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研究生:陳怡儒
論文名稱:以大腸桿菌表現抗菌蛋白PR-39及其功能分析
論文名稱(外文):E. coli expression and functional study of antimicrobial peptide PR-39
指導教授:毛嘉洪
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫微生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
中文關鍵詞:PR-39 抗菌蛋白
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PR-39為抗菌蛋白cathelicidin家族之一員,preproPR-39在豬的骨髓中合成,並經彈力蛋白酵素切割後成為有抗菌活性的PR-39。PR-39在免疫系統中具有多種功能,包括對嗜中性球具有化學趨化性,以及抑制超氧化物形成等等。本研究目的是利用大腸桿菌表現系統,表現perproPR-39之衍生抗菌蛋白,期能大量製備此抗菌蛋白,以應用於仔豬下痢症狀之改善。本研究使用反轉錄聚合酵素連鎖反應技術(RT-PCR),由仔豬之嗜中性球分別選殖出P31(prepro-region of PR-39)、preproPR-39、preproPR-26、PR-39及PR-26基因,再嵌入pET32a載體中,分別轉形至大腸桿菌之表現菌株中進行重組抗菌蛋白之表現,結果只有P31蛋白能大量表現,其他融合蛋白之表現量均不佳。將純化後之preproPR-39衍生蛋白經腸激酵素去除融合蛋白後,定量並進行抗菌活性試驗。結果顯示P31蛋白並無抑菌效果,而PR-26蛋白在1.9 mM時能有效抑制標準大腸桿菌菌株(E. coli, ATCC 25922)生長,PR-39蛋白則受限於低表現量與低回收率,而無法定出最低抗菌濃度,但結果顯示PR-26與PR-39蛋白都能使大腸桿菌菌株和金黃色葡萄球菌(S. aureus, ATCC 25923)生長遲緩。本研究證明確能利用大腸桿菌表現系統產製具有活性的抗菌蛋白,但如何提高蛋白產量和穩定性,則需要再進一步研究。
目 次
頁次
中文摘要i
英文摘要ii
目 次iii
圖 次viii
表 次xi
壹、緒 言………………………………………………………..01
貳、文獻探討……………………………………………………02
一、Cathelicidins家族……………………………………….02
二、PR-39之發現與表現……………………………………03
三、PR-39之基因與蛋白結構特性…………………………05
四、PR-39之抗菌功能………………………………………07
五、PR-39之相似基因與蛋白…………………………...09
六、PR-39之其他生物作用………………………………...10
1. PR-39對細胞的毒性……………………………………10
2. PR-39之化學趨化作用…………………………………10
3. PR-39與疾病的關係……………………………………11
3.1防止缺血後微血管不良症…………………………...11
3.2調控血管生成………………………………………...12
3.2與癌細胞之間的關係………………………………...14
參、材料與方法 15
一、TA/preproPR-39重組質體之構築與確認15
(一)白血球總核醣核酸之萃取15
(二) preproPR-39基因之製備15
(三) TA/preproPR-39重組質體之構築17
(四)勝任細胞之製備19
(五) TA/preproPR-39重組質體之轉形與確認19
(六) TA/preproPR-39重組質體DNA的萃取與純化20
二、重組pET32a/PR-39系列基因表現載體之構築與確認22
(一) PR-39系列基因片段之製備與確認22
(二) pET32a載體DNA片段之製備與確認24
(三) pET32a/PR-39系列基因表現載體之構築25
(四)勝任細胞之pET32a/PR-39系列基因表現載體的轉形
…………………………………………………………27
(五)含有pET32a/PR-39系列基因表現載體之菌株的篩選
…………………………………………………………27
三、各表現系統之pET32a/PR-39系列基因表現載體的轉形
與表現29
(一) 轉形用pET32a/PR-39系列基因表現載體的製備29
(二)各表現系統之pET32a/PR-39系列基因表現載體的
轉形29
(三)pET32a/PR-39系列基因於各表現系統的表現與分析
29
(四)PR-39系列表現蛋白之純化、確認與定量30
四、抗菌蛋白活性試驗32
(一)影響宿主細胞生長之試驗32
(二)抗菌試驗32
肆、結 果34
一、TA/preproPR-39重組質體之構築與確認34
(一)preproPR-39基因之製備34
(二)TA/preproPR-39重組質體之確認34
二、重組pET32a/PR-39系列基因表現載體之構築與確認39
(一) PR-39系列基因片段之製備與確認39
(二) pET32a/PR-39系列基因表現載體之確認39
三、各表現系統之pET32a/PR-39系列基因表現載體的轉形
