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猪圆环病毒2型细胞培养适应毒株的培育和鉴定
引用本文:刘长明,张超范,危艳武,谭斌,陆月华,谷守林.猪圆环病毒2型细胞培养适应毒株的培育和鉴定[J].中国预防兽医学报,2006,28(3):248-252.
作者姓名:刘长明  张超范  危艳武  谭斌  陆月华  谷守林
作者单位:中国农业科学院哈尔滨兽医研究所,兽医生物技术国家重点实验室,黑龙江,哈尔滨,150001
摘    要:从临床表现为仔猪断奶后多系统衰竭综合征(PMWS)淋巴组织病料,经聚合酶链式反应(PCR)证实为猪圆环病毒2型(PCV2)感染,采用无污染的猪肾细胞系(PK15)分离培养,并连续传代培育成一株细胞培养适应毒,命名为PCV2/LG株。分离毒株经细胞培养,于第25代后毒价显著升高,于第35代毒价可达10^5.6TCID 50/mL。采用免疫过氧化物酶单层细胞染色法(IPMA)、免疫电镜技术、分子克隆及核酸序列分析等鉴定表明,分离株感染细胞后病毒抗原主要分布在细胞核及细胞质中;病毒感染的阳性细胞呈散在分布,阳性细胞数可达50%以上;免疫电镜观察到与PCV2特异抗体结合形成的病毒免疫复合物呈实心小颗粒样粒子团,病毒粒子直径约为17nm;病毒抗原基因组由1768个核苷酸组成,与GenBank登录的8个PCV2基因组序列同源性达96.2%以上。用2mL的病毒细胞培养物(10^5.6TCID 50/mL)接种30日龄PCV2抗体阴性仔猪3头,可引起典型PMWS临床症状。本研究为进一步开展该病毒的致病性、疫苗免疫、诊断及分子生物学等研究奠定了基础。

关 键 词:猪圆环病毒2型  病毒分离鉴定  毒株培育
文章编号:1008-0589(2006)03-0248-05
收稿时间:2005-03-21
修稿时间:2005年3月21日

Cultivation and identification of porcine circovirus type 2(PCV2)cell culture-adapted strain
LIU Chang-ming,ZHANG Chao-fan,WEI Yan-wu,TAN Bin,LU Yue-hua,GU Shou-lin.Cultivation and identification of porcine circovirus type 2(PCV2)cell culture-adapted strain[J].Chinese Journal of Preventive Veterinary Medicine,2006,28(3):248-252.
Authors:LIU Chang-ming  ZHANG Chao-fan  WEI Yan-wu  TAN Bin  LU Yue-hua  GU Shou-lin
Institution:National Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China
Abstract:A porcine circovirus type 2(PCV2) was isolated from a piglet lymph node material with postweaning multsystemic wasting syndrome(PMWS) using PCV1-free pig kidney cell line(PK15).The isolate was cultivated by a series of passages in the cells,and a cell culture-adapted strain was obtained,which was termed as PCV2/LG.The virus titer of PCV2/LG increased markedly after the 25 th passage,and harvested 105.6 TCID50/mL at the 35th passage.The virus antigen distributed mai-nly in the infected cell nucleolus and cytoplasm,the antigen positive cells showed spot distribution and accounted for more than 50 % by immunoperoxidase monolayer assay(IPMA).The immune complex formed by the virus particles with anti-PCV2 antibodies was observed by electron microscope,which showed multiple sold virus particles.The diameter of single particle was measured about 17 nm.The virus genome sequence was determined to be 1 768 nucleotides,more than 96.2 % identical rate in comparison with other eight PCV2 sequences of GenBank.Three of 30-days-old piglets were inoculated with PCV2/LG culture liquid with 2 mL dose of 105.6 TCID50/mL,these piglets showed typical PMWS.The study establishes a base for further research on the virus pathogenicity,vaccination,diagnosis and molecular biology.
Keywords:porcine circovirus type 2  virus isolation and identification  seed virus cultivation
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