β-防御素130在大肠杆菌中的串联表达、纯化及生物活性分析
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摘要
为了研究抗菌肽β-防御素130的生物学活性和实现大规模制备,通过改良其分子结构,构建表达载体pET28a-3×β-defensin130,利用大肠杆菌BL21 (DE3)作为宿主细胞诱导表达后为水溶性蛋白。对纯化后抗菌肽进行抑菌实验、稳定性实验、MTT实验和溶血性实验确定其生物活性。最终成功制备出25 kDa的重组蛋白,对金黄色葡萄球菌(ATCC25923)(45μg/mL)和单增李斯特菌(ATCC221633)(80μg/mL)等革兰氏阴性和阳性菌都表现出极强的抗菌活性,且其抗菌活性不受温度、pH值和蛋白酶消化等影响,MTT细胞毒性实验显示其对HEK293细胞无毒性且对兔源红细胞具有极低的溶血性。这将为新型抗菌肽的开发提供理论基础并推动抗生素替代产业快速发展。
To improve and broaden the antimicrobial activity of β-defensin130, 3 copies of β-defensin130 encoding sequences were synthesized and cloned into pET28 a(+) expression vector, and expressed in Escherichia coli BL21(DE3) as a25 kDa soluble protein. The affinity purified 3×β-defensin 130 displayed antimicrobial activity against not only Gram-positive strains including Staphylococcus aureus(ATCC 25923)(45 μg/mL) and Listeria monocytogenes(ATCC 221633)(80 μg/mL)but also Gram-negative strains. Furthermore, the antimicrobial activity of β-defensin130 was not affected by temperature, pH and proteinase digestion. In addition, E. coli-derived 3×β-defensin130 was not toxic to HEK 293 cells and showed a relatively low hemolytic activity against rabbit erythrocytes. Our study proves 3×β-defensin130 expressed in E. coli is stable,non-cytotoxic and low-hemolytic active with great potential as alternative antibiotics.
To improve and broaden the antimicrobial activity of β-defensin130, 3 copies of β-defensin130 encoding sequences were synthesized and cloned into pET28 a(+) expression vector, and expressed in Escherichia coli BL21(DE3) as a25 kDa soluble protein. The affinity purified 3×β-defensin 130 displayed antimicrobial activity against not only Gram-positive strains including Staphylococcus aureus(ATCC 25923)(45 μg/mL) and Listeria monocytogenes(ATCC 221633)(80 μg/mL)but also Gram-negative strains. Furthermore, the antimicrobial activity of β-defensin130 was not affected by temperature, pH and proteinase digestion. In addition, E. coli-derived 3×β-defensin130 was not toxic to HEK 293 cells and showed a relatively low hemolytic activity against rabbit erythrocytes. Our study proves 3×β-defensin130 expressed in E. coli is stable,non-cytotoxic and low-hemolytic active with great potential as alternative antibiotics.
引文
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[2]Conlon JM,Sonnevend A.Antimicrobial peptides in frog skin secretions//Giuliani A,Rinaldi A.Methods in Molecular Biology.Totowa,NJ:Humana Press,2010.
[3]Li C,Blencke HM,Paulsen V,et al.Powerful workhorses for antimicrobial peptide expression and characterization.Bioeng Bugs,2010,1(3):217-220.
[4]Radek K,Gallo R.Antimicrobial peptides:natural effectors of the innate immune system.Semin Immunopathol,2007,29(1):27-43.
[5]Zasloff M.Antimicrobial peptides of multicellular organisms.Nature,2002,415(6870):389-395.
[6]Terkawi MA,Takano R,Furukawa A,et al.Involvement ofβ-defensin 130(DEFB130)in the macrophage microbicidal mechanisms for killing Plasmodium falciparum.Sci Rep,2017,7:41772.
[7]Luan C,Xie YG,Yu TP,et al.Recombinant expression of antimicrobial peptides using a novel self-cleaving aggregation tag in Escherichia coli.Canad J Microbiol,2014,60(3):113-120.
[8]Meng DM,Zhao JF,Ling X,et al.Recombinant expression,purification and antimicrobial activity of a novel antimicrobial peptide PaDef in Pichia pastoris.Protein Expr Purif,2017,130:90-99.
[9]Dong B,Cheng RQ,Liu QY,et al.Multimer of the antimicrobial peptide Mytichitin-A expressed in Chlamydomonas reinhardtii exerts a broader antibacterial spectrum and increased potency.J Biosci Bioeng,2017,125(2):175-179.
[10]Rao XC,Hu JS,Li S,et al.Design and expression of peptide antibiotic hPAB-βas tandem multimers in Escherichia coli.Peptides,2005,26(5):721-729.
[11]Ren HY,Dong B,Fan ZC,et al.Prokaryotic expression and purification of Chlamydomonas reinhardtii intraflagellar transport protein 46(IFT46)and preparation of polyclonal antibody.Chin J Biotech,2016,32(8):1124-1132(in Chinese).任海月,董彬,樊振川,等.莱茵衣藻纤毛内运输蛋白IFT46的原核表达纯化及其多克隆抗体的制备.生物工程学报,2016,32(8):1124-1132.
[12]Meng DM,Dai HX,Gao XF,et al.Expression,purification and initial characterization of a novel recombinant antimicrobial peptide Mytichitin-A in Pichia pastoris.Protein Expr Purif,2016,127:35-43.
[13]Meng DM,Lv YJ,Zhao JF,et al.Efficient production of a recombinant Venerupis philippinarum defensin(VpDef)in Pichia pastoris and characterization of its antibacterial activity and stability.Protein Expr Purif,2018,147:78-84.
[14]Wanmakok M,Orrapin S,Intorasoot A,et al.Expression in Escherichia coli of novel recombinant hybrid antimicrobial peptide AL32-P113 with enhanced antimicrobial activity in vitro.Gene,2018,671:1-9.
[15]Cheng KT,Wu CL,Yip BS,et al.High level expression and purification of the clinically active antimicrobial peptide P-113 in Escherichia coli.Molecules,2018,23(4):800.
[16]Xi D,Teng D,Wang XM,et al.Design,expression and characterization of the hybrid antimicrobial peptide LHP7,connected by a flexible linker,against Staphylococcus and Streptococcus.Proc Biochem,2013,48(3):453-461.