mCherry红色荧光标记乳酸菌的融合表达系统构建及应用
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摘要
为开发新型荧光蛋白标记乳酸菌以填补国内研究空白,利用pSIP载体,构建了以红色荧光蛋白mCherry为标记,并以乳酸菌胆盐水解酶基因bsh为报告基因的乳酸菌融合表达系统。在4种不同启动子(P_(sppA)、P_(ldhL)、P_(32)和P_(slpA))调节下,相继实现了融合基因的诱导型和组成型表达,表达的融合重组蛋白mCherry-BSH同时检测出红色荧光活性和胆盐水解酶BSH活性。mCherry红色荧光标记的乳酸菌融合表达系统的成功构建不仅为研究乳酸菌在生物体内的分布、定植及存活情况从而揭示其益生功能的作用机理提供有利条件,也为更多活性蛋白在乳酸菌宿主中的表达、细胞定位、功能鉴定的研究奠定基础。
In order to enrich the library of domestic research about new red fluorescent marker in lactic acid bacteria(LAB),we described a new fusion expression system in Lactobacillus plantarum WCFS1 based on the pSIP vector. This system contained red fluorescent protein mCherry as a marker and bile salt hydrolase gene(bsh) as a reporter gene. Moreover, in this study, four different promoters(P_(sppA)、P_(ldhL)、P_(32)and P_(slpA)) were used to regulate the expression of the fusion protein mCherry-BSH, completing the inducible and constitutive expression in lactic acid bacteria. The recombinant protein mCherry-BSH presented activity of red fluorescence and bile salt hydrolase(BSH). The successful construction of the fusion expression system in LAB using a red fluorescent protein mCherry provides favorable conditions for the distribution, intestinal colonization and survival rate of lactic acid bacteria, so as to reveal the function mechanism of its probiotic characteristics; and the system also could lay the foundation for researches on protein expression, cellular localization and properties identification of active protein in lactic acid bacteria.
In order to enrich the library of domestic research about new red fluorescent marker in lactic acid bacteria(LAB),we described a new fusion expression system in Lactobacillus plantarum WCFS1 based on the pSIP vector. This system contained red fluorescent protein mCherry as a marker and bile salt hydrolase gene(bsh) as a reporter gene. Moreover, in this study, four different promoters(P_(sppA)、P_(ldhL)、P_(32)and P_(slpA)) were used to regulate the expression of the fusion protein mCherry-BSH, completing the inducible and constitutive expression in lactic acid bacteria. The recombinant protein mCherry-BSH presented activity of red fluorescence and bile salt hydrolase(BSH). The successful construction of the fusion expression system in LAB using a red fluorescent protein mCherry provides favorable conditions for the distribution, intestinal colonization and survival rate of lactic acid bacteria, so as to reveal the function mechanism of its probiotic characteristics; and the system also could lay the foundation for researches on protein expression, cellular localization and properties identification of active protein in lactic acid bacteria.
引文
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[2]Bai FL,Zhang BL,Zhao HF.Progress in protein metabolism of lactic acid bacteria.Food Sci,2010,31(19):381-384(in Chinese).白凤翎,张柏林,赵宏飞.乳酸菌蛋白代谢研究进展.食品科学,2010,31(19):381-384.
[3]van Pijkeren JP,Barrangou R.Genome editing of food-grade lactobacilli to develop therapeutic probiotics.Microbiol Spectr,2017,5(5):BAD-0013-2016.
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[6]Cui YQ,Wang JR,Wang YP.A review of research on lactic acid bacteria vectors for gene expression and their applications.Food Sci,2015,36(9):224-229(in Chinese).崔月倩,王菁蕊,王艳萍.乳酸菌基因表达载体及其应用研究进展.食品科学,2015,36(9):224-229.
[7]Renye Jr JA,Somkuti GA.Nisin-induced expression of pediocin in dairy lactic acid bacteria.J Appl Microbiol,2010,108(6):2142-2151.
[8]Abdullah-Al-Mahin,Sugimoto S,Higashi C,et al.Improvement of multiple-stress tolerance and lactic acid production in Lactococcus lactis NZ9000 under conditions of thermal stress by heterologous expression of Escherichia coli DnaK.Appl Environ Microbiol,2010,76(13):4277-4285.
