利用信息肽诱导和Cre/LoxP系统构建猪链球菌基因缺失株
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摘要
为建立一种高效的猪链球菌(Streptococcus suis)基因缺失方法,本研究利用信息肽诱导DNA片段转化和Cre/LoxP系统去除抗性基因这两种技术,通过在S.suis 05ZYH33的ssu05_1921基因上、下游片段和壮观霉素抗性基因(spc)片段之间引入LoxP位点,采用信息肽GE9诱导构建的DNA片段转化S.suis并发生重组,经抗性筛选快速获得spc替代ssu05_1921基因的缺失株;进一步引入pSET6s/P_(tufA-cre)质粒表达Cre重组酶作用于LoxP位点,去除spc基因,产生无痕ssu05_1921基因缺失株,经测序和RT-PCR验证缺失正确。本研究建立的该方法简单、快捷、阳性率高,为构建S.suis基因缺失株、研究S.suis致病机制提供了新的选择。
In the present study, an efficient method for gene-deletion of Streptococcus suis was established by combination of recently reported pheromone-induced DNA fragment transformation and Cre/LoxP system. Briefly, a DNA fragment was prepared by fusion of the upstream fragment of ssu05_1921 gene of S.suis, the spectinomycin resistance(spc) marker gene and the downstream fragment of ssu05_1921 gene. During the fusion processing, LoxP sites were introduced sequentially at 5' and 3' ends of the spc gene. Next, the DNA fragment was transformed into S.suis 05 ZYH33 strain induced by the pheromone for recombination, resulting in ssu05_1921-deleted from the S.suis strain in which the ssu05_1921 gene was substituted by spc marker gene.Then, the spc marker gene flanked with LoxP sites was removed, leading to a marker-less ssu05_1921-deleted strain by introducing the Cre recombinase expressing recombinant plasmid of pSET6s/P_(tufA-cre). It was further verified that the ssu05_1921 gene was deleted by sequencing and RT-PCR. The results showed that this method is simple and rapid, with high positive rate. It provides a efficient alternative protocol for gene-deleted strain construction of S.suis, facilitatinge further pathogenesis study of the pathogen.
In the present study, an efficient method for gene-deletion of Streptococcus suis was established by combination of recently reported pheromone-induced DNA fragment transformation and Cre/LoxP system. Briefly, a DNA fragment was prepared by fusion of the upstream fragment of ssu05_1921 gene of S.suis, the spectinomycin resistance(spc) marker gene and the downstream fragment of ssu05_1921 gene. During the fusion processing, LoxP sites were introduced sequentially at 5' and 3' ends of the spc gene. Next, the DNA fragment was transformed into S.suis 05 ZYH33 strain induced by the pheromone for recombination, resulting in ssu05_1921-deleted from the S.suis strain in which the ssu05_1921 gene was substituted by spc marker gene.Then, the spc marker gene flanked with LoxP sites was removed, leading to a marker-less ssu05_1921-deleted strain by introducing the Cre recombinase expressing recombinant plasmid of pSET6s/P_(tufA-cre). It was further verified that the ssu05_1921 gene was deleted by sequencing and RT-PCR. The results showed that this method is simple and rapid, with high positive rate. It provides a efficient alternative protocol for gene-deleted strain construction of S.suis, facilitatinge further pathogenesis study of the pathogen.
引文
[1]Staats J J,Feder I,Okwumabua O,et al.Streptococcus suis:Past and present[J].Vet Res Commun,1997,21(6):381-407.
[2]Lun Zhao-Rong,Wang Qiao-Ping,Chen Xiao-Guang,et al.Streptococcus suis:an emerging zoonotic pathogen[J].Lancet Infect Dis,2007,7(3):201-209.
[3]Huong V T L,Ha N,Huy N T,et al.Epidemiology,clinical manifestations,and outcomes of Streptococcus suis infection in humans[J].Emerg Infect Dis,2014,20(7):1105-1114.
[4]陆承平,姚火春,范红结,等.猪链球菌致病性研究及其公共卫生意义[J].中国预防医学杂志,2006,(04):361-362.
[5]Shanker E,Morrison D A,Talagas A,et al.Pheromone recognition and selectivity by ComR proteins among Streptococcus species[J].PLoS Pathog,2016,12(12):e1005979.
[6]Claverys J P,Dintilhac A,Mortier-Barriere I,et al.Regulation of competence for genetic transformation in Streptococcus pneumoniae[J].J Appl Microbiol,1997,83(S1):32S-41S.
[7]Pestova E V,Havarstein L S,Morrison D A.Regulation of competence for genetic transformation in Streptococcus pneumoniae by an auto-induced peptide pheromone and a two-component regulatory system[J].Mol Microbiol,1996,21(4):853-862.
[8]Mashburn-Warren L,Morrison D A,Federle M J.The cryptic competence pathway in Streptococcus pyogenes is controlled by a peptide pheromone[J].J Bacteriol,2012,194(17):4589-4600.
[9]Ahn S J,Wen Z T,Burne R A.Multilevel control of competence development and stress tolerance in Streptococcus mutans UA159[J].Infect Immun,2006,74(3):1631-1642.
[10]Kowalko J E,Sebert M E.The Streptococcus pneumoniae competence regulatory system influences respiratory tract colonization[J].Infect Immun,2008,76(7):3131-3140.
[11]Zaccaria E,van Baarlen P,de Greeff A,et al.Control of competence for DNA transformation in Streptococcus suis by genetically transferable pherotypes[J].PLoS One,2014,9(6).
