鸡Gal-11和Gal-12基因的克隆、表达及活性检测
|
摘要
B-防御素是鸡体内最重要的抗菌肽,除了具有直接的杀菌、抗病毒作用外,它还参与机体免疫反应,有重大的开发利用潜力。本文构建了表达鸡B-防御素-11和β-防御素-12(Gal-11和Gal-12)的大肠杆菌表达系统,研究了重组鸡β-防御素在大肠杆菌系统中高水平表达规律和蛋白纯化技术,为其进一步研究开发奠定了基础。
本研究共分四部分:
①鸡Gal-11基因cDNA的克隆根据genebank中AY621326序列设计了一对引物,运用RT-PCR技术,从固始鸡L系-崇仁麻鸡肾脏总RNA中扩增出片断大小为315bp的鸡Gal-11编码cDNA序列全长。测序结果与AY621326比对,同源性达99%。
②鸡Gal-12成熟肽DNA的克隆根据genebank中AY621327设计上下游引物,从基因组DNA中扩增出大小为159bp的目的片断测序结果EF044309与序列AY621327,AY534898和DQ858309比对,序列完全一致;
③鸡Gal-11和Gal-12基因的原核表达以pET32a为表达质粒构建了重组质粒pET32a-Gal-11和pET32a-Gal-12,并在BL21(DE3)pLySs中进行融合表达。SDS-PAGE电泳结果表明在目标位置约27.175KD和22.896KD出现条带。诱导剂IPTG在0.2mM-1.2mM的对重组蛋白的产量无明显影响,加入诱导剂1h开始有可检出量重组蛋白质的表达,在2h时其表达水平已接近最大量。
④鸡Gal-11和Gal-12基因表达产物的活性检测融合表达蛋白经过初步分离,变性,组氨酸亲和层析柱纯化,复性,对纯化的表达蛋白进行琼脂糖弥散试验检测生物学活性。结果表明:蛋白进行抑菌试验对大肠杆菌、白色念球菌、金黄色葡萄球菌没有产生抗菌活性。
鸡Gal-11和Gal-12在pET32a质粒表达体系中成功表达为鸡防御素多肽下一步的研究奠定基础。
β-defensin Gallinacin is the most important member of a large family of antimicrobial peptides in chicken.β-defensin Gallinacin displays a strong antimicrobial activity against bacteria and fungi.Furthermore,it also plays indispensable function in triggering adaptive immune responses.So it could act as a substitute for antibiotic.In this paper,systems for high-level expression of theβ-defensin fusions in Escherichia toll were constructed,and expression regulation and the technology of protein purification were investigated.
There are four parts in this paper:
①Cloning of chicken Gal-ll gene:A pair of primers were designed and synthesized based on the publised gene sequence of Gal-11(AY621326).The target DNA,which contains 315bps,were amplified by RT-PCR on the template of tatal RNA isolated from kidney.The sequencing result displaied the target DNA was homologous with AY621326 by 99%.
②Cloning of chicken Gal-12 mature peptides gene:A pair of primers were designed and synthesized based on the publised gene sequence of Gal-12(AY621327). The target DNA were amplified by PCR on the template of genome DNA isolated from blood.The sequencing result was completely consistent with AY621327,AY534898 and DQ858309.
③Expression of chicken Gal-11 and Gal-12 gene:Gal-11 and Gal-12 gene was expressed in the form of fusion protein useing pET32a as expression vectors and BL21 (DE3)pLySs as expression host.Expression products with molecular weight of approximately 27.175KD and 22.896KD were examined by SDS-PAGE.Top yield of recombinant Gal-11 and Gal-12 could be achieved with as less as 0.2 mmol/L IPTG induction after 2 hours.
④Biological activity detection of the purified protein:Recombinant fusion protein was extracted roughly from dissolved BL21 and purified with Ni-NTA resin affinity column.The biological activity was measured by the minimum inhibitory concentrations (MICs)the radial diffusion plate assay method.The result demonstrated that the purified protein has no antimicrobial activity against Escherichia.coli O78,Candida.albicans ATCC10231 and Staphylococcus.aureus 1056MRSA.
Further study will be continued based on the results of this research.
