东北林蛙皮肤抗微生物肽真核表达体系构建与抗菌活性研究
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摘要
抗微生物肽(Antimicrobial peptides,AMPs)作为生物体天然免疫屏障的先锋效应因子,可通过病原体诱导的免疫应答迅速合成,表现为广谱抗微生物活性,且两栖类皮肤中抗微生物肽的种类及数量最为丰富。本项目以东北地区两栖类代表物种东北林蛙(Rana dybowskii)为研究对象,构建其皮肤AMPs分泌型毕赤酵母表达系统,获得重组AMPs,进行抗菌活性鉴定,为筛选抗菌效果显著的新药候选分子以及探讨其作用机制奠定基础。
方法:本实验采用PCR技术在重组pMD18-T质粒上扩增AMPs基因,在基因两端引入酶切位点,首次利用AMPs前体中天然EKR序列设计成熟肽AMPs表达程序,PCR产物经EcoRⅠ和NotⅠ双酶切,克隆入分泌型表达载体pPIC9K,PCR鉴定、DNA测序检测重组质粒,确定正确插入的重组真核表达载体。真核表达载体经LiCl转化和PEG1000转化导入毕赤酵母后涂于MD平板,挑取菌落,G418加压筛选多拷贝株,进行表型鉴定、PCR鉴定,确定获得正确插入的高拷贝重组酵母株。筛选优化重组酵母株培养条件,摇瓶培养,培养液经葡聚糖G-25层析脱盐、冻干,Bradford法检测重组AMPs含量,比浊法测定重组AMPs对标准质控G-大肠杆菌(E.coli)、G~+金黄色葡萄球菌(Saureus)最低抑菌浓度(MIC)和协同效应。
结果:(1)获得六种正确插入的重组真核分泌型表达载体pPIC9K-DB_1、pPIC9K-DB_2、pPIC9K-DB_3、pPIC9K-CS_1、pPIC9K-CS_2、pPIC9K-AM_3;并成功获得AMP-DB_2、AMP-CS_2、AMP-AM_3三种重组分泌型毕赤酵母高拷贝株,分别为5、11、1株;重组酵母摇瓶培养最优条件为0.5%甲醇,诱导36h。(2)重组AMP-DB_2、AMP-CS_2、AMP-AM_3对G~+S.aureus的MIC分别为7.44、14.88、14.88(mg/ml),而对Ecoli的MIC则分别为59.52、29.76、59.52(mg/ml);AMP-DB_2与AMP-CS_2的组合对G~-E.coli的MIC比单独作用时分别降低至75%和50%,但重组AMPs对G~+S.aureus无明显协同作用。
结论:本项目首次利用AMPs前体中天然EKR序列设计成熟肽AMPs表达载体,建立了东北林蛙皮肤成熟AMPs的分泌型毕赤酵母表达系统。并通过重组酵母诱导成功生成对G~+和G~-菌有抑杀作用的重组AMPs,对G~-E.coli活性的抑制有明显协同作用。
Antimicrobial peptides(AMPs)which are pioneer effectors in natural immunity defense line,synthesize rapidly through the immune response inducted by pathogen.The AMPs has broad-spectrum antimicrobial activity.The type and quantity of AMPs from the amphibian skin and the secretion are richest in all Organisms.We use the Pichia pastoris expression system to express several AMPs from the skin of Rana dybowskii which is one of main amphibian species in northeast of china,they could become the experimental referece for the production of a new speciall antimicrobial drug,and be used to research the mechanism of antimicrobial effect and synergetic effect.
Methods:Using the six recombinant pMD18-T as the template,six AMPs genes were amplified with PCR and double digested with EcoRⅠand NotⅠ.These genes were ligated into vector pPIC9K digested with EcoRⅠand NotⅠ.Then they were transformed into host strain GS115,several His+cell line was screened and multicopy transformants were screened by various G418 concentrations.The transformants were cultivated in flasks,and firstly used the nature EKR sequence in AMPs Precursors to get mature peptides in culture medium.The supernatant were desalted by Sephadex G-25,and then lyophilized.We tested minimal inhibitory concentrations(MICs)of AMPs on G~+(Escherichia coli)、G~-(Staphyloccocus aureus) in vitro,and they are combinated with Each other for the test of synergetic effect.
The results were as follows:(1)we got six recombinant expression plasmids pPIC9K-DB_1、pPIC9K-DB_2、pPIC9K-DB_3、pPIC9K-CS_1、pPIC9K-CS_2、pPIC9K-AM_3,and got three recombined P.pastoris cell line which can successfully express AMP-DB_2、AMP-CS_2、AMP-AM3,respectively are five、eleven one transformants.The optimum condition is inducted by 0.5%methanol for 36 hour.(2)The antimicrobial effect on G~+S.aureus of all AMPs is better than G~-E.coli,and the average MIC of G~+S.aureus is 25%of G'E.coli.The mixture of AMP-DB_2 and AMP-CS_2can suppress G~+E.coli better,the MIC is reduced by 75% and 50%respectively.The synergetic effect of AMPs on S.aureus is inapparent.
Conclutions:This study has constructed P.pastoris expression system of AMPs from the skin of Rana dybowskii.The recombined AMPs have antimicrobial activity,and some of them displayed synergetic effect on G~-E.coli.
方法:本实验采用PCR技术在重组pMD18-T质粒上扩增AMPs基因,在基因两端引入酶切位点,首次利用AMPs前体中天然EKR序列设计成熟肽AMPs表达程序,PCR产物经EcoRⅠ和NotⅠ双酶切,克隆入分泌型表达载体pPIC9K,PCR鉴定、DNA测序检测重组质粒,确定正确插入的重组真核表达载体。真核表达载体经LiCl转化和PEG1000转化导入毕赤酵母后涂于MD平板,挑取菌落,G418加压筛选多拷贝株,进行表型鉴定、PCR鉴定,确定获得正确插入的高拷贝重组酵母株。筛选优化重组酵母株培养条件,摇瓶培养,培养液经葡聚糖G-25层析脱盐、冻干,Bradford法检测重组AMPs含量,比浊法测定重组AMPs对标准质控G-大肠杆菌(E.coli)、G~+金黄色葡萄球菌(Saureus)最低抑菌浓度(MIC)和协同效应。
结果:(1)获得六种正确插入的重组真核分泌型表达载体pPIC9K-DB_1、pPIC9K-DB_2、pPIC9K-DB_3、pPIC9K-CS_1、pPIC9K-CS_2、pPIC9K-AM_3;并成功获得AMP-DB_2、AMP-CS_2、AMP-AM_3三种重组分泌型毕赤酵母高拷贝株,分别为5、11、1株;重组酵母摇瓶培养最优条件为0.5%甲醇,诱导36h。(2)重组AMP-DB_2、AMP-CS_2、AMP-AM_3对G~+S.aureus的MIC分别为7.44、14.88、14.88(mg/ml),而对Ecoli的MIC则分别为59.52、29.76、59.52(mg/ml);AMP-DB_2与AMP-CS_2的组合对G~-E.coli的MIC比单独作用时分别降低至75%和50%,但重组AMPs对G~+S.aureus无明显协同作用。
结论:本项目首次利用AMPs前体中天然EKR序列设计成熟肽AMPs表达载体,建立了东北林蛙皮肤成熟AMPs的分泌型毕赤酵母表达系统。并通过重组酵母诱导成功生成对G~+和G~-菌有抑杀作用的重组AMPs,对G~-E.coli活性的抑制有明显协同作用。
Antimicrobial peptides(AMPs)which are pioneer effectors in natural immunity defense line,synthesize rapidly through the immune response inducted by pathogen.The AMPs has broad-spectrum antimicrobial activity.The type and quantity of AMPs from the amphibian skin and the secretion are richest in all Organisms.We use the Pichia pastoris expression system to express several AMPs from the skin of Rana dybowskii which is one of main amphibian species in northeast of china,they could become the experimental referece for the production of a new speciall antimicrobial drug,and be used to research the mechanism of antimicrobial effect and synergetic effect.
Methods:Using the six recombinant pMD18-T as the template,six AMPs genes were amplified with PCR and double digested with EcoRⅠand NotⅠ.These genes were ligated into vector pPIC9K digested with EcoRⅠand NotⅠ.Then they were transformed into host strain GS115,several His+cell line was screened and multicopy transformants were screened by various G418 concentrations.The transformants were cultivated in flasks,and firstly used the nature EKR sequence in AMPs Precursors to get mature peptides in culture medium.The supernatant were desalted by Sephadex G-25,and then lyophilized.We tested minimal inhibitory concentrations(MICs)of AMPs on G~+(Escherichia coli)、G~-(Staphyloccocus aureus) in vitro,and they are combinated with Each other for the test of synergetic effect.
