抗菌肽FAPs在毕赤酵母中的重组表达研究
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摘要
将4种抗菌肽Fowlicidin-2、Cecropin B、Cecropin A(1-8)-Melittin(1-18)和Thanatin串联,并在每种抗菌肽N-末端加上Kex2蛋白酶裂解位点,根据毕赤酵母密码子偏爱性,化学合成四种抗菌肽(Fusion Antimicrobial Peptides,FAPs)编码基因,连接到pPICZαC载体中,构建分泌型重组表达载体并转化毕赤酵母。经甲醇诱导后,Tricine-SDS-PAGE分析获得与目蛋白大小一致特异表达蛋白条带,每升发酵液上清含量达到32 mg。初步鉴定FAPs对大肠杆菌(E.coli)ATCC25922、金黄色葡萄球菌(S.aureus)ATCC25923、鼠伤寒沙门氏菌(S.syphimurium)C77-31和绿脓杆菌(P.aeruginosa)ATCC27853均有抑菌活性,且对S.aureus ATCC25923活性最强,最小抑菌浓度MIC为8.0μg·mL-1。
Four antimicrobial peptides(FAPs), Fowlicidin-2, Cecropin B, Cecropin A(1-8)-Melittin(1-18) and Thanatin were joint successively, and the recognition site of Kex2 protease was added at the NTerminus of each peptide. The gene encoding FAPs was designed according to the biased codon usage of pichia pastoris and synthesized by chemical method. The gene was cloned into the pPICZαC vector and transformed into Pichia pastoris. After induction, FAPs were successfully expressed by Tricine-SDS-PAGE analysis. The production of FAPs was 32 mg per 1 liter of culture supernatant. The recombinant FAPs possessed antibacterial activity against E. coli ATCC25922, S. aureus ATCC25923, S. typhimurium C77-31and P. aeruginosa ATCC27853, and showed the strongest antimicrobial activity against S. aureus ATCC25923 among them, for which the MIC was 8.0 μg· mL-1.
Four antimicrobial peptides(FAPs), Fowlicidin-2, Cecropin B, Cecropin A(1-8)-Melittin(1-18) and Thanatin were joint successively, and the recognition site of Kex2 protease was added at the NTerminus of each peptide. The gene encoding FAPs was designed according to the biased codon usage of pichia pastoris and synthesized by chemical method. The gene was cloned into the pPICZαC vector and transformed into Pichia pastoris. After induction, FAPs were successfully expressed by Tricine-SDS-PAGE analysis. The production of FAPs was 32 mg per 1 liter of culture supernatant. The recombinant FAPs possessed antibacterial activity against E. coli ATCC25922, S. aureus ATCC25923, S. typhimurium C77-31and P. aeruginosa ATCC27853, and showed the strongest antimicrobial activity against S. aureus ATCC25923 among them, for which the MIC was 8.0 μg· mL-1.
引文
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[16]冯兴军,李静,赵晓宇,等.牛乳铁蛋白素-马盖宁杂合抗菌肽的设计、合成及抑菌活性[J].东北农业大学学报,2011,42(3):105-109.
[17]Van Den Hazel H B,Kielland-Brandt M C,Winther J R.Review:biosynthesis and function of yeast vacuolar proteases[J].Yeast,1996,12(1):1-16.
[2]Hancock R E.Cationic peptides:effectors in innate immunity and novel antimicrobials[J].The lancet Infect Dis,2001,1(3):156-164.
[3]Jin F L,Xu X X,Zhang W Q,et al.Expression and characteriza tion of a housefly Cecropin gene in the methylotrophic yeast,Pich ia pastoris[J].Protein Expr Purif,2006,49(1):39-46.
[4]Lapis K.Physiologic and path physiologic significance of antimi crobial(host defensive)small peptides[J].Orv Hetil,2008,149(51):2419-2424.
[5]陈福,罗玉萍,龚反熹,等.抗菌肽耐药性研究进展[J].微生物学通报,2008,35(11):1186-1790.
[6]Bommineni Y R,Dai H,Gong Y X,et al.Fowlicidin-3 is aβ-he lical cationic host defense peptide with potent antibacterial and li popolysaccharide-neutralizing activities[J].FEBS J,2007,274(2):418-428
[7]Gankovskaia O A.Kovalchuk L V,Gankovskaia LV,et al.Role of Toll-like receptors and defensins in antimicrobial protetion of urogenital tract in females[J].Zh Mikrobiol Epidemiol Immunobiol,2008,(1):46-54.
[8]Merrifield R B,Merrifield E L,Juvvadi P,et al.Design and syn thesis of antimicrobial peptides[J].Ciba Fond Symp,1994,186:5-20.
[9]奥斯伯F,布伦特R,金斯顿R E.精编分子生物学实验指南[M].北京:科学出版社,1998.
[10]刘秋云,罗樨,何康泽,等.快速制备酵母质粒和基因组DNA PCR模板[J].微生物学通报,2011,28(5):77-79.
[11]Schagger H,von Jagow G.Tricine-sodium dodecyl sulfate-poly acrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 ku[J].Anal Biochem,1987,166(2):368-379.
[12]张昊,牛海涛,李改瑞,等.抗菌肽分子结构对其活性的影响[J].中国抗生素杂志,2010,35(12):892-897.
[13]Feng X J,Liu C L,Guo J Y,et al.Expression and Purification of an antimicrobial Peptide,Bovine Lactoferricin Derivative LfcinBW10 in Escherichia coli[J].Curr Microbiol,2009,60(3):179-184.
[14]杨细媚,文阳安,万祥辉,等.天蚕素A截短肽在大肠埃希菌中的高效表达及活性分析[J].中国抗生素杂志,2010,35(1):20-23.
[15]Schlamadinger D E,Wang Y,McCammon J A,et al.Spectroscop ic and Computational Study of Melittin,Cecropin A,and the Hy brid Peptide CM15[J].J Phys Chem B,2012,116(35):10600-10608.
[16]冯兴军,李静,赵晓宇,等.牛乳铁蛋白素-马盖宁杂合抗菌肽的设计、合成及抑菌活性[J].东北农业大学学报,2011,42(3):105-109.
[17]Van Den Hazel H B,Kielland-Brandt M C,Winther J R.Review:biosynthesis and function of yeast vacuolar proteases[J].Yeast,1996,12(1):1-16.