家蚕抗菌肽Cecropin-XJ的原核优化表达及活性检测

Cecropin-XJ是一种从家蚕幼虫体内分离纯化的具有很强热稳定性、酸碱适应性和广谱抗菌性的新型家蚕抗菌肽。以融合不同标签的表达载体构建pET28a-Cecropin-XJ、pET30a-Cecropin-XJ、pET32a-Cecropin-XJ、pMAL-p2X-Cecropin-XJ重组质粒,转化E.coli BL21(DE3)感受态细胞,并优化诱导时间、诱导温度、诱导剂IPTG浓度等条件,通过对目的蛋白表达的检测分析,选择、建立Cecropin-XJ在大肠杆菌中高效、可溶性表达的技术体系。试验结果表明:采用构建的重组原核表达载体pET32a-Cecropin-XJ在IPTG终浓度为0.8 mmol/L、培养温度为37℃的条件下诱导5 h,目的蛋白的表达量可达10 mg/L,重组蛋白主要以可溶性表达产物形式存在,可溶性蛋白的表达量约占菌体总蛋白的35%,经金属螯合层析进一步纯化后的Cecropin-XJ融合蛋白纯度可达90%以上。体外抑菌试验显示Cecropin-XJ融合蛋白对金黄色葡萄球菌具有较强的抑菌活性。

Optimization of Prokaryotic Expression and Activity Determination of Bombyx mori Antimicrobial Peptide Cecropin-XJ

Cecropin-XJ,a novel antibacterial peptide isolated and purified from silkworm(Bombyx mori) larvae,had the characteristics of high thermal stability and pH adaptability,and broad spectrum of antibacterial activity.Recombinant plasmids pET28a-Cecropin-XJ,pET30a-Cecropin-XJ,pET32a-Cecropin-XJ and pMAL-p2X-Cecropin-XJ were constructed by using expression vectors fused with different tags.After these recombinant plasmids were transferred into E.coli strain BL21(DE3) competent cells,the expression conditions including induction time,induction temperature,and concentration of inducer IPTG were optimized to select and establish an efficient expression system for preparation of soluble Cecropin-XJ protein in E.coli through detection and analysis to the expressed target protein.The results showed that the amount of target protein could reach 10 mg/L after IPTG induction at 0.8 mmol/L final concentration for 5 h at 37 ℃ by using the recombinant prokaryotic expression vector pET32a-Cecropin-XJ.The recombinant protein was expressed mainly in soluble form and the soluble protein accounted for about 35% in total bacterial proteins.After purified by metal chelate chromatography,purity of fusion protein Cecropin-XJ was over 90%.In vitro antimicrobial test showed that fusion protein Cecropin-XJ had strong antibacterial activity against Staphylococcus aureus.