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[10]Rodriguez EA,Campbell RE,Lin JY,et al.The growing and glowing toolbox of fluorescent and photoactive proteins.Trends Biochem Sci,2017,42(2):111-129.
[11]Piscitelli A,Pennacchio A,Cicatiello P,et al.Rapid and ultrasensitive detection of active thrombin based on the Vmh2 hydrophobin fused to a Green Fluorescent Protein.Biosens Bioelectron,2017,87:816-822.
[12]Kono J,Konno K,Talukder AH,et al.Distribution of corticotropin-releasing factor neurons in the mouse brain:a study using corticotropin-releasing factor-modified yellow fluorescent protein knock-in mouse.Brain Struct Funct,2017,222(4):1705-1732.
[13]Vieira J,O'Hearn PM.Use of the red fluorescent protein as a marker of Kaposi's sarcoma-associated herpesvirus lytic gene expression.Virology,2004,325(2):225-240.
[14]Lippincott-Schwartz J,Patterson GH.Development and use of fluorescent protein markers in living cells.Science,2003,300(5616):87-91.
[15]Martinez-Jaramillo E,Garza-Morales R,Loera-Arias MJ,et al.Development of Lactococcus lactis encoding fluorescent proteins,GFP,mCherry and iRFP regulated by the nisin-controlled gene expression system.Biotech Histochem,2017,92(3):167-174.
[16]Chen XL,Yang GL,Wang CF.Construction of GFPlabeling shuttle expression vector and preparation of restitution Lactobacillus.J Jilin Agric Univ,2009,31(3):325-329(in Chinese).陈晓雷,杨桂连,王春凤.绿色荧光蛋白标记穿梭表达载体构建及重组乳酸菌制备.吉林农业大学学报,2009,31(3):325-329.
[17]Xu YK.Construction of expressing enhanced green fluorescent protein in lactic acid bacteria.Biotechworld,2014,(4):4-6(in Chinese).徐一轲.表达增强绿色荧光蛋白重组乳酸菌的构建.生物技术世界,2014,(4):4-6.
[18]Kou TT,Bei TT,Li C,et al.Establishment of red fluorescent protein fusion expression system of lactic acid bacteria.J Chin Inst Food Sci Technol,2017,17(3):235-240(in Chinese).寇田田,北婷婷,李晨,等.乳酸菌红色荧光蛋白融合表达系统的构建.中国食品学报,2017,17(3):235-240.
[19]Fink D,Wohrer S,Pfeffer M,et al.Ubiquitous expression of the monomeric red fluorescent protein mCherry in transgenic mice.Genesis,2010,48(12):723-729.
[20]Chapagain PP,Regmi CK,Castillo W.Fluorescent protein barrel fluctuations and oxygen diffusion pathways in mCherry.J Chem Phys,2011,135(23):235101.
[21]Ransom EM,Ellermeier CD,Weiss DS.Use of mCherry Red fluorescent protein for studies of protein localization and gene expression in Clostridium difficile.Appl Environ Microbiol,2015,81(5):1652-1660.
[22]Tauer C,Heinl S,Egger E,et al.Tuning constitutive recombinant gene expression in Lactobacillus plantarum.Microb Cell Fact,2014,13:150.
[23]Russo P,Iturria I,Mohedano ML,et al.Zebrafish gut colonization by mCherry-labelled lactic acid bacteria.Appl Microbiol Biotechnol,2015,99(8):3479-3490.
[24]García-Cayuela T,de Cadi?anos LPZ,Mohedano ML,et al.Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli.Appl Microbiol Biotechnol,2012,96(1):171-181.
[25]Mohedano ML,García-Cayuela T,Pérez-Ramos A,et al.Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus.J Ind Microbiol Biotechnol,2015,42(2):247-253.
[26]van Zyl WF,Deane SM,Dicks LMT.Use of the mCherry fluorescent protein to study intestinal colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in mice.Appl Environ Microbiol,2015,81(17):5993-6002.
[27]van den Nieuwboer M,van Hemert S,Claassen E,et al.Lactobacillus plantarum WCFS1 and its host interaction:a dozen years after the genome.Microb Biotechnol,2016,9(4):452-465.
