鲎素Tachyplesin Ⅰ在大肠杆菌中的重组表达及其抑菌活性

Recombinant Expression of TachyplesinⅠin E.coli and Its Antibacterial Activity

为了高效制备抗菌肽,本文将鲎素抗菌肽目的基因Tachyplesin-1(TP1)在大肠杆菌BL21中进行表达。首先构建表达质粒pET32a-TP1并转化大肠杆菌BL21(DE3),在IPTG诱导下进行目的融合蛋白表达,并利用His标签与镍柱亲和层析对融合蛋白进行纯化。纯化后的融合蛋白经羟胺裂解液切割和质谱分析后,获得单一的TP1重组蛋白,最后对其抑菌活性进行表征。结果表明,重组菌在37℃经IPTG诱导后,融合蛋白表达成功,TrxA-TP1融合蛋白的分子量在20 kDa左右。经羟胺裂解得到的重组蛋白TP1对于金黄色葡萄球菌(Staphylococcus aureus)与枯草芽孢杆菌(Bacillus subtilis)均具有良好抑菌活性,最小抑菌浓度分别为6和10 mg/L。本研究为鲎素抗菌肽TP1的开发应用和大量生产奠定了基础。

In order to obtain antimicrobial peptide efficiently,the recombinant expression of antimicrobial peptide target gene TachyplesinⅠ(TP1)in Escherichia coli BL21(DE3)was studied.The expression vector pET32a-TP1 was constructed and converted to Escherichia coli BL21(DE3).IPTG induced the expression of target genes,and the fusion protein was purified by NI-NTA affinity chromatography.The purified TrxA-TP1 fusion peptide was released by hydroxylamine and recombinant TP1 was analyzed by mass spectrometry.Results showed that TrxA-TP1 fusion protein was induced successfully with IPTG at 37℃.The molecular weight of TrxA-TP1 was about 20 kDa.The recombinant TP1 obtained by hydroxylamine cleavage showed strong antibacterial bioactivity against S.aureus and B.subtilis,The minimal inhibitory concentration was 6 and 10 mg/L respectively.These experiments established a useful system for further studies,application and mass production of antimicrobial peptide TP1.