中国畜牧兽医2025，Vol.52Issue(6)：2772-2780,9.DOI:10.16431/j.cnki.1671-7236.2025.06.029
鸡补体受体Ⅱ蛋白兔多克隆抗体制备及鉴定
Preparation and Identification of Rabbit Polyclonal Antibody of Chicken Complement Receptor 2
摘要
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-△TM recombinant plasmid was transfected into HEK293F cells to express the recombinant chCR2 protein(His-chCR2-△TM)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-△TM protein could be expressed in HEK293F cell line.The concentration of recombinant His-chCR2-△TM 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.关键词
鸡补体受体Ⅱ/表达纯化/多克隆抗体Key words
chicken complement receptor 2/expression and purification/polyclonal antibody分类
农业科技引用本文复制引用
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):2772-2780,9.基金项目
国家自然科学基金青年基金(32202793) （32202793）
北京市农林科学院创新能力建设(KJCX20230424、KJCX20240338) （KJCX20230424、KJCX20240338）
