布鲁氏菌Omp16蛋白的原核表达与鉴定
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摘要
为实现布鲁氏菌Omp16蛋白在大肠杆菌中的高效表达,根据GenBank发表的Omp16(登录号为JF918760. 1)基因序列,在不改变Omp16蛋白氨基酸序列的情况下,根据大肠杆菌密码子偏好性优化基因序列,全基因合成Omp16基因,并将其克隆到pET28a(+)载体中,构建重组质粒pET28a(+)-Omp16,转化至BL21工程菌进行诱导表达,利用His-tag镍柱纯化Omp16重组蛋白,并对纯化后的重组蛋白进行鉴定。结果表明:当重组菌株pET28a (+)-Omp16/BL21(DE3)诱导条件为37℃、IPTG浓度为0. 5 mmol/L诱导6 h时,Omp16蛋白表达量最高; SDS-PAGE电泳显示经镍柱纯化后Omp16蛋白达到了电泳纯,经Western blot分析,目的蛋白具有抗原特异性,在21ku处出现阳性条带,与预期蛋白分子大小相符,因此成功在大肠杆菌BL21(DE3)中高效表达出布鲁氏菌Omp16重组蛋白,为后续单克隆抗体的制备和布鲁氏菌病的检测试剂的研发提供了参考。
In order to realize the Omp16 protein high expression in E. coli,this paper aimed to construct the recombinant plasmid with Omp16 gene of Brucella and prepare the Omp16 protein by prokaryotic expression system. The gene sequences of Omp16 were optimized and synthesized without changing the amino acid sequence of Omp16 protein. The Omp16 gene was amplified by PCR and cloned into the p ET28 a( +) vector to construct recombinant plasmid p ET28 a( +)-Omp16,which was transformed into E. coli BL21( DE3),then IPTG was used as an inducer to induce the expression of the fusion protein. The recombinant Omp16 protein was purified by Ni affinity chromatography,the protein was proved correct by SDS-PAGE,and the obtained Omp16 protein had immunogenicity by Westernblot. When the induction conditions of recombinant strain p ET28 a( +)-Omp16/BL21( DE3) was37 ℃ and IPTG concentration was 0. 5 mmol/L for 6 h,the expression of Omp16 protein was highest.SDS-PAGE electrophoresis showed that purified Omp16 protein by nickel-affinity chromatography column reached electrophoretic purity. Western blot analysis showed that the target protein had antigen specificity,there are positive bands at 21 ku,in line with the expected. The Omp16 protein was highly expressed in E. coli BL21( DE3),which provided an effective immunogen for detection of Brucella.
In order to realize the Omp16 protein high expression in E. coli,this paper aimed to construct the recombinant plasmid with Omp16 gene of Brucella and prepare the Omp16 protein by prokaryotic expression system. The gene sequences of Omp16 were optimized and synthesized without changing the amino acid sequence of Omp16 protein. The Omp16 gene was amplified by PCR and cloned into the p ET28 a( +) vector to construct recombinant plasmid p ET28 a( +)-Omp16,which was transformed into E. coli BL21( DE3),then IPTG was used as an inducer to induce the expression of the fusion protein. The recombinant Omp16 protein was purified by Ni affinity chromatography,the protein was proved correct by SDS-PAGE,and the obtained Omp16 protein had immunogenicity by Westernblot. When the induction conditions of recombinant strain p ET28 a( +)-Omp16/BL21( DE3) was37 ℃ and IPTG concentration was 0. 5 mmol/L for 6 h,the expression of Omp16 protein was highest.SDS-PAGE electrophoresis showed that purified Omp16 protein by nickel-affinity chromatography column reached electrophoretic purity. Western blot analysis showed that the target protein had antigen specificity,there are positive bands at 21 ku,in line with the expected. The Omp16 protein was highly expressed in E. coli BL21( DE3),which provided an effective immunogen for detection of Brucella.
引文
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[15] CLOECKAERT A,VIZCAINO N,PAQUET J Y,et al.Major outer membrane proteins of Brucella spp:past,present and future[J]. Veterinary Microbiology(Vet Mi-crobiol),2002,90(1/4):229-247.
[16]熊流新,黄健,张涛,等.布鲁氏菌外膜蛋白Omp16的原核表达、纯化及多克隆抗体制备[J].现代医药卫生,2018(5):32-37.
[17]王勇,南文龙,巩明霞,等.布鲁氏菌5种蛋白的原核表达及其作为间接ELISA检测用抗原的适用性比较[J].中国动物检疫,2015(10):72-75.
[18]王艺娟.家畜布鲁氏菌病的危害及防控措施[C]//福建省猪病学术研讨会论文集. 2016.
[19] WELLINGHAUSEN N,NOCKER K,SIGGE A,et al.Rapid Detection of Brucella spp. in Blood Cultures byFluorescence In Situ Hybridization[J]. Journal of Clini-cal Microbiology(J CLIN MICROBIOL),2006,44(5):1828.
[20]朱其太.用FPA试验诊断牛布鲁氏菌病[J].畜牧兽医科技信息,2000(5):62-66.