The results were as follows:(1)we got six recombinant expression plasmids pPIC9K-DB_1、pPIC9K-DB_2、pPIC9K-DB_3、pPIC9K-CS_1、pPIC9K-CS_2、pPIC9K-AM_3,and got three recombined P.pastoris cell line which can successfully express AMP-DB_2、AMP-CS_2、AMP-AM3,respectively are five、eleven one transformants.The optimum condition is inducted by 0.5%methanol for 36 hour.(2)The antimicrobial effect on G~+S.aureus of all AMPs is better than G~-E.coli,and the average MIC of G~+S.aureus is 25%of G'E.coli.The mixture of AMP-DB_2 and AMP-CS_2can suppress G~+E.coli better,the MIC is reduced by 75% and 50%respectively.The synergetic effect of AMPs on S.aureus is inapparent.
Conclutions:This study has constructed P.pastoris expression system of AMPs from the skin of Rana dybowskii.The recombined AMPs have antimicrobial activity,and some of them displayed synergetic effect on G~-E.coli.
引文
[1]Boman HG.Antimicrobial Peptides:Key Components Needed in Immunity [J].Cell,1991,65:205-207
[2]Man AK,Gooderham JW,Hancock EW.Antibacterial Peptides for Therapeutic use:Obstacles and Realistic Outlook [J].Curr Opin Pharmacol,2006,6:468-472
[3]Barraa D,Simmacob M,Boman HG.Gene-encoded Peptide Antibiotics and Innate Immunity.Do "Animalcules" have Defence Budgets? [J]FEBS Lett,1998,430:130-134
[4]Lupetti A,Nibbering HP,Welling MM.et al.Radiopharmaceuticals:New Antimicrobial Agents [J].Trends Biotechnol,2003,21(2):70-73
[5]Boman HG.Gene-encoded Peptide Antibiotics and the Concept of Innate Immunity:A Update Review [J].Scand T Immunol.1998,48:15-25
[6]Brown KL,Hancock EW.Cationic Host Defense (Antimicrobial) Peptides [J].Curr Opin Immunol,2006,18:24-30
[7]Hancock EW,Diamond G.The Role of Cationic Antimicrobial Peptides in Innate Host Defences [J].Trends Microbiol,2000,8(9):402-410
[8]王可洲,李浩,刘学玲．抗菌肽的分类及作用机理研究进展[J]．科学技术与工程,2005,5(7)：415-420
[9]Powers JPS,Hancock EW.The Relationship between Peptide Structure and Antibacterial Activity [J].Peptides,2003,24:1681-1691
[10]Michael RY,Nannette YY.Mechanisms of Antimicrobial Peptide Action and Resistance [J].Pharmacol Rev,2003,55(1):27-55
[11]Harder J,Bartels J,Christophers E,et al.Isolation and Characterization of Human betadefensin 3,a Novel Human Inducible Peptide Antibiotic [J].J Biol Chem,2001,276 (8):5707-5713
[12]Bobek LA,Situ H.MUC7:Investingation of Antimicrobial Activity,Secondary Structure,and Possible Mechanism of Antifungal Action [J].Antimicrob Agents Chemother,2003,47 (2):643-652
[13]Chen Y,Xu X,Hong S,et al.RGD Tachyples Ininhibits Tumor Growth [J].Cancer Res,2001,61 (6):2434-2438
[14]Fehlbaum P,Bulet P,Chernysh S,et al.Structure Activity Analysis of Thanatin,a 21-Residueinduc Inducible Insect Defense Peptide with Sequence Homology to Frog Skin Antimicrobial Peptides [J].Proc Natl Acad Sci USA,1996,93 (3):1221-1225
[15]Huang HW.Action of Antimicrobial Peptides:Two State Model [J].Biochem,2000,39:8347-8352
[16]Lee DG,Sin SY,Meng CY.Cecropin A-melittin Hybrid Peptide Exerts its Antifungal Effects by Damaging on the Plasma Membranes of Trichosporon beigelii [J].Biotechnol Lett,1998,20(3):211-214
[17]Fehllbaum P,Bulet P.Structure-activity Analysis of Thanatin,a 21-residue Inducible Insect Defense Peptide with Sequence Homology to Frog Skin Antimicrobial Peptides [J].Proc Natl Acad Sci,1996,93(3):1221-1225
[18]Park CB,Kim HS,Kim SC.Mechanism of Action of the Antimicrobial Peptide Buforin Ⅱ:Buforin Ⅱ Kills Microorganism by Penetrating the Cell Membrane and Inhibiting Cellular Functions [J].Biochem Biophys Res Common,1998,244:253-257
[19]Chen HM,Leung KW,Thakur NN.Distinguishing between different pathways of bilayer disrup tion by the related antimicrobial peptides cecrop in B,Bl and B3 [J].Eur J Biochem,2003,270 (5):911-920
[20]Mai JC,Mi Z,Kim SH.Aproapoptotic Peptide for the Treatment of Solid Tumors [J].Cancer Res,2001,61 (21):7709-7712
[21]Hariton Gazal E,Fede rR,MorA,et al.Targeting of Fonokaryophilic Cell-permeable Peptides into the Nuclei of Intact Cells by Covalently Attached Nuclear Localization Signals [J].Biochem,2002,41(29):9208-9214
[22]DekkerN,Cox RC,Kramer RA,et al.Substrate Specificity of the Integral Membrane Protease OmpT Determined by Spatially Addressed Peptide Libraries [J].Biochem,2001,40 (6):1694-1701
[23]Aboudy Y,Mendelson E,Shalit I,et al.Activity of two Synthetic Amphiphilic Peptides and Magainin-2 against Herpes Simplex Virus Types 1 and 2 [J].Int J Pept Protein Res,1994,43(6):573-582
[24]Wachinger M,Kleinschmidt A,Winder D,et al.Antimicrobial Peptides Melttin and Cecropin Inhibit Replication of Human Immunodeficiency Virus I by Suppressing Viral Gene Expression [J].Gen Virol,1998,79:731-740
[25]Andreu D,Rivas L.Animal Antimicrobial Peptides:an Overview [J].Biopolymers,1999,47:413-433
[26]Mangoni ML,Saugar JM,Dellisanti M,et al.Temporins,Small Antimicrobial Peptides with Leishmanicidal Activity [J].Biochem,2005,280:984-990
[27]Vizioli J,Salzet M.Antimicrobial Peptides versus Parasitic Infections [J].Trends Parasitol,2002,18(11):475-476
[28]Shahabuddin M,Phillipe B,Jules A,et al.Plasmodium gallinaceum:Differential Killing of Some Mosquito Stages of the Parasite by Insect Defensin [J].Experimental Parasitogy,1998,89(1):103-112
[29]Bulet P,cklin RS,Menin L.Anti-microbial Peptides:from Invertebrates to Vertebrates [J].Immunol Rev,2004,198:169-184
[30]Takumi O,Fujitoshi Y,Masao T,et al.Production and Purification of a Bacteriocin Pep tide Produced by Lactococcus sp Strain GM005,Isolated from Misopaste [J].Int J Food M icrob,2003,87 (1-2):153-159
[31]Lehrer RI,Ganz T.Antimicrobial Peptides in Mammalian and Insect Host Defence [J].Curr Opin Immunol,1999,11:23-30
[32]尹文,徐晨,马戈甲．抗菌肽的研究进展[J]．细胞与分子免疫学杂志,2005,21(1)：136-138
[33]Li J,Xu X,Xu C,et al.Anti-infection Peptidomics of Amphibian Skin [J].Mol Cell Proteomics.2007,l(6):882-894
[34]Gudmundsson GH,Lidholm DA,Asling B,et al.The Cecropin Locus.Cloning and Expression of a Gene Cluster Encoding Three Antibacterial Peptides in Hyalophora cecropia [J].J Biol Chem,1991,266:11510-11517
[35]Ganz T.Defensins and other Antimicrobial Peptides:a Historical Perspective and an Update [J].Comb Chem High Throughput Screen,2005,8:209-217
[36]Zanetti M.The Role of Cathelicidins in the Innate Host Defenses of Mammals [J].Curr Issues Mol Biol,2005,7:179-196
[37]Pasquier LD.Meeting the Demand for Innate and Adaptive Immunities during Evolution [J].Scandinavian J Immunol,2005,62:39-48
