中国林蛙皮肤抗菌肽temporin-1CEa基因的串联融合表达
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摘要
抗菌肽(antibacterial peptides,简称ABP)是一类小分子肽类物质,由生物体特定基因编码,具有高效的杀菌能力和广谱的抗菌活性。皮肤抗菌肽是两栖类先天防御系统的主要组成部分,是两栖类抵御病原菌的武器。在当今抗生素滥用而引发大量耐药菌株产生的情况下,具有独特抗菌作用机理的两栖类皮肤抗菌肽具有广阔的开发和应用前景。然而,从两栖类皮肤中提取天然抗菌肽工艺复杂,难以规模化生产,用基因工程重组表达两栖类皮肤抗菌肽无疑是最经济的方法,是生产抗菌肽的最佳选择。由于两栖类皮肤抗菌肽对细菌有很强的杀伤作用,因此不宜在原核系统中直接进行表达,一般采用融合表达策略或在真核细胞中进行表达。
本研究采用串联融合表达策略在大肠杆菌表达系统中表达中国林蛙皮肤抗菌肽temporin-1CEa基因。人工合成中国林蛙皮肤抗菌肽temporin-1CEa基因序列,用PCR方法将6个temporin-1CEa基因同向串联连接,并在每个基因前面加上3个单核苷酸ATG,最后在6个串联temporin-1CEa基因片段的5′和3′端分别加入两个酶切位点:Sma I/ Hind III。目的片段全长339bp,将目的基因插入表达载体pET-42a(+),形成融合表达,测序结果证实temporin-1CEa基因6串联体正确插入表达载体pET-42a(+)中。并将阳性克隆转化至大肠杆菌表达菌BL21(DE3)。
IPTG诱导融合蛋白表达,SDS-PAGE凝胶电泳检测目的蛋白的表达情况。经过培养基pH,诱导剂浓度,诱导时间和诱导温度四种条件的筛选,最终确定在培养基pH为7.2,菌体OD600达到7-0.9时,IPTG至终浓度为0. 8mmo1/L,诱导温度30℃,诱导时间4小时条件下,目的蛋白表达情况最好,表达产物相对分子量约43KD,占菌体总蛋白量的10.8%。此时目的蛋白主要以可溶性蛋白的形式存在于细菌裂解液中。
将可溶性蛋白用GST亲和层析柱、Ni2 +-NTA亲和层析柱分别进行纯化,纯化后的目的蛋白纯度达到60%。Xa因子切割去除融合标签后,用滤纸片法对酶切产物进行了抗菌肽活性检测。为基因工程方法制备抗菌肽提供新的思路。
Antibacterial peptides (ABPs),small molecule peptides encoded by the specific gene, have broad-spectrum antimicrobial activity. Amphibian skin peptides are a kind of ABPs considered to be a main element of innate immunity, which are also the weapon for fighting back the pathogenic bacteria. For the emergence of multiple-antibiotic resistant strains, ABPs from amphibian skin play an important role in the development of novel antibiotics because their broad-spectrum antimicrobial activities and specific antimicrobial mechanism. However, the isolation and purification of natural bioactive peptides from amphibian skin is too difficult to meet the needs of large-scale production. So, it is an inevitable choice to produce bioactive peptides by the gene expression in prokaryotes or eukaryotes. Amphibian skin recombination peptides can not be expressed directly in prokaryotic expression system because the high antimicrobial activity. Therefore, the fusion expression system, or expression in eukaryotic cells for recombination anti-bacterial peptides expression often is used.
In this reseach ,we use recombinant technology and the fusion expression system to express six temporin-1CEa genes from the skin of Chinese brown frogs. Based on the amino acid sequence of temporin-1CEa,We synthesize temporin-1CEa gene sequence .We use PCR method to connect the six of temporin-1CEa genes, of which each gene is preceded by ATG codon. Lastly, at the 5 'and 3' end, two restriction sites of Sma I / Hind III were added, respectively. The 339bp fragment was constructed into the expressive vector pET-42a(+) for the expression of GST-temporin-1CEa fusion protein after six of temporin-1CE gene were synthesized in tandem.The sequencing analysis confirmed that the six of temporin-1CEa genes had been correctly inserted into vector pET-42a(+). The recombinant vectors were transformed into E.coIi BL21.
