摘要
为建立布鲁菌的快速检测方法,通过引物设计、PCR扩增、表达载体构建、SDS-PAGE和Western-blot分析对牛布鲁菌3种外膜蛋白OMP19、OMP22、OMP28基因进行原核表达,利用亲和层析法纯化3种重组蛋白,将3种重组蛋白混合作为包被抗原,经重复性、灵敏度和特异性试验建立了布鲁菌抗体检测间接ELISA,并对300份牛血清样品进行检测应用,同时使用进口的商品化试剂盒进行平行检测。结果显示,成功构建了p Cold-TF/OMP19、p Cold-TF/OMP22和p Cold-TF/OMP28原核表达载体,诱导后分别表达大小分别为69、74和80 ku的可溶性且具有良好反应原性的重组蛋白r OMP19、r OMP22和r OMP28。基于此3种重组蛋白混合抗原建立的间接ELISA具有良好的重复性和特异性,其灵敏度达1∶1 600。临床样品检测试验结果显示,与进口试剂盒总符合率高达96%(288/300),其中阳性符合率为93.5%(58/62),阴性样品符合率为96.64%(230/238)。将3种重组外膜蛋白联合作为包被抗原进行牛布鲁菌抗体检测,为我国牛布鲁菌病的诊断检测技术相关研究提供了新的思路和参考。
In order to establish a rapid method for the detection of brucellosis ,we expressed three kinds of Brucella abortus outer membrane proteins OMP19, OMP22, OMP28 in prokaryotic cells by carrying out the experiments of primer design,PCR amplification,expression vector construction,SDS-PAGE and Western blot analysis and utilized affinity chromatography to purify three recombinant proteins.The purified proteins were combined as coating antigens ,then we established Brucella abortus antibody detection indirect ELISA by carrying out the experiments of reproducibility, sensitivity and specificity,300 of bovine serum samples were tested,while the samples were tested by the imported commercial kits in parallel.The results showed that we successfully constructed the pCold-TF/OMP19,pCold-TF/OMP22 and pCold-TF/OMP28 prokaryotic expression vector,respectively,induced and expressed soluble recombinant proteins with a size of 69 ku, 74 ku and 80 ku which had a good reactogenicity.The indirect ELISA based on the three kinds of recombinant proteins showed good reproducibility,specificity and the sensitivity up to 1 ： 1 600.The results of clinical test showed the total coincidence rate was 96% （ 288/300）,in which the positive rate was 93.5% （58/62） and the negative rate was 96.64% （230/238）.The study is an attempt to utilize rOMPs as coating antigens ,to develop a new way in the serodiagnosis of brucellosis.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2016年第8期946-952,共7页
Chinese Veterinary Science
基金
质检公益性行业科研专项(201510017)
四川省科技计划项目(2016NZYZF0059)
关键词
牛布鲁菌
外膜蛋白
重组抗原
间接ELISA
Brucella abortus
outer membrane protein
recombinant antigens
indirect ELISA