本研究共分四部分:
①鸡Gal-11基因cDNA的克隆根据genebank中AY621326序列设计了一对引物,运用RT-PCR技术,从固始鸡L系-崇仁麻鸡肾脏总RNA中扩增出片断大小为315bp的鸡Gal-11编码cDNA序列全长。测序结果与AY621326比对,同源性达99%。
②鸡Gal-12成熟肽DNA的克隆根据genebank中AY621327设计上下游引物,从基因组DNA中扩增出大小为159bp的目的片断测序结果EF044309与序列AY621327,AY534898和DQ858309比对,序列完全一致;
③鸡Gal-11和Gal-12基因的原核表达以pET32a为表达质粒构建了重组质粒pET32a-Gal-11和pET32a-Gal-12,并在BL21(DE3)pLySs中进行融合表达。SDS-PAGE电泳结果表明在目标位置约27.175KD和22.896KD出现条带。诱导剂IPTG在0.2mM-1.2mM的对重组蛋白的产量无明显影响,加入诱导剂1h开始有可检出量重组蛋白质的表达,在2h时其表达水平已接近最大量。
④鸡Gal-11和Gal-12基因表达产物的活性检测融合表达蛋白经过初步分离,变性,组氨酸亲和层析柱纯化,复性,对纯化的表达蛋白进行琼脂糖弥散试验检测生物学活性。结果表明:蛋白进行抑菌试验对大肠杆菌、白色念球菌、金黄色葡萄球菌没有产生抗菌活性。
鸡Gal-11和Gal-12在pET32a质粒表达体系中成功表达为鸡防御素多肽下一步的研究奠定基础。
β-defensin Gallinacin is the most important member of a large family of antimicrobial peptides in chicken.β-defensin Gallinacin displays a strong antimicrobial activity against bacteria and fungi.Furthermore,it also plays indispensable function in triggering adaptive immune responses.So it could act as a substitute for antibiotic.In this paper,systems for high-level expression of theβ-defensin fusions in Escherichia toll were constructed,and expression regulation and the technology of protein purification were investigated.
There are four parts in this paper:
①Cloning of chicken Gal-ll gene:A pair of primers were designed and synthesized based on the publised gene sequence of Gal-11(AY621326).The target DNA,which contains 315bps,were amplified by RT-PCR on the template of tatal RNA isolated from kidney.The sequencing result displaied the target DNA was homologous with AY621326 by 99%.
②Cloning of chicken Gal-12 mature peptides gene:A pair of primers were designed and synthesized based on the publised gene sequence of Gal-12(AY621327). The target DNA were amplified by PCR on the template of genome DNA isolated from blood.The sequencing result was completely consistent with AY621327,AY534898 and DQ858309.
③Expression of chicken Gal-11 and Gal-12 gene:Gal-11 and Gal-12 gene was expressed in the form of fusion protein useing pET32a as expression vectors and BL21 (DE3)pLySs as expression host.Expression products with molecular weight of approximately 27.175KD and 22.896KD were examined by SDS-PAGE.Top yield of recombinant Gal-11 and Gal-12 could be achieved with as less as 0.2 mmol/L IPTG induction after 2 hours.
④Biological activity detection of the purified protein:Recombinant fusion protein was extracted roughly from dissolved BL21 and purified with Ni-NTA resin affinity column.The biological activity was measured by the minimum inhibitory concentrations (MICs)the radial diffusion plate assay method.The result demonstrated that the purified protein has no antimicrobial activity against Escherichia.coli O78,Candida.albicans ATCC10231 and Staphylococcus.aureus 1056MRSA.
Further study will be continued based on the results of this research.
引文
[1]BALS R.Epithelial antimicrobial peptides in host defense against infection[J].Respir Res,2000,1(3):141-150.
[2]HOFFMANN J A,KAFATOS F C,JANEWAY C A,et al.Phylogenetic perspectives in innate immunity[J].Science,1999,284:1313-1318.
[3]ZAMBON R A,NANDAKUMAR M,VAKHARIA V N,et at.The toll pathway is important for an antiviral response in Drosophila[J].Proc Natl Acad Sci USA,2005,102(20):7257-7262.
[4]REDDY K V R,YEDERY R D,ARANHA C.Antimicrobial peptides:premises and promises[J].Int J Antimicrob Agents,2004,24(6):536-547.
[5]梁永利,天然抗菌肽的来源及分类[J],安徽农业科学,2006,34(38):4728-4734
[6]BULET P,HETRU C,DIMARCQ J,et al.Antimicrobial peptides from insects:structure and function[J].Dev Comp Immunol,1999,23:329-344.
[7]Bellm L,Lehrer RI Ganz T.Protegrins:new antibiotics of mammalian origin.Expert Opin Investig Drugs[J],2000,9(8):1731-1742.
[8]Hancock RE.Cationic antimicrobial peptides:towards clinical applications.Expert Opinlnvestig Drugs[J],2000,9(8):1723-1729.
[9]朱颐申,王卫国等.海洋抗菌肽研究进展[J].高技术通讯,2004(2):105-110.
[10]Robert E W Hancock.Cationic peptides:effectors in innate immunity and novel antimicrobials[J].The Lancet infectious Disease,2001(1):156-164.
[11]马卫明.猪小肠抗菌肽分离鉴定及其活性研究[D].中国农业大学博士学位论文,2004:10-11.
[12]MichaelR,et al.Mechanism of antimicrobial peptide Action and resistance[J].Pharmacological Review,2003,55(1):27-55.