[28]Nguyen TT,Mathiesen G,Fredriksen L,et al.Afood-grade system for inducible gene expression in Lactobacillus plantarum using an alanine racemase-encoding selection marker.J Agric Food Chem,2011,59(10):5617-5624.
[29]Sak-Ubol S,Namvijitr P,Pechsrichuang P,et al.Secretory production of a beta-mannanase and a chitosanase using a Lactobacillus plantarum expression system.Microb Cell Fact,2016,15:81.
[30]Reverón I,Jiménez N,Curiel JA,et al.Differential gene expression by Lactobacillus plantarum WCFS1 in response to phenolic compounds reveals new genes involved in Tannin Degradation.Appl Environ Microbiol,2017,83(7):e03387-16.
[31]Huang Q,Huang L,Pan DD,et al.Expression in Escherichia coli,purification and functional characterization of recombination fusion protein MBP-BSH.Food Sci,2012,33(7):198-203(in Chinese).黄茜,黄璐,潘道东,等.重组融合蛋白MBP-BSH在大肠杆菌中的表达及其纯化、功能鉴定.食品科学,2012,33(7):198-203.
[32]Huang Q,Huang L,Pan DD,et al.Molecular cloning and expression of the bile salt hydrolase gene(bsh)from Lactobacillus plantarum Y1.JNanjing Norm Univ:Nat Sci Ed,2010,33(3):91-96(in Chinese).黄茜,黄璐,潘道东,等.植物乳杆菌Lactobacillu plantarum Y1菌株胆盐水解酶基因(bsh)的克隆及重组表达.南京师范大学学报:自然科学版,2010,33(3):91-96.
[33]S?rvig E,Gr?nqvist S,Naterstad K,et al.Construction of vectors for inducible gene expression in Lactobacillus sakei and L.plantarum.FEMS Microbiol Lett,2003,229(1):119-126.
[34]Moser SA,Savage DC.Bile salt hydrolase activity and resistance to toxicity of conjugated bile salts are unrelated properties in lactobacilli.Appl Environ Microbiol,2001,67(8):3476-3480.
[35]Landete JM,Langa S,Revilla C,et al.Use of anaerobic green fluorescent protein versus green fluorescent protein as reporter in lactic acid bacteria.Appl Microbiol Biotechnol,2015,99(16):6865-6877.
[36]Landete JM,Peiroténá,Rodríguez E,et al.Anaerobic green fluorescent protein as a marker of Bifidobacterium strains.Int Food Microbiol,2014,175:6-13.
[37]Geoffroy MC,Guyard C,Quatannens B,et al.Use of green fluorescent protein to tag lactic acid bacterium strains under development as live vaccine vectors.Appl Environ Microbiol,2000,66(1):383-391.
[38]Fredriksen L,Moen A,Adzhubei AA,et al.Lactobacillus plantarum WCFS1 O-linked protein glycosylation:an extended spectrum of target proteins and modification sites detected by mass spectrometry.Glycobiology,2013,23(12):1439-1451.
[39]Fujii T,Ingham C,Nakayama J,et al.Two homologous Agr-like quorum-sensing systems cooperatively control adherence,cell morphology,and cell viability properties in Lactobacillus plantarum WCFS1.J Bacteriol,2008,190(23):7655-7665.
[2]Bai FL,Zhang BL,Zhao HF.Progress in protein metabolism of lactic acid bacteria.Food Sci,2010,31(19):381-384(in Chinese).白凤翎,张柏林,赵宏飞.乳酸菌蛋白代谢研究进展.食品科学,2010,31(19):381-384.
[3]van Pijkeren JP,Barrangou R.Genome editing of food-grade lactobacilli to develop therapeutic probiotics.Microbiol Spectr,2017,5(5):BAD-0013-2016.
[4]Linares DM,Gómez C,Renes E,et al.Lactic acid bacteria and bifidobacteria with potential to design natural biofunctional health-promoting dairy foods.Front Microbiol,2017,8:846.
[5]Saez-Lara MJ,Gomez-Llorente C,Plaza-Diaz J,et al.The role of probiotic lactic acid bacteria and bifidobacteria in the prevention and treatment of inflammatory bowel disease and other related diseases:a systematic review of randomized human clinical trials.BioMed Res Int,2015,2015:505878.
