specialty,Chinese Academy of Agricultural Sciences,Changehun 130112,China;North West Agriculture and Forestry University,College of Veterinary Medicine,Yangling 712100.china)

机构地区：[1]中国农业科学院特产研究所,长春130112 [2]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《黑龙江畜牧兽医》2018年第24期194-197,264,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金青年科学基金项目(31502102)

摘　　要：为了研究当前犬瘟热的流行毒株,试验选取来自辽宁省大连市疑似犬瘟热发病死亡水貂的肺脏、脾脏、肾脏等组织,将研磨匀浆后的上清液接种于稳定表达犬瘟热SLAM受体的非洲猕猴肾细胞系-Vero/DogSLAM(VDS),通过病料的处理、VDS细胞的复苏与培养、病毒的分离与培养、病毒TCID50的测定、反转录-聚合酶链式反应(RT-PCR)的方法检测感染细胞中的病毒核酸,用序列比对的方法与已发表的犬瘟热病毒H蛋白(CDV-H)和SLAM受体结合区(RBS)序列进行比对分析,用CDV-H蛋白间接免疫荧光(IFA)的方法检测荧光蛋白表达。结果表明:在Vero/DogSLAM(VDS)细胞上接种病毒培养24h后,80%~90%的VDS细胞出现合胞体病变(CPE),分离到的病毒TCID50达到1×10^5.23/(100μL),同时检测的CDV-H和SLAM受体结合区(RBS)序列比对分析一致,同源性为98%以上,在细胞质中可观察到抗CDV-H蛋白的特异性绿色荧光,RT-PCR成功扩增出H基。因部分片段。说明本试验成功分离并鉴定出1株犬瘟热毒株,将其命名为LNDL(17)M4株。In order to investigate the current CD epidemic strains,epidemic samples of lung,spleen,kidney and other tissues were collected from the susceptible diseased minks in Dalian and Liaoning Province.The supernatant of the grinding homogenate was inoculated into the African macaque kidney cell line in which the SLAM receptor-Vero/DogSLAM(VDS)of canine distemper can be stably expressed.Then the viral nucleic acid of infected cells was detected by the pathological material treatment and the methods of resuscitation and culture of VDS cells,virus isolation and culture,determination of virus TCID50,reverse transcription-polymerase chain reaction (RT-PCR).Also,sequence alignment method was used to compare the identified sequences with the published H protein (CDV-H)and SLAM receptor binding domain (RBS)sequences of canine distemper virus.In addition,fluorescent protein expression was detected by indirect immunofluoreseence (IFA).The results showed that after 24 hours' culture of transfeeted Vero/DogSLAM (VDS)cells,syncytial lesions (CPE)was observed in 80% to 90% VDS cells.TCID50 of isolated virus reached to 1×10^5.23/(100μL).Meanwhile,the sequence alignment analysis of CDV-H and SLAM receptor binding domain (RBS)was consistent,with a homology of more than 98%.Furthermore,specific green fluorescence of CDV-H protein was observed in cytoplasm,and partial fragments of H gene were successfully amplified by RT-PCR.The results indicated that one strains of canine distemper virus was successfully isolated and identified,and named as LNDL(17)M4-VDS strain.

关 键 词：犬瘟热 犬瘟热病毒 病毒分离 非洲猕猴肾细胞系-Vero/DogSLAM(VDS)细胞 间接免疫荧光 反转录-聚合酶链式反应(RT-PCR) 

分 类 号：S852.659.5[农业科学—基础兽医学] S865.22[农业科学—兽医学]