Chen Tao, Zang Yuhui, Zhu Jie, Lu Haiqin, Han Junhai, Qin Junchuan
School of Life Science and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China.
Protein Expr Purif. 2005 Jun;41(2):402-8. doi: 10.1016/j.pep.2005.02.009.
A novel human stem cell factor (SCF)/macrophage colony-stimulating factor (M-CSF) fusion protein gene was constructed, in which the coding regions of human SCF cDNA (1-165aa) and the truncated M-CSF cDNA (1-149aa) were connected by a linker sequence encoding a short peptide GGGGSGGGGSGG. The SCF/M-CSF gene was cloned into baculovirus transfer vector pVL1392 under the control of polyhedrin promoter and expressed in the Sf9 cells (Spodoptera frugiperda). SDS-PAGE and Western blot analysis showed that the purified fusion protein was a homodimer with a molecular weight about 84kDa under non-reducing conditions or a monomer about 42kDa under reducing conditions. The specific activity of rhSCF/M-CSF was 17 times as high as that of monomeric rhSCF to stimulate the proliferation of TF-1 cell. The results of macrophages colony-forming (CFU-M) assay performed with human bone marrow mononuclear cells demonstrated that rhSCF/M-CSF was more potent in promoting CFU-M than the equimolar of SCF, M-CSF or that of two cytokines mixture.
构建了一种新型人干细胞因子（SCF）/巨噬细胞集落刺激因子（M-CSF）融合蛋白基因，其中人SCF cDNA（1-165aa）的编码区与截短的M-CSF cDNA（1-149aa）通过编码短肽GGGGSGGGGSGG的接头序列连接。将SCF/M-CSF基因克隆到多角体蛋白启动子控制下的杆状病毒转移载体pVL1392中，并在Sf9细胞（草地贪夜蛾）中表达。SDS-PAGE和Western印迹分析表明，纯化的融合蛋白在非还原条件下是分子量约为84kDa的同二聚体，在还原条件下是约42kDa的单体。重组人SCF/M-CSF刺激TF-1细胞增殖的比活性是单体重组人SCF的17倍。用人骨髓单个核细胞进行巨噬细胞集落形成（CFU-M）试验的结果表明，重组人SCF/M-CSF在促进CFU-M方面比等摩尔的SCF、M-CSF或两种细胞因子混合物更有效。
