鱼类融合抗菌肽重组表达载体构建及蛋白表达研究
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摘要
内源性的抗菌肽是天然免疫的重要组成部分,抗菌肽以其广谱的杀菌作用、高效的抗菌活性、靶菌株难以产生抗药性以及天然、安全等优点,有潜力开发成为具有一种优质、高效、无公害、无残留的新型天然抗生素,应用于海洋水产动物病害防治。
论文第一部分设计并合成了三种海洋鱼类抗菌肽Moronecidin-Hepcidin-Pleurocidin融合基因的全序列,在设计过程中考虑了外源基因在原核、真核表达系统中表达所需的一系列因素。成功构建了融合抗菌肽基因的表达载体,包括酵母真核表达载体和原核表达载体,分别在酵母表达体系中和原核表达体系进行表达,以期获得高活性、低成本的水产动物抗菌制剂。
论文第二部分对河豚的TLR1胞外区进行了原核表达研究,同时采用改良的重叠延伸PCR方法,成功获得鱼类抗菌肽hepcidin与TLR1的胞外区相融合的重组分子,并构建了重组表达载体,实现在原核表达系统中的表达。研究工作为利用模式识别受体高效识别病原相关分子模式的特点进行构建能够泛特异性识别并富集于病原菌的高效抗菌肽,打下了基础。
Endogenous antimicrobial peptides are one of the most important effectors in the innate immunity. Antimicrobial peptides have a broad-spectrum antimicrobial activity and the bacteria are hardly resistant to them. So it provides a novel native antibiotic in prevention and cure of the fish diseases.
In the first part of the research, in combination of the common character between the eukaryotic and prokaryotic expression system, we desighed and synthesized the fusion gene, cascade connection of three marine antimicrobial peptide, which is coined as Moronecidin-Hepcidin-Pleurocidin. After successfully constructed the eukaryotic and prokaryotic expression vectors containing the fusion antimicrobial peptide, we expressed the fusion protein in the eukaryotic and prokaryotic expression systems to obtain a new antimicrobial bioproduct.
In the second part of the research, we expressed the extracellular part of TLR1 protein in prokaryotic expression system. Meanwhile, utilizing the modified methods of over-lapping extension PCR, we constructed the fusion gene harboring the hepcidin gene and extracellular part of TLR1 gene. After restructuring the recombinant plasmid which was inserted with the fusion gene, we expressed the fusion protein in the prokaryotic expression system. This research presents a novel method to gain efficient and targeting effect antimicrobial peptides.
论文第一部分设计并合成了三种海洋鱼类抗菌肽Moronecidin-Hepcidin-Pleurocidin融合基因的全序列,在设计过程中考虑了外源基因在原核、真核表达系统中表达所需的一系列因素。成功构建了融合抗菌肽基因的表达载体,包括酵母真核表达载体和原核表达载体,分别在酵母表达体系中和原核表达体系进行表达,以期获得高活性、低成本的水产动物抗菌制剂。
论文第二部分对河豚的TLR1胞外区进行了原核表达研究,同时采用改良的重叠延伸PCR方法,成功获得鱼类抗菌肽hepcidin与TLR1的胞外区相融合的重组分子,并构建了重组表达载体,实现在原核表达系统中的表达。研究工作为利用模式识别受体高效识别病原相关分子模式的特点进行构建能够泛特异性识别并富集于病原菌的高效抗菌肽,打下了基础。
Endogenous antimicrobial peptides are one of the most important effectors in the innate immunity. Antimicrobial peptides have a broad-spectrum antimicrobial activity and the bacteria are hardly resistant to them. So it provides a novel native antibiotic in prevention and cure of the fish diseases.
In the first part of the research, in combination of the common character between the eukaryotic and prokaryotic expression system, we desighed and synthesized the fusion gene, cascade connection of three marine antimicrobial peptide, which is coined as Moronecidin-Hepcidin-Pleurocidin. After successfully constructed the eukaryotic and prokaryotic expression vectors containing the fusion antimicrobial peptide, we expressed the fusion protein in the eukaryotic and prokaryotic expression systems to obtain a new antimicrobial bioproduct.
In the second part of the research, we expressed the extracellular part of TLR1 protein in prokaryotic expression system. Meanwhile, utilizing the modified methods of over-lapping extension PCR, we constructed the fusion gene harboring the hepcidin gene and extracellular part of TLR1 gene. After restructuring the recombinant plasmid which was inserted with the fusion gene, we expressed the fusion protein in the prokaryotic expression system. This research presents a novel method to gain efficient and targeting effect antimicrobial peptides.
引文
1. Lauth X, Babon JJ, Stannard JA, Singh S, Nizet V, Carlberg JM, Ostland VE, Pennington MW, Norton RS, Westerman ME: Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections. J Biol Chem 2005, 280(10):9272-9282.
2. Salerno G, Parrinello N, Roch P, Cammarata M: cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax. Comp Biochem Physiol B Biochem Mol Biol 2007, 146(4):521-529.
3. Lauth X, Shike H, Burns JC, Westerman ME, Ostland VE, Carlberg JM, Van Olst JC, Nizet V, Taylor SW, Shimizu C et al: Discovery and characterization of two isoforms of moronecidin, a novel antimicrobial peptide from hybrid striped bass. J Biol Chem 2002,277(7):5030-5039.
4. Moon WJ, Hwang DK, Park EJ, Kim YM, Chae YK: Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin. Protein Expr Purif 2007,51(2):141-146.
5. Lee SA, Kim YK, Lim SS, Zhu WL, Ko H, Shin SY, Hahm KS, Kim Y: Solution structure and cell selectivity of piscidin 1 and its analogues. Biochemistry 2007, 46(12):3653-3663.
6. Xu Q, Cheng CH, Hu P, Ye H, Chen Z, Cao L, Chen L, Shen Y, Chen L: Adaptive evolution of hepcidin genes in antarctic notothenioid fishes. Mol Biol Evol 2008,25(6):1099-1112.
7. Chen J, Shi YH, Li MY: Changes in transferrin and hepcidin genes expression in the liver of the fish Pseudosciaena crocea following exposure to cadmium. Arch Toxicol 2008, 82(8):525-530.
8. Hu X, Camus AC, Aono S, Morrison EE, Dennis J, Nusbaum KE, Judd RL, Shi J: Channel catfish hepcidin expression in infection and anemia. Comp Immunol Microbiol Infect Dis 2007,30(1):55-69.
9. Chen SL, Xu MY, Ji XS, Yu GC, Liu Y: Cloning, characterization, and expression analysis of hepcidin gene from red sea bream (Chrysophrys major). Antimicrob Agents Chemother 2005,49(4): 1608-1612.
10. Hu X, Ward C, Aono S, Lan L, Dykstra C, Kemppainen RJ, Morrison EE, Shi J: Comparative analysis of Xenopus tropicalis hepcidin I and hepcidin II genes. Gene 2008,426(1-2):91-97.
11. Harrison-Findik DD, Schafer D, Klein E, Timchenko NA, Kulaksiz H, Clemens D, Fein E, Andriopoulos B, Pantopoulos K, Gollan J: Alcohol metabolism-mediated oxidative stress down-regulates hepcidin transcription and leads to increased duodenal iron transporter expression. J Biol Chem 2006, 281(32):22974-22982.
12. Rodrigues PN, Vazquez-Dorado S, Neves JV, Wilson JM: Dual function of fish hepcidin: response to experimental iron overload and bacterial infection in sea bass (Dicentrarchus labrax). Dev Comp Immunol 2006, 30(12):1156-1167.
13. Padhi A, Verghese B: Evidence for positive Darwinian selection on the hepcidin gene of Perciform and Pleuronectiform fishes. Mol Divers 2007, 11(3-4):119-130.
14. Kang CJ, Xue JF, Liu N, Zhao XF, Wang JX: Characterization and expression of a new subfamily member of penaeidin antimicrobial peptides (penaeidin 5) from Fenneropenaeus chinensis. Mol Immunol 2007, 44(7):1535-1543.
15. Segat L, Pontillo A, Milanese M, Tossi A, Crovella S: Evolution of the hepcidin gene in primates. BMC Genomics 2008, 9:120.
16. Srinivasulu B, Syvitski R, Seo JK, Mattatall NR, Knickle LC, Douglas SE: Expression, purification and structural characterization of recombinant hepcidin, an antimicrobial peptide identified in Japanese flounder, Paralichthys olivaceus. Protein Expr Purif 2008,61(1):36-44.
17. Kim YO, Hong S, Nam BH, Lee JH, Kim KK, Lee SJ: Molecular cloning and expression analysis of two hepcidin genes from olive flounder Paralichthys olivaceus. Biosci Biotechnol Biochem 2005, 69(7): 1411-1414.
18. Hilton KB, Lambert LA: Molecular evolution and characterization of hepcidin gene products in vertebrates. Gene 2008, 415(l-2):40-48.
19. Cuesta A, Meseguer J, Esteban MA: The antimicrobial peptide hepcidin exerts an important role in the innate immunity against bacteria in the bony fish gilthead seabream. Mol Immunol 2008, 45(8):2333-2342.
20. Mason AJ, Chotimah IN, Bertani P, Bechinger B: A spectroscopic study of the membrane interaction of the antimicrobial peptide Pleurocidin. Mol Membr Biol 2006, 23(2): 185-194.
21. Cole AM, Weis P, Diamond G: Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter flounder. J Biol Chem 1997,272(18): 12008-12013.
22. Brocal I, Falco A, Mas V, Rocha A, Perez L, Coll JM, Estepa A: Stable expression of bioactive recombinant pleurocidin in a fish cell line. Appl Microbiol Biotechnol 2006, 72(6): 1217-1228.
23. Chen P, Novak J, Kirk M, Barnes S, Qi F, Caufield PW: Structure-activity study of the lantibiotic mutacin II from Streptococcus mutans T8 by a gene replacement strategy. Appl Environ Microbiol 1998,64(7):2335-2340.
24. Patrzykat A, Zhang L, Mendoza V, Iwama GK, Hancock RE: Synergy of histone-derived peptides of coho salmon with lysozyme and flounder pleurocidin. Antimicrob Agents Chemother 2001, 45(5):1337-1342.
25. Wang JX, Zhao XF, Liang YL, Li L, Zhang W, Ren Q, Wang LC, Wang LY: Molecular characterization and expression of the antimicrobial peptide defensin from the housefly (Musca domestica). Cell Mol Life Sci 2006, 63(24):3072-3082.
26. Patrzykat A, Friedrich CL, Zhang L, Mendoza V, Hancock RE: Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli. Antimicrob Agents Chemother 2002, 46(3):605-614.
27. Lee JH, Kim MS, Cho JH, Kim SC: Enhanced expression of tandem multimers of the antimicrobial peptide buforin II in Escherichia coli by the DEAD-box protein and trxB mutant. Appl Microbiol Biotechnol 2002, 58(6):790-796.
28. Chen SL, Li W, Meng L, Sha ZX, Wang ZJ, Ren GC: Molecular cloning and expression analysis of a hepcidin antimicrobial peptide gene from turbot (Scophthalmus maximus). Fish Shellfish Immunol 2007,22(3):172-181.
29. Dong ZY, Zhang YZ: Molecular cloning and expression of yak (Bos grunniens) lactoferrin cDNA in Pichia pastoris. Biotechnol Lett 2006, 28(16):1285-1292.
30. Tian ZG, Teng D, Yang YL, Luo J, Feng XJ, Fan Y, Zhang F, Wang JH: Multimerization and fusion expression of bovine lactoferricin derivative LfcinB15-W4,10 in Escherichia coli. Appl Microbiol Biotechnol 2007, 75(1):117-124.
31. Zhou Y, Cao W, Wang J, Ma Y, Wei D: Comparison of expression of monomeric and multimeric adenoregulin genes in Escherichia coli and Pichia pastorias. Protein Pept Lett 2005,12(4):349-355.
32. Morin KM, Arcidiacono S, Beckwitt R, Mello CM: Recombinant expression of indolicidin concatamers in Escherichia coli. Appl Microbiol Biotechnol 2006, 70(6):698-704.
33. Zhong Z, Xu Z, Peng L, Huang L, Fang X, Cen P: Tandem repeat mhBD2 gene enhance the soluble fusion expression of hBD2 in Escherichia coli. Appl Microbiol Biotechnol 2006, 71(5):661-667.
34. Wang YQ, Cai JY: High-level expression of acidic partner-mediated antimicrobial peptide from tandem genes in Escherichia coli. Appl Biochem Biotechnol 2007, 141(2-3):203-213.
35. Lee JH, Minn I, Park CB, Kim SC: Acidic peptide-mediated expression of the antimicrobial peptide buforin II as tandem repeats in Escherichia coli. Protein Expr Purif 1998, 12(1):53-60.
36. Kim HK, Chun DS, Kim JS, Yun CH, Lee JH, Hong SK, Kang DK: Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli. Appl Microbiol Biotechnol 2006, 72(2):330-338.
37. Xu J, Li W, Wu J, Zhang Y, Zhu Z, Liu J, Hu Z: Stability of plasmid and expression of a recombinant gonadotropin-releasing hormone (GnRH) vaccine in Escherichia coli. Appl Microbiol Biotechnol 2006, 73(4):780-788.
38. Chen H, Xu Z, Xu N, Cen P: Efficient production of a soluble fusion protein containing human beta-defensin-2 in E. coli cell-free system. J Biotechnol 2005,115(3):307-315.
39. Zhang H, Yuan Q, Zhu Y, Ma R: Expression and preparation of recombinant hepcidin in Escherichia coli. Protein Expr Purif 2005, 41(2):409-416.
40. Munoz M, Vandenbulcke F, Gueguen Y, Bachere E: Expression of penaeidin antimicrobial peptides in early larval stages of the shrimp Penaeus vannamei. Dev Comp Immunol 2003,27(4):283-289.
41. Destoumieux D, Bulet P, Strub JM, Van Dorsselaer A, Bachere E: Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp. Eur J Biochem 1999, 266(2):335-346.
42. Ogawa M, Nakamura S, Atsuchi T, Tamiya T, Tsuchiya T, Nakai S: Macromolecular antimicrobial glycoprotein, achacin, expressed in a methylotrophic yeast Pichia pastoris. FEES Lett 1999, 448(1):41-44.
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2. Salerno G, Parrinello N, Roch P, Cammarata M: cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax. Comp Biochem Physiol B Biochem Mol Biol 2007, 146(4):521-529.
3. Lauth X, Shike H, Burns JC, Westerman ME, Ostland VE, Carlberg JM, Van Olst JC, Nizet V, Taylor SW, Shimizu C et al: Discovery and characterization of two isoforms of moronecidin, a novel antimicrobial peptide from hybrid striped bass. J Biol Chem 2002,277(7):5030-5039.
4. Moon WJ, Hwang DK, Park EJ, Kim YM, Chae YK: Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin. Protein Expr Purif 2007,51(2):141-146.
5. Lee SA, Kim YK, Lim SS, Zhu WL, Ko H, Shin SY, Hahm KS, Kim Y: Solution structure and cell selectivity of piscidin 1 and its analogues. Biochemistry 2007, 46(12):3653-3663.
6. Xu Q, Cheng CH, Hu P, Ye H, Chen Z, Cao L, Chen L, Shen Y, Chen L: Adaptive evolution of hepcidin genes in antarctic notothenioid fishes. Mol Biol Evol 2008,25(6):1099-1112.
7. Chen J, Shi YH, Li MY: Changes in transferrin and hepcidin genes expression in the liver of the fish Pseudosciaena crocea following exposure to cadmium. Arch Toxicol 2008, 82(8):525-530.