The GST-temporin-1CEa fusion proteins expressed in the host bacteria under the IPTG induction,,and analyzed with SDS-PAGE. Different induced temperature, pH, IPTG concentrations and the induction time were screened to determine the optimum inducing conditions. At last, we choose the condition as following: after bacteria growing in LB medium of pH7.2 to saturation, induced recombinant protein for 4h at 30℃with 0.8mM IPTG. The experimental results showed that the recombinant protein molecular weight is 43KD, which occupy 10.8% content of total bacterial protein and present in the form of soluble protein in bacterial lysates.
Fusion protein was purified with GSTrap FF affinity chromatography and HisTrap HP affinity chromatography. The purity of fusion protein is about 60% after purifying. After the fusion tag was cleaved by factor Xa, the antibacterial activity was tested. The construction of prokaryotic expressive plasmid of temporin-1CEa gene establishes a solid basis for further studying the amphibian skin antibacterial.
本研究采用串联融合表达策略在大肠杆菌表达系统中表达中国林蛙皮肤抗菌肽temporin-1CEa基因。人工合成中国林蛙皮肤抗菌肽temporin-1CEa基因序列,用PCR方法将6个temporin-1CEa基因同向串联连接,并在每个基因前面加上3个单核苷酸ATG,最后在6个串联temporin-1CEa基因片段的5′和3′端分别加入两个酶切位点:Sma I/ Hind III。目的片段全长339bp,将目的基因插入表达载体pET-42a(+),形成融合表达,测序结果证实temporin-1CEa基因6串联体正确插入表达载体pET-42a(+)中。并将阳性克隆转化至大肠杆菌表达菌BL21(DE3)。
IPTG诱导融合蛋白表达,SDS-PAGE凝胶电泳检测目的蛋白的表达情况。经过培养基pH,诱导剂浓度,诱导时间和诱导温度四种条件的筛选,最终确定在培养基pH为7.2,菌体OD600达到7-0.9时,IPTG至终浓度为0. 8mmo1/L,诱导温度30℃,诱导时间4小时条件下,目的蛋白表达情况最好,表达产物相对分子量约43KD,占菌体总蛋白量的10.8%。此时目的蛋白主要以可溶性蛋白的形式存在于细菌裂解液中。
将可溶性蛋白用GST亲和层析柱、Ni2 +-NTA亲和层析柱分别进行纯化,纯化后的目的蛋白纯度达到60%。Xa因子切割去除融合标签后,用滤纸片法对酶切产物进行了抗菌肽活性检测。为基因工程方法制备抗菌肽提供新的思路。
Antibacterial peptides (ABPs),small molecule peptides encoded by the specific gene, have broad-spectrum antimicrobial activity. Amphibian skin peptides are a kind of ABPs considered to be a main element of innate immunity, which are also the weapon for fighting back the pathogenic bacteria. For the emergence of multiple-antibiotic resistant strains, ABPs from amphibian skin play an important role in the development of novel antibiotics because their broad-spectrum antimicrobial activities and specific antimicrobial mechanism. However, the isolation and purification of natural bioactive peptides from amphibian skin is too difficult to meet the needs of large-scale production. So, it is an inevitable choice to produce bioactive peptides by the gene expression in prokaryotes or eukaryotes. Amphibian skin recombination peptides can not be expressed directly in prokaryotic expression system because the high antimicrobial activity. Therefore, the fusion expression system, or expression in eukaryotic cells for recombination anti-bacterial peptides expression often is used.
