人干细胞因子模拟肽与c-JUN亮氨酸拉链融合蛋白的原核表达和生物学功能鉴定

Expression of Human Stem Cell Factor Mimetic Peptides Fused with c-jun Leucine Zipper in E. coli and Identification of Its Bio-activity

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。

The two human stem cell factor（hSCF）mimetic peptides（CS2 and LS2）fused with the c-jun leucine zipper respectively were expressed in E.coli and the bio-activity of the fusion proteins was identified.The three expression plasmids pET30a-CSJ,pET30a-LSJ and pET30a-Jun,which contain fusion proteins（CSJ and LSJ）and c-jun gene respectively,were constructed by using overlapping PCR.Results showed that the CSJ,LSJ and c-Jun（CJ）were expressed about 20% of total protein in E.coli BL21.After purified by using affinity Ni-NTA and Sephadex G-50 column chromatography,the purity of the CSJ,LSJ and CJ were over 95% identified by using SDS-PAGE analysis.Their molecular mass was 7336.0 8,7991.54 and 6672.74 respectively determined by mass spectrometry.The bioactivity of the fusion proteins was evaluated by using cell proliferation assay with MTT in UT-7 cell.Compared to CS2 and LS2,the bioactivities of CSJ and LSJ were dramatically improved and it is about 1000 fold higher than that of CS2 and LS2.