滑液囊支原体NADH氧化酶单克隆抗体的制备  

Preparation of a Monoclonal Antibody Against NADH Oxidase of Mycoplasma synoviae

作　　者：刘晓涵 祁晶晶[2] 胡增金 李浩然 宋翠萍[2] 尚原冰 王少辉[2] 田明星 李涛[2] 丁铲[2] 蔡玉梅[1] 于圣青[2] LIU Xiaohan;QI Jingjing;HU Zengjin;LI Haoran;SONG Cuiping;SHANG Yuanbing;WANG Shaohui;TIAN Mingxing;LI Tao;DING Chan;CAI Yumei;YU Shengqing(College of Animal Science and Technology,Shandong Agricultural University,Taian 271000,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China)

机构地区：[1]山东农业大学动物科技学院,泰安271000 [2]中国农业科学院上海兽医研究所,上海200241 [3]安徽农业大学动物科技学院,合肥230036

出　　处：《中国动物传染病学报》2023年第6期20-27,共8页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(31902244);国家重点研发项目(2017YFD0500705);中央级公益性科研院所基本科研业务费专项资金(2019JB11)。

摘　　要：为制备滑液囊支原体NADH氧化酶(NOX)的单克隆抗体。将本实验室前期构建好的重组菌株E.coliBL21(pET28a-NOX),进行诱导表达,经过纯化后免疫BALB/c小鼠,取小鼠脾细胞与SP2/0骨髓瘤细胞融合,经间接ELISA筛选结合有限稀释法克隆化,得到1株稳定分泌抗MSNOX抗体的杂交瘤细胞株,命名为2F4。单克隆抗体亚型鉴定结果显示,该单克隆抗体为IgG2b亚类,间接ELISA测定单克隆抗体腹水效价为1∶2048000。经Westernblot和悬浮免疫荧光试验鉴定,该株单克隆抗体与MS不同分离株均发生特异性反应,与其他禽致病菌不发生反应。通过染色体分析,该杂交瘤细胞株2F4有98±5条染色体,符合杂交瘤细胞染色体的数目,证实细胞融合成功。本研究成功制备NADH氧化酶单克隆抗体,为建立滑液囊支原体的检测方法及进一步研究NADH氧化酶在MS致病机制中的作用提供了重要的生物材料。To prepare monoclonal antibodies(MAbs)against NADH oxidase(NOX)from Mycoplasmasynoviae(MS),the recombinant MSNOX protein was expressed and purified from a prokaryotic expression system and used as an antigen to immunize BALB/c mice.The spleen cells from the immunized mice were fused with mouse myeloma cell line SP2/0.The hybridoma cells secreting MAbs against MSNOX was selected by indirect enzyme linked immunosorbent assay(ELISA)and cloned based on limited dilution method.The monoclonal cell line was obtained and designated as 2F4.In Western blot and immunofluorescence test,the MAb was proved to react with MS but not with other avian pathogens.After chromosome staining,the monoclonal cell line 2F4 was confirmed to have 98±5 chromosomes,which was consistent with the chromosome number of a hybridoma cell.The MAb 2F4 was determined as subtype IgG2b and the asciticfluid was titrated to be 1∶2,048,000 in indirect ELISA.In this study,the MAb 2F4 against MSNOX was successfully prepared,which provided an important biological material for developing detection method and further investigating the role of NOX in the pathogenesis of MS.

关 键 词：滑液支原体 NADH氧化酶 单克隆抗体 

分 类 号：S852.4[农业科学—基础兽医学]