幽门螺杆菌多靶点疫苗工程菌BIB发酵及纯化工艺研究

目的初步建立重组幽门螺杆菌多靶点疫苗工程菌（BIB）的高密度发酵及其蛋白纯化工艺。方法以摇瓶发酵结果为基础,放大工艺至50L发酵罐中,对影响目的蛋白收率的因素如发酵培养基、工作种子接种量、诱导剂浓度、诱导起始时间、诱导持续时间及诱导剂添加方式等进行优化验证;并利用rBIB蛋白高等电点特性（pI=9.05）,在pH 7.0-7.5的磷酸盐缓冲液中目的蛋白带正电荷,采用阳离子交换层析进行纯化,对阳离子交换层析填料及纯化缓冲液的pH进行优化。结果经高密度发酵后BIB菌体产量为70g/L,蛋白表达量为32%;rBIB蛋白经阳离子交换层析纯化后其纯度为91.8%。结论该工艺的初步建立为深入研究rBIB蛋白性质及其规模化生产奠定了基础。

Research on High-Density Fermentation and Purification of BIB

Objective To basically establish the optimal high cell density fermentation and purification conditions of the recombinant engineering bacterium BIB of Helicobacter pylori.Method Based on the experimental results obtained in culture bottle,the BIB was transferred to fermenter culture.The effects of the culture medium,concentrations of lactose,activation time,and induction time were analyzed and optimized.After that,bacteria were collected,inclusion body was washed effectivity,and the cation-exchange chromatography was used to purify.The buffer systems was also optimized.Results Under the optimal conditions mentioned above,a highest yield of over 70g/L BIBbacteria was achieved,and the BIB was 32% of the total BIBbacterial protein.Compared with the other cation-anion-cation exchange methods,the SP Sepharose XL of this method is superior,and the purity was about 91.8%.Conclusion The method is reasonable and suitable for rBIB massproduction.