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

摘要/Abstract

摘要： 【目的】补体受体Ⅱ(complement receptor 2，CR2)与其生理学配体C3d相互作用能桥连先天性免疫反应和适应性免疫反应，放大下游信号的转导现象，显著降低抗原刺激B细胞活化的阈值，可将B细胞对抗原的敏感性提高1 000～10 000倍，对于激活B细胞及启动免疫反应至关重要。本研究旨在制备兔源鸡CR2(chCR2)多克隆抗体，为检测chCR2蛋白的表达情况提供工具，也为深入探究chCR2蛋白的功能和作用奠定基础。【方法】将pTT5-His-chCR2-ΔTM重组质粒转染至HEK293F细胞中，在真核表达系统HEK293F细胞中表达重组chCR2蛋白(His-chCR2-ΔTM)，经Western blotting对蛋白表达进行分析，利用镍柱亲和层析纯化系统纯化chCR2蛋白并进行SDS-PAGE鉴定。用纯化出的chCR2蛋白免疫新西兰大白兔制备抗chCR2血清多克隆抗体；利用ELISA方法检测chCR2兔血清的效价；利用Western blotting和间接免疫荧光试验(indirect immunofluorescence assay，IFA)检测chCR2血清多克隆抗体的特异性和灵敏度。【结果】重组His-chCR2-ΔTM蛋白可在HEK293F细胞系表达，利用镍柱亲和层析法纯化得到的重组His-chCR2-ΔTM 蛋白浓度为4.439 mg/mL，分子质量为45 ku。ELISA检测结果显示，chCR2血清多克隆抗体效价最高为1∶32 768 000；Western blotting和IFA结果表明，chCR2蛋白可被chCR2血清多抗体特异性识别；Western blotting检测时，chCR2血清多克隆抗体的稀释度为1∶5 000时特异性最好；IFA检测时，chCR2血清多克隆抗体最大稀释度为1∶1 600。【结论】本研究成功制备了chCR2蛋白兔多克隆抗体，该抗体效价较高且特异性较好，为后续检测chCR2蛋白及研究chCR2蛋白的功能提供了试验基础。

关键词: 鸡补体受体Ⅱ; 表达纯化; 多克隆抗体

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

中图分类号:

S852.4⁺3

孟昭英, 靳换, 涂敏, 胡格, 章振华. 鸡补体受体Ⅱ蛋白兔多克隆抗体制备及鉴定[J]. 中国畜牧兽医, 2025, 52(6): 2772-2780.

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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