摘要
为研发非洲猪瘟病毒(ASFV)的免疫诊断学试剂,利用大肠杆菌系统表达ASFV重组K145R蛋白,制备并鉴定K145R蛋白单克隆抗体。结果显示,成功构建pET28a-K145R表达载体,表达得到大小约为17 ku可溶性的重组K145R蛋白。Western blot分析证实,重组K145R蛋白与ASFV阳性血清有良好的反应性。经细胞融合、筛选,得到单克隆抗体1D4;ELISA检测其效价为1∶5120000;亚类鉴定其重链为IgG2a,轻链为κ;Western blot鉴定其能特异性识别重组K145R蛋白;IFA检测其能与ASFV反应。综上,实现K145R蛋白在大肠杆菌中的可溶性表达并制备单克隆抗体。
In order to develop an immunodiagnostic reagent for African swine fever virus(ASFV),the recombinant ASFV K145R protein was expressed by E.coli system,and the monoclonal antibody against K145R protein was prepared and identified.The results showed that the pET28a‑K145R expression vector was successfully constructed,and the soluble recombinant protein of K145R with the size of about 17 ku was obtained.Western blot analysis confirmed that recombinant K145R protein had a good reactivity with ASFV positive serum.A monoclonal antibody 1D4 was obtained by cell fusion and screening.The titer was 1∶5120000 by ELISA.The heavy chain was identified as IgG2a and the light chain asκ.Western blot analysis showed that it could specifically recognize recombinant K145R protein.IFA test showed that it could react with ASFV.In summary,the soluble expression of K145R protein in E.coli and the preparation of monoclonal antibody were achieved.
作者
耿笑林
孙杰
王彦伟
黄甜
刘鹏
曹红梅
逄文强
郝丽影
邓均华
黄玉欣
田克恭
GENG Xiaolin;SUN Jie;WANG Yanwei;HUANG Tian;LIU Peng;CAO Hongmei;PANG Wenqiang;HAO Liying;DENG Junhua;HUANG Yuxin;TIAN Kegong(National Research Center for Veterinary Medicine,Luoyang 471000,China;Publike Bioengineering Co.,Ltd.,Luoyang 471000,China;Luoyang Putai Biotechnology Co.,Ltd.,Luoyang 471000,China)
出处
《河南农业科学》
北大核心
2021年第8期154-159,共6页
Journal of Henan Agricultural Sciences
基金
洛阳市重大科技专项(1901029A)。