[38]Cellura C,Toubiana M,Parrinello N,et al.Specific Expression of Antimicrobial Peptide and HSP70 Genes in Response to Heat-shock and Several Bacterial Challenges in Mussels [J].Fish Shellfish Immunol.2007,22:340-350
[39]Mitta G,Hubert F,Dyrynda EA,et al.Mytilin B and MGD2,two Antimicrobial Peptides of Marine Mussels:Gene Structure and Expression Analysis [J].Developmental Comparative Immunol.2000,24(4):381-393
[40]Menzies BE,Kenoyer A.Staphylococcus aureus Infection of Epidermal Keratinocytes Promotes Expression of Innate Antimicrobial Peptides [J].Infection and Immunity.2005,73(4):5241-5244
[41]Lemaitre B,Reichhart JM,Hoffmann JA.Drosophila Host Defense:Differential Induction of Antimicrobial Peptide Genes after Infection by Various Classes of Micro organisms [J].Proc Natl Acad Sci USA.1997,94:14614-14619
[42]Engstrom Y.Induction and Regulation of Antimicrobial Peptides in Drosophila [J].Developmental Comparative immunol.1999,23:345-358
[43]Medzhitov R,Janeway CA.Innate Immunity:The Virtues of a Nonclonal System of Recognition [J].Cell.1997,91:295-298
[44]Lee WJ,Lee JD,Kravchenko VV,et al.Purification and Molecular Cloning of an Inducible Gram-negative Bacteria-binding Protein from the Silkworm,Bombyx mori[J].Proc Natl Acad Sci USA.1996,93:7888-7893
[45]Kang D,Liu G,LundstroE m A,et al.A Peptidoglycan Recognition Protein in Innate Immunity Conserved from Insects to Humans [J].Proc Natl Acad Sci USA.1998,95:10078-10082
[46]Rollins-Smith LA,Woodhams DC,Reinert LK,et al.Antimicrobial Peptide Defenses of the Mountain Yellow-leged Frog (Rana muscosa) [J].Developmental Comparative Immunol.2006,30:831-842
[47]Levashina EA,Ohresser S,Lemaitre B,et al.Two Distinct Pathways can Control Expression of the Gene Encoding the Drosophila Antimicrobial Peptide Metchnikowin [J].J Mol Biol.1998.278(3):515-527
[48]Mor A,Hani K,Nicolas P.The Vertebrate Peptide Antibiotics Dermaseptins Have Overlapping Structural Features but Target Specific Microorganisms [J].J Biol Chem,1994,269:31635-31641
[49]Giacometti A,Cirioni O,Kamysz W,et al.In vitro Activity of Amphibian Peptides alone and in Combination with Antimicrobial Agents against Multidrug-resistant Pathogens Isolated from Surgical wound infection [J].Peptides,2005,26:2111-2116
[50]Mor A,Hani K,Nicolas P.The Vertebrate Peptide Antibiotics Dermaseptins Have Overlapping Structural Features but Target Specific Microorganisms [J].J Biol Chem,1994,269:31635-31641
[51]Chen XJ,Niyonsaba F,Ushio H,et al.Synergistic Effect of Antibacterial Agents Human 6-defensins,Cathelicidin LL-37 and Lysozyme against Staphylococcus aureus and Escherichia coli [J].Dermatol Sci,2005,40:123-132
[52]Kumar M,Chaturvedi AK,Kavishwar A,et al.Identification of a Novel Antifungal Nona Peptide Generated by Combinatorial Approach [J].Int J Antimicrob Agen,2005,25:313-320
[53]Moore AJ,Beazley WD,Bibby MC,et al.Antimicrobial Activity of Cecropins [J].J Antimicrob Chemother,1996,37:1077-1089
[54]Miele R,Ponti D,Boman HG,et al.Molecular Cloning of a Bombinin Gene from Bombina orientalis:Detection of NF-kB and NF-IL6 Binding Sites in its Promoter [J].FEBS Lett,1998,431:23-28
[55]Liu L,Zhao C,Heng HH,et al.The Human B-defensin-1 and a-defensins are Encoded by djacent Genes:Two Peptide Families with Differing Disulfide Topology Share a CommonAncestry [J].Genomics,1997,43:316-320
[56]Marco S,Shenglun W,Margherita Z.Structural Organization of the Bovine Cathelicidin Gene Family and Identification of a Novel Member [J].FEBS Lett,1997,417:311-315
[57]Vanhoye D,Bruston F,Nicolas P,et al.Antimicrobial Peptides from Hylid and Ranid Frogs Originated from a 150-million-year-old Ancestral Precursor with a Conserved Signal Peptidebut a Hypermutable Antimicrobial Domain [J].Eur J Biochem,2003,270:2068-2081
[58]Michaelson D,Rayner J,Conto M,et al.Cationic Defensins Arise from Charge-neutralized ropeptides:a Mechanism for Avoiding Leukocyte Autotoxicity [J].J Leukoc Biol,1992,51:32-639
[59]Oren Z,Shai YA.Class of Highly Potent Antibacterial Peptides Derived from Pardaxin,a Pore-forming Peptide Isolated from Mosesesole Fish pardachirus marmoratus [J].Eur J Biochem,1996,237(1):303-310
[60]Swendsen CL,Sphepard DA.Study of Antimicrobial Effects of Mountain Dusky salamander mucus [J].Proc Nat Acad Sci USA,1998,86:24-31
[61]Barra D,Simmaco M,Boman HG.Gene-encoded Peptide Antibiotics and Innate Immunity.Do'Animalcules'have Defence Budgets? [J]FEBS Lett,1998,430(1-2):130-134
[62]Hancock REW,Chappie DS.Peptides Antibiotics [J].Antimicrob Agents Chemother,1999,43(6):1317-1323
[63]Simmaco M,Mignogna G,and Barra D.Antimicrobial Peptides from Amphibian Skin:What do they tell us? [J].Biopolymers,1998,47(6):425-450
[64]Magainins MZ.a Class of Antimicrobial Peptides from Xenopus Skin:Isolation、Characterzation of two Active Forms,and Partial cDNA Sequence of a Precursor [J].Proc Nat Acad Sci,1987,84(15):5449-5453
[65]Boman HG.Peptide Antibiotics and their Role in Innate Immunity [J].Annu Rev Immunol,1995,13:61-92
[66]Steinborner ST,Currie GJ,Bowie JH,et al.New Antibiotic Cearinl Peptides from the Skin Secretion of the Australian Tree Frog Litoriachloris,Comparison of the Activities of the Caerinl Peptides from the Genus Litoria [J].J Peptide Research,1985,51:121-126
[67]Wegene KL,Wabnitz PA,Carver JA.Host Defense Peptides from the Skin Glands of the Australian Blue Mountains Tree Frog Litoriacitropa [J].Eur J Biochem,1999,265:627-637
[68]Giovannini MG,Poulter L,Gibson BW,et al.Biosynthesis and Egradation of Peptides Derived from Xenopus laevis prohormones [J].Biochem J,1987,243(1):113-120
[69]Chia BCS,Carver JA,Mulhern TD,et al.The Solution Structure of Upperin3.6,an Antibiotic Peptide from the Granular Dorsal Glands of the Australian Toad let,Uperoleiam jobergii[J].J Peptide Reseach,1999,54:137-145
[70]Chia BCS,Carver JA,Mulhern TD,et al.Maculatinll,an Antimicrobial Peptide from the Australian Tree Frog,Litoriageni Maculata Solution Struncture and Biological Activity[J].Eur J Biochem,2000,267:1894-1908
[71]Gibson BW,Tang DZ,Mandrell R,et al.Bombinin like Peptides with Antimicrobial Activity from Skin Secretion of the Asian Toad,Bombinaorientalis[J].J Biol Chem,1991,266(34):23103-23111
[72]Conlon JM,Kolodziejek J,Nowotny N.Antimicrobial Peptides from Ranid Frogs:Taxonomic and Phylogenetic Markers and a Potential Source of new Therapeutic Agents [J].Biochim Biophys Acta.2004,1696(1):1-14
