新型抗菌肽与磷脂膜的相互作用研究
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摘要
抗菌肽的主要作用位点是细菌细胞膜的骨架——磷脂双分子层,由于磷脂酰胆碱(DMPC)和磷脂酰甘油(DMPG)分别为构成动植物细胞膜和细菌细胞膜的重要磷脂,因此本论文分别采用DMPC和DMPG来模拟动植物细胞膜和细菌细胞膜,研究抗菌肽对不同细胞的破膜效果。本研究从头设计了三组新型抗菌肽,并采用电化学法,荧光淬灭法以及理论计算模拟的方法对三组抗菌肽与磷脂之间的相互作用进行了研究,研究结果如下：
采用KFNFK、KFSFK、KFTFK为活性片段设计含有单活性序列的短链多肽、含有双活性序列的线型肽和环状肽,并对该组多肽和磷脂的相互作用进行了研究。计算模拟的结果表明,双活性序列抗菌肽能够提供较单活性序列肽多的正电荷氨基酸与DMPG负电头基形成静电吸引,因此具有较高的结合能,而环状肽因为其较为固定的构型和序列较短使得其结合能低于线型肽。以固体支撑双层膜模拟生物膜,采用电化学方法研究了所设计多肽与磷脂之间的相互作用,结果显示,抗菌肽无论与DMPC还是DMPG进行作用,具有双活性序列的抗菌肽较单活性序列抗菌肽更易对磷脂发生破坏作用；抗菌肽对DMPG的破坏作用的程度较DMPC更易受到抗菌肽浓度的影响。
采用从抗菌肽库中搜索所得到的短链多肽RRWWRF和FRWWHR为活性序列设计含单活性序列的短链肽和含双活性序列的线型肽和环状肽,并对多肽与磷脂的相互作用进行研究。理论计算的结果显示,双活性序列抗菌肽的结合能大于单活性序列抗菌肽,双活性序列抗菌肽更易于DMPG结合；电化学实验结果显示,第二组抗菌肽只能对DMPC产生破坏作用,而对DMPG基本上不具有破坏作用。因此推测第二组抗菌肽并不是通过对细菌细胞膜产生破坏作用而杀菌,而是与细菌细胞膜上的磷脂产生相互作用并进入到细胞膜内部,与细胞内物质产生相互作用而使细菌死亡。
采用RFTFR、RWTWR、KWTWK为活性片段设计含有单活性序列的短链多肽和含有双活性序列的线型肽,并对该组多肽和磷脂的相互作用进行了研究。计算模拟的结果显示双活性序列的抗菌肽具有较单活性序列抗菌肽高的结合能,更易与DMPG结合。电化学实验结果显示,双活性序列抗菌肽能够与DMPC和DMPG发生作用使膜破裂,而单活性序列抗菌肽则基本不与DMPC和DMPG产生作用；抗菌肽对DMPG的破坏作用较DMPC大。荧光淬灭实验结果显示,短链肽基本不能插入到DMPG和DMPC产生相互作用,而线型肽均能插入到DMPG中却不能插入到DMPC中。
The most important property of antimicrobial peptide is their special sterilization mechanism. The antimicrobial peptide mainly targets the backbone of bacterial cell membrane-phospholipid bilayer. Because DMPC and DMPG are the main phospholipids to constitute the membrane of flora and fauna and bacterium respectively, the DMPC and DMPG are used to simulate the flora and fauna and bacterium cell membrane to investigate the interaction between antimicrobial peptides with different phospholipids. Three groups of antimicrobial peptides are designed in this research. The interaction between these three groups peptide and phospholipids are investigated by electrochemistry, fluorescence quenching and compute simulation method. The results are as follows.
The sequences KFNFK, KFSFK, KFTFK were chosen in the first group as single bioactive sequence, and according to these sequences, linear and cyclic peptides with dual bioactive sequences were designed and the interaction between peptides and phospholipids were studied. The result of compute simulation showed that peptides with dual bioactive sequences could offer more positive charged amino acids than short peptides with single bioactive sequence to facilitate the electrostatic attraction with negative charged head group of DMPG, which leads to higher binding energy with DMPG for linear and cyclic peptides. Because of the rigid configuration and short sequence of cyclic peptides, so they have a lower binding energy with DMPG compared to linear peptides. The interaction between peptides and phospholipid was further investigated by electrochemistry method by using solid supported bilayer as membrane mimic. The results showed that peptides with dual bioactive sequences have a higher destructive effect against phospholipid bilayer than peptide with single bioactive sequence regardless of DMPC or DMPG. The influence of peptides concentration on the interaction with DMPG was more sensitive than DMPC.
RRWWRF and FRWWHR which are from antimicrobial peptides database were chosen as single bioactive sequences for designing linear and cyclic antimicrobial peptides with dual bioactive sequences, and their interaction with phospholipids was further investigated. Compute simulation results showed that peptides with dual bioactive sequences had a higher binding energy with DMPG than peptides with single bioactive sequence, which indicates that peptides with dual bioactive sequences are easier to bind to DMPG. The electrochemistry experiment results showed that this second group peptide couldn't destruct DMPG membrane though they had a destructive effect to DMPC. So we deduced that the second group peptides could interact with bacterial cell membrane, enter the interior of cell, interact with the substance in cell and finally kill the bacterium, instead of disrupting cell membrane.
Peptides with single bioactive sequence and dual bioactive sequences using RFTFR, RWTWR, KWTWK as bioactive segments has been designed and their interaction with phospholipids was further studied. The compute simulation results showed that linear peptides with dual bioactive sequences had higher binding energy than peptides with single bioactive sequence, indicating a stronger binding of linear peptides with DMPG. The electrochemistry experiment results showed that peptides with dual bioactive sequences could disrupt DMPC or DMPG membrane while peptides with single bioactive sequence hardly disrupt phospholipid membrane. Peptides showed stronger disrupting ability on DMPG membrane compared to DMPC membrane. The results of fluorescence quenching experiment showed that short peptides hardly inserted into DMPC or DMPG lipsomes. However linear peptides could inserted into DMPG lipsomes but nearly have no interaction with DMPC.
