摘要
为建立牛布鲁氏菌病血清学诊断方法,本研究以牛种布鲁氏菌B.abortus2308基因组为模板,PCR扩增其HSP70基因,构建重组表达质粒pET32a-HSP70,转化大肠杆菌BL21进行诱导表达并纯化,SDS-PAGE结果显示,得到重组HSP70蛋白,HSP70主要以包涵体形式表达。经条件优化建立以该蛋白为包被抗原的间接ELISA(iELISA)方法,结果显示,iELISA最佳反应条件为:抗原HSP70包被浓度为0.2μg/孔,血清稀释度为1200,封闭液为5%脱脂奶粉,兔抗牛IgG-HRP稀释度为14000,显色10 min;该iELISA方法的临界值为0.186。特异性试验结果显示,除布鲁氏菌病阳性血清外,该iELISA与IBR、BVD和FMD阳性血清无明显交叉反应;敏感性试验结果显示,血清1400稀释时仍能有效检出布鲁氏菌;重复性试验结果显示。批内批间变异系数均小于10%,重复性好。利用本实验建立的iELISA方法与传统的虎红凝集和试管凝集试验同时对血清样品进行检测,比分析检测结果,结果显示该iELISA方法与其它两者符合率较高。利用本方法对秦皇岛未进行布鲁氏菌病免疫的规模化肉牛养殖场血清样品进行检测,布病抗体阳性率为2.5%。本研究建立的iELISA为牛布鲁氏菌病的血清学诊断提供了可行方法。
To establish a serological method for diagnosis of Bovine Brucellosis,B.abortus2308 gene was used as template to amplify HSP70 gene,HSP70 protein was expressed mainly in the form of inclusion bodies in a prokaryotic system and obtain the purified protein.An indirect ELISA(iELISA)method for detecting Bovine Brucellosis was established using the recombinant HSP70 protein as coating antigen.The optimum reaction conditions were determined as follows:antigen coating concentration was0.2μg/well,serum dilution was 1200,blocking solution was 5%skimmed milk,secondary antibody dilution was 14000,and chromogenic reaction was 10 minutes.The critical value of iELISA was 0.186.The results of specificity test showed that there was no obvious cross reaction with the positive serum of IBR,BVD and FMD,sensitivity test showed that Brucella could be detected effectively when the serum was diluted with 1400.The variation coefficients of the intra-batch and inter-batch were lower than 10%,which indicated that the method was reproducible.This method showed a high coincidence rate with the traditional tiger red agglutination test and tube agglutination test.The method was used to detect Brucellosis antibody in Qinhuangdao beef cattle farm and positive rate was 2.5%.The i ELISA established in this study provides an important method for the serodiagnosis of Brucellosis.
作者
吴同垒
冀梦瑶
于秀剑
杜万年
张志强
吴清民
史秋梅
WU Tong-lei;JI Meng-yao;YU Xiu-jian;DUWan-nian;ZHANG Zhi-qiang;WU Qing-min;SHI Qiu-mei(Hebei Key Laboratory of Preventive Veterinary Medicine,Hebei Normal University of Science&Technology,Qinhuangdao 066004,China;China Agricultural University,Key Laboratory of Animal Epidemiology and Zoonosis of the Ministry of Agriculture,Beijing 100193,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2020年第4期366-370,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然基金(31902310)
秦皇岛市科学技术研究与发展计划项目(201803B006)
河北省重点研发计划(19226628D、19226629D)