[73]Ohnuma A,Conlon JM,Yamaguchi K,et al.Antimicrobial Peptides from the Skin of the Japanese Mountain Brown Frog Rana ornativentris:Evidence for Polymorphism among preprotemporin mRNAs[J].Peptides.2007,28(3):524-532
[74]Clarke BT.The Natural History of a Amphibian Skin Secretions,their Normal Function and Potential Medical Applications[J].Biol Rev.1997,72(3):365-379
[75]谢锋,叶昌媛,费梁,等.中国东北地区林蛙属物种的分类学研究(两栖纲:蛙科)[J].动物分类学报.1999,24(2):224-231
[76]Conlona JM,Kolodziejekb J,Nowotny N.Antimicrobial Peptides from Ranid Frogs:Taxonomic and Phylogenetic Markers and a Potential Source of New Therapeutic Agents [J].Biochimi Biophys Acta,1696(2004):1-14
[77]Marra AK,Gooderhama WJ,Hancock REW.Antibacterial Peptides for Therapeutic Use:Obstacles and Realistic Outlook[J].Curr Opin Pharmacol,2006,6(5):468-472
[78]Hancock REW,Lehrer R.Cationic Peptides:a New Source of Antibiotics[J].Trends Biotechnol,1998,16(7):82-88
[79]陆莹瑾,王禾,屈贤铭.抗菌肽的固相合成、分离纯化与构效关系的研究[J].生物工程学报,1997,13(1):35-41
[80]Schmidt FR.Recombinant Expression Systems in the Pharmaceutical Industry[J].Appl Microb Biotech,2004,65:363-372
[81]Shigeyuki Yokoyama.Protein Expression Systems for Structural Genomics and Proteomics[J].curr opin Chem Biol,2003,7:39-43
[82]Better M,Chang CP,Robinson RR,et al.Escherichia coli Secretion of an Active Chimeric Antibody Fragment[J].Science,1988,240(4855):1041-1043
[83]Yedery RD,Reddy KV.Antimicrobial Peptides as Microbicidal Contraceptives:Prophecies for Prophylactics[J].Eur J Contracept Reprod Health Care,2005,10(1):32
[84]Lange CF,Hancock RE,Samuel J,et al.In Vitro Aerosol Delivery and Regional Airway Surface Liquid Concentration of a Liposomal Cationic Peptide[J].J Pharm Sci,2001,90(10):1647
[85]Zhang L,Falla T,Wu M,et al.Determinants of Recombinant Production of Antimicrobial Cationic Peptides and Creation of Peptide Variants in Bacteria [J].Biochem Biophys Res Commun,1998,247(3):674
[86]Lee JH,Minn Ⅱ,Park CB,et al.Acidic Peptide-Mediated Expression of the Antimicrobial Peptide Buforin Ⅱ as Tandem Repeats in Escherichia coli [J].Protein Expr Purif,1998,12(1):53
[87]Harder J,Bartels J,Christophers E,et al.Isolation and Characterization of Human beta-defensin-3,a Novel Human Inducible Peptide Antibiotic [J].J Biol Chem,2001,276( 8):5707
[88]Peng L,Xu Z,Fang X,et al.High-level Expression of Soluble Human β-defensin-2 in Escherichia coli [J].Process Biochem,2004,39(12):2199
[89]Moon JY,Henzler Wildman KA,Ramamoorfhy A.Expression and Purification of a Recombinant LL-37 from Escherichia coli [J].Biochim Biophys Acta,2006,1758(9):1351
[90]Rao X,Hu J,Li S,et al.Design and Expression of Peptide Antibiotic hPAB-beta as Tandem Multimers in Escherichia coli [J].Peptides,2005,26(5):721
[91]Erik Boer,Gerhard S,Gotthard K,et al.Yeast Expression Platforms [J].Appl Microb Biotech,2007,77:513-523
[92]Vilas Alves AL,Samblanx GW,Terras FRG,et al.Expression of Functional Raphanus Sativus Antifungal Protein in Yeast [J].FEBS Lett,1994,384 (3):228-232
[93]Destoumieux D,Bule P,Strub JM,et al.Recombiant Expression and Range of Activity of Penaeidins,Antimicrobial Peptides from Penaeid Shrimp [J].Eur J Biochem,1999,266 (2):335-346
[94]Cereghino JL,Cregg JM.Heterologous Protein Expression in the Methylotrophic Yeast Pichia pastoris [J],FEMS Microb Rev,2000,24:45-66
[95]Ogata K,Nishikawa H,Ohsugi M.A Yeast Capable of Utilizing Methanol [J].Agric Biol Chem,1969,33:1519-1520
[96]Davidson JF.The Origin of Insights in Chemical Engineering:Planned and Unplanned research [J].Chem Eng Sci,1995,50(23):3661-3684
[97]Gellissen G.Heterologous Protein Production in Methylotrophic Yeasts [J].Appl Microb Biotech.2000,54(6):741-750
[98]Daly R,Hearn MTW.Expression of Heterologous Proteins in Pichia pastoris:a Useful Experimental Tool in Protein Engineering and Production [J].J Mol Recognit.2005,18:119-138
[99]Rosenfeld SA,Nadeau D,Tirado J,et al.Production and Purification of Recombinant Hirudin Expressed in the Methylotrophic Yeast Pichia pastoris [J].Protein Expre Purif,1996,8:476
[100]Kristensen AK,Brunstedt J,Nielsen JE,et al.Processing,Disulfide Pattern,and Biological Activity of a Sugar Beet Defensin,AX2,Expressed in Pichia pastoris[J].Protein Expr Purif,1999,16(3):377-387
[101]Almeida MS,Cabral KS,de Medeiros LN,et al.cDNA Cloning and Heterologous Expression of Functional Cysteine-rich Antifungal Protein Psd1 in the Yeast Pichia pastoris[J].Arch Biochem Biophys,2001,395(2):199
[102]Cabral KM,Almeida MS,Valente AP,et al.Production of the Active Antifungal Pisum Sativum Defensin 1(Psdl) in Pichia pastoris:Overcoming the Inefficiency of the STE13Protease[J].Protein Expr Purif,2003,31(1):115
[103]Chen JJ,Chen GH,Hsu HC,et al.Cloning and Functional Expression of a Mungbean Defensin VrD1 in Pichia pastoris[J].J Agric Food Chem,2004,52(8):2256
[104]Hong IP,Lee SJ,Choi SG.Recombinant Expression of Human Cathelicidin(hCAP18/LL-37) in Pichia pastoris[J].Biotechnol Lett,2007,29(1):73
[105]张素芳,曹瑞兵,贾赟,等.杂合抗菌肽 CecA-mil 的改造及在毕赤酵母中的分泌表达[J].微生物学报,2005,45(2):218-222
[106]赵亚华,董竟南,崔红,等.蜂毒素分子的改造及其基因在毕赤酵母中的表达[J].中国生物工程杂志,2005,25(2):45-48
[107]陆建荣,王惠民,吴萍,等.人源抗菌肽 LL-37表达载体的构建及其在毕赤酵母中的表达[J].第二军医大学学报,2007,28(8):833-837
[108]Branduardi P,Valli M,Brambilla L,et al.The Yeast Zygosaccharomyces bailii:a new Host for Heterologous Protein Production,Secretion and for Metabolic Engineering Applications[J].FEMS Yeast Research,2004,4:493-504
[109]Hasemann CA,Capra JD,Hasemann CA,et al.High-level Production of a Functional Immunoglobulin Heterodimer in a Baculovirus Expression System[J].Proc Natl Acad Sci,1990,87:3942-3946
[110]Smith GE,Summers MD,Fraser MJ.Production of Human Beta Interferon in Insectcell Infected with a Baculovirus Express Vector[J].Mol Cell Biol,1983(3):2156-2165
[111]Hellers M,Gunne H,Steiner H.Expression and Posttranslational Processing of Preprocecropin A Using a Baculovirus Vector[J].Eur Biochem,1991,199(2):435-439
[112]Andersons D,Engstrom A,Josephsom S,et al.Biologically Active and Amidated Cecropin Produced in a Baculovirus Expression System from a Fusion Construct Containing the Antibody-binding Part of Protein A[J].Biochem J,1991,280(Pt1):291-224
[113]Jelkmann W.Control of Erythropoietin Gene Expression and its use in Medicine[J].Methods Enzymol,2007,435:179-197
[114]Devasahayam M.Factors Affecting the Expression of Recombinant Glycoproteins[J]. Indian J Med Res.2007,126(1):22-27
