非洲猪瘟病毒CD2v胞外区蛋白的真核表达及其单克隆抗体的制备与功能分析  

Eukaryotic expression of CD2v extracellular domain protein of African swine fever virus and preparation and functional analysis of its monoclonal antibody

作　　者：魏泽良 涂凯 张萌萌 王学军[3] 陈国江 冯健男 王升启 石艳春[1] 王晶 郑源强[1] WEI Ze-liang;TU Kai;ZHANG Meng-meng;WANG Xue-jun;CHEN Guo-jiang;FENG Jian-nan;WANG Sheng-qi;SHI Yan-chun;WANG Jing;ZHENG Yuan-qiang(Inner Mongolia Autonomous Region Key Laboratory of Molecular Biology,Inner Mongolia Medical University,Hohhot 010058,China;Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]内蒙古医科大学内蒙古自治区分子生物学重点实验室,呼和浩特010058 [2]军事科学院军事医学研究院毒物药物研究所,北京100850 [3]军事科学院军事医学研究院微生物流行病研究所,北京100850

出　　处：《军事医学》2023年第5期346-352,共7页Military Medical Sciences

基　　金：北京市自然科学基金资助项目(7222262);国家自然科学基金资助项目(31900676)。

摘　　要：目的利用真核表达系统制备非洲猪瘟病毒(ASFV)CD2v分子N端胞外区融合蛋白,并作为抗原免疫和筛选特异性识别CD2v单克隆抗体。方法以pcDNA3.1-CD2v-HA质粒(含ASFV-BA71v株EP402R基因全长编码序列和HA标签)为模板,扩增CD2v蛋白胞外区碱基序列,构建真核表达载体pcDNA3.1-N-CD2v-His,通过Expi-293表达系统获得重组N-CD2v-His蛋白。将纯化后的蛋白免疫小鼠,利用杂交瘤细胞技术制备特异识别CD2v分子单克隆抗体,对获得的抗体经酶联免疫吸附实验(ELISA)、Western印迹以及间接免疫荧光等方法进行鉴定。结果N-CD2v-His融合蛋白基因被成功构建到真核表达载体上,转染Expi-293F细胞收获重组蛋白,纯蛋白经SDS-PAGE分析发现,N-CD2v-His融合蛋白条带呈高度糖基化修饰的梯状分布。用该融合蛋白免疫小鼠,经杂交瘤细胞筛选,获得2株高亲合活性单克隆抗体,2株抗体经间接ELISA、Western印迹及免疫荧光实验等检测均能特异识别糖基化修饰的CD2v全长蛋白。结论成功制备了抗CD2v特异性单克隆抗体,为进一步开发新型ASFV抗原检测或治疗方法奠定了基础。Objective To obtain the specific monoclonal antibodies targeting the capsid protein CD2v of African swine fever virus(ASFV)by expressing the N-terminal extracellular domain of CD2v with the eukaryotic expression system.Methods The eukaryotic expression vector(pcDNA3.1-N-CD2v-His)of the extracellular segment of CD2v protein was constructed using the CD2v plasmid pcDNA3.1-CD2v-HA as the template(containing full length of EP402R gene and HA Taq in C-terminal,ASFV-BA71v stain).The recombinant N-CD2v-His protein was obtained through the Expi-293 expression system.After immunization with N-CD2v-His,the specific monoclonal antibodies anti-CD2v were obtained using the hybridoma technique.The functions of the antibodies were then analyzed by enzyme-linked immunosorbent assay(ELISA),Western blotting and indirect immunofluorescence.Results The N-CD2v-His protein gene was constructed into the eukaryotic expression vector,and the recombinant protein was harvested after transfection into Expi-293F cells.SDS-PAGE showed that the N-CD2v-His fusion protein bands appeared highly glycosylation-modified and ladder-shaped.After the mice were immunized with the fusion protein,two monoclonal antibodies with high-affinity were obtained by screening hybridoma cells.The antibodies were found to be able to specifically recognize glycosylated CD2v protein in indirect ELISA,Western blotting and immunofluorescence assays.Conclusion Two specific monoclonal antibodies targeting the extracellular domain of CD2v protein have been prepared,which is expected to facilitate the development of novel detection or treatments of ASFV.

关 键 词：非洲猪瘟病毒 单克隆抗体 CD2v蛋白 真核表达系统 EP402R基因 抗体鉴定 

分 类 号：S855.3[农业科学—临床兽医学]