中国兽医科学2025，Vol.55Issue(11)：1502-1508,7.DOI:10.16656/j.issn.1673-4696.2025.0203
非洲猪瘟病毒M1249L蛋白的表达及单克隆抗体制备
Expression of African swine fever virus M1249L protein and preparation of its monoclonal antibodies
摘要
Abstract
This research aimed to develop monoclonal antibodies against the M1249L protein of the African swine fever virus(ASFV)and to analyze their subtype,sensitivity,and reactivity.First,the M1249L(673-1 249 aa)sequence was codon-optimized,and the fragment was inserted into the pET-28a vector to construct a prokaryotic expression plasmid.Subsequently,transformation,monoclonal colony selection,protein expression and purification were carried out.The purified M1249L truncated protein was used to immunize mice.Hybridoma cell lines secreting antibodies were obtained through cell fusion and limiting dilution,followed by subtype identification and ascites preparation.The specificity of the monoclonal antibody was evaluated using Western-blot and indirect immunofluorescence assay(IFA).The findings showed that the heavy chain of the monoclonal antibody 1D6 belongs to the IgG1 subtype,while the light chain is of the Kappa type.The ascites antibody titer reached up to 1∶1 024 000.Western-blot results indicated that this monoclonal antibody could specifically recognize the M1249L protein expressed by porcine alveolar macrophages(PAMs)at different time points after ASFV inf ection,as well as the M1249L protein expressed by HEK 293T cells transfected with various amounts of plasmid.IFA results showed the presence of green fluorescence in ASFV-infected PAMs,indicating high reactivity.Overall,this study successfully prepared the M1249L monoclonal antibody,providing a foundation for subsequent research on the biological functions of the M1249L protein and ASFV diagnostic testing.关键词
非洲猪瘟病毒/单克隆抗体/M1249L蛋白Key words
African swine fever virus/monoclonal antibody/M1249L protein分类
畜牧业引用本文复制引用
孙树凯,席飞,郑海学,朱国梁,朱紫祥..非洲猪瘟病毒M1249L蛋白的表达及单克隆抗体制备[J].中国兽医科学,2025,55(11):1502-1508,7.基金项目
国家生猪产业技术体系项目(CARS-35) （CARS-35）
中央高校基本科研业务费专项资金项目(lzujbky-2022-ct02) （lzujbky-2022-ct02）
"十四五"广东省农业科技创新十大主攻方向"揭榜挂帅"项目(2024KJ14) （2024KJ14）
甘肃省联合科研基金重大项目(24JRRA813) （24JRRA813）