[115]Oka MS,Rupp RG.Large-scale Animal Cell Culture:a Biological Perspective [J].Bioprocess Technol,1990,10:71-92
[116]Xu J,Wang H.Gene Cloning and Expression of Human Bactericidal/Permeabilityincreasing Protein[J].Wei Sheng Wu Xue Bao,2001,41(6):669-673
[117]刘水平,杨宇.重组人类杀菌肽基因在CHO细胞中的表达[J].中国医学工程,2006,04
[118]袁红艳,韩艳非,朱明光,等.人源阳离子抗菌肽hCAP218真核表达载体的构建与表达[J],中国生物制品学杂志,2007,20(2):87-90
[119]Sharma A,Khoury Christianson AM,White SP,et al.High-efficiency Synthesis of Human Aendorphin and Magainin in the Erythrocytes of Transgenic Mice:A Production System for Therapeutic Peptides[J].Proc Natl Acad Sci USA,1994,91(20):9337-9341
[120]Yarus S,Rosen JM,Cole AM,et al.Production of Active Bovine Tracheal Antimicrobial Peptied in Milk of Transgenic Mice[J].Proc Natl Acad Sci USA.1996,93(11):14118-14121
[121]Sharma A,Sharma R,Imarmrua M,et al.Transgenic Expression of Cecropin B,an Antibacterial Peptide from Bombyx mori,Confers Enganced Resistance to Bacterial Leaf Blight in Rice[J].FEBS Lett,2000,484(1):7211
[122]Morassutti C,Amicis FD,Skerlavaj B,et al.Production of a Recombinant Antimicrobial Peptide in Transgenic Plants using a Modified VMA Intein Expression System[J].FEBS Lett,2002,519(123):141-146
[123]Huang HW.Molecular Mechanism of Antimicrobial Peptides:The Origin of Cooperativity[J].Biochim Biophysi Acta-Biomembranes.2006,1758(9):1292-1302
[124]Yeaman MR,Yount NY.Mechanisms of Antimicrobial Peptide Action and Resistance [J].Pharmacolog Rev.2003,55:27-55
[125]朱倩.东北林蛙皮肤抗菌肽mRNA多样性的研究.东北林业大学硕士毕业论文,2007
[126]Bradford MM.A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein Dye Binding[J].Anal Biochem.1976,(72):248-254
[127]刘忠渊,张富春,蔡伦,等.酵母菌中表达的新疆家蚕抗菌肽(Cecropin-XJ)的特性研究[J].微生物学报,2003,43(5):635-641
[128]McCafferty DG,Cudic P,Yu MK,et al.Synergy and Duality in Peptide Antibiotic Mechanisms[J].Curr Opin Chem Biol.1999,3(6):672-680
[129]Sawyer JG,Martin NL,Hancock RE.Interaction of Macrophage Cationic Proteins with the Outer Membrane of Pseudomonas aeruginosa[J].Infect Immun,1988,56:693-698
[130]Hiromi Sato,Jimmy B.Peptide-membrane Interactions and Mechanisms of Membrane Destruction by Amphipathic a-helical Antimicrobial Peptides [J].Biochim Biophys Acta,2006,1758(9):1245-1256
[131]王云起,蔡继业,马淑媛,等．抗菌肽magainin对E．coli杀伤作用的AFM观察[J]．电子显微学报．2006,25(1)：52-56
[132]Rosenfeld Y,Barra D,Simmaco M,et al.A Synergism between Temporins toward Gramnegative Bacteria Overcomes Resistance Imposed by the Lipopolysaccharide Protective Layer [J].Biol Chem,2006,281(39):28565-28574
[133]Hong Y,Hancock REW.Synergistic Interactions between Mammalian Antimicrobial Defense Peptides [J].Antimicrob Agents Chemoth,2001,5(45):1558-1560
[134]Kobayashi S.Bacteria-Selective Synergism between the Antimicrobial Peptides Magainin2 and Tachyplesin I:Toward Cocktail Therapy [J].Pharma Socie,2002,122(11):967-973
[135]Matsuzaki K.Magainins as Paradigm for the Mode of Action of Pore Forming Polypeptides [J].Biochim Biophys Acta,1998,1376:391-400
[136]Wieprecht T,Apostolov O,Beyermann M,et al.Membrane Binding and Pore Formation of the Antibacterial Peptide PGLa:Thermodynamic and Mechanistic Aspects [J].Biochem,2000,39(2):442-452
[137]Westerhoff HV,Zasloff M,Rosner JL,et al.Functional Synergism of the Magainins PGLa and Magainin-2 in Escherichia coli,Tumor Cells and Liposomes [J].Eur J Biochem,1995,228:257-264
[138]Hara T,Mitani Y,Tanaka K,et al.Matsuzaki.Heterodimer Formation between the Antimicrobial Peptides Magainin 2 and PGLa in Lipid Bilayers:a Cross-linking Study [J].Biochem,2001,40(41):12395-12399
[139]Matsuzaki K,Mitani Y,Akada KY,et al.Mechanism of Synergism between Antimicrobial Peptides Magainin 2 and PGLa [J].Biochem,1998,37:15144-15153
[2]Man AK,Gooderham JW,Hancock EW.Antibacterial Peptides for Therapeutic use:Obstacles and Realistic Outlook [J].Curr Opin Pharmacol,2006,6:468-472
[3]Barraa D,Simmacob M,Boman HG.Gene-encoded Peptide Antibiotics and Innate Immunity.Do "Animalcules" have Defence Budgets? [J]FEBS Lett,1998,430:130-134
[4]Lupetti A,Nibbering HP,Welling MM.et al.Radiopharmaceuticals:New Antimicrobial Agents [J].Trends Biotechnol,2003,21(2):70-73
[5]Boman HG.Gene-encoded Peptide Antibiotics and the Concept of Innate Immunity:A Update Review [J].Scand T Immunol.1998,48:15-25
[6]Brown KL,Hancock EW.Cationic Host Defense (Antimicrobial) Peptides [J].Curr Opin Immunol,2006,18:24-30
[7]Hancock EW,Diamond G.The Role of Cationic Antimicrobial Peptides in Innate Host Defences [J].Trends Microbiol,2000,8(9):402-410
[8]王可洲,李浩,刘学玲．抗菌肽的分类及作用机理研究进展[J]．科学技术与工程,2005,5(7)：415-420
[9]Powers JPS,Hancock EW.The Relationship between Peptide Structure and Antibacterial Activity [J].Peptides,2003,24:1681-1691
[10]Michael RY,Nannette YY.Mechanisms of Antimicrobial Peptide Action and Resistance [J].Pharmacol Rev,2003,55(1):27-55
[11]Harder J,Bartels J,Christophers E,et al.Isolation and Characterization of Human betadefensin 3,a Novel Human Inducible Peptide Antibiotic [J].J Biol Chem,2001,276 (8):5707-5713
[12]Bobek LA,Situ H.MUC7:Investingation of Antimicrobial Activity,Secondary Structure,and Possible Mechanism of Antifungal Action [J].Antimicrob Agents Chemother,2003,47 (2):643-652
[13]Chen Y,Xu X,Hong S,et al.RGD Tachyples Ininhibits Tumor Growth [J].Cancer Res,2001,61 (6):2434-2438
[14]Fehlbaum P,Bulet P,Chernysh S,et al.Structure Activity Analysis of Thanatin,a 21-Residueinduc Inducible Insect Defense Peptide with Sequence Homology to Frog Skin Antimicrobial Peptides [J].Proc Natl Acad Sci USA,1996,93 (3):1221-1225
[15]Huang HW.Action of Antimicrobial Peptides:Two State Model [J].Biochem,2000,39:8347-8352
[16]Lee DG,Sin SY,Meng CY.Cecropin A-melittin Hybrid Peptide Exerts its Antifungal Effects by Damaging on the Plasma Membranes of Trichosporon beigelii [J].Biotechnol Lett,1998,20(3):211-214
[17]Fehllbaum P,Bulet P.Structure-activity Analysis of Thanatin,a 21-residue Inducible Insect Defense Peptide with Sequence Homology to Frog Skin Antimicrobial Peptides [J].Proc Natl Acad Sci,1996,93(3):1221-1225
[18]Park CB,Kim HS,Kim SC.Mechanism of Action of the Antimicrobial Peptide Buforin Ⅱ:Buforin Ⅱ Kills Microorganism by Penetrating the Cell Membrane and Inhibiting Cellular Functions [J].Biochem Biophys Res Common,1998,244:253-257
[19]Chen HM,Leung KW,Thakur NN.Distinguishing between different pathways of bilayer disrup tion by the related antimicrobial peptides cecrop in B,Bl and B3 [J].Eur J Biochem,2003,270 (5):911-920