[6]Cui YQ,Wang JR,Wang YP.A review of research on lactic acid bacteria vectors for gene expression and their applications.Food Sci,2015,36(9):224-229(in Chinese).崔月倩,王菁蕊,王艳萍.乳酸菌基因表达载体及其应用研究进展.食品科学,2015,36(9):224-229.
[7]Renye Jr JA,Somkuti GA.Nisin-induced expression of pediocin in dairy lactic acid bacteria.J Appl Microbiol,2010,108(6):2142-2151.
[8]Abdullah-Al-Mahin,Sugimoto S,Higashi C,et al.Improvement of multiple-stress tolerance and lactic acid production in Lactococcus lactis NZ9000 under conditions of thermal stress by heterologous expression of Escherichia coli DnaK.Appl Environ Microbiol,2010,76(13):4277-4285.
[9]Enterina JR,Wu LS,Campbell RE.Emerging fluorescent protein technologies.Curr Opin Chem Biol,2015,27:10-17.
[10]Rodriguez EA,Campbell RE,Lin JY,et al.The growing and glowing toolbox of fluorescent and photoactive proteins.Trends Biochem Sci,2017,42(2):111-129.
[11]Piscitelli A,Pennacchio A,Cicatiello P,et al.Rapid and ultrasensitive detection of active thrombin based on the Vmh2 hydrophobin fused to a Green Fluorescent Protein.Biosens Bioelectron,2017,87:816-822.
[12]Kono J,Konno K,Talukder AH,et al.Distribution of corticotropin-releasing factor neurons in the mouse brain:a study using corticotropin-releasing factor-modified yellow fluorescent protein knock-in mouse.Brain Struct Funct,2017,222(4):1705-1732.
[13]Vieira J,O'Hearn PM.Use of the red fluorescent protein as a marker of Kaposi's sarcoma-associated herpesvirus lytic gene expression.Virology,2004,325(2):225-240.
[14]Lippincott-Schwartz J,Patterson GH.Development and use of fluorescent protein markers in living cells.Science,2003,300(5616):87-91.
[15]Martinez-Jaramillo E,Garza-Morales R,Loera-Arias MJ,et al.Development of Lactococcus lactis encoding fluorescent proteins,GFP,mCherry and iRFP regulated by the nisin-controlled gene expression system.Biotech Histochem,2017,92(3):167-174.
[16]Chen XL,Yang GL,Wang CF.Construction of GFPlabeling shuttle expression vector and preparation of restitution Lactobacillus.J Jilin Agric Univ,2009,31(3):325-329(in Chinese).陈晓雷,杨桂连,王春凤.绿色荧光蛋白标记穿梭表达载体构建及重组乳酸菌制备.吉林农业大学学报,2009,31(3):325-329.
[17]Xu YK.Construction of expressing enhanced green fluorescent protein in lactic acid bacteria.Biotechworld,2014,(4):4-6(in Chinese).徐一轲.表达增强绿色荧光蛋白重组乳酸菌的构建.生物技术世界,2014,(4):4-6.
[18]Kou TT,Bei TT,Li C,et al.Establishment of red fluorescent protein fusion expression system of lactic acid bacteria.J Chin Inst Food Sci Technol,2017,17(3):235-240(in Chinese).寇田田,北婷婷,李晨,等.乳酸菌红色荧光蛋白融合表达系统的构建.中国食品学报,2017,17(3):235-240.
[19]Fink D,Wohrer S,Pfeffer M,et al.Ubiquitous expression of the monomeric red fluorescent protein mCherry in transgenic mice.Genesis,2010,48(12):723-729.
[20]Chapagain PP,Regmi CK,Castillo W.Fluorescent protein barrel fluctuations and oxygen diffusion pathways in mCherry.J Chem Phys,2011,135(23):235101.
[21]Ransom EM,Ellermeier CD,Weiss DS.Use of mCherry Red fluorescent protein for studies of protein localization and gene expression in Clostridium difficile.Appl Environ Microbiol,2015,81(5):1652-1660.
[22]Tauer C,Heinl S,Egger E,et al.Tuning constitutive recombinant gene expression in Lactobacillus plantarum.Microb Cell Fact,2014,13:150.
