【作者】 杨青；

【导师】 张玉奎；

【作者基本信息】 中国科学院研究生院（大连化学物理研究所） ， 分析化学， 2005， 博士

【摘要】 人组织激肽释放酶是一种丝氨酸蛋白酶。它在人体内重要的生理作用说明它有可能成为治疗肾脏病的有效药物。此外,它在血液中的含量与缺血性脑中风的相关性研究显示它可以作为该疾病体外诊断的重要指标。本论文选用Sf-9及Hi-5两种昆虫细胞为载体,用基因工程的手段表达制备了人组织激肽释放酶原。对两种昆虫细胞的无血清适应及悬浮适应条件进行了系统研究,将基因工程人组织激肽释放酶原的表达量提高到25mg/L。对载有人组织激肽释放酶cDNA的CHO细胞进行了无血清悬浮适应。采用色谱技术对细胞表达产物进行了分离纯化。探讨了不同流动相组成、pH值、离子强度以及分离介质的影响。分离纯化后的样品经检测为纯品,分子量约为40961Da,回收率约为57%。对检测未知样本中组织激肽释放酶含量的ELISA方法进行了系统优化。开发了缺血性脑中风早期诊断试剂盒。考查了试剂盒的各种指标,并将其用于660例健康人及21例脑中风患者血液中组织激肽释放酶含量的检测。此外,与大连医科大学附属二院合作,用分离纯化后得到的人组织激肽释放酶原对糖尿病肾病大鼠进行了实验。结果显示,组织激肽释放酶对肾脏具有较好的保护作用。更多还原

【Abstract】 Tissue kallikrein is a kind of serine proteinase, and its important biological functions indicate that it might be an effective drug against renal disease. In addition, the relationship between the tissue kallikrein content in blood and ischamia stroke makes it as a possible biomarker for the early diagnosis of such a disease.In this dissertation, baculovirus-insect cell system was employed to express human tissue prokallikrein. Sf-9 cell was used to amply recombinant baculovirus, whilst Hi-5 cell was taken to express the target protein. These two cells were recultured in medium without FBS, and further suspended in a spinner flask. After systematic studies on the effects of MOI, infection and the harvesting time, the expression level of recombinant human prokallikrein was as high as 25mg/L. In addition, CHO cell which harboring human tissue kallikrein cDNA was also recultured and suspended in the medium free of FBS.Tissue prokallikrein excreted from insect cells was purified by liquid chromatography. The influences of pH, buffer composition, ionic strength and separation matrix on purification were studied to obtain the optimal conditions for the purification of recombinant prokallikrein. Furthermore, HPLC/MS analysis and amino acid sequencing were performed to validate the purified protein. By HPLC, SDS-PAGE and CIEF analysis, the obtained protein was found pure, and the recovery was about 57%.更多还原