杂合抗菌肽BM16的原核表达及其生物学活性分析
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摘要
为了在在大肠杆菌中融合表达杂合抗菌肽BM16并研究其生物学活性,本试验利用生物信息学分析软件设计出1条杂合抗菌肽BM16,运用同源重组的方法构建重组表达质粒p ET-28a(+)-Ub-BM16,在大肠杆菌BL21(DE3)中诱导表达,采用Ni2+亲和层析纯化获得目的蛋白。结果显示,表达出13.2 ku的Ub-BM16融合蛋白,经SUMO酶切后获得2.0 ku的目的蛋白,纯化后纯度大于95%,该多肽对大肠杆菌、沙门菌和金黄色葡萄球菌等均有抑制作用。上述研究结果为开发新型抗菌肽提供了思路。
The study aims to investigate the fusion expression system and biological activity of the hybrid antimicrobial peptide BM16 expressed from E.coli.The hybrid antimicrobial peptide BM16 was designed by bioinformatics analysis software and the recombinant expression plasmid p ET28 a-Ub-BM16 was constructed using the homologous recombination method.The recombinant plasmid was induced to express in E.coli BL 21(DE3) and the recombinant protein was successfully expressed and purified using Ni2+affinity chromatography,which was 13.2 ku.The target protein of 3.0 ku was obtained by SUMO enzyme digestion of the recombinant protein.The purity of the target protein is higher than 95%.The polypeptide protein exhibited an inhibitory effect on Escherichia coli,Salmonella and Staphylococcus aureus.The above-mentioned results provide an idea for the development of new antimicrobial peptides.
The study aims to investigate the fusion expression system and biological activity of the hybrid antimicrobial peptide BM16 expressed from E.coli.The hybrid antimicrobial peptide BM16 was designed by bioinformatics analysis software and the recombinant expression plasmid p ET28 a-Ub-BM16 was constructed using the homologous recombination method.The recombinant plasmid was induced to express in E.coli BL 21(DE3) and the recombinant protein was successfully expressed and purified using Ni2+affinity chromatography,which was 13.2 ku.The target protein of 3.0 ku was obtained by SUMO enzyme digestion of the recombinant protein.The purity of the target protein is higher than 95%.The polypeptide protein exhibited an inhibitory effect on Escherichia coli,Salmonella and Staphylococcus aureus.The above-mentioned results provide an idea for the development of new antimicrobial peptides.
引文
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[16]LI Y.Carrier proteins for fusion expression of antimicrobial peptides in Escherichia coli[J].Biotechnology&Applied Biochemistry,2009,54(1):1.
[2]THEURETZBACHER U.Global antibacterial resis tance:The never-ending story[J].Journal of Global Antimicrobial Resistance,2013,1(2):63-69.
[3]王臣,廖成水,牛明福,等.Metalnikowin类抗菌肽MLP的设计及其抗菌活性的测定[J].中国兽医科学,2016,46(7):841-846.WANG Chen,LIAO Chen-shui,NIU Ming-fu,et al.Molecular design and antibacterial activity determination of Metalnikowin-like antibacterial peptide[J].Chinese Veterinary Science,201,46(7):841-846.
[4]PEI Z,SUN X,TANG Y,et al.Cloning,expression,and purification of a new antimicrobial peptide gene from Musca domestica larva[J].Gene,2014,549(1):41.
[5]HAO G,GUO-WEI L E,SHI Y H.The action mechanism of two analogues of the antimicrobial peptide buforinⅡon Staphylococcus aureus membrane[J].Journal of Genetics,2013,89(4):417-23.
[6]XU X,JIN F,YU X,et al.High-level expression of the recombinant hybrid peptide cecropin A(1-8)-magainin2(1-12)with an ubiquitin fusion partner in Escherichia coli[J].Protein Expression&Purification,2007,55(1):175.
[7]TAN J,HUANG J,HUANG Y,et al.Effects of single amino acid substitution on the biophysical properties and biological activities of an amphipathicα-helical antibacterial peptide against Gram-negative bacteria[J].Molecules,2014,19(8):10803.
[8]HOFFMANN J A,REICHHART J,HETRU C.Innate immunity in higher insects[J].Current Opinion in Immunology,1996,8(1):8-13
[9]CHEN Y,MANT C T,FARMER S W,et al.Rational Design ofα-helical antimicrobial peptides with enhanced activities and specificity/therapeutic index[J].Journal of Biological Chemistry,2005,280(13):12316.
[10]MAROTIG,KERESZT A,KONDOROSI E,et al.Natural roles of antimicrobial peptides in microbes,plants and animals[J].Research in Microbiology,2011,162(4):363-374.
[11]CHE Y,LU Y,ZHA X,et al.Higher efficiency soluble prokaryotic expression,purification,and structural analysis of antimicrobial peptide G13[J].Protein Expression&Purification,2016,119:45-50.
[12]YI T,SUN S,HUANG Y,et al.Prokaryotic expression and mechanism of action ofα-helical antimicrobial peptide A20L using fusion tags[J].BMC Biotechnology,2015,15(1):69.
[13]王利娜,余冰,韩国全,等.Attacin-Thanatin杂合抗菌肽的融合表达及其菌内抑菌活性[J].中国兽医科学,2009,39(6):531-537.WANG Li-na,YU Bing,HAN Guo-quan,et al.Fusion expression and bacteriostatic activity in vivo of hybrid peptide attacin-Thanatin[J].Chinese Veterinary Science,2009,39(6):531-537.
[14]刘诚,黎满香,卢帅,等.抗菌肽Dermadistinctin-K的原核表达及生物活性鉴定[J].中国兽医科学,2012,42(5):517-522.LIU Cheng,LI Man-xiao,LU Shua,et al.Prokaryotic expression and biological activity of antibacterial peptide Dermadistinctin-K[J].Chinese Veterinary Science,2012,42(5):517-522.
[15]HUANG W,LU L,SHAO X,et al.Anti-melanoma activity of hybrid peptide P18 and its mechanism of action[J].Biotechnology Letters,2010,32(4):463-469.
[16]LI Y.Carrier proteins for fusion expression of antimicrobial peptides in Escherichia coli[J].Biotechnology&Applied Biochemistry,2009,54(1):1.
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