[23]Russo P,Iturria I,Mohedano ML,et al.Zebrafish gut colonization by mCherry-labelled lactic acid bacteria.Appl Microbiol Biotechnol,2015,99(8):3479-3490.
[24]García-Cayuela T,de Cadi?anos LPZ,Mohedano ML,et al.Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli.Appl Microbiol Biotechnol,2012,96(1):171-181.
[25]Mohedano ML,García-Cayuela T,Pérez-Ramos A,et al.Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus.J Ind Microbiol Biotechnol,2015,42(2):247-253.
[26]van Zyl WF,Deane SM,Dicks LMT.Use of the mCherry fluorescent protein to study intestinal colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in mice.Appl Environ Microbiol,2015,81(17):5993-6002.
[27]van den Nieuwboer M,van Hemert S,Claassen E,et al.Lactobacillus plantarum WCFS1 and its host interaction:a dozen years after the genome.Microb Biotechnol,2016,9(4):452-465.
[28]Nguyen TT,Mathiesen G,Fredriksen L,et al.Afood-grade system for inducible gene expression in Lactobacillus plantarum using an alanine racemase-encoding selection marker.J Agric Food Chem,2011,59(10):5617-5624.
[29]Sak-Ubol S,Namvijitr P,Pechsrichuang P,et al.Secretory production of a beta-mannanase and a chitosanase using a Lactobacillus plantarum expression system.Microb Cell Fact,2016,15:81.
[30]Reverón I,Jiménez N,Curiel JA,et al.Differential gene expression by Lactobacillus plantarum WCFS1 in response to phenolic compounds reveals new genes involved in Tannin Degradation.Appl Environ Microbiol,2017,83(7):e03387-16.
[31]Huang Q,Huang L,Pan DD,et al.Expression in Escherichia coli,purification and functional characterization of recombination fusion protein MBP-BSH.Food Sci,2012,33(7):198-203(in Chinese).黄茜,黄璐,潘道东,等.重组融合蛋白MBP-BSH在大肠杆菌中的表达及其纯化、功能鉴定.食品科学,2012,33(7):198-203.
[32]Huang Q,Huang L,Pan DD,et al.Molecular cloning and expression of the bile salt hydrolase gene(bsh)from Lactobacillus plantarum Y1.JNanjing Norm Univ:Nat Sci Ed,2010,33(3):91-96(in Chinese).黄茜,黄璐,潘道东,等.植物乳杆菌Lactobacillu plantarum Y1菌株胆盐水解酶基因(bsh)的克隆及重组表达.南京师范大学学报:自然科学版,2010,33(3):91-96.
[33]S?rvig E,Gr?nqvist S,Naterstad K,et al.Construction of vectors for inducible gene expression in Lactobacillus sakei and L.plantarum.FEMS Microbiol Lett,2003,229(1):119-126.
[34]Moser SA,Savage DC.Bile salt hydrolase activity and resistance to toxicity of conjugated bile salts are unrelated properties in lactobacilli.Appl Environ Microbiol,2001,67(8):3476-3480.
[35]Landete JM,Langa S,Revilla C,et al.Use of anaerobic green fluorescent protein versus green fluorescent protein as reporter in lactic acid bacteria.Appl Microbiol Biotechnol,2015,99(16):6865-6877.
[36]Landete JM,Peiroténá,Rodríguez E,et al.Anaerobic green fluorescent protein as a marker of Bifidobacterium strains.Int Food Microbiol,2014,175:6-13.
[37]Geoffroy MC,Guyard C,Quatannens B,et al.Use of green fluorescent protein to tag lactic acid bacterium strains under development as live vaccine vectors.Appl Environ Microbiol,2000,66(1):383-391.
[38]Fredriksen L,Moen A,Adzhubei AA,et al.Lactobacillus plantarum WCFS1 O-linked protein glycosylation:an extended spectrum of target proteins and modification sites detected by mass spectrometry.Glycobiology,2013,23(12):1439-1451.
[39]Fujii T,Ingham C,Nakayama J,et al.Two homologous Agr-like quorum-sensing systems cooperatively control adherence,cell morphology,and cell viability properties in Lactobacillus plantarum WCFS1.J Bacteriol,2008,190(23):7655-7665.