[13]Fehlbaum P,Bullet P,et al.Structure-activity analysis of thanatin,a21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptide[J].Proc Natl Acad sci USA,1996,93(3):1221-1225.
[14]王军,庞广昌。抗菌肽抗菌机理的研究现状及趋势[J].食品科学,2005,26(8):526-529.
[15]Daher KA,Seksted ME,Lehrer RI,et al.Direct inactivation of viruses by human granulocyte denfensins[J].J Virol,1988:1068-1074.
[16]陈继南,陈红霞.抗菌肽及其应用研究进展[J].生物技术通讯,2006,17(4):669-672.
[17]Toney JH.Iseganan(IntraBiotics pharmaceuticals)[J].Curr Opin Investing Drugs,2002,3(2):225
[18]Wachinger M,Kleinschmidtet A,Winder D,et al.Antimicrobial Peptides Melittin and Cecropin Inhibit Relication of Human Immunodeficiency Virus by Suppressing Viral Gene Expression[J].J.General Virology,1998,79(4):731-740.
[19]Yarus S,Rosen JM,Cole AM,et al.Production of active bovine tracheal antimicrobial peptide in milk of transgenic mice.Proc Natl Acad Sci,USA,1996,93:14118-14121.
[20]Jaynes JM,Nagpala P,Destefano Beltrant L.Expression of a Cecropin B lyric peptide analog in transgenic tobacco confers enhanced resistance to bacterial with caused by Pseudomonas solanacearum.Plant Sci.,1993,89:43-53.
[21]崔艳红,黄现青,抗菌肽的抗菌机理及其应用[J].中国兽医杂志.2006,42(9):51-52
[22]齐珂珂,固始鸡、丝毛乌鸡和鸵鸟中性粒细胞中抗菌肽的分离纯化和部分性质研究[M].河南农业大学硕士学位论文,2006,27-31
[23]温刘发,何丹林,张常明等.抗菌肽酵母制剂的生产及其作饲料添加剂应用价值的探讨.广东蚕业,2001,35(2):34-36.
[24]温刘发,何丹林,张常明等.抗菌肽酵母制剂作为饲料添加剂的应用前景.中国饲料,2001,23:22-23.
[25]Harwig S S,Swiderek K M,Kokryakov V N,et al.Gallinacins:cysteine-rich antimicrobial peptides of chicken leukocytes[J].FEBS Lett,1994,342(3):281-285
[26]Evans E W,Beach G G,Wunderlich J,et al.Isolation of antimicrobial peptides from avian heterophils[J]J.Leukoc.Biol,1994,56(5):661-665
[27]Evans E W,Beach F G,Moore K M,et al.Antimicrobialactivity of chicken and turkey heterophil peptides CHP1,CHP2,THP1,and THP3[J].Vet,Microbiol,1995,47(3-4):295-303.
[28]Lynn DJ et al,Immunogenetics,2004,56(3):170-177.
[29]Xiao Y,et al.A genome-wide screen identifies a single β-defensin gene cluster in the chicken:implications for the origin and evolution of mammalian defensins[J].BMC Genomics,2004,5:56.
[30]Ganz T.,Selsted M E.,Lehrer R I.Defensins.Eur.J.Haematol,1991,44:1-8.
[31]Gill Diomand,Michael Zmloff,Howard ECK,et al.Tracheal antimicrobial peptides,a cysteine-rich peptides from mammalian tracheal mucosa.Proc.Natl.Acad.Sci.USA,1991,88:3952-3956.
[32]Wu Z B,Hoover D M,Yang D,et al.Engineering disulfide bridges to dissect antimicrobial and chemotactie activities of human β-defensin 3[J].PNAS,2003, 100:8880-8885.
[33]Ganu T,Cowland J B.Liu L,et al.Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes,a consequence of common growth factors[J].Immunol,2003,170:5583-5589.
[34]Tang Y Q,Yann J,Osapay G,et al.A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins[J].Science,1999,286:498-502.
[35]余兴邦,郭锁链等.防御素研究进展[J].动物医学进展,2006,27(8):47-51
[36]G.Zasloff,Antimicrobial peptides of multicellular organisms,Molecular cloning and tissue expression of porcine β-defensin-1[J].FEBS Lett.1998,424:37-40.
[37]Harwig S S,et al.Primary structure of Gallinacin-1,an antimicrobial β-Defensins from chicken leukocytes[J].Techniques in Protein Chemistry,1994,(1):81-88.
[38]P.L.Yu,S.D.Choudhury,K.Ahrens,Purification and characterization of antimicrobial peptides[J],ostricain,Biotechnol.Lett.2001,23:207-210.
[39]Zhao C,Nguyen T,Liu L,et al.Gallinacin-3,an inducible epthelial beta-defensin in the chicken[J].Infect Immun,2001,69(4):2684-2691.
