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

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.