采用KFNFK、KFSFK、KFTFK为活性片段设计含有单活性序列的短链多肽、含有双活性序列的线型肽和环状肽,并对该组多肽和磷脂的相互作用进行了研究。计算模拟的结果表明,双活性序列抗菌肽能够提供较单活性序列肽多的正电荷氨基酸与DMPG负电头基形成静电吸引,因此具有较高的结合能,而环状肽因为其较为固定的构型和序列较短使得其结合能低于线型肽。以固体支撑双层膜模拟生物膜,采用电化学方法研究了所设计多肽与磷脂之间的相互作用,结果显示,抗菌肽无论与DMPC还是DMPG进行作用,具有双活性序列的抗菌肽较单活性序列抗菌肽更易对磷脂发生破坏作用；抗菌肽对DMPG的破坏作用的程度较DMPC更易受到抗菌肽浓度的影响。
采用从抗菌肽库中搜索所得到的短链多肽RRWWRF和FRWWHR为活性序列设计含单活性序列的短链肽和含双活性序列的线型肽和环状肽,并对多肽与磷脂的相互作用进行研究。理论计算的结果显示,双活性序列抗菌肽的结合能大于单活性序列抗菌肽,双活性序列抗菌肽更易于DMPG结合；电化学实验结果显示,第二组抗菌肽只能对DMPC产生破坏作用,而对DMPG基本上不具有破坏作用。因此推测第二组抗菌肽并不是通过对细菌细胞膜产生破坏作用而杀菌,而是与细菌细胞膜上的磷脂产生相互作用并进入到细胞膜内部,与细胞内物质产生相互作用而使细菌死亡。
采用RFTFR、RWTWR、KWTWK为活性片段设计含有单活性序列的短链多肽和含有双活性序列的线型肽,并对该组多肽和磷脂的相互作用进行了研究。计算模拟的结果显示双活性序列的抗菌肽具有较单活性序列抗菌肽高的结合能,更易与DMPG结合。电化学实验结果显示,双活性序列抗菌肽能够与DMPC和DMPG发生作用使膜破裂,而单活性序列抗菌肽则基本不与DMPC和DMPG产生作用；抗菌肽对DMPG的破坏作用较DMPC大。荧光淬灭实验结果显示,短链肽基本不能插入到DMPG和DMPC产生相互作用,而线型肽均能插入到DMPG中却不能插入到DMPC中。
The most important property of antimicrobial peptide is their special sterilization mechanism. The antimicrobial peptide mainly targets the backbone of bacterial cell membrane-phospholipid bilayer. Because DMPC and DMPG are the main phospholipids to constitute the membrane of flora and fauna and bacterium respectively, the DMPC and DMPG are used to simulate the flora and fauna and bacterium cell membrane to investigate the interaction between antimicrobial peptides with different phospholipids. Three groups of antimicrobial peptides are designed in this research. The interaction between these three groups peptide and phospholipids are investigated by electrochemistry, fluorescence quenching and compute simulation method. The results are as follows.
The sequences KFNFK, KFSFK, KFTFK were chosen in the first group as single bioactive sequence, and according to these sequences, linear and cyclic peptides with dual bioactive sequences were designed and the interaction between peptides and phospholipids were studied. The result of compute simulation showed that peptides with dual bioactive sequences could offer more positive charged amino acids than short peptides with single bioactive sequence to facilitate the electrostatic attraction with negative charged head group of DMPG, which leads to higher binding energy with DMPG for linear and cyclic peptides. Because of the rigid configuration and short sequence of cyclic peptides, so they have a lower binding energy with DMPG compared to linear peptides. The interaction between peptides and phospholipid was further investigated by electrochemistry method by using solid supported bilayer as membrane mimic. The results showed that peptides with dual bioactive sequences have a higher destructive effect against phospholipid bilayer than peptide with single bioactive sequence regardless of DMPC or DMPG. The influence of peptides concentration on the interaction with DMPG was more sensitive than DMPC.
RRWWRF and FRWWHR which are from antimicrobial peptides database were chosen as single bioactive sequences for designing linear and cyclic antimicrobial peptides with dual bioactive sequences, and their interaction with phospholipids was further investigated. Compute simulation results showed that peptides with dual bioactive sequences had a higher binding energy with DMPG than peptides with single bioactive sequence, which indicates that peptides with dual bioactive sequences are easier to bind to DMPG. The electrochemistry experiment results showed that this second group peptide couldn't destruct DMPG membrane though they had a destructive effect to DMPC. So we deduced that the second group peptides could interact with bacterial cell membrane, enter the interior of cell, interact with the substance in cell and finally kill the bacterium, instead of disrupting cell membrane.
Peptides with single bioactive sequence and dual bioactive sequences using RFTFR, RWTWR, KWTWK as bioactive segments has been designed and their interaction with phospholipids was further studied. The compute simulation results showed that linear peptides with dual bioactive sequences had higher binding energy than peptides with single bioactive sequence, indicating a stronger binding of linear peptides with DMPG. The electrochemistry experiment results showed that peptides with dual bioactive sequences could disrupt DMPC or DMPG membrane while peptides with single bioactive sequence hardly disrupt phospholipid membrane. Peptides showed stronger disrupting ability on DMPG membrane compared to DMPC membrane. The results of fluorescence quenching experiment showed that short peptides hardly inserted into DMPC or DMPG lipsomes. However linear peptides could inserted into DMPG lipsomes but nearly have no interaction with DMPC.
