布鲁氏菌VirB12蛋白纯化及其抗体ELISA方法的初步建立
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摘要
为获得高纯度布鲁氏菌VirB12蛋白,对VirB12分泌表达蛋白先后进行亲和纯化和离子交换纯化。将其作为抗原包被ELISA板,优化反应条件后初步建立了布鲁氏菌抗体的ELISA检测方法。结果表明:蛋白纯化效果良好,重组蛋白纯度可达到98%以上。在建立的ELISA中,纯化重组蛋白的最佳包被浓度为1μg·mL-1,最适封闭剂为5%脱脂奶粉,血清最佳稀释浓度为1∶25,酶标二抗最佳工作浓度为1∶1500。对20份阳性血清及12份阴性血清的检测结果表明,其与SUANOVIR Brucella-Ab C-ELISA试剂盒的总体符合率达到90.6%,表明本试验建立的以VirB12重组蛋白为抗原的ELISA方法可以检测布氏杆菌感染抗体,有望研制成试剂盒用于养殖场布病的检测和净化。
To obtain high purity VirB12 protein of Brucella, affinity purification and ion-exchange purification were conducted.The purified recombinant VirB12 protein was used for coating antigen to establish the ELISA method for the detection of Brucella antibodiesin after optimizing the reaction conditions. The results showed that the purity of the recombined protein could reach more than 98% after purification. In the established ELISA, the optimum coating concentration of purified protein was1 μg·m L-1, the optimum sealing solution was 5% skimmed milk powder, the optimum dilution of serum was 1 ∶25 and the optimum working dilution of enzyme-labeled secondary antibody was 1 ∶1500. Detection results of 20 positive sera and 12 negative sera indicated that the coincidence rate between the established ELISA method and SUANOVIR Brucella-Ab C-ELISA kit was 94.7%. The results showed that the ELISA method using VirB12 protein as antigen can detect Brucella antibody and be applied to the detection of brucellosis in farms.
To obtain high purity VirB12 protein of Brucella, affinity purification and ion-exchange purification were conducted.The purified recombinant VirB12 protein was used for coating antigen to establish the ELISA method for the detection of Brucella antibodiesin after optimizing the reaction conditions. The results showed that the purity of the recombined protein could reach more than 98% after purification. In the established ELISA, the optimum coating concentration of purified protein was1 μg·m L-1, the optimum sealing solution was 5% skimmed milk powder, the optimum dilution of serum was 1 ∶25 and the optimum working dilution of enzyme-labeled secondary antibody was 1 ∶1500. Detection results of 20 positive sera and 12 negative sera indicated that the coincidence rate between the established ELISA method and SUANOVIR Brucella-Ab C-ELISA kit was 94.7%. The results showed that the ELISA method using VirB12 protein as antigen can detect Brucella antibody and be applied to the detection of brucellosis in farms.
引文
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[14]马晓菁,易新萍,付湘芸,等.羊种布鲁氏菌Rev.1疫苗株Vir B12基因缺失株的构建及鉴定[J].中国预防兽医学报,2017,39(10):794-798.
[15] MARCHESINI MI,HERRMANN CK,SALCEDO SP,et al.In search of Brucella abortus typeIV secretion substrates:screening and identification of four proteins translocation into host cells through Vir B system[J]. CellMicrobiol,2011,13(8):1261-1274.
[16] MARCHESINI M I,HERRMANN C K,SALCEDO S P,et al.In search of Brucella abortus type IV secretion substrates:screening and identification of four proteins translocated into host cells through Vir B system[J].Cell Microbiol,2011,1(8):1261-1274.
[17] WANG Y,CHEN Z,QIAO F,et al.The type IV secretion system affects the expression of Omp25/Omp31 and the outer membrane properties of Brucella melitensis[J].FEMS Microbiol Lett,2010,303(1):92-100.
[18] BOSCHIROLI M L,OUAHRANI-BETTACHE S,FOULONGNE V,et al.The Brucella suis vir B operon is induced intracellularly in macrophages[J].Proc Natl Acad Sci U S A,2002,99(3):1544-1549.
