抗菌肽Cecropin AD在毕赤酵母中的组成型表达及中试发酵研究  

Large-scale constitutive expression and pilot scale fermentation of antibacterial peptide cecropin AD in pichia pastoris

作　　者：江学斌[1,2] 张敏静[3] 林健聪[2] 梁世中[1] 

机构地区：[1]华南理工大学生物科学与工程学院 [2]广州大学生命科学学院 [3]暨南大学医药生物技术研究开发中心

出　　处：《饲料工业》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]

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.

关 键 词：抗菌肽 CECROPIN AD 毕赤酵母 发酵 抑菌活性 

分 类 号：S816.6[农业科学—饲料科学]