幽门螺杆菌定植因子HpPrtC胶原蛋白酶的异源表达及其功能活性研究
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摘要
目的异源表达幽门螺杆菌HpPrtC胶原蛋白酶,并检测其对胶原蛋白的降解活性。方法根据幽门螺杆菌基因组信息中编号hp0169基因的序列设计引物,PCR扩增HpPrtC基因的核苷酸序列,经NdeI和XhoI酶切后构建原核表达载体pET-22b(+)-HpPrtC,转化至E.coli BL21(DE3),经IPTG诱导表达后采用亲和层析法纯化重组蛋白,通过检测重组酶处理胶原蛋白后L-亮氨酸的产生量表征其胶原蛋白降解活性。结果 HpPrtC基因编码422个氨基酸,与已知PrtC家族蛋白酶氨基酸序列比对最高相似性<40%。克隆HpPrtC基因连接入表达质粒pET-22b(+),将测序正确的表达质粒pET-22b(+)-HpPrtC转化至感受态细胞BL21(DE3),经IPTG诱导表达相对分子质量约为47×103的可溶性蛋白,与预期相符。经亲和层析纯化后的重组蛋白酶降解热变性I型胶原蛋白(可溶、不可溶)和天然I型胶原蛋白的活性分别为922.2±36.2、1168.8±65.6和674.0±13.4,与幽门螺杆菌培养液中胶原蛋白酶对可溶热变性I型胶原蛋白、不可溶热变性I型胶原蛋白和天然I型胶原蛋白活性分别为205.6±21.5、228.4±19.3和123.2±14.5一致。结论幽门螺杆菌定植因子HpPrtC为胶原蛋白酶。通过大肠埃希菌异源表达的重组HpPrtC蛋白具有胶原蛋白酶解活性,为探索该酶在幽门螺杆菌感染定植中的作用机制奠定了基础。
Objectives To induce heterogenous expression of the protease HpPrtC from Helicobacter pylori and to detect its collagenolytic activity in order to analyze its pathogenic role. Methods The HpPrtC gene was amplified with PCR using primers designed in accordance with the hp0169 gene in the H.pylori genome.After digestion with NdeI and XhoI,the fragment was inserted into a vector pET-22 b(+)to construct the recombinant expression plasmid pET-22 b(+)-HpPrtC.After the plasmid was verified as correct with sequencing,the plasmid was transformed into E.coli BL21(DE3).Expression of the recombinant protein was induced with IPTG,and the recombinant protein was purified using affinity chromatography.The purity of the protein was examined using SDS-PAGE.The collagenolytic activity of the purified protein was indicated by the amount of L-leucine in its products of collagen hydrolysis. Results The total length of the cloned HpPrtC gene was 1 269 bp,and the gene encoded 422 amino acids.Among collagenases,HpPrtCwas most similar(32%)to PrtC fromSalmonella enterica,indicating that HpPrtCis a novel collagenase.According to sequencing,HpPrtCis a stable(instability index(II)= 36.98)hydrophilic protein(grand average of hydropathicity,GRAVY=-0.132)without a typical signal peptide.After the recombinant plasmid pET-22 b(+)-HpPrtC was constructed and transformed into E.coli BL21(DE3),expression of a large amount of soluble recombinant proteins with an expected mass of 47×103 was induced with IPTG.After purification with affinity chromatography,recombinant HpPrtC dis-played collagenolytic activity against both thermally treated type I collagen(soluble:922.2±36.2,insoluble:1168.8±65.6)and natural type I collagen(674.0±13.4).H.pylori culture supernatant displayed collagenolytic activity against soluble thermal denatured type I collagen(205.6±21.5),insoluble thermal denatured type I collagen(228.4±19.3),and natural type I collagen(123.2±14.5).H.pylori culture supernatant displayed less collagenolytic activity than recombinant HpPrtC,but that activity followed the same pattern. Conclusion HpPrtCis a novel collagenase that is a factor for H.pylori colonization.When it was expressed in E.coli,recombinant HpPrtCdisplayed a high level of collagenolytic activity.These results have laid the foundation for study of this enzyme's role in the pathogenesis of H.pylori.
Objectives To induce heterogenous expression of the protease HpPrtC from Helicobacter pylori and to detect its collagenolytic activity in order to analyze its pathogenic role. Methods The HpPrtC gene was amplified with PCR using primers designed in accordance with the hp0169 gene in the H.pylori genome.After digestion with NdeI and XhoI,the fragment was inserted into a vector pET-22 b(+)to construct the recombinant expression plasmid pET-22 b(+)-HpPrtC.After the plasmid was verified as correct with sequencing,the plasmid was transformed into E.coli BL21(DE3).Expression of the recombinant protein was induced with IPTG,and the recombinant protein was purified using affinity chromatography.The purity of the protein was examined using SDS-PAGE.The collagenolytic activity of the purified protein was indicated by the amount of L-leucine in its products of collagen hydrolysis. Results The total length of the cloned HpPrtC gene was 1 269 bp,and the gene encoded 422 amino acids.Among collagenases,HpPrtCwas most similar(32%)to PrtC fromSalmonella enterica,indicating that HpPrtCis a novel collagenase.According to sequencing,HpPrtCis a stable(instability index(II)= 36.98)hydrophilic protein(grand average of hydropathicity,GRAVY=-0.132)without a typical signal peptide.After the recombinant plasmid pET-22 b(+)-HpPrtC was constructed and transformed into E.coli BL21(DE3),expression of a large amount of soluble recombinant proteins with an expected mass of 47×103 was induced with IPTG.After purification with affinity chromatography,recombinant HpPrtC dis-played collagenolytic activity against both thermally treated type I collagen(soluble:922.2±36.2,insoluble:1168.8±65.6)and natural type I collagen(674.0±13.4).H.pylori culture supernatant displayed collagenolytic activity against soluble thermal denatured type I collagen(205.6±21.5),insoluble thermal denatured type I collagen(228.4±19.3),and natural type I collagen(123.2±14.5).H.pylori culture supernatant displayed less collagenolytic activity than recombinant HpPrtC,but that activity followed the same pattern. Conclusion HpPrtCis a novel collagenase that is a factor for H.pylori colonization.When it was expressed in E.coli,recombinant HpPrtCdisplayed a high level of collagenolytic activity.These results have laid the foundation for study of this enzyme's role in the pathogenesis of H.pylori.
