抗人分化抑制因子3蛋白单克隆抗体的制备和鉴定  

Preparation and identification of monoclonal antibody against human Id3

作　　者：仲爱芳[1,2] 李晓军[1] 贾丽[1] 陈芳芳[1] 虞伟[1] 

机构地区：[1]南京军区南京总医院解放军临床检验医学研究所,南京210002 [2]解放军第一0二医院检验科

出　　处：《医学研究生学报》2010年第9期920-923,共4页Journal of Medical Postgraduates

基　　金：江苏省"科教兴卫"医学重点人才基金(RC2007117);南京军区医药卫生科研项目重点课题(07Z032)

摘　　要：目的分化抑制因子3(inhibitor of differentiation3,Id3)是重要的细胞生长调控分子,在细胞增殖、分化等过程中发挥重要作用。文中探讨在大肠杆菌中表达及纯化重组人Id3蛋白,制备抗人Id3单克隆抗体(McAb),以进一步研究Id3的生物学功能及其临床应用。方法将重组表达载体Id3/pET32a转化大肠杆菌BL21(DE3),用异丙基β-D硫代半乳糖苷(IPTG)诱导转化菌表达Id3重组融合蛋白(Trx-His-hId3),通过镍亲和层析柱纯化Trx-His-hId3。以纯化的Id3重组蛋白为免疫原,免疫BALB/c小鼠,采用杂交瘤技术制备Id3McAb,应用酶联免疫吸附试验(ELISA)及Western blot进行抗体鉴定。结果经免疫小鼠、脾细胞分离、细胞融合及克隆筛选等步骤,筛选出2株分泌抗人Id3McAb的杂交瘤细胞株,分别命名为McAb6G7和6G9,免疫球蛋白亚类鉴定均为IgG1亚类。Western blot分析表明,McAb6G7和6G9能与重组人Id3蛋白特异性结合。间接ELISA法检测2株细胞冻融前后所产生上清中的抗体效价均分别为1∶1000、1∶5000。结论成功制备了抗人Id3McAb,为研究该蛋白的功能提供了有利工具。Objective The inhibitor of differentiation 3（Id3）,as an important cell growth regulator,plays a significant role in cell proliferation and differentiation.This study aimed to express and purify the recombinant human Id3 protein in E coli and prepare a monoclonal antibody（McAb） against human Id3 for further investigating the biological function and clinical application of Id3.Methods The recombinant vector Id3/pET32a was transformed into E coli BL21 and recombinant human Id3 was expressed after induction with IPTG.The Id3 fusion protein was purified through immobilized Ni2＋ absorption chromatographic column and used to immunize BALB/c mice.The McAb was produced by the conventional hybridoma cell method and characterized by ELISA and Western blot.Results Two hybridoma cells,named 6G7 and 6G9,were obtained after 3-4 rounds of cloning,which could stably secrete McAbs against the Id3 recombinant protein and belonged to the IgG1 subtype.Western blot analysis showed that the McAbs specifically identified with the Id3 recombinant protein.Results of indirect ELISA demonstrated that the titers of the two McAbs,secreted by 6G7 and 6G9 before or after freeze and thaw were 1∶1000 and 1∶5000,respectively.Conclusion The Id3-specific McAb against the recombinant protein was successfully prepared,which may provide an important tool for further study of the Id3 protein.

关 键 词：分化抑制因子3 单克隆抗体 免疫印迹 

分 类 号：Q819[生物学—生物工程]