【摘要】 为了制备牛冠状病毒（Bovine coronavirus,BCoV）结构蛋白N的单克隆抗体，将N基因克隆到原核表达载体pCOLD I中，将构建好的重组质粒转化进大肠埃希氏菌BL21(DE3)中，诱导目的蛋白表达后纯化，用纯化的蛋白免疫小鼠制备杂交瘤细胞。对阳性杂交瘤细胞进行3次亚克隆后，用筛选鉴定正确的阳性杂交瘤细胞，腹腔注射小鼠制备腹水并进行初步纯化，利用Western blot检测腹水中抗体的特异性，利用间接免疫荧光试验（IFA）检测腹水中抗体的反应性。Western blot、IFA显示制备的腹水中抗体的特异性和反应性均良好。成功制备出一株能稳定分泌牛冠状病毒N蛋白单克隆抗体的杂交瘤细胞，为进一步研究牛冠状病毒和N蛋白的功能提供了材料。更多还原

【Abstract】 In order to prepare monoclonal antibodies against bovine coronavirus(BCoV)structural protein N,the N gene was cloned into the prokaryotic expression vector pCOLD I,the constructed recombinant plasmid was transformed into Escherichia coli BL21(DE3),the expressed target protein was purified,and mice were immunized with purified protein to prepare hybridoma cells.After three subcloning experiments of positive hybridoma cells,ascites were prepared from mice with selected hybridoma cells,ascites reactivity was detected by Western blot,and ascites reactivity was determined by indirect immunofluorescence assay(IFA).Western blot and IFA showed the good reactivity and potency of ascites.A hybridoma cell line can stably secrete monoclonal antibody against bovine coronavirus N protein,which provides materials for further study of the function of bovine coronavirus and N protein.更多还原