响应面法优化产H因子结合蛋白的重组大肠埃希菌发酵条件  

Optimization of fermentation conditions for recombinant Escherichia coli producing factor H-binding protein by response surface methodology

作　　者：张宇 郭通 陈浩 杨飞鸽 苏桂民 朱卫华 杜琳 ZHANG Yu;GUO Tong;CHEN Hao;YANG Feige;SU Guimin;ZHU Weihua;DU Lin(Research and Development Center,Beijing Zhifei Lvzhu Biopharmaceutical Co.,Ltd.,Beijing Bacterial Vaccine Engineering Research Center,Beijing 100176,China)

机构地区：[1]北京智飞绿竹生物制药有限公司研发中心,北京市细菌性疫苗工程技术研究中心,北京100176

出　　处：《微生物学免疫学进展》2024年第2期43-51,共9页Progress In Microbiology and Immunology

摘　　要：目的 使用响应面法(response surface methodology, RSM)对重组大肠埃希菌fHBP-B-1/BL21(DE3)的发酵条件进行优化,以提高目的蛋白表达量。方法 在单因素试验的基础上,利用Plackett-Burman试验筛选出对目的蛋白表达有显著影响的因素;再用最陡爬坡试验及Box-Behnken设计进一步优化试验条件并进行验证。结果 单因素试验结果显示,在酵母抽提物质量浓度为12.500 g/L、硫酸镁质量浓度为0.250 g/L、初始pH为6.00、诱导培养时间为8 h、诱导温度为35.00℃时,目的蛋白表达量最高,为363.04μg/mL。酵母抽提物质量浓度、硫酸镁质量浓度及诱导培养温度是影响目的蛋白表达量的显著因素。发酵条件优化后,酵母抽提物质量浓度为10.500 g/L、硫酸镁质量浓度为0.220 g/L、初始pH为6.00、诱导培养时间为4 h、诱导培养温度为37.50℃时,可以获得425.62μg/mL的目的蛋白,较单因素试验结果提高了17.25%。结论 重组大肠埃希菌fHBP-B-1/BL21(DE3)的发酵条件优化后,目的蛋白的表达量明显提升,为重组B群脑膜炎球菌疫苗后续工艺的开发奠定基础。Objective The fermentation conditions of recombinant Escherichia coli fHBP-B-1/BL21(DE3) were optimized to increase the expression level of target protein by response surface methodology. Methods Based on the single factor experiment, the Plackett-Burman design was used to screen out the factors that had significant influence on the expression level of target protein. Then the experiment conditions were further optimized and verified through the steepest ascent design and Box-Behnken design. Results The results of single factor experiment showed that the highest expression level of target protein was 363.04 μg/mL when the concentration of yeast extract was 12.500 g/L,the concentration of magnesium sulfate was 0.250 g/L,the initial pH value was pH 6.00,the induction culture time was 8 h, and the induction temperature was 35.00 ℃.Yeast extract concentration, magnesium sulfate concentration and induced culture temperature were significant factors that affected the expression level of target protein. The optimized fermentation conditions were as follows: yeast extract concentration was 10.500 g/L,magnesium sulfate concentration was 0.220 g/L,initial pH was 6.00,induction culture time was 4 h and induction culture temperature was 37.50 ℃.Under these conditions, 425.62 μg/mL target protein could be obtained, with an expression level improvement of 17.25% compared to the single factor experiment results. Conclusion The expression level of target protein is significantly increased while optimizing the fermentation conditions of recombinant Escherichia coli fHBP-B-1/BL21(DE3),laying the foundation for the development of subsequent processes in recombinant group B meningococcal vaccine.

关 键 词：重组大肠埃希菌 响应面法 PLACKETT-BURMAN设计 最陡爬坡试验 H因子结合蛋白 B群脑膜炎奈瑟菌 

分 类 号：TQ920.6[轻工技术与工程—发酵工程]