[20]Mai JC,Mi Z,Kim SH.Aproapoptotic Peptide for the Treatment of Solid Tumors [J].Cancer Res,2001,61 (21):7709-7712
[21]Hariton Gazal E,Fede rR,MorA,et al.Targeting of Fonokaryophilic Cell-permeable Peptides into the Nuclei of Intact Cells by Covalently Attached Nuclear Localization Signals [J].Biochem,2002,41(29):9208-9214
[22]DekkerN,Cox RC,Kramer RA,et al.Substrate Specificity of the Integral Membrane Protease OmpT Determined by Spatially Addressed Peptide Libraries [J].Biochem,2001,40 (6):1694-1701
[23]Aboudy Y,Mendelson E,Shalit I,et al.Activity of two Synthetic Amphiphilic Peptides and Magainin-2 against Herpes Simplex Virus Types 1 and 2 [J].Int J Pept Protein Res,1994,43(6):573-582
[24]Wachinger M,Kleinschmidt A,Winder D,et al.Antimicrobial Peptides Melttin and Cecropin Inhibit Replication of Human Immunodeficiency Virus I by Suppressing Viral Gene Expression [J].Gen Virol,1998,79:731-740
[25]Andreu D,Rivas L.Animal Antimicrobial Peptides:an Overview [J].Biopolymers,1999,47:413-433
[26]Mangoni ML,Saugar JM,Dellisanti M,et al.Temporins,Small Antimicrobial Peptides with Leishmanicidal Activity [J].Biochem,2005,280:984-990
[27]Vizioli J,Salzet M.Antimicrobial Peptides versus Parasitic Infections [J].Trends Parasitol,2002,18(11):475-476
[28]Shahabuddin M,Phillipe B,Jules A,et al.Plasmodium gallinaceum:Differential Killing of Some Mosquito Stages of the Parasite by Insect Defensin [J].Experimental Parasitogy,1998,89(1):103-112
[29]Bulet P,cklin RS,Menin L.Anti-microbial Peptides:from Invertebrates to Vertebrates [J].Immunol Rev,2004,198:169-184
[30]Takumi O,Fujitoshi Y,Masao T,et al.Production and Purification of a Bacteriocin Pep tide Produced by Lactococcus sp Strain GM005,Isolated from Misopaste [J].Int J Food M icrob,2003,87 (1-2):153-159
[31]Lehrer RI,Ganz T.Antimicrobial Peptides in Mammalian and Insect Host Defence [J].Curr Opin Immunol,1999,11:23-30
[32]尹文,徐晨,马戈甲．抗菌肽的研究进展[J]．细胞与分子免疫学杂志,2005,21(1)：136-138
[33]Li J,Xu X,Xu C,et al.Anti-infection Peptidomics of Amphibian Skin [J].Mol Cell Proteomics.2007,l(6):882-894
[34]Gudmundsson GH,Lidholm DA,Asling B,et al.The Cecropin Locus.Cloning and Expression of a Gene Cluster Encoding Three Antibacterial Peptides in Hyalophora cecropia [J].J Biol Chem,1991,266:11510-11517
[35]Ganz T.Defensins and other Antimicrobial Peptides:a Historical Perspective and an Update [J].Comb Chem High Throughput Screen,2005,8:209-217
[36]Zanetti M.The Role of Cathelicidins in the Innate Host Defenses of Mammals [J].Curr Issues Mol Biol,2005,7:179-196
[37]Pasquier LD.Meeting the Demand for Innate and Adaptive Immunities during Evolution [J].Scandinavian J Immunol,2005,62:39-48
[38]Cellura C,Toubiana M,Parrinello N,et al.Specific Expression of Antimicrobial Peptide and HSP70 Genes in Response to Heat-shock and Several Bacterial Challenges in Mussels [J].Fish Shellfish Immunol.2007,22:340-350
[39]Mitta G,Hubert F,Dyrynda EA,et al.Mytilin B and MGD2,two Antimicrobial Peptides of Marine Mussels:Gene Structure and Expression Analysis [J].Developmental Comparative Immunol.2000,24(4):381-393
[40]Menzies BE,Kenoyer A.Staphylococcus aureus Infection of Epidermal Keratinocytes Promotes Expression of Innate Antimicrobial Peptides [J].Infection and Immunity.2005,73(4):5241-5244
[41]Lemaitre B,Reichhart JM,Hoffmann JA.Drosophila Host Defense:Differential Induction of Antimicrobial Peptide Genes after Infection by Various Classes of Micro organisms [J].Proc Natl Acad Sci USA.1997,94:14614-14619
[42]Engstrom Y.Induction and Regulation of Antimicrobial Peptides in Drosophila [J].Developmental Comparative immunol.1999,23:345-358
[43]Medzhitov R,Janeway CA.Innate Immunity:The Virtues of a Nonclonal System of Recognition [J].Cell.1997,91:295-298
[44]Lee WJ,Lee JD,Kravchenko VV,et al.Purification and Molecular Cloning of an Inducible Gram-negative Bacteria-binding Protein from the Silkworm,Bombyx mori[J].Proc Natl Acad Sci USA.1996,93:7888-7893
[45]Kang D,Liu G,LundstroE m A,et al.A Peptidoglycan Recognition Protein in Innate Immunity Conserved from Insects to Humans [J].Proc Natl Acad Sci USA.1998,95:10078-10082
[46]Rollins-Smith LA,Woodhams DC,Reinert LK,et al.Antimicrobial Peptide Defenses of the Mountain Yellow-leged Frog (Rana muscosa) [J].Developmental Comparative Immunol.2006,30:831-842
[47]Levashina EA,Ohresser S,Lemaitre B,et al.Two Distinct Pathways can Control Expression of the Gene Encoding the Drosophila Antimicrobial Peptide Metchnikowin [J].J Mol Biol.1998.278(3):515-527
[48]Mor A,Hani K,Nicolas P.The Vertebrate Peptide Antibiotics Dermaseptins Have Overlapping Structural Features but Target Specific Microorganisms [J].J Biol Chem,1994,269:31635-31641
[49]Giacometti A,Cirioni O,Kamysz W,et al.In vitro Activity of Amphibian Peptides alone and in Combination with Antimicrobial Agents against Multidrug-resistant Pathogens Isolated from Surgical wound infection [J].Peptides,2005,26:2111-2116
[50]Mor A,Hani K,Nicolas P.The Vertebrate Peptide Antibiotics Dermaseptins Have Overlapping Structural Features but Target Specific Microorganisms [J].J Biol Chem,1994,269:31635-31641
[51]Chen XJ,Niyonsaba F,Ushio H,et al.Synergistic Effect of Antibacterial Agents Human 6-defensins,Cathelicidin LL-37 and Lysozyme against Staphylococcus aureus and Escherichia coli [J].Dermatol Sci,2005,40:123-132
[52]Kumar M,Chaturvedi AK,Kavishwar A,et al.Identification of a Novel Antifungal Nona Peptide Generated by Combinatorial Approach [J].Int J Antimicrob Agen,2005,25:313-320
[53]Moore AJ,Beazley WD,Bibby MC,et al.Antimicrobial Activity of Cecropins [J].J Antimicrob Chemother,1996,37:1077-1089
[54]Miele R,Ponti D,Boman HG,et al.Molecular Cloning of a Bombinin Gene from Bombina orientalis:Detection of NF-kB and NF-IL6 Binding Sites in its Promoter [J].FEBS Lett,1998,431:23-28
[55]Liu L,Zhao C,Heng HH,et al.The Human B-defensin-1 and a-defensins are Encoded by djacent Genes:Two Peptide Families with Differing Disulfide Topology Share a CommonAncestry [J].Genomics,1997,43:316-320
[56]Marco S,Shenglun W,Margherita Z.Structural Organization of the Bovine Cathelicidin Gene Family and Identification of a Novel Member [J].FEBS Lett,1997,417:311-315
[57]Vanhoye D,Bruston F,Nicolas P,et al.Antimicrobial Peptides from Hylid and Ranid Frogs Originated from a 150-million-year-old Ancestral Precursor with a Conserved Signal Peptidebut a Hypermutable Antimicrobial Domain [J].Eur J Biochem,2003,270:2068-2081
