抗SARS-CoV-2 S蛋白IgM单克隆抗体的表达及鉴定  

Expression and Identification of Anti-SARS-CoV-2 S Protein IgM Monoclonal Antibody

作　　者：张瑞雪 殷强玲 黄涛[1] 钱振 芜为[1] 王世文[1] 梁米芳[1] 孙丽娜[1] 王晓芳[1] ZHANG Ruixue;YIN Qiangling;HUANG Tao;QIAN Zhen;WU Wei;WANG Shiwen;LIANG Mifang;SUN Lina;WANG Xiaofang(National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Center for Disease Control and Prevention of Hubei Province,Wuhan 430079,China;Department of Microbiology,School of Basic Medical Sciences,AnhuiMedicalUniversity,Hefei230032,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京102206 [2]湖北省疾病预防控制中心,武汉430079 [3]安徽医科大学基础医学院微生物学教研室,合肥230032

出　　处：《病毒学报》2023年第2期305-312,共8页Chinese Journal of Virology

摘　　要：为了寻求SARS-CoV-2 IgM抗体阳性血清质控品的替代品,本研究成功表达纯化出抗SARS-CoV-2 S蛋白IgM单克隆抗体。研究确定了IgM单克隆抗体最佳表达载体为含增强子sp163和信号肽2的组合以及第5d为目的蛋白收获的最佳时间。此外在研究J链的作用时发现转染细胞时不添加J链(nCoV-163-IgM3)的表达量明显高于添加J链(nCoV-163-IgM3 J)时的表达量,J链对蛋白稳定性也无明显影响,且nCoV-163-IgM3和nCoV-163-IgM3 J抗原结合活性之间的差别无统计学意义(P>0.05),nCoV-163-IgM3和nCoV-163-IgM3 J对本研究所检测的WTS1、Alpha-S1、Beta-S1、Delta-S1、Omicron-S1、BA.2-S1、BF.7-RBD、XBB.1-S1、BQ1.1-S1等九种抗原均有结合活性,只有一株(BA.2.75-RBD)逃逸。纯化后的目的蛋白经还原剂(β-ME)还原后显示重链分子量大小为70kD左右,轻链分子量大小为25kD左右。目的蛋白均可与新型冠状病毒抗体检测试剂盒(胶体金,INNOVITA)、SARS-CoV-2 S蛋白IgM抗体酶联免疫吸附测定试剂盒(Elabscience)、抗SARS-CoV-2 S-RBD蛋白人IgM抗体酶联免疫吸附测定试剂盒(Proteintech)等三种试剂盒反应。In order to create a substitute for the positive serum control of SARS-CoV-2 IgM antibody, a monoclonal IgM antibody against SARS-CoV-2 S protein was successfully expressed and purified in this study.The optimal expression vector for IgM monoclonal antibody was determined to be a combination containing enhancer sp163 and signal peptide 2. The fifth day of culture was the optimal time for target protein harvest. In addition, when investigating the effect of J chain, this study found that the expression level of transfected cells without J chain(nCoV-163-IgM3) was significantly higher than that with J chain(nCoV-163-IgM3 J), and J chain showed no significant effect on protein stability. There was no significant difference on antigen-binding activity between nCoV-163-IgM3 and nCoV-163-IgM3 J(P>0.05). Both nCoV-163-IgM3 and nCoV-163-IgM3 J showed antigen-binding activity against nine antigens tested in this study, including WT-S1, Alpha-S1, Beta-S1, Delta-S1, Omicron-S1, BA.2-S1, BF.7-RBD, XBB.1-S1, and BQ1.1-S1, but only one strain(BA.2.75-RBD) escaped. The target protein showed the heavy chain molecular weight was about 70 kD and the light chain molecular weight was about 25 kD after reduction with reducing agent(β-ME). The target protein can react with three new coronavirus antibody detection kits,including 2019-nCoV Ab Test(colloidal gold, INNOVITA)、SARS-CoV-2 Spike Protein IgM ELISA Kit(Elabscience) and Anti-SARS-CoV-2 SRBD protein Human IgM ELISA Kit(Proteintech).

关 键 词：SARS-CoV-2 IgM单克隆抗体 J链 重组蛋白 

分 类 号：R392.7[医药卫生—免疫学] R373.1[医药卫生—基础医学]