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研究生:蔡佳蓉
研究生(外文):Chia-Jung Tsai
論文名稱:埃及斑蚊抗菌蛋白(Attacin)之基因表現及功能性分析
論文名稱(外文):Gene Characterization and Functional Analysis of an Antimicrobial Peptide-Attacin from Aedes aegypti
指導教授:卓文隆
指導教授(外文):Wen-Long Cho
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:臨床醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:103
中文關鍵詞:抗菌蛋白埃及斑蚊
外文關鍵詞:Aedes aegyptiAttacinAntimicrobial peptide
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本研究中已成功選殖出埃及斑蚊的Attacin全長cDNA,其大小為798 bp,可推演出265個胺基酸的完整轉譯區 (open reading frame) 。在埃及斑蚊體內 (in vivo) 和埃及斑蚊細胞株Aag2 (in vitro) 的實驗中,AaeAttacin mRNA可以被革蘭氏陽性菌- Staphylococcus aureus (S. aureus)和革蘭氏陰性菌- Escherichia coli (E. coli /LE392) 刺激而誘發表現,但不會被黴菌- Candida albicans (C. albicans) 所誘發;利用細菌細胞壁單一成分進行刺激,發現AaeAttacin mRNA可以被革蘭氏陽性菌中的台口酸 (lipoteichoic acid , LTA)和革蘭氏陰性菌中的脂多醣(lipopolysaccharide , LPS)所誘發表現,但對黴菌中的胜肽多醣(peptidoglycan , PGN)的刺激則沒有反應。
此外,利用大腸桿菌表現系統大量表現及純化AaeAttacin融合蛋白質,並成功地製備出抗AaeAttacin的多株抗體。利用西方墨點法可以在蚊子及細胞受E. coli刺激後,於蚊子血液及細胞培養液中偵測到AaeAttacin的蛋白存在,顯示AaeAttacin是一種分泌性蛋白質。利用AaeAttacin重組蛋白質已證明可以抑制E .coli的生長,但對S. aureus的生長則無抑制作用。
為了瞭解AaeAttacin基因調控的機制, AaeAttacin基因的上游調控因子區域被選殖連結到紅色螢光 (DsRed) 或冷光 (Luciferase) 報導基因的前面,構築成一個調控報導系統,經轉染至C6/36細胞株中進行基因調控因子的活性分析,實驗結果發現AaeAttacin基因調控因子可受到細菌刺激而誘發報導基因的表現;經過序列刪除分析後,發現此基因調控因子上的STAT和Dorsal 轉錄因子結合位可能對調控AaeAttacin的表現是重要的。另外,利用雙股RNA干擾法,發現Relish-2可能是調控AaeAttacin基因表現的主要途徑。本研究的結果提供埃及斑蚊Attacin 在 mRNA、蛋白質表現層級以及基因調控方面更深入的了解。
In this study, the expression, regulation and antibacterial function of an Aedes aegypti Attacin
(AaeAttacin) were investigated intensively.
The full length cDNA of AaeAttacin was cloned and sequenced. It is 798 bp bearing an open reading frame of 265 amino acids. The transcript of Aa Attacin was induced by Gram positive bacteria Staphylococcus aureus and Gram negative bacteria Escherichia coli (LE 392), but not by fungi in vivo and in vitro. The mRNA of AaeAttacin was also induced by bacterial cell wall components of Gram positive lipoteichoic acid (LTA) and Gram negative lipopolysaccharide (LPS), but not by peptidoglycan (PGN) of fungi in vivo and in vitro.
Polyclonal anti-AaeAttacin antibodies were produced by purified recombinant protein from E. coli expression system. AaeAttacin protein was detected in mosquito henolymphe and cell medium after bacterial challenge useing western blot analysis. These results indicate that AaeAttacin is a secreted protein. For the antibacterial assay, the purified recombination protein of AaeAttacin was proved to suppress the growth of E. coli, but not to S. aureus.
