牙龈卟啉单胞菌clpP基因缺失株和回补株的构建和鉴定
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摘要
目的构建并鉴定牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)W83的丝氨酸蛋白酶(caseinolytic protease,Clp)基因缺失株和回补株,为探索clpP基因在P.g致病过程中的作用和机制奠定基础。方法设计clpP基因的引物对,PCR扩增其上下游同源片段,克隆入质粒pUC18中,插入红霉素抗性基因ermB作为筛选标记,构建重组质粒,电转化入P.g W83构建缺失株;PCR扩增clpP基因片段与重组质粒pUC18-ΔclpP-ermB连接,电转化入P.g W83缺失株中构建回补株。采用PCR和酶切电泳鉴定序列的正确性,利用抗性培养基筛选高表达菌株。结果 clpP基因克隆成功并顺利导入质粒pUC18中,P.g W83的clpP基因缺失株和回补株构建成功,并能在体外稳定传代。结论本研究运用同源重组技术构建了P.g W83的clpP基因缺失株,并建立了以pUC18质粒为载体的clpP基因回补方法,为进一步研究clpP基因在P.g致病过程中的作用打下了基础。
Objective To construct and identify caseinolytic protease(Clp) gene deletion strains and complemented strains of Porphyromonas gingivalis(P.g) W83 in order to make a foundation for exploring the role and mechanism of clpP gene in the pathogenesis of P.g W83. Methods The primer pair of clpP gene was designed and its upstream and downstream homologous fragments were amplified by PCR, which were cloned into plasmid pUC18. And the erythromycin resistance gene ermB from plasmid pMG36 e was inserted as a screening marker to construct a recombinant plasmid,which was electrotransfered into P.g W83 to get the corresponding clpP deficient recombinant strain. The clpP gene fragment was amplified by PCR and ligated with the recombinant plasmid pUC18-ΔclpP-ermB and electrotransfered into the recombinant strain of P.g so as to construct the corresponding complemented recombinant strain. PCR and electrophoresis were used to identify the correctness of the sequence, and the high-expression strain was screened and selected using a resistant medium. Results The clpP gene fragment was successfully cloned and introduced into the plasmid vector pUC18. The clpP gene deletion strain and the complemented strain of P.g W83 were successfully constructed and could have a stable passage in vitro. Conclusion In this study, the clpP gene deletion strain of P.g W83 is constructed using homologous recombination technology, and the method of using pUC18 plasmid as a vector to construct the complemented strains is also established, making a foundation for the further study on the role of clpP gene in the pathogenesis of P.g.
Objective To construct and identify caseinolytic protease(Clp) gene deletion strains and complemented strains of Porphyromonas gingivalis(P.g) W83 in order to make a foundation for exploring the role and mechanism of clpP gene in the pathogenesis of P.g W83. Methods The primer pair of clpP gene was designed and its upstream and downstream homologous fragments were amplified by PCR, which were cloned into plasmid pUC18. And the erythromycin resistance gene ermB from plasmid pMG36 e was inserted as a screening marker to construct a recombinant plasmid,which was electrotransfered into P.g W83 to get the corresponding clpP deficient recombinant strain. The clpP gene fragment was amplified by PCR and ligated with the recombinant plasmid pUC18-ΔclpP-ermB and electrotransfered into the recombinant strain of P.g so as to construct the corresponding complemented recombinant strain. PCR and electrophoresis were used to identify the correctness of the sequence, and the high-expression strain was screened and selected using a resistant medium. Results The clpP gene fragment was successfully cloned and introduced into the plasmid vector pUC18. The clpP gene deletion strain and the complemented strain of P.g W83 were successfully constructed and could have a stable passage in vitro. Conclusion In this study, the clpP gene deletion strain of P.g W83 is constructed using homologous recombination technology, and the method of using pUC18 plasmid as a vector to construct the complemented strains is also established, making a foundation for the further study on the role of clpP gene in the pathogenesis of P.g.
引文
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[22] Gispert S,Parganlija D,Klinkenberg M,et al.Loss of mitochondrial peptidase Clpp leads to infertility,hearing loss plus growth retardation via accumulation of CLPX,mtDNA and inflammatory factors[J].Hum Mol Genet,2013,22(24):4871-4887.
[23] Selvaraj G,Iyer VN.Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria[J].J Bacteriol,1983,156(3):1292-1300.
[24] Li C,Yang X,Pan Y,et al.A Sialidase-deficient porphyromonas gingivalis mutant strain induces less interleukin-1β and tumor necrosis Factor-α in Epi4 cells than W83 strain through regulation of the JNK Pathway[J].J Periodontology,2017,88(8):e129-e139.
[25] 韩海红,汪俊卿,王腾飞,等.一种基于单交换原理的地衣芽孢杆菌基因敲除方法及应用[J].中国生物工程杂志,2016,36(11):63-69.
[26] Huang J,Wang X,Cao Q,et al.ClpP participates in stress tolerance and negatively regulates biofilm formation in Haemophilus parasuis[J].Vet Microbiol,2016,182:141-149.
[27] Li C,Kurniyati,Hu B,et al.Abrogation of neuraminidase reduces biofilm formation,capsule biosynthesis,and virulence of Porphyromonas gingivalis[J].Infect Immun,2012,80(1):3-13.
