摘要
为了制备胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)Ⅰ型ApxⅠ毒素(ApxⅠ)AI2蛋白特异性单克隆抗体,进而建立用于评估亚单位疫苗免疫效果的阻断ELISA方法,以实验室前期筛选得到的ApxⅠ抗原优势决定簇AI2重组蛋白为免疫原,免疫6周龄BALB/c雌鼠。利用常规细胞融合和间接ELISA筛选阳性杂交瘤细胞,获得4株单克隆抗体,命名为2C2、4E4、5E7和6F2,上清液效价分别为1∶6400、1∶6400、1∶12800和1∶6400。ELISA与Western blot结果表明,制备的4株单克隆抗体均与重组AI2蛋白有良好的反应性。其中5E7可与APP天然ApxⅠ毒素蛋白特异性结合,同时4株单克隆抗体均不与多杀性巴氏杆菌、支气管败血波氏菌、猪丹毒丝菌、副猪格拉菌和猪链球菌2型反应。亚类分型结果表明,2C2、4E4、5E7属于IgG1亚类,6F2属IgG2a亚类,4株单抗轻链均为κ链。通过构建AI2重组蛋白截短体,初步确定线性表位在74~93 aa之间。本研究成功制备了4株针对AI2蛋白单克隆抗体,为APP亚单位疫苗免疫效果评估方法的建立奠定了基础。
This study was to prepare monoclonal antibodies against AI2 protein of APP and to establish a blocking ELISA method for evalu-ating the immune effect of subunit vaccine.6-week-old female BALB/c mice were immunized with the recombinant AI2 protein,an antigen dominant determinant for ApxⅠ.The positive hybridoma cells were prepared and screened by conventional cell fusion and indirect ELISA,and 4 monoclonal antibodies were obtained,which named 2C2,4E4,5E7 and 6F2,with supernatant titers of 1∶6400,1∶6400,1∶12800 and 1∶6400,respectively.The results of ELISA and Western blot showed that all the 4 monoclonal antibodies(McAbs)reacted well with the recombinant AI2 protein.Among them,5E7 could specifically bind to the native ApxⅠtoxin protein of APP,but none of the 4 McAbs could react with Pasteurella multocida,Bordetella brcmchiseptica,Erysipelothrix rhusiopathiae,Glaesserella parasuis and Streptococcus suis serotype 2.The subtyping results showed that 2C2,4E4,5E7 belonged to the IgG1 subclass,and 6F2 belonged to the IgG2a subclass.And,all the light chains of the 4 monoclonal antibodies strains were of theκ-chain.The linear epitope between 74 and 93 aa was prelimina-rily determined by constructing a truncated recombinant protein of AI2.In this study,4 monoclonal antibodies against AI2 protein were suc-cessfully prepared,which laid a foundation for the establishment of the immune evaluation method of APP subunit vaccine.
作者
蔡金双
耿琰
张宝戈
车巧林
丁文傒
李玉峰
CAI Jinshuang;GENG Yan;ZHANG Baoge;CHE Qiaoin;DING Wenxi;LI Yufeng(Nanjing Agricultural University/Key Laboratory of Animal Bacteriology of Ministry of Agriculture and Rural Affairs,Nanjing 210095,China;Jiangsu Nannong High-tech Co.,Ltd.,Wuxi 214400,China)
出处
《畜牧与兽医》
CAS
北大核心
2024年第5期119-126,共8页
Animal Husbandry & Veterinary Medicine
基金
国家重点研发计划(2022YFD1800902)
横向课题(江苏南农高科技股份有限公司,090HMQY21042)。