In this reseach ,we use recombinant technology and the fusion expression system to express six temporin-1CEa genes from the skin of Chinese brown frogs. Based on the amino acid sequence of temporin-1CEa,We synthesize temporin-1CEa gene sequence .We use PCR method to connect the six of temporin-1CEa genes, of which each gene is preceded by ATG codon. Lastly, at the 5 'and 3' end, two restriction sites of Sma I / Hind III were added, respectively. The 339bp fragment was constructed into the expressive vector pET-42a(+) for the expression of GST-temporin-1CEa fusion protein after six of temporin-1CE gene were synthesized in tandem.The sequencing analysis confirmed that the six of temporin-1CEa genes had been correctly inserted into vector pET-42a(+). The recombinant vectors were transformed into E.coIi BL21.
The GST-temporin-1CEa fusion proteins expressed in the host bacteria under the IPTG induction,,and analyzed with SDS-PAGE. Different induced temperature, pH, IPTG concentrations and the induction time were screened to determine the optimum inducing conditions. At last, we choose the condition as following: after bacteria growing in LB medium of pH7.2 to saturation, induced recombinant protein for 4h at 30℃with 0.8mM IPTG. The experimental results showed that the recombinant protein molecular weight is 43KD, which occupy 10.8% content of total bacterial protein and present in the form of soluble protein in bacterial lysates.
Fusion protein was purified with GSTrap FF affinity chromatography and HisTrap HP affinity chromatography. The purity of fusion protein is about 60% after purifying. After the fusion tag was cleaved by factor Xa, the antibacterial activity was tested. The construction of prokaryotic expressive plasmid of temporin-1CEa gene establishes a solid basis for further studying the amphibian skin antibacterial.
引文
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[2] Boman H G. Antibacterial peptides: key components needed in immunity. Cell, 1991, 65:205-207.
[3] Boman H G. Peptide antibiotics and their role innate immunity. Annau Revlmmunol,1995, 13:62
[4] Faye I, Pye A,Rasmuson B et a1.Insect Immunty.simultaneous induction of antibacterial activity and selective synthesis of some haemolymph protein in diapausing pupae of hyalophora cecropia and samia cythia. Infect Immun: 1975,12:1426-1438.
[5] Anna Christina C. Nascimento, Wagner Fontes, et al. Antimicrobial peptides from Anurans skin secretions. Protein and Peptide Letters, 2003, 10(3):227-238.
[6] Takumi O, Fujitoshi Y, Masco T, et al. Production and purification of a bacteriocin peptide produced by Lactococcus sp strain GM005, isolated from Miso-paste. Int J Food Microbiol, 2003, 87(1-2):153-159.
[7] Lehrer R I, Ganz T. Antimicrobial peptides in mammalian and insect host defence. CurrOpin Immunol, 1999, 11: 23-30.
[8] Orsi N. The antimicrobial activity of lactoferrin: current status and perspectives. Biometals. 2004 Jun;17(3):189-196.
[9] Schibli DJ, Epand RF, Vogeletal. Tryptophan-rich antimicrobial peptides: comparative properties and membrane interactions. Biochem Cell Biol. 2002. 80(5): 667-677.
[10] Yamanchi K. Tomita M Giehl, et al. Ahbacterial activity of lacteoferrin and a pepsin-derived lactoferrin peptide fragment. Infection and Immunity. 1993, 61(2): 719-728
[11] Zasl of M, et al. Proc Natl Acad Sci USA, 1987, 4(84):5449-5455.
[12]李孝东,袁建华.抗菌肽的研究进展「J].山东医药工业,2003 (1) :27-28.
[13] Tomita M , Bellamy W ,Takase M,et al. Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin. J Dairy Sci. 1991.
[14] Lee DG,Park Y,Kim H N,et a1.Antifungal mechanism of an antimicrobial peptide,HP(2-20),derived from N-terminus of Helicobacter pylori ribosomal protein L1 against Candida albicans.Biochem Biophys Res Commun,2002,291(4):1006-1013.