引文
[1]杜剑春.浅议抗生素药物的不良反应[J].中国药物经济学,2012,42(6)：362-363.
[2]仇妍虹.家蝇防御素抗菌肽在毕赤酵母中分泌表达及活性研究[D].南京：南京农业大学硕士论文,2006.
[3]H.I.Zeya,J.K.Spitznagel.Cationic protein-bearing granules of polymorphonuclear leukocytes: separation from enzyme-rich granules[J].Science,1969,163(3871):1069-1071.
[4]Christian Mayer,Ralf Moritz,Carolin Kirschner.et al.The role of inter- molecular interactions: studies on model systems for bacterial biofilms[J]. Int J Biol Macromol,1999,26(1):3-16.
[5]Dan Hultmark,Hakan Steriner,Torgny Rasmuson,et al.Insect immunity,purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalohora cecropia[J].Eur J Biochemistry,1980,106(1):7-16.
[6]吴甜甜,杨洁.天然抗菌肽的研究进展及应用前景[J].生物技术通报,2009,(1)：27-30.
[7]梁永利.天然抗菌肽的来源及分类[J].安徽农业科学,2006,34(18)：4728-4734.
[8]江龙法,谢慧,邬敏辰.阳离子抗菌肽的作用机理及构效关系[J].中国医药工业杂志,2005,36(4)：244-249.
[9]王威,王联结.阳离子抗菌肽分子设计的研究现状[J].食品工业科技,2010,31(1)：442-445.
[10]潘秋冬,夏明.抗菌肽的研究现状及应用前景[J].农产品加工·学刊,2011,2235(2)：81-83.
[11]刘汉灵,潘扬昌,王孝英等.黄粉虫蛋白抗菌肽酶解工艺研究[J].食品科学,2009,30(15)：156-159.
[12]吴琼英,徐金玲.酶解反应和膜分离耦合制备乳源抗菌肽[J].江苏科技大学学报(自然科学版),2010,24(4)：386-390.
[13]冯兴军,李静,赵晓宇等.牛乳铁蛋白素·马盖宁杂合抗菌肽的设计、合成及抑菌活性[J].东北农业大学学报,2011,42(3)：105-109.
[14]赵亦静,倪密,诺林等.人工合成抗菌肽对棉花黄萎病菌的抑菌效果[J].浙江大学学报,2013,39(1)：11-17.
[15]张召兄,潘晓亮,任耀军.抗菌肽概述及抗菌分子机理的研究进展[J].草食家畜,2006,133(4)：7-10.
[16]赵冬梅,陶妍.斑点叉尾鲴hepcidin抗菌肽在大肠杆菌中的融合表达[J].东北农业大学学报,2012,43(12)：114-120.
[17]单艳菊,胡艳,徐文娟等,鸡p防御素1基因密码子优化及其在真核细胞中的表达[J].云南农业大学学报,2012,27(6)：833-839.
[18]付登峰,胡建和,刘兴友.抗菌肽基因工程表达技术研究进展[J].中国畜牧兽医,2010,37(9)：124-126.
[19]M Zaslof. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, charact-erization of two active forms, and partial cDNA sequence of a precursor[J].Proc Natl Acad Sci:USA,1987,84(15):5449-5453.
[20]H Steiner,D Hultmark,A Engstrom,et al. Sequence and specificity of two antibacterial proteins involved in insect immunity[J].Nature,1981,292:246-248.
[21]Howard N. Hunter,A. Ross Demcoe,Hayard Jenssen,et al.Human lactoferricin is partially folded in aqueous solution and is better stabilized in a membrane mimetic solvent[J].Antimicrob Agents Chemother,2005,49:3387-3395.
[22]M E Selsted,D M Brown,R J DeLange,et al.Primary structure of Mcp-1 and Mcp-2.natural peptide antibiotics of rabbit lung macrophages[J].J Biol Chem,1983,258(23):14485-14493.
[23]Bulet P,Stocklin R Menin L.Anti-microbial peptides:from invertebrates to vertebrates[J]. Immunol Rev,2004,198:169-184.
[24]Trabi M.Schirra HJ,Craik DJ.Three-dimensional structure of RTD-l,a cyclic antimicrobial defensin from Rhesus macaque leukocvtes[J].Biochemistry,2001,40(14):4211-4221.
[25]王燕.抗菌肽cecropins的研究进展[J].广东饲料,2007,16(6)：27-28.
[26]Sorenson G D,Bloom S R.Bombesin production by human small cell carcinoma of the lung [J].Regul Pept,1982,4(2):59-66.
[27]Price J,Kruseman A C, Donicach I,et al.Bombesin-like peptides in human endocrine tumors: quantitation, biochemical characterization, and secretion [J].J Clin Endocrino Metab,1985,60(6): 1097-1103.
[28]王东旺.蛙皮素及其同类物与肿瘤的关系[J].现代诊断与治疗,2005,16(4)：225-227.
[29]Carretero M,Escamez M J,Garcia M,et al.In vitro and in vivo Wound Healing promoting Activities of Human Cathelicidin LL-37[J].J Invest Dermatol,2008,128(1):223-236.
[30]Otte J M,Zdebik A E,Brand S,et al. Effects of the Cathelicidin LL-37 Oil Intestinal Epithelial Barrier Integrity[J].Regul Pept,2009.156(1/3):104-117.
[31]杨铭,结构生物学与药学研究[M].科学出版社,北京,2006,p.142-162.
[32]江黎丽.生物小分子对模拟生物膜作用的电化学研究[D].[硕士学位论文].辽宁：辽宁大学,2007.
[33]牛真真.电化学法研究硫酸多粘菌素B与磷脂的相互作用[D].郑州,郑州大学硕士论文,2011.
