表达牛分枝杆菌esat-6和cfp-10基因的重组流产布氏杆菌S19疫苗株的构建及毒力评估
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摘要
通过overlap PCR扩增了含有bp26基因上游片段、bp26基因下游片段及牛分枝杆菌esat6-cfp10基因的目的片段Δbp26-ec,利用含有sacB基因的自杀质粒pRE112为载体,通过等位基因交换的方法,将构建好的自杀质粒电转入已经构建好的缺失bp26基因的流产布氏杆菌减毒活疫苗S19突变株中,构建了具有非抗性基因标记的突变株S19-Δbp26-ec,并对构建好的新型基因突变株进行了生物学特性及毒力的鉴定及分析。PCR及核苷酸序列测序结果显示,S19-Δbp26-ec构建成功。与亲本株相比,疫苗株S19-Δbp26-ec的生长特性没有显著性差异;体外连续传至20代后,菌落PCR和核苷酸测序结果显示,S19-Δbp26-ec株具有良好的遗传稳定性。被感染小鼠脾重和脾中菌落数表明,S19-Δbp26-ec的毒力最弱,S19和S19-Δbp26次之。上述结果表明,本试验获得了毒力减弱的基因突变株S19-Δbp26-ec,为牛布氏杆菌病-结核病二联疫苗的研制奠定了基础。
The upstream and downstream fragments of bp26 gene were amplified fromBrucella abortus S19 strain,respectively,and esat6-cfp10 was amplified from Mycobacterium bovis.Then,a DNA fragment was amplified from these three fragments through fusion PCR and subcloned into the suicide plasmid pRE112.The modified plasmid was transferred into the B.abortus S19-Δbp26mutant strains.Under the selection of chloramphenicol and sucrose,a novel mutant strain S19-Δbp26-ec was constructed through homologous recombination.The results of PCR amplification and sequencing showed that the mutant was constructed successfully.Compared to the parental strain S19 and S19-Δbp26,S19-Δbp26-ec mutant strains had similar growing characteristics and the sequence of S19-Δbp26-ec was verified after 20 passages,which indicated that it has stable genetic generation.The animal experiments based on the weight and counts of spleen of BALB/c showed that S19-Δbp26-ec exhibited the lowest virulence compared with S19 and S19-Δbp26strains.In conclusion,we have constructed a novel mutant strain S19-Δbp26-ec with less virulence,and it might be developed to a new candidate vaccine.At the same time,the above-mentioned result might offer the basic theory for the development of vaccines against both bovine brucellosis and tuberculosis.
The upstream and downstream fragments of bp26 gene were amplified fromBrucella abortus S19 strain,respectively,and esat6-cfp10 was amplified from Mycobacterium bovis.Then,a DNA fragment was amplified from these three fragments through fusion PCR and subcloned into the suicide plasmid pRE112.The modified plasmid was transferred into the B.abortus S19-Δbp26mutant strains.Under the selection of chloramphenicol and sucrose,a novel mutant strain S19-Δbp26-ec was constructed through homologous recombination.The results of PCR amplification and sequencing showed that the mutant was constructed successfully.Compared to the parental strain S19 and S19-Δbp26,S19-Δbp26-ec mutant strains had similar growing characteristics and the sequence of S19-Δbp26-ec was verified after 20 passages,which indicated that it has stable genetic generation.The animal experiments based on the weight and counts of spleen of BALB/c showed that S19-Δbp26-ec exhibited the lowest virulence compared with S19 and S19-Δbp26strains.In conclusion,we have constructed a novel mutant strain S19-Δbp26-ec with less virulence,and it might be developed to a new candidate vaccine.At the same time,the above-mentioned result might offer the basic theory for the development of vaccines against both bovine brucellosis and tuberculosis.
引文
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[16]CLOECKAERT A,JACQUES IGRILLO M J,MARIN C M,et al.Development and evaluation as vaccines in mice of Brucella melitensis Rev.1single and double deletion mutants of the bp26and omp31genes coding for antigens of diagnostic significance in ovine brucellosis[J].Vaccine,2004,2(21-22):2827-2835.
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[19]潘文,王佳莹,赵明秋,等.布氏杆菌bp26基因缺失株的构建[J].中国兽医科学,2011,41(3):280-286.PAN Wen,WANG Jia-ying,ZHAO Ming-qiu,et al.Construction of unmarked bp26gene-deleted strains of Brucella spp.[J].Chinese Veterinary Science,2011,41(3):280-286.(in Chinese)
[20]刘林涛.表达结核分枝杆菌Ag85B抗原重组牛布氏杆菌S19疫苗株的构建[D].北京:中国农业科学院研究生院,2010.LIU Lin-tao.Recombinant Brucella abortus S19expressing Mycobacterium tuberculosis Ag85B antigen[D].Beijing:Graduate School of Chinese Academy of Agricultural Sciences,2010.(in Chinese)
[21]郑孝辉.流产布氏杆菌S19株bp26基因标记疫苗株的构建及免疫原性初步研究[D].哈尔滨:东北农业大学,2009.ZHENG Xiao-hui.Construction of bp26gene marker strain Brucella abortus S19and evaluation of the immunogenicity in mice[D].Harbin:Northeast Agricultural University,2009.(in Chinese)
[2]GARNIER T,EIGLMEIER K,CAMUS J C,et al.The complete genome sequence of Mycobacterium bovis[J].Proc Natl Acad Sci USA,2003,100(13):7877-7882.
