摘要
构建表达抗菌肽Cecropin AD蛋白的真核重组表达质粒pGAPZα-A-CAD,并转化酵母菌Pichia pastoris X-33,Zeocin抗性筛选阳性重组子,通过PCR鉴定、Tricine-SDS-PAGE分析和琼脂孔穴扩散法筛选获得抗菌肽CAD组成型表达菌株,并采用pH-溶氧监控策略进行工程菌Pichia pastoris X-33/pGAPZα-A-CAD的50 L中试发酵研究。结果表明,本实验成功实现了抗菌肽CAD在毕赤酵母Pichiapastoris X-33中的组成型表达pGAPZα-A,Tricine-SDS-PAGE分析显示在4.0 kDa处有明显的目的条带,工程菌经50 L发酵罐培养48 h后收获的上清液,100℃热处理10 min后,对金黄色葡萄球菌ATCC25923、大肠杆菌ATCC25922等均有明显的抑杀作用。抗菌肽的组成型表达方法在本研究中得到了成功应用,中试研究为以后的规模化生产和应用奠定了基础。
DNA fragment coded antibacterial peptide Cecropin AD protein was cloned into pGAPZα-A-CAD and the recombinant plasmid was transformated into Pichia pastoris X-33.The transformants were screened by Zeocin and PCR amplification of yeast colonies.The expression supernatant was analyzed by Tricine-SDS-PAGE.The antibacterial activity of expression product was analyzed by agarose diffusion assay.Datas showed that the genetic engineering Pichia pastoris X-33/pGAPZαA-CAD,constitutive expressed the recombinant antibacterial peptide CAD,was successful constructed.The result of Tricine-SDS-PAGE analysis of expression supernatants showed that the line with molecular weight of 4.0 kDa was corresponding to CAD.After cultured for 48 h in 50 L fermenter,the expression supernatant（100 μl） of the Pichia pastoris X-33/pGAPZαA-CAD,treated with 100 ℃ 5 min,had obvious antibacterial activity on S.aureus ATCC25923 and E.coli ATCC25922,exceed 2 μg ampicillin.Constitutive expression and pilot scale of antibacterial peptide Cecropin AD was applied successfully in this study,which set up the fundament for the future large-scale production.
出处
《饲料工业》
北大核心
2011年第19期25-29,共5页
Feed Industry
基金
温州科技计划项目[No.20090279]
广东省科技攻关项目[2007A020300007-11]
关键词
抗菌肽
CECROPIN
AD
毕赤酵母
发酵
抑菌活性
antibacterial peptide
cecropin AD
pichia pastoris
fermentation
antibacterial activity
