杂合抗菌肽Mel-MytB在毕赤酵母中的表达及其抗菌活性测定

doi:10.16431/j.cnki.1671-7236.2018.11.032

摘要/Abstract

摘要：

为了获得抗菌活性较强的抗菌肽，将蜂毒肽（Melittin）与贻贝素B （Mytilin-B）的核心功能序列杂合，以Melittin （3-14）和Mytilin-B （13-27）的成熟肽段作为模板序列，根据巴斯德毕赤酵母（Pichia pastoris）偏好密码子，采用SOE方法合成杂合抗菌肽Mel-MytB（MEM）基因。改造后的基因克隆到pPICZα-A质粒，构建分泌型重组酵母表达载体pPICZα-A-MEM，而后经Sac Ⅰ酶切线性化后电转入毕赤酵母受体菌X-33。结果表明，在醇氧化酶（AOX）启动子调控下，分子质量约3.0 ku的Mel-MytB杂合抗菌肽获得表达，具有热稳定性和酸稳定性，煮沸40 min、pH 2.0~10.0范围内抑菌活性基本不变。抗菌特性研究结果表明，该表达产物具有广谱抗菌活性，其对大肠杆菌ATCC25922、金黄色葡萄球菌ATCC25923/ATCC6538、肠道沙门氏菌ATCC13076、鼠伤寒沙门氏菌ATCC14028、副溶血弧菌ATCC17802、创伤弧菌ATCC27562、枯草芽孢杆菌ATCC6633的MIC值分别为5.1、2.2、2.0、4.6、7.6、9.4、13.2和59.4 μg/mL。因此，重组抗菌肽Mel-MytB在疾病防治和动物饲料添加剂等方面具备较好的应用前景。

关键词: 杂合抗菌肽; 蜂毒肽; 贻贝素B; 毕赤酵母; 抑菌活性

Abstract:

In order to abtain a high activity antimicrobial peptide,an expression vector was constructed with a tandem of Melittin and Mytilin-B.According to the partiality codon of Pichia pastoris,the Melittin(3-14)-Mytilin-B(13-27) hybrid antimicrobial peptide,Mel-MytB (MEM) gene was synthesized by SOE method,and cloned into pPICZa-A to construct the recombinant expression vector pPICZa-A-MEM.pPICZα-A-MEM was linearized by Sac Ⅰ and electroporated into Pichia pastoris X-33.The results showed that under the control of the promoter alcohol oxidase (AOX),an approximately 3.0 ku Mel-MytB protein was expressed,which had thermal stability and acid stability.The antibacterial activity was basically unchanged after boiling for 40 min and pH 2.0-10.0.Antibacterial assays demonstrated that Mel-MytB had broad-spectrum antibacterial activity.The MIC to Escherichia coli ATCC25922,Staphylococcus aureus ATCC25923,Staphylococcus aureus ATCC6538,Salmonella enterica ATCC13076,Salmonella Typhimurium ATCC14028,Vibrio parahaemolyticus ATCC17802,Vibrio vulnificus ATCC27562 and Bacillus subtilis ATCC6633 were 5.1,2.2,2.0,4.6,7.6,9.4,13.2 and 59.4 μg/mL,respectively.Therefore,the recombinant antimicrobial peptide Mel-MytB had a good application prospect in disease prevention and animal feed additives.

Key words: hybrid antimicrobial peptide; Melittin; Mytilin-B; Pichia pastoris; antibacterial activity

中图分类号:

S859.79⁺7

张宏刚, 吴剑良, 李莉. 杂合抗菌肽Mel-MytB在毕赤酵母中的表达及其抗菌活性测定[J]. 《中国畜牧兽医》, 2018, 45(11): 3246-3252.

ZHANG Honggang, WU Jianliang, LI Li. Expression of Hybrid Antimicrobial Peptide Mel-MytB in Pichia pastoris and Detection of Its Antibacterial Activity[J]. , 2018, 45(11): 3246-3252.

