猪圆环病毒2d亚型病毒样颗粒的制备与鉴定  

Preparation and identification of porcine circovirus type 2d Cap protein as virus-like particles in insect cells

作　　者：侯强[1] 侯绍华[1] 贾红[1] 姜一曈[1] 鑫婷[1] 李明[1] 陈青[2] 朱鸿飞[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]北京农学院农业部都市农业(北方)重点实验室,北京102206

出　　处：《中国兽医科学》2017年第11期1385-1391,共7页Chinese Veterinary Science

基　　金：国家重点发展计划项目(2017YFD0502302);北京市教育委员会科技计划面上项目(KM201410020002);中国农业科学院科技创新工程(ASTIP-IAS-11)

摘　　要：本研究从临床病料中扩增获得猪圆环病毒2型(PCV2)Cap基因,并进行序列分析和抗原性比对,然后利用杆状病毒表达系统在昆虫细胞内将Cap基因进行表达,目的蛋白进行Western-blot、IFA、质谱鉴定以及透射电镜和免疫电镜检测。结果表明,扩增获得的PCV2 Cap基因属于PCV2d亚型,在抗原性方面与国内PCV2a、PCV2b代表性毒株及疫苗株存在4个部位的差异;在昆虫细胞内PCV2d Cap蛋白成功表达并具有良好的免疫原性;PCV2d Cap蛋白在昆虫细胞内完成自组装,形成与天然病毒结构相似的病毒样颗粒(VLP)。本研究首次在昆虫细胞内制备出PCV2d亚型病毒样颗粒,为PCV2d亚单位疫苗研制及诊断抗原开发奠定了基础。The capsid protein（Cap） comprises the major structural protein of porcine circovirus type 2（PCV2）.In this study,a full-length of Cap gene was cloned and sequenced for phylogenic analysis of PCV2 strain and antigenicity analysis of Cap protein,and then Cap gene was expressed via Bac to Bac expression system in insect cells.The target protein was identified by Western blot,IFA,MS,EM and Immuno-EM methods in following work.It was shown that the cloned Cap gene was classified into a PCV2d subtype gene cluster as the reference strains through phylogenic analysis.By antigenicity analysis,four domains were found to be different with those from domestic PCV2a,PCV2b and PCV2 vaccine strains.By the Bac to bac expression system,PCV2d Cap protein was highly expressed in insect cells.The target protein was identified with anti-Cap monoclonal antibody by WB and analyzed by MS method.Antigenicity was analyzed with PCV2 positive serum through IFA assay.Finally,the target protein was checked as virus-like particles Via electron microscopy and immuno-electron microscopy.All together,PCV2 d VLP was successfully prepared and identified in insect cells and had good immunogenicity as a PCV2d sub-unit vaccine candidate or diagnostic reagent in future work.

关 键 词：猪圆环病毒2d亚型 核衣壳蛋白 杆状病毒表达系统 病毒样颗粒 

分 类 号：S852.66[农业科学—基础兽医学]