[40]C.Thouzeau,Y,et al.avian beta-defensin in preserved stomach contents of the king penguin,Aptenodytes patagonicus,J.Biol.Chem.2003,278:51053-510058.
[41]吴金梅等,禽类抗菌肽的最新研究进展[J].家禽科学,2005.5:43-44.
[42]陈燕珊.禽类的重要免疫因子-鸡β-防御素[J].生命的化学,2005,25(6):502-504.
[43]Jotwani R,Muthukuru M,Cutler.C.W.Increase in HIV receptors / co-receptors /alpha-defensins in inflamed human gingiva[J].J Dent Res,2004,83(5):371-377
[44]唐博,骆驼β-防御素caBD-1 cDNA的克隆及序列分析[M],内蒙古农业大学硕士学位文.2004,12-15
[45]Yoshioka D.Laks H.Gans T,et al.lncreased bronchoalveolar lavage human beta -defensin type 2 in bronchiolitis obliterans syndrome after lung transplantation[J].Transplantation,2004,78(8):1222-1224.
[46]Aley S,Zimmerman M,Hetsko M et al.1994.Killing of Giardia lumblia by cryptdins and cationic neutrophil peptides[J].Infect.Immun.62:5397-5403
[47]Lehrer R 1,Ganz T,Szlarek E et al.1988.Modulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations[J].J C1ini.Investi..81:1829-1835
[48]Yang D,Biragyn A,Kwak L W et al.2002.Mammalian defensins in immunity:more than just microbicidal[J].Tren.Immuno.23:291-296
[49]Sun LL,Finnegan CM,Tina K C,et al.Defensins suppress human immunodeficiency virus infection;potential role in mucosal protection[J].Journal of Virology,2005,79:14318-14329.
[50]Papo N,Y Shai.Host defense peptides as new weapons in Cancer treatment[J].Cell Mol Life Sci,2005,62(7-8):784-790
[51]Mosey C,Weiner DJ,Lysenko E,et al.β-defensin 1 contributes to pulmonary innate immunity in mice.[J].Infect Immun.,2002,70:3068-3072.
[52]B.G.Harmon,Avain heterophils in inflammation and disease resistance[J],Poult.Sci.1998,77:972-977.
[53]Epand R M.Vogel H J.Diversity of antlmicrobial peptides and their mechanisms of action[J].Biochimica et Biophysica Acts,1999,1462:11-28.
[54]Biragyn A.Defensins-non-antibiotic use for vaccine development[J].Curt Protein Pept Sci,2005,6(1):53-60
[55]Bajaj-ElliottM,Fedeli P,Smith G V,etal.Modulation of host antimicrobial peptide (beta-defensins 1 and 2)expression during gastritis[J].Gut,2002,51(3):356-361.
[56]Ganu T,Cowland J B.Liu L,et al.Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes,a consequence of common growth factors[J].Immunol,2003,170:5583-5589.
[57]Ganz T.Dcfensins:antimicrobial peptides of innate immunity[J].Nat Rev Immunol,2003,3:710-720
[58]Lehrer RI,Ganz T.Defensins of vertebrate animals[J].Curr Opin Immunol,2002,14:96-102
[59]Schutte BC.McCray P.B.Jr.Beta-defensins in lung host defense[J].Annu Rev Physiol,2002,64:709-748
[60]Sugiarto H.Yu P L.Avian antimicrobial peptides:the defense role of β-defensins [J].Biochemical and Biophysical Research Communications,2004.323:721-727
[61]Lehrer R I.Ganz T.Defensins of vertebrate animals[J].Curr Opin Immunol,2002,14:96-102.
[62]刘进,奚涛.人β-防御索-3的研究进展[J].药物生物技术,2005.12(5)I 337-341.