與表現45
(一)pET32a/PR-39系列基因於各表現系統的表現與分析
45
(二) PR-39系列表現蛋白之定量52
四、抗菌蛋白活性試驗56
(一)影響宿主細胞生長之試驗56
(二)抗菌試驗56
伍、討 論61
一、TA/preproPR-39重組質體之構築61
二、重組pET32a/PR-39表現載體之構築61
三、表現PR-39的系列蛋白質62
四、抗菌蛋白活性測試66
(一)影響宿主細胞生長之試驗66
(二)抗菌試驗67
陸、結 論70
柒、參考文獻71
附錄77
附錄一:萃取白血球總核醣核酸之實驗步驟77
附錄二:製備preproPR-39基因所使用之4組引子78
附錄三:M13 (+)和M13 (-)引子之序列79
附錄四:Gene-SpinTM Miniprep Purification Kit步驟80
附錄五:選殖preproPR-39、preproPR-26、PR-39及PR-26
基因序列引子81
附錄六:Gene-SpinTMGel Extraction Kit步驟82
附錄七:pET32a載體上S(+)和T7(-)引子之序列83
附錄八:限制酵素Aat II截切之核酸序列84
附錄九:LB broth與M9 broth的比較84
附錄十:pET expression system之蛋白質純化步驟85
附錄十一:BCA蛋白質分析方法之實驗步驟86
附錄十二:測試菌種之細菌生長曲線對照表87
附錄十三:P31與preproPR-26基因序列的比較圖88
圖 次
頁次
圖一:Cathelicidins家族的基因構造圖02
圖二:preproPR-39的基因結構圖06
圖三:在炎症發生時PR-39產生的反應13
圖四:TA/preproPR-39重組質體之構築流程示意圖18
圖五:pET32a 載體構造圖26
圖六:pET32a/PR-39系列基因表現載體內各基因構築簡圖26
圖七:以RT-PCR製備preproPR-39基因之瓊脂凝膠電泳
分析35
圖八:已轉形TA/preproPR-39重組質體成功之菌落,應用
M13 (+)和M13 (-)引子進行PCR後之電泳分析36
圖九:選殖得到preproPR-39基因之核酸序列與已發表序列
之比較分析37
圖十:preproPR-39基因序列經人工轉譯為胺基酸序列後與
已發表的preproPR-39胺基酸序列之比較分析38
圖十一:以PCR製備PR-39系列基因片段之瓊脂凝膠電泳
分析41
圖十二:已轉形pET32a/PR-39系列基因表現載體成功之菌落,
應用S(+)和T7(-)引子進行PCR後之電泳分析42
圖十三:以核酸限制酵素Aat II及Fsp I分別截切分析各載體
樣本之電泳分析43
圖十四:pET32a/PR-39表現載體於BL21 (DE3)之表現分析
47
圖十五:pET32a/PR-39系列基因表現載體於BL21 (DE3)之
表現分析48
圖十六:pET32a及pET32a/PR-39載體於BL21 (DE3)之表現
49
圖十七:pET32a及pET32a/PR-39載體於AD494 (DE3)pLys
之表現分析50
圖十八:pET32a及pET32a/PR-39載體於BL21 (DE3)中
表現,使用M9培養液培養51
圖十九:pET32a與pET32a/P31載體於BL21 (DE3)中之表現
、純化分析53
圖二十:pET32a/preproPR-39與pET32a/preproPR-26載體於
BL21 (DE3)中之表現、純化分析54
圖二十一:pET32a/PR-39與pET32a/PR-26載體於BL21 (DE3)
中之表現、純化分析55
圖二十二:preproPR-39衍生之系列抗菌蛋白於BL21 (DE3)
表現時,各組細菌之生長曲線圖58
圖二十三:preproPR-39衍生之系列抗菌蛋白於AD494 (DE3)
pLys表現時,各組細菌之生長曲線圖59
圖二十四:於不同誘導條件下,pET32a與PR-39融合蛋白於
BL21 (DE3)表現時,各組細菌之生長曲線圖59
表 次
頁次
表一:用於製備preproPR-39基因之4組寡核酸引子組16
表二:分別用於選殖preproPR-39、preproPR-26、PR-39及
PR-26基因序列之4組核酸引子23
表三:PR-39與其他抗菌蛋白相似序列之比較33
表四:本次實驗所表現各PR-39系列之基因序列44
表五:本次實驗所表現各PR-39系列表現蛋白之大小及胺
基酸序列46
表六:融合蛋白未腸激酵素切割前之抗菌活性測試60
表七:融合蛋白經腸激酵素切割後之抗菌活性測試60
柒、參考文獻
Agerberth, B., J. Grunewald, E. Castanos-Velez, B. Olsson, H. Jornvall, H. Wigzell, A. Eklund, and G. H. Gudmundsson. 1999. Antibacterial components in bronchoalveolar lavage fluid from healthy individuals and sarcoidosis patients. American Journal of Respiratory & Critical Care Medicine 160: 283-290.
