鸡源抗菌肽Fowlicidin-2在大肠杆菌中的 重组表达及其生物学活性鉴定

Recombinant expression of the chicken antimicrobial peptide fowlicidin-2 in Escherichia coli and identification of its biological activity

摘要:

【目的】对抗菌肽Fowlicidin-2基因进行克隆与表达, 并鉴定其生物学活性。【方法】根据抗菌肽Fowlicidin-2氨基酸序列, 依照大肠杆菌(E. coli)密码子的偏爱性, 人工设计合成其编码基因。与质粒pET-32a连接, 构建重组表达载体, 转化表达宿主菌E. coli BL21(DE3), IPTG诱导表达, 融合蛋白经溴化氰裂解后进行纯化, 测定重组抗菌肽的抑菌活性。【结果】Fowlicidin-2融合蛋白以包涵体形式表达, 经溴化氰裂解后, 成功释放出Fowlicidin-2, 获得的重组Fowlicidin-2对革兰氏阳性菌和革兰氏阴性菌均有明显的抑菌效果。【结论】实现了抗菌肽Fowlicidin-2的重组表达, 为抗菌肽的重组量化制备提供了理论基础与技术手段。

Abstract:

[Objective] The study was to clone, express the gene of chicken antimicrobial peptide fowlicidin-2 and characterize its bioactivity. [Methods] The gene encoding fowlicidin-2 with the condon preference of E. coli was designed based on the amino acids of fowlicidin-2 and synthesized in vitro. The gene was ligated into the plasmid pET32a, and transformed into E. coli BL21(DE3). The transformant E. coli BL21(DE3) was induced by IPTG. Cyanogen bromide (CNBr) was used to cleave the inclusion body of fusion protein and molecular sieve chromatography was used to purify the released fowlicidin-2. Bioactivity of the recombinant fowlicidin-2 was tested. [Results] The fowlicidin-2 fusion protein was expressed as inclusion body. Fowlicidin-2 was effectively released after digestion of the fusion protein with CNBr. The recombinant fowlicidin-2 exhibited high antibacterial activity against the Gram-positive and Gram-negative bacteria. [Conclusion] Fowlicidin-2 was successfully expressed in E. coli. The study provides theoretical foundation and technical means for scale-up preparation of antimicrobial peptides by engineering method.