巴斯德毕赤酵母表达的人抑瘤素M发酵条件及发酵产物分析  

Fermentation conditions of human oncostatin M expressed in Pichia pastoris and analysis of fermentation products

作　　者：孔宁[1] 高海成[2] 刘国民[3] 陈月[1] 周余来[1] 颜炜群[1] 

机构地区：[1]吉林大学药学院医用生物材料学教研室,吉林长春130021 [2]吉林大学药学院临床药学与药事管理学教研室,吉林长春130021 [3]吉林大学第二医院骨科,吉林长春130041

出　　处：《吉林大学学报（医学版）》2012年第5期943-947,共5页Journal of Jilin University：Medicine Edition

基　　金：吉林省卫生厅科研基金资助课题(3D511AJ83432)

摘　　要：目的:研究巴斯德毕赤酵母表达的人抑瘤素M(hOSM)大规模发酵条件。方法:利用hOSM毕赤酵母高表达菌株,于试管水平进行发酵pH条件的摸索,以补料分批培养方式在80L发酵罐中对hOSM工程菌进行3个批次的高密度发酵,控制和优化各种发酵条件,通过SDS-PAGE对发酵产物进行分析。结果:最终确定在pH 5.0的FM21培养基中扩增菌体,待菌体密度达190g.L-1时添加甲醇诱导,在发酵液pH 5.5的条件下,发酵温度稳定在28℃,通入空气的流量为2vvm,转速为650r.min-1,罐内压力为10P,溶解氧保持在25%左右,甲醇流加速度在9.3mL.h-1.L-1初始发酵液体积,诱导36h时结束发酵,hOSM占分泌总蛋白的28%,产量达到280mg.L-1。结论:应用hOSM工程菌能够进行稳定的发酵,为进行下一步生物学功能测定提供充足的物质基础。Objective To optimize the fermentation condition of large scale fermentation of human oncostatin M（hOSM） with engineering strain of Pichia pastoris.Methods The optimal pH condition of hOSM expressed in the methylotrophic yeast Pichia pastoris X-33 in tube-scale was determined and fermentation of hOSM was performed in an 80 L fermentor in a fed-batch mode for three times.The pH value,culture medium,dissolved oxygen,temperature,methanol feeding speed,initial biomass and etc were focused mainly.hOSM was analyzed by SDS-PAGE.Results In the FM21 medium of pH 5.0,when a cell yield of 190 g·L-1 wet weight was achieved,methanol induction phase began.In the fermentation broth of pH 5.5,the temperature was controlled at 28 ℃,a stirring speed of up to 650 r·min-1,the pressure of fermentor was 10 P,dissolved oxygen above 25% and the supply speed of methanol was 9.3 mL·h-1·L-1 initial fermentation volume,after methanol induction for about 36 h,the expression level of hOSM reached 280 mg·L-1.Conclusion OSM is successfully obtained through genetic engineering for large scale production of hOSM for following biological functions.

关 键 词：抑瘤素M 毕赤酵母 发酵 

分 类 号：Q78[生物学—分子生物学]