[58]Michaelson D,Rayner J,Conto M,et al.Cationic Defensins Arise from Charge-neutralized ropeptides:a Mechanism for Avoiding Leukocyte Autotoxicity [J].J Leukoc Biol,1992,51:32-639
[59]Oren Z,Shai YA.Class of Highly Potent Antibacterial Peptides Derived from Pardaxin,a Pore-forming Peptide Isolated from Mosesesole Fish pardachirus marmoratus [J].Eur J Biochem,1996,237(1):303-310
[60]Swendsen CL,Sphepard DA.Study of Antimicrobial Effects of Mountain Dusky salamander mucus [J].Proc Nat Acad Sci USA,1998,86:24-31
[61]Barra D,Simmaco M,Boman HG.Gene-encoded Peptide Antibiotics and Innate Immunity.Do'Animalcules'have Defence Budgets? [J]FEBS Lett,1998,430(1-2):130-134
[62]Hancock REW,Chappie DS.Peptides Antibiotics [J].Antimicrob Agents Chemother,1999,43(6):1317-1323
[63]Simmaco M,Mignogna G,and Barra D.Antimicrobial Peptides from Amphibian Skin:What do they tell us? [J].Biopolymers,1998,47(6):425-450
[64]Magainins MZ.a Class of Antimicrobial Peptides from Xenopus Skin:Isolation、Characterzation of two Active Forms,and Partial cDNA Sequence of a Precursor [J].Proc Nat Acad Sci,1987,84(15):5449-5453
[65]Boman HG.Peptide Antibiotics and their Role in Innate Immunity [J].Annu Rev Immunol,1995,13:61-92
[66]Steinborner ST,Currie GJ,Bowie JH,et al.New Antibiotic Cearinl Peptides from the Skin Secretion of the Australian Tree Frog Litoriachloris,Comparison of the Activities of the Caerinl Peptides from the Genus Litoria [J].J Peptide Research,1985,51:121-126
[67]Wegene KL,Wabnitz PA,Carver JA.Host Defense Peptides from the Skin Glands of the Australian Blue Mountains Tree Frog Litoriacitropa [J].Eur J Biochem,1999,265:627-637
[68]Giovannini MG,Poulter L,Gibson BW,et al.Biosynthesis and Egradation of Peptides Derived from Xenopus laevis prohormones [J].Biochem J,1987,243(1):113-120
[69]Chia BCS,Carver JA,Mulhern TD,et al.The Solution Structure of Upperin3.6,an Antibiotic Peptide from the Granular Dorsal Glands of the Australian Toad let,Uperoleiam jobergii[J].J Peptide Reseach,1999,54:137-145
[70]Chia BCS,Carver JA,Mulhern TD,et al.Maculatinll,an Antimicrobial Peptide from the Australian Tree Frog,Litoriageni Maculata Solution Struncture and Biological Activity[J].Eur J Biochem,2000,267:1894-1908
[71]Gibson BW,Tang DZ,Mandrell R,et al.Bombinin like Peptides with Antimicrobial Activity from Skin Secretion of the Asian Toad,Bombinaorientalis[J].J Biol Chem,1991,266(34):23103-23111
[72]Conlon JM,Kolodziejek J,Nowotny N.Antimicrobial Peptides from Ranid Frogs:Taxonomic and Phylogenetic Markers and a Potential Source of new Therapeutic Agents [J].Biochim Biophys Acta.2004,1696(1):1-14
[73]Ohnuma A,Conlon JM,Yamaguchi K,et al.Antimicrobial Peptides from the Skin of the Japanese Mountain Brown Frog Rana ornativentris:Evidence for Polymorphism among preprotemporin mRNAs[J].Peptides.2007,28(3):524-532
[74]Clarke BT.The Natural History of a Amphibian Skin Secretions,their Normal Function and Potential Medical Applications[J].Biol Rev.1997,72(3):365-379
[75]谢锋,叶昌媛,费梁,等.中国东北地区林蛙属物种的分类学研究(两栖纲:蛙科)[J].动物分类学报.1999,24(2):224-231
[76]Conlona JM,Kolodziejekb J,Nowotny N.Antimicrobial Peptides from Ranid Frogs:Taxonomic and Phylogenetic Markers and a Potential Source of New Therapeutic Agents [J].Biochimi Biophys Acta,1696(2004):1-14
[77]Marra AK,Gooderhama WJ,Hancock REW.Antibacterial Peptides for Therapeutic Use:Obstacles and Realistic Outlook[J].Curr Opin Pharmacol,2006,6(5):468-472
[78]Hancock REW,Lehrer R.Cationic Peptides:a New Source of Antibiotics[J].Trends Biotechnol,1998,16(7):82-88
[79]陆莹瑾,王禾,屈贤铭.抗菌肽的固相合成、分离纯化与构效关系的研究[J].生物工程学报,1997,13(1):35-41
[80]Schmidt FR.Recombinant Expression Systems in the Pharmaceutical Industry[J].Appl Microb Biotech,2004,65:363-372
[81]Shigeyuki Yokoyama.Protein Expression Systems for Structural Genomics and Proteomics[J].curr opin Chem Biol,2003,7:39-43
[82]Better M,Chang CP,Robinson RR,et al.Escherichia coli Secretion of an Active Chimeric Antibody Fragment[J].Science,1988,240(4855):1041-1043
[83]Yedery RD,Reddy KV.Antimicrobial Peptides as Microbicidal Contraceptives:Prophecies for Prophylactics[J].Eur J Contracept Reprod Health Care,2005,10(1):32
[84]Lange CF,Hancock RE,Samuel J,et al.In Vitro Aerosol Delivery and Regional Airway Surface Liquid Concentration of a Liposomal Cationic Peptide[J].J Pharm Sci,2001,90(10):1647
[85]Zhang L,Falla T,Wu M,et al.Determinants of Recombinant Production of Antimicrobial Cationic Peptides and Creation of Peptide Variants in Bacteria [J].Biochem Biophys Res Commun,1998,247(3):674
[86]Lee JH,Minn Ⅱ,Park CB,et al.Acidic Peptide-Mediated Expression of the Antimicrobial Peptide Buforin Ⅱ as Tandem Repeats in Escherichia coli [J].Protein Expr Purif,1998,12(1):53
[87]Harder J,Bartels J,Christophers E,et al.Isolation and Characterization of Human beta-defensin-3,a Novel Human Inducible Peptide Antibiotic [J].J Biol Chem,2001,276( 8):5707
[88]Peng L,Xu Z,Fang X,et al.High-level Expression of Soluble Human β-defensin-2 in Escherichia coli [J].Process Biochem,2004,39(12):2199
[89]Moon JY,Henzler Wildman KA,Ramamoorfhy A.Expression and Purification of a Recombinant LL-37 from Escherichia coli [J].Biochim Biophys Acta,2006,1758(9):1351
[90]Rao X,Hu J,Li S,et al.Design and Expression of Peptide Antibiotic hPAB-beta as Tandem Multimers in Escherichia coli [J].Peptides,2005,26(5):721
[91]Erik Boer,Gerhard S,Gotthard K,et al.Yeast Expression Platforms [J].Appl Microb Biotech,2007,77:513-523
[92]Vilas Alves AL,Samblanx GW,Terras FRG,et al.Expression of Functional Raphanus Sativus Antifungal Protein in Yeast [J].FEBS Lett,1994,384 (3):228-232
[93]Destoumieux D,Bule P,Strub JM,et al.Recombiant Expression and Range of Activity of Penaeidins,Antimicrobial Peptides from Penaeid Shrimp [J].Eur J Biochem,1999,266 (2):335-346
[94]Cereghino JL,Cregg JM.Heterologous Protein Expression in the Methylotrophic Yeast Pichia pastoris [J],FEMS Microb Rev,2000,24:45-66
[95]Ogata K,Nishikawa H,Ohsugi M.A Yeast Capable of Utilizing Methanol [J].Agric Biol Chem,1969,33:1519-1520
[96]Davidson JF.The Origin of Insights in Chemical Engineering:Planned and Unplanned research [J].Chem Eng Sci,1995,50(23):3661-3684
[97]Gellissen G.Heterologous Protein Production in Methylotrophic Yeasts [J].Appl Microb Biotech.2000,54(6):741-750