[15] Dekker N,Cox RC,Kramer RA,et al. Substrate specificity of the Integral membrane protease OmpT determined by spatially addressed peptide libraries. Biochemistry,2001,40(6): 1694-1701.
[16]谭显胜,李巍巍,王志明等.抗菌肽的研究进展.现代生物医学进展,2009,9(16):3153-3156.
[17] Fehlbaum P,Bulet P,Chernysh S ,et al. Structure-activity analysis of thanatin a 21-residue inducible insect defense peptide with sequence homology to frog skinantimicrobial peptides Proc Natl Acad Sci USA ,1996, (37):388-400.
[18] Bobek LA,Situ H. MUC7 20 -Mer:Investigation of Antimicrobial Activity secondary Structure and Possible Mechanism of Antifungal Action .Antimicrob Agents Chemother,2003,47(2):643-652.
[19] Christensen B, Fink H, Merrifield RB, Mauzerall D. Channel-forming properties of cecropins and related midel compounds incorporated into planar lipid membranes. Proc. Natl. Acad. Sci. USA. 1988, 85: 5072-5076.
[20]赵东红,戴祝英,周开亚.昆虫抗菌肽的功能、作用机理与分子生物学研究最新进展.生物工程进展,1999,19(5):14-17.
[21] Blazyk J, Wiegand R, Klein J, et al. A novel linear amphipathic beta-sheet cationic antimicrobial peptide with enhanced selectivity for bacterial lipids. J Biol Chem, 2001, 276(30): 27899-27906.
[22] Toney JH. Iseganan (Intra Biotics pharmaceuticals). Curr Opin Investig Drugs. 2002, 3(2): 225-228.
[23] Chen J, Falla TJ, Liu H, et al. Development of protegrins for the treatment and prevention flf oral mucositis: structure-activity relationships of synthetic protegrin analogues. Biopolumers, 2000, 55(1): 88-98.
[24]黄自然,廖富拨,郑青等.昆虫抗菌肽在医药上的应用.天然产品研究与开发.2000 Vo1.13 No.2:79-83.
[25] Park S, Park .S H, Ahn H C, Kim S, Kim S S, Lee B J. Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata. FEES Letters. 2001, 507:95-100.
[26] Issacson T, Soto A, Iwamuro S, et al. Antimicrobial peptides with atypical structural feature from the skin of the Japanese brown frogRana japonica. Peptides, 2002, 23(3) :419-425.
[27] Maloy WL, Kari UP. Structure-avtivity studies on magainins and other host defense peptides [J].Biopolymers (Peptide Science),1995, 37:105-122.
[28] Andreu D, Rivas L. Animal antimicrobial peptides:an overview [J].Biopolymers (Peptide Science),1998, 47(6):415-433.
[29] Lai R , Ye WJ , Ran YL , LüSQ , Yang DT. Antimicrobial peptide from the skin of Bombina maxima . Zool.Res., 1998,19(4):257-262.
[30] Barry D,Simmaco M,Boman H G. Gene-encoded peptide antibiotics and innate immunity Do 'animalcules 'have defence budgets?FEBS Letters ,1998,430(1-2): 130-134.
[31] Swendsen C L,Sphepard D. A study of antimicrobial effects of mountain dus匆salamander Mucus Jon Hasfjord:Nat Sci Seminar Abstracts ,1998.
[32] Hancock R E W Chapple D S. Peptide antibiotics. Antimicrobial Agents and Chemotherapy,1999,43 (6):1317-1323.
[33]乔红伟.中国林蛙基因工程抗菌肚制备及生物学特性研究:(博士学位论文).吉林:吉林大学,2006.
[34]徐进署,张双全.昆虫抗菌肽对病原微生物作用的研究进展[J].昆虫学报,2002, 45 (5):673-678.