[63]Yount N Y,et al.Cloning and expression of bovine neutrophil β-defensins[J]. The Journal of Biological Chemistry,1999,274:26249-26258
[64]Maxwell A I,Morrison G M and Dorin J R.Rapid sequence divergence in mammalian β-defensins by adaptive evolution[J].Molecular Immunology,2003,40:413-421
[65]Zhang G,et al.Cloning and characterization of the gene for a new epithelial beta-defensin.Genomic structure,chromosomal localization,and evidence for its constitutive expression[J].The Journal of Biological Chemistry,1999,274:24031-24037
[66]Higgs R et al.Immunogenetics,2005,57(1-2):90-98
[67]Brockus CW et al.Anita Genet,1998,29(4):283-289
[68]Robert E W,Hancock R E W and Diamond G.The role of cationic antimicrobial peptides in innate host defences[J].Trends in Microbiology,2000,8:402-410
[69]Morrison G M,Davidson D J,et al.Mouse beta-defensin-1 is a functional homolog of human beta- defensin-1[J].Mammalian Genome,1998,9:453-457
[70]Becker M N,Diamond G,Verghese M W and Randell S H.CD14 dependent LPS-induced β-defensin-2 expression in human tracheobronchial epithelium[J].The Journal of Biological Chemistry,2000,275:29731-29736
[71]Diamond G,Kaiser V,Rhodes J,Russell J P and Bevins C L.Transcriptional regulation of β-defensin gene expression in tracheal epithelial cells[J].Infection and Immunity,2000,68:113-119
[72]O' Neil D A,Porter E M,Elewaut D,Anderson G M,EckmannL,Ganz T and Kagnoff M F.Expression and regulation of the human beta-defensin hBD-1 and hBD-2 in intestinal epithelium[J].The Journal of Immunology,1999,163:6718-6724
[73]Diamond G and Bevins C L.β-defensin:Endogenous antibiotics in the innate host defense response[J].Clinical Immunology and Immunopathology,1998,88:221-225
[74]张辉华等.鸡β-防御素-3的克隆与诱导表达[J].中国预防兽医学报,2006,26(3):401-404
[75]Yang D,Biragyn A,Kwak L W,et al.Mammalian defensins in immunity:more than lust microbicidal[J].Trends immuno,2002,23(6):291-296.
[76]Ypunt N Y,Yuan J,Talver A,et al.Cloning and Expression of bovine neutrophil β-defensins[J].J Biolo Chemi,1999,274(37):26249-26258.
[77]Ganz T,Rayner J R,Valore E V,et al.The structure of the rabbit macrophage defensin genes and their organ-specific expression[J].J.Immunol,1989,143(4):1358-1365.
[78]Boman H G,Agerberth 8,Boman A.Mechanisms of action on Escherichia coli of cecropin P1 and PR-39,two antibacterial peptides from pig intestine[J].Infect Immun,1993,61(7):2978-2984
[79]张辉华,鸡β-防御素基因的克隆及在毕赤酵母中的表达[M].华南农业大学博士学位论文,2004.
[80]邢海云,鸡β-防御素在大肠杆菌中的高效表达及复性工艺研究[M].山东农业大学硕士学位论文.2005.
[81]赵建增等,重组Gallinacin-3的克隆及在大肠杆菌中的表达[J].中国兽药杂志2006,40(2):1-4,34.
[82]吴静,李玉峰等,鸡β-防御素Gal-1cDNA的克隆及序列分析[J].家禽科学,2006,12:11-14
[83]李春丽.人β防御素3和植物甜蛋白des-pGlul-Brazzein基因的重组表达研究[J].浙江大学博士学位文.2005.61-73.
[84]方敏,黄华梁.包涵体蛋白体外复性的研究进展[J].生物工程报.2001,17(6):608-612.
[85]Lehrer R I,Rosenman M,Harwig SSL,et al.Ulteasensitive assay for endogenous antimicrobial peptides.J Immunol Meth,1991,137:167.
[86]陈姗,何凤田,董燕麟,等.人β-防御素3融合蛋白在大肠杆菌中的表达、纯化与活性分析[J].生物工程学报,2004,20(4):490-495.
[87]张辉华等,鸡β-防御素-1基因(Gal-1)在大肠杆菌中的融合表达与纯化[J].中国兽医杂志2006,42(9):9-12.
[88]刘伯华等.原核表达的猪瘟病毒E2蛋白抗原多肽的复性和纯化[J].中国兽医学报.2003,3(23):145-148.
[89]纪剑飞等.包涵体重组蛋白的纯化和复性[J].沈阳药科大学学报.1998,10(15):303-307.
[90]冯岚等.大肠杆菌表达人重组IL-3的纯化[J].生物化学杂志.1993,9(3):135-139.
[91]高永贵等.包涵体蛋白的变性复性研究[J].科技通讯.2003,1(19):10-15.
[92]李会成等.大肠杆菌表达的真核蛋白产物的复性[J].基因工程进展.1994,14(5):36-39.
[2]HOFFMANN J A,KAFATOS F C,JANEWAY C A,et al.Phylogenetic perspectives in innate immunity[J].Science,1999,284:1313-1318.
[3]ZAMBON R A,NANDAKUMAR M,VAKHARIA V N,et at.The toll pathway is important for an antiviral response in Drosophila[J].Proc Natl Acad Sci USA,2005,102(20):7257-7262.
[4]REDDY K V R,YEDERY R D,ARANHA C.Antimicrobial peptides:premises and promises[J].Int J Antimicrob Agents,2004,24(6):536-547.
[5]梁永利,天然抗菌肽的来源及分类[J],安徽农业科学,2006,34(38):4728-4734
[6]BULET P,HETRU C,DIMARCQ J,et al.Antimicrobial peptides from insects:structure and function[J].Dev Comp Immunol,1999,23:329-344.