In regulation study of AaeAttacin gene, the regulation region (1.7 kb) containing promoters of AaeAttacin gene was cloned and sequenced. The AaeAttacin regulation region was ligated with florescent reporter genes, DeRed and luciferase, and then was transfected into a C6/36 cell line for expression activity assay. The results displayed inducible properties after bacterial stimulations. Serial deletions of the AaeAttacin regulation region were conducted for reporter assay. Two immune related elements, STAT and Dorsal, were demonstrated to be important for the regulation of AaeAttacin gene. Double stranded RNA interference (dsRNAi) results showed that AaeAttacin gene was regulated mainly by the A. aegypti Relish-2 (AaeRelish-2) immune pathway. Our study provides better understanding of A. aegypti Attacin at gene expression and regulation levels.
目錄
頁次
致謝……………………………………………………… i
縮寫表…………………………………………………… ii
中文摘要………………………………………………… iii
英文摘要………………………………………………… v
目錄……………………………………………………… vii
壹、緒論………………………………………………… 1
貳、材料與方法………………………………………… 14
參、結果………………………………………………… 29
肆、討論………………………………………………… 42
伍、參考文獻…………………………………………… 50
陸、圖…………………………………………………… 58
柒、附錄………………………………………………… 95
Agaisse, H.,Petersen, U. M.,Boutros, M.,Mathey-Prevot, B., and Perrimon, N. (2003). Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury. Dev Cell 5, 441-450.
Andre’s, E., Dimarcq , J.L. (2001). Les peptides antimicrobiens cationiqueset leurs applications otentielles. Antibiotiques 3,201-205.
Arbouzova, N. I., and Zeidler, M. P. (2006). JAK/STAT signalling in Drosophila: insights into conserved regulatory and cellular functions. Development 133, 2605-2616.
Bevins, C. L. and Zasloff, M. (1990). Peptides from frog skin. Annu Rev Biochem 59, 395-414.
Boman, H.G., Faye, I., Gudmundsson, G.H., Lee, J.Y., and Lidholm, D.A., (1991). Cell-free immunity in cecropia. A model system for antibacterial proteins. Eur J Biochem 201, 849–854.
Boman, H.G.,and Steiner, H. (1981). Humoral immunity in Cecropia pupae. Curr Top Microbiol Immunol 94-95,75-91. Review.
Boman, H. G., and Hultmark, D. (1987). Cell-free immunity in insects. Annu Rev Microbiol 41, 103-126.
Carlsson, A., Engström, P.,Palva, E.T.,and Bennich,H.,(1991). Attacin, anantibacterial protein from Hyalophora cecropia, inhibits synthesis of outer membrane proteins in Escherichia coli by interfering with omp gene transcription. Infect Immun 59, 1040–1045.
Chen,C.C., and Chen, C. S. (1995). Brugia pahangi: effects of melanization on the uptake of nutrients by microfilariae in vitro. Exp Parasitol 81, 72-78.
Chen, H. W., Chen, X., Oh, S. W., Marinissen, M. J., Gutkind, J. S., and Hou, S. X. (2002). Mom identifies a receptor for the Drosophila JAK/STAT signal transduction pathway and encodes a protein distantly related to the mammalian cytokine receptor family. Genes Dev 16, 388-398.
Chemysh, S.,Kim, S.I.,and Bekker, G.(2002).Ativiral and antitumor peptides from insects. Roc Natl Acad Sci USA 2, 628- 632.
Cole, A. M., Weis, P., and Diamond, G. (1997). Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter flounder. J Biol Chem 272, 12008-12013.
Destoumieux, D., Bulet, P., Loew, D., Van Dorsselaer, A., Rodriguez, J., and Bachere,E. (1997). Penaeidins, a new family of antimicrobial peptides isolated from the shrimp Penaeus vannamei (Decapoda). J Biol Chem 272, 28398-28406.