[34]马垧玻,张玉明,倪志华,等.抗茵肽的结构与功能研究[J].安徽农业科学,2009,37(23)：10878-10880.
[35]张召兄,潘晓亮,任耀军.抗菌肽概述及抗菌分子机理的研究进展[J].草食家畜(季刊),2006,133(4)：7-10.
[36]P.Li,M.Sun,B.Ho,J.L.Ding.The specificity of Sushi peptides for endotoxin and anionic phospholipids: potential application of POPG as an adjuvant for anti-LPS strategies[J].Biochem-ical Society Transactions,2006,34(2):270-272.
[37]V Frecer,B Ho,JL Ding.De Nove Design of Potent Antimicrobial Peptides[J].Antimicrobial Agents and Chemotherapy,2004(9):3349-3357.
[38]申吉泓.抗菌肽作用机制研究现状[J].国外医学肿瘤学分册,2003,10(5)：347-350.
[39]Bessin Y,Saint N, Marri L,et al.Antibacterial activity and pore-forming properties of ceratotoxins:a mechanism of action based on the barrel model [J].Biochim Biophys,Acta,2004, 1667(2):148-156.
[40]苏琦,孙燕,李治.抗菌肽对细菌胞内杀伤作用的分子机制[J].中国生物制品学杂志,2010,23(3)：325-328.
[41]Brogden KA.Antimicrobial peptides :pore formers or metabolic inhibitors in bacterial Nat Rev Microbiol.2005,3(3):238-250.
[42]Peng Li,Miao Sun,Thorsten Wohland. Molecular Mechanisms that Govern the Specificity of Sushi Peptide for Gram-Negative Bacterial Membrance Lipids[J].Biochemistry,2006,45(35): 10554-10562.
[43]郝刚,施用晖,唐亚丽.抗菌Buforin Ⅱ衍生物与大肠杆菌基因组DNA的作用机制[J].微生物学报,2010,50(3)：328-333.
[44]刘忠渊,徐涛,郑树涛等.新疆家蚕抗菌肽Cecropin-XJ与细菌DNA相互作用的光谱研究[J].光谱学与光谱分析,2008,28(3)：612-616.
[45]Carlsson A,Engstrm P,Palva ET.et al.Attacin,an antibacterial protein from Hyalophora cecropia, inhibits synthesis of outer membrane proteins in Escherichia coli by interfering with omp gene transcription [J].Infect Immun,1991,59(9):3040-3045.
[46]Hasper HE,Kramer NE.Smith JL,et al.An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II[J].Science,2006,313(5793):1636-1637.
[47]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):5707-5713.
[48]Martinez B,B6ttiqer T,Schneider T,et al.Specific interaction of the unmodified bacteriocin Lactococcin 972 with the cell wall precursor lipid Ⅱ[J].Appl Environ Microbiol,2008,74(15): 4666-4670.
[49]吴希,张双全.抗菌肽对细菌杀伤作用的分子机制[J].生物化学与生物物理进展,2005,32(12)：1109-1113.
[50]Gennaro R,Zanetti M,Benincasa M,et al.Pro-rich antimicrobial peptides from animals: structure, biological functions and mechanism of action[J].Curr Pharm Des,2002,8(9):763-778.
[51]Brogden KA,Ackermann M,McCray PB Jr,et al. Antimicrobial peptides in animals and their role in host defences[J].Antimicrobial Agents,2003,22(4):465-478.
[52]Park CB,Yi KS,Matsuzaki K,et al.Structure-activity analysis of buforin II,a histone H2A-derived antimicrobial peptide:the proline hinge is responsible for the cell-penetrating ability of buforin II[J].Proc Natl Acad Sci,2000,97(15):8245-8251.
[53]程琼侠,代培芳,罗景星.家蝇幼虫抗菌肽对肿瘤细胞K562作用的扫描电镜观察[J].中华卫生杀虫药械,2007,13(4)：259-261.
[54]佘锐萍,靳红,彭芳珍等.猪小肠抗菌肽的抗菌作用研究[J].中国兽医杂志,2005,41(1)：3-7.
[55]董小卫,宋晓妍,石梅.Peptaibols类抗茵肽mTrichokoninVI在不同溶剂中构象变化的圆二色谱研究[J].光谱学与光谱分析,2010,30(2)：458-461.
[56]李顺子,阎虎生,刘国栋.蜂毒肽类似物的合成和生物活性研究[J].高等学校化学学报,2003,24(3)：449-453.
[57]Yamaquchi S, Hong T, Waring A, et al. Solid-state NMR investigations of peptide-lipid interaction and orientation of a beta-sheet antimicrobial peptide, protegrin[J].Biochemistry,2002, 41(31):9852-9862.
[58]夏顺霞,徐国华,曹淑芬.抗菌肽Maximin3溶液结构的NMR研究,中国化学会第十四届有机分析及生物分析学术研讨会会议论文摘要集[C]：2007年.
[59]Bechinger B,Zasloff M,Opella SJ.Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy[J].Protein Sci,1993,2(12): 2077-2084.
[60]Yamaquchi S,Huster D,Waring A,et al.Orientation and dynamics of an antimicrobial peptide in the lipid bilayer by solid-state NMR spectroscopy[J].Biophys J,2001,81(2):2203-2214.
[61]Henzler Wildman KA,Lee DK,Ramamoorthy A. Mechanism of lipid bilayer disruption by the human antimicrobial peptide,LL-37[J].Biochemistry,2003,42(21):6545-6558.
[62]肖勇梅,温浙盛,刘汝青.体外抑菌试验评价核糖体表达筛选的抗菌肽活性[J].中华生物医学工程杂志,2007,13(5)：306-309.
