摘要
为建立检测流产布鲁氏菌(B.abortus)抗体的间接ELISA方法,本研究将表达该菌BvrS蛋白的重组质粒pET28a-BvrS转化大肠杆菌BL21(DE3)感受态细胞,经诱导后获得了可溶性表达的重组BvrS蛋白(rBvrS)。Western blot检测结果显示,BvrS能与布鲁氏菌阳性血清结合,具有较强的反应原性,可作为建立间接ELISA(iELISA)检测方法的包被抗原。采用棋盘滴定法对以该蛋白为包被抗原的iELISA方法各条件优化,结果显示,最优的反应条件为:BvrS包被浓度为3.125μg/mL,4℃包被过夜;商品化封闭液37℃封闭2 h;待检血清稀释度为1∶100,37℃孵育1 h;辣根过氧化酶(HRP)标记的兔抗鼠SPG稀释度为1∶8000,37℃孵育40 min。特异性试验结果显示,除布鲁氏菌阳性血清外,该iELISA与大肠杆菌、沙门氏菌、霍乱孤菌、军团菌、结核杆菌的阳性血清均无明显交叉反应;将牛布鲁氏菌阳性血清2倍倍比稀释(1∶50~1∶6400)后,利用建立的iELISA检测,结果显示,该iELISA方法对阳性血清的检测限为1∶400,敏感性高于试管凝集试验(SAT)检测结果(1∶100),该方法敏感性较高;分别利用同一批次和不同批次rBvrS包被的酶标板,按照本研究建立的方法对已知血清进行重复性试验,结果显示,批内批间重复性试验的变异系数均小于10%,该方法重复性较好。利用本实验建立的iELISA方法与国外商品化试剂盒ID-VET同时检测220份临床牛血清样品,结果显示,二者检测结果符合率为99.5%。本研究建立的iELISA方法为牛布鲁氏菌病的血清学诊断提供了可行方法。
In order to establish an indirect ELISA serological assay for Brucella abortus(B.abortus),the recombinant plasmid pET28a-BvrS expressing BvrS protein was transformed into E.coli BL21(DE3)receptor cells in this study,and the soluble expressed recombinant BvrS protein(rBvrS)was obtained after induction.Western blot results showed that BvrS showed good reactogenicity with brucellosis positive serum and could be used as an encapsulated antigen for the establishment of indirect ELISA(iELISA)assay.The conditions were optimized to establish an iELISA method using this protein as the encapsulated antigen.The optimal reaction conditions were as follows:BvrS encapsulation concentration of 3.125μg/mL,overnight encapsulation at 4℃;commercialized blocking solution at 37℃for 2 hours;serum to be tested at a dilution of 1∶100 and incubated at 37℃for 1 hour;horseradish peroxidase(HRP)-labeled rabbit anti-mouse SPG at a dilution of 1∶8000 and incubated at 37℃for 40 minutes.The results of the specificity test showed that the iELISA had no significant cross-reactivity with positive sera of E.coli,Salmonella,Cholera isolates,Legionella and Mycobacterium tuberculosis,except for brucellosis positive sera;the results of the sensitivity test showed that the detection of brucellosis positive sera was negative at 1∶200 dilution of the sera,indicating a high sensitivity;the results of the reproducibility test showed that the intra-batch and inter-batch coefficients of variation were less than 10%,which were good reproducible.The results of the clinical samples showed that the iELISA method established in this experiment for BvrS protein antibodies was 99.5%consistent with the results of the foreign commercial kit ID-VET.The iELISA method established in this study provides a feasible method for the serological diagnosis of bovine brucellosis.
作者
田雪
焉鑫
范颖
孙明军
南文龙
刘建柱
孙淑芳
胡莉萍
TIAN Xue;YAN Xin;FAN Ying;SUN Ming-jun;NAN Wen-long;LIU Jian-zhu;SUN Shu-fang;HU Li-ping(Division of zoonosis diseases surveillance,China Animal Health and Epidemiology Center,Qingdao 266032,China;College of Animal Science and Technology,Shandong Agricultural University,Taian 271000,China;Shandong Provincial Center for Animal Disease Control and Prevention,Jinan 250100,China;Key Laboratory of Animal Biosafety Risk Prevention and Control(South),Ministry of Agriculture and Rural Affairs,Qingdao 266032,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2023年第11期1135-1140,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
中心科技创新基金项目(DW2021002)
国家现代农业(奶牛)产业技术体系建设专项(CARS-36)。