[3]SABIO J,BIGI F,ROSSETTI O,et al.Expression of MPB83from Mycobacterium bovis in Brucella abortus S19induces specific cellular immune response against the recombinant antigen in BALB/c mice[J].Microbes Infect,2010,12(14-15):1236-1243.
[4]RENSHAW P S,PANAGIOTIDOU P,WHELAN A,et al.Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6and CFP-10form a tight,1∶1 complex and characterization of the structural properties of ESAT-6,CFP-10,and the ESAT-6CFP-10complex-implications for pathogenesis and virulence[J].J Biol Chem,2002,277(24):21598-21603.
[5]BRODIN P,ROSENKRANDS I,ANDERSEN P,et al.ESAT-6proteins:protective antigens and virulence factors?[J].Trends Microbiol,2004,12(11):500-508.
[6]YOUNG D B,KAUFMAN S H E,HERMANS P W,et al.Mycobacterial protein antigens:a compilation[J].Mol Microbiol,1992,6(2):133-145.
[7]PYM A S,BRODIN P,MAJLESSI L,et al.Recombinant BCG exporting ESAT-6confers enhanced protection against tuberculosis[J].Nat Med,2003,9(5):533-539.
[8]CORBEL M J.Brucellosis:an overview[J].Emerg Infect Dis,1997,3(2):213-221.
[9]DUBRAY G.Protective antigens in brucellosis[J].Ann Inst Pasterur Microbiol,1987,138(1):84-87.
[10]VEMULAPALLI R,HE Y,BOYLE S M,et al.Brucella abortus strain RB51as a vector for heterologous protein expression and induction of specific Th1type immune responses[J].Infect Immun,2000,68(6):3290-3296.
[11]SELEEM M N,JAIN N,AIQUBLAN H,et al.Activity of native vs synthetic promoters in Brucella[J].FEMS Microbiol Lett,2008,288(2):211-215.
[12]BANDARA A B,POFFREICHOW S A,NIKOLICH M,et al.Simultaneous expression of homologous and heterologous antigens in rough,attenuated Brucella melitensis[J].Microbes Infect,2009,11(3):424-428.
[13]RAMAMOORTHY S,SANAKKAYALA N,VEMULAPALLI R,et al.Prevention of vertical transmission of Neospora caninum in C57BL/6 mice vaccinated with Brucella abortus strain RB51expressing N.caninum protective antigens[J].Int J Parasitol,2007,37(13):1531-1538.
[14]COMERCI D J,POLLEVICK G D,VIGLIOCCO A M,et al.Vector development for the expression of foreign proteins in the vaccine strain Brucella abortus S19[J].Infect Immun,1998,66(8):3862-3866.
[15]SABIO J,FARBER M,CARRICA M,et al.Expression of Babesia bovis rhoptry-associated protein 1(RAP1)in Brucella abortus S19[J].Microbes Infect,2008,10(6):635-641.
[16]CLOECKAERT A,JACQUES IGRILLO M J,MARIN C M,et al.Development and evaluation as vaccines in mice of Brucella melitensis Rev.1single and double deletion mutants of the bp26and omp31genes coding for antigens of diagnostic significance in ovine brucellosis[J].Vaccine,2004,2(21-22):2827-2835.
[17]JACQUES I,VERGER J M,LAROUCAU K,et al.Immunological responses and protective efficacy against Brucella melitensis induced by bp26and omp31 B.melitensis Rev.1deletion mutants in sheep[J].Vaccine,2007,25(5):794-805.
[18]BOSCHIROLI M L,CRAVERO S L,ARESE A I,et al.Protection against infection in mice vaccinated with a Brucella abortus mutant[J].Infect Immun,1997,65(2):798-800.
[19]潘文,王佳莹,赵明秋,等.布氏杆菌bp26基因缺失株的构建[J].中国兽医科学,2011,41(3):280-286.PAN Wen,WANG Jia-ying,ZHAO Ming-qiu,et al.Construction of unmarked bp26gene-deleted strains of Brucella spp.[J].Chinese Veterinary Science,2011,41(3):280-286.(in Chinese)
[20]刘林涛.表达结核分枝杆菌Ag85B抗原重组牛布氏杆菌S19疫苗株的构建[D].北京:中国农业科学院研究生院,2010.LIU Lin-tao.Recombinant Brucella abortus S19expressing Mycobacterium tuberculosis Ag85B antigen[D].Beijing:Graduate School of Chinese Academy of Agricultural Sciences,2010.(in Chinese)
[21]郑孝辉.流产布氏杆菌S19株bp26基因标记疫苗株的构建及免疫原性初步研究[D].哈尔滨:东北农业大学,2009.ZHENG Xiao-hui.Construction of bp26gene marker strain Brucella abortus S19and evaluation of the immunogenicity in mice[D].Harbin:Northeast Agricultural University,2009.(in Chinese)