Dostert, C., Jouanguy, E., Irving, P., Troxler, L., Galiana-Arnoux, D., Hetru, C., Hoffmann,J.A., and Imler,J.L.(2005). The Jak-STAT signaling pathway is required but not sufficient for the antiviral response of Drosophila. Nat Immunol 6, 946-953.
Engström Y.(1999).Induction and regulation of antimicrobial peptides in Drosophila.Dev Comp Immunol 23,345-58. Review.
Engström, P., Arlsson, A., Engström, Å., Tao, Z.J., and Bennich, H., (1984). The antibacterial effect of attacins from the silk moth Hyalophora cecropia is directed against the outer membrane of Escherichia coli. EMBO J 3, 3347–3351.
Flyg, C., Dalhammar ,G., Rasmuson ,B. and Boman, H. G. (1987).Insect immunity: inducible antibacterial activity in Drosophila. Insect Biochem 17, 153-160.
Gilbert, M. M., Weaver, B. K., Gergen, J. P., and Reich, N. C. (2005). A novel functional activator of the Drosophila JAK/STAT pathway, unpaired2, is revealed by an in vivo reporter of pathway activation. Mech Dev 122, 939-948.
Gottar,M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, J.A.,Ferrandon, D., and Royet, J.(2002). The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein.Nature 416,640-4.
Giacometti, A., Cirioni, O., Del Prete, M. S., Barchiesi, F., Paggi, A. M., Petrelli, E., and Scalise, G. (2000). Comparative activities of polycationic peptides and clinically used antimicrobial agents against multidrug-resistant nosocomial isolates of Acinetobacter baumannii. J Antimicrob Chemother 46, 807-810.
Ganz, T., Selsted, M.E., Szklarek, D., Harwig, S.S., Daher, K., Bainton, D.F., and Lehrer, R.I.(1985). Defensins. Natural peptide antibiotics of human neutrophils. J Clin Invest 4,1427-35.
Hancock, R.E., and Scott, M.G.(2000). The role of antimicrobial peptides in animal defenses. Proc Natl Acad Sci USA 97,8856-61. Review.
Huang ,H.W.(2000). Action of antimicrobial peptides: two-state model. Biochemistry 39,8347-52.
Huang, S.J., Wu, R.C., Shaio, M.F., Wang, P.S. and Cho, W.L. (2003). An immune signalling kinase AaMEK3 from mosquitoes: c DNA cloning and characterization. Insect Mol Biol ,12, 595–603.
Hultmark, D.,(1993). Immune reaction in Drosophila and other insects: a model for innate immunity. Trends Genet 9, 178–183.
Hultmark, D., Engstrom ,A., Anderson ,K., Steiner ,H., Bennich ,H. and Boman, H. G. (1983). Insect immunity. Attacins, a family of antibacterial proteins from Hyalophora cecropia. EMBO J 2, 571-576.
Hultmark, D., Steiner ,H., Rasmuson ,T. and Boman ,H. G. (1980). Insect immunity,purification and properties of three inducible bactericidal proteins fromhemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem 106,7-16.
Ihler, C. F., Rootwelt, V., Heyeraas, A., and Dolvik, N. J. (1995). The prevalence and epidemiology of Anoplocephala perfoliata infection in Norway. Vet Res Commun 19, 487-494.
Kadalayil, L., Petersen, U.M., and Engström, Y.(1997). Adjacent GATA and kappa B-like motifs regulate the expression of a Drosophila immune gene. Nucleic Acids Res 25,1233-9.
Kaneko, T., Golenbock, D., and Silverman, N.(2005). Peptidoglycan recognition by the Drosophila Imd pathway. J Endotoxin Res 11,383-9.
Kaneko, T., Yano, T,, Aggarwal, K., Lim. J.H., Ueda, K., Oshima, Y., Peach, C., Erturk-Hasdemir,D., Goldman, W.E., Oh, B.H., Kurata, S., and Silverman, N.(2006). PGRP-LC and PGRP-LE have essential yet distinct functions in the Drosophila immune response to monomeric DAP-type peptidoglycan. Nat Immunol 7,715-23.
