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

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

目的制备新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)核衣壳蛋白重组抗原及其单克隆抗体(monoclonal antibody,mAb)。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EscherichiacoliBL21(DE3)进行原核表达,酶联免疫吸附测定(enzyme linked immunosorbent assay,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(biofilm layer interference,BLI)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,其纯度可达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^(-9)mol/L,可作为抗体元件应用于新冠病毒的快速免疫分析领域。结论本研究获得高特异性的SARS-CoV-2核衣壳蛋白重组抗原及其单克隆抗体。

Objective To prepare the recombinant antigen and monoclonal antibody(mAb)of nucleocapsid protein of severe acute respiratory syndrome coronavirus 2(SARA-CoV-2).Methods According to the published SARS-CoV-2 genome sequence,the expression vector pET-28 a-N of its nucleocapsid protein was constructed and transformed into Escherichia coli BL21(DE3),the nucleocapsid protein was obtained by prokaryotic expression,the specificity of the purified recombinant nucleocapsid protein antigen was identified by enzyme linked immunosorbent assay(ELISA)and Western blot,and Balb/c mice were immunized,a cell line that could continuously secrete mAb to the SARS-CoV-2 nucleocapsid protein was screened and obtained based on hybridoma technology,the specificity and affinity of the mAb were identified by ELISA and biofilm layer interference(BLI)technology.Results The recombinant antigen of the SARS-CoV-2 nucleocapsid protein with a purity reaching 90%was obtained through prokaryotic expression in E.coli,7 strains of hybridoma cells that could secrete the nucleocapsid protein specifically binding to the SARS-CoV-2 were screened and obtained,and the Western blot,ELISA and molecular interaction analysis and other methods confirmed that the obtained mAb had excellent binding specificity and binding activity with the nucleocapsid protein,the affinity constants of the 7E11 and 7E8 mAb with the nucleocapsid protein antigen was 1.69×10^(-9) and 2.031×10^(-9) mol/L,respectively,which could be used as antibody components in the field of rapid immunoassay of the SARS-CoV-2.Conclusion The recombinant antigen of the SARS-CoV-2 nucleocapsid protein and its monoclonal antibody with high specificity are obtained.