猪圆环病毒Ⅱ型Cap蛋白重组腺病毒的构建与鉴定  

Construction and Identification of Recombinant Adenoviruses Containing the Cap Protein of Porcine Circovirus 2

作　　者：王先炜[1] 姜平[1] 李玉峰[1] 蒋文明[1] 汤景元[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点开放实验室,江苏南京210095

出　　处：《中国病毒学》2006年第1期29-33,共5页Virologica Sinica

基　　金：浙江省攻关项目(021102529);国家自然科学基金(30270990);江苏省高新技术项目(BG2002317);教育部博士点基金项目(20030307012);教育部新世纪优秀人才支持计划(NCET-04-0502)

摘　　要：猪圆环病毒Ⅱ型(PCV2)是近年来新发现的引起仔猪多系统衰弱综合症的必需病原,含有两个主要的阅读框ORF1和ORF2,分别编码复制相关蛋白(Rep)和核衣壳蛋白(Cap),其中Cap含有病毒主要抗原表位,是研究PCV2基因工程苗的主要候选目的基因。本研究利用PCR技术将Cap蛋白基因克隆入人5型腺病毒穿梭载体(pShuttle-CMV),与腺病毒骨架载体(pAdEasyTM)共转化大肠杆菌BJ5183进行同源重组并转染HEK-293A细胞,经多次亚克隆获得了重组腺病毒rAd-Cap,测定其TCID50为1013.7/mL。用RT-PCR、间接ELISA、Westernblot和IPMA等方法证明了Cap蛋白在腺病毒中获得表达。该研究为发展PCV2的重组腺病毒基因工程疫苗奠定了基础。Porcine circovirus 2 （PCV2） has been implicated as the etiological agent of a new disease, named postweaning multisystemic wasting syndrome （PMWS）. PCV2 contains two major open reading frames （ORFs）： ORF1 and ORF2, encoding replication-associated （Rep） protein and capsid （Cap） protein, respectively. The cap protein is a candidate 2 for recombinant PCV vaccine, because it possesses several potential antigen epitopes. In the study, the cap gene was amplified by PCR, and cloned into the transfer vector pShuttle-CMV subsequentially. The recombinant plasmid and bone vector pAdEasy-1 were cotransformed into BJ5183 to generate the recombinant plasmid rAd-ORF2 pAd-Cap. The recombinant adenovirus （rAd-Cap） was generated by transfection and plaque purification in in 293 cells. The transcription and expression of Cap protein in rAd-Cap infected 293 cells were confirmed by RT-PCR, indirect-ELISA, Western blot and IPMA. The study laid the foundation for development of the recombinant vaccine against PCV2.

关 键 词：Ⅱ型猪圆环病毒 CAP蛋白 重组 腺病毒 构建 

分 类 号：S852.65[农业科学—基础兽医学]