汉逊酵母高密度发酵表达乙型肝炎表面抗原的甲醇流加控制策略  

Strategy for Control of Methanol Feeding During High Density Fermentation of Hansenula polymorpha for Expression of HBsAg

作　　者：杨喆 刘红岩 马波 郭仁 代云波 李昌军[2] 侯峰 谢飞 

机构地区：[1]云南沃森生物技术有限公司基因工程疫苗项目组,昆明650118 [2]华中师范大学生命科学学院昆虫学研究所,武汉430074

出　　处：《中国生物制品学杂志》2007年第11期851-854,共4页Chinese Journal of Biologicals

基　　金：国家863计划资助项目(编号:2006AA02Z487).

摘　　要：目的考察不同甲醇流加策略对汉逊酵母（Hansenula polymorpha）高密度发酵表达重组乙型肝炎表面抗原的影响。方法在细胞生长阶段采用DO/pH在线测量的控制方法,使酵母细胞密度达到一个较高水平,诱导期比较DO-Stat/pH-Stat法和离线检测甲醇两种控制流加甲醇的方法,同时也比较用甘油和甲醇双碳源间断流加法与单纯流加甲醇法对目的蛋白表达的影响。结果诱导期用DO-Stat和pH-Stat法不能有效地提高乙肝表面抗原表达量,而离线检测甲醇的控制方法,在诱导阶段可将甲醇浓度控制在0~5g/L的范围,乙肝表面抗原表达量比DO-Stat/pH-Stat法提高了20%。甲醇氧化酶（MOX）酶活性的变化与乙肝表面抗原表达量变化存在对应关系,交替加入甘油和甲醇双碳源刺激,可以使乙肝表面抗原表达量达到395mg/L。结论已筛选出优化工艺配置,并建立了切实可行的规模化生产工艺。Objective To explore the effect of strategy for methanol feeding during high density fermentation of Hansenula polymorpha on the expression of recombinant HBsAg.Methods Control the methanol feeding in growth phase of Hansenula polymorpha by online monitoring.In induction phase,the expression levels of HBsAg when the methanol feeding were controlled by DO-Stat/pH-Stat method and by offline gas chromatography,as well as those when only methanol was fed and glycerol and metyanol were fed alternatively,were compared.Results In induction phase,the control of methanol feeding by both DO-Stat and pH-Stat methods failed to increase the expression level of HBsAg effectively.However,by off-line gas chromatography,the methanol concentration was controlled at 0～5 g/L,and the expression level of HBsAg increased by 20% as compared with those by DO-Stat/pH-Stat method.In addition,the expression level of HBsAg increased with the increasing activity of methanol oxidase.The alternative feeding of glycerol and methanol increased the expression level of HBsAg to 395 mg/L.ConclusionAn optimal procedure for fermentation of Hansenula polymorpha and large-scale production of hepatitis B vaccine was developed.

关 键 词：汉逊酵母 高密度发酵 表达 乙肝表面抗原 甲醇 流加 

分 类 号：Q78[生物学—分子生物学]