[63]Frecer V,Ho B,Ding JL.Interpretation of biological activity data of bacterial endotoxins by simple molecular models of mechanism of action[J]Eur. J. Biochem.2000,267(3): 837-52.
[64]Yang ST,Shin SY,Hahm KS et al.Design of perfectly symmetric Trp-rich peptides with potent and broad-spectrum antimicrobial activities[J].International Journal of Antimicrobial Agents, 2006,27(4):325-330.
[65]Li P,Wohland T,Ho B,et al.Perturbation of lipopolysaccharide (LPS) micelles by Sushi 3 (S3) antimicrobial peptide [J]. The Journal of Biological Chemistry,2004,279(48):50150-50156.
[66]Gottler LM,Ramamoorthy A.Structure,membrane orientation,mechanism and function of pexiganan-a highly potent antimicrobial peptide designed from magainin[J].Biochimica et Biophysica Acta (BBA)-Biomembranes,2009,1788(8):1680-1686.
[67]杨丽敏,黄燕,张日俊.新抗菌肽设计及活性与功能预测[J].计算机与现代化,2013,210(2)：138-142.
[68]宫霞,乐国伟.家蝇幼虫抗菌肽MDL-1的构象分析[J].高等学校化学学报,2008,29(4)：745-748.
[69]P. Muller,D.O.Rudin,H.T.Tien,et al.Reconstitution of cell membrane structure in vitro and its transformation into an excitable system[J].Nature,1962,194:979-980.
[70]Tien H.T.,Salamon Z..Formation of self-assembled lipid bilayers on solid substrates [J].Bioelectrochemistry and Bioenergetic,1989,3(22):211-218.
[71]GONG Jing Ming,LIN Xiang Qin.Ion Channel Behavior of a Supported Bilayer Lipid Membrane Composed of 5,5-Ditetradecyl-2-(2-trimethyl-ammonioethyl)-1,3-dioxane Bromide Modified Glassy Carbon Electrode[J].Chinese Journal of Chemistry,2003,7(21):756-760.
[72]Yan Li Zhang, James Dunlop,Thai Phung,et al.Supported bilayer lipid membranes modified with a phosphate ionophore[J].Biosensors and Bioelectronics,2006,12(21):2311-2314.
[73]Yu-Feng Zhang,Xin-Hou,Gang Wu,et al.Study on selectivity of permeating chiral complexes in bilayer lipid membranes[J].Electrochemistry Communications,1999,6(1):238-241.
[74]Li Wang,Xin-Pu Hou,Angelica Ottova,et al.Receptor-ligand interactions in a reconstituted bilayer lipid membrane[J].Electrochemistry Communications,2000,5(2):287-289.
[75]赵艳,方炎,双层类脂膜成膜过程的电化学方法研究[J].电化学,2004,10(1)：70-74.
[76]高宏,罗国安,冯军.硫醇-磷脂混合双层膜的电化学性质及其与蜂毒素的相互作用研究[J].化学学报,2001,59(2)：220-223.
[77]Rezansoff A.J., Hunter H.N.,Jing W.et al.Interactions of the antimicrobial peptide Ac-FRWWHR-NH2 with model membrane systems and bacterial cells[J].J.Peptide Res,2005, 65(5):491-501.
[78]任锦,钦传光,徐春兰等.细胞穿膜肽作为药物载体的研究进展[J].药学学报,2010,45(1)：17-25.
[79]P.Sospedra,C.Mestres,I.Haro, et al.Effect of Amino Acid Sequence Change on Peptide-Membrane Interaction[J].Langmuir,2002,4(18),1231-1237.
[80]H.Zhao,R.Sood,A.Jutila,et al. Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: implications for a novel mechanism of action[J].Biochim.Biophys. Acta,2006,9(1758):1461-1474.
[81]S.Butler,R.Wang,S.L.Wunder,et al. Perturbing effects of carvedilol on a model membrane system:Role of lipophilicity and chemical structure[J].Biophys.Chem.,2006,3(119):307-315.
[82]许倩影,刘忠芳,胡小莉.Pd(Ⅱ)与色氨酸、酪氨酸及苯丙氨酸相互作用的荧光光谱[J].高等学校化学学报,2011,32(7)：1492-1496.
[83]蔡开聪,王建平.乙醇醛的分子动态结构[J].物理化学学报,2009,25(4)：677-683.
[84]Alexander Pertsin,Dmitry Platonov,Michael Grunze.Origin of Short-Range Repulsion between Hydrated Phospholipid Bilayers:A Computer Simulation Study[J].Langmuir,2007,23: 1388-1393.
[85]张彤,李宗圣,金宏威等.异核苷掺人DNA:DNA双链体系的分子动力学模拟研究[J].中国药物化学杂志,2008,18(3)：161-169.
[86]吴晓敏,祖元刚,杨志伟.温控分子动力学研究微管蛋白活性肽链的折叠机制[J].物理化学学报,2009,25(4)：773-782.
[87]孟煊宇,楚慧郢,郑清川.人类丁酰胆碱酯酶(BuChE)与抑制剂小分子的对接研究[J].中国科技论文在线,2009,4(9)：627-631.
[88]刘黎,黄庆生,方颖.抗菌肽HP(2-20)及其类似物与POPE膜相互作用的分子动力学模拟[J].生物物理学报,2009,25(S1)：234-235.
[89]Qian Wang,Gongyi Hong,Glenn R.Johnson,et al.Biophysical Properties of Membrane-Active Peptides Based on Micelle Modeling:A Case Study of Cell-Penetrating and Antimicrobial Peptides[J].J Phys Chem B,2010,114(43):13726-13735.
[90]Jing W., Hunter H.N., Hagel J..The structure of the antimicrobial peptide Ac-RRWWRF-NH2 bound to micelles and its interactions with phospholipid bilayers[J].J.Peptide Res.,2003,61(5): 219-229.