参考文献

[1] HANCOCK R E W,SAHL H G.Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies[J].Nature Biotechnology,2006,24(12):1551-1557.
[2] KANG H K,KIM C,SEO C H,et al.The therapeutic applications of antimicrobial peptides (AMPs):A patent review[J].Journal of Microbiology,2017,55(1):1-12.
[3] ZASLOFF M.Antimicrobial peptides of multicellular organisms[J].Nature,2002,415(6870):389-395.
[4] 江学斌,陈嘉蔚,杨军,等.猪源抗菌肽PBD-1在毕赤酵母中的表达及鉴定[J].中国畜牧兽医,2016,43(3):644-649. JIANG X B,CHEN J W,YANG J,et al.Expression and identification of porcine antimicrobial peptide PBD-1 in Pichia pastoris[J].China Animal Husbandry & Veterinary Medicine,2016,43(3):644-649.(in Chinese)
[5] LI Y,XIANG Q,ZHANG Q,et al.Overview on the recent study of antimicrobial peptides:Origins,functions,relative mechanisms and application[J].Peptides,2012,37(2):207-215.
[6] WANG S,ZENG X F,YANG Q,et al.Antimicrobial peptides as potential alternatives to antibiotics in food animal industry[J].International Journal of Molecular Sciences,2016,17(5):603.
[7] XIAO H,WU M M,TAN B E,et al.Effects of composite antimicrobial peptides in weanling piglets challenged with deoxynivalenol:Ⅰ.Growth performance,immune function,and antioxidation capacity[J].Journal of Animal Science,2013,91(10):4772-4780.
[8] XIONG X,YANG H S,LI L,et al.Effects of antimicrobial peptides in nursery diets on growth performance of pigs reared on five different farms[J].Livestock Science,2014,167:206-210.
[9] XIAO H,TAN B E,WU M M,et al.Effects of composite antimicrobial peptides in weanling piglets challenged with deoxynivalenol:Ⅱ.Intestinal morphology and function[J].Journal of Animal Science,2013,91(10):4750.
[10] 祁丽,姜宁,张爱忠,等.抗菌肽研发现状及其改造策略[J].中国畜牧兽医,2016,43(2):450-456. QI L,JIANG N,ZHANG A Z,et al.Research present status and reform strategy of antimicrobial peptide[J].China Animal Husbandry & Veterinary Medicine,2016,43(2):450-456.(in Chinese)
[11] FOX M A,THWAITE J E,ULAETO D O,et al.Design and characterization of novel hybrid antimicrobial peptides based on Cecropin A,LL-37 and magainin Ⅱ[J].Peptides,2012,33(2):197-205.
[12] ZHANG S K,SONG J W,GONG F,et al.Design of an α-helical antimicrobial peptide with improved cell-selective and potent anti-biofilm activity[J].Scientific Reports,2016,6:27394.
[13] 刘晓明,姜宁,张爱忠,等.杂合抗菌肽在毕赤酵母中的表达及其活性测定[J].中国生物工程杂志,2016,36(2):81-89. LIU X M,JIANG N,ZHANG A Z,et al.Expression of hybrid antimicrobial peptides in Pichia yeast and identification of its biological activity[J].China Biotechnology,2016,36(2):81-89.(in Chinese)
[14] LUO X F,LIU W,QIN Y,et al.Design,characterization and antimicrobial activity of novel antimicrobial peptides from Temporin-Pta[J].Journal of Biomedical Nanotechnology,2017,13(9):1124-1133.
[15] 谢秀枝,刘丽华,皮雄娥,等.抗菌肽Caseicin A氨基酸排列顺序改变对其抑菌活性的影响[J].浙江农业学报,2013,2:280-283. XIE X Z,LIU L H,PI X E,et al.Effect of Caseicin A's amino acid sequence alteration on its antimicrobial activity[J].Acta Agriculturae Zhengjiangensis,2013,2:280-283.(in Chinese)
[16] SILVA T,CLARO B,SILVA B B,et al.Unravelling a mechanism of action for a Cecropin A-Melittin hybrid antimicrobial peptide:The induced formation of multilamellar lipid stacks[J].Langmuir,2018,34(5):2158-2170.
[17] CAO Y,YU R Q,LIU Y,et al.Design,recombinant expression,and antibacterial activity of the Cecropins-Melittin hybrid antimicrobial peptides[J].Current Microbiology,2010,61(3):169-175.
[18] WU R,WANG Q,ZHENG Z,et al.Design,characterization and expression of a novel hybrid peptides melittin (1-13)-LL37(17-30)[J].Molecular Biology Reports,2014,41(7):4163.
[19] MITTA G,HUBERT F,DYRYNDA E A,et al.Mytilin B and MGD2,two antimicrobial peptides of marine mussels:Gene structure and expression analysis[J].Developmental & Comparative Immunology,2000,24(4):381-393.
[20] 吴静,梁永利,李玉峰,等.重组鸡Cathelicidins类抗菌肽的融合表达及其对金黄色葡萄球菌抗菌活性的研究[J].中国畜牧兽医,2013,40(6):19-26. WU J,LIANG Y L,LI Y F,et al.Fusion expression of recombinant Cathelicidins genes in Escherichia coli and study on its antibacterial activity against Staphylococcus aureus in vivo[J].China Animal Husbandry & Veterinary Medicine,2013,40(6):19-26.(in Chinese)
[21] JINDAL H M,LE C F,YUSOF M Y M,et al.Antimicrobial activity of novel synthetic peptides derived from indolicidin and ranalexin against Streptococcus pneumoniae [J].PLoS One,2015,10(6):e0128532.
[22] ALMAAYTA H,TARAZ I,FANDI M A.The design and functional characterization of the antimicrobial and antibiofilm activities of BMAP27-Melittin,a rationally designed hybrid peptide[J].International Journal of Peptide Research & Therapeutics,2015,21(2):165-177.
[23] XU W,ZHU X,TAN T,et al.Design of embedded-hybrid antimicrobial peptides with enhanced cell selectivity and anti-biofilm activity[J].PLoS One,2014,9(6):e98935.