Kim T, and Kim YJ. (2005).Overview of innate immunity in Drosophila. J Biochem Mol Biol 2,121-7. Review.
Kockum, K., Faye,I., von Hofsten,P., Lee, J.-Y., Xanthopoulos, K. G. and Boman ,H. G. (1984). Insect immunity, isolation and sequence of two cDNA clones corresponding to acidic and basic attacins from Hyalophora cecropia. EMBO J 3, 2071-2075.
Kwon , Y.M. Kim,H.J. Kim,Y.I. Kang , Y.J. Lee , I.H. Jin ,B.R. Han ,Y.S. Cheonf, H.M. Ha ,N.G. , and Seo ,S.J. (2008) . Comparative analysis of two attacin genes from Hyphantria cunea. Comp Biochem Physiol B Biochem Mol Biol 151, 213–220.
Kragol, G., Lovas, S., Varadi, G., Condie, B.A., Hoffmann, R., and Otvos L, J.(2001).The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. Biochemistry 40,3016-26.
Lamprou, I., Tsakas, S., Theodorou, G.L., Karakantza, M., Lampropoulou, M. and Marmaras, V.J. (2005). Uptake of LPS/ E. coli/latex beads via distinct signalling pathways in medfly hemocytes: the role of MAP kinases activation and protein secretion. Biochim Biophys Acta 1744, 1–10.
Leclerc V, Reichhart J.M. (2004). The immune response of Drosophila melanogaster. Immunol Rev 198, 59-71. Review.
Lee, J. Y., Boman ,A., Chuanxin, S., Anderson ,M., Jounvall, H., Mutt ,V. and Boman ,H. G. (1989).Antibacterial peptides from pig intestine: isolation of a mammalian cecropin. Proc Natl Acad Sci USA 86, 9159-9162.
Lemaitre, B., and Hoffmann, J. (2007). The host defense of Drosophila melanogaster. Annu Rev Immunol 25, 697-743.
Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M., and Hoffmann, J. A. (1996). The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973-983.
Lemos, F. J. A., Ribeiro ,A. F. and Terra ,W. R. (1993). A bacteria-digesting midgut-lysozyme from Musca domestica (Diptera) larvae, purification, properties and secretory mechanism. Insect Biochem Mol Biol 23, 533-541.
Lesch C, Goto A, Lindgren M, Bidla G, Dushay M.S., and Theopold U. (2007) . A role for Hemolectin in coagulation and immunity in Drosophila melanogaster. Comp Immunol 12, 1255-63.
Leulier, F., Rodriguez, A., Khush, R.S., Abrams, J.M., and Lemaitre, B.(2000). The Drosophila caspase Dredd is required to resist gram-negative bacterial infection. EMBO Rep 1,353-8.
Levashina, E. A., Langley, E., Green, C., Gubb, D., Ashburner, M., Hoffmann, J. A., and Reichhart, J. M. (1999). Constitutive activation of toll-mediated antifungal defense in serpin-deficient Drosophila. Science 285, 1917-1919.
Matsumoto, N., Okada ,M., Takahashi ,H., Ming ,Q. X., Nakanishi, Y., Nakajima, Y., Komano ,H. and Natori ,S. (1986). Molecular cloning of a cDNA and assignment of the C-terminal of sarcotoxin IA, a potent antibacterial protein of Sarcophaga peregrina. Biochem J 239, 717-722.
Matsuzaki, K. (1999). Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim Biophys Acta 1462,1-10.
Mavrouli, M.D., Tsakas, S., Theodorou, G.L., Lampropoulou, M.,and Marmaras, V.J. (2005). MAP kinases mediate phagocytosis and melanization via prophenoloxidase activation in medfly hemocytes. Biochim Biophys Acta 1744,145–156.
Meng, X., Khanuja, B. S., and Ip, Y. T. (1999). Toll receptor-mediated Drosophila immune response requires Dif, an NF-kappaB factor. Genes Dev 13, 792-797.