Agerberth, B., H. Gunne, J. Odeberg, P. Kogner, H. G. Boman, and G. H. Gudmundsson. 1995. FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis. Proceedings of the National Academy of Sciences of the United States of America 92: 195-199.
Agerberth, B., H. Gunne, J. Odeberg, P. Kogner, H. G. Boman, and G. H. Gudmundsson. 1996. PR-39, a proline-rich peptide antibiotic from pig, and FALL-39, a tentative human counterpart. Veterinary Immunology and Immunopathology 54: 127-131.
Agerberth, B., J. Y. Lee, T. Bergman, M. Carlquist, H. G. Boman, V. Mutt, and H. Jornvall. 1991. Amino acid sequence of PR-39 isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides. European Journal of Biochemistry 202: 849-854.
Al-Mehdi, A. B., G. Zhao, C. Dodia, K. Tozawa, K. Costa, V. Muzykantov, C. Ross, F. Blecha, M. Dinauer, and A. B. Fisher. 1998. Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+. Circulation Research 83: 730-737.
Bals, R., X. Wang, Z. Wu, T. Freeman, V. Bafna, M. Zasloff, and J. M. Wilson. 1998. Human beta-defensin 2 is a salt-sensitive peptide antibiotic expressed in human lung. Journal of Clinical Investigation 102: 874-880.
Bals, R., D. J. Weiner, R. L. Meegalla, and J. M. Wilson. 1999. Transfer of a cathelicidin peptide antibiotic gene restores bacterial killing in a cystic fibrosis xenograft model. Journal of Clinical Investigation 103: 1113-1117.
Bernet-Camard, M. F., M. H. Coconnier, S. Hudault, and A. L. Servin. 1996. Differentiation-associated antimicrobial functions in human colon adenocarcinoma cell lines. Experimental Cell Research 226: 80-89.
Boman, H. G., B. Agerberth, and A. Boman. 1993. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine. Infection and Immunity 61: 2978-2984.
Bonetto, V., M. Andersson, T. Bergman, R. Sillard, A. Norberg, V. Mutt, and H. Jornvall. 1999. Spleen antibacterial peptides: high levels of PR-39 and presence of two forms of NK-lysin. Cellular and Molecular Life Sciences 56: 174-178.
Cabiaux, V., B. Agerberth, J. Johansson, F. Homble, E. Goormaghtigh, and J. M. Ruysschaert. 1994. Secondary structure and membrane interaction of PR-39, a Pro rich antibacterial peptide. European Journal of Biochemistry 224: 1019-1027.
Castle, M., A. Nazarian, S. S. Yi, and P. Tempst. 1999. Lethal effects of apidaecin on Escherichia coli involve sequential molecular interactions with diverse targets. Journal of Biological Chemistry 274: 32555-32564.
Chan, Y. R., and R. L. Gallo. 1998. PR-39, a syndecan-inducing antimicrobial peptide, binds and affects p130 (Cas). Journal of Biological Chemistry 273: 28978-28985.
Chan, Y. R., M. Zanetti, R. Gennaro, and R. L. Gallo. 2001. Anti-microbial activity and cell binding are controlled by sequence determinants in the anti-microbial peptide PR-39. The Journal of Investigative Dermatology 116: 230-235.
Frohm, M., B. Agerberth, G. Ahangari, M. Stahle-Backdahl, S. Liden, H. Wigzell, and G. H. Gudmundsson. 1997. The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders. Journal of Biological Chemistry 272: 15258-15263.
Gallo, R. L., M. Ono, T. Povsic, C. Page, E. Eriksson, M. Klagsbrun, and M. Bernfield. 1994. Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds. Proceedings of the National Academy of Sciences of the United States of America 91: 11035-11039.
