基于奶牛乳腺炎无乳链球菌rSip-PGK-FbsA融合蛋白的间接ELISA检测方法的建立  

Establishment of indirect ELISA for bovine mastitis Streptococcus agalactiae based on rSip-PGK-FbsA fusion protein

作　　者：吴金花[1,2] 布日额[1,2] 王金良[3] 陈金龙 锡林高娃[1,2] 孙立杰[1,2] 王华[1,2] 王学理[2] 刘燕[1,2] 杜长智[1,2] 朝洛蒙[1,2] 于辰龙[1,2] 白文丽[1,2] 

机构地区：[1]内蒙古民族大学生命科学学院,内蒙古通辽028043 [2]内蒙古民族大学乳源性致病菌研究所,内蒙古通辽028043 [3]山东省滨州畜牧兽医研究院,山东滨州256600 [4]山东绿都生物科技有限公司,山东滨州256600

出　　处：《中国预防兽医学报》2016年第3期230-234,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金项目(31260600;31560689);内蒙古民族大学与通辽市政府合作项目(SXYB2012086)

摘　　要：为建立一种快速而准确的奶牛乳腺炎无乳链球菌抗体检测方法,本研究以原核表达并纯化的无乳链球菌的膜表面相关蛋白SIP、磷酸甘油激酶PGK及纤连蛋白FbsA 3种串联表达的融合蛋白rSip-PGK-FbsA为包被抗原,建立了检测奶牛无乳链球菌抗体的间接ELISA方法。该方法与化脓性链球菌阳性血清、大肠杆菌阳性血清、金黄色葡萄球菌阳性血清、表皮葡萄球菌阳性血清均无交叉反应,特异性良好;阳性血清稀释至1颐12 800仍检测为阳性,具有较高的敏感性;批内和批间试验变异系数均小于10%,重复性好。利用该方法与以SIP、PGK单一蛋白建立的ELISA方法对160份已知样品进行检测,结果显示,该方法的阳性符合率达到98.6%,高于SIP、PGK单一蛋白建立的ELISA方法的阳性符合率。对389份临床样品进行奶牛无乳链球菌抗体检测,其阳性检出率为46.53%。本研究建立的间接ELISA方法为奶牛无乳链球菌的快速检测提供了一种更加准确、可靠的血清学方法。Streptococcus agalactiae is the one of the important caused agent for mastitis in cow. To establish a rapid and sensitive assay for detection of the antibody against S.agalactiae, a indirect ELISA was developed with prokaryotic expressed three proteins （rSip-PGK-FbsA） of S.agalactiae as the coating antigen. The reacted condition was optimized, including 2 p,g/mL of coating rSip-PGK-FbsA, the 1：160 dilution of the serum to be detected, and detecting with 1：6,000 dilution of IgG-HRP. The results of specificity, sensitivity and repeatability tests showed that the assay had no reactions with the positive serum of S.pyo- genes, E.coli, S.aureus and S.epidermidis. The positive coincidence rate of this method was 98.6%, higher than that ELISA method coating with the SIP and PGK protein. This method had high sensitivity. The detection rate was 46.53% of 389 clinical samples. This indirect ELISA method is a more accurate and reliable serological method for the rapid detection of antibody against S.agalactiae.

关 键 词：无乳链球菌 多表位融合抗原rSip-PGK-FbsA 纯化 ELISA检测 

分 类 号：S852.61[农业科学—基础兽医学]