猪丹毒杆菌SpaA单克隆抗体的制备及抗原表位鉴定  

Monoclonal antibody against SpaA protein of Erysipelothrix rhusiopathiae and its antigenic epitope identification

作　　者：黄雅琴 蔡金双 缪芬芳 李玉峰[1] HUANG Yaqin;CAI Jinshuang;MIAO Fenfang;LI Yufeng(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Jiangsu Nannong Hi-Tech Co.,Ltd.,Wuxi 214405,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]江苏南农高科技股份有限公司,江苏无锡214405

出　　处：《畜牧与兽医》2024年第9期84-90,共7页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发项目(2022YFD1800902);江苏南农高科技股份有限公司项目(090HMQY21042)。

摘　　要：旨在制备猪丹毒杆菌单克隆抗体,并确定其识别的抗原表位。采用原核表达、细胞融合试验、间接ELISA、亚克隆技术、Western blot、抗原表位以及抗体分型鉴定来研究猪丹毒杆菌表面保护性抗原A(SpaA)的单克隆抗体。结果:制备了SpaA重组蛋白,得到4株稳定分泌的单抗,且这4株单抗均能与SpaA蛋白发生特异性反应,其中1E8、5B3杂交瘤细胞培养上清液抗体效价为1∶1600,5B5、9B5杂交瘤细胞培养上清液抗体效价为1∶3200;1E8重链为IgG 2b,5B3、9B5重链为IgG 1,5B5重链为IgG 2a,轻链均为Kappa;1E8、5B5识别的抗原表位因SpaA蛋白C端的重复序列无法确认,5B3识别的抗原表位为258~267 aa,9B5识别的抗原表位为462~472 aa。综上,本研究制备的单克隆抗体将为猪丹毒杆菌ELISA检测方法的建立奠定基础。This study was to prepare monoclonal antibody against protein of Erysipelothrix rhusiopathiae and identify its antigentic epitope.The SpaA gene was cloned into the prokaryotic expression vector pCold I to construct the plasmid pCold I-SpaA.The recombinant SpaA protein was successfully expressed and purified by inducing Rosetta-engineered bacteria.Six to eight week BALB/c mice were immunized with the purified SpaA protein,and four hybridoma cell clones were stably screened after cell fusion using the sub-cloning technique and indirect ELISA.These four monoclonal antibodies were subjected to Western blot,epitope mapping and antibody subtype identification.The results showed that all four monoclonal antibodies specifically reacted with the SpaA protein.The antibody titer for hybridoma supernatants against the SpaA protein for 1E8 and 5B3 cells was 1∶1600,and for 5B5 and 9B5 was 1∶3200.The subtype identification revealed that the heavy chain of 1E8 was IgG2b,the heavy chains of 5B3,9B5 were IgG1,and the heavy chain of 5B5 was IgG2a.The light chains for all of them were kappa.Western blot showed that all four monoclonal antibodies reacted specifically with the SpaA protein.Finally,the antigenic epitope recognized by 1E8 and 5B5 could not be identified due to the repetition of the SpaA protein at the C-terminus.The epitope targeted by the 5B3 monoclonal antibody was identified as 258-267 aa,and the antigenic epitope recognized by 9B5 was identified as 462-472 aa.In summary,the monoclonal antibodies against the SpaA protein of Erysipelothrix rhusiopathiae might serve as a foundation for the development of ELISA detection assay.

关 键 词：猪丹毒杆菌 SpaA蛋白 原核表达 单克隆抗体 

分 类 号：S855.1[农业科学—临床兽医学]