(3.235.236.13) 您好!臺灣時間:2021/05/15 03:43
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:::

: 
twitterline
研究生:洪怡瑄
研究生(外文):Yi-Hsuan Hung
論文名稱:Xanthomonas細菌素與抗菌胜肽Cecropin A在大腸桿菌表現之探討
論文名稱(外文):Expression of Xanthomonas Bacteriocins and Antimicrobial Peptide Cecropin A in Escherichia coli
指導教授:楊明德楊明德引用關係
指導教授(外文):Ming-Te Yang
口試委員:李晏忠李天雄
口試委員(外文):Yen-Chung LeeTien-Hsiung Li
口試日期:2016-07-27
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
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:2016
畢業學年度:104
語文別:中文
論文頁數:77
中文關鍵詞:細菌素抗菌胜肽
外文關鍵詞:XanthomonasALBmALBAntimicrobial peptidescecropin A
相關次數:
  • 被引用被引用:0
  • 點閱點閱:73
  • 評分評分:
  • 下載下載:16
  • 收藏至我的研究室書目清單書目收藏:0
由細菌生成可殺死親緣性相近菌株的胜肽或蛋白稱為細菌素,先前的研究中,本實驗室已將由植物病原菌Xanthomonas axonopodis pv. glycines YR32 (Xag YR32) 選殖得到之glyA與glyB細菌素基因,利用linker方式連接並在 E. coli表現,所得之融合蛋白ALB為不可溶性。將此蛋白進行變性與復性的過程中,可形成具有殺菌活性的mature ALB (mALB)。本研究進一步對ALB、mALB及抗菌胜肽 cecropin A 於E. coli表現及純化進行探討。首先為了增加ALB與mALB 在 E. coli表現時的可溶性,於蛋白N 端接上 thioredoxin-, CBD-intein- 與MalE- 進行融合蛋白之構築,其中MalE-fused ALB 與mALB在表現後可得到可溶的蛋白;然而經 TVMV protease 切除 MalE 蛋白之後,又回復成為不可溶蛋白。利用pBBad載體於X. axonopodis pv. glycines 12609 (Xag 12609) 進行ALB 與 mALB 表現,表現 mALB 之 Xag 12609,胞內蛋白萃取液與胞外培養液之殺菌活性並無提升,但表現 ALB 之 Xag 12609 因有 mALB 生成,則可以提升其胞內蛋白萃取液之殺菌活性至26 x 100 AU/ml。本研究之第二部分,利用四種不同改造的 cecropin A,即在 cecropin N 端有或無GWL以及C 端有或無his tag與可自我切割的CBD-intein進行融合蛋白構築。結果顯示,從pTWIN-cecA-SN與pTWIN-cecA-SC 構築表現之 CBD-intein 融合蛋白,在 cecropin A 之 N 端多含有GWL 3個胺基酸,有較高的自我切割活性,經Ni2+-NTA管柱純化後,可得到對DH5α有抗菌活性的 cecropin A。此外,為了提高 cecropin A 在 E. coli之生產量,本研究亦將 cecropin A 利用串聯的形式表現,完成了1至7套 cecropin A 基因的構築並與 CBD-intein-, SUMO 與 thioredoxin 形成融合蛋白的形式表現。結果顯示,與 SUMO 及 thioredoxin形成之融合蛋白,在表現時可得到可溶性蛋白,含1至2套 cecropin A 基因之融合蛋白之最高表現量,表現量可達總蛋白之13.2 %。

