毒害艾美耳球虫配子体抗原EnGAM22单克隆抗体的制备与鉴定

doi:10.11843/j.issn.0366-6964.2022.03.020

Abstract

Abstract: 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 1:256 000 (2F3) and 1:64 000 (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.

Key words: Eimeria necatrix, gametocyte protein, monoclonal antibodies, preparation, characterization

CLC Number:

S855.9

CAI Weimin, LI Wenjing, WANG Lele, SU-DING Zeyang, ZHU Yu, LIU Dandan, XU Jinjun, TAO Jianping. Preparation and Characterization of Monoclonal Antibodies against the Gametocyte Antigen EnGAM22 of Eimeria necatrix[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 875-882.

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

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