[98]Daly R,Hearn MTW.Expression of Heterologous Proteins in Pichia pastoris:a Useful Experimental Tool in Protein Engineering and Production [J].J Mol Recognit.2005,18:119-138
[99]Rosenfeld SA,Nadeau D,Tirado J,et al.Production and Purification of Recombinant Hirudin Expressed in the Methylotrophic Yeast Pichia pastoris [J].Protein Expre Purif,1996,8:476
[100]Kristensen AK,Brunstedt J,Nielsen JE,et al.Processing,Disulfide Pattern,and Biological Activity of a Sugar Beet Defensin,AX2,Expressed in Pichia pastoris[J].Protein Expr Purif,1999,16(3):377-387
[101]Almeida MS,Cabral KS,de Medeiros LN,et al.cDNA Cloning and Heterologous Expression of Functional Cysteine-rich Antifungal Protein Psd1 in the Yeast Pichia pastoris[J].Arch Biochem Biophys,2001,395(2):199
[102]Cabral KM,Almeida MS,Valente AP,et al.Production of the Active Antifungal Pisum Sativum Defensin 1(Psdl) in Pichia pastoris:Overcoming the Inefficiency of the STE13Protease[J].Protein Expr Purif,2003,31(1):115
[103]Chen JJ,Chen GH,Hsu HC,et al.Cloning and Functional Expression of a Mungbean Defensin VrD1 in Pichia pastoris[J].J Agric Food Chem,2004,52(8):2256
[104]Hong IP,Lee SJ,Choi SG.Recombinant Expression of Human Cathelicidin(hCAP18/LL-37) in Pichia pastoris[J].Biotechnol Lett,2007,29(1):73
[105]张素芳,曹瑞兵,贾赟,等.杂合抗菌肽 CecA-mil 的改造及在毕赤酵母中的分泌表达[J].微生物学报,2005,45(2):218-222
[106]赵亚华,董竟南,崔红,等.蜂毒素分子的改造及其基因在毕赤酵母中的表达[J].中国生物工程杂志,2005,25(2):45-48
[107]陆建荣,王惠民,吴萍,等.人源抗菌肽 LL-37表达载体的构建及其在毕赤酵母中的表达[J].第二军医大学学报,2007,28(8):833-837
[108]Branduardi P,Valli M,Brambilla L,et al.The Yeast Zygosaccharomyces bailii:a new Host for Heterologous Protein Production,Secretion and for Metabolic Engineering Applications[J].FEMS Yeast Research,2004,4:493-504
[109]Hasemann CA,Capra JD,Hasemann CA,et al.High-level Production of a Functional Immunoglobulin Heterodimer in a Baculovirus Expression System[J].Proc Natl Acad Sci,1990,87:3942-3946
[110]Smith GE,Summers MD,Fraser MJ.Production of Human Beta Interferon in Insectcell Infected with a Baculovirus Express Vector[J].Mol Cell Biol,1983(3):2156-2165
[111]Hellers M,Gunne H,Steiner H.Expression and Posttranslational Processing of Preprocecropin A Using a Baculovirus Vector[J].Eur Biochem,1991,199(2):435-439
[112]Andersons D,Engstrom A,Josephsom S,et al.Biologically Active and Amidated Cecropin Produced in a Baculovirus Expression System from a Fusion Construct Containing the Antibody-binding Part of Protein A[J].Biochem J,1991,280(Pt1):291-224
[113]Jelkmann W.Control of Erythropoietin Gene Expression and its use in Medicine[J].Methods Enzymol,2007,435:179-197
[114]Devasahayam M.Factors Affecting the Expression of Recombinant Glycoproteins[J]. Indian J Med Res.2007,126(1):22-27
[115]Oka MS,Rupp RG.Large-scale Animal Cell Culture:a Biological Perspective [J].Bioprocess Technol,1990,10:71-92
[116]Xu J,Wang H.Gene Cloning and Expression of Human Bactericidal/Permeabilityincreasing Protein[J].Wei Sheng Wu Xue Bao,2001,41(6):669-673
[117]刘水平,杨宇.重组人类杀菌肽基因在CHO细胞中的表达[J].中国医学工程,2006,04
[118]袁红艳,韩艳非,朱明光,等.人源阳离子抗菌肽hCAP218真核表达载体的构建与表达[J],中国生物制品学杂志,2007,20(2):87-90
[119]Sharma A,Khoury Christianson AM,White SP,et al.High-efficiency Synthesis of Human Aendorphin and Magainin in the Erythrocytes of Transgenic Mice:A Production System for Therapeutic Peptides[J].Proc Natl Acad Sci USA,1994,91(20):9337-9341
[120]Yarus S,Rosen JM,Cole AM,et al.Production of Active Bovine Tracheal Antimicrobial Peptied in Milk of Transgenic Mice[J].Proc Natl Acad Sci USA.1996,93(11):14118-14121
[121]Sharma A,Sharma R,Imarmrua M,et al.Transgenic Expression of Cecropin B,an Antibacterial Peptide from Bombyx mori,Confers Enganced Resistance to Bacterial Leaf Blight in Rice[J].FEBS Lett,2000,484(1):7211
[122]Morassutti C,Amicis FD,Skerlavaj B,et al.Production of a Recombinant Antimicrobial Peptide in Transgenic Plants using a Modified VMA Intein Expression System[J].FEBS Lett,2002,519(123):141-146
[123]Huang HW.Molecular Mechanism of Antimicrobial Peptides:The Origin of Cooperativity[J].Biochim Biophysi Acta-Biomembranes.2006,1758(9):1292-1302
[124]Yeaman MR,Yount NY.Mechanisms of Antimicrobial Peptide Action and Resistance [J].Pharmacolog Rev.2003,55:27-55
[125]朱倩.东北林蛙皮肤抗菌肽mRNA多样性的研究.东北林业大学硕士毕业论文,2007
[126]Bradford MM.A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein Dye Binding[J].Anal Biochem.1976,(72):248-254
[127]刘忠渊,张富春,蔡伦,等.酵母菌中表达的新疆家蚕抗菌肽(Cecropin-XJ)的特性研究[J].微生物学报,2003,43(5):635-641
[128]McCafferty DG,Cudic P,Yu MK,et al.Synergy and Duality in Peptide Antibiotic Mechanisms[J].Curr Opin Chem Biol.1999,3(6):672-680
[129]Sawyer JG,Martin NL,Hancock RE.Interaction of Macrophage Cationic Proteins with the Outer Membrane of Pseudomonas aeruginosa[J].Infect Immun,1988,56:693-698
[130]Hiromi Sato,Jimmy B.Peptide-membrane Interactions and Mechanisms of Membrane Destruction by Amphipathic a-helical Antimicrobial Peptides [J].Biochim Biophys Acta,2006,1758(9):1245-1256
[131]王云起,蔡继业,马淑媛,等．抗菌肽magainin对E．coli杀伤作用的AFM观察[J]．电子显微学报．2006,25(1)：52-56
[132]Rosenfeld Y,Barra D,Simmaco M,et al.A Synergism between Temporins toward Gramnegative Bacteria Overcomes Resistance Imposed by the Lipopolysaccharide Protective Layer [J].Biol Chem,2006,281(39):28565-28574
[133]Hong Y,Hancock REW.Synergistic Interactions between Mammalian Antimicrobial Defense Peptides [J].Antimicrob Agents Chemoth,2001,5(45):1558-1560
[134]Kobayashi S.Bacteria-Selective Synergism between the Antimicrobial Peptides Magainin2 and Tachyplesin I:Toward Cocktail Therapy [J].Pharma Socie,2002,122(11):967-973
[135]Matsuzaki K.Magainins as Paradigm for the Mode of Action of Pore Forming Polypeptides [J].Biochim Biophys Acta,1998,1376:391-400
[136]Wieprecht T,Apostolov O,Beyermann M,et al.Membrane Binding and Pore Formation of the Antibacterial Peptide PGLa:Thermodynamic and Mechanistic Aspects [J].Biochem,2000,39(2):442-452
[137]Westerhoff HV,Zasloff M,Rosner JL,et al.Functional Synergism of the Magainins PGLa and Magainin-2 in Escherichia coli,Tumor Cells and Liposomes [J].Eur J Biochem,1995,228:257-264
[138]Hara T,Mitani Y,Tanaka K,et al.Matsuzaki.Heterodimer Formation between the Antimicrobial Peptides Magainin 2 and PGLa in Lipid Bilayers:a Cross-linking Study [J].Biochem,2001,40(41):12395-12399
[139]Matsuzaki K,Mitani Y,Akada KY,et al.Mechanism of Synergism between Antimicrobial Peptides Magainin 2 and PGLa [J].Biochem,1998,37:15144-15153