鼠疫耶尔森菌基因组无痕修饰方法的建立
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摘要
目的构建鼠疫耶尔森菌(鼠疫菌)中基因组无痕修饰的技术平台,深入研究鼠疫菌相关基因的功能及作用机制。方法通过不对称PCR扩增抗性盒片段(含修饰靶标区域上下游同源臂),将其导入含pKD46质粒的鼠疫菌中。在阿拉伯糖的诱导下,pKD46质粒可表达与同源重组相关的3个酶(Exo,Beta和Gam蛋白),诱导重组后再导入p KSI-1质粒与目的基因的重组载体,与抗性盒进行第二次同源重组。通过加入阿拉伯糖和IPTG诱导pREDTKI表达重组酶和I-SceⅠ酶,可对未发生重组的抗性盒片段和p KSI-1质粒上的I-SceⅠ位点酶切,从而起到消除抗性盒与质粒作用,达到无痕修饰的目的。结果与结论成功构建了鼠疫菌的Δwaa A和waa A(△9nt)两个突变株。根据不同的实验目的选择相应的基因敲除方法,得到的无痕修饰菌株为鼠疫菌相关基因的功能研究奠定了基础。
Objective To construct a technical platform for scarless gene modification of Yersinia pestis and to study the functions of its specific genes. Methods The resistance fragment,including upstream and downstream homologous arms of targeted regions,was reamplified by asymmetric PCR. The amplicons were introduced into Y. pestis harboring plasmid p KD46. With the induction of L-arabinose,the recombinant related enzymes: Exo,Beta and Gam,were expressed to guide the homologous recombination. A donor plasmid,p KSI-1,which carried the desired modification fragment flanking by I-SceⅠrecognition sites,was introduced into Y. pestis as the second step of λ-Red recombination with the help of pREDTKI.Results and Conclusion Two mutant strains: △waa A and waa A( △9nt),were successfully constructed for Y. pestis strain201. Scarless modification introduces no extra modification to the genome,and it is ideal for comprehensive functional genomic studies.
Objective To construct a technical platform for scarless gene modification of Yersinia pestis and to study the functions of its specific genes. Methods The resistance fragment,including upstream and downstream homologous arms of targeted regions,was reamplified by asymmetric PCR. The amplicons were introduced into Y. pestis harboring plasmid p KD46. With the induction of L-arabinose,the recombinant related enzymes: Exo,Beta and Gam,were expressed to guide the homologous recombination. A donor plasmid,p KSI-1,which carried the desired modification fragment flanking by I-SceⅠrecognition sites,was introduced into Y. pestis as the second step of λ-Red recombination with the help of pREDTKI.Results and Conclusion Two mutant strains: △waa A and waa A( △9nt),were successfully constructed for Y. pestis strain201. Scarless modification introduces no extra modification to the genome,and it is ideal for comprehensive functional genomic studies.
引文
[1]Prentice MB,Rahalison L.Plague[J].Lancet,2007,369(9568):1196-1207.
[2]Makoveichuk E,Cherepanov P,Lundberg S,et al.p H6 antigen of Yersinia pestis interacts with plasma lipoproteins and cell membranes[J].J Lipid Res,2003,44(2):320-330.
[3]Keeling MJ,Gilligan CA.Bubonic plague:a metapopulation model of a zoonosis[J].Proc Biol Sci,2000,267(1458):2219-2230.
[4]于心.新疆准噶尔盆地荒漠大沙鼠鼠疫自然疫源地调查研究概述[J].地方病通报,2007,22(2):57-60.
[5]祁美英,魏柏青,杨晓艳,等.青海省2008~2012年鼠疫菌生物学特性及流行病学意义[J].中国人兽共患病学报,2013,29(8):826-828.
[6]Mc Donough KA,Falkow S.A Yersinia pestis-specific DNA fragment encodes temperature-dependent coagulase and fibrinolysinassociated phenotypes[J].Mol Microbiol,1989,3(6):767-775.
[7]吴海莲,赵海红,张青雯,等.鼠疫耶尔森氏菌噬菌体耐受株的选育及稳定性评价[J].中国病原生物学杂志,2015,10(7):582-586.
[8]Zhao X,Cui Y,Yan Y,et al.Outer membrane proteins ail and Omp F of Yersinia pestis are involved in the adsorption of T7-related bacteriophage Yep-phi[J].J Virol,2013,87(22):12260-12269.
