菲律宾蛤仔二种I型溶菌酶的重组表达和抑菌活性
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摘要
为研究病原微生物刺激后,菲律宾蛤仔I型溶菌酶的免疫应答反应,实验采用重组表达技术,从菲律宾蛤仔中得到2种I型溶菌酶的重组蛋白rVpILYZ-1和rVpILYZ-2,发现其均具有较为广谱的抑菌活性;其中,rVpILYZ-1对副溶血弧菌、灿烂弧菌和藤黄微球菌等具较强的抑菌活性,而rVpILYZ-2对副溶血弧菌抑菌活性较强,且二者均通过非酶抑菌活性和胞壁酸酶活性来发挥抑菌作用。结果显示,rVpILYZ-1最适pH和温度分别为6.5和20°C,而rVpILYZ-2最适pH和温度则分别为4.5和10°C。此外,rVpILYZ-1和rVpILYZ-2均具免疫调理活性,能够促进血细胞对病原菌的吞噬作用。研究表明,VpILYZ-1和VpILYZ-2在菲律宾蛤仔清除病原微生物的免疫应答反应中发挥重要作用。
Lysozyme is an important immune effector present in phylogenetically diverse organisms, and plays important roles in bacteria elimination. In this study, two I-type lysozymes from Ruditapes philippinarum(designated as VpILYZ-1 and VpILYZ-2, respectively) were recombinantly expressed in Escherichia coli BL21(DE3). The recombinant proteins rVpILYZ-1 and rVpILYZ-2 showed broad spectrum of antimicrobial activities.Especially, the rVpILYZ-1 displayed high lytic activities against V. parahaemolyticus, V. splendidus and M. luteus,while rVpILYZ-2 showed strong microbicidal activities against V. parahaemolyticus. The antibacterial activities of rVpILYZs may be performed by both the enzymatic way and non-enzymatic ways. The optimum pH and temperature for the lytic activities of rVpILYZ-1 were 6.5 and 20 °C, and the highest antimicrobial activities of rVpILYZ-2 was detected at pH 4.5 and 10 °C. Moreover, both rVpILYZ-1 and rVpILYZ-2 possessed the opsonic activities, and the phagocytic activities of hemocytes were boosted after being incubated with rVpILYZ-1 or rVpILYZ-2. These results suggested that VpILYZ-1 and VpILYZ-2 played important roles in the immune responses of R. philippinarum.
Lysozyme is an important immune effector present in phylogenetically diverse organisms, and plays important roles in bacteria elimination. In this study, two I-type lysozymes from Ruditapes philippinarum(designated as VpILYZ-1 and VpILYZ-2, respectively) were recombinantly expressed in Escherichia coli BL21(DE3). The recombinant proteins rVpILYZ-1 and rVpILYZ-2 showed broad spectrum of antimicrobial activities.Especially, the rVpILYZ-1 displayed high lytic activities against V. parahaemolyticus, V. splendidus and M. luteus,while rVpILYZ-2 showed strong microbicidal activities against V. parahaemolyticus. The antibacterial activities of rVpILYZs may be performed by both the enzymatic way and non-enzymatic ways. The optimum pH and temperature for the lytic activities of rVpILYZ-1 were 6.5 and 20 °C, and the highest antimicrobial activities of rVpILYZ-2 was detected at pH 4.5 and 10 °C. Moreover, both rVpILYZ-1 and rVpILYZ-2 possessed the opsonic activities, and the phagocytic activities of hemocytes were boosted after being incubated with rVpILYZ-1 or rVpILYZ-2. These results suggested that VpILYZ-1 and VpILYZ-2 played important roles in the immune responses of R. philippinarum.
引文
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[19]Ye X,Zhang L L,Tian Y Y,et al.Identification and expression analysis of the g-type and c-type lysozymes in grass carp Ctenopharyngodon idella[J].Developmental&Comparative Immunology,2010,34(5):501-509.
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[22]Nash J A,Ballard T N S,Weaver T E,et al.The peptidoglycan-degrading property of lysozyme is not required for bactericidal activity in vivo[J].Journal of Immunology,2006,177(1):519-526.
[23]吴丹.褶纹冠蚌i-型溶菌酶基因表达及蛋白活性分析[D].南昌:南昌大学,2012.Wu D.Expression analysis of i-type lysozyme gene from Cristaria plicata and enzymatic activity analysis[D].Nanchang:Nanchang University,2012(in Chinese).
[24]Ibrahim H R,Thomas U,Pellegrini A.A helix-loophelix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action[J].Journal of Biological Chemistry,2001,276(47):43767-43774.
[25]Masschalck B,Deckers D,Michiels C W.Lytic and nonlytic mechanism of inactivation of gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure[J].Journal of Food Protection,2002,65(12):1916-1923.
[26]Masschalck B,Michiels C W.Antimicrobial properties of lysozyme in relation to foodborne vegetative bacteria[J].Critical Reviews in Microbiology,2003,29(3):191-214.
[2]Supungul P,Rimphanitchayakit V,Aoki T,et al.Molecular characterization and expression analysis of a c-type and two novel muramidase-deficient i-type lysozymes from Penaeus monodon[J].Fish&Shellfish Immunology,2010,28(3):490-498.
[3]Jiménez-Cantizano R M,Infante C,Martin-Antonio B,et al.Molecular characterization,phylogeny,and expression of c-type and g-type lysozymes in brill(Scophthalmus rhombus)[J].Fish&Shellfish Immunology,2008,25(1-2):57-65.
[4]Jollès J,Jollès P.The lysozyme from Asterias rubens[J].European Journal of Biochemistry,1975,54(1):19-23.
