牛乳腺炎无乳链球菌PI-2a菌毛岛屿BP亚基单克隆抗体胶体金标记试纸条制备  

Preparation of colloidal gold-labeled strip of monoclonal antibody against BP subunit of PI-2a pili islands of bovine mastitis Streptococcus agalactiae

作　　者：布日额 吴金花[1,2,3] 锡林高娃 陈金龙[4] 王金良 BU Ri-e;WU Jin-hua;XI Lin-gao-wa;CHEN Jin-long;WANG Jin-liang(Inner Mongolia Autonomous Region Engineering Technology Research Center of Prevention And Control the Pathogenic Bacteriain Milk,Tongliao 028043,China;College of Life Science,Inner Mongolia University for Nationalities,Tongliao 028043,China;Research Institute for Pathogenic in Milk of Inner Mongolia University for Nationalities,Tongliao 028043,China;Shandong Lvdu Bio-sciences and Technology Limited company,Binzhou 256600,China)

机构地区：[1]内蒙古自治区乳源性致病菌防控工程技术研究中心,通辽028043 [2]内蒙古民族大学生命科学学院,通辽028043 [3]内蒙古民族大学乳源性致病菌研究所,通辽028043 [4]山东绿都生物科技有限公司,滨州256600

出　　处：《中国人兽共患病学报》2020年第1期45-49,共5页Chinese Journal of Zoonoses

基　　金：内蒙古自治区科技厅社会发展领域计划项目（No.kjJH1701）;国家自然科学基金项目（No.31560689）;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（No.MDK2018005、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~~

摘　　要：目的本研究通过优化牛乳腺炎无乳链球菌PI-2a菌毛岛屿BP亚单位单克隆抗体的性能,制备胶体金标记检测牛乳腺炎无乳链球菌的试纸条。方法利用pET30a(+)-BP重组质粒表达蛋白纯化物免疫小鼠,无菌取脾细胞与sp2/0骨髓瘤细胞融合,筛选杂交瘤细胞,制备单克隆抗体,对单克隆抗体进行胶体金标记后制备检测试剂盒,并确定其灵敏度、特异性等技术指标。结果经数轮筛选,获得1株阳性杂交瘤细胞株,抗体效价达到1∶25600。胶体金标记试纸条灵敏度量达到10~6 CFU/mL。特异性测试结果表明,胶体金标记试纸条与金黄色葡萄球菌、大肠杆菌、沙门氏菌、副结核杆菌、化脓性链球菌培养物人工污染的牛乳样均无阳性反应,只与无乳链球菌乳样出现阳性结果。结论该胶体金标记免疫层析试纸条,具有实际应用价值,将为牛乳腺炎的筛查检测提供新型、便捷的技术产品。In this experiment, by optimizing performance of the monoclonal antibody against PI-2 a pili island BP subunit of Streptococcus agalactiae, the colloidal gold labeling for the detection of S.agalactiae of mastitis was prepared. The mice were firstly immunized with purified protein expressed from pET30 a(+)-BP recombinant plasmid. Then mice spleen cells were taken aseptically for fusion with sp2/0 myeloma cells. The hybridoma cells were screened and monoclonal antibodies were prepared. The monoclonal antibody was labeled with colloidal gold for the detection kit preparation, and its sensitivity and specificity were evaluated. After screening, a positive hybridoma cell line was obtained, and the antibody titer reached 1∶256 00. The sensitivity measurement of colloidal gold marking strip reached 10~6 CFU/mL. Specificity test results showed that there was no positive reaction between colloidal gold label test strip and bovine milk samples artificially contaminated by S. aureus, Escherichia coli, Salmonella paraturium, Mycobacterium paratuberculosis and Streptococcus suppurate. The positive results were only found in the milk samples of Streptococcus agalactiea. In conclusion, the colloidal gold labeled immune-chromatographic strip has practical application value and will provide a new and convenient technical product for the screening and detection of bovine mastitis.

关 键 词：无乳链球菌 BP基因 单克隆抗体 胶体金 

分 类 号：R378.1[医药卫生—病原生物学]