[35] Fiordalisi J J, Fetter C H, TenHarmselA, et al. Synthesis and expression in Escherichia coli of a gene for x -bungarotoxin[J].Biochemistry, 1991, 30:10337-10343.
[36] Latham P W. Therapeutic peptides revisited Nature Biotechnology, 1999, 17:755-777.
[37] Simmaco M, Mignogna G, Barra D. Antimicrobial peptides from amphibian skin : what do they tell us ? Biopolymers ,1998 , 47 (6) : 435-450.
[38]陈晓平,詹冬玲,贾惠文等.抗茵肽MagaininⅡ基因的克隆及其在E.coil BLP中的融合表达.吉林农业大学学报,2008,30(6):797-804.
[39]栗学清,韩跃武.抗菌肽Aure in112基因的同向串联及其克隆载体的构建和鉴定.第四军医大学学报(J FourthMilMed Univ) .2005,26 (10):875-877.
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[2] Boman H G. Antibacterial peptides: key components needed in immunity. Cell, 1991, 65:205-207.
[3] Boman H G. Peptide antibiotics and their role innate immunity. Annau Revlmmunol,1995, 13:62
[4] Faye I, Pye A,Rasmuson B et a1.Insect Immunty.simultaneous induction of antibacterial activity and selective synthesis of some haemolymph protein in diapausing pupae of hyalophora cecropia and samia cythia. Infect Immun: 1975,12:1426-1438.
[5] Anna Christina C. Nascimento, Wagner Fontes, et al. Antimicrobial peptides from Anurans skin secretions. Protein and Peptide Letters, 2003, 10(3):227-238.
[6] Takumi O, Fujitoshi Y, Masco T, et al. Production and purification of a bacteriocin peptide produced by Lactococcus sp strain GM005, isolated from Miso-paste. Int J Food Microbiol, 2003, 87(1-2):153-159.
[7] Lehrer R I, Ganz T. Antimicrobial peptides in mammalian and insect host defence. CurrOpin Immunol, 1999, 11: 23-30.
[8] Orsi N. The antimicrobial activity of lactoferrin: current status and perspectives. Biometals. 2004 Jun;17(3):189-196.
[9] Schibli DJ, Epand RF, Vogeletal. Tryptophan-rich antimicrobial peptides: comparative properties and membrane interactions. Biochem Cell Biol. 2002. 80(5): 667-677.
[10] Yamanchi K. Tomita M Giehl, et al. Ahbacterial activity of lacteoferrin and a pepsin-derived lactoferrin peptide fragment. Infection and Immunity. 1993, 61(2): 719-728
[11] Zasl of M, et al. Proc Natl Acad Sci USA, 1987, 4(84):5449-5455.
[12]李孝东,袁建华.抗菌肽的研究进展「J].山东医药工业,2003 (1) :27-28.
[13] Tomita M , Bellamy W ,Takase M,et al. Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin. J Dairy Sci. 1991.
[14] Lee DG,Park Y,Kim H N,et a1.Antifungal mechanism of an antimicrobial peptide,HP(2-20),derived from N-terminus of Helicobacter pylori ribosomal protein L1 against Candida albicans.Biochem Biophys Res Commun,2002,291(4):1006-1013.
[15] Dekker N,Cox RC,Kramer RA,et al. Substrate specificity of the Integral membrane protease OmpT determined by spatially addressed peptide libraries. Biochemistry,2001,40(6): 1694-1701.
[16]谭显胜,李巍巍,王志明等.抗菌肽的研究进展.现代生物医学进展,2009,9(16):3153-3156.
[17] Fehlbaum P,Bulet P,Chernysh S ,et al. Structure-activity analysis of thanatin a 21-residue inducible insect defense peptide with sequence homology to frog skinantimicrobial peptides Proc Natl Acad Sci USA ,1996, (37):388-400.
[18] Bobek LA,Situ H. MUC7 20 -Mer:Investigation of Antimicrobial Activity secondary Structure and Possible Mechanism of Antifungal Action .Antimicrob Agents Chemother,2003,47(2):643-652.
