新型冠状病毒核衣壳蛋白抗原的重组表达及其单克隆抗体的制备

目的 制备新型冠状病毒SARS-CoV-2核衣壳蛋白重组抗原及其单克隆抗体。方法 根据公布的新冠病毒全基因组序列,构建其核衣壳蛋白的表达载体pET-28a-N, 并转化至E. coil BL21(DE3)进行原核表达, ELISA及Western blot鉴定纯化后的核衣壳蛋白重组抗原的特异性并免疫Balb/c系小鼠, 基于杂交瘤融合技术筛选获得可持续分泌新冠病毒核衣壳蛋白单克隆抗体的细胞株, 并基于ELISA及生物膜层干涉技术鉴定单克隆抗体的特异性和亲和力。结果 基于大肠杆菌原核表达获得新冠病毒核衣壳蛋白的重组抗原, 其纯度大于90%, 筛选获得7株可分泌特异性结合新冠病毒核衣壳蛋白的杂交瘤细胞株, 经Western blot、ELISA及分子相互作用分析等方法证实所获得的单克隆抗体与核衣壳蛋白具有优良的结合特异性和结合活性, 其中的7E11、7E8单克隆抗体与核衣壳蛋白抗原的亲和力常数分别为1.69×10-9、2.031×10-9M, 可作为抗体元件应用于新冠病毒的快速免疫分析领域。结论 获得高特异性的新型冠状病毒SARS-CoV-2核衣壳蛋白重组抗原及其单克隆抗体。

Preparation of recombinant antigen and monoclonal antibody of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein

Objective Preparation of recombinant antigen and monoclonal antibody of nucleocapsid protein of SARA-CoV-2. Methods According to the published SARS-CoV-2 genome sequence, the expression vector pET-28a-N of its nucleocapsid protein was constructed and transformed into E.coil BL21 (DE3). The nucleocapsid protein was obtained by prokaryotic expression, identify the specificity of the purified recombinant nucleocapsid protein antigen by ELISA and Western blot, and use it to immunize Balb/c mice. Based on hybridoma technology, a cell line that could continuously secrete mAb to the SARS-CoV-2 nucleocapsid protein was screened and obtained. The specificity and affinity of the mAb were identified by ELISA and BLI. Results The recombinant antigen of the SARS-CoV-2 nucleocapsid protein with a purity greater than 90% was obtained through prokaryotic expression in Escherichia coli, Screened and obtained 7 hybridoma cell that can secrete the nucleocapsid protein specifically binding to the SARS-CoV-2. After Western blot, ELISA and molecular interaction analysis and other methods confirmed that the obtained mAb has excellent binding specificity and binding activity with the nucleocapsid protein, the affinity constant of the 7E11 mAb with the nucleocapsid protein antigen is 1.69×10-9M, and 7E8 mAb is 2.031×10-9 M, which can be used as an antibody component in the field of rapid immunoassay of the SARS-CoV-2. Conclusion Obtained a highly specific recombinant antigen of the SARS-CoV-2 nucleocapsid protein and its monoclonal antibody.