【作者】 陈其亮；

【导师】 金坚；

【作者基本信息】 江南大学 ， 微生物与生化药学， 2008， 硕士

【摘要】 本文利用构建好的重组Pichia Pastoris KM71/(GLP-1_A2G)₂-HSA酵母工程菌株表达系统进行了发酵条件优化,并对目标蛋白进行了较深入的纯化工艺研究和简单的体内、体外活性研究。将生产菌株增菌培养至一定菌体量后,用BMMY培养基悬浮菌体后定时添加甲醇进行诱导培养。研究表明重组Pichia Pastoris KM71/(GLP-1_A2G)₂-HSA的最佳诱导条件为温度30℃、pH6.0、甲醇浓度2.0 %、诱导时间为60 h。此最优化条件既节省反应材料,又可以提高效率,目标蛋白的最高产量可达到210 mg/L。发酵液经过4℃,10000rpm,15min离心除菌离心后,采用截留分子量为10KDa的Biomax超滤膜对发酵上清进行浓缩,浓缩倍数可达到20倍,回收率最高可达到90%。在精细分离过程中,首先将浓缩后的发酵液上Q-Seperose Fast Flow阴离子交换层析,上样后进行阶段洗脱,目标蛋白在盐浓度达到0.1mol/L时开始被洗脱下来,回收率达74%。所得到的洗脱成分处理后上HPLC检测,结果显示纯度为90%,SDS-PAGE显示为一条带。将Q-Seperose阴离子交换层析后的洗脱液浓缩后上Sephacryl S-200凝胶过滤,结果出现两个蛋白吸收峰,经检测后一个峰为目标蛋白分子吸收峰。将蛋白活性峰上HPLC检测,结果显示纯度达98%以上,经I125标记后,采用TCA沉淀法测定放射化学纯度达97%,纯度优于药代动力学研究的要求,达到了预期纯化目标。本纯化工艺由发酵上清经超滤、离子交换、凝胶过滤共3步组成,与文献报道的重组人血清白蛋白纯化工艺相比,具有流程简单、回收率高和成本较低等优点,为本产品的工业化生产奠定了基础。融合蛋白rh(GLP-1_A2G)₂-HSA在体外对胰岛原代细胞生长有较好的刺激作用,在浓度40 nmol/L时增殖率为35.4%,与单体的GLP-1有相似的增殖作用。更多还原

【Abstract】 Intensive studies on the process of fermentation, purification and bioactivity were conducted in this thesis.During the fermentation, the producing stain Pichia Pastoris KM71/(GLP-1_A2G)₂-HSA was induced after 60 hours processing, then the concentration of (GLP-1_A2G)₂-HSA in the fermentation liquid reached 210 mg/L. The optimized expression condition consisted temperature 30℃, pH 6.0 and the methanol induced concentration 2.0 %.This research mainly dealt with purification of recombinant protein rh(GLP-1_A2G)₂-HSA from fermentation broth. Highly purified rh(GLP-1_A2G)₂-HSA was separated from fermentation broth by ultrafiltration concentration, ion exchange chromatography and gel filtration. The purified product was conformed as one single band by SDS-PAGE and its purity was identified as 98% by HPLC while its radioactive purity was identified as 97% by TCA deposition method after marked with 125I. The purity of rh(GLP-1_A2G)₂-HSA met the requirement of drug activity and drug metabolism research. The total recovery yield reached 48.5 %. Highly purified rh(GLP-1_A2G)₂-HSA could be gained by this purification method and layed a foundation for advanced drug activity and drug metabolism research.Cell proliferative assay showed that the activity of rh(GLP-1_A2G)₂-HSA remained well during the purification while the purified product had similar proliferative activity of isletβ-cell with GLP-1. When the concentration of rh(GLP-1_A2G)₂-HSA is 40 nmol/L, the growth rate is 35.4%.更多还原