幽门螺杆菌CagL蛋白单克隆抗体的制备与鉴定  

Preparation and identification of monoclonal antibody against Helicobacter pylori CagL protein

作　　者：朱虹[1] 陈珵[1] 凌峰[1] 邵晨[1] 余敏[1] 邵世和[1] 

机构地区：[1]江苏大学基础医学与医学技术学院,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2013年第3期238-242,共5页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(81271795);江苏省省级科技创新与成果转化专项基金资助项目(BL2012047);高等学校博士学科点专项科研基金资助项目(20123227110008)

摘　　要：目的:制备幽门螺杆菌(Helicobacter pylori,H.pylori)CagL蛋白单克隆抗体(monoclonal antibody,mAb),并鉴定其特性。方法:用原核表达并纯化的CagL融合蛋白免疫BALB/c小鼠,6周后取小鼠脾细胞与骨髓瘤细胞融合,经亚克隆筛选获得阳性杂交瘤细胞株;ELISA法鉴定mAb的亚型;间接ELISA法测定杂交瘤细胞培养上清和腹腔积液效价及其相对亲和力;相加ELISA法分析mAb的识别表位;蛋白质印迹法鉴定mAb特异性;冻溶法检测杂交瘤细胞株的稳定性。结果:获得3株能稳定分泌抗CagL的杂交瘤细胞株,其中两株单抗属IgM类,一株单抗属IgG2a类,轻链型别均为κ型;杂交瘤细胞培养上清效价在1∶64～1∶128之间,腹腔积液的效价均达1∶16 000以上;相对亲和力较高;3株mAb识别3种不同的抗原表位;3株mAb都能与融合蛋白反应,其中两株能与H.pylori全菌蛋白发生特异性反应;杂交瘤细胞经冻存复苏后,体外传代仍能稳定分泌mAb。结论:成功制备了抗H.pylori CagL蛋白的单克隆抗体,为深入研究幽门螺杆菌CagL蛋白的功能和Ⅳ型分泌系统的致病机制奠定了基础。Objective： To prepare and characterize the monoclonal antibody（mAb） against Helicobacter pylori（H.pylori） CagL protein.Methods： The CagL fusion protein was induced to express by IPTG in E.coli BL21（DE3） which contains recombinant expression vector pET-28a（＋） and then purified by Ni2＋-NTA resin.Purified CagL fusion protein was used to immunize 6-week BALB/c mice,and then the spleen cells were fused with myeloma SP2/0 cells;the positive hybridoma cell lines were obtained by means of several rounds of subclone.Immunoglobulin isotype of mAb was identified by ELISA method.The titer of mAbs in cell culture supernatant and ascites,as well as the relative affinity of these antibodies,were measured by indirect ELISA method.The antigenic epitopes of mAbs were evaluated with ELISA additivity test.The specificity for antibody was determined by Western blotting.The stability of hybridoma cell lines specifically secreting mAbs was tested by cryopreservation and resuscitation.Results： Three hybridoma cell lines which could secrete stably mAbs against CagL were successfully obtained.The isotype of two mAbs was classified as IgM,another IgG2a,and the light chain subset of three mAbs was classified as type κ.The titers of three mAbs in cell culture supernatant were 1∶64～1∶128,while those in ascites reached more than 1∶16 000.The affinities of mAbs were found to be high.Three different antigenic epitopes might be recognized by the mAbs.Western blotting analysis showed that all three mAbs could bind to the CagL fusion protein;however,only two of them had specific reaction with the whole protein extracted from H.pylori strains.After cryopreservation,resuscitation and subculture,the titers of mAbs secreted by three hybridoma cell lines were still high.Conclusion： The monoclonal antibody against CagL protein was successfully prepared,which would play a significant role for the further study in the mechanisms of CagL as well as H.pylori type Ⅳ secretion system.

关 键 词：CagL 幽门螺杆菌 单克隆抗体 杂交瘤 

分 类 号：R392-33[医药卫生—免疫学] R378-99[医药卫生—基础医学]