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

作　　者：苏林[1] 孔燕[2] 刘长征[1] 杨克恭[1] 陈松森[1] 

机构地区：[1]中国医学科学院北京协和医学院基础医学研究所医学分子生物学国家重点实验室,北京100005 [2]北京大学临床肿瘤学院北京肿瘤医院暨北京市肿瘤防治研究所,北京100036

出　　处：《中国生物工程杂志》2010年第12期5-9,共5页China Biotechnology

基　　金：国家"863"计划重大专项基金(2003AAZ3360)资助项目

摘　　要：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.

关 键 词：人干细胞因子 模拟肽 融合蛋白 亮氨酸拉链 

分 类 号：R542.220.5[医药卫生—心血管疾病]