Flt3配体的同源二聚体构建及其生物学特性

Flt3配体 (FL)是一种早期造血生长因子 ,与相应受体结合后 ,驱动受体二聚化 ,从而启动细胞内信号转导。体内 ,可溶性的FL主要以单体形式存在。将 2个相同的编码可溶性FL的基因片段 ,通过连接序列以首尾相连的方式构建成FL FL基因 ,并在毕氏酵母 (Pichiapastoris)中表达。SDS PAGE试验表明 ,FL FL融合蛋白 (rFL FL)分子量约为 4 3kD ,与预期相符 ,并为Western印迹试验所证实。骨髓祖细胞体外集落形成试验提示 ,rFL FL具有更强的生物学活性 ,约是单体FL(rhFL)的 10倍之多。另外 ,FL FL融合蛋白还能促进和延长饥饿的Raji细胞存活 ,与rhFL相比 ,也表现出较强的生物学功能。

Increasing Bioactivity of Flt3 Ligand by Fusing Two Identical Soluble Domains

Flt3 ligand (FL) is a hematopoietic growth factor, initiating its intracellular signaling cascade by binding to counterpart receptor and driving receptor dimerization. The native form of soluble FLin vivois mainly monomeric. In this study, we constructed a rFL-FL fusion protein cDNA by linking two copies of cDNA encoding the soluble domain of FL in tandem and expressed it inPichia pastoris. On SDS-polyacrylamide gel electrophoresis, the rFL-FL fusion protein showed a molecular weight of 43 kD, agreeing well with the predicted value. The 43 kD protein was further confirmed by Western blot using polyclonal rabbit anti-human FL antibody. The rFL-FL fusion protein exhibited about 10-fold increment in its activity on colony formation of bone marrow progenitor cells. rFL-FL fusion protein also exerted more potent effect than monomeric FL on extending the survival of starving Raji cells.