Bacteriocins are proteins or peptides produced by bacteria that have bactericidal activities and often against the closely related bacteria. In our previous studies, the genes of GlyA and GlyB from plant pathogen X. axonopodis pv. glycines YR32 (Xag YR32), were linked together and is capable of forming an insoluable fusion protein ALB after expression in E. coli. In addition, during renaturation of the the ALB fusion protein, a processed mature mALB protein with bactericidal activity was observed. In this study, expression and purification of ALB, mALB, and antimicrobial peptide cecropin A in Escherichia coli were carried out. To enhance the soluability of the ALB and mALB proteins, the thioredoxin-, CBD-intein-, and MalE- fused proteins were constructed. Soluable form fusion proteins were obtained for MalE-fused ALB and mALB; however, these fusion proteins convert to inclusion bodies after removing of the MalE fusion tag by TVMV protease digestion. Expression of the mALB in X. axonopodis pv. glycines 12609 (Xag 12609) showed no difference in intercellular and extracellular bactericidal activities against Xv66. Enhancing the intercellular but not extracellular bactericidal activity was observed for ALB expressed in Xag 12609. In the second part of this study, four plasmids containing the antimicrobial peptide cecropin A and a self-cleavageable CBD-intein as fusion tag were constructed. Results showed that the CBD-intein fusion proteins expressed from pTWIN-cecA-SN and pTWIN-cecA-SC plasmids have a higher self-cleavage activity. After Ni2+-NTA column purification, cecropin A with detectable bactericidal activity against DH5αwas obtained. Moreover, in order to improve the production of active cecropin peptide, expression plasmids containing one to seven copies of tandem cecropin A genes and individually fused with thioredoxin, SUMO, and CBD-intein were constructed. Results showed that the majority of the fusion proteins expressed from tandem multimers of cecropin A with SUMO and thioredoxin fusion tags were soluble and the fusion monomer and dimer had the highest expression level at about 13.2 % of the total protein.

中文摘要 i
Abstract ii
目錄 iii
表目錄 vi
圖目錄 vii
縮寫字對照表 ix
前言 1
(一) GlyA-GlyB細菌素 (Bacteriocin) 1
1. 細菌素起源 1
2. 細菌素的分類 1
3. 細菌素的殺菌機制 1
4. 宿主的免疫機制 2
5. 細菌素的應用 2
6. Glycinecin A 2
(二) Cecropin 抗菌胜肽 (Antimicrobial peptides, AMPs) 4
1. 抗菌胜肽起源 4
2. 抗菌胜肽的分類 4
3. 抗菌胜肽的作用機制 4
4. 抗菌胜肽的應用 4
5. Cecropin 5
(三) 研究動機 6
材料與方法 7
(一) 材料 7
1. 菌種及質體 7
2. 藥品 7
3. 酵素 7
4. 純化管柱 7
5. 引子 8
6. 培養基與緩衝溶液 8
(二) 實驗方法 8
1. 小量製備E. coli質體 DNA 8
2. 聚合酶鏈鎖反應 ( Polymerase chain reaction, PCR) 8
3. E. coli勝任細胞製備 9
4. 轉形作用(熱休克法) 9
5. 蛋白質凝膠體電泳 (SDS-PAGE) 9
6. Tricine SDS-PAGE 電泳 10
7. 西方墨點轉漬法 10
8. 利用不同載體表現ALB 與mALB 蛋白 10
9. 以 MalE 融合蛋白形式表現ALB 與mALB蛋白 10
10. ALB 與 mALB 於Xag 12609 表現 11
11. 蛋白變性與復性 11
12. Cecropin 載體之構築與表現 11
13. 增幅多套 cecropin A 基因 12
14. 串聯基因 cecropin A 基因之構築 13
15. 多套數 cecropin 之蛋白表現與純化 14
16. 細菌素 ALB、mALB與抗菌胜肽cecropin的活性測試 14
17. 膠體上蛋白質之定量方法 14
結果 15
(一) 細菌素ALB與mALB 15
1. 利用不同載體表現 ALB 與 mALB 蛋白 15
2. 以MalE 融合蛋白形式表現 ALB 與mALB蛋白 15
3. ALB 與 mALB 於 Xag 12609 表現 16
(二) 抗菌胜肽cecropin A 16
1. pTWIN-cecA-dN載體之構築與表現 17
2. pTWIN-cecA-SN載體之構築與表現 17
3. pTWIN-cecA-dC載體之構築與表現 17
4. pTWIN-cecA-SC載體之構築與表現 17
5. pMT21N-cecA-dN與 pMT21N-cecA-SN載體之構築與表現 18
6. 串聯式cecropin A基因之構築與表現 18
討論 20
(一) 細菌素 ALB 與 mALB 20
(二) 抗菌胜肽 cecropin A 21
參考文獻 24
表一、本實驗所使用之菌株 29
表二、本實驗所使用與構築之質體 30
表三、本實驗所使用的引子 32
表四、於不同溫度誘導之his-Trx-cecA x1-7 與his-SUMO-cecA x1-7目標蛋白產量與可溶率 33
圖 34-68
附錄一、CIA7、ALB 與 mALB蛋白之胺基酸序列 69
附錄二、Mature Cecropin A 胺基酸序列 70
附錄三、藥品配方及培養基的組成 71


