新型长效EPO的构建、表达及生物活性研究

发布时间：2018-07-05 06:31

本文选题：EPO + 贫血　；参考：《吉林大学》2005年博士论文

【摘要】：红细胞生成素(EPO,erythropoietin)是一种刺激骨髓造血的糖蛋白类激素,基因工程方法生产的重组人红细胞生成素在治疗肾性贫血及其它类型的贫血等方面有很好的疗效。为了增加其生物活性,及延长其半衰期,作者构建了三种不同启动子的重组人红细胞生成素(rhEPO)二聚体真核表达载体pBT-1c、pBT-2s、pBT-3c,及两个rhEPO 单体表达载体pBT-2se,pBT-3ce。比较其转染效率后,选择一种真核表达载体pBT-2s 稳定转染CHO-dhfr-细胞,经MTX加压,筛选到稳定高效表达rhEPO 二聚体融合蛋白的细胞株CHO-BTE,表达量达到33.3μg /106cell?d。经纯化得到rhEPO 二聚体融合蛋白,经检测分子量约为70KD,其体内及体外生物活性均显著高于rhEPO 单体。经大鼠体内的初步药代动力学试验,结果表明,此rhEPO 二聚体融合蛋白在大鼠体内的半衰期比rhEPO 单体半衰期延长。
[Abstract]:Erythropoietin (EPO) is a glycoprotein hormone that stimulates bone marrow hematopoiesis. Recombinant human erythropoietin produced by genetic engineering has a good effect in the treatment of renal anemia and other types of anemia. In order to increase its biological activity and prolong its half-life, the authors constructed three recombinant human erythropoietin (rhEPO) dimer eukaryotic expression vectors pBT-1, pBT-2spBT-3cand two rhEPO monomeric expression vectors pBT-2sesepBT-3ce. After the transfection efficiency was compared, a eukaryotic expression vector pBT-2s was selected to transfect CHO-dhfr- cells stably. The cell line CHO-BTE-1, which expressed rhEPO dimer fusion protein stably and efficiently, was screened by MTX pressurization. The expression level reached 33.3 渭 g / 10 6 cell line. RhEPO dimer fusion protein was purified and its molecular weight was about 70 KD.The bioactivity of rhEPO was significantly higher than that of rhEPO monomer in vivo and in vitro. The preliminary pharmacokinetic test in rats showed that the half-life of rhEPO dimer fusion protein in rats was longer than that of rhEPO monomer.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346

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