猪圆环病毒2型病毒样颗粒的制备及其免疫原性研究  

Preparation of porcine circovirus type 2 virus-like particles and its immunogenicity research

作　　者：边海桥 黄立平[1] 危艳武[1] 夏德利[1] 朱红振 吴洪丽[1] 于冲 刘建行 冯力[1] 刘长明[1] BIAN Hai-qiao;HUANG Li-ping;WEI Yan-wu;XIA De-li;ZHU Hong-zhen;WU Hong-li;YU Chong;LIU Jian-hang;FENG Li;LIU Chang-ming(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]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150069

出　　处：《中国兽医科学》2021年第8期1007-1014,共8页Chinese Veterinary Science

基　　金：国家自然科学基金面上项目(31873012)。

摘　　要：利用真核细胞表达系统制备了猪圆环病毒2型(PCV2)重组Cap蛋白,旨在探索病毒样颗粒(VLPs)的形成及其免疫原性。以pCAGGS为表达载体构建了重组质粒pCAGGS-Cap-Flag,瞬时转染293T细胞,利用Flag标签对表达的Cap蛋白进行纯化。通过电镜观察重组Cap蛋白形成的VLPs形态表明,纯化的重组Cap蛋白以VLPs形式存在,其直径约17 nm,与自然PCV2病毒粒子一致。SDS-PAGE和Western-blot鉴定表明,重组Cap蛋白为28.7 ku,分子质量符合预期,它与PCV2特异性单抗具有良好的免疫活性反应。捕获ELISA检测显示,重组Cap蛋白所组装的VLPs与纯化的自然PCV2具有相似的抗原活性。用制备的VLPs与经典的PCV2灭活疫苗分别免疫小鼠,这两种疫苗均能诱导良好的PCV2抗体应答反应,VLPs免疫组小鼠的抗体效价最高可达1∶12800,是PCV2灭活疫苗免疫组抗体效价的2倍。采用PCR检测免疫攻毒后小鼠肺组织中PCV2核酸,结果显示,VLPs免疫组和PCV2灭活疫苗免疫组均未检测到PCV2核酸,判定100%保护。本研究通过真核细胞表达系统制备的PCV2 VLPs具有与自然病毒粒子相似的形态结构,免疫小鼠可产生良好的免疫反应,可用于新型PCV2疫苗的研制。Porcine circovirus type 2(PCV2)recombinant Cap protein was expressed using an eukaryotic cell expression system to explore the formation and immunogenicity of virus-like particles(VLPs).The recombinant plasmid p CAGGS-Cap-Flag was constructed with p CAGGS as the expression vector and transfected transiently into 293 T cells,and the expressed Cap protein was then purified by the Flag tag.Observation of the VLPs morphology formed by the recombinant Cap protein by electron microscopy showed that the purified recombinant Cap protein could form VLPs with a diameter of approximately 17 nm,which was consistent with natural PCV2 virus particles.The recombinant Cap protein was 28.7 ku and the molecular weight was in line with expectations by SDS-PAGE.A good immune response of the recombinant Cap protein with PCV2 specific monoclonal antibodies was shown by Western-blot.It was found that the VLPs assembled by the recombinant Cap protein had the similar antigen activity as the purified natural PCV2 by capture ELISA.The prepared VLPs and the classic PCV2 inactivated vaccine were used to immunize mice respectively.Both of the two vaccines could induce a good PCV2 antibody response,and the antibody titers of mice immunized with VLPs was tested by IPMA to reach up to 12800,which was twice the antibody titer of mice immunized with PCV2 inactivated vaccine.The PCV2 nucleic acid in the lung tissue of the mice was tested by PCR after immunization and challenge.The results showed that neither the VLPs immunized group nor the PCV2 inactivated vaccine immunized group had PCV2 nucleic acid detected,and it was judged to be 100%protected.PCV2-VLPs prepared by the eukaryotic cell expression system in this study had a similar morphology to that of natural virus particles and induced a good immune response in mice.It may be used for the development of new PCV2 vaccines.

关 键 词：猪圆环病毒2型 病毒样颗粒 免疫原性 

分 类 号：S852.659.2[农业科学—基础兽医学]