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。
The synthesized AvBD2 gene according to mature peptide mRNA was cloned into pGHKα expression 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 re- sponse surface methodology. Tricine-SDS-PAGE and Western-blot experiments showed that AvBD2 protein was about 5.8 ku in size. Agar diffusion experiment indicated that the recombinant protein had obviously antibacterial activity against Escherich ia coli（ CMCC44102）, Enterococcus f aecalis （ ATCC29212 ）, Staphy- lococcus aureus （ATCC25923） and Pasteurella. The hemolytic activity of fermentation production with the concentration of 500 mg/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 528 mg/100 mL inoculum and liquid volume 8.43%. These results showed that the recombinant AvBD2 with obviously antimierobial activity was expressed in P. pastoris and the optimal conditions of fer- mentation were achieved,which provide theoretical foundation for the application of AvBD2 as a new feed additive.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2015年第7期693-699,共7页
Chinese Veterinary Science
基金
国家自然科学基金项目(31372402)
国家高技术研究发展计划(863)项目(2006AA10Z320)
