人造血增效因子C末端结构与其趋化活性的关系

C-terminal structures of human hematopoiotic synergistic factor and its chemotaxis activity

目的研究人造血增效因子(hHSF)C末端结构与其趋化活性的关系。方法用PCR方法合成编码3种hHSFC末端截短变异体DNA片段,分别重组到pET30a或pET42a中,并在E.coliBL21(DE3)中进行表达。hHSF1-66和hHSF1-59用凝胶过滤和阳离子交换层析法进行分离纯化,融合蛋白GST-hHSF1-5 3经亲和层析、肠激酶(EK)酶切和凝胶过滤法进行分离纯化。hHSF3种缺失变异体进行Western blot鉴定,用飞行时间质谱法测定其相对分子质量,用改良的Boyden小室法测定其对人外周血中性粒细胞(PMN)趋化活性。结果测序证实合成的hHSF 1-66、hHSF1-59和hHSF1-53序列与设计一致,hHSF1-66/1-59表达量占菌体总蛋白的20%左右,主要以包涵体形式存在。GST-hHSF1-53表达量占菌体总蛋白的30%以上,主要以可溶形式存在。3种目的蛋白纯度均达95%左右,它们的相对分子质量分别为7206.0、6401.2和5697.3 D,均具有全长hHSF的免疫学活性。与全长hHSF相比,hHSF1-66(缺失3个氨基酸)对人PMN最大趋化活性(CI)(50 nmol/L)没有明显差异,hHSF1-59(缺失10个氨基酸)和hHSF1-53(缺失16个氨基酸,α螺旋完全缺失)最大趋化活性分别下降34.3%和70.5%。结论hHSF C末端结构,特别是α螺旋结构对维持和稳定hHSF的空间构象起着重要作用。

Objective To study the relationship between the C-terminal structure of hHSF and its chemotaxis activity. Methods The artificial DNA fragments encoding three C terminal truncated hHSF analogs were obtained by PCR,then cloned into the vector pET30a or pET42a, respectively, and expressed in E. coli BL21 （DE3）through IPTG induction subsequently, hHSF1-66 and hHSF1-59 were purified by gel filtration and cation exchange chromatography and then subjected to refolding, hHSF1-53 was purified by affinity gel chromatography, EK cleavage, and gel filtration. The molecular weight of three hHSF analogs and its immunity were measured by MALDI-TOF Mass Spectroscopy and Western blot respectivelly. The chemotaxis activity of hHSF and its mutants for human neutroplil was detected by Boyden chamber method with modification. Results The sequences of hHSF1-66, hHSF1-59 and hHSF1-53 were correct as shown by gene sequencing. The expression level of hHSF1-66 and hHSF1-59 was about 20% of total cell proteins and most of them were present in inclusion bodies form. The yield of fusion protein GST-hHSF1-53 ver 30% of total cell proteins, most of which is soluble. The purifity of target proteins was over 95%. The molecular weight of hHSF1-66, hHSF1-59 and hHSF1-53 was 7206. 0,6401.2 and 5697.3D, respectively and had hHSF immuno-activity. In comparison with the full length hHSF, the maxium chemotaxis activity （50 nmol/L） of hHSF1-66 （less 3 residues） and hHSF for their efficacy to stimulate human PMN was not significantly different. But the maxium chemataxis activity of hHSF1-59 （less 10 residues） and hHSF1-53 （less 16 residues ,α-helix） was decreased 34. 3% and 70. 5% ,respectively. Conclusion The α-helix at C-terminus of hHSF is important for the stabilization of its molecular stereo-conformation.