[9]Filippov AA,Sergueev KV,He Y,et al.Bacteriophage-resistant mutants in Yersinia pestis:identification of phage receptors and attenuation for mice[J].PLo S One,2011,6(9):e25486.
[10]Yang J,Sun B,Huang H,et al.High-efficiency scarless genetic modification in Escherichia coli by using lambda red recombination and I-SceⅠcleavage[J].Appl Environ Microbiol,2014,80(13):3826-3834.
[11]Liu L,Fang H,Yang H,et al.CRP is an activator of Yersinia pestis biofilm formation that operates via a mechanism involving gmh A and waa AE-coa D[J].Front Microbiol,2016,7:295.
[12]Hua Y,Sun Q,Wang X,et al.Construction of enterohemorrhagic Escherichia coli O157∶H7 strains with esp F gene deletion and complementation[J].Nan Fang Yi Ke Da Xue Xue Bao,2015,35(11):1546-1551.
[13]陈泽良.鼠疫耶尔森氏菌密度感应系统与毒力关系研究[D].北京:军事医学科学院,2005.
[14]刘霞,高鹤,杨琳,等.副溶血性弧菌基因敲除方法的建立及应用[J].中国实验动物学报,2011,19(3):188-192.
[15]Philippe N,Alcaraz JP,Coursange E,et al.Improvement of p CVD442,a suicide plasmid for gene allele exchange in bacteria[J].Plasmid,2004,51(3):246-255.
[2]Makoveichuk E,Cherepanov P,Lundberg S,et al.p H6 antigen of Yersinia pestis interacts with plasma lipoproteins and cell membranes[J].J Lipid Res,2003,44(2):320-330.
[3]Keeling MJ,Gilligan CA.Bubonic plague:a metapopulation model of a zoonosis[J].Proc Biol Sci,2000,267(1458):2219-2230.
[4]于心.新疆准噶尔盆地荒漠大沙鼠鼠疫自然疫源地调查研究概述[J].地方病通报,2007,22(2):57-60.
[5]祁美英,魏柏青,杨晓艳,等.青海省2008~2012年鼠疫菌生物学特性及流行病学意义[J].中国人兽共患病学报,2013,29(8):826-828.
[6]Mc Donough KA,Falkow S.A Yersinia pestis-specific DNA fragment encodes temperature-dependent coagulase and fibrinolysinassociated phenotypes[J].Mol Microbiol,1989,3(6):767-775.
[7]吴海莲,赵海红,张青雯,等.鼠疫耶尔森氏菌噬菌体耐受株的选育及稳定性评价[J].中国病原生物学杂志,2015,10(7):582-586.
[8]Zhao X,Cui Y,Yan Y,et al.Outer membrane proteins ail and Omp F of Yersinia pestis are involved in the adsorption of T7-related bacteriophage Yep-phi[J].J Virol,2013,87(22):12260-12269.
[9]Filippov AA,Sergueev KV,He Y,et al.Bacteriophage-resistant mutants in Yersinia pestis:identification of phage receptors and attenuation for mice[J].PLo S One,2011,6(9):e25486.
[10]Yang J,Sun B,Huang H,et al.High-efficiency scarless genetic modification in Escherichia coli by using lambda red recombination and I-SceⅠcleavage[J].Appl Environ Microbiol,2014,80(13):3826-3834.
[11]Liu L,Fang H,Yang H,et al.CRP is an activator of Yersinia pestis biofilm formation that operates via a mechanism involving gmh A and waa AE-coa D[J].Front Microbiol,2016,7:295.
[12]Hua Y,Sun Q,Wang X,et al.Construction of enterohemorrhagic Escherichia coli O157∶H7 strains with esp F gene deletion and complementation[J].Nan Fang Yi Ke Da Xue Xue Bao,2015,35(11):1546-1551.
[13]陈泽良.鼠疫耶尔森氏菌密度感应系统与毒力关系研究[D].北京:军事医学科学院,2005.
[14]刘霞,高鹤,杨琳,等.副溶血性弧菌基因敲除方法的建立及应用[J].中国实验动物学报,2011,19(3):188-192.
[15]Philippe N,Alcaraz JP,Coursange E,et al.Improvement of p CVD442,a suicide plasmid for gene allele exchange in bacteria[J].Plasmid,2004,51(3):246-255.