【摘要】 合成鸡β防御素2(AvBD2)成熟肽基因,构建重组质粒pGHK-AvBD2;将其电转化至毕赤酵母GS115;Tricine-SDS-PAGE和Western-blot检测表达产物,琼脂扩散法测定产物的抑菌活性,并测定产物的溶血活性;响应面法优化接种量、装液量和发酵温度。Tricine-SDS-PAGE和Western blot检测结果显示,在5.8ku位置出现目的蛋白质条带;抑菌活性检测表明,表达产物对粪肠球菌(ATCC29212)、大肠杆菌(CMCC44102)、金黄色葡萄球菌(ATCC25923)和巴氏杆菌具有明显的抑菌活性;发酵产物在500mg/L时的溶血活性仅2.17%。由建立的响应面模型得到重组酵母最佳发酵条件为:接种量528mg/100mL、装液量8.43%、发酵温度27.8℃,产物蛋白浓度较优化前提高20%。本研究实现了AvBD2在毕赤酵母中的组成型表达,表达产物有较好的抗菌活性,为AvBD2作为新型饲料添加剂的应用奠定了基础。更多还原

【Abstract】 The synthesized AvBD2 gene according to mature peptide mRNA was cloned into pGHKαexpression vector and electrotransformed into Pichia pastoris GS115.After fermentation,the supernatant was analyzed by Tricine-SDS-PAGE,Western-blot,agar diffusion test and hemolytic activity test.Then,fermentation conditions of inoculation quantity,medium volume and temperature were optimized by response surface methodology.Tricine-SDS-PAGE and Western-blot experiments showed that AvBD2 protein was about 5.8ku in size.Agar diffusion experiment indicated that the recombinant protein had obviously antibacterial activity against Escherichia coli(CMCC44102),Enterococcus faecalis(ATCC29212),Staphylococcus aureus(ATCC25923)and Pasteurella.The hemolytic activity of fermentation production with the concentration of 500mg/L was only 2.17%.After optimizing the fermentation conditions,the production protein concentration had increased by 20% when GS115-AvBD2 was cultivated in YPD at 27.8 ℃,with addition of 528mg/100 mL inoculum and liquid volume 8.43%.These results showed that the recombinant AvBD2 with obviously antimicrobial activity was expressed in P.pastoris and the optimal conditions of fermentation were achieved,which provide theoretical foundation for the application of AvBD2 as a new feed additive.更多还原