跳到主要內容

臺灣博碩士論文加值系統

(34.226.244.254) 您好!臺灣時間:2021/08/01 04:13
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:黃俊儒
研究生(外文):Jun-Ru Huang
論文名稱:以人造油體蛋白質純化表達系統純化蜂王漿抗菌蛋白質-royalisin
論文名稱(外文):Overexpression and purification of an antimicrobial peptide “royalisin” via artificial oil body system
指導教授:黃秀珍黃秀珍引用關係
指導教授(外文):Hsiou-Chen Huang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:49
中文關鍵詞:Royalisin抗菌蛋白質抗生素人造油體表達純化系統抗菌活性測試
外文關鍵詞:Royalisinartificial oil body systemMIC testMBC test
相關次數:
  • 被引用被引用:2
  • 點閱點閱:208
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
Royalisin為蜜蜂工蜂下咽頭腺分泌的胜肽,其大小為51個胺基酸,內含三個雙硫鍵,結構穩定;royalisin是抗菌蛋白質的一種。抗菌蛋白質是種小分子蛋白質,其主要特徵有:一、分子量不大,大小約在十來個胺基酸到五十來個胺基酸之間,二、富含帶有正電荷的胺基酸,三、為兩性分子;抗菌蛋白是藉由物理性力量破壞菌體的細胞壁,而非像傳統抗生素一樣藉由結合特定受體進而影響細菌生長,所以不易產生抗藥性,有替代傳統抗生素的潛力。但由於蜂王乳產量稀少,且極難從中純化royalisin,為了能夠大量取得royalisin,我們把目標放在能夠快速繁殖、大量表現蛋白質的大腸桿菌上,希望能利用大腸桿菌繁殖迅速的優點,來大量表現royalisin。本研究利用人造油體蛋白質純化系統來純化royalisin,首先將抗菌蛋白royalisin、intein及油體膜蛋白構築在一起,利用微生物大量表現此蛋白,並利用人造油體蛋白質純化方法來純化此抗菌蛋白royalisin,這方式有著方便和便宜的優點。 本研究藉由最小抑菌濃度測試和最小殺菌濃度測試的方法來測試確認純化的royalisin的活性。結果發現純化的royalisin對於S.aureus、S. intermedius B、S. xylosus、S. alactolyticus、S. cholearasuis、V.parahamelytics CCRC10806等菌株具有抗菌活性。
Royalisin, a small peptide isolated from the royal jelly of honey bee. Royalisin was composed of 51 amino acid residues, being a cationic protein with a net charge of +2 as calculated from the amino acid composition. The structural novelty of rayalisin is in its high cysteine content (6 residues) and connection by three intramolecular disulfide linkages. Royalisin have an antifungal activity and its antibacterial activity against honeybee pathogen Paenibacillus larvae larvae, which is the cause of a serious disease of honeybee larvae, American Foulbrood. Royalisin is a kind of antibacterial protein, antibacterial proteins have three characteristics: (1) low molecular weight, there are usually 14~50 amino acids, (2) Rich of positive charge and (3) it is an amphipathic molecule. Its bactericide way is destroying bacteria membrane with physical strength, not like traditional antibiotics that were interact with ligand and receptor of bacteria. Therefore it is a potentiality reagent to replace traditional antibiotics. In this study, we used artificial oil body (AOBs) system to purify the royalisin. Construct royalisin into the oleosin fusion intein plasmids and overexpress the rayalisin in E. coli and purified it by artificial oil body protein purification system. Royalisin purfied via AOB protein purification system display antibacterial activity for S. aureus、S. intermedius B、S. xylosus、S. alactolyticus、S. cholearasuis、V. parahamelytics CCRC10806. In this result, the purified royalisin had higher antibacterial activity for gram positive than gram negative bacteria in lower protein concentration.
中文摘要......i
Abstract......ii
壹、緒論......1
貳、材料與方法......11
一、供試菌株、質體及菌體生長環境......11
二、製備 E.coli DH10B 和AD494的勝任細胞......11
三、構築含融合蛋白 oleosin-intein-royalisin序列的質體(pBP05)...12
四、以T7-RNA polymerase-dependent方式表現融合蛋白質oleosin- intein-royalisin.......15
五、回溶菌體形成的包涵體.......16
六、人造油體的備製.............16
七、誘導intein系統作用及回收純化royalisin.......16
八、利用Tricine gel分析作用前後的油體和澄清液......17
九、最小抑菌濃度測試(MIC assay).......17
十、最小殺菌濃度測試(MBC assay).......17
參、結果.......19
一、構築含融合蛋白oleosin-intein-royalisin gene的序列(pBP05)..19
二、大量表達融合蛋白 oleosin-intein-royalisin.......20
三、人造油體蛋白質表達系統(Artificial Oil Body Protein Expression and Purification System)表達並純化Royalisin.......20
四、以人造油體蛋白質表達純化系統回收純化後royalisin的抗菌測試分析.......21
肆、討論......25
伍、參考文獻......29
陸、圖表......36
附錄一......48
Ando, K., Natori, S. (1988) Inhibitory effect of sarcotoxin IIA, an antibacterial protein of Sarcophaga peregrina, on growth of Escherichia coli. J Biochem. 103, 735-739.

Bachanova, K., Klaudiny, J., Kopernicky, J., Simuth, J. (2002) Identification of honeybee peptide active against Paenibacillus larvae larvae through bacterial growth-inhibition assay on polyacrylamide gel. Apidologie 33, 259-269.

Bilikova, K., Gusui, W., Simuth, J. (2001) Isolation of a peptide fraction from honeybee royal jelly as apotential antifoulbrood factor. Apidologie 32, 275-283.

Boman,H.G., Steiner, H. (1980) Humoral immunity in Cecropia pupae. Curr Top Microbiol Immunol. 1981;94-95:75-91.

Boman, H. G. (1995) Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13, 61-92.

Bonmatin, J.M., Bonnat, J.L., Gallet, X., Vovelle, F., Ptak, M.,Reichart, J.M., Hoffmann,J.A., Keppi, E., Legrain, M., Achstetter, T.,(1992). Two-dimensional 1H NMR study of recombinant insect defensin A in water: resonance assignments, secondary structure and global folding. J. Biomol. 2, 235–256.

Bulet, P., Hetru, C., Dimarcq, J. L., Hoffmann, D. (1999) Antimicrobial peptides in insects; structure and function. Dev. Comp.Immunol. 23, 329-344.

Carlsson, A., Engström, P., Palva, E.T., Bennich, H. (1991) Attacin, an antibacterial protein from Hyalophora cecropia, inhibits synthesis of outer membrane proteins in Escherichia coli by interfering with omp gene transcription. Infect Immun. 59, 3040-3045.

Carlsson, A., Nyström, T., de Cock, H., Bennich, H. (1998) Attacin-an insect immune protein – binds LPS and triggers the specific inhibition of bacterial outer-membrane protein synthesis. Microbiology , 144, 2179–2188.

Casteels, P., Ampe, C., Jacobs, F., Vaek, M., Tempst, P., (1989) Apidaecins: antibacterial peptides from honeybees. EMBO J. 8, 2387-2391.

Casteels, P., Ampe, C., Riviere, L., Damme, J.V., Elicone, C., Fleming, M., Jacobs, F., Tempst, P., (1990) Isolation and characterization of abaecin, a major antibacterial peptide in the honeybee (Apis mellifera). Eur. J. Biochem. 187, 381-386.

Casteels, P., Ampe, C., Jacobs, F., Tempst, P., (1993) Functional and chemical characterization of hymenoptaecin, an antibacterial polypeptide that is infection-inducible in the (Apis mellifera). J. Biol. Chem. 268, 7704-7054.

Casteels-Jasson, K., Zhang, W., Capaci, T., Casteels, P., Tempst, P., (1994) Acute transcriptional response of the honeybee peptide-antibiotics gene repertorire and required post-translational conversion of the precursor structures. J. Biol. Chem. 269, 28569-28575.

Chen, E.C., Tai, S.S., Peng, C.C., Tzen. J.T. (1998) Identification of three novel unique proteins in seed oil bodies of sesame. Plant Cell Physiol. 39, 935-941.

Chiang, C.J., Chen, H.C., Chao, Y.P., Tzen, J.T. (2005) Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli. J. Agric. Food Chem. 53, 4799-4804.

Christensen, B., Fink, J., Merrifield, R. B., and Mauzerall, D. (1988) Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes .Proc. Natl. Acad. Sci. U.S.A.85, 5072-5076.

Cociancich, S., Ghazi, A., Hetru, C., Hoffmann, J.A., Letellier, L. (1993) Insect defensin an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus. J. Biol. Chem. 268, 19239-19245.

Cornet, B., Bonmatin, J.M., Hetru, C., Hoffmann, J.A., Ptak, M., Vovelle, F.,(1995) Refined three-dimensional solution structure of insect defensin A. Structure 3, 435–448.

Fehlbaum, P., Bulet, P., Chernysh, S., Briand, J.P., Roussel, J.P., Letellier, L., Hetru, C., Hoffmann, J.A.(1996) Structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides. Proc Natl Acad Sci U S A. 93, 1221-1225.

Fink, J., Boman, A., Boman, H.G., Merrifield,R.B. (1989) Design, synthesis and antibacterial activity of cecropin-like model peptides. Int J Pept Protein Res. 33, 412-421.

Fujiwara, S., Imai, J., Fujiwara, M., Yaeshima, T., Kawashima, T., Kobayashi, K.(1990) A potent antibacterial protein in royal jelly. J. Biol. Chem. 265, 11333-11337.

Hancock, R.E., Lehrer, R.(1998) Cationic peptides: a new source of antibiotics. Trends Biotechnol. 16, 82-88

Hancock, R.E., Sahl, H.G. (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Rev Nat Biotechnol. 24, 1551-1557.

Hanzawa, H., Shimada, I., Kuzuhara, T., Komano, H., Kohda, D., Inagaki, F., Natori, S., Arata, Y., 1990. 1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin. FEBS Lett. 269, 413–420.

Hetru, C., Hoffmann, D., Bulet, P., (1998) Antimicrobail peptides from insects. In: Brey, P.T., Hultmark, D(Eds.), Molecular Mechanusms of Immune Responses in Insects. Chapman & Hall, London, pp. 40-66.

Hilpert, K., Volkmer-Engert, R., Walter, T., Hancock, R.E. (2005) High-throughput generation of small antibacterial peptides with improved activity. Nat Biotechnol. 23, 1008-1012.
Hultmark, D, Engström, A, Andersson, K, Steiner, H., Bennich, H., Boman, H.G. (1983) Insect immunity. Attacins, a family of antibacterial proteins from Hyalophora cecropia. EMBO J. 2, 571-576.


Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J. (1990) PCR protocols.San Diego:Academic Press.

Klaudiny, J., Albert, S., Bachanova, K., Kopernicky, J., Simuth, J. (2005) Two structurally different defensin genes, one of them encoding a novel defensin isoform, are expressed in honeybee Apis mellifera. Insect Biochemistry and Molecular biology 35, 11-22.

Lau, Y.E., Rozek, A., Scott, M.G., Goosney, D.L., Davidson, D.J., Hancock, R.E.(2005) Interaction and cellular localization of the human host defense peptide LL-37 with lung epithelial cells. Infect Immun. 73, 583-591.

Li ,M., Lai, Y., Villaruz ,A.E., Cha, D.J., Sturdevant, D.E., Otto, M. (2007) Gram-positive three-component antimicrobial peptide-sensing system. Proc Natl Acad Sci U S A. 104, 9469-9474.

