重组鹅β-防御素12蛋白的原核表达及生物学特性

Recombinant expression and biological characteristics of goose β-defensin 12

【目的】研究重组鹅β-防御素12蛋白的原核表达并探究其生物学特性。【方法】采用His标签蛋白原核表达系统,将鹅防御素12(Av BD12)基因亚克隆到表达载体p Pro EX-HTa上,构建重组表达质粒。将重组表达质粒转化到大肠杆菌Rosseta感受态中,用IPTG进行诱导表达,并对该重组蛋白进行纯化。进一步采用菌落计数法测定其体外抗菌活性和盐离子稳定性。【结果】经Tricine-SDS-PAGE电泳分析,诱导表达的鹅Av BD12重组蛋白分子量约为12 k D,大部分以包涵体形式存在。该重组蛋白对大肠杆菌、鸡白痢沙门氏菌、金黄色葡萄球菌、四联球菌、枯草芽孢杆菌均具有抗菌活性,高浓度盐离子显著抑制重组蛋白的抗菌活性。此外,该重组蛋白对鸡红细胞没有溶血活性。【结论】该重组蛋白具有广谱抗菌活性,高浓度盐离子显著降低其抗菌活性,且该重组蛋白不具有溶解鸡红细胞的活性。

[Objective] The study was to express and characterize the fusion protein of goose β-defensin（Av BD） 12 in Escherichia coli. [Methods] The c DNA of goose Av BD12 was cloned into Eco R I and Xho I sites of p Pro EX-HTa vector. The recombinant expression plasmid was translated into E. coli Rosseta and the bacteria were induced by isopropyl β-D-1-thiogalactopyranoside（IPTG）. The recombinant protein was purified. Furthermore, antibacterial activity and salt ionic stability of the recombinant protein were determined by colony-counting assays. [Results] It was demonstrated by Tricine-SDS-PAGE that the recombinant protein（molecular weight, 12 k D） was produced as insoluble bodies in the cells. The recombinant protein showed extensive antibacterial activity against bacteria, including E. coli, Micrococcus tetragenus, Salmonella pullorum, Bacillus subtilis, and Staphylococcus aureus. At high Na Cl concentrations, the antibacterial activity of goose Av BD12 decreased significantly. In addition, goose Av BD12 showed little hemolytic activity. [Conclusion] Recombinant goose Av BD12 exhibited extensive antibacterial activity. High salt concentration significantly decreased its antibacterial activity. In addition, Av BD12 showed little hemolytic activity.