| 牛分枝杆菌MPB70蛋白单克隆抗体的制备及检测 |
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| 引用本文： | 周曼莉,周玉成,张海威,乔连江,杨艳玲.牛分枝杆菌MPB70蛋白单克隆抗体的制备及检测[J].中国畜牧兽医,2019,46(10):3075-3083. |
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| 作者姓名： | 周曼莉 周玉成 张海威 乔连江 杨艳玲 |
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| 作者单位： | 1. 中国农业科学院特产研究所, 长春 130112;2. 宁夏大学, 西部特色生物资源保护与利用教育部重点实验室, 银川 750021 |
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| 基金项目： | “十三五”重点研发项目“牛羊重要病原分子诊断新技术”（2016YFD00500900）；宁夏回族自治区重点研发计划项目（东西部合作）“牛羊重要传染病快速诊断与检测关键技术研究”（2017BN041） |
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| 摘 要： | 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9.95×109和9.53×108；抗体效价分别为1∶102 400和1∶12 800；抗体亚类分别为IgG2b和IgG1，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。
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| 关 键 词： | 结核分枝杆菌 MPB70蛋白 单克隆抗体 |
| 收稿时间： | 2019-05-05 |
Preparation and Detection of Monoclonal Antibodies Against Mycobacterium bovis MPB70 Protein |
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| ZHOU Manli,ZHOU Yucheng,ZHANG Haiwei,QIAO Lianjiang,YANG Yanling.Preparation and Detection of Monoclonal Antibodies Against Mycobacterium bovis MPB70 Protein[J].China Animal Husbandry & Veterinary Medicine,2019,46(10):3075-3083. |
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| Authors: | ZHOU Manli ZHOU Yucheng ZHANG Haiwei QIAO Lianjiang YANG Yanling |
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| Institution: | 1. Institute of Special Animal and Plant Sciences of CAAS, Changchun 130112, China;2. Key Laboratory of Western Characteristic Biological Resources Protection and Utilization, Ministry of Education, Ningxia University, Yinchuan 750021, China |
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| Abstract: | 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 9.95×109 and 9.53×108,respectively;The antibody titers were 1:102 400 and 1:12 800,respectively;The antibody subclasses were IgG2b and IgG1,respectively,and the light chain types were κ type.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. |
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| Keywords: | Mycobacterium tuberculosis MPB70 protein monoclonal antibody |
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