中和表位串联的PCV2b病毒样颗粒在昆虫细胞中的表达与鉴定  

Expression and Identification of PCV2b Virus-Like Particles with Neutralizing Epitopes in Tandem in Insect Cells

作　　者：任春晓 冯华[1] 张腾[1,3] 蒋敏 刘运超 金前跃[1] 张改平[1,2] REN Chunxiao;FENG Hua;ZHANG Teng;JIANG Min;LIU Yunchao;JIN Qianyue;ZHANG Gaiping(Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;College of Life Sciences,Henan Agricultural University,Zhengzhou 450002,China;School of Veterinary Medicine,Northwest A&F University,Yangling 712000,China;College of Life Sciences,Zhengzhou University,Zhengzhou 450001,China)

机构地区：[1]河南省农业科学院动物免疫学重点实验室,河南郑州450002 [2]河南农业大学生命科学学院,河南郑州450002 [3]西北农林科技大学动物医学院,陕西杨凌712000 [4]郑州大学生命科学学院,河南郑州450001

出　　处：《河南农业科学》2021年第7期154-160,共7页Journal of Henan Agricultural Sciences

基　　金：河南省农业科学院自主创新专项(2020ZC55);。

摘　　要：为了研究一种高效的预防猪圆环病毒2b亚型(PCV2b)感染的亚单位疫苗,针对其主要免疫原Cap蛋白的基因进行分析,将Cap蛋白上重要中和表位226LKDPPLNP233以重复串联的方式连接于cap基因C端(capE2),并构建于pFastBacTMI载体上,借助昆虫杆状病毒表达系统进行表达,利用SDS-PAGE、Western blot分析和动态光散射及透射电镜观察等对重组蛋白CapE2进行鉴定。将纯化的CapE2蛋白免疫小鼠,同时以未连接中和表位的Cap蛋白、PCV2亚单位疫苗和PBS为对照,收集免疫血清分析目的重组蛋白的免疫原性以及连接中和表位对Cap蛋白免疫原性的影响。结果表明,Cap及CapE2蛋白成功在sf21细胞内表达;纯化后的目的蛋白能够有效地与PCV2单克隆抗体产生特异性反应,并且可以在体外自组装形成均一的、直径约17 nm的病毒样颗粒;血清抗体检测结果显示,在第2次免疫后,CapE2免疫组抗体水平迅速提高,并在3免之后显著高于其他免疫组;中和抗体测定结果表明,Cap及CapE2组血清抗体效价均显著高于疫苗免疫对照组,且CapE2免疫组血清的中和效价可达1:29。通过昆虫杆状病毒表达系统表达的连接2个中和表位的Cap蛋白,能自组装形成病毒样颗粒,并且具有较好的免疫原性,为PCV2感染的预防提供了新的疫苗候选。In order to develop a subunit vaccine that is more efficient to prevent porcine circovirus 2 b subtype(PCV2 b)infection,the important neutralizing epitope 226LKDPPLNP233 on the Cap protein was connected to the C-terminal of the cap gene(capE2)in a repeated tandem manner,and the corresponding recombinant proteins were expressed in insect baculovirus expression system.The recombinant proteins CapE2 were identified by means of SDS-PAGE,Western blot,dynamic light scattering and transmission electron microscopy observations.Then mice were immunized with purified CapE2 protein,and Cap without the neutralizing epitope,PCV2 subunit vaccine,and PBS were used as controls.Tthe serums post immunization were collected for analyzing the immunogenicity of these target recombinant proteins and the effect of linking neutralizing epitopes on the immunogenicity of Cap protein.The results showed that Cap and CapE2 proteins were successfully expressed in sf21 cells.These purified target proteins could effectively react specifically with the PCV2 monoclonal antibody,and could self-assemble to form uniform virus-like particles in vitro with a diameter of about 17 nm.Serums antibody test results showed that after the second immunization,the antibody level of CapE2 group increased rapidly,and was significantly higher than other immunization groups after the third immunization.The neutralizing antibody test results showed that the serum antibody titres of the Cap and CapE2 groups were significantly higher than the positive vaccine group,and the neutralization titer of the serum in the CapE2 immunization group could reach up to 1:29.The recombinant Cap proteins that were connected to two repeating neutralizing epitopes were successfully expressed in insect baculovirus expression system and could self-assemble to form virus-like particles,which had good immunogenicity and provided a new vaccine candidate for the prevention of PCV2 infection.

关 键 词：猪圆环病毒2型 CAP蛋白 病毒样颗粒 免疫原性 亚单位疫苗 

分 类 号：S855.3[农业科学—临床兽医学]