引文
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[10]Zhang YZ,Ran LY,Li CY,et al.Diversity,structures,and collagen-degrading mechanisms of bacterial collagenolytic proteases[J].Appl Environ Microbiol,2015(81):6098-107.
[11]李娇娇,荣倩玉,季晓飞,等.幽门螺杆菌hp0788基因对GES-1细胞功能影响的初步研究[J].中国病原生物学杂志,2017,12(7):614-8.
[12]荣倩玉,李娇娇,季晓飞,等.幽门螺杆菌在两种液体培养基中生长规律的探讨[J].中国病原生物学杂志,2017,12(8):738-40.
[13]Lower M,Weydig C,Metzler D,et al.Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19protein HtrA[J].PLoS One,2008,3(10):e3510.
[14]Hoy B,Lower M,Weydig C,et al.Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion[J].EMBO Rep,2010,11(10):798-804.
[15]Kato T,Takahashi N,Kuramitsu HK.Sequence analysis and characterization of the Porphyromonas gingivalis prtC gene,which expresses a novel collagenase activity[J].J Bacteriol,1992,174(12)3889-95.
[16]Wu MT,Carlson SA,Meyerholz DK.Cytopathic effects observed upon expression of a repressed collagenase gene present in Salmonellaand related pathogens:mimicry of a cytotoxin from multiple antibiotic-resistant Salmonella enterica serotype Typhimurium phagetype DT104[J].Microb Pathog,2002,33(6):279-87.
[2]Keilberg D,Ottemann KM.How Helicobacter pylori senses,targets and interacts with the gastric epithelium[J].Environ Microbiol,2016,18(3):791-806.
[3]Kao CY,Sheu BS,Wu JJ.Helicobacter pylori infection:An overview of bacterial virulence factors and pathogenesis[J].Biomed J,2016,39(1):14-23.
[4]胡伏莲.幽门螺杆菌致病因子与胃黏膜屏障[J].临床药物治疗杂志,2007,5(3):1-4.
[5]Backert S,Neddermann M,Maubach G,et al.Pathogenesis of Helicobacter pylori infection[J].Helicobacter,2016,21(Suppl1):19-25.
[6]Tegtmeyer N,Wessler S,Backert S.Role of the cag pathogenicity island encoded type IV secretion system in Helicobacter pylori pathogenesis[J].FEBS J,2011,278(8):1190-202.
[7]Anazco C,Delgado-Lopez F,Araya P,et al.Lysyl oxidase isoforms in gastric cancer[J].Biomark Med,2016,10(9):987-98.
[8]Kavermann H,Burns BP,Angermuller K,et al.Identification and characterization of Helicobacter pylori genes essential for gastric colonization[J].J Exp Med,2003,197(7):813-22.
[9]Zhao GY,Chen XL,Zhao HL,et al.Hydrolysis of insoluble collagen by Deseasin MCP-01from Deep-sea Pseudoalteromonas sp.SM9913:collagenolytic characters,collagen-binding ability of Cterminal polycystic kidney disease domain,and implication for its novel role in deep-sea sedimentary particulate organic nitrogen degradation[J].J Biol Chem,2008,283(52):36100-7.
[10]Zhang YZ,Ran LY,Li CY,et al.Diversity,structures,and collagen-degrading mechanisms of bacterial collagenolytic proteases[J].Appl Environ Microbiol,2015(81):6098-107.
[11]李娇娇,荣倩玉,季晓飞,等.幽门螺杆菌hp0788基因对GES-1细胞功能影响的初步研究[J].中国病原生物学杂志,2017,12(7):614-8.
[12]荣倩玉,李娇娇,季晓飞,等.幽门螺杆菌在两种液体培养基中生长规律的探讨[J].中国病原生物学杂志,2017,12(8):738-40.
[13]Lower M,Weydig C,Metzler D,et al.Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19protein HtrA[J].PLoS One,2008,3(10):e3510.
[14]Hoy B,Lower M,Weydig C,et al.Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion[J].EMBO Rep,2010,11(10):798-804.
[15]Kato T,Takahashi N,Kuramitsu HK.Sequence analysis and characterization of the Porphyromonas gingivalis prtC gene,which expresses a novel collagenase activity[J].J Bacteriol,1992,174(12)3889-95.
[16]Wu MT,Carlson SA,Meyerholz DK.Cytopathic effects observed upon expression of a repressed collagenase gene present in Salmonellaand related pathogens:mimicry of a cytotoxin from multiple antibiotic-resistant Salmonella enterica serotype Typhimurium phagetype DT104[J].Microb Pathog,2002,33(6):279-87.