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将两种人干细胞因子(hSCF)模拟肽(CS2和LS2)分别与c-jun亮氨酸拉链在大肠杆菌中融合表达,比较模拟肽与融合蛋白的生物学活性。通过搭桥PCR的方法,构建分别含有CS2和LS2与c-jun亮氨酸拉链融合蛋白及单独c-jun亮氨酸拉链编码序列的三种原核表达质粒pET30a-CSJ,pET30a-LSJ和pET30a-Jun,在E.coli BL21中进行表达,经镍柱和Sephadex G-50柱纯化后,SDS-PAGE和质谱法检测重组融合蛋白(CSJ,LSJ)和c-Jun的理化性质,MTT法检测融合蛋白刺激UT-7细胞增殖的活性。结果显示CSJ,LSJ和c-jun在E.coli中均呈20%左右表达,经纯化后其纯度达95%以上,分子量分别为7336.08,7991.54和6672.74。细胞学活性实验显示:与CS2和LS2相比,CSJ和LSJ促进UT-7细胞增殖的活性提高约1000倍。在大肠杆菌中成功表达了hSCF模拟肽与c-jun亮氨酸拉链融合蛋白,融合蛋白活性显著高于合成的hSCF模拟肽。
Two human stem cell factor (hSCF) mimic peptides (CS2 and LS2) were respectively fused with c-jun leucine zipper in Escherichia coli to compare the biological activity of mimic peptide and fusion protein. By using PCR, three prokaryotic expression plasmids pET30a-CSJ, pET30a-LSJ and pET30a-Jun containing zipcode sequence of CS2 and LS2, c-jun leucine zipper and c-jun leucine zipper respectively , And expressed in E.coli BL21. The purified proteins were purified by Ni-Sephadex G-50 and Sephadex G-50 column. The physicochemical properties of recombinant fusion protein (CSJ, LSJ) and c-Jun were detected by SDS-PAGE and mass spectrometry. Stimulation of UT-7 cell proliferation activity. The results showed that the expression of CSJ, LSJ and c-jun was about 20% in E.coli. The purity of CSJ, LSJ and c-jun was over 95% after purification. The molecular weights of CSJ, LSJ and c-jun were 7336.08, 7991.54 and 6672.74, respectively. Cytological activity experiments showed that compared with CS2 and LS2, CSJ and LSJ increased the proliferation activity of UT-7 cells by about 1000-fold. The hSCF mimic peptide and c-jun leucine zipper fusion protein were successfully expressed in E. coli, and the activity of the fusion protein was significantly higher than that of the synthetic hSCF mimetic peptide.