8. Hu X, Camus AC, Aono S, Morrison EE, Dennis J, Nusbaum KE, Judd RL, Shi J: Channel catfish hepcidin expression in infection and anemia. Comp Immunol Microbiol Infect Dis 2007,30(1):55-69.
9. Chen SL, Xu MY, Ji XS, Yu GC, Liu Y: Cloning, characterization, and expression analysis of hepcidin gene from red sea bream (Chrysophrys major). Antimicrob Agents Chemother 2005,49(4): 1608-1612.
10. Hu X, Ward C, Aono S, Lan L, Dykstra C, Kemppainen RJ, Morrison EE, Shi J: Comparative analysis of Xenopus tropicalis hepcidin I and hepcidin II genes. Gene 2008,426(1-2):91-97.
11. Harrison-Findik DD, Schafer D, Klein E, Timchenko NA, Kulaksiz H, Clemens D, Fein E, Andriopoulos B, Pantopoulos K, Gollan J: Alcohol metabolism-mediated oxidative stress down-regulates hepcidin transcription and leads to increased duodenal iron transporter expression. J Biol Chem 2006, 281(32):22974-22982.
12. Rodrigues PN, Vazquez-Dorado S, Neves JV, Wilson JM: Dual function of fish hepcidin: response to experimental iron overload and bacterial infection in sea bass (Dicentrarchus labrax). Dev Comp Immunol 2006, 30(12):1156-1167.
13. Padhi A, Verghese B: Evidence for positive Darwinian selection on the hepcidin gene of Perciform and Pleuronectiform fishes. Mol Divers 2007, 11(3-4):119-130.
14. Kang CJ, Xue JF, Liu N, Zhao XF, Wang JX: Characterization and expression of a new subfamily member of penaeidin antimicrobial peptides (penaeidin 5) from Fenneropenaeus chinensis. Mol Immunol 2007, 44(7):1535-1543.
15. Segat L, Pontillo A, Milanese M, Tossi A, Crovella S: Evolution of the hepcidin gene in primates. BMC Genomics 2008, 9:120.
16. Srinivasulu B, Syvitski R, Seo JK, Mattatall NR, Knickle LC, Douglas SE: Expression, purification and structural characterization of recombinant hepcidin, an antimicrobial peptide identified in Japanese flounder, Paralichthys olivaceus. Protein Expr Purif 2008,61(1):36-44.
17. Kim YO, Hong S, Nam BH, Lee JH, Kim KK, Lee SJ: Molecular cloning and expression analysis of two hepcidin genes from olive flounder Paralichthys olivaceus. Biosci Biotechnol Biochem 2005, 69(7): 1411-1414.
18. Hilton KB, Lambert LA: Molecular evolution and characterization of hepcidin gene products in vertebrates. Gene 2008, 415(l-2):40-48.
19. Cuesta A, Meseguer J, Esteban MA: The antimicrobial peptide hepcidin exerts an important role in the innate immunity against bacteria in the bony fish gilthead seabream. Mol Immunol 2008, 45(8):2333-2342.
20. Mason AJ, Chotimah IN, Bertani P, Bechinger B: A spectroscopic study of the membrane interaction of the antimicrobial peptide Pleurocidin. Mol Membr Biol 2006, 23(2): 185-194.
21. Cole AM, Weis P, Diamond G: Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter flounder. J Biol Chem 1997,272(18): 12008-12013.
22. Brocal I, Falco A, Mas V, Rocha A, Perez L, Coll JM, Estepa A: Stable expression of bioactive recombinant pleurocidin in a fish cell line. Appl Microbiol Biotechnol 2006, 72(6): 1217-1228.
23. Chen P, Novak J, Kirk M, Barnes S, Qi F, Caufield PW: Structure-activity study of the lantibiotic mutacin II from Streptococcus mutans T8 by a gene replacement strategy. Appl Environ Microbiol 1998,64(7):2335-2340.
