人干细胞生长因子Ca^(2+)依赖糖识别结构域缺失突变体的重组表达及生物学活性的初步探讨  

Expression of CRD Domain Deletion Variant of Human Stem Cell Growth Factor and Preliminary Study on its Biological Activity

作　　者：原野[1] 要晖宇[1] 李秀森[1] 郭子宽[1] 吴英[1] 江小霞[1] 毛宁[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850

出　　处：《生物技术通讯》2005年第3期235-238,共4页Letters in Biotechnology

基　　金：国家高技术研究发展计划项目(2003AA205170)

摘　　要：本文作者已有的研究结果证明,完整的人干细胞生长因子(hSCGF)没有种属特异性,即可以作用于小鼠骨髓造血细胞。这一点与在Ca2+依赖糖识别结构域(CRD)缺失了78个氨基酸残基的截短分子(hSCGFβ)有所不同。本研究从hSCGF全长cDNA中完全删除了CRD结构域编码序列,进一步探讨CRD结构域的生物学功能。由于该突变体序列GC含量较高,因此将该缺失突变体序列克隆在GST融合表达载体中进行融合表达。通过低温(28℃)诱导,表达产物主要以可溶蛋白的形式存在。利用亲和层析纯化CRD结构域完全缺失的hSCGF突变体融合蛋白,通过检测重组突变分子的协同刺激造血活性有无改变来初步探讨CRD结构域在hSCGF分子中的生物学功能。研究结果表明,去掉完整CRD结构域的突变分子仍然具有造血刺激活性。据此推断CRD结构域在hSCGF分子中可能对于受体配体结合起辅助作用。The authors' previous clonogenic assay result revealed that GST-hSCGF fusion protein has the granulococyte/ macrophage (GM)-promoting activity (GPA) for murine bone marrow GM progenitor. This result implied that the calcium-dependent carbohydrate-recognition domain (CRD) in which 78 aa was lose in human SCGFβ may play some roles in human stem cell growth factor. In order to further investigate the biological function of CRD domain in hSCGF, the CRD coding sequence was fully deleted by means of PCR. The mutation sequence was then fused to downstream of GST sequence inside plasmid pGEX4T-2 and expressed in E.coli. The low-temperature(28℃) induction expression strategy was carried out. SDS-PAGE analysis indicated that the recombinant protein mainly existed in soluble form. The fusion recombinant protein was purified by affinity chromatography. The preliminary study on the mutant suggested that CRD domain completely deletion mutant maintained its stimulating activity on human BM progenitor cells in vitro. This result, combining the authors' previous result that the intact molecular hSCGF has no species-specificity whereas the truncate form hSCGFβhas, implicate that this carbohydrate binding domain in hSCGF may work as a helper domain to stabilize ligand-receptor reaction.

关 键 词：细胞生长因子 缺失突变体 Ca^2+ 结构域 糖识别 生物学活性 重组表达 生物学功能 全长cDNA 融合表达载体 研究结果 CRD 种属特异性 氨基酸残基 造血细胞 小鼠骨髓 编码序列 GC含量 可溶蛋白 表达产物 融合蛋白 层析纯化 

分 类 号：Q516[生物学—生物化学] S565.1[农业科学—作物学]