[19] Christensen B, Fink H, Merrifield RB, Mauzerall D. Channel-forming properties of cecropins and related midel compounds incorporated into planar lipid membranes. Proc. Natl. Acad. Sci. USA. 1988, 85: 5072-5076.
[20]赵东红,戴祝英,周开亚.昆虫抗菌肽的功能、作用机理与分子生物学研究最新进展.生物工程进展,1999,19(5):14-17.
[21] Blazyk J, Wiegand R, Klein J, et al. A novel linear amphipathic beta-sheet cationic antimicrobial peptide with enhanced selectivity for bacterial lipids. J Biol Chem, 2001, 276(30): 27899-27906.
[22] Toney JH. Iseganan (Intra Biotics pharmaceuticals). Curr Opin Investig Drugs. 2002, 3(2): 225-228.
[23] Chen J, Falla TJ, Liu H, et al. Development of protegrins for the treatment and prevention flf oral mucositis: structure-activity relationships of synthetic protegrin analogues. Biopolumers, 2000, 55(1): 88-98.
[24]黄自然,廖富拨,郑青等.昆虫抗菌肽在医药上的应用.天然产品研究与开发.2000 Vo1.13 No.2:79-83.
[25] Park S, Park .S H, Ahn H C, Kim S, Kim S S, Lee B J. Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata. FEES Letters. 2001, 507:95-100.
[26] Issacson T, Soto A, Iwamuro S, et al. Antimicrobial peptides with atypical structural feature from the skin of the Japanese brown frogRana japonica. Peptides, 2002, 23(3) :419-425.
[27] Maloy WL, Kari UP. Structure-avtivity studies on magainins and other host defense peptides [J].Biopolymers (Peptide Science),1995, 37:105-122.
[28] Andreu D, Rivas L. Animal antimicrobial peptides:an overview [J].Biopolymers (Peptide Science),1998, 47(6):415-433.
[29] Lai R , Ye WJ , Ran YL , LüSQ , Yang DT. Antimicrobial peptide from the skin of Bombina maxima . Zool.Res., 1998,19(4):257-262.
[30] Barry D,Simmaco M,Boman H G. Gene-encoded peptide antibiotics and innate immunity Do 'animalcules 'have defence budgets?FEBS Letters ,1998,430(1-2): 130-134.
[31] Swendsen C L,Sphepard D. A study of antimicrobial effects of mountain dus匆salamander Mucus Jon Hasfjord:Nat Sci Seminar Abstracts ,1998.
[32] Hancock R E W Chapple D S. Peptide antibiotics. Antimicrobial Agents and Chemotherapy,1999,43 (6):1317-1323.
[33]乔红伟.中国林蛙基因工程抗菌肚制备及生物学特性研究:(博士学位论文).吉林:吉林大学,2006.
[34]徐进署,张双全.昆虫抗菌肽对病原微生物作用的研究进展[J].昆虫学报,2002, 45 (5):673-678.
[35] Fiordalisi J J, Fetter C H, TenHarmselA, et al. Synthesis and expression in Escherichia coli of a gene for x -bungarotoxin[J].Biochemistry, 1991, 30:10337-10343.
[36] Latham P W. Therapeutic peptides revisited Nature Biotechnology, 1999, 17:755-777.
[37] Simmaco M, Mignogna G, Barra D. Antimicrobial peptides from amphibian skin : what do they tell us ? Biopolymers ,1998 , 47 (6) : 435-450.
[38]陈晓平,詹冬玲,贾惠文等.抗茵肽MagaininⅡ基因的克隆及其在E.coil BLP中的融合表达.吉林农业大学学报,2008,30(6):797-804.
[39]栗学清,韩跃武.抗菌肽Aure in112基因的同向串联及其克隆载体的构建和鉴定.第四军医大学学报(J FourthMilMed Univ) .2005,26 (10):875-877.
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