重组牛乳铁蛋白肽衍生肽设计及其在毕赤酵母中的表达
|
摘要
利用抗菌肽数据库和生物信息学分析工具,基于抗菌肽结构和功能的关系,设计了LFcin B衍生肽LFcin B-W4,10。为了验证衍生肽设计的合理性及表达产物功能正确性,将优化设计的基因序列经限制性内切酶酶切后,用T4 DNA连接酶连接到分泌型表达载体p PIC9K中,获得的重组表达载体p PIC9K-LFcinB-W 4,10经线性化后电转入毕赤酵母GS115中,经过G418浓度梯度筛选得到高拷贝转化子。经PCR鉴定,目的基因与酵母基因组稳定整合。阳性转化子经体积分数为2. 5%的甲醇诱导表达,表达产物用Tricine-SDS-PAGE检测到相对分子质量约为3. 2 k Da的衍生肽LFcin B-W4,10。每24 h取样检测发酵上清液抑菌活性,结果表明,抗菌肽LFcin BW4,10对金黄色葡萄球菌具有良好的抑菌活性,诱导表达96 h时其抑菌圈直径最大。实验为探究牛乳铁蛋白肽功能-结构关系奠定了基础。
A LfcinB-derived peptide LfcinB-W4,10 was designed based on the structure-function relationship of antimicrobial peptides based on antimicrobial peptide database and bioinformatics analysis. In order to verify the rationality of the designed derivative peptide and the functional correctness of the expression product,the optimized gene sequence was digested with restriction endonucleases and ligated into the secretory expression vector p PIC9 K with T4 DNA ligase. The recombinant expression vector p PIC9 K-LFcinB-W4,10 was linearized by Sac I and transformed into Pichia pastoris GS115 by electroporation,and G418 was used to obtain high copy transformants. The results showed the target gene was stably integrated with the yeast genome. The expression of positive transformants were induced by2. 5%( v/v) methanol,and the expressed product was LfcinB-W4,10 with a relative molecular weight of 3. 2 kDa detected by Tricine-SDS-PAGE. Moreover,the antibacterial activity of the fermentation supernatant was measured every24 h. The results showed the derived peptide LfcinB-W4,10 had good antibacterial activity against Staphylococcus aureus,and the bacteriostatic ring diameter was the largest after 96 h induction. In conclusion,this study lays a foundation to further explore the relationship between function and structure of bovine lactoferrin peptides.
A LfcinB-derived peptide LfcinB-W4,10 was designed based on the structure-function relationship of antimicrobial peptides based on antimicrobial peptide database and bioinformatics analysis. In order to verify the rationality of the designed derivative peptide and the functional correctness of the expression product,the optimized gene sequence was digested with restriction endonucleases and ligated into the secretory expression vector p PIC9 K with T4 DNA ligase. The recombinant expression vector p PIC9 K-LFcinB-W4,10 was linearized by Sac I and transformed into Pichia pastoris GS115 by electroporation,and G418 was used to obtain high copy transformants. The results showed the target gene was stably integrated with the yeast genome. The expression of positive transformants were induced by2. 5%( v/v) methanol,and the expressed product was LfcinB-W4,10 with a relative molecular weight of 3. 2 kDa detected by Tricine-SDS-PAGE. Moreover,the antibacterial activity of the fermentation supernatant was measured every24 h. The results showed the derived peptide LfcinB-W4,10 had good antibacterial activity against Staphylococcus aureus,and the bacteriostatic ring diameter was the largest after 96 h induction. In conclusion,this study lays a foundation to further explore the relationship between function and structure of bovine lactoferrin peptides.
引文
[1] GONZALEZ-CHAVEZ S A, AREVALO-GALLEGOS S,RASCON-CRUZ Q. Lactoferrin:Structure,function and applications[J]. Int J Antimicrob Ag,2009,33(4):1-8.
[2] PANTELEEV P V,BOLOSOV I A,BALANDIN S V,et al. Structure and biological functions ofβ-hairpin antimicrobial peptides[J]. Acta Naturae, 2015, 7(1):37-47.
[3] SHESTAKOV A,JENSSEN H,NORDSTROM I,et al.Lactoferricin but not lactoferrin inhibit herpes simplex virus type 2 infection in mice[J]. Antiviral Res,2012,93(3):340-345.
[4] BO L Y,LI T J,ZHAO X H. Effect of Cu/Mn-fortification on in vitro activities of the peptic hydrolysate of bovine lactoferrinagainst human gastric cancer BGC-823 cells[J].Molecules,2019,24(7):1 195.