廖珮鑾 (2006)。Xanthomonas細菌素基因的篩選及在 E. coli中表現。國立國立中興大學分子生物研究所碩士論文。

郭乃瑜 (2007)。 Xanthomonas albilineans及 Xanthomonas campestris pv. glycines 細菌素之探討。國立國立中興大學分子生物研究所碩士論文。

羅右瑋 (2010)。Xanthomonas albilineans細菌素基因選殖及Xanthomonas campestris pv. glycines細菌素基因表現之探討。國立國立中興大學分子生物研究所碩士論文。

吳致淵 (2012)。Xanthomonas細菌素基因及抗菌胜肽於大腸桿菌表現及純化之探討。國立國立中興大學分子生物研究所碩士論文。

林韋辰 (2013)。大豆斑疹病菌細菌素基因表現及純化之探討。國立國立中興大學分子生物研究所碩士論文。

Bechinger, B., and Lohner, K. (2006). Detergent-like actions of linear amphipathic cationic antimicrobial peptides. Biochim. Biophys. Acta1 758, 1529-1539.

Berdy, J. (1974). Recent developments of antibiotic research and classification of antibiotics according to chemical structure. Adv. Appl. Microbiol. 18, 309-406.

Boman, H. G. (1991). Antibacterial peptides: key components needed in immunity. Cell. 65, 205-207.

Bommarius, B., Jenssen, H., Elliott, M., Kindrachuk, J., Pasupuleti, M., Gieren, H., Jaeger, K. E., Hancock, R. E. & Kalman, D. (2010). Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. Peptides. 31, 1957-1965.

Brogden, K.A. (2005). Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria. Nature reviews. Microbiology. 3, 238-250.

Callaway, J.E., Lai, J., Haselbeck, B., Baltaian, M., Bonnesen, S.P., Weickmann, J., Wilcox, G., and Lei, S.P. (1993). Modification of the C terminus of cecropin is essential for broad-spectrum antimicrobial activity. Antimicrob. Agents Chemother. 37, 1614-1619.
Cao, Y., Yu, R. Q., Liu, Y., Zhou, H. X., Song, L. L., Cao, Y. & Qiao, D. R. (2010). Design, recombinant expression, and antibacterial activity of the cecropins-melittin hybrid antimicrobial peptides. Curr Microbiol. 61, 169-175.

Chong, S., Montello, G. E., Zhang, A., Cantor, E. J., Liao, W., Xu, M. Q. & Benner, J. (1998). Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucleic Acids Res. 26, 5109-5115.

Cotter, P. D., Hill, C. & Ross, R. P. (2005). Bacteriocins: developing innate immunity for food. Nat Rev Microbiol. 3, 777-788.

Diao, H., Guo, C., Lin, D. & Zhang, Y. (2007). Intein-mediated expression is an effective approach in the study of beta-defensins. Biochem Biophys Res Commun. 357, 840-846.

