抗菌肽Shepherin的表达纯化和抗菌活性研究

Expression and Purification of Shepherin in E. coli and its Antimicrobial Activity

多肽Shepherin是一种最早在荠菜中发现的抗菌肽。研究显示Shepherin具有良好的抗菌活性,能抑制植物病原微生物的生长。通过构建Shepherin的表达质粒在大肠杆菌中生产抗菌肽并研究其对常见临床菌的抗菌活性。首先构建大肠杆菌表达质粒pET32a-Trx-His-Erk-Shepherin,并在大肠杆菌BL21中进行表达。经过菌体破碎、镍柱亲和层析、酶切、柱层析等分离纯化步骤,最终获得高纯度的Shepherin抗菌肽。选取临床常见细菌研究Shepherin的抗菌活性,并初步探讨抗菌肽Shepherin对脂质体和细菌外膜的作用。pET32-Trx-His-Erk-Shepherin在大肠杆菌BL21中成功表达,融合蛋白主要以可溶性形式表达,约占总蛋白30%。经过酶切和多步柱层析,最终目的蛋白Shepherin纯度达到90%以上。抗菌实验显示Shepherin对常见的临床病菌均显示有良好的抗菌活性,但是Ca^(2+)离子能显著降低抗菌肽的杀菌活性。杀菌曲线显示抗菌肽能在2 min内快速杀灭99%的E.coli,并在半小时内杀灭所有细菌。钙黄氯素泄露实验和杀菌曲线类似,证明Shepherin能破坏磷脂双分子层并在数分钟内造成胞质的快速泄露。透射电镜结果显示Shepherin能破坏E.coli细菌的外膜结构致使胞质泄露。Shepherin在大肠杆菌能以融合蛋白的形式成功进行表达;Shepherin蛋白对大多数临床细菌具有较好的杀菌活性;其杀菌机制主要是通过破坏细菌E.coli的外膜的完整性造成胞内物质外流。

Shepherin,an antimicrobial peptide was first isolated from Capsella bursa-pastoris. It was reported to have good activity against plant pathogens. This study was aimed to express Shepherin in E. coli cells and next to purify the fusion protein from the cell lysate. Its antimicrobial activity was further evaluated by testing its MICs against standard bacteria and clinic isolates. To express the shephrin in E. coli cells,a plasmid pET32a-Trx-His-Erk-Shepherin was first constructed accordingly. The constructed plasmid was then transfered into E. coli BL21 cells for expression with LB medium. The expressed Shepherin was a fusion protein accompanied with a fusion chaperon of thioredoxin. The fusion protein was separated from the impurity by Nickel affinity chromatography after cell lysis. To separate shepherin from its fusion partner,the fusion protein was treated with enterokinase to cleave the fusion protein. A high purity was obtained after another round of chromatography. Some of the clinic pathogens were chosen for activity study. The effect of Ca～（2＋）and the interaction between the peptide and the liposome were investigated accordingly. The results indicated that Shepherin was successfully expressed in pET32-Trx-His-Erk-Shepherin/BL21 mainly as a soluble fusion protein. The fusion protein was accounted 30% of the total protein. The peptide was obtained with a purity over 90% after separation and purification. Antimicrobial assay indicated that Shepherin had a broad antimicrobial activity against normal bacteria including Gram positive and negative strains,but was sensitive to Ca～（2＋）. Electronic microscope study indicated that Shepherin could break the out membrane of the E. coli cells. Calcein leakage assay indicated that Shepherin could break the phosphate lipid bilayer in a concentration dependent manner. In conclusion,Shepherin was expressed successfully in the E. coli cells mainly as a fusion form. It was active for most of the clinic pathogens through breaking the phosphate lipid bilayer and causing vital materials leakage subsequently.