[21]张付贤,李晓艳,闫广谋,等.鉴别布鲁菌病自然感染动物与疫苗免疫动物胶体金检测试纸的研制[J].中国畜牧兽医,2009,36(7):79-82.
[22]张太翔,凌宗帅,赵立娜,等.基于Taq Man探针的牛布鲁氏菌实时荧光定量PCR的特异性鉴定[J].中国动物检疫,2011,28(11):29-31.
[23]徐军,陈创夫,刘建平,等.巢式PCR检测乳中布氏杆菌[J].畜牧与兽医,2008,40(4):67-69.
[2] PAPPAS G,PANAGOPOULOU L C,AKRITIDIS N. Bru-cella as a biological weapon[J]. Cell Mel Life Sci,2006,63:2229-2236.
[3] CLOECKAERT A,KERKHOFS P,LIMET J N. Antibodyresponse to Brucella outer membrane proteins in bovinebrucellosis; Immunoblotanalysis and competitive enzyme-linked immunosorbent assay using monoclonal antibodies[J]. J Clin Microbiol,1992,30:3168-3174.
[4] HUSON J M. Selection of protective epitopes for Brucellamelitensis by DNA Vaccination[J]. Infect Immun,2005,73(3):7297-7303.
[5] VIZCAINO N,CLOECKAERT A,ZYGMUNT M S. Mo-lecular characterization of a Brucella species large DNAfragment deleted in Brucella abortus strains:evidence fora locus involved in the synthesis of a polysaccharide[J].Infect Immun,1999,67(3):2700-2712.
[6]徐楠,李娜.布鲁氏菌病流行病学及诊治研究进展[J].中国实用医药,2010,5(20):246-247.
[7] BALDWIN C L,WINTER A J. Macrophages and Brucel-la[J]. Immunol Ser,1994,60:363-380.
[8]胡剑飞,崔步云,关平原,等.布鲁氏菌外膜蛋白的研究进展[J].疾病监测,2010,25(5):380-389.
[9] PASQUEVICH K A,ESTEIN S M,SAMARTINO C G,etal. Immunization with recombinant Brucella species outermembrane protein Omp16 or Omp19 in adjuvant inducesspecific CD4+and CD8+T cells as well as systemicand oral protection against Brucella abortus infection[J].Infection&Immunity(Infect Immun),2009,77(1):436.
[10] PASQUEVICH K A,GARCIA S C,CORIA LM,et al. Theprotein moiety of Brucella abortus outer membrane pro-tein 1 6 is a new bacterial pathogen-associated molecularpattern that activates dendritic cells in vivo,induces a Thlimmune response,and is a promising self-adjuvantingvaccine against systemic and oral acquired brucellosis[J]. J Immunol,2010,184(9):5200-5212.
[11]张福林.布鲁氏菌病的危害及其防治[J].中国畜牧兽医文摘,2012,28(9):92-93.
[12]江明静,赵世刚.布鲁氏菌病及其诊断和药物治疗研究进展[J].医学动物防制,2016,32(12):1372-1380.
[13] JSABIO Y,FARBER M. Expression of Babesia bovisrhoptry-associated protein 1(RAP1)in Brucella abortusSI9[J]. Microbes Infection,2008,10:635-641.
[14]赵忠鹏,王希良.布鲁菌毒力因子研究进展[J].微生物免疫学展,2007,35(1):50-53.
[15] CLOECKAERT A,VIZCAINO N,PAQUET J Y,et al.Major outer membrane proteins of Brucella spp:past,present and future[J]. Veterinary Microbiology(Vet Mi-crobiol),2002,90(1/4):229-247.
[16]熊流新,黄健,张涛,等.布鲁氏菌外膜蛋白Omp16的原核表达、纯化及多克隆抗体制备[J].现代医药卫生,2018(5):32-37.
[17]王勇,南文龙,巩明霞,等.布鲁氏菌5种蛋白的原核表达及其作为间接ELISA检测用抗原的适用性比较[J].中国动物检疫,2015(10):72-75.
[18]王艺娟.家畜布鲁氏菌病的危害及防控措施[C]//福建省猪病学术研讨会论文集. 2016.
[19] WELLINGHAUSEN N,NOCKER K,SIGGE A,et al.Rapid Detection of Brucella spp. in Blood Cultures byFluorescence In Situ Hybridization[J]. Journal of Clini-cal Microbiology(J CLIN MICROBIOL),2006,44(5):1828.
[20]朱其太.用FPA试验诊断牛布鲁氏菌病[J].畜牧兽医科技信息,2000(5):62-66.
[21]张付贤,李晓艳,闫广谋,等.鉴别布鲁菌病自然感染动物与疫苗免疫动物胶体金检测试纸的研制[J].中国畜牧兽医,2009,36(7):79-82.
[22]张太翔,凌宗帅,赵立娜,等.基于Taq Man探针的牛布鲁氏菌实时荧光定量PCR的特异性鉴定[J].中国动物检疫,2011,28(11):29-31.
[23]徐军,陈创夫,刘建平,等.巢式PCR检测乳中布氏杆菌[J].畜牧与兽医,2008,40(4):67-69.