鳗鲡疱疹病毒免疫原性蛋白ORF36的鉴定、表达及特征分析  

Identification,Expression,and Characterization Analysis Immunogenic Protein ORF36 of Anguillid herpesvirus

作　　者：陈曦[1] 杨金先[1] 陈华[1] 葛均青[1] CHEN Xi;YANG Jin-Xian;CHEN Hua;GE Jun-Qing(Institute of Biotechnology,Fujian Academy of Agricultural Sciences,Fuzhou 350003,China)

机构地区：[1]福建省农业科学院生物技术研究所,福州350003

出　　处：《农业生物技术学报》2023年第8期1710-1718,共9页Journal of Agricultural Biotechnology

基　　金：福建省公益类科研院所基本科研专项(2021R1027007);福建省科技厅星火项目(2021S0001);福建省农业科学院“5511”协同创新工程建设项目(XTCXGC2021013)。

摘　　要：鳗鲡疱疹病毒(Anguillid herpesvirus,AngHV)是鳗鲡(Anguilla)的一种重要病毒性病原,可引起养殖鳗鲡暴发“脱黏败血综合征”,给养殖者造成巨大的经济损失。开展AngHV的免疫原性蛋白研究对于开发AngHV的免疫学诊断技术及亚单位疫苗具有重要意义。为了获得鳗鲡疱疹病毒的免疫原性蛋白,本研究通过质谱分析AngHV病毒粒子结构蛋白,筛选到ORF36,对其氨基酸序列进行生物信息学分析,克隆至原核表达载体进行诱导表达;用纯化的重组表达蛋白制备兔抗多克隆抗体,评价该抗体检测AngHV的特异性、灵敏性以及病毒中和效果,并对ORF36进行病毒粒子结构定位。结果显示,通过质谱鉴定出病毒的主要免疫原性蛋白ORF36;序列分析显示,ORF36无跨膜结构域,不含有信号肽,具有13个B细胞抗原表位,免疫原性良好;克隆ORF36至原核表达载体pET-30a,实现了其在大肠杆菌(Escherichia coli)BL21(DE3)中的高效表达,重组表达蛋白大小约为40 kD;ELISA检测显示,制备的兔抗ORF36多克隆抗体效价为1∶8000;Western blot结果显示,该抗体能特异性识别感染AngHV的鳗鲡卵巢细胞系(eel ovary cell line,EO)及鳗鲡组织,最低可检测的内脏组织的病毒量为1000 PFU;抗体中和试验表明,制备的兔抗ORF36多克隆抗体具有中和作用,能显著降低AngHV的病毒滴度;ORF36的病毒粒子定位分析表明,ORF36是病毒粒子的结构蛋白且定位于核衣壳上。本研究鉴定出病毒的主要免疫原性蛋白ORF36,制备了具有病毒中和作用的ORF36多克隆抗体,明确了ORF36为AngHV核衣壳结构蛋白,为阐明ORF36在AngHV侵染过程中的作用及开发病毒亚单位疫苗提供了参考。Anguillid herpesvirus(AngHV)is an important viral pathogen of eel(Anguilla),it causes“mucus sloughing and hemorrhagic septicemia disease”on cultured eels,which brought huge economic losses to the farmers.The study of immunogenic protein of AngHV is of great significance for the development of immunological diagnostic technology and subunit vaccine of AngHV.In order to obtain the immunogenic proteins of AngHV,in this study,mass spectrometry analysis on the viron proteins was conducted.The obtained protein ORF36 was further analysed by bioinformatics software.Then the sequence of ORF36 was cloned into expression vector pET-30a,transformed into Escherichia coli BL21,and induced by isopropylβ-Dthiogalactoside(IPTG)for prokaryotic expression.After that,the anti-ORF36 polyclonal antibody was prepared by immunizing rabbits(Oryctolagus cuniculus)using the expressed recombinant protein.The titer of the polyclonal antibody was tested,followed by the specific detection on the prepared antibody by the eel ovary cell line(EO)cells and host tissues infected by AngHV.The sensitivity and neutralization effect of the antibody on AngHV was evaluated,then the virion localization analysis of ORF36 was conducted.According to the results of mass spectrometry,the main immunogenic protein of the virion was identified to be ORF36.Bioinformatics analysis showed that ORF36 protein had no transmembrane domain or signal peptide.Thirteen B cell epitopes were predicted,which indicated that ORF36 had good immunogenicity.ORF36 was cloned into prokaryotic expression vector pET-30a,and induced for protein expression.SDS-PAGE results showed that high level of expressed ORF36 was achieved in E.coli BL21(DE3),with the size of 40 kD and mainly existed in the form of inclusion body.ELISA results showed that the titer of the anti-ORF36 polyclonal antibody was 1:8000.Western blot results showed that the prepared antibody could specifically recognize infected EO cells,as well as the gill,fin and skin mucus tissues of eels infected by AngHV.Sensitivit

关 键 词：鳗鲡疱疹病毒(AngHV) ORF36 原核表达 多克隆抗体 中和作用 定位 

分 类 号：S941.41[农业科学—水产养殖]