中国农业大学学报2025，Vol.30Issue(6)：101-111,11.DOI:10.11841/j.issn.1007-4333.2025.06.10
鸡源JAK1蛋白单克隆抗体的制备及在禽流感病毒感染中的应用
Preparation of monoclonal antibody to chicken JAK1 protein and its application during avian influenza virus infection
摘要
Abstract
To investigate the biological functions of chicken JAK1(chJAK1)protein,the full-length chJAK1 ORF was RT-PCR amplified using total RNA from chicken embryo fibroblast cell line(DF-1)as template and subjected to construct the eukaryotic expression plasmid pcDNA3.1-His-chJAK1.The truncated fragment chJAK1-286-608 aa(ΔchJAK1)was used to construct the recombinant prokaryotic expression plasmid pET-32a-ΔchJAK1.The monoclonal antibodies against chJAK1 protein were generated by immunizing BALB/c mice with the recombinant protein expressed in E.coli,and subsequently utilized to preliminarily analyze chJAK1 expression level in avian influenza virus infection of DF-1 cells.The results showed that:1)Two monoclonal antibodies 2E11 and 3F4 were obtained by ELISA and Western blot screening.They belonged to the subclasses IgG 1 and lgG2b in the heavy chain with κ as the light chain.Their ascites titers were determined to be 6.4×104 and 1.6×104,respectively.2)Both strains of monoclonal antibody could recognize chicken,duck and goose JAK1 by indirect immunofluorescence and Western blot assay.Additionally,2E11 could react with human JAK1,whereas 3F4 did not react with human derived JAK1.3)By truncating the protein to identify the antigen epitope,it was determined that the antigenic epitopes recognized by 2E11 was as 367HIVIKESTVSINKQDNKKMELKLSSHDEALSFASLI403 and the antigen epitope recognized by 3F4 was 586RTQIYSGILNYKD-DENEGYQNE608,respectively.4)Avian influenza virus infection with DF-1 can induce down-regulation of endogenous chJAK1 protein expression.In summary,this study successfully prepared monoclonal antibodies against chJAK1 and confirmed that the chJAK1 protein plays an important role in the AIV infection process.The preparation of anti-chJAK1 monoclonal antibodies provides a valuable tool for studying the biology and antiviral function of this protein.关键词
鸡源JAK1/蛋白表达/单克隆抗体/抗原表位/禽流感病毒Key words
Chicken JAK1/protein expression/monoclonal antibody/antigenic epitope/avian influenza virus分类
农业科技引用本文复制引用
霍臣智,刘秀梵,杨辉,余玉霞,董宇睿,卞盈,朱春成,秦涛,陈素娟,彭大新..鸡源JAK1蛋白单克隆抗体的制备及在禽流感病毒感染中的应用[J].中国农业大学学报,2025,30(6):101-111,11.基金项目
国家自然科学基金项目(32373042) （32373042）
