中国畜牧兽医2024，Vol.51Issue(7)：3008-3019,12.DOI:10.16431/j.cnki.1671-7236.2024.07.026
鸡γ-干扰素的原核表达及其单克隆抗体制备与鉴定
Prokaryotic Expression of Chicken Interferon-γ and Preparation and Identification of Its Monoclonal Antibodies
摘要
Abstract
[Objective]The purpose of this study was to express chicken interferon-gamma(ChIFN-y)by prokaryotic expression system and prepare its monoclonal antibody to provide crucial reagents for the detection of natural ChIFN-γ.[Method]Recombinant plasmid pET21a-ChIFN-γ was constructed by cloning codon optimized ChIFN-γ gene into pET-21a(+)vector,and then transformed into Escherichia coli BL21(DE3)competent cells.The recombinant protein was induced by IPTG.BALB/c mice were immunized with this purified protein.The splenocytes of immune-qualified mouse was hybridized with myeloma cells(SP2/0)by cell fusion technique.Positive hybridoma secreting specific antibodies against ChIFN-γ were identified through screening using indirect ELISA,and prepare ascites,purified ascites with Protein A/G to obtain specific antibodies.The classes and subclasses of monoclonal antibodies were identified.Western blotting and indirect immunofluorescence assay(IFA)were used to detect the reactivity,affinity,and specific cross reactivity of monoclonal antibodies.Monoclonal antibodies were labeled with horseradish peroxidase(HRP),and antibody pairing were conducted using pairwise methods.[Result]Soluble recombinant ChIFN-γ protein were successfully prepared.Western blotting showed that ChIFN-γ binded to the corresponding antibody,presenting specific bands(16 ku).Seven hybridoma cell lines specific to ChIFN-γ were obtained which were 3E8,5F8,2G12,5 A10,3D8,3B5 and 3G2,respectively.Western blotting and IFA results showed that all 7 monoclonal antibodies had good reactivity,and could specifically recognize eukaryotic expressed ChIFN-γ.The results of class and subclass identification of monoclonal antibodies showed that the heavy chains of 3E8,5F8,3D8,3B5 and 2G12 were all IgG1.The heavy chains of 5A10 and 3G2 were IgG2a and IgM,respectively.The light chains of the 7 monoclonal antibodies were all Kappa type.The 7 monoclonal antibodies obtained had dissociation constants(Kd)between 1.04 and 58.33 nmol/L,indicating high affinity.Five pairs of paired antibodies were also obtained.[Conclusion]This study successfully prepared recombinant ChIFN-γ protein and its monoclonal antibodies,laying the foundation for further research on ChIFN-γ and its related studies in avian immunology,virology and vaccine efficacy evaluation.关键词
鸡γ-干扰素/单克隆抗体/亲和力/抗体配对Key words
chicken IFN-γ/monoclonal antibodies/affinity/antibody pairing分类
农业科技引用本文复制引用
崔锦蔷,程晶,江波,周林宜,刘文晓,李永清,宋勤叶..鸡γ-干扰素的原核表达及其单克隆抗体制备与鉴定[J].中国畜牧兽医,2024,51(7):3008-3019,12.基金项目
北京市农林科学院创新能力建设(KJCX20220422) （KJCX20220422）
北京市农林科学院畜牧兽医研究所改革与发展(XMS202324) （XMS202324）