24. Patrzykat A, Zhang L, Mendoza V, Iwama GK, Hancock RE: Synergy of histone-derived peptides of coho salmon with lysozyme and flounder pleurocidin. Antimicrob Agents Chemother 2001, 45(5):1337-1342.
25. Wang JX, Zhao XF, Liang YL, Li L, Zhang W, Ren Q, Wang LC, Wang LY: Molecular characterization and expression of the antimicrobial peptide defensin from the housefly (Musca domestica). Cell Mol Life Sci 2006, 63(24):3072-3082.
26. Patrzykat A, Friedrich CL, Zhang L, Mendoza V, Hancock RE: Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli. Antimicrob Agents Chemother 2002, 46(3):605-614.
27. Lee JH, Kim MS, Cho JH, Kim SC: Enhanced expression of tandem multimers of the antimicrobial peptide buforin II in Escherichia coli by the DEAD-box protein and trxB mutant. Appl Microbiol Biotechnol 2002, 58(6):790-796.
28. Chen SL, Li W, Meng L, Sha ZX, Wang ZJ, Ren GC: Molecular cloning and expression analysis of a hepcidin antimicrobial peptide gene from turbot (Scophthalmus maximus). Fish Shellfish Immunol 2007,22(3):172-181.
29. Dong ZY, Zhang YZ: Molecular cloning and expression of yak (Bos grunniens) lactoferrin cDNA in Pichia pastoris. Biotechnol Lett 2006, 28(16):1285-1292.
30. Tian ZG, Teng D, Yang YL, Luo J, Feng XJ, Fan Y, Zhang F, Wang JH: Multimerization and fusion expression of bovine lactoferricin derivative LfcinB15-W4,10 in Escherichia coli. Appl Microbiol Biotechnol 2007, 75(1):117-124.
31. Zhou Y, Cao W, Wang J, Ma Y, Wei D: Comparison of expression of monomeric and multimeric adenoregulin genes in Escherichia coli and Pichia pastorias. Protein Pept Lett 2005,12(4):349-355.
32. Morin KM, Arcidiacono S, Beckwitt R, Mello CM: Recombinant expression of indolicidin concatamers in Escherichia coli. Appl Microbiol Biotechnol 2006, 70(6):698-704.
33. Zhong Z, Xu Z, Peng L, Huang L, Fang X, Cen P: Tandem repeat mhBD2 gene enhance the soluble fusion expression of hBD2 in Escherichia coli. Appl Microbiol Biotechnol 2006, 71(5):661-667.
34. Wang YQ, Cai JY: High-level expression of acidic partner-mediated antimicrobial peptide from tandem genes in Escherichia coli. Appl Biochem Biotechnol 2007, 141(2-3):203-213.
35. Lee JH, Minn I, Park CB, Kim SC: Acidic peptide-mediated expression of the antimicrobial peptide buforin II as tandem repeats in Escherichia coli. Protein Expr Purif 1998, 12(1):53-60.
36. Kim HK, Chun DS, Kim JS, Yun CH, Lee JH, Hong SK, Kang DK: Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli. Appl Microbiol Biotechnol 2006, 72(2):330-338.
37. Xu J, Li W, Wu J, Zhang Y, Zhu Z, Liu J, Hu Z: Stability of plasmid and expression of a recombinant gonadotropin-releasing hormone (GnRH) vaccine in Escherichia coli. Appl Microbiol Biotechnol 2006, 73(4):780-788.
38. Chen H, Xu Z, Xu N, Cen P: Efficient production of a soluble fusion protein containing human beta-defensin-2 in E. coli cell-free system. J Biotechnol 2005,115(3):307-315.
39. Zhang H, Yuan Q, Zhu Y, Ma R: Expression and preparation of recombinant hepcidin in Escherichia coli. Protein Expr Purif 2005, 41(2):409-416.
40. Munoz M, Vandenbulcke F, Gueguen Y, Bachere E: Expression of penaeidin antimicrobial peptides in early larval stages of the shrimp Penaeus vannamei. Dev Comp Immunol 2003,27(4):283-289.
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