迟眼蕈蚊抗菌肽BhSGAMP-1-S在大肠杆菌中的优化表达及其活性分析

Expression of a modified antimicrobial peptide BhSGAMP-1-S (Bradysia hygida) in Escherichia coli and characterization of its activity

摘要:

摘要：【目的】BhSGAMP-1是迟眼蕈蚊唾液腺抗菌肽，为了能够更好的了解其分子特性，我们将其表达、纯化并进行了活性测定。【方法】依据大肠杆菌稀有密码子设计并合成了抗菌肽基因BhSGAMP-1-S，以pMAL-c2X作为表达载体在大肠杆菌TB1中进行融合表达，融合蛋白通过麦芽糖亲和层析柱进行纯化，获得的融合蛋白经肠激酶切割后，混合物通过分子筛凝胶层析和反相高效液相色谱来获得单体重组抗菌肽BhSGAMP-1-S，对获得的抗菌肽进行活性测定。【结果】在最优的表达条件下融合蛋白以可溶的形式表达，100 mL诱导

Abstract:

bstract: [Objective] Bradysia hygida salivary glands antimicrobial peptide 1 (BhSGAMP-1) is one of the antimicrobial peptides involved in a preventive mechanism of defense of the fly Bradysia hygida. To know better about the molecular characterization of this antimicrobial peptide, we expressed and purified the modified BhSGAMP-1-S and investigated its antimicrobial activity. [Methods] We synthesized the gene of BhSGAMP-1-S designed with preferred codons of Escherichia coli and expressed it as a fusion protein in E.coli TB1 by using pMAL-c2X as vector. We purified the fusion protein using amylase resin affinity chromatography. In addition, we cleaved the fusion protein by enterokinase and the released recombinant BhSGAMP-1-S was separated by size exclusion chromatography, then followed by reversed-phase high performance liquid chromatography. We analyzed the antimicrobial activities of the purified recombinant BhSGAMP-1-S by bioassay. [Results] The fusion protein was mostly expressed in soluble form under the optimized conditions. The recombinant BhSGAMP-1-S was produced with a pure yield of 0.38 mg/100 mL culture medium. Antimicrobial assays demonstrated that the recombinant BhSGAMP-1-S was active against several Gram-positive and Gram-negative bacteria and fungi. [Conclusion] It appears to be first successful production of the recombinant BhSGAMP-1-S from fly Bradysia hygida. Data presented here confirm that the recombinant BhSGAMP-1-S is now ready for further studying and characterize their antimicrobial properties.