牛结核分枝杆菌MPB70+83MAb的制备及胶体金免疫层析试纸条的初步研制  

Preparation of monoclonal antibody colloidal gold immunochromatographic test strip for Mycobacterium bovis MPB70+83 fusion protein

作　　者：叶俊 布日额[1,2,3,4] 吴金花 锡林高娃[1,2,3,4] 陈金龙 王金良 刘思国[6] 崔子寅[6] 王思珍[1,2] YE Jun;BU Ri-e;WU Jin-hua;XI Lin gao-wa;CHEN Jin-long;WANG Jin-liang;LIU Si-guo;CUI Zi-yin;WANG Si-zhen(Animal Science college,Inner Mongolia University for Nationalities,Tongliao 028043,China;College of Life Science,Inner Mongolia University for Nationalities,Tongliao 028043,China;Inner Mongolia Autonomous Region Engineering Technology Research Center of Prevention and Control the Pathogenic Bacteria in Milk,Tongliao 028043,China;Research Institute for Pathogenic in Milk of Inner Mongolia University for Nationalities,Tongliao 028043,China;Shandong Binzhou Animal Science and Veterinary Medicine Academy,Binzhou 256600,China;Harbin Institute of Veterinary Medicine,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[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

出　　处：《中国预防兽医学报》2019年第11期1131-1136,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：内蒙古自治区科技创新引导项目(批准号：KCBJ2018026);内蒙古自治区乳源性致病菌防控工程技术研究中心开放课题(MDK2018004;MDK2018001);内蒙古自治区第七批“草原英才”人才工程产业创新团队建设项目(第七批)

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p ET30a(+)-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∶12800,1∶12800,1∶25600。制备的腹水MAb均经纯化后浓度分别为2.1 mg/m L、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∶2500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。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 39 ku,the purity was more than 95%.Three positive hybridoma cell lines were obtained from MPB70+83 immunized mice,namely 4 H6,5 A2 and 4 D11 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/m L and 2.5 mg/m L,respectively.MAb production maintains stable after fifteen serial passage of hybridoma cells.To develop the test strip,MAb 4 D11 was used to label gold particles and MAb 4 H6 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.

关 键 词：结核分枝杆菌 MPB70 mpb83 单克隆抗体 

分 类 号：S852.61[农业科学—基础兽医学]