摘要
本研究运用硫酸铵盐析法结合Protein A亲和层法从大菱鲆血清中分离、纯化免疫球蛋白IgM。SDS-PAGE分析结果显示,纯化的血清IgM主要有2条蛋白带,分子量分别为78和27kDa,分别为大菱鲆IgM的重链与轻链。以纯化的IgM免疫BALB/c小鼠,取免疫小鼠脾细胞与骨髓瘤细胞进行细胞融合,将融合后的细胞置于含HAT的培养基中培养。利用ELISA和Western blotting筛选杂交瘤,并利用有限稀释法对阳性杂交瘤进行单克隆,最终共获得3株抗大菱鲆血清IgM单克隆抗体,分别命名为1B2、1G4和2A1,抗体分型结果显示3株单抗均属IgG。Western blotting结果显示,单抗1B2和1G4可特异性识别大菱鲆IgM重链蛋白,而单抗2A1则特异性识别轻链蛋白。同时,以制备的腹水单抗1G4结合免疫荧光检测显示,单抗1G4可识别大菱鲆外周血、脾及前肾组织中表面IgM阳性(sIgM^+)细胞;流式细胞术分析显示,大菱鲆外周血、脾和前肾白细胞中sIgM^+细胞比例分别为48.07%,20.05%和18.45%。研究结果显示,制备的抗大菱鲆血清IgM单抗特异性高、反应性强,可为研究大菱鲆免疫系统及免疫应答动态提供有力的工具。
Immunoglobulins（Ig）paly vital roles in fish adaptive humoral immune system.In order to investigate the characters of Ig M and surface IgM positive（sIgM^＋）cells in turbot（Scophthalmus maximus）,serum IgM was isolated from turbot with salting out and protein A affinity chromatography in combination.In SDS-PAGE,the purified protein showed two main bands with the molecular weight of 78 kDa and 27 kDa,which corresponded to the heavy chain and light chain of turbot IgM,respectively.The purified IgM was used to immunize BALB/c mice.Three days after the last immunization,spleen cells were collected and fused with myeloma cells.The hybridomas were cultured in the medium containing HAT,and screened with indirect ELISA and western blotting.Hybridomas giving positive results were cloned by limiting dilution,and three MAbs,named 1B2,1G4 and 2A1,were produced.Isotype analyzing showed that all the three MAbs belonged to IgG isotype.Western blotting showed that MAb 1B2 and MAb 1G4 specifically recognized the heavy chain of turbot IgM while MAb 2A1 recognized the light chain.MAb 1G4 was largely produced in mouse ascites and applied in immunofluorescence assay（IFA）and flow cytometry assay（FCA）.In IFA,obvious fluorescence signals were observed at the membrane of partial lymphocyte,suggesting that MAb1G4 recognized the sIgM-＋lymphocytes in peripheral blood,spleen and anterior kidney.FCA revealed that 48.07%of leukocyte in the peripheral blood,20.05%in the spleen and 18.45%in the anterior kidney were reactive to MAb1G4.These results demonstrated that obtained MAbs could be used as tools to detect the soluble and membrane bounded IgM molecules of turbot.In this study,MAbs that specifically recognized the serum IgM and sIgM-＋lymphocytes of turbot were produced,which may be employed to investigate the immune system and immune defense mechanisms of turbot.
出处
《中国海洋大学学报（自然科学版）》
CAS
CSCD
北大核心
2016年第1期27-32,共6页
Periodical of Ocean University of China
基金
国家自然科学基金项目(31302216
31172429
31472295)
国家重点基础研究发展规划项目(2012CB114406)
国家科技支撑计划项目(2012BAD17B01)资助~~
关键词
大菱鲆
血清IGM
单克隆抗体
免疫荧光
流式细胞术
Scophthalmus maximus
serum immunoglobulin M
monoclonal antibody
immunofluorescence
flow cytometry
