重组毕赤酵母高密度发酵表达H5N1禽流感病毒糖蛋白  

Expression of H5N1 avian influenza virus haemagglutinin protein in Pichia pastoris by high-density cell fermentation

作　　者：杨坤宇[1] 何芳萍[1] 李少伟[1] 张佳鸿[1] 林庆山[1] 陈振钦[1] 李仲艺[1] 张军[1] 夏宁邵[1] 

机构地区：[1]厦门大学国家传染病诊断试剂与疫苗工程技术研究中心生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室,厦门361005

出　　处：《生物工程学报》2009年第5期773-778,共6页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.2006AA020905);国家自然科学基金(No.30500092);科技部工程中心建设项目(No.2005DC105006);教育部新世纪优秀人才培养计划(No.NCET-05-0567);厦门市科技计划社会发展项目(No.3502Z20073001)资助~~

摘　　要：在10L发酵罐中,对高致病性禽流感病毒H5N1糖蛋白HA1在重组毕赤酵母中的表达发酵工艺进行了研究。通过分批补料培养方法探讨不同培养温度、诱导温度、补料方式、微量元素等因素对菌体的生长以及重组蛋白表达和活性的影响。结果表明,菌种培养和诱导温度均为25oC时,菌体的生长、分泌表达量和与广谱中和抗体的反应活性较好;微量元素是影响重组HA1蛋白生物活性的重要因素;通过优化高密度发酵工艺,H5N1病毒糖蛋白HA1在发酵罐中的表达量比摇瓶培养提高10.5倍,达到约120mg/L,为大规模制备高致病性禽流感病毒的HA1蛋白奠定了基础。We produced high pathogenic avian influenza H5N1 haemagglutinin protein HA1 in recombinant Pichia pastoris in a 10 L fermentor, to establish a high-density cell fermentation method. We studied the effects of different factors such as culture temperature, induced temperature, methanol feeding methods, trace elements on the growth of Pichia pastoris, the yield and the biologic activity of recombinant HA1 protein. The culture temperature in pre-induced and induced stage were optimized at 25~C to adapt cell growth and recombinant protein expression, and induced temperature at 25~C also resulted in higher biologic activity of rHA1 than at 30~C. The binding activity of rHA1 against a wide-spectrum neutralizing antibody was susceptible to the presence of any trace elements, although trace elements would essentially benefit for the cell fermentation. As a conclusion, the expression level of rHA1 produced with optimized fermentation process reached 120 mg/L, which was 10.5 times higher than the one produced in regular shaking flask. The resultant high-density cell fermentation can likely produce rHA1 of H5N1 in large scale.

关 键 词：毕赤酵母 高致病性禽流感病毒 H5N1 HA1 发酵 

分 类 号：S852.65[农业科学—基础兽医学]