Dubois, J. Y., Kouwen, T. R., Schurich, A. K., Reis, C. R., Ensing, H. T., Trip, E. N., Zweers, J. C. & van Dijl, J. M. (2009). Immunity to the bacteriocin sublancin 168 Is determined by the SunI (YolF) protein of Bacillus subtilis. Antimicrob Agents Chemother. 53, 651-661.

Fasano, O., Alfdrich, T., Tamanoi, F., Taparowsky, E., Furth, M., and Wigler, M. (1984) Analysis of the transformingpotential of the human H-ras gene by random mutagenesis. Proc. Natl. Acad. Sci. USA 81, 4008.

Feng, X. J., Xing, L. W., Liu, D., Song, X. Y., Liu, C. L., Li, J., Xu, W. S. & Li, Z. Q. (2014). Design and high-level expression of a hybrid antimicrobial peptide LF15-CA8 in Escherichia coli. J Ind Microbiol Biotechnol. 41, 527-534.

Fimland, G., Johnsen, L., Dalhus, B. & Nissen-Meyer, J. (2005). Pediocin-like antimicrobial peptides (class IIa bacteriocins) and their immunity proteins: biosynthesis, structure, and mode of action. J Pept Sci. 11, 688-696.

Gratia, A.(1925). Compt. rend. soc. biol. (Paris), 93, 1040.

Hames, B.D. (1990) One-dimensional polyacrylamide gel electrophoresis. In: Hames, B.D. and Rickwood, D., eds.Gel electrophoresis of proteins: a practical approach. Oxford: Oxford University Press, p 1.

Heu, S., Oh, J., Kang, Y., Ryu, S., Cho, S. K., Cho, Y. & Cho, M. (2001). gly gene cloning and expression and purification of glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines 8ra. Appl Environ Microbiol. 67, 4105-4110.

Hultmark, D., Engstrom, A., Bennich, H., Kapur, R. & Boman, H. G. (1982). Insect Immunity - Isolation and Structure of Cecropin-D and 4 Minor Antibacterial Components from Cecropia Pupae. Eur. J. Biochem. 127, 207-217.

Jacob, F., Siminovitch, L. & Wollman, E. (1952). Biosynthesis of a colicin and its mode of action. Ann Inst Pasteur. (Paris) 83, 295-315.

Jan, P. S., Huang, H. Y. & Chen, H. M. (2010). Expression of a synthesized gene encoding cationic peptide cecropin B in transgenic tomato plants protects against bacterial diseases. Appl Environ Microbiol. 76, 769-775.

Kim, H. K., Chun, D. S., Kim, J. S., Yun, C. H., Lee, J. H., Hong, S. K. & Kang, D. K. (2006). Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli. Appl Microbiol Biotechnol. 72, 330-338.

Lavermicocca, P., Lonigro, S. L., Valerio, F., Evidente, A. & Visconti, A. (2002). Reduction of olive knot disease by a bacteriocin from Pseudomonas syringae pv. ciccaronei. Appl Environ Microbiol. 68, 1403-1407.

Lee, J. H., Minn, I., Park, C. B. & Kim, S. C. (1998). Acidic peptide-mediated expression of the antimicrobial peptide buforin II as tandem repeats in Escherichia coli. Protein Expr Purif. 12, 53-60.

Li, Y. (2011). Recombinant production of antimicrobial peptides in Escherichia coli: a review. Protein Expr Purif. 80, 260-267.

Li, Y., Li, X. & Wang, G. (2006). Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies. Protein Expr Purif. 47, 498-505.

Lubelski, J., Rink, R., Khusainov, R., Moll, G. N. & Kuipers, O. P. (2008). Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin. Cell Mol Life Sci. 65, 455-476.

Miroux, B. & Walker, J. E. (1996). Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol. 260, 289-298.

Morassutti, C., De Amicis, F., Bandiera, A. & Marchetti, S. (2005). Expression of SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated system. Protein Expr Purif. 39, 160-168.

