[1]叶 俊,布日额*,吴金花,等.牛结核分枝杆菌MPB70+83 MAb的制备及胶体金免疫层析试纸条的初步研制[J].中国预防兽医学报,2019,(11):1131-1136.[doi:0.3969/j.issn.1008-0589.201904021]
 YE Jun,BU Ri-e*,WU Jin-hua,et al.Preparation of monoclonal antibody colloidal gold immunochromatographic test strip for Mycobacterium bovis MPB70+83 fusion protein[J].Chinese journal of preventive veterinary medicine,2019,(11):1131-1136.[doi:0.3969/j.issn.1008-0589.201904021]
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牛结核分枝杆菌MPB70+83 MAb的制备及胶体金免疫层析试纸条的初步研制()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年11
页码:
1131-1136
栏目:
诊断和检测技术
出版日期:
2019-12-25

文章信息/Info

Title:
Preparation of monoclonal antibody colloidal gold immunochromatographic test strip for Mycobacterium bovis  MPB70+83 fusion protein
文章编号:
1008-0589(2019)11-1131-06
作者:
 

叶 俊1234布日额1234*吴金花1234锡林高娃1234陈金龙5王金良5刘思国6崔子寅6王思珍12*

 (1. 内蒙古民族大学 动物科技学院,内蒙古 通辽 028043;2. 内蒙古民族大学 生命科学学院,内蒙古 通辽 028043;
3. 内蒙古自治区乳源性致病菌防控工程技术研究中心,内蒙古 通辽 028043;4. 内蒙古民族大学 乳源性致病菌研究所,内蒙古
通辽 028043;5. 山东省滨州畜牧兽医研究院,山东 滨州 256600;6. 中国农业科学院 哈尔滨兽医研究所,黑龙江 哈尔滨 150069)
Author(s):
 

YE Jun1234 BU Ri-e1234* WU Jin-hua1234 XI Lin gao-wa1234 CHEN Jin-long5WANG Jin-liang5 LIU Si-guo6 CUI Zi-yin6 WANG Si-zhen12*

 (1. Animal Science college, Inner Mongolia University for Nationalities, Tongliao 028043, China; 2. College of Life Science, Inner Mongolia University for Nationalities, Tongliao 028043, China; 3. Inner Mongolia Autonomous Region Engineering Technology Research
Center of Prevention and Control the Pathogenic Bacteria in Milk, Tongliao 028043, China;
4. Research Institute for Pathogenic in Milk of Inner Mongolia University for Nationalities, Tongliao 028043, China;
5. Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, China;
6. Harbin Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences, Harbin 150069, China)
关键词:
 结核分枝杆菌mpb70mpb83单克隆抗体
Keywords:
Mycobacterium bovis  MPB70  MPB83  monoclonal antibody
分类号:
S852.61
DOI:
0.3969/j.issn.1008-0589.201904021
文献标志码:
A
摘要:
为制备牛结核分枝杆菌(MB)感染早期分泌性蛋白MPB70+83的胶体金标记免疫层析试剂条,本研究将构建的重组大肠杆菌pET30a(+)-MPB70+83/BL21诱导表达,经纯化后免疫小鼠,制备MPB70+83融合蛋白的单克隆抗体(MAb),对MAb标记胶体金后制备检测试纸,并确定其特异性、灵敏度等技术指标。结果显示,获得分子量为39 ku的MPB70+83融合蛋白,经纯化后重组蛋白的纯度>95 %,经4次免疫BALB/c小鼠后,最终获得3株阳性杂交瘤细胞株,分别命名为4H6、5A2、4D11,小鼠腹水MAb效价分别达到1∶12 800,1∶12 800,1∶25 600。制备的腹水MAb均经纯化后浓度分别为2.1 mg/mL、1.8 mg/mL、2.5 mg/mL。对杂交瘤细胞经15代培养,MAb均具有良好的稳定性。利用4D11 MAb标记金颗粒,4H6 MAb包被检测线,并对不同培养物人工污染健康牛血清样品进行检测,结果显示,胶体金试纸对人工污染MB培养物的健康牛血清出现阳性反应,对无乳链球菌、金黄色葡萄球菌、大肠杆菌、沙门氏菌、副结核杆菌培养物人工污染的健康牛血清的检测均为阴性,最低可以检测到1∶2 500倍稀释的MB培养上清。本研究为牛结核分枝杆菌感染的快速检测提供可行技术手段。
Abstract:
Abstract: In this study, the colloidal gold immunochromatographictest strip was developed to detect secretory protein MPB70+MPB83 in early stage of Mycobacterium bovis (MB) infection. The prokaryotic fusion protein MPB70+83 was expressed and purified in BL21 E.coli cells. Monoclonal antibody (MAb) against MPB70+83 was prepared in the ascites of MPB70+83 immunized mice. After MAb was labeled with colloidal gold, the test strip was prepared and its specificity, sensitivity and repeatability were evaluated. The results showed that MB70+83 fusion protein had a molecular weight of 39ku, the purity was more than 95%. Three positive hybridoma cell lines were obtained from MPB70+83 immunized mice, namely 4H6, 5A2 and 4D11 respectively. The MAb titers of the cell culture supernatants of the three hybridoma cells were 1∶1,024, 1∶1,024, 1∶2,048, respectively. MAb titers in ascites of mice reached 1∶12,800, 1∶12,800 and 1∶25,600, respectively. The concentration of MAb in purified ascites was 2.1 mg/mL, 1.8 mg/mL and 2.5 mg/mL, respectively. MAb production maintains stable after fifteen serial passage of hybridoma cells. To develop the test strip, MAb 4D11 was used to label gold particles and MAb 4H6 was used to coat the detection line. Results showed that colloidal gold test strip successfully detected the MB antigen in bovine serum with the lower detection limit of 1∶2,500. While the test strip was negative for Streptococcus agalactis, Staphylococcus aureus, Escherichia coli, Salmonella and Paratuberculosis antigen containing bovine serum. Our results provide a useful tool for rapid detection of Mycobacterium tuberculosisearly infection in cattle.

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(责任编辑:彭永刚;英文编辑:温志远)

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更新日期/Last Update: 2019-12-09