[19]易新萍,叶锋,姚刚,等.牛布鲁氏菌A19突变株的构建及在BALB/c鼠中的免疫保护评估[J].微生物学报,2013,53(11):1213-1220.
[2]周莉,候权书,黄诚,等.布鲁氏菌病的危害与个人防护[J].中国动物检疫,2016,33(6):52-54,76.
[3] AL D S,TOMASO H,NCKLER K,et al.Laboratory-based diagnosis of brucellosis-a review of the literature.Part I:Techniques for direct detection and identification of Brucella spp.[J].Clin Lab,2003,49(9-10):487-505.
[4] KOYUNCU I,KOCYIGIT A,OZER A,et al.Diagnostic potential of Brucella melitensis Rev1 native Omp28 precursor in human brucellosis[J].Cent Eur J Immunol,2018,43(1):81-89.
[5] MOHSENI K,MIRNEJAD R,PIRANFAR V,et al.A comparative evaluation of ELISA,PCR,and serum agglutination tests for diagnosis of Brucella using human serum[J].Iran J Pathol,2017,12(4):371-376.
[6] PROCH V,SINGH B B,SCHEMANN K,et al.Risk factors for occupational Brucella infection in veterinary personnel in India[J].Transbound Emerg Dis,2018,65(3):791-798.
[7] ROLN H G,TSOLIS R M.Mice lacking components of adaptive immunity show increased Brucella abortus virB mutant colonization[J].Infect Immun,2007,75(6):2965-2973.
[8] ROUX C M,ROLN H G,SANTOS R L,et al.Brucella requires a functional Type IV secretion system to elicit innate immune responses in mice[J].Cell Microbiol,2007,9(7):1851-1869.
[9] ROLN H G,TSOLIS R M.Inactivation of the type IV secretion system reduces the Th1 polarization of the immune response to Brucella abortus infection[J].Infect Immun,2008,76(7):3207-3213.
[10] SUN Y H,ROLN H G,den HARTIGH AB,et al.Brucella abortus vir B12 is expressed during infection but is not an essential component of the type IV secretion system[J].Infect Immun,2005,73(9):6048-6054.
[11] ROLN H G,den HARTIGH AB,KAHL-MCDONAGH M,et al.VirB12 is a serological marker of Brucella infection in experimental and natural hosts[J].Clin Vaccine Immunol,2008,15(2):208-214.
[12] YI X,YE F,YAO G,et al.Construction of Brucella abortus A19-delta Vir B12 mutant and evaluation of its protective efficacy against 2308 strain challenge in BALB/c mice[J].2013,53(11):1213-1220.
[13]易新萍,谷文喜,吴冬玲,等.流产布鲁氏菌疫苗A19-ΔVir B12突变株生物学特性研究[J].中国人兽共患病学报,2013,29(9):836-840.
[14]马晓菁,易新萍,付湘芸,等.羊种布鲁氏菌Rev.1疫苗株Vir B12基因缺失株的构建及鉴定[J].中国预防兽医学报,2017,39(10):794-798.
[15] MARCHESINI MI,HERRMANN CK,SALCEDO SP,et al.In search of Brucella abortus typeIV secretion substrates:screening and identification of four proteins translocation into host cells through Vir B system[J]. CellMicrobiol,2011,13(8):1261-1274.
[16] MARCHESINI M I,HERRMANN C K,SALCEDO S P,et al.In search of Brucella abortus type IV secretion substrates:screening and identification of four proteins translocated into host cells through Vir B system[J].Cell Microbiol,2011,1(8):1261-1274.
[17] WANG Y,CHEN Z,QIAO F,et al.The type IV secretion system affects the expression of Omp25/Omp31 and the outer membrane properties of Brucella melitensis[J].FEMS Microbiol Lett,2010,303(1):92-100.
[18] BOSCHIROLI M L,OUAHRANI-BETTACHE S,FOULONGNE V,et al.The Brucella suis vir B operon is induced intracellularly in macrophages[J].Proc Natl Acad Sci U S A,2002,99(3):1544-1549.
[19]易新萍,叶锋,姚刚,等.牛布鲁氏菌A19突变株的构建及在BALB/c鼠中的免疫保护评估[J].微生物学报,2013,53(11):1213-1220.
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