Cu/Zn SOD蛋白在布鲁菌S19疫苗株中的过表达及鉴定
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摘要
以Cu/Zn SOD为目的基因,通过基因工程技术分别构建了组成型过表达系统pBBR-trc-sod和诱导型过表达系统pBBR-lacPtrc-sod,在布鲁菌S19疫苗株中进行了Cu/Zn SOD的过表达。同时,以大肠杆菌为宿主表达纯化的重组Cu/Zn SOD蛋白免疫家兔制备多克隆抗体,对2种过表达系统中Cu/Zn SOD蛋白的表达量进行分析。结果显示,诱导型Cu/Zn SOD过表达系统的表达量更高,且能与所制备的多抗发生特异性反应。结果表明,构建的过表达系统能够实现Cu/Zn SOD蛋白在布鲁菌S19疫苗株中过表达;同时,该试验也提示我们这种表达系统可应用于布鲁菌S19疫苗的抗原改造。
Brucella abortus strain 19,as a live attenuated vaccine naturally attenuated from a virulent culture of B.abortus,has not been reported about its antigen modification at present.Due to the crucial role of Cu-Zn superoxide dismutase(Cu/Zn SOD) in immunoprotection of B.abortus strain 19,a constitutive Cu/Zn SOD expression system pBBR-trc-sod and an inducible Cu/Zn SOD expression system pBBR-lacPtrc-sod were constructed by genetic engineering in this research.Furthermore,these systems were used in overexpressing Cu/Zn SOD in B.abortus S19 for the first time.Meanwhile,the antiserum was prepared in mice by immunization of the purified recombinant protein.The level of Cu/Zn SOD expression in B.abortus S19 was analyzed by SDS-PAGE.Our data indicated that Cu/Zn SOD which expressed through the inducible protein expression system pBBR-lacPtrc-sod had high expression level and good specificity.In conclusion,Cu/Zn SOD was overexpressed successfully in B.abortus S19 in this research.Additionally,these results also implied that our expression system can be applied in antigen modification of B.abortus S19.
Brucella abortus strain 19,as a live attenuated vaccine naturally attenuated from a virulent culture of B.abortus,has not been reported about its antigen modification at present.Due to the crucial role of Cu-Zn superoxide dismutase(Cu/Zn SOD) in immunoprotection of B.abortus strain 19,a constitutive Cu/Zn SOD expression system pBBR-trc-sod and an inducible Cu/Zn SOD expression system pBBR-lacPtrc-sod were constructed by genetic engineering in this research.Furthermore,these systems were used in overexpressing Cu/Zn SOD in B.abortus S19 for the first time.Meanwhile,the antiserum was prepared in mice by immunization of the purified recombinant protein.The level of Cu/Zn SOD expression in B.abortus S19 was analyzed by SDS-PAGE.Our data indicated that Cu/Zn SOD which expressed through the inducible protein expression system pBBR-lacPtrc-sod had high expression level and good specificity.In conclusion,Cu/Zn SOD was overexpressed successfully in B.abortus S19 in this research.Additionally,these results also implied that our expression system can be applied in antigen modification of B.abortus S19.
引文
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[3] 王文杰,王力.布鲁菌病的研究与防控进展[J].中国卫生产业,2017(15):193-194.
[4] 玉花,陈敏洁,王晶妍,等.布鲁菌免疫保护抗原与抗病疫苗的研究进展[J].广东农业科学,2011(24):102-104.
[5] ZHU J,LARSON C B,RAMAKER M A,et al.Characterization of recombinant B.abortus strain RB51SOD toward understanding the uncorrelated innate and adaptive immune responses induced by RB51SOD compared to its parent vaccine strain RB51[J].Front Cell Infect Microbiol,2011,25(1):10.doi:10.3389/fcimb.2011.00010.eCollection 2011.
[6] VEMULAPALLI R,HE Y,CRAVERO S,et al.Overexpression of protective antigen as a novel approach to enhance vaccine efficacy of Brucella abortus strain RB51[J].Infect Immun,2000,68(6):3286-3289.
[7] LIU X,ZHOU M,YANG Y,et al.Overexpression of Cu-Zn SOD in Brucella abortus suppresses bacterial intracellular replication via down-regulation of Sar1 activity[J].Oncotarget,2018,9(11):9596-9607.
[8] 覃晓琳,乔祖健,胡森,等.布鲁菌Cu/Zn-SOD基因的原核表达及其免疫原性分析[J].中国预防兽医学报,2013(4):317-320.
[9] KOVACH M E,ELZER P H,HILL D S,et al.Four new derivatives of the broad-host-range cloning vector pBBR1MCS,carrying different antibiotic-resistance cassettes[J].Gene,1995,166(1):175-176.
[10] 苗玉强.布鲁菌免疫磁珠及免疫荧光检测方法的研究[D].吉林长春:吉林农业大学,2016.
[11] 冯宇.牛布鲁菌病诊断技术研究[D].山东泰安:山东农业大学,2017.
[12] 欧阳伟强,陈盛文,周柱辉,等.动物布鲁菌病疫苗的研究进展[J].畜牧兽医科技信息,2018(6):4-5.
[13] 高玉环,王慧军,赵鲁,等.牛种布鲁菌病疫苗的研究进展[J].畜牧兽医科技信息,2017(8):6-7.
[14] GEE J M,VALDERAS M W,KOVACH M E,et al.The Brucella abortus Cu,Zn superoxide dismutase is required for optimal resistance to oxidative killing by murine macrophages and wild-type virulence in experimentally infected mice[J].Infect Immun,2005,73(5):2873-2880.
[15] ONATE A A,CESPEDES S,CABRERA A,et al.A DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus induces protective immunity in BALB/c mice[J].Infect Immun,2003,71(9):4857-4861.
[16] ELZER P H,KOVACH M E,PHILLIPS R W,et al.In vivo and in vitro stability of the broad-host-range cloning vector pBBR1MCS in six Brucella species[J].Plasmid,1995,33(1):51-57.
[17] SELEEM M N,JAIN N,ALQUBLAN H,et al.Activity of native vs.synthetic promoters in Brucella[J].FEMS Microbiol Lett,2008,288(2):211-215.