[7]Bellm L,Lehrer RI Ganz T.Protegrins:new antibiotics of mammalian origin.Expert Opin Investig Drugs[J],2000,9(8):1731-1742.
[8]Hancock RE.Cationic antimicrobial peptides:towards clinical applications.Expert Opinlnvestig Drugs[J],2000,9(8):1723-1729.
[9]朱颐申,王卫国等.海洋抗菌肽研究进展[J].高技术通讯,2004(2):105-110.
[10]Robert E W Hancock.Cationic peptides:effectors in innate immunity and novel antimicrobials[J].The Lancet infectious Disease,2001(1):156-164.
[11]马卫明.猪小肠抗菌肽分离鉴定及其活性研究[D].中国农业大学博士学位论文,2004:10-11.
[12]MichaelR,et al.Mechanism of antimicrobial peptide Action and resistance[J].Pharmacological Review,2003,55(1):27-55.
[13]Fehlbaum P,Bullet P,et al.Structure-activity analysis of thanatin,a21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptide[J].Proc Natl Acad sci USA,1996,93(3):1221-1225.
[14]王军,庞广昌。抗菌肽抗菌机理的研究现状及趋势[J].食品科学,2005,26(8):526-529.
[15]Daher KA,Seksted ME,Lehrer RI,et al.Direct inactivation of viruses by human granulocyte denfensins[J].J Virol,1988:1068-1074.
[16]陈继南,陈红霞.抗菌肽及其应用研究进展[J].生物技术通讯,2006,17(4):669-672.
[17]Toney JH.Iseganan(IntraBiotics pharmaceuticals)[J].Curr Opin Investing Drugs,2002,3(2):225
[18]Wachinger M,Kleinschmidtet A,Winder D,et al.Antimicrobial Peptides Melittin and Cecropin Inhibit Relication of Human Immunodeficiency Virus by Suppressing Viral Gene Expression[J].J.General Virology,1998,79(4):731-740.
[19]Yarus S,Rosen JM,Cole AM,et al.Production of active bovine tracheal antimicrobial peptide in milk of transgenic mice.Proc Natl Acad Sci,USA,1996,93:14118-14121.
[20]Jaynes JM,Nagpala P,Destefano Beltrant L.Expression of a Cecropin B lyric peptide analog in transgenic tobacco confers enhanced resistance to bacterial with caused by Pseudomonas solanacearum.Plant Sci.,1993,89:43-53.
[21]崔艳红,黄现青,抗菌肽的抗菌机理及其应用[J].中国兽医杂志.2006,42(9):51-52
[22]齐珂珂,固始鸡、丝毛乌鸡和鸵鸟中性粒细胞中抗菌肽的分离纯化和部分性质研究[M].河南农业大学硕士学位论文,2006,27-31
[23]温刘发,何丹林,张常明等.抗菌肽酵母制剂的生产及其作饲料添加剂应用价值的探讨.广东蚕业,2001,35(2):34-36.
[24]温刘发,何丹林,张常明等.抗菌肽酵母制剂作为饲料添加剂的应用前景.中国饲料,2001,23:22-23.
[25]Harwig S S,Swiderek K M,Kokryakov V N,et al.Gallinacins:cysteine-rich antimicrobial peptides of chicken leukocytes[J].FEBS Lett,1994,342(3):281-285
[26]Evans E W,Beach G G,Wunderlich J,et al.Isolation of antimicrobial peptides from avian heterophils[J]J.Leukoc.Biol,1994,56(5):661-665
[27]Evans E W,Beach F G,Moore K M,et al.Antimicrobialactivity of chicken and turkey heterophil peptides CHP1,CHP2,THP1,and THP3[J].Vet,Microbiol,1995,47(3-4):295-303.
[28]Lynn DJ et al,Immunogenetics,2004,56(3):170-177.
[29]Xiao Y,et al.A genome-wide screen identifies a single β-defensin gene cluster in the chicken:implications for the origin and evolution of mammalian defensins[J].BMC Genomics,2004,5:56.
[30]Ganz T.,Selsted M E.,Lehrer R I.Defensins.Eur.J.Haematol,1991,44:1-8.
[31]Gill Diomand,Michael Zmloff,Howard ECK,et al.Tracheal antimicrobial peptides,a cysteine-rich peptides from mammalian tracheal mucosa.Proc.Natl.Acad.Sci.USA,1991,88:3952-3956.
[32]Wu Z B,Hoover D M,Yang D,et al.Engineering disulfide bridges to dissect antimicrobial and chemotactie activities of human β-defensin 3[J].PNAS,2003, 100:8880-8885.
[33]Ganu T,Cowland J B.Liu L,et al.Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes,a consequence of common growth factors[J].Immunol,2003,170:5583-5589.
[34]Tang Y Q,Yann J,Osapay G,et al.A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins[J].Science,1999,286:498-502.
