血小板衍生生长因子BB亚型在毕赤酵母中的表达及纯化  

Expression and Purification of Recombinant Human Platelet-Derived Growth Factor-BB in Pichia pastoris

作　　者：房婷[1] 张晓鹏[1] 章晟[2] 戴萌萌[1] 杨秀旭[1] 付玲[1] 于长明[1] 

机构地区：[1]军事医学科学院科学院生物工程研究所,北京100071 [2]北京军区总医院附属八一儿童医院,北京100700

出　　处：《生物技术通讯》2015年第5期605-610,共6页Letters in Biotechnology

摘　　要：目的:用毕赤酵母表达系统表达重组人血小板衍生生长因子BB亚型(PDGF-BB)。方法:采用PT-PCR从人早幼粒白血病细胞系HL-60细胞中获得目的基因,克隆到表达载体p MEX9K中,质粒线性化后转化酵母表达菌株GS115,筛选后的酵母表达菌株经BMGY/BMMY培养基体系诱导表达后,通过疏水作用、离子交换、凝胶过滤纯化获得目的蛋白,采用SDS-PAGE、Western印迹、N端氨基酸序列和MTT增殖活性测定等方法检测目的蛋白性质及生物活性。结果:重组人PDGF-BB为分泌表达,表达量大于100 mg/L;经三步纯化,获得纯度高于95%且具有较高活性(5.0×105IU/mg)的目标蛋白。结论:利用毕赤酵母表达系统表达重组人PDGF-BB,表达产量高,成本低,工艺简单,易于工业化放大,有良好的市场前景。Objective： To clone, express the recombinant human platelet-derived growth factor-BB（rhPDGF-BB） protein in Pichia pastoris system. Methods： Human PDGF-BB gene was cloned by RT-PCR from human promyelo- cytic leukemia cells HL-60 via extraction of total RNA, and then inserted into the expression vector pMEX9K. Re- combinant plasmids were linearized and integrated into P.pastoris expression strain GSllS, and expression strains of mutant proteins were obtained after screening on MD plate. Finally, the yeast strains were induced to express protein in BMGY/BMMY culture medium, and rhPDGF-BB proteins were obtained after three steps of purifica- tion process including hydrophobie interaction chromatography, ion-exchange chromatography and gel chromatogra- phy. The purified proteins were analyzed by SDS-PAGE, Western blot, N-terminal sequencing and MTT prolifera- tion assay to detect protein properties and biological activity. Results： The rhPDGF-BB was expressed in secretory form with a final yield more than 100 mg/L, and obtained with high purity（〉95%） and high biological activity （5.0×10^5 IU/mg） after three steps of purification process. Conclusion： The rhPDGF-BB was expressed by P.pasto- ris system with high yield, the whole process was simple, economic, easy to scale up and showed a distinct eco- nomics benefits.

关 键 词：重组人血小板衍生生长因子BB亚型 巴斯德毕赤酵母表达系统 表达与纯化 

分 类 号：Q78[生物学—分子生物学]