溶氧反馈-补料分批技术高密度培养基因重组MAGE1/HSP70/MAGE3工程菌  

Preparation of gene recombinant MAGE1/HSP70/MAGE3 by high cell density culture of E.coli.with DO fed-back control of nutrient feeding

作　　者：胡沛臻[1] 温江田[2] 路凡[3] 王孝功[3] 李侠[1] 司少艳[1] 葛伟[1] 黄杨[1] 张秀敏[1] 隋延仿[1] 

机构地区：[1]第四军医大学西京医院病理科,西安710032 [2]第四军医大学西京医院超声诊断科,西安710032 [3]第四军医大学基础部生物化学与分子生物学教研室,西安710032

出　　处：《第三军医大学学报》2007年第16期1576-1579,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30271464)~~

摘　　要：目的建立人黑色素瘤抗原MAGE1/HSP70/MAGE3融合蛋白基因重组工程菌E.coli.BL21/pET-MHM的高密度发酵工艺。方法以摇瓶发酵结果为基础,扩大至NBS BiofloⅣ20L发酵罐发酵,利用溶氧反馈-补料分批培养技术,对影响工程菌生长及目的蛋白表达的因素如发酵培养基、活化时间、诱导浓度及时间、pH值及分批补加营养物质等进行优化。结果保持培养过程中40%的溶解氧,采用半合成培养基、活化至对数生长期时0.2mmol/LIPTG诱导5h以及以甘油为碳源连续流加补料的条件发酵,连续3批重复发酵,最终菌密度D(600)均达45～50时,菌体量可提高至70g/L以上,目的蛋白的表达量占菌体蛋白总量的38%以上。结论确定了周期短、产率高且稳定可靠的发酵工艺。Objective To investigate the optimal high cell density fermentation conditions of recombinant E. coll. BL21/pET-MHM to produce recombinant MAGE1/HSP70/MAGE3 fusion protein. Methods Based on the experimental results obtained in shake flask cultivation, the cells were transferred to NBS Bioflo 20 L DO feed-back fed-batch culture system. The effects of the composition of the fermentation medium, activation time, induction time, the range of pH and fed-batch carbon sources on the expression level of MAGE1/HSP70/ MAGE3 and cell output were analyzed. Results By keeping dissolved oxygen at 40%, culturing in semi-defined medium and inducing for 5 h in the presence of 0.2 mmoL/L IPTG at 37℃ with glycerol as the carbon sources during fed-batch cultivation, we performed a three-time repeated fermentation. The final cell density was D（600） 45 to 50 and more than 70 g of wet bacteria per liter. The expression level of recombinant protein MAGE1/HSP70/MAGE3 was over 38% of the total protein in E. coli. Conclusion A Short cycle, high expression and stabilized fermentation process of recombinant MAGE1/HSP70/MAGE3 has been established, which founds the basis for further purification and large-scale production of MAGE1/HSP70/MAGE3.

关 键 词：黑色素瘤抗原 重组 大肠杆菌 发酵 

分 类 号：Q784[生物学—分子生物学] Q93-335