[5] AHMADINIA K,YAN D,ELLMAN M,et al. The anticatabolic role of bovine lactoferricin in cartilage[J]. Biomolecular Concepts,2013,4(5):495-500.
[6] STR?M M B,REKDAL O,SVENDSEN J S. Antibacterial activity of 15-residue lactoferricin derivatives[J]. Chem Biol Drug Des,2000,56(5):265-274.
[7] VEGA S C,MARTINEZ D A,CHALAM D S,et al. Design,synthesis and evaluation of branched RRWQWRbased peptides as antibacterial agents against clinically relevant gram-positive and gram-negative pathogens[J].Front Microbiol,2018,2(9):329.
[8] SINHA M,KAUSHIK S,KAUR P,et al. Antimicrobial lactoferrin peptides:The hidden players in the protective function of a multifunctional protein[J]. Int J Pept,2013:1-12.
[9] PRITCHARD S R,KAILASAPATHY K. Dairy Ingredients for Food Processing[M]. Oxford,U K:Wiley-Blackwell,2011:3-33.
[10] CHOUH T,KUO T Y,CHIANG J C,et al. Design and synthesis of cationic antimicrobial peptides with improved activity and selectivity against Vibrio spp.[J]. Int J Antimicrob Agents,2008,32(2):130-138.
[11] EDWARDS I A,ELLIOTT A G,KAVANAGH A M,et al. Contribution of amphipathicity and hydrophobicity to the antimicrobial activity and cytotoxicity ofβ-hairpin peptides[J]. Acs Infectious Diseases,2016,2(6):442-450.
[12] HOLLMANN A,MARTA-NEZ M,NOGUERA M E,et al. Role of amphipathicity and hydrophobicity in the balance between hemolysis and peptide-membrane interactions of three related antimicrobial peptides[J]. Colloid Surf. B:Biointerfaces,2016,141:528-536.
[13] SUN C,LI Y,CAO S,et al. Antibacterial activity and mechanism of action of bovine lactoferricin derivatives with symmetrical amino acid sequences[J]. Int J Mol Sci,2018,19(10):76.
[14] DRAGO-SERRANO M E, CAMPOS-RODRIGUEZ R,CARRERO J C,et al. Lactoferrin and peptide-derivatives:Antimicrobial agents with potential use in nonspecific immunity modulation[J]. Curr Pharm Des,2018,24(10):1 067-1 078.
[15] BRANDENBURG L O,MERRES J,ALBRECHT L J,et al. Antimicrobial peptides:Multifunctional drugs for different applications[J]. Polymers,2012,4(1):539-560.
[16] HAMMAMI R,FLISS I. Current trends in antimicrobial agent research:Chemo-and bioinformatics approaches[J]. Drug Discov Today,2010,15(13-14):540-546.
[17] WANG G,XIA L,ZHE W. APD3:The antimicrobial peptide database as a tool for research and education[J].Nucleic Acids Res, 2016, 44(Database issue):D1 087-D1 093.
[18] BERTONI M,KIEFER F,BIASINI M,et al. Modeling protein quaternary structure of homo-and hetero-oligomers beyond binary interactions by homology[J]. Sci Rep,2017,7(1):10 480.
[19] HANIF W F,SHANKAR B R,PRATIMA G,et al.CAMPR3:A database on sequences,structures and signatures of antimicrobial peptides[J]. Nucleic Acids Res,2016,44(Database issue):D1 094-D1 097.
[20] CREGG J M. DNA-mediated transformation[J]. Methods Mol Biol,2007,389:27-42.
[21] SEMPLE J I,LEHNER B. Single and dual drug selection for transgenes following bombardment of Caenorhabditis species[J]. Methods,2014,68(3):409-416.
[22] JAYANTA S,PLANTZ B A,MEHMET I,et al. Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris:Case study with recombinant ovine interferon-tau[J]. Biotechnol. Bioeng,2010,89(1):102-112.
[23] NI Z,ZHOU X,SUN X,et al. Decrease of hirudin degradation by deleting the KEX1 gene in recombinant Pichia pastoris[J]. Yeast,2008,25(1):1-8.
[2] PANTELEEV P V,BOLOSOV I A,BALANDIN S V,et al. Structure and biological functions ofβ-hairpin antimicrobial peptides[J]. Acta Naturae, 2015, 7(1):37-47.