[2]仇妍虹.家蝇防御素抗菌肽在毕赤酵母中分泌表达及活性研究[D].南京：南京农业大学硕士论文,2006.
[3]H.I.Zeya,J.K.Spitznagel.Cationic protein-bearing granules of polymorphonuclear leukocytes: separation from enzyme-rich granules[J].Science,1969,163(3871):1069-1071.
[4]Christian Mayer,Ralf Moritz,Carolin Kirschner.et al.The role of inter- molecular interactions: studies on model systems for bacterial biofilms[J]. Int J Biol Macromol,1999,26(1):3-16.
[5]Dan Hultmark,Hakan Steriner,Torgny Rasmuson,et al.Insect immunity,purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalohora cecropia[J].Eur J Biochemistry,1980,106(1):7-16.
[6]吴甜甜,杨洁.天然抗菌肽的研究进展及应用前景[J].生物技术通报,2009,(1)：27-30.
[7]梁永利.天然抗菌肽的来源及分类[J].安徽农业科学,2006,34(18)：4728-4734.
[8]江龙法,谢慧,邬敏辰.阳离子抗菌肽的作用机理及构效关系[J].中国医药工业杂志,2005,36(4)：244-249.
[9]王威,王联结.阳离子抗菌肽分子设计的研究现状[J].食品工业科技,2010,31(1)：442-445.
[10]潘秋冬,夏明.抗菌肽的研究现状及应用前景[J].农产品加工·学刊,2011,2235(2)：81-83.
[11]刘汉灵,潘扬昌,王孝英等.黄粉虫蛋白抗菌肽酶解工艺研究[J].食品科学,2009,30(15)：156-159.
[12]吴琼英,徐金玲.酶解反应和膜分离耦合制备乳源抗菌肽[J].江苏科技大学学报(自然科学版),2010,24(4)：386-390.
[13]冯兴军,李静,赵晓宇等.牛乳铁蛋白素·马盖宁杂合抗菌肽的设计、合成及抑菌活性[J].东北农业大学学报,2011,42(3)：105-109.
[14]赵亦静,倪密,诺林等.人工合成抗菌肽对棉花黄萎病菌的抑菌效果[J].浙江大学学报,2013,39(1)：11-17.
[15]张召兄,潘晓亮,任耀军.抗菌肽概述及抗菌分子机理的研究进展[J].草食家畜,2006,133(4)：7-10.
[16]赵冬梅,陶妍.斑点叉尾鲴hepcidin抗菌肽在大肠杆菌中的融合表达[J].东北农业大学学报,2012,43(12)：114-120.
[17]单艳菊,胡艳,徐文娟等,鸡p防御素1基因密码子优化及其在真核细胞中的表达[J].云南农业大学学报,2012,27(6)：833-839.
[18]付登峰,胡建和,刘兴友.抗菌肽基因工程表达技术研究进展[J].中国畜牧兽医,2010,37(9)：124-126.
[19]M Zaslof. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, charact-erization of two active forms, and partial cDNA sequence of a precursor[J].Proc Natl Acad Sci:USA,1987,84(15):5449-5453.
[20]H Steiner,D Hultmark,A Engstrom,et al. Sequence and specificity of two antibacterial proteins involved in insect immunity[J].Nature,1981,292:246-248.
[21]Howard N. Hunter,A. Ross Demcoe,Hayard Jenssen,et al.Human lactoferricin is partially folded in aqueous solution and is better stabilized in a membrane mimetic solvent[J].Antimicrob Agents Chemother,2005,49:3387-3395.
[22]M E Selsted,D M Brown,R J DeLange,et al.Primary structure of Mcp-1 and Mcp-2.natural peptide antibiotics of rabbit lung macrophages[J].J Biol Chem,1983,258(23):14485-14493.
[23]Bulet P,Stocklin R Menin L.Anti-microbial peptides:from invertebrates to vertebrates[J]. Immunol Rev,2004,198:169-184.
[24]Trabi M.Schirra HJ,Craik DJ.Three-dimensional structure of RTD-l,a cyclic antimicrobial defensin from Rhesus macaque leukocvtes[J].Biochemistry,2001,40(14):4211-4221.
[25]王燕.抗菌肽cecropins的研究进展[J].广东饲料,2007,16(6)：27-28.
[26]Sorenson G D,Bloom S R.Bombesin production by human small cell carcinoma of the lung [J].Regul Pept,1982,4(2):59-66.
[27]Price J,Kruseman A C, Donicach I,et al.Bombesin-like peptides in human endocrine tumors: quantitation, biochemical characterization, and secretion [J].J Clin Endocrino Metab,1985,60(6): 1097-1103.
[28]王东旺.蛙皮素及其同类物与肿瘤的关系[J].现代诊断与治疗,2005,16(4)：225-227.
[29]Carretero M,Escamez M J,Garcia M,et al.In vitro and in vivo Wound Healing promoting Activities of Human Cathelicidin LL-37[J].J Invest Dermatol,2008,128(1):223-236.
[30]Otte J M,Zdebik A E,Brand S,et al. Effects of the Cathelicidin LL-37 Oil Intestinal Epithelial Barrier Integrity[J].Regul Pept,2009.156(1/3):104-117.
[31]杨铭,结构生物学与药学研究[M].科学出版社,北京,2006,p.142-162.
[32]江黎丽.生物小分子对模拟生物膜作用的电化学研究[D].[硕士学位论文].辽宁：辽宁大学,2007.
[33]牛真真.电化学法研究硫酸多粘菌素B与磷脂的相互作用[D].郑州,郑州大学硕士论文,2011.
[34]马垧玻,张玉明,倪志华,等.抗茵肽的结构与功能研究[J].安徽农业科学,2009,37(23)：10878-10880.
[35]张召兄,潘晓亮,任耀军.抗菌肽概述及抗菌分子机理的研究进展[J].草食家畜(季刊),2006,133(4)：7-10.
[36]P.Li,M.Sun,B.Ho,J.L.Ding.The specificity of Sushi peptides for endotoxin and anionic phospholipids: potential application of POPG as an adjuvant for anti-LPS strategies[J].Biochem-ical Society Transactions,2006,34(2):270-272.
[37]V Frecer,B Ho,JL Ding.De Nove Design of Potent Antimicrobial Peptides[J].Antimicrobial Agents and Chemotherapy,2004(9):3349-3357.
[38]申吉泓.抗菌肽作用机制研究现状[J].国外医学肿瘤学分册,2003,10(5)：347-350.
[39]Bessin Y,Saint N, Marri L,et al.Antibacterial activity and pore-forming properties of ceratotoxins:a mechanism of action based on the barrel model [J].Biochim Biophys,Acta,2004, 1667(2):148-156.
[40]苏琦,孙燕,李治.抗菌肽对细菌胞内杀伤作用的分子机制[J].中国生物制品学杂志,2010,23(3)：325-328.
[41]Brogden KA.Antimicrobial peptides :pore formers or metabolic inhibitors in bacterial Nat Rev Microbiol.2005,3(3):238-250.
[42]Peng Li,Miao Sun,Thorsten Wohland. Molecular Mechanisms that Govern the Specificity of Sushi Peptide for Gram-Negative Bacterial Membrance Lipids[J].Biochemistry,2006,45(35): 10554-10562.
[43]郝刚,施用晖,唐亚丽.抗菌Buforin Ⅱ衍生物与大肠杆菌基因组DNA的作用机制[J].微生物学报,2010,50(3)：328-333.
[44]刘忠渊,徐涛,郑树涛等.新疆家蚕抗菌肽Cecropin-XJ与细菌DNA相互作用的光谱研究[J].光谱学与光谱分析,2008,28(3)：612-616.
[45]Carlsson A,Engstrm P,Palva ET.et al.Attacin,an antibacterial protein from Hyalophora cecropia, inhibits synthesis of outer membrane proteins in Escherichia coli by interfering with omp gene transcription [J].Infect Immun,1991,59(9):3040-3045.
[46]Hasper HE,Kramer NE.Smith JL,et al.An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II[J].Science,2006,313(5793):1636-1637.
[47]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):5707-5713.
[48]Martinez B,B6ttiqer T,Schneider T,et al.Specific interaction of the unmodified bacteriocin Lactococcin 972 with the cell wall precursor lipid Ⅱ[J].Appl Environ Microbiol,2008,74(15): 4666-4670.
[49]吴希,张双全.抗菌肽对细菌杀伤作用的分子机制[J].生物化学与生物物理进展,2005,32(12)：1109-1113.
[50]Gennaro R,Zanetti M,Benincasa M,et al.Pro-rich antimicrobial peptides from animals: structure, biological functions and mechanism of action[J].Curr Pharm Des,2002,8(9):763-778.
[51]Brogden KA,Ackermann M,McCray PB Jr,et al. Antimicrobial peptides in animals and their role in host defences[J].Antimicrobial Agents,2003,22(4):465-478.
[52]Park CB,Yi KS,Matsuzaki K,et al.Structure-activity analysis of buforin II,a histone H2A-derived antimicrobial peptide:the proline hinge is responsible for the cell-penetrating ability of buforin II[J].Proc Natl Acad Sci,2000,97(15):8245-8251.
[53]程琼侠,代培芳,罗景星.家蝇幼虫抗菌肽对肿瘤细胞K562作用的扫描电镜观察[J].中华卫生杀虫药械,2007,13(4)：259-261.
[54]佘锐萍,靳红,彭芳珍等.猪小肠抗菌肽的抗菌作用研究[J].中国兽医杂志,2005,41(1)：3-7.
[55]董小卫,宋晓妍,石梅.Peptaibols类抗茵肽mTrichokoninVI在不同溶剂中构象变化的圆二色谱研究[J].光谱学与光谱分析,2010,30(2)：458-461.
[56]李顺子,阎虎生,刘国栋.蜂毒肽类似物的合成和生物活性研究[J].高等学校化学学报,2003,24(3)：449-453.
[57]Yamaquchi S, Hong T, Waring A, et al. Solid-state NMR investigations of peptide-lipid interaction and orientation of a beta-sheet antimicrobial peptide, protegrin[J].Biochemistry,2002, 41(31):9852-9862.
[58]夏顺霞,徐国华,曹淑芬.抗菌肽Maximin3溶液结构的NMR研究,中国化学会第十四届有机分析及生物分析学术研讨会会议论文摘要集[C]：2007年.
[59]Bechinger B,Zasloff M,Opella SJ.Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy[J].Protein Sci,1993,2(12): 2077-2084.
[60]Yamaquchi S,Huster D,Waring A,et al.Orientation and dynamics of an antimicrobial peptide in the lipid bilayer by solid-state NMR spectroscopy[J].Biophys J,2001,81(2):2203-2214.
[61]Henzler Wildman KA,Lee DK,Ramamoorthy A. Mechanism of lipid bilayer disruption by the human antimicrobial peptide,LL-37[J].Biochemistry,2003,42(21):6545-6558.
[62]肖勇梅,温浙盛,刘汝青.体外抑菌试验评价核糖体表达筛选的抗菌肽活性[J].中华生物医学工程杂志,2007,13(5)：306-309.
[63]Frecer V,Ho B,Ding JL.Interpretation of biological activity data of bacterial endotoxins by simple molecular models of mechanism of action[J]Eur. J. Biochem.2000,267(3): 837-52.
