鸡β-防御素7在大肠杆菌中的表达及活性分析

【目的】根据鸡β-防御素7(Gal-7)的成熟肽基因序列合成基因,构建表达Gal-7的大肠杆菌工程菌,研究重组鸡防御素Gal-7成熟肽的体外生物活性。【方法】将合成的gal-7基因克隆到大肠杆菌表达载体pGEX-6p-1中,得到重组质粒pGEX-6p-gal7,转化大肠杆菌BL21(DE3),经IPTG诱导表达得到含GST标签的融合蛋白GST-Gal7;之后用Prescission蛋白酶将GST标签切除,并对成熟肽进行质谱分析;再利用琼脂打孔扩散法检测Gal-7成熟肽的体外抑菌活性,用2倍稀释法测定对指示菌的最低抑菌浓度。【结果】成功构建Gal-7大肠杆菌异源表达工程菌,表达纯化的重组Gal-7成熟肽质谱鉴定分子量为5 516 Da,其对黄色微球菌(NCIB 8166)、金黄色葡萄球菌(ATCC 25923)、粪肠球菌(ATCC 29212)、大肠杆菌(CMCC 44102)均有抑菌活性,最低抑菌浓度分别为16.875、67.5、67.5、135 mg/L。【结论】获得表达鸡Gal-7成熟肽的大肠杆菌工程菌,并且切除GST标签的Gal-7成熟肽具有生物活性。

Heterologous expression of chicken beta-defensin-7 in Escherichia coli and antimicrobial activity analysis

[Objective] In order to study the antimicrobial activity of Gal-7 mature peptide in vitro, we constructed an engineered E. coli strain contains chicken beta-defensin-7 (gal-7) gene encoding Gal-7 mature peptide published previously. [Methods] Based on the gene sequence, we synthesized the gal-7 gene, cloned into pGEX-6p-1 prokaryotic expression vector and subsequently transformed into the E. coli BL21(DE3). The E. coli strain contains the expression vector was induced with IPTG and the fusion protein GST-Gal7 was purified by affinity chromatograph, then Prescission protease was used to remove GST-label to form Gal-7 mature peptide. The molecular weight of purified Gal-7 mature peptide was monitored by MALDI-TOF-MS and antimicrobial activity was tested by the agar well diffusion assay. Besides 2-fold dilution method was used to determine the minimum inhibitory concentration of Gal-7 mature peptide. [Results] We successfully constructed the engineered E. coli strain expressing pGEX-6p-Gal7 and GST-label was cleaved by Prescission protease. The molecule weight of purified Gal-7 mature peptide was 5 516 D, it exhibited antimicrobial activity against Micrococcus flavus (NCIB 8166), S. aureus (ATCC 25923), E. faecalis (ATCC 29212), E. coli (CMCC 44102) with MICs 16.875, 67.5, 67.5, 135 mg/L respectively. [Conclusion] The GST-Gal7 protein was successfully expressed in E. coli, besides the Gal-7 mature peptide without GST-label showed antimicrobial activity against several indicator strains.