布鲁氏菌OMP25与OMP31蛋白的表达及其间接ELISA诊断试剂盒研制
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摘要
目的为建立布鲁氏杆菌间接ELISA检测方法。方法本研究根据GenBank中已登录猪种布鲁氏菌S2株全基因组序列,分别针对omp25和omp31设计一对引物,分别扩增并回收573 bp与783 bp目的基因片段,将其分别克隆入pET-28a原核表达载体中,构建重组质粒pET-OMP25与pET-OMP31,进行酶切鉴定与基因序列测定后,转化BL21表达菌,经IPTG诱导表达,OMP25与OMP31蛋白主要以包涵体形式存在;将目的蛋白纯化,应用Western blot进行蛋白活性的检测。结果纯化的目的蛋白具有良好的免疫学活性。以纯化的重组蛋白为包被抗原,在确定了OMP25与OMP31蛋白最佳配比为4∶1、抗原最佳包被浓度为10μg/mL、血清的最佳稀释倍数为1∶50倍稀释、临界值为0.314的基础上,建立羊布鲁氏菌病抗体间接ELISA诊断方法并组装成诊断试剂盒,并将试剂盒进行了特异性、敏感性、重复性、符合率试验、血清交叉反应性等检测。结论研制的羊布鲁氏菌病抗体ELISA诊断试剂盒具有良好的特异性、敏感性,可重复性,可应用于临床样本检测。
The primers were designed according to the OMP25 and OMP31 gene sequence from the genome of Brucella suis S2 strain in GenBank, and the 573 bp and 783 bp predicted gene fragments were amplified and cloned respectively into a prokaryotic expression vector pET-28. The recombinant plasmid pET-OMP25 and pET-OMP31 were sucessfully constructed and transformed to bacterium BL21. inducted by IPTG, the recombinant OMP25 and OMP31 protein were expressed mainly in the form of inclusion body. The target proteins were purified and loaded to SDS-PAGE gel for Western blot analysis. The results showed that the purified proteins had good immunological activity. Coating with the two recombinant proteins, the conditions of an indirect ELISA tests were optimized as follows: the best ratio of OMP31 and OMP25 proteins is 4∶1, the antigen concentration is 10μg/mL, the best dilution of serum is 1∶50, and the critical value is 0.314. based on the conditions above, the sheep brucellosis antibody indirect ELISA diagnostic method was established, the diagnostic kit was assembled and further tested for specificity, sensitivity, repeatability, coincidence rate, cross reactivity, etc. In conclusion, the sheep brucellosis antibody ELISA diagnostic kit has good specificity,sensitivity, and repeatability, and it can reach the quality of similar commercial kits. Therefore, it can be applied in field detection.
The primers were designed according to the OMP25 and OMP31 gene sequence from the genome of Brucella suis S2 strain in GenBank, and the 573 bp and 783 bp predicted gene fragments were amplified and cloned respectively into a prokaryotic expression vector pET-28. The recombinant plasmid pET-OMP25 and pET-OMP31 were sucessfully constructed and transformed to bacterium BL21. inducted by IPTG, the recombinant OMP25 and OMP31 protein were expressed mainly in the form of inclusion body. The target proteins were purified and loaded to SDS-PAGE gel for Western blot analysis. The results showed that the purified proteins had good immunological activity. Coating with the two recombinant proteins, the conditions of an indirect ELISA tests were optimized as follows: the best ratio of OMP31 and OMP25 proteins is 4∶1, the antigen concentration is 10μg/mL, the best dilution of serum is 1∶50, and the critical value is 0.314. based on the conditions above, the sheep brucellosis antibody indirect ELISA diagnostic method was established, the diagnostic kit was assembled and further tested for specificity, sensitivity, repeatability, coincidence rate, cross reactivity, etc. In conclusion, the sheep brucellosis antibody ELISA diagnostic kit has good specificity,sensitivity, and repeatability, and it can reach the quality of similar commercial kits. Therefore, it can be applied in field detection.
引文
[1] 赵建东,解松林,项朝荣,等.山羊不同途径免疫S2株布鲁氏菌病活疫苗后的抗体消长规律[J].黑龙江畜牧兽医,2018(4):136-137.
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[4] Tukana A,Warner J,Hedlefs R,et al.The history of brucellosis in the Pacific Island Countries and Territories and its ree-mergence[J].Prev Vet Med,2015,122(1/2):14-20.
[5] Peck D,Bruce M.The economic efficiency and equity of government policies on brucellosis:comparative insights from Albania and the United States of America[J].Rev Sci Tech,2017,36(1):291-302.
[6] Paul S,Peddayelachagiri BV,Nagaraj S,et al.Recombinant outer membrane protein 25c from Brucella abortus induces Th1 and Th2 mediated protection against Brucella abortus infection in mouse model[J].Mol Immunol,2018,99:9-18.
[7] Yousefi S,Abbassi-Daloii T,Sekhavati MH,et al.Evaluation of immune responses induced by polymeric OMP25-BLS Brucella antigen[J].Microb Pathog,2018,115:50-56.
[8] Li J,Hu F,Chen S,et al.Characterization of novel Omp31 antigenic epitopes of Brucella melitensis by monoclonal antibodies[J].BMC Microbiol,2017,17(1):115.
[9] Zheng WY,Wang Y,Zhang ZC,et al.Immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody[J].Genet Mol Res,2015,14(4):11965-11974.
[10] Ducrotoy MJ,Muňoz PM,Conde-álvarez R,et al.A systematic review of current immunological tests for the diagnosis of cattle brucellosis[J].Prev Vet Med,2018,151:57-72.
[11] Cosford KL.Brucella canis:An update on research and clinical management[J].Can Vet J,2018,59(1):74-81.
[2] Olsen SC,Palmer MV.Advancement of knowledge of Brucella over the past 50 years[J].Vet Pathol,2014,51(6):1076-1089.
[3] Viana M,Shirima GM,John KS,et al.Integrating serological and genetic data to quantify cross-species transmission:brucellosis as a case study[J].Parasitology,2016,3:1-14.
[4] Tukana A,Warner J,Hedlefs R,et al.The history of brucellosis in the Pacific Island Countries and Territories and its ree-mergence[J].Prev Vet Med,2015,122(1/2):14-20.
[5] Peck D,Bruce M.The economic efficiency and equity of government policies on brucellosis:comparative insights from Albania and the United States of America[J].Rev Sci Tech,2017,36(1):291-302.
[6] Paul S,Peddayelachagiri BV,Nagaraj S,et al.Recombinant outer membrane protein 25c from Brucella abortus induces Th1 and Th2 mediated protection against Brucella abortus infection in mouse model[J].Mol Immunol,2018,99:9-18.
[7] Yousefi S,Abbassi-Daloii T,Sekhavati MH,et al.Evaluation of immune responses induced by polymeric OMP25-BLS Brucella antigen[J].Microb Pathog,2018,115:50-56.
[8] Li J,Hu F,Chen S,et al.Characterization of novel Omp31 antigenic epitopes of Brucella melitensis by monoclonal antibodies[J].BMC Microbiol,2017,17(1):115.
[9] Zheng WY,Wang Y,Zhang ZC,et al.Immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody[J].Genet Mol Res,2015,14(4):11965-11974.
[10] Ducrotoy MJ,Muňoz PM,Conde-álvarez R,et al.A systematic review of current immunological tests for the diagnosis of cattle brucellosis[J].Prev Vet Med,2018,151:57-72.
[11] Cosford KL.Brucella canis:An update on research and clinical management[J].Can Vet J,2018,59(1):74-81.