Gao, Y., S. Lecker, M. J. Post, A. J. Hietaranta, J. Li, R. Volk, M. Li, K. Sato, A. K. Saluja, M. L. Steer, A. L. Goldberg, and M. Simons. 2000. Inhibition of ubiquitin-proteasome pathway-mediated I kappa B alpha degradation by a naturally occurring antibacterial peptide. The Journal of Clinical Investigation 106: 439-448.
Gudmundsson, G. H., B. Agerberth, J. Odeberg, T. Bergman, B. Olsson, and R. Salcedo. 1996. The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes. European Journal of Biochemistry 238: 325-332.
Gudmundsson, G. H., and B. Agerberth. 1999. Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system. Journal of Immunological Methods 232: 45-54.
Gudmundsson, G. H., K. P. Magnusson, B. P. Chowdhary, M. Johansson, L. Andersson, and H. G. Boman. 1995. Structure of the gene for porcine peptide antibiotic PR-39, a cathelin gene family member: comparative mapping of the locus for the human peptide antibiotic FALL-39. Proceedings of the National Academy of Sciences of the United States of America 92: 7085-7089.
Hoffmeyer, M. R., R. Scalia, C. R. Ross, S. P. Jones, and D. J. Lefer. 2000. PR-39, a potent neutrophil inhibitor, attenuates myocardial ischemia-reperfusion injury in mice. American Journal of Physiology: Heart and Circulatory Physiology 279: 2824-2828.
Huang, H. J., C. R. Ross, and F. Blecha. 1997. Chemoattractant properties of PR-39, a neutrophil antibacterial peptide. Journal of Leukocyte Biology 61: 624-629.
Hugosson, M., D. Andreu, H. G. Boman, and E. Glaser. 1994. Antibacterial peptides and mitochondrial presequences affect mitochondrial coupling, respiration and protein import. European Journal of Biochemistry 223: 1027-1033.
Ikeda, Y., L. H. Young, R. Scalia, C. R. Ross, and A. M. Lefer. 2001. PR-39, a proline/arginine-rich antimicrobial peptide, exerts cardioprotective effects in myocardial ischemia-reperfusion. Cardiovascular Research 49: 69-77.
Korthuis, R. J., D. C. Gute, F. Blecha, and C. R. Ross. 1999. PR-39, a proline/arginine-rich antimicrobial peptide, prevents postischemic microvascular dysfunction. American Journal of Physiology 277: 1007-1013.
Li, J., L. F. Brown, R. J. Laham, R. Volk, and M. Simons. 1997. Macrophage-dependent regulation of syndecan gene expression. Cardiovascular Research 81: 785-796.
Li, J., M. Post, R. Volk, Y. Gao, M. Li, C. Metais, K. Sato, J. Tsai, W. Aird, R. D. Rosenberg, T. G. Hampton, F. Sellke, P. Carmeliet, and M. Simons. 2000. PR39, a peptide regulator of angiogenesis. Nature Medicine 6: 49-55.
Linde, C. M., S. E. Hoffner, E. Refai, and M. Andersson. 2001. In vitro activity of PR-39, a proline-arginine-rich peptide, against susceptible and multi-drug-resistant Mycobacterium tuberculosis. The Journal of Antimicrobial Chemotherapy 47: 575-580.
Nagaoka, I., S. Hirota, S. Yomogida, A. Ohwada, and M. Hirata. 2000. Synergistic actions of antibacterial neutrophil defensins and cathelicidins. Inflammation Research 49: 73-79.
Ohtake, T., Y. Fujimoto, K. Ikuta, H. Saito, M. Ohhira, M. Ono, and Y. Kohgo. 1999. Proline-rich antimicrobial peptide, PR-39 gene transduction altered invasive activity and actin structure in human hepatocellular carcinoma cells. British Journal of Cancer 81: 393-403.
Piter, J., and Branes. 1997. Molecules in focus Nuclear Factor-kB. Cell Biology International 29: 867-870.
Piers, K. L., M. H. Brown, and R. E. Hancock. 1993. Recombinant DNA procedures for producing small antimicrobial cationic peptides in bacteria. Gene 134: 7-13.
Pleskach, V. A., G. M. Aleshina, I. V. Artsybasheva, O. V. Shamova, I.V. Kozhukharova, T. A. Goilo, and V. N. Kokriakov. 2000. Cytotoxic and mitogenic effect of antimicrobial peptides from neutrophils on cultured cells. Tsitologiia 42: 228-234.
