杆状病毒-昆虫细胞系统表达的戊型肝炎病毒衣壳蛋白p495的纯化、结构解析以及免疫原性分析

Expression,Purification,Structure Determination and Immunogenicity Assay of Hepatitis E Virus Capsid Protein p495 Derived from Baculovirus-based Insect Cell

本研究目的是利用杆状病毒-昆虫细胞表达系统建立戊型肝炎病毒(HEV)p495蛋白的高效稳定表达和可放大纯化的方法,研究其理化性质和解析三维结构,与上市戊肝疫苗益可宁(Hecolin)进行免疫原性的比较。本研究选择基因1型HEV毒株的开放读码框2(ORF2)的aa112-606片段构建杆状病毒,感染昆虫细胞进行重组蛋白的表达,经过PEG沉淀和阴离子交换层析的纯化,获得纯度为95%以上的p495蛋白,每升细胞培养液最终获得15mg的目的蛋白。经ELISA、分析超离、分子排阻色谱和电镜负染等分析显示p495蛋白具有良好的抗原性且呈现为均一的病毒样颗粒(VLP),沉降系数为51.3S,颗粒直径约20nm。冷冻电镜三维结构解析获得分辨率为3.8的三维结构,揭示p495形成了T=1的等二十面体结构,与已报道的晶体结构相一致。小鼠免疫实验表明p495蛋白与益可宁中p239抗原的免疫原性相当。本研究为进一步开展HEV的受体鉴定、表位结构研究以及疫苗改进等奠定良好的基础。

Our objective was to establish a robust method for the expression and purification of hepatitis E virus（HEV）p495protein using a baculovirus-based insect cell expression system;to determine the properties and cryo-EM structure of the resulting virus-like particles（VLPs）;and to compare their immunogenicity with p239 particles in the commercial hepatitis E vaccine（Hecolin）.The sequence spanning HEV ORF2 amino acids 112-606 in the genotype I HEV isolate was cloned into baculovirus to express recombinant p495 protein.ELISA,analytical ultracentrifugation,size-exclusion chromatography and negativestaining transmission electron microscopy（TEM）were carried out to characterize the physicochemical properties of p495.Recombinant p495 VLPs were obtained successfully from the insect cell expression system with purity of〉95%and yield of 15mg/L.The recombinant HEV p495 protein was homogeneous in solutions.The 3Dstructure of p495 VLPs was determined by cryo-EM;it was icosahedral with T=1arrangement,and showed good congruency with the crystal structure in the literature（PDB ID：2ZZQ）.In mouse vaccination experiments,p495 conferred comparable immunogenicity with that of p239 antigen in Hecolin.Thus,a robust and scalable approach to obtain homogeneous,immunogenic HEV p495 VLPs has been established.This study may assist investigations of HEV receptors,epitope mapping,vaccine improvement and so on.