[35]余兴邦,郭锁链等.防御素研究进展[J].动物医学进展,2006,27(8):47-51
[36]G.Zasloff,Antimicrobial peptides of multicellular organisms,Molecular cloning and tissue expression of porcine β-defensin-1[J].FEBS Lett.1998,424:37-40.
[37]Harwig S S,et al.Primary structure of Gallinacin-1,an antimicrobial β-Defensins from chicken leukocytes[J].Techniques in Protein Chemistry,1994,(1):81-88.
[38]P.L.Yu,S.D.Choudhury,K.Ahrens,Purification and characterization of antimicrobial peptides[J],ostricain,Biotechnol.Lett.2001,23:207-210.
[39]Zhao C,Nguyen T,Liu L,et al.Gallinacin-3,an inducible epthelial beta-defensin in the chicken[J].Infect Immun,2001,69(4):2684-2691.
[40]C.Thouzeau,Y,et al.avian beta-defensin in preserved stomach contents of the king penguin,Aptenodytes patagonicus,J.Biol.Chem.2003,278:51053-510058.
[41]吴金梅等,禽类抗菌肽的最新研究进展[J].家禽科学,2005.5:43-44.
[42]陈燕珊.禽类的重要免疫因子-鸡β-防御素[J].生命的化学,2005,25(6):502-504.
[43]Jotwani R,Muthukuru M,Cutler.C.W.Increase in HIV receptors / co-receptors /alpha-defensins in inflamed human gingiva[J].J Dent Res,2004,83(5):371-377
[44]唐博,骆驼β-防御素caBD-1 cDNA的克隆及序列分析[M],内蒙古农业大学硕士学位文.2004,12-15
[45]Yoshioka D.Laks H.Gans T,et al.lncreased bronchoalveolar lavage human beta -defensin type 2 in bronchiolitis obliterans syndrome after lung transplantation[J].Transplantation,2004,78(8):1222-1224.
[46]Aley S,Zimmerman M,Hetsko M et al.1994.Killing of Giardia lumblia by cryptdins and cationic neutrophil peptides[J].Infect.Immun.62:5397-5403
[47]Lehrer R 1,Ganz T,Szlarek E et al.1988.Modulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations[J].J C1ini.Investi..81:1829-1835
[48]Yang D,Biragyn A,Kwak L W et al.2002.Mammalian defensins in immunity:more than just microbicidal[J].Tren.Immuno.23:291-296
[49]Sun LL,Finnegan CM,Tina K C,et al.Defensins suppress human immunodeficiency virus infection;potential role in mucosal protection[J].Journal of Virology,2005,79:14318-14329.
[50]Papo N,Y Shai.Host defense peptides as new weapons in Cancer treatment[J].Cell Mol Life Sci,2005,62(7-8):784-790
[51]Mosey C,Weiner DJ,Lysenko E,et al.β-defensin 1 contributes to pulmonary innate immunity in mice.[J].Infect Immun.,2002,70:3068-3072.
[52]B.G.Harmon,Avain heterophils in inflammation and disease resistance[J],Poult.Sci.1998,77:972-977.
[53]Epand R M.Vogel H J.Diversity of antlmicrobial peptides and their mechanisms of action[J].Biochimica et Biophysica Acts,1999,1462:11-28.
[54]Biragyn A.Defensins-non-antibiotic use for vaccine development[J].Curt Protein Pept Sci,2005,6(1):53-60
[55]Bajaj-ElliottM,Fedeli P,Smith G V,etal.Modulation of host antimicrobial peptide (beta-defensins 1 and 2)expression during gastritis[J].Gut,2002,51(3):356-361.
[56]Ganu T,Cowland J B.Liu L,et al.Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes,a consequence of common growth factors[J].Immunol,2003,170:5583-5589.
[57]Ganz T.Dcfensins:antimicrobial peptides of innate immunity[J].Nat Rev Immunol,2003,3:710-720
[58]Lehrer RI,Ganz T.Defensins of vertebrate animals[J].Curr Opin Immunol,2002,14:96-102
[59]Schutte BC.McCray P.B.Jr.Beta-defensins in lung host defense[J].Annu Rev Physiol,2002,64:709-748
[60]Sugiarto H.Yu P L.Avian antimicrobial peptides:the defense role of β-defensins [J].Biochemical and Biophysical Research Communications,2004.323:721-727
[61]Lehrer R I.Ganz T.Defensins of vertebrate animals[J].Curr Opin Immunol,2002,14:96-102.
[62]刘进,奚涛.人β-防御索-3的研究进展[J].药物生物技术,2005.12(5)I 337-341.