Parker,M.S., and Uth,D.D.(1999). Specific Binding of Human Interferon-gamma to Particulates from Hemolymph and Protocerebrum of Tobacco Hornworm (Manduca sexta) larvae.Comp Biochem Physiol B Biochem Mol Biol 122 ,155-163.
Phipps, D. J., Chadwick ,J. S. and Aston, W. P. (1994). Gallysin-l, an antibacterial protein isolated from hemolymph of Galleria mellonella. Dev. Comp. Immunol 18, 13-23.
Reichhart, .JM., Meister, M., Dimarcq, J.L., Zachary, D., Hoffmann, D., Ruiz, C., Richards, G., and Hoffmann, J.A.(1992).Insect immunity: developmental and inducible activity of the Drosophila diptericin promoter.EMBO J 11,1469-77.
Richman A, and Kafatos F.C. (1996).Immunity to eukaryotic parasites in vector insects. Curr Opin Immunol 1,14-9. Review
Richman, A., and Kafatos, F. C. (1996). Immunity to eukaryotic parasites in vector insects. Curr Opin Immunol 8, 14-19.
Robertson, M. and Postlethwait, J. H. (1986). The humoral antibacterial response of Drosophila adults. Dev. Comp. Immunol 10, 167-179.
Samakovlis, C., Kimbrell ,D. A., Kylsten, P., Engstrom A. and Hultmark D. (1990).The immune response in Drosophila: pattern of cecropin expression and biological activity. EMBO J 9, 2969-2976.
Sawyer, J.G., Martin, N.L., Hancock, R.E.(1988). Interaction of macrophage cationic proteins with the outer membrane of Pseudomonas aeruginosa. Infect Immun 56,693-8.
Schneider, D. S., Hudson, K. L., Lin, T. Y., and Anderson, K. V. (1991). Dominant and recessive mutations define functional domains of Toll, a transmembrane protein required for dorsal-ventral polarity in the Drosophila embryo. Genes Dev 5, 797-807.
Shai, Y.(1995). Molecular recognition between membrane-spanning polypeptides. Trends Biochem Sci 20,460-4. Review.
Shai, Y. (1999). Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta 1462, 55-70.
Shin, S.W., Kokoza, V., Bian, G., Cheon, H.M., Kim, Y.J. and Raikhel, A.S. (2005). REL1, a homologue of Drosophila dorsal, regulates toll antifungal immune pathway in the female mosquito Aedes aegypti. J Biol Chem 280, 16499–16507.
Shin, S.W., Park, S.S., Park, D.S., Kim, M.G., Kim, S.C., Brey, P.T., and Park, H.Y., (1998). Isolation and characterization of immune-related genes from the fall webworm, Hyphantria cunea, using PCR-based differential display and subtractive cloning. Insect Biochem Mol Biol 28, 827–837.
Soderhall, K., and Cerenius, L. (1998). Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 10, 23-28
Stoven, S., Silverman, N., Junell, A., Hedengren-Olcott, M., Erturk, D., Engstrom, Y., Maniatis, T., and Hultmark, D.(2003). Caspase-mediated processing of the Drosophila NF-kappaB factor Relish.Proc Natl Acad Sci U S A 100,5991-6.
Sumida, M., Ichimori, H., Johchi , S., Takaoka , A., Yuhki, T. , Mori ,H. and Matsubara, F. (1992a). Antibacterial activity inducible in the haemolymph of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 during the fifth larval instar and pharate adult development. Comp. Biochem. Physiol 101B,165-171.
Sugiyama, M., Kuniyoshi, H., Kotani, E., Taniai, K., Kadono-Okuda, K., Kato, Y., Yamamoto,M., Shimabukuro, M., Chowdhury, S., and Xu, J. .(1995). Characterization of a Bombyx mori cDNA encoding a novel member of the attacin family of insect antibacterial proteins. Insect Biochem Mol Biol 25, 385–392.