Nallamsetty, S., Kapust, R. B., Tozser, J., Cherry, S., Tropea, J. E., Copeland, T. D. & Waugh, D. S. (2004). Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro. Protein Expr Purif. 38, 108-115.

Nakayama, K., Takashima, K., Ishihara, H., Shinomiya, T., Kageyama, M., Kanaya, S., Ohnishi, M., Murata, T., Mori, H. & Hayashi, T. (2000). The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage. Mol Microbiol. 38, 213-231.

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.

Sakthivel, N. & Mew, T. W. (1991). Efficacy of bacteriocinogenic strains of Xanthomonas-oryzae pv oryzae on the incidence of bacterial-blight disease of rice (oryza-sativa l). Can. J. Microbiol. 37, 764-768.

Singh, S.M., Sharma, A., Upadhyay, A.K., Singh, A., Garg, L.C., and Panda, A.K. (2012). Solubilization of inclusion body proteins using n-propanol and its refolding into bioactive form.Protein Expr Purif. 81, 75-82.

Smarda, J. & Benada, O. (2005). Phage tail-like (high-molecular-weight) bacteriocins of Budvicia aquatica and Pragia fontium (Enterobacteriaceae). Appl Environ Microbiol. 71, 8970-8973.

Strauch, E., Kaspar, H., Schaudinn, C., Dersch, P., Madela, K., Gewinner, C., Hertwig, S., Wecke, J. & Appel, B. (2001). Characterization of enterocoliticin, a phage tail-like bacteriocin, and its effect on pathogenic Yersinia enterocolitica strains. Appl Environ Microbiol. 67, 5634-5642.

Sukchawalit, R., Vattanaviboon, P., Sallabhan, R. & Mongkolsuk, S. (1999). Construction and characterization of regulated L-arabinose-inducible broad host range expression vectors in Xanthomonas. FEMS Microbiol Lett 181, 217-223.

Tagg, J. R., Dajani, A. S. & Wannamaker, L. W. (1976). Bacteriocins of Gram-Positive Bacteria. Bacteriol Rev. 40, 722-756.

Tellez, G. A. & Castano-Osorio, J. C. (2014). Expression and purification of an active cecropin-like recombinant protein against multidrug resistance Escherichia coli. Protein Expr Purif. 100, 48-53.

Tsumoto, K., Ejima, D., Kumagai, I. & Arakawa, T. (Morassutti et al., 2005). Practical considerations in refolding proteins from inclusion bodies. Protein Expr Purif. 28, 1-8.

Wang, H., Meng, X.L., Xu, J.P., Wang, J., Wang, H., and Ma, C.W. (2012). Production, purification, and characterization of the cecropin from Plutella xylostella, pxCECA1, using an intein-induced self-cleavable system in Escherichia coli. Appl Microbiol Biotechnol. 94, 1031-1039.

Wang, Y. Q. & Cai, J. Y. (2007). High-level expression of acidic partner-mediated antimicrobial peptide from tandem genes in Escherichia coli. Appl Biochem Biotechnol 141, 203-213.

Widjaja, R., Suwanto, A. & Tjahjono, B. (1999). Genome size and macrorestriction map of Xanthomonas campestris pv. glycines YR32 chromosome. FEMS Microbiol Lett. 175, 59-68.

Wright, O., Yoshimi, T. & Tunnacliffe, A. (2012). Recombinant production of cathelicidin-derived antimicrobial peptides in Escherichia coli using an inducible autocleaving enzyme tag. New Biotechnology. 29, 352-358.

Xu, M. Q. & Evans, T. C., Jr. (2003). Purification of recombinant proteins from E. coli by engineered inteins. Methods Mol Biol. 205, 43-68.

Xu, X., Jin, F., Yu, X., Ji, S., Wang, J., Cheng, H., Wang, C., and Zhang, W. (2007). Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli. Protein Expr Purif. 53, 293-301.

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