[63]Yount N Y,et al.Cloning and expression of bovine neutrophil β-defensins[J]. The Journal of Biological Chemistry,1999,274:26249-26258
[64]Maxwell A I,Morrison G M and Dorin J R.Rapid sequence divergence in mammalian β-defensins by adaptive evolution[J].Molecular Immunology,2003,40:413-421
[65]Zhang G,et al.Cloning and characterization of the gene for a new epithelial beta-defensin.Genomic structure,chromosomal localization,and evidence for its constitutive expression[J].The Journal of Biological Chemistry,1999,274:24031-24037
[66]Higgs R et al.Immunogenetics,2005,57(1-2):90-98
[67]Brockus CW et al.Anita Genet,1998,29(4):283-289
[68]Robert E W,Hancock R E W and Diamond G.The role of cationic antimicrobial peptides in innate host defences[J].Trends in Microbiology,2000,8:402-410
[69]Morrison G M,Davidson D J,et al.Mouse beta-defensin-1 is a functional homolog of human beta- defensin-1[J].Mammalian Genome,1998,9:453-457
[70]Becker M N,Diamond G,Verghese M W and Randell S H.CD14 dependent LPS-induced β-defensin-2 expression in human tracheobronchial epithelium[J].The Journal of Biological Chemistry,2000,275:29731-29736
[71]Diamond G,Kaiser V,Rhodes J,Russell J P and Bevins C L.Transcriptional regulation of β-defensin gene expression in tracheal epithelial cells[J].Infection and Immunity,2000,68:113-119
[72]O' Neil D A,Porter E M,Elewaut D,Anderson G M,EckmannL,Ganz T and Kagnoff M F.Expression and regulation of the human beta-defensin hBD-1 and hBD-2 in intestinal epithelium[J].The Journal of Immunology,1999,163:6718-6724
[73]Diamond G and Bevins C L.β-defensin:Endogenous antibiotics in the innate host defense response[J].Clinical Immunology and Immunopathology,1998,88:221-225
[74]张辉华等.鸡β-防御素-3的克隆与诱导表达[J].中国预防兽医学报,2006,26(3):401-404
[75]Yang D,Biragyn A,Kwak L W,et al.Mammalian defensins in immunity:more than lust microbicidal[J].Trends immuno,2002,23(6):291-296.
[76]Ypunt N Y,Yuan J,Talver A,et al.Cloning and Expression of bovine neutrophil β-defensins[J].J Biolo Chemi,1999,274(37):26249-26258.
[77]Ganz T,Rayner J R,Valore E V,et al.The structure of the rabbit macrophage defensin genes and their organ-specific expression[J].J.Immunol,1989,143(4):1358-1365.
[78]Boman H G,Agerberth 8,Boman A.Mechanisms of action on Escherichia coli of cecropin P1 and PR-39,two antibacterial peptides from pig intestine[J].Infect Immun,1993,61(7):2978-2984
[79]张辉华,鸡β-防御素基因的克隆及在毕赤酵母中的表达[M].华南农业大学博士学位论文,2004.
[80]邢海云,鸡β-防御素在大肠杆菌中的高效表达及复性工艺研究[M].山东农业大学硕士学位论文.2005.
[81]赵建增等,重组Gallinacin-3的克隆及在大肠杆菌中的表达[J].中国兽药杂志2006,40(2):1-4,34.
[82]吴静,李玉峰等,鸡β-防御素Gal-1cDNA的克隆及序列分析[J].家禽科学,2006,12:11-14
[83]李春丽.人β防御素3和植物甜蛋白des-pGlul-Brazzein基因的重组表达研究[J].浙江大学博士学位文.2005.61-73.
[84]方敏,黄华梁.包涵体蛋白体外复性的研究进展[J].生物工程报.2001,17(6):608-612.
[85]Lehrer R I,Rosenman M,Harwig SSL,et al.Ulteasensitive assay for endogenous antimicrobial peptides.J Immunol Meth,1991,137:167.
[86]陈姗,何凤田,董燕麟,等.人β-防御素3融合蛋白在大肠杆菌中的表达、纯化与活性分析[J].生物工程学报,2004,20(4):490-495.
[87]张辉华等,鸡β-防御素-1基因(Gal-1)在大肠杆菌中的融合表达与纯化[J].中国兽医杂志2006,42(9):9-12.
[88]刘伯华等.原核表达的猪瘟病毒E2蛋白抗原多肽的复性和纯化[J].中国兽医学报.2003,3(23):145-148.
[89]纪剑飞等.包涵体重组蛋白的纯化和复性[J].沈阳药科大学学报.1998,10(15):303-307.
[90]冯岚等.大肠杆菌表达人重组IL-3的纯化[J].生物化学杂志.1993,9(3):135-139.
[91]高永贵等.包涵体蛋白的变性复性研究[J].科技通讯.2003,1(19):10-15.
[92]李会成等.大肠杆菌表达的真核蛋白产物的复性[J].基因工程进展.1994,14(5):36-39.