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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。
Objective To establish a high-density fermentation of recombinant Helicobacter pylori multi-target vaccine engineering bacteria (BIB) and its protein purification technology. Methods Based on the results of shake flask fermentation, the process was enlarged to 50L fermenter. Factors affecting the yield of target protein such as fermentation medium, inoculation amount of working seed, concentration of inducer, duration of induction, duration of induction and inducer (PI = 9.05). The target protein was positively charged in pH 7.0-7.5 phosphate buffer and purified by cation exchange chromatography. The cation exchange layer Optimize the pH of the packing and purification buffer. Results After high density fermentation, the yield of BIB was 70g / L and the protein level was 32%. The purity of rBIB protein was 91.8% when purified by cation exchange chromatography. Conclusion The preliminary establishment of this process lays the foundation for further research on the nature of rBIB protein and its large-scale production.