Scocchi, M., D. Romeo, and M. Zanetti. 1994. Molecular cloning of Bac7, a proline- and arginine-rich antimicrobial peptide from bovine neutrophils. FEBS Letters 352: 197-200.
Shi, J., C. R. Ross, M. M. Chengappa, and F. Blecha. 1994. Identification of a proline-arginine-rich antibacterial peptide from neutrophils that is analogous to PR-39, an antibacterial peptide from the small intestine. Journal of Leukocyte Biology 56: 807-811.
Shi, J., C. R. Ross, M. M. Chengappa, M. J. Sylte, D. S. McVey, and F. Blecha. 1996. Antibacterial activity of a synthetic peptide (PR-26) derived from PR-39, a proline-arginine-rich neutrophil antimicrobial peptide. Antimicrobial Agents and Chemotherapy 40: 115-121.
Shi, J., C. R. Ross, T. L. Leto, and F. Blecha. 1996. PR-39, a proline-rich antibacterial peptide that inhibits phagocyte NADPH oxidase activity by binding to Src homology 3 domains of p47 phox. Proceedings of the National Academy of Sciences of the United States of America 93: 6014-6018.
Shi, J., G. Zhang, H. Wu, C. Ross, F. Blecha, and T. Ganz. 1999. Porcine epithelial beta-defensin 1 is expressed in the dorsal tongue at antimicrobial concentrations. Infection and Immunity 67: 3121-3127.
Steiner, H., D. Hultmark, A. Engstrom, H. Bennich, and H. G. Boman. 1981. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292: 246-248.
Storici, P., and M. A. Zanetti. 1993. cDNA derived from pig bone marrow cells predicts a sequence identical to the intestinal antibacterial peptide PR-39. Biochemical and Biophysical Research Communications 196: 1058-1065.
Tossi, A., M. Scocchi, M. Zanetti, P. Storici, and R. Gennaro. 1995. PMAP-37, a novel antibacterial peptide from pig myeloid cells. cDNA cloning, chemical synthesis and activity. European Journal of Biochemistry 228: 941-946
Toyoshima, E., Y. Ohsaki, Y. Nishigaki, Y. Fujimoto, Y. Kohgo, and K. Kikuchi. 2001. Expression of syndecan-1 is common in human lung cancers independent of expression of epidermal growth factor receptor. Lung Cancer 31: 193-202.
Vunnam, S., P. Juvvadi, and R. B. Merrifield. 1997. Synthesis and antibacterial action of cecropin and proline-arginine-rich peptides from pig intestine. Journal of Peptide Research 49: 59-66.
Weinberg, A., S. Krisanaprakornkit, and B. A. Dale. 1998. Epithelial antimicrobial peptides: review and significance for oral applications. Critical Reviews in Oral Biology & Medicine 9: 399-414.
Wu, H., G. Zhang, J. E. Minton, C. R. Ross, and F. Blecha. 2000. Regulation of cathelicidin gene expression: induction by lipopolysaccharide, interleukin-6, retinoic acid, and Salmonella enterica serovar typhimurium infection. Infection and Immunity 68: 5552-5558.
Wu, H., G. Zhang, C. R. Ross, and F. Blecha. 1999. Cathelicidin gene expression in porcine tissues: roles in ontogeny and tissue specificity. Infection and Immunity 67: 439-442.
Zanetti, M., R. Gennaro, and D. Romeo. 1995. Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain. FEBS Letters 374: 1-5.
Zanetti, M., L. Litteri, R. Gennaro, H. Horstmann, and D. Romeo. 1990. Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor molecules stored in the large granules. Journal of Cell Biology 111: 1363-1371.
Zhang, L., T. Falla, M. Wu, S. Fidai, J. Burian, W. Kay, and R. E. Hancock. 1998. Determinants of recombinant production of antimicrobial cationic peptides and creation of peptide variants in bacteria. Biochemical & Biophysical Research Communications 247: 674-680.
Zhang, G., C. R. Ross, S. S. Dritz, J. C. Nietfeld, and F. Blecha. 1997. Salmonella infection increases porcine antibacterial peptide concentrations in serum. Clinical and Diagnostic Laboratory Immunology 4: 774-777.
Zhao, C., T. Ganz, and R. I. Lehrer. 1995. Structures of genes for two cathelin-associated antimicrobial peptides: prophenin-2 and PR-39. FEBS Letters 376: 130-134.
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