鸡补体受体Ⅱ蛋白兔多克隆抗体制备及鉴定

doi:10.16431/j.cnki.1671-7236.2025.06.029

Abstract

Abstract: 【Objective】 The interaction between complement receptor 2 (CR2) interacted with its physiological ligand C3d could bridge innate immune response and adaptive immune response,amplify the signal transduction downstream,significantly decrease the threshold for B cell activation by antigen stimulation,and increase the sensitivity of B cells to antigen by 1 000-10 000 times,which was crucial for activating B cells and initiating the immune response.This study aimed to prepare rabbit polyclonal antibody against chicken CR2 (chCR2),provide a tool for detecting the expression of chCR2 protein,and lay a foundation for further exploring the function and role of chCR2 protein.【Method】 The pTT5-His-chCR2-ΔTM recombinant plasmid was transfected into HEK293F cells to express the recombinant chCR2 protein (His-chCR2-ΔTM) in the eukaryotic expression system of HEK293F cells.The protein expression was analyzed by Western blotting.The chCR2 protein was then purified using a nickel column affinity chromatography purification system,followed by SDS-PAGE identification.The purified chCR2 protein was used to immunize New Zealand White rabbits to prepare polyclonal antibodies against chCR2.The titer of these polyclonal antibodies against chCR2 was quantified through an enzyme-linked immunosorbent assay (ELISA).The specificity and sensitivity of the chCR2 polyclonal antibody were determined through Western blotting and indirect immunofluorescence assay (IFA).【Result】 The results showed that recombinant His-chCR2-ΔTM protein could be expressed in HEK293F cell line.The concentration of recombinant His-chCR2-ΔTM protein purified by nickel affinity chromatography was 4.439 mg/mL,and the molecular weight between 45 ku.ELISA results showed that the highest titer of chCR2 polyclonal antibody was 1∶32 768 000.The Western blotting and IFA results demonstrated that the chCR2 protein could be specifically recognized by the chCR2 polyclonal antibody.The optimal dilution ratio of chCR2 polyclonal antibody for Western blotting detection was 1∶5 000,and the maximum dilution ratio for IFA detection was 1∶1 600.【Conclusion】 In this study,a rabbit polyclonal antibody against chCR2 protein was successfully prepared.The antibody had high titer and good specificity,which provided an experimental basis for the subsequent detection of chCR2 protein and the study of the function of chCR2 protein.

Key words: chicken complement receptor 2; expression and purification; polyclonal antibody

CLC Number:

S852.4⁺3

MENG Zhaoying, JIN Huan, TU Min, HU Ge, ZHANG Zhenhua. Preparation and Identification of Rabbit Polyclonal Antibody of Chicken Complement Receptor 2[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2772-2780.

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Preparation and Identification of Rabbit Polyclonal Antibody of Chicken Complement Receptor 2

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