柯萨奇病毒A6病毒样颗粒的制备及免疫原性初步研究  

Preparation and Humoral Immunogenicity Analysis of Coxsackievirus A6 Virus-Like Particles

作　　者：陈静[1] 李波[2] 李鹏飞[3] 陈易 吴杰[3] 王泽鋆[3] 孟胜利[3] 申硕[3] CHEN Jing;LI Bo;LI Pengfei;CHEN Yi;WU Jie;WANG Zejun;MENG Shengli;SHEN Shuo(Medical College,China Three Gorges University,Yichang 443002,China;Affiliated Renhe Hospital of China Three Gorges University,Yichang 443003,China;Viral Vaccine Research Laboratory,Wuhan Institute of Biological Products,Wuhan 430207,China)

机构地区：[1]三峡大学医学院,宜昌443002 [2]三峡大学附属仁和医院,宜昌443003 [3]武汉生物制品研究所病毒性疫苗研究一室,武汉430207

出　　处：《病毒学报》2020年第6期1067-1074,共8页Chinese Journal of Virology

基　　金：湖北省知识创新专项(项目号:2019CFC865),题目:柯萨奇病毒A6病毒样颗粒的免疫原性及保护性研究;湖北省技术创新专项重大项目(项目号:141),题目:手足口病双价灭活疫苗的研制;国家“重大新药创制”科技重大专项(项目号:2015ZX09102021),题目:人肠道病毒71型及柯萨奇病毒A16型双价灭活疫苗的研制;三峡大学人才专项经费(项目号:20171227)。

摘　　要：手足口病(Hand,foot,and mouth disease,HFMD)是一种常见传染病,以婴幼儿发病为主,柯萨奇病毒A组6型(Coxsackievirus A6,CV-A6)是引起HFMD暴发的主要病原体,其疫苗开发有待深入的研究,而病毒样颗粒(Virus-like particles,VLPs)是潜在安全的候选疫苗。为制备CV-A6 VLPs,并研究其免疫原性,本研究利用Bac-toBac昆虫细胞杆状病毒表达系统共表达P1和3CD蛋白,制备并纯化CV-A6 VLPs,应用间接免疫荧光试验(Indirect immunofluscent assay,IFA)、SDS-PAGE、Western Blot、透射电镜法等方法鉴定。以Al(OH)3为佐剂,三种不同剂量CV-A6 VLPs免疫BALB/c小鼠,采用微量中和试验法检测小鼠血清中和抗体滴度。结果显示,重组有P1和3CD的杆状病毒质粒转染Sf9细胞,可形成CV-A6类病毒颗粒,直径约为26nm。SDS-PAGE和Western Blot结果说明P1前体蛋白被3CD作用切割产生了VP0、VP1和VP3,CV-A6 VLPs由这三个病毒结构蛋白构成。小鼠免疫实验结果显示,CV-A6 VLPs可以刺激产生较高水平抗CV-A6的特异性中和抗体,维持至少12周,并且抗体中和效价与免疫剂量和免疫次数呈正相关。本研究获得重组杆状病毒rBac-CV-A6-P1-3CD,并在Sf9细胞内成功表达CV-A6 VLPs,可以刺激小鼠产生较高水平和持久的体液免疫反应。Hand,foot,and mouth disease(HFMD)is a common infectious disease,which mostly affects young children.Coxsackievirus A6(CV-A6)is one of the main pathogens causing HFMD outbreaks.The related vaccine needs more research,and virus-like particles(VLPs)are the potentially safe vaccine candidates.To prepare purified CV-A6 VLPs and study its humoral immunogenicity,insect-baculovirus expression system was applied in this research.Recombinant baculovirus plasmid containing CV-A6 P1 and 3 CD genes were used to transfect Sf9 insect cells,and a recombinant baculovirus was obtained.CV-A6 VLPs were prepared and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),Western Blot and transmission electron microscopy(TEM).Mice were immunized with purified VLPs and the humoral immune response was identified by neutralization assays.SDS-PAGE,Western Blot showed that P1 protein was cleaved by 3 CD to produce VP0,VP1 and VP3,and formed the recombinant CV-A6 VLPs.Spherical particles of about 26 nm in diameter were seen under electron microscope.Mice immunized with purified VLPs could elicit CV-A6-specific antibodies in blood.Moreover,the titers of neutralizing antibody were increased in a dose-dependent manner and maintained at the same level in blood for≥12 weeks.The present study suggested that CV-A6 VLPs were produced through insect-baculovirus expression system which consisted of VP0,VP1 and VP3.The VLPs could elicit CV-A6-specific humoral immunogenicity in mice and maintained it for≥12 weeks.

关 键 词：柯萨奇病毒A组6型(CV-A6) 病毒样颗粒(VLPs) 昆虫细胞杆状病毒表达系统 

分 类 号：R373.2[医药卫生—病原生物学]