[2] Koziel J,Mydel P,Potempa J.The link between periodontal disease and rheumatoid arthritis:an updated review[J].Curr Rheumatol Rep,2014,16(3):408-414.
[3] Dou Y,Robles A,Roy F,et al.The roles of RgpB and Kgp in late onset gingipain activity in the vimA-defective mutant of Porphyromonas gingivalis W83[J].Mol Oral Microbiol,2015,30(5):347-360.
[4] 潘亚萍,刘静波.牙龈卟啉单胞菌研究进展[J].国际口腔医学杂志,2011,38(2):125-127.
[5] Kuboniwa M,Tribble GD,Hendrickson EL,et al.Insights into the virulence of oral biofilms:discoveries from proteomics[J].Expert Rev Proteomics,2012,9(3):311-323.
[6] Naito Y,Tohda H,Okuda K,et al.Adherence and hydrophobicity of invasive and noninvasive strains of Porphyromonas gingivalis[J].Oral Microbiol Immunol,1993,8(4):195-202.
[7] Barbosa GM,Colombo AV,Rodrigues PH,et al.Intraspecies variability affects heterotypic biofilms of Porphyromonas gingivalis and Prevotella intermedia:evidences of strain-dependence biofilm modulation by physical contact and by released soluble factors[J].PLoS One,2015,10(9):1-14.
[8] 李红,纪海,吴杉杉,等.基于比较蛋白质组学技术的牙髓卟啉单胞菌毒力因子分析[J].中华口腔医学杂志,2016,51(12):746-752.
[9] Alexopoulos JA,Guarné A,Ortega J.ClpP:a structurally dynamic protease regulated by AAA+ proteins[J].J Struct Biol,2012,179(2):202-210.
[10] Kress W,Maglica Z,Weber-Ban E.Clp chaperone-proteases:Structure and function[J].Res Microbiol,2009,160(9):618-628.
[11] Hou XH,Zhang JQ,Song XU,et al.Contribution of ClpP to stress tolerance and virulence properties of Streptococcus mutans[J].J Basic Microbiol,2014,54(11):1222-1232.
[12] Xie F,Zhang YH,Li G,et al.The ClpP protease is required for the stress tolerance and biofilm formation in Actinobacillus pleuropneumoniae[J].PLoS One,2013,8(1):e53600.
[13] Freesa D,Ulf Gerthb U,Ingmera H.Clp chaperones and proteases are central in stress survival,virulence and antibiotic resistance of Staphylococcus aureus[J].Int J Med Microbiol,2014,304(2):142-149.
[14] 陈林,蓝林华,何海栋,等.ClpP 蛋白酶研究进展:从细菌到人线粒体[J].中国细胞生物学学报,2014,36(6):717-725.
[15] Zhang Y,Wang T,Chen W,et al.Differential protein expression by Porphyromonas gingivalis in response to secreted epithelial cell components[J].Proteomics,2005,5(1):198-211.
[16] Senanayake SD,Brian DA.Precise large deletions by the PCR-based overlap extension method[J].Mol Biotechnol,1995,4:13-15.
[17] Abaibou H,Chen Z,Olango GJ,et al.vimA gene downstream of recA is involved in virulence modulation in Porphyromonas gingivalis W83[J].Infect Immun,2001,69(1):325-335.
[18] Wang C,Li M,Dong D,et al.Role of ClpP in biofilm formation and virulence of Staphylococcus epidermidis[J].Microbes Infect,2007,9(11):1376-1383.
[19] Br?tz-Oesterhelt H,Sass P.Bacterial caseinolytic proteases as novel targets for antibacterial treatment[J].Int J Med Microbiol,2014,304(1):23-30.
[20] Xie F,Zhang YH,Li G,et al.The ClpP protease is required for the stress tolerance and biofilm formation in Actinobacillus pleuropneumoniae[J].PLoS One,2013,8(1):e53600.
[21] Hou XH,Zhang JQ,Song XU,et al.Contribution of ClpP to stress tolerance and virulence properties of Streptococcus mutans[J].J Basic Microbiol,2014,54(11):1222-1232.
[22] Gispert S,Parganlija D,Klinkenberg M,et al.Loss of mitochondrial peptidase Clpp leads to infertility,hearing loss plus growth retardation via accumulation of CLPX,mtDNA and inflammatory factors[J].Hum Mol Genet,2013,22(24):4871-4887.
[23] Selvaraj G,Iyer VN.Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria[J].J Bacteriol,1983,156(3):1292-1300.
[24] Li C,Yang X,Pan Y,et al.A Sialidase-deficient porphyromonas gingivalis mutant strain induces less interleukin-1β and tumor necrosis Factor-α in Epi4 cells than W83 strain through regulation of the JNK Pathway[J].J Periodontology,2017,88(8):e129-e139.
[25] 韩海红,汪俊卿,王腾飞,等.一种基于单交换原理的地衣芽孢杆菌基因敲除方法及应用[J].中国生物工程杂志,2016,36(11):63-69.
[26] Huang J,Wang X,Cao Q,et al.ClpP participates in stress tolerance and negatively regulates biofilm formation in Haemophilus parasuis[J].Vet Microbiol,2016,182:141-149.
[27] Li C,Kurniyati,Hu B,et al.Abrogation of neuraminidase reduces biofilm formation,capsule biosynthesis,and virulence of Porphyromonas gingivalis[J].Infect Immun,2012,80(1):3-13.