表达乙肝表面抗原结合蛋白的毕赤酵母工程菌遗传稳定性研究  

Study on the Genetic Stability of the Recombinant Pichia Pastoris Strain Expressing HBsAg Binding Protein

作　　者：李津[1] 范长胜[1] 朱乃硕[1] 

机构地区：[1]复旦大学生命科学学院生物医学研究院,上海200433

出　　处：《药物生物技术》2014年第1期18-21,共4页Pharmaceutical Biotechnology

基　　金：国家自然科学基金资助项目(No.30571650;31370927);国家科技重大专项资助项目(No.2012ZX10002006-002-003);863生物医药高技术课题资助项目(No.2011AA02A114);上海市科委科学基金资助项目(No.13431900602)

摘　　要：研究表达乙肝表面抗原结合蛋白的毕赤酵母工程菌的遗传稳定性,为今后可能的大规模生产奠定基础。在无选择压力下,将分泌表达乙肝表面抗原结合蛋白SBP的毕赤酵母工程菌在YPD固体平板上进行连续传代培养,并对工程菌的菌落和细胞形态、SBP的分泌表达情况以及目的基因的拷贝数等进行检测。结果显示,传代至50代过程中,工程菌的菌落特征和菌体形态与典型的毕赤酵母菌保持一致,其基因组中SBP基因未发生突变,并且原代和第30,50代工程菌基因组中SBP基因的整合拷贝数都为1;目的蛋白SBP的分泌表达量基本不变,SBP在发酵液可溶性蛋白中所占比例保持在50%左右。该毕赤酵母工程菌具有良好的遗传稳定性,适合大规模生产。The genetic stability of the SBP-expressing Pichia pastoris recombinant strain was studied,which may lay a foundation for the large-scale production of SBP in the future. The recombinant strain was cultured on YPD solid medium under non-selection pressure and subcultured serially for 50 generations. During this period, cell morphology, DNA sequence and integrated copy number of the target gene were analyzed. The expression of SBP was identified by SDS-PAGE. It was found that the generation 50 recombi- nant strain had the same appearances of cells and colonies on YPD medium as the original recombinant strain. SBP gene sequence did not change at all. The proportion of SBP in total soluble proteins secreted by the recombinant strain was about 50% throughout the period, indicating that the production of SBP was relatively stable. Furthermore, the integrated copy number of SBP gene in the genome of Generation 50 recombinant strain was just 1, which was also identical to that of the original recombinant strain. All the above results show that this SBP-expressing Pichia pastoris recombinant strain has a very good genetic stability and is suitable for large-scale production.

关 键 词：乙肝表面抗原结合蛋白 毕赤酵母 遗传稳定性 

分 类 号：R575.1[医药卫生—消化系统]