重组人干细胞因子

Recombinant Human Stem Cell Factor

选择大肠杆菌的偏性密码，合成了可溶形式的人干细胞因子（ｈＳＣＦ）的ｃＤＮＡ。通过ＰＣＲ完成了合成片段的一次性组装与克隆，并在Ｅ．ｃｏｌｉ中进行了温控型的高效表达，目的蛋白可占菌体总蛋白的４０％左右。表达产物复性后，经离子交换、凝胶过滤层析，得到了电泳纯的ｒｈＳＣＦ。经测定，ｒｈＳＣＦ氨基端序列及其它理化性质与天然ｈＳＣＦ完全一致，并可刺激人骨髓细胞的增殖，导致粒、巨核系集落（ＣＦＵ－ＧＭ）大小、数量的明显增加，显示天然ｈＳＣＦ的生物活性。

We report here the chemical synthesis cloning and expression of the cDNA for human stem cell factor in Escherichia coli. The synthetic oligonucleotides were assembled in only one step by a PCR approach and inserted into pUC19 for cloning. The sequence of the whole synthetic cDNA was confirmed by DNA sequencing. The synthetic hSCF cDNA was also expressed at a high level in E.coli and the hSCF protein consisted about 40% of the total soluble protein as detected by the SDS PAGE assay. After oxidation and refolding, the crude extract was applied on to anion exchange and gel filtration chromatography. With these procedures, a homogeneous rhSCF was obtained, which has the same amino terminal sequence and other physical chemical characters to that of natural hSCF. The purified rhSCF also has the ability to stimulate the proliferation of human bone marrow progenitor cells, resulting in increased numbers and size of granulocyte macrophage colony and showing the natural hSCF′s bioactivity.