[3] SHESTAKOV A,JENSSEN H,NORDSTROM I,et al.Lactoferricin but not lactoferrin inhibit herpes simplex virus type 2 infection in mice[J]. Antiviral Res,2012,93(3):340-345.
[4] BO L Y,LI T J,ZHAO X H. Effect of Cu/Mn-fortification on in vitro activities of the peptic hydrolysate of bovine lactoferrinagainst human gastric cancer BGC-823 cells[J].Molecules,2019,24(7):1 195.
[5] AHMADINIA K,YAN D,ELLMAN M,et al. The anticatabolic role of bovine lactoferricin in cartilage[J]. Biomolecular Concepts,2013,4(5):495-500.
[6] STR?M M B,REKDAL O,SVENDSEN J S. Antibacterial activity of 15-residue lactoferricin derivatives[J]. Chem Biol Drug Des,2000,56(5):265-274.
[7] VEGA S C,MARTINEZ D A,CHALAM D S,et al. Design,synthesis and evaluation of branched RRWQWRbased peptides as antibacterial agents against clinically relevant gram-positive and gram-negative pathogens[J].Front Microbiol,2018,2(9):329.
[8] SINHA M,KAUSHIK S,KAUR P,et al. Antimicrobial lactoferrin peptides:The hidden players in the protective function of a multifunctional protein[J]. Int J Pept,2013:1-12.
[9] PRITCHARD S R,KAILASAPATHY K. Dairy Ingredients for Food Processing[M]. Oxford,U K:Wiley-Blackwell,2011:3-33.
[10] CHOUH T,KUO T Y,CHIANG J C,et al. Design and synthesis of cationic antimicrobial peptides with improved activity and selectivity against Vibrio spp.[J]. Int J Antimicrob Agents,2008,32(2):130-138.
[11] EDWARDS I A,ELLIOTT A G,KAVANAGH A M,et al. Contribution of amphipathicity and hydrophobicity to the antimicrobial activity and cytotoxicity ofβ-hairpin peptides[J]. Acs Infectious Diseases,2016,2(6):442-450.
[12] HOLLMANN A,MARTA-NEZ M,NOGUERA M E,et al. Role of amphipathicity and hydrophobicity in the balance between hemolysis and peptide-membrane interactions of three related antimicrobial peptides[J]. Colloid Surf. B:Biointerfaces,2016,141:528-536.
[13] SUN C,LI Y,CAO S,et al. Antibacterial activity and mechanism of action of bovine lactoferricin derivatives with symmetrical amino acid sequences[J]. Int J Mol Sci,2018,19(10):76.
[14] DRAGO-SERRANO M E, CAMPOS-RODRIGUEZ R,CARRERO J C,et al. Lactoferrin and peptide-derivatives:Antimicrobial agents with potential use in nonspecific immunity modulation[J]. Curr Pharm Des,2018,24(10):1 067-1 078.
[15] BRANDENBURG L O,MERRES J,ALBRECHT L J,et al. Antimicrobial peptides:Multifunctional drugs for different applications[J]. Polymers,2012,4(1):539-560.
[16] HAMMAMI R,FLISS I. Current trends in antimicrobial agent research:Chemo-and bioinformatics approaches[J]. Drug Discov Today,2010,15(13-14):540-546.
[17] WANG G,XIA L,ZHE W. APD3:The antimicrobial peptide database as a tool for research and education[J].Nucleic Acids Res, 2016, 44(Database issue):D1 087-D1 093.
[18] BERTONI M,KIEFER F,BIASINI M,et al. Modeling protein quaternary structure of homo-and hetero-oligomers beyond binary interactions by homology[J]. Sci Rep,2017,7(1):10 480.
[19] HANIF W F,SHANKAR B R,PRATIMA G,et al.CAMPR3:A database on sequences,structures and signatures of antimicrobial peptides[J]. Nucleic Acids Res,2016,44(Database issue):D1 094-D1 097.
[20] CREGG J M. DNA-mediated transformation[J]. Methods Mol Biol,2007,389:27-42.
[21] SEMPLE J I,LEHNER B. Single and dual drug selection for transgenes following bombardment of Caenorhabditis species[J]. Methods,2014,68(3):409-416.
[22] JAYANTA S,PLANTZ B A,MEHMET I,et al. Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris:Case study with recombinant ovine interferon-tau[J]. Biotechnol. Bioeng,2010,89(1):102-112.
[23] NI Z,ZHOU X,SUN X,et al. Decrease of hirudin degradation by deleting the KEX1 gene in recombinant Pichia pastoris[J]. Yeast,2008,25(1):1-8.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |