牛分枝杆菌MPB70蛋白单克隆抗体的制备及检测

Preparation and Detection of Monoclonal Antibodies Against Mycobacterium bovis MPB70 Protein

为了制备牛分枝杆菌的单克隆抗体,本试验利用制备的原核表达的牛分枝杆菌重要抗原蛋白MPB70作为免疫原皮下多点注射免疫BALB/c小鼠。利用细胞融合技术,经过5次亚克隆与筛选,取已免疫好的小鼠脾细胞与SP2/0骨髓瘤细胞在PEG3500的作用下进行细胞融合。筛选得到了2株分泌抗牛分枝杆菌MPB70蛋白的杂交瘤细胞,分别命名为Anti-B-MPB70:8和Anti-B-MPB70:12。采用间接ELISA方法对获得的单克隆抗体进行亲和常数检测、效价分析及亚类鉴定,通过SDS-PAGE凝胶试验对单克隆抗体进行纯度分析,并检测其浓度,通过Western blotting方法对单克隆抗体的反应特性进行鉴定。结果显示,纯化后的MPB70蛋白分子质量大小为20 ku,浓度为0.5 mg/mL。两株单克隆抗体的亲和常数分别为9.95×10^9和9.53×10^8;抗体效价分别为1∶102 400和1∶12 800;抗体亚类分别为IgG 2b和IgG 1,轻链类型均为κ型;纯度>90%;浓度分别为3.0和2.2 mg/mL;且具有良好的反应特性。以上研究结果表明,本试验成功制备了抗MPB70蛋白单克隆抗体,可为牛结核病病原和抗体检测技术研究奠定基础。

In order to prepare monoclonal antibodies against Mycobacterium bovis(M.bovis),this experiment was carried out by immunizing BALB/c mice by subcutaneous injection of the prokaryotic expression of M.bovis important antigen protein MPB70.Using cell fusion technology,after 5 subclones and screening,the spleen cells of the well-immunized mice and the myeloma cells SP2/0 were subjected to cell fusion under the action of PEG3500.Two hybridoma cells secreting anti-M.bovis MPB70 protein were screened and named as Anti-B-MPB70:8 and Anti-B-MPB70:12,respectively.The indirect ELISA method was used to detect the affinity constant of the collected ascites,the titer analysis and the subclass identification.The purity of the monoclonal antibody was analyzed by SDS-PAGE test,the concentration of monoclonal antibodies was detected and the reaction characteristics of monoclonal antibodies were identified by Western blotting.The results showed that the purified MPB70 protein had a molecular mass of 20 ku and a concentration of 0.5 mg/mL.The affinity constants of the two monoclonal antibodies were 9.95×10^9 and 9.53×10^8,respectively;The antibody titers were 1∶102 400 and 1∶12 800,respectively;The antibody subclasses were IgG 2b and IgG 1,respectively,and the light chain types wereκtype.The purity was greater than 90%;The concentrations were 3.0 and 2.2 mg/mL,respectively,and had good reactivity.The above results indicated that the monoclonal antibody against MPB70 protein had been successfully prepared,which could lay a foundation for the research of bovine tuberculosis pathogen and antibody detection technology.