GST-hDSL重组蛋白的原核表达纯化及对人造血祖细胞的扩增作用

Expression and Purification of GST-hDSL Fusion Protein and Its Effects on the Expansion of Human Hematopoietic Progenitor Cells

目的:原核表达DSL与谷胱甘肽S转移酶(GST)的融合蛋白并研究观察GST-hDSL对人脐带血CD34^+造血祖细胞的体外扩增作用。方法:将人DSL cDNA的蛋白编码序列克隆入原核表达载体pGEX-2T中,在大肠杆茵DH5α中诱导表达融合蛋白,用DEAE阴离子交换柱纯化目的蛋白。然后分离、纯化脐带血CD34^+造血祖细胞,不加细胞因子或加入SCF(干细胞生长因子,stem cell factor)和GM-CSF(粒-巨噬细胞集落刺激因子,granulocyte-macrophage colony-stimulating factor),经过14天的培养,检测GST-hDSL对细胞总数、CD^(34+)细胞百分率、以及集落形成的影响。结果:当SCF和GM-CSF存在时,GST-hDSL组的CD34^+细胞百分率,集落(colony forming cells,CFC)数以及高增值潜能集落(high proliferative potential colonyformning cells,HPP-CFC)数分别是对照组的1.9、1.2、5.3倍。结论:当与SCF和GM-CSF联合作用时,重组GST-hDSL蛋白对造血祖细胞具有扩增作用。

Objective： To investigate the expression of human DSL （hDSL） fusion protein in E. coli and its effects on expansion of human cord blood hematopoietic progenitor cells in vitro. Methods： hDSL sequence was cloned into expression vector pGEX-2T containing GST coding sequences, and transformed into E. coli DH5α. Fusion protein GST-hDSL was expressed in E. coli via IPTG induction. The expressed protein was purified using DEAE anion exchange column. After culture for two weeks with GST-hDSL in absence or in combination with SCF and GM-CSF, the output of total cells,percentage of CD^34＋ cells, colony forming cells （CFC）, high proliferation potential CFC （HPP-CFC） were evaluated. Results： In presence of SCFand GM-CSF, the percentage of CD^34＋ cells, the number of CFC and HPP-CFC in the culture system with GST-hDSL were 1.9, 1.2, 5.3 times as much as the control group, respectively. Conclusion： GST-hDSL in combination with SCF and GM-CSF promotes the expansion of cord blood CD^34＋ hematopoietic progenitor cells.