人干细胞生长因子cDNA克隆、表达和纯化及其造血种属特异性

Cloning, Expression and Purification of Human Stem Cell Growth Factor cDNA and Its Species-specificity in Hematopoiesis

人干细胞生长因子(humanstemcellgrowthfactor,hSCGF)是一种早期造血调控因子。已知人干细胞生长因子具有两种形式,包括全长分子hSCGF以及在Ca2+依赖糖识别结构域(calcium-dependentcarbohydraterecognitiondomain,CRD)内缺失78氨基酸的截短型分子hSCGFβ。hSCGFβ的造血刺激活性具有严格的种属特异性。本研究目的在于探讨hSCGF能否协同刺激小鼠粒/单系祖细胞增殖。为克服hSCGF的cDNAGC含量较高的困难,本研究以两步PCR的方法从人胎肝cDNA文库(Clontech)中成功克隆hSCGFcDNA,进而将hSCGF成熟肽编码序列亚克隆于原核表达载体pGEX4T-2中,融合表达。研究结果表明,通过低温诱导(28℃),重组表达产物主要以可溶蛋白的形式存在于裂解上清中,利用亲和层析纯化融合表达蛋白。对重组蛋白的造血刺激活性分析表明,不同于截短型分子hSCGFβ,全长分子hSCGF能够刺激小鼠骨髓粒/单系造血祖细胞增殖。结论:hSCGFCRD不直接结合受体,可能具有其他生物学功能。

Stem cell growth factor （SCGF） is an early-acting hematopoitic cytokine that has two isoforms including hSCGF with full length molecules and hSCGFβ,78 amino acids of which lost in the conserved calcium-dependent carbohydrate-recognition domain （CRD）. It has been demonstrated that hSCGFβ is strictly species-specific in regulating he matopoiesis. This study was aimed to explore whether human SCGF can exert synergistic stimulatory effect on heterogenous murine CFU-GM progenitor. Firstly, hSCGF cDNA was amplified from human fetal liver cDNA library by using two-step PCR. The hSCGF mature peptide coding sequence was subsequently placed at downstream of glutathione Stransferase （GST） sequence in GST gene fusion expression vector. The results indicated that there existed an additional 60 kD protein compared with mock BL21 when the cells hosting recombinant plasmid were induced with IPTG at 37℃. SDS-PAGE analysis demonstrated that the GST-hSCGF fusion protein mainly existed in insoluble form. When induced at low temperature （28℃）, the recombinant protein was mostly soluble. The GST-fusion recombinant protein was subsequently purified by using affinity chromatography. The clonogenic assay revealed that, unlike hSCGFβ, hSCGF had the granulocyte/macrophage promoting activity （GPA） for murine bone marrow GM progenitor. It is concluded that, in contrast to human SCGFβ, the intact molecular hSCGF may have no species specificity, implying that CRD domain in human SCGFβ does not directly bind to corresponding SCGF receptor, but may have certain biological function.