一种靶向性阳离子多肽载体的表达纯化  

Expression and Purification of a Targeted Cationic Polypeptide Vector

作　　者：王海[1] 王华茂[1] 李锦军[1] 石必枝[1] 李宗海[1] 

机构地区：[1]上海交通大学医学院上海市肿瘤研究所癌基因及相关基因国家重点实验室,上海200023

出　　处：《中国生物工程杂志》2007年第11期57-60,共4页China Biotechnology

基　　金：国家"973"计划资助项目(2004CB518802);国家自然科学基金资助项目(50603012)

摘　　要：目的:原核表达靶向性阳离子多肽(KH)20-EGF,并对其DNA包装能力进行检验。方法:构建pET28a-(KH)20-EGF原核表达载体。进行酶切和测序鉴定。转化BL21(DE3)后,经过IPTG诱导表达和NTA树脂纯化得到粗提蛋白产物,SDS-PAGE和Western blot鉴定后,将蛋白与