东方泰勒虫P33蛋白单克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2025.08.028

Abstract

Abstract: 【Objective】 P33 protein is an important surface protein of Theileria orientalis,possessing good antigenicity and immunogenicity.The present study aimed to develop specific monoclonal antibodies against P33 protein using hybridoma technology,systematically evaluate their immunological characteristics,and screen monoclonal cell lines with stable antibody-secreting capacity,thereby laying a material foundation for establishing a P33 antigen-based serological diagnostic method for Theileria orientalis. 【Method】 The position of the signal peptide and B cell epitopes of P33 protein was predicted and analyzed by online software.P33 gene without signal peptide but with dominant antigenic epitopes was cloned into prokaryotic expression vectors pET-30a and pGEX-4T-1 to construct recombinant expression vectors pET-30a-P33 and pGEX-4T-1-P33,respectively.The recombinant plasmids were transformation into Escherichia coli BL21 (DE3) and BL21 competent cells for induction of expression.The expressed products were analyzed by SDS-PAGE and Western blotting.BALB/c mice were immunized with the purified pET-30a-P33 recombinant protein,and the specific monoclonal antibody was screened with the pGEX-4T-1-P33 recombinant protein.Antibody subclasses and titer were identified by ELISA.The antigenicity and the specificity of the monoclonal antibody were detected by Western blotting and indirect immunofluorescence assay (IFA). 【Result】 PCR and restriction enzyme digestion results showed that the two expression vectors pET-30a-P33 and pGEX-4T-1-P33 were successfully constructed.The results of SDS-PAGE and Western blotting showed that the recombinant proteins pET-30a-P33 and pGEX-4T-1-P33 with sizes of approximately 43 and 57 ku were successfully prepared and had good antigenicity.A hybridoma cell line (2H10A6) that stably secretes monoclonal antibodies against P33 protein was established through screening with the pGEX-4T-1-P33 recombinant protein.Monoclonal antibody subclass identification results showed that the antibody secreted by 2H10A6 was IgG1 subtype,and the antibody titer was 1∶6 553 600.Western blotting and IFA results showed that the 2H10A6 hybridoma cell line could specifically bind to the P33 protein of Theileria orientalis. 【Conclusion】 The recombinant P33 protein of Theileria orientalis was successfully obtained through a prokaryotic expression system in this study,and monoclonal antibody specifically recognizing the P33 protein was successfully generated.

Key words: Theileria orientalis; P33 protein; prokaryotic expression; monoclonal antibody

CLC Number:

WANG Xianjiong, JIN Yu, XIA Ying, ZHANG Bingyin, DONG Siyan, JIN Chunmei, YU Longzheng. Preparation and Identification of Monoclonal Antibodies Against Theileria orientalis P33 Protein[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3800-3809.

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Preparation and Identification of Monoclonal Antibodies Against Theileria orientalis P33 Protein

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