[64]Yang ST,Shin SY,Hahm KS et al.Design of perfectly symmetric Trp-rich peptides with potent and broad-spectrum antimicrobial activities[J].International Journal of Antimicrobial Agents, 2006,27(4):325-330.
[65]Li P,Wohland T,Ho B,et al.Perturbation of lipopolysaccharide (LPS) micelles by Sushi 3 (S3) antimicrobial peptide [J]. The Journal of Biological Chemistry,2004,279(48):50150-50156.
[66]Gottler LM,Ramamoorthy A.Structure,membrane orientation,mechanism and function of pexiganan-a highly potent antimicrobial peptide designed from magainin[J].Biochimica et Biophysica Acta (BBA)-Biomembranes,2009,1788(8):1680-1686.
[67]杨丽敏,黄燕,张日俊.新抗菌肽设计及活性与功能预测[J].计算机与现代化,2013,210(2)：138-142.
[68]宫霞,乐国伟.家蝇幼虫抗菌肽MDL-1的构象分析[J].高等学校化学学报,2008,29(4)：745-748.
[69]P. Muller,D.O.Rudin,H.T.Tien,et al.Reconstitution of cell membrane structure in vitro and its transformation into an excitable system[J].Nature,1962,194:979-980.
[70]Tien H.T.,Salamon Z..Formation of self-assembled lipid bilayers on solid substrates [J].Bioelectrochemistry and Bioenergetic,1989,3(22):211-218.
[71]GONG Jing Ming,LIN Xiang Qin.Ion Channel Behavior of a Supported Bilayer Lipid Membrane Composed of 5,5-Ditetradecyl-2-(2-trimethyl-ammonioethyl)-1,3-dioxane Bromide Modified Glassy Carbon Electrode[J].Chinese Journal of Chemistry,2003,7(21):756-760.
[72]Yan Li Zhang, James Dunlop,Thai Phung,et al.Supported bilayer lipid membranes modified with a phosphate ionophore[J].Biosensors and Bioelectronics,2006,12(21):2311-2314.
[73]Yu-Feng Zhang,Xin-Hou,Gang Wu,et al.Study on selectivity of permeating chiral complexes in bilayer lipid membranes[J].Electrochemistry Communications,1999,6(1):238-241.
[74]Li Wang,Xin-Pu Hou,Angelica Ottova,et al.Receptor-ligand interactions in a reconstituted bilayer lipid membrane[J].Electrochemistry Communications,2000,5(2):287-289.
[75]赵艳,方炎,双层类脂膜成膜过程的电化学方法研究[J].电化学,2004,10(1)：70-74.
[76]高宏,罗国安,冯军.硫醇-磷脂混合双层膜的电化学性质及其与蜂毒素的相互作用研究[J].化学学报,2001,59(2)：220-223.
[77]Rezansoff A.J., Hunter H.N.,Jing W.et al.Interactions of the antimicrobial peptide Ac-FRWWHR-NH2 with model membrane systems and bacterial cells[J].J.Peptide Res,2005, 65(5):491-501.
[78]任锦,钦传光,徐春兰等.细胞穿膜肽作为药物载体的研究进展[J].药学学报,2010,45(1)：17-25.
[79]P.Sospedra,C.Mestres,I.Haro, et al.Effect of Amino Acid Sequence Change on Peptide-Membrane Interaction[J].Langmuir,2002,4(18),1231-1237.
[80]H.Zhao,R.Sood,A.Jutila,et al. Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: implications for a novel mechanism of action[J].Biochim.Biophys. Acta,2006,9(1758):1461-1474.
[81]S.Butler,R.Wang,S.L.Wunder,et al. Perturbing effects of carvedilol on a model membrane system:Role of lipophilicity and chemical structure[J].Biophys.Chem.,2006,3(119):307-315.
[82]许倩影,刘忠芳,胡小莉.Pd(Ⅱ)与色氨酸、酪氨酸及苯丙氨酸相互作用的荧光光谱[J].高等学校化学学报,2011,32(7)：1492-1496.
[83]蔡开聪,王建平.乙醇醛的分子动态结构[J].物理化学学报,2009,25(4)：677-683.
[84]Alexander Pertsin,Dmitry Platonov,Michael Grunze.Origin of Short-Range Repulsion between Hydrated Phospholipid Bilayers:A Computer Simulation Study[J].Langmuir,2007,23: 1388-1393.
[85]张彤,李宗圣,金宏威等.异核苷掺人DNA:DNA双链体系的分子动力学模拟研究[J].中国药物化学杂志,2008,18(3)：161-169.
[86]吴晓敏,祖元刚,杨志伟.温控分子动力学研究微管蛋白活性肽链的折叠机制[J].物理化学学报,2009,25(4)：773-782.
[87]孟煊宇,楚慧郢,郑清川.人类丁酰胆碱酯酶(BuChE)与抑制剂小分子的对接研究[J].中国科技论文在线,2009,4(9)：627-631.
[88]刘黎,黄庆生,方颖.抗菌肽HP(2-20)及其类似物与POPE膜相互作用的分子动力学模拟[J].生物物理学报,2009,25(S1)：234-235.
[89]Qian Wang,Gongyi Hong,Glenn R.Johnson,et al.Biophysical Properties of Membrane-Active Peptides Based on Micelle Modeling:A Case Study of Cell-Penetrating and Antimicrobial Peptides[J].J Phys Chem B,2010,114(43):13726-13735.
[90]Jing W., Hunter H.N., Hagel J..The structure of the antimicrobial peptide Ac-RRWWRF-NH2 bound to micelles and its interactions with phospholipid bilayers[J].J.Peptide Res.,2003,61(5): 219-229.