GⅡ.4诺如病毒P结构域单克隆抗体的制备与鉴定  

Preparation and characterization of monoclonal antibodies against GⅡ.4 norovirus P domain

作　　者：侯玉珍 张婷 胡贵方[1] 张绪富 郭伦爱 戴迎春[1] HOU Yuzhen;ZHANG Ting;HU Guifang;ZHANG Xufu;GUO Lunai;DAI Yingchun(Department of Epidemiology,School of Public Health and Tropical Medicine,Southern Medical University,Guangzhou 510515,China;Department of Integrated Traditional Chinese and Western Medicine,School of Traditional Chinese Medicine,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]南方医科大学公共卫生学院流行病学系,广东广州510515 [2]南方医科大学中医药学院中西医结合基础教研室,广东广州510515

出　　处：《细胞与分子免疫学杂志》2020年第8期734-739,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31771007,81773975,81473402);广东省科技创新战略专项资金(2019A1515010951)。

摘　　要：目的通过多个基因簇变异株联合免疫的方式制备抗GⅡ.4型诺如病毒(NoV)P结构域单克隆抗体(mAb)。方法用复合抗原GⅡ.4型NoV/1996(VA387)、2004、2006b和2010基因簇P颗粒免疫BALB/c小鼠,将成功免疫的小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,通过ELISA筛选出阳性杂交瘤细胞,采用有限稀释法制备mAb并进行纯化,ELISA检测mAb效价和特异性,亚类试剂盒鉴定抗体亚类,NoV P颗粒-HBGA结合阻断实验检测mAb中和能力。结果筛选出13株稳定分泌抗GⅡ.4型NoV P颗粒mAb的杂交瘤细胞株,纯化后的13株mAb效价均在10-4以上,属于IgG1型,且均与不同GⅡ.4型NoV变异株具有结合能力;其中5株mAb与GⅡ.17、GⅡ.3和GⅡ.6型NoV具有结合能力,3株mAb与GⅡ.2型NoV具有结合能力且具有人类组织血型抗原(HBGA)受体阻断能力。结论通过NoV多个基因簇变异株联合免疫,成功制备出抗GⅡ.4型NoV P结构域的mAb,为基于P颗粒的GⅡ.4型NoV诊断、疫苗及抗体药物的研发奠定了基础。Objective To prepare monoclonal antibodies(mAbs)against GⅡ.4 norovirus P domain by multiple antigens in an immunization program.Methods BALB/c mice were immunized with the multiple GⅡ.4 NoV P domain,namely 1996cluster(VA387),2004cluster,2006b cluster and 2010 cluster.The spleen cells from the immunized mice were fused with SP2/0 cells and the hybridoma cells were screened by ELISA.The supernatant of the mAbs was collected and purified by the limiting dilution assay.Its subtype was identified,and the specificity and neutralization were analyzed by indirect ELISA and HBGA blocking,respectively.Results We obtained thirteen hybridoma cell lines that stably secreted mAbs against GⅡ.4 NoV P domain.Their titers reached above 10-4 after purification.The subtypes of the mAbs were identified as IgG1.Indirect ELISA showed that all the mAbs specifically bound to all GⅡ.4 norovirus variants.Five mAbs specifically bound to GⅡ.17,GⅡ.3 and GⅡ.6 variants.Three mAbs specifically bound to GⅡ.2 variants and strongly blocked NoV P particle from binding to the histo-blood group antigen(HBGA)receptors.Conclusion The mAbs against GⅡ.4 norovirus P domain have been obtained by combined antigens immunization program.Multi-antigen immunization can enhance immune response significantly and cross-react with other GⅡ.4 norovirus variants.The findings provide a basis for further development of novel GⅡ.4 norovirus vaccines and for the optimization of the immunization programs of combined multi-antigen vaccine candidates.

关 键 词：GⅡ.4诺如病毒 变异株 单克隆抗体(mAb) 联合免疫 

分 类 号：Q813.2[生物学—生物工程] R392.1[医药卫生—免疫学]