Lockey, T. D., Ourth, D. D.(1996) Formation of pores in Escherichia coli cell membranes by a cecropin isolated from hemolymph of Heliothis virescens larvae. Eur J Biochem, 236, 263-271

McAuliffe,O., Ross, R.P., Hill C.(2001) Lantibiotics: structure, biosynthesis and mode of action. FEMS Microbiol Rev. 25, 285-308.

Okada,M., Natori S.(1985) Ionophore activity of sarcotoxin I, a bactericidal protein of Sarcophaga peregrina. Biochem J. 229, 453-458.

Peng,C.C., Lin,I.P., Lin,C.K., Tzen,J.T. (2003) Size and stability of reconstituted sesame oil bodies. Biotechnol. Prog. 19, 1623-1626.

Peng,C.C., Shyu,D.J., Chou,W.M., Chen,M.J., Tzen,J.T. (2004). Method for bacterial expression and purification of sesame cystatin via artificial oil bodies. J. Agri. Food Chem. 52, 3115-3119.

Peng, C.C., Chen, J.C., Shyu, D.J., Chen, M.J., Tzen, J.T., (2004) A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides. J. Biotechnology 111, 51-57.

Peng, C.C. (2006) Application of antimicrobial peptides in biotechnology. Plant Pathol.Bull.15,69-75

Perler, F.B., Davis,E.O., Dean, G.E., Gimble, F.S., Jack,W.E., Neff, N., Noren,C.J., Thorner, J., Belfort, M. (1994) Protein splicing elements: inteins and exteins--a definition of terms and recommended nomenclature. Nucleic Acids Res. 22, 1125-1127.

Radermacher, S.W., Schoop, V.M., Schluesener, H.J. (1993) Bactenecin, a leukocytic antimicrobial peptide, is cytotoxic to neuronal and glial cells. J Neurosci Res. 36, 657-662.

Sambrook,J., Russell,D.W.(2001) Molecular Cloning:A Laboratory Manual, 3rd edn.Cold Spring Harbor,NY:Cold Spring Harbor Laboratory Press.

Schagger, H.(2006) Tricine-SDS-PAGE. Nat Protoc,1,16-22.

Shin, S.Y., Kang, J.H., Lee, M.K., Kim, S.Y., Kim Y., Hahm, K.S. (1998) Cecropin A - magainin 2 hybrid peptides having potent antimicrobial activity with low hemolytic effect. J Pept Res. 53, 82-90.

Tzen, J.T., Huang, A.H. (1992) Surface structure and properties of plant seed oil bodies. J. Cell Biol. 117, 327-335.

Tzen J.T., Lie G.C., Huang A.H. (1992) Characterization of the charged components and their topology on the surface of plant seed oil bodies. J. Biol. Chem. 267, 15626-15634.

Tzen, J.T., Chuang, R.L., Chen, J.C., Wu, L.S. (1998) Coexistence of both oleosin isoforms on the surface of seed oil bodies and their individual stabilization to the organelles. J. Biochem. 123, 319-324.

Tzen J.T., Peng C.C., Cheng, D.J., Chen, E.C., Chiu J.M. (1997) A new method for seed oil body purification and examination of oil body integrity following germination. J. Biochem. 121, 762-768.

Van Rooijen, G.J.H., Moloney, M.M. (1995) Plant seed oil-bodies as carriers for foreign proteins. Bio/technology. 13, 72-77.

Wong, J.H., Ng, T.B. (2005) Sesquin, a potent defensin-like antimicrobial peptide from ground beans with inhibitory activities toward tumor cells and HIV-1 reverse transcriptase. Peptides. 26, 1120-1126.

Yamauchi, H. (2001) Two novel insect defensins from larve of the cupreous chafer, Anomala cuprea: purification, amino acid sequences and antibacterial activity. Insect Biochem. Mol. Biol. 32, 75-78.

Yeaman, M.R., Yount, N.Y..(2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev. 55, 27-55.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top