毒害艾美耳球虫配子体抗原EnGAM22单克隆抗体的制备与鉴定

Preparation and Characterization of Monoclonal Antibodies against the Gametocyte Antigen EnGAM22 of Eimeria necatrix

旨在制备毒害艾美耳球虫(Eimeria necatrix)配子体抗原EnGAM22单克隆抗体,用Ni-NTA亲和层析纯化的重组抗原rEnGAM22免疫BALB/c小鼠,免疫3次后,取脾细胞与骨髓瘤细胞SP2/0进行融合,用预先建立的ELISA方法筛选阳性杂交瘤细胞,经有限稀释法对阳性杂交瘤细胞进行4次亚克隆后,获得2株能稳定分泌特异性抗体的杂交瘤细胞株并命名为2F3和3D3;对单抗亚类和特异性进行鉴定,应用单抗识别天然蛋白和虫体定位。结果显示,单克隆抗体2F3和3D3亚类分别为IgG2a、IgG2b,纯化后腹水效价分别为1∶256000、1∶64000,能特异性识别重组抗原rEnGAM22和毒害艾美耳球虫天然配子体蛋白;免疫荧光定位显示单克隆抗体2F3和3D3能特异性识别大配子体的成壁体和卵囊壁,表明EnGAM22抗原参与卵囊壁的形成。制备的单克隆抗体为研究球虫卵囊壁形成的分子机制奠定了基础。

To prepare monoclonal antibodies(McAbs)against the gametocyte antigen EnGAM22 of Eimeria necatrix,BALB/c mice were immunized with recombinant gametocyte antigen rEnGAM22 purified by Ni-NTA affinity chromatography.After three times of immunization,the spleen cells of the immunized mice were fused with myeloma cells SP2/0.Hybridoma culture supernatant were examined by the pre established ELISA assay.Two hybridomas stably secreting specific antibody were obtained after four times of subcloning by limiting dilution method,namely 2F3 and 3D3 respectively.The subclasses and specificities of McAbs were identified and the recognition of McAbs to natural proteins and the localization of McAbs in E.necatrix were studied.The results showed that the subclasses of McAbs 2F3 and 3D3 were identified as IgG2a and IgG2b,respectively.The titer of purified ascites were 1∶256000(2F3)and 1∶64000(3D3),respectively.The McAbs 2F3 and 3D3 could recognize rEnGAM22 and the native gametocyte protein of E.necatrix.The indirect immunofluorescence assay with McAbs 2F3 and 3D3 showed that EnGAM22 were located in wall-formating bodies of macrogametocytes and the walls of oocyst,which indicated that EnGAM22 protein was involved in the formation of oocyst wall.The McAbs 2F3 and 3D3 might be used for studying the molecular mechanism of oocyst wall formation in coccidia.