粒细胞巨噬细胞集落刺激因子/白细胞介素-3融合蛋白的表达研究

Studies on the Expression of the Recombinant Human GM-CSF/IL-3 Fusion Protein

利用PCR扩增得到粒细胞 巨噬细胞集落刺激因子 (GM CSF)、白细胞介素 3(IL 3)完整基因片段 ,将其分别克隆至pGEM T ,构建成GM CSF/IL 3融合蛋白基因 ,DNA序列与设计预期一致。将得到的融合蛋白基因克隆至T7RNA聚合酶表达载体pT7zz,得到表达质粒pFu ,经转化至表达宿主E .coliBL2 1(DE3) ,在IPTG诱导下获得融合蛋白目的产物的直接表达。经SDS PAGE电泳鉴定扫描分析 ,目的基因产物表达量占菌体总蛋白量的 30 %以上 ,目的基因表达产物以包涵体的形式表达。Western blot鉴定表明 ,该表达产物可以与GM CSF抗体及IL 3抗体特异性结合。目的基因表达产物经过包涵体变性、透析复性及柱层析纯化 ,用GM CSF、IL 3依赖细胞株TF 1检测 。

A human granulocyte macrophage colony stimulating factor (GM CSF)/interleukin 3(IL 3) fusion gene with a short linker between the GM CSF and IL 3 gene has been successfully constructed and expressed in E.coli under the control of T7 promoter.The recombinant fusion protein was expressed as inclusion bodies after the IPTG induction.The yield of the GM CSF/IL 3 fusion protein was over 30% of the total cellular proteins.Western blotting results showed that the fusion protein could specifically combined with GM CSF antibody and IL 3 antibody.The biological activity was detected by the GM CSF and IL 3 dependent cell line TF 1.After solubilizing with 8mol/L urea and renaturing with dialysis against Tris.HCl solution,the refolded fusion protein showed obvious activities to maintain the growth of TF 1 cell.