[5]JoskováR,?ilerováM,ProcházkováP,et al.Identification and cloning of an invertebrate-type lysozyme from Eisenia andrei[J].Developmental&Comparative Immunology,2009,33(8):932-938.
[6]Cong L N,Yang X J,Wang X X,et al.Characterization of an i-type lysozyme gene from the sea cucumber Stichopus japonicus,and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein[J].Journal of Bioscience and Bioengineering,2009,107(6):583-588.
[7]Beckert A,Wiesner J,Schmidtberg H,et al.Expression and characterization of a recombinant i-type lysozyme from the harlequin ladybird beetle Harmonia axyridis[J].Insect Molecular Biology,2016,25(3):202-215.
[8]Carrillo W,García-Ruiz A,Recio I,et al.Antibacterial activity of hen egg white lysozyme modified by heat and enzymatic treatments against oenological lactic acid bacteria and acetic acid bacteria[J].Journal of Food Protection,2014,77(10):1732-1739.
[9]Yang D L,Wang Q,Chen L Z,et al.Molecular characterization and antibacterial activity of a phagetype lysozyme from the Manila clam,Ruditapes philippinarum[J].Fish&Shellfish Immunology,2017,65:17-24.
[10]Xue Q G,Itoh N,Schey K L,et al.A new lysozyme from the eastern oyster(Crassostrea virginica)indicates adaptive evolution of i-type lysozymes[J].Cellular and Molecular Life Sciences,2007,64(1):82-95.
[11]Yang D L,Wang Q,Cao R W,et al.Molecular characterization,expression and antimicrobial activities of two c-type lysozymes from Manila clam Venerupis philippinarum[J].Developmental&Comparative Immunology,2017,73:109-118.
[12]Jollès P,Jollès J.What's new in lysozyme research?Always a model system,today as yesterday[J].Molecular and Cellular Biochemistry,1984,63(2):165-189.
[13]Zhang K X,Gao R,Zhang H X,et al.Molecular cloning and characterization of three novel lysozyme-like genes,predominantly expressed in the male reproductive system of humans,belonging to the c-type lysozyme/alphalactalbumin family[J].Biology of Reproduction,2005,73(5):1064-1071.
[14]Bathige S D N K,Umasuthan N,Kasthuri S R,et al.Abifunctional invertebrate-type lysozyme from the disk abalone,Haliotis discus discus:genome organization, transcriptional profiling and biological activities of recombinant protein[J].Developmental&Comparative Immunology,2013,41(2):282-294.
[15]Yue X,Liu B Z,Xue Q G.An i-type lysozyme from the Asiatic hard clam Meretrix meretrix potentially functioning in host immunity[J].Fish&Shellfish Immunology,2011,30(2):550-558.
[16]Zhou J,Zhao S,Fang W H,et al.Newly identified invertebrate-type lysozyme(Splys-i)in mud crab(Scylla paramamosain)exhibiting muramidase-deficient antimicrobial activity[J].Developmental&Comparative Immunology,2017,74:154-166.
[17]Dai W J,Wu D,Zhang M,et al.Molecular cloning and functional characterization of a novel i‐type lysozyme in the freshwater mussel Cristaria plicata[J].Microbiology and Immunology,2015,59(12):744-755.
[18]祝艳霞,母昌考,王春琳.温度、盐度变化及鳗弧菌刺激对菲律宾蛤仔Ⅰ型溶菌酶基因表达的影响[J].水产学报,2016,40(12):1813-1822.Zhu Y X,Mu C K,Wang C L.Expression analysis of two I-type lysozyme genes from Ruditapes philippinarum under temperature and salinity stress and Vibrio anguillarum challenge[J].Journal of Fisheries of China,2016,40(12):1813-1822(in Chinese).
[19]Ye X,Zhang L L,Tian Y Y,et al.Identification and expression analysis of the g-type and c-type lysozymes in grass carp Ctenopharyngodon idella[J].Developmental&Comparative Immunology,2010,34(5):501-509.
[20]Olsen?M,Nilsen I W,Sletten K,et al.Multiple invertebrate lysozymes in blue mussel(Mytilus edulis)[J].Comparative Biochemistry and Physiology-Part B:Bioch- emistry and Molecular Biology,2003,136(1):107-115.
[21]Salih E,Malthouse J P,Kowlessur D,et al.Differences in the chemical and catalytic characteristics of two crystallographically‘identical’enzyme catalytic sites.Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment[J].The Biochemical Journal,1987,247(1):181-193.
[22]Nash J A,Ballard T N S,Weaver T E,et al.The peptidoglycan-degrading property of lysozyme is not required for bactericidal activity in vivo[J].Journal of Immunology,2006,177(1):519-526.
[23]吴丹.褶纹冠蚌i-型溶菌酶基因表达及蛋白活性分析[D].南昌:南昌大学,2012.Wu D.Expression analysis of i-type lysozyme gene from Cristaria plicata and enzymatic activity analysis[D].Nanchang:Nanchang University,2012(in Chinese).
[24]Ibrahim H R,Thomas U,Pellegrini A.A helix-loophelix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action[J].Journal of Biological Chemistry,2001,276(47):43767-43774.
[25]Masschalck B,Deckers D,Michiels C W.Lytic and nonlytic mechanism of inactivation of gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure[J].Journal of Food Protection,2002,65(12):1916-1923.
[26]Masschalck B,Michiels C W.Antimicrobial properties of lysozyme in relation to foodborne vegetative bacteria[J].Critical Reviews in Microbiology,2003,29(3):191-214.