Sumida, M., Ichimori, H., Johchi , S., Takaoka , A., Yuhki, T. , Mori ,H. and Matsubara, F. (1992b). Induction of antibacterial activity in the haemolymph of the silkworm, Bontbyx mori, by injection of formalin-treated Escherichia coli K-12 in the anterior and posterior body part of the ligated larvae. Comp Biochem Phys 101B, 173-178.
Sun, S.-C., Asling ,B., and Faye, I. (1991). Organization and expression of the immunoresponsive lysozyme gene in the giant silk moth, Hyalophora cecropia. J Biol Chem 266, 6644-6649.
Tanji T, and Ip YT.,(2005).Regulators of the Toll and Imd pathways in the Drosophila innate immune response. Trends Immunol 24,193-8. Review.
Theopold, U., Li, D., Fabbri, M., Scherfer, C., and Schmidt, O. (2002). The coagulation of insect hemolymph. Cell Mol Life Sci 59, 363-372.
Thevissen, K., Osborn, R.W., Acland, D.P., and Broekaert, W.F.(1997).S pecific, high affinity binding sites for an antifungal plant defensin on Neurospora crassa hyphae and microsomal membranes. J Biol Chem 272,32176-81.
Tzou, P., De Gregorio, E., and Lemaitre, B. (2002). How Drosophila combats microbial infection: a model to study innate immunity and host-pathogen interactions. Curr Opin Microbiol 5, 102-110.
van't Hof, W., Veerman ,E. C., Helmerhorst ,E. J. and Amerongen ,A. V. (2001).Antimicrobial peptides: properties and applicability. Biol Chem 382, 597-619.
Vlak, J.M., and Keus, R.J.(1990). Baculovirus expression vector system for production of viral vaccines. Adv Biotechnol Processes14,91-128. Review.
Wang, L., and Ligoxygakis, P. (2006). Pathogen recognition and signalling in the Drosophila innate immune response. Immunobiology 211, 251-261.
Wang ,L., Weber ,A.N., Atilano, M.L., Filipe, S.R., Gay, N.J., and Ligoxygakis, P. (2006). Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA. EMBO J 20,5005-14.
Wachinger,M.,Leinschiniidt,A.K., and Winder, D. (1998). Antimicrobial Peptides Melittin and Cecropin Inhibit Replication of Human Immunodeficiency Virus 1 by Suppressing Viral Gene Expression. Gen Virol 79 ,31-740.
Weber, A. N., Tauszig-Delamasure, S., Hoffmann, J. A., Lelievre, E., Gascan, H., Ray, K. P., Morse, M. A., Imler, J. L., and Gay, N. J. (2003). Binding of the Drosophila cytokine Spatzle to Toll is direct and establishes signaling. Nat Immunol 4, 794-800.
Wu ,R. Chen-Chih, Shaio, M.F. and Cho, W.L.(2007). A p38 MAP kinase regulates the expression of theAedes aegypti defensin gene in mosquito cells. Insect Molecular Biology 16, 389–399.
Antonova ,Y. ,Alvarez ,K. S. ,Kim, Y. J., Kokoza , V., and Raikhel, A. S. (2009).The role of NF-kB factor REL2 in the Aedes aegypti immune response. Insect Biochemistry and Molecular Biology 39 , 303–314.
Yan, R., Small, S., Desplan, C., Dearolf, C. R., and Darnell, J. E., Jr. (1996). Identification of a Stat gene that functions in Drosophila development. Cell 84, 421-430.
Ye, J.S., Zheng, X.J., Leung ,and K.W.(2004) .Induction of Transiention Channel-like Pores in a Cancer Cell by Antibiotic Peptide. Biochem (Tokyo)136,255-259.
Zasloff, M. (1987). Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci USA 84, 5449-5453.
Zasloff, M. (2002). Antimicrobial peptides of multicellular organisms. Nature 415,389-395.
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