[12]Sternberg N,Hamilton D,Hoess R.Bacteriophage P1 site-specific recombination.II.Recombination between loxP and the bacterial chromosome[J].J Mol Biol,1981,150(4):487-507.
[13]Sternberg N,Hamilton D.Bacteriophage P1 site-specific recombination.I.Recombination between loxP sites[J].J Mol Biol,1981,150(4):467-486.
[14]Albert H,Dale E C,Lee E,et al.Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome[J].Plant J,1995,7(4):649-659.
[15]Koczula A,Willenborg J,Bertram R,et al.Establishment of a Cre recombinase based mutagenesis protocol for markerless gene deletion in Streptococcus suis[J].J Microbiol Meth,2014,10780-83.
[16]Chen Chen,Tang Jia-Qi,Dong Wei,et al.A glimpse of Streptococcal toxic shock syndrome from comparative genomics of S-suis 2 Chinese isolates[J].PLoS One,2007,2(3).
[17]Takamatsu D,Osaki M,Sekizaki T.Thermosensitive suicide vectors for gene replacement in Streptococcus suis[J].Plasmid,2001,46(2):140-148.
[18]Rocha D J,Santos C S,Pacheco L G.Bacterial reference genes for gene expression studies by RT-q PCR:survey and analysis[J].Anton Leeuw,2015,108(3):685-693.
[19]任建鸾,陆承平.猪链球菌9型GZ0565株体外传代的生物学稳定性[J].中国兽医科学,2010,40(11):1115-1118.
[20]Robb M,Hobbs J K,Woodiga S A,et al.Molecular characterization of N-glycan degradation and transport in Streptococcus pneumoniae and its contribution to virulence[J].PLo S Pathog,2017,13(1):e1006090.
[2]Lun Zhao-Rong,Wang Qiao-Ping,Chen Xiao-Guang,et al.Streptococcus suis:an emerging zoonotic pathogen[J].Lancet Infect Dis,2007,7(3):201-209.
[3]Huong V T L,Ha N,Huy N T,et al.Epidemiology,clinical manifestations,and outcomes of Streptococcus suis infection in humans[J].Emerg Infect Dis,2014,20(7):1105-1114.
[4]陆承平,姚火春,范红结,等.猪链球菌致病性研究及其公共卫生意义[J].中国预防医学杂志,2006,(04):361-362.
[5]Shanker E,Morrison D A,Talagas A,et al.Pheromone recognition and selectivity by ComR proteins among Streptococcus species[J].PLoS Pathog,2016,12(12):e1005979.
[6]Claverys J P,Dintilhac A,Mortier-Barriere I,et al.Regulation of competence for genetic transformation in Streptococcus pneumoniae[J].J Appl Microbiol,1997,83(S1):32S-41S.
[7]Pestova E V,Havarstein L S,Morrison D A.Regulation of competence for genetic transformation in Streptococcus pneumoniae by an auto-induced peptide pheromone and a two-component regulatory system[J].Mol Microbiol,1996,21(4):853-862.
[8]Mashburn-Warren L,Morrison D A,Federle M J.The cryptic competence pathway in Streptococcus pyogenes is controlled by a peptide pheromone[J].J Bacteriol,2012,194(17):4589-4600.
[9]Ahn S J,Wen Z T,Burne R A.Multilevel control of competence development and stress tolerance in Streptococcus mutans UA159[J].Infect Immun,2006,74(3):1631-1642.
[10]Kowalko J E,Sebert M E.The Streptococcus pneumoniae competence regulatory system influences respiratory tract colonization[J].Infect Immun,2008,76(7):3131-3140.
[11]Zaccaria E,van Baarlen P,de Greeff A,et al.Control of competence for DNA transformation in Streptococcus suis by genetically transferable pherotypes[J].PLoS One,2014,9(6).
[12]Sternberg N,Hamilton D,Hoess R.Bacteriophage P1 site-specific recombination.II.Recombination between loxP and the bacterial chromosome[J].J Mol Biol,1981,150(4):487-507.
[13]Sternberg N,Hamilton D.Bacteriophage P1 site-specific recombination.I.Recombination between loxP sites[J].J Mol Biol,1981,150(4):467-486.
[14]Albert H,Dale E C,Lee E,et al.Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome[J].Plant J,1995,7(4):649-659.
[15]Koczula A,Willenborg J,Bertram R,et al.Establishment of a Cre recombinase based mutagenesis protocol for markerless gene deletion in Streptococcus suis[J].J Microbiol Meth,2014,10780-83.
[16]Chen Chen,Tang Jia-Qi,Dong Wei,et al.A glimpse of Streptococcal toxic shock syndrome from comparative genomics of S-suis 2 Chinese isolates[J].PLoS One,2007,2(3).
[17]Takamatsu D,Osaki M,Sekizaki T.Thermosensitive suicide vectors for gene replacement in Streptococcus suis[J].Plasmid,2001,46(2):140-148.
[18]Rocha D J,Santos C S,Pacheco L G.Bacterial reference genes for gene expression studies by RT-q PCR:survey and analysis[J].Anton Leeuw,2015,108(3):685-693.
[19]任建鸾,陆承平.猪链球菌9型GZ0565株体外传代的生物学稳定性[J].中国兽医科学,2010,40(11):1115-1118.
[20]Robb M,Hobbs J K,Woodiga S A,et al.Molecular characterization of N-glycan degradation and transport in Streptococcus pneumoniae and its contribution to virulence[J].PLo S Pathog,2017,13(1):e1006090.