rhTpo／GM-CSF融合蛋白的构建、表达及活性测定

本研究的目的是找出一个治疗由于放化疗等原因造成的造血组织损伤导致的贫血、感染和出血的有效方法。通过RT-PCR的方法从人胎肝中克隆了重组人血小板生成素(rhTpo)的基因,利用基因工程的手段将其与重组人粒-巨噬细胞集落刺激因子(rhGM-CSF)的基因相融合,并在原核细胞中表达。研究结果表明,大肠杆菌JM101表达的融合蛋白rhTpo/GM-CSF在体外小鼠骨髓造血祖细胞的培养中保留了Tpo对巨核细胞系和红细胞系的刺激作用,并增加了GM-CSF对粒细胞系的刺激活性。结论提示,原核细胞表达的融合蛋白rhTpo/GM-CSF具有刺激骨髓红细胞系、粒细胞系及巨核细胞系造血的活性。

Construction, expression and biological activity of recombinant human fusion protein Tpo/GM-CSF

In order to find a new biological therapy for the secondary hematopoietic failure including anemia, infection and hemorrhage after irradiation and administration of chemiotheraputic drugs etc, we ligated hGM-CSF gene with hTpo gene isolated from human fetal liver mRNA. After expressing this constructed gene in E. coli JM101, we purified the expressed inclusion body from the SDS-PAGE and obtained a new fusion protein rhTpo/ GM-CSF. For delineating the biologic function of fusion protein rhTpo/GM-CSF, the bone marrow cells of normal mice were cultivated in vitro with purified rhTpo/GM-CSF. As a result, rhTpo/GM-CSF significantly stimulated the production of erythrocyte, granulocyte and megakaryocyte colonies. These resuls suggested that the fusion protein rhTpo/GM-CSF has the both biological activity of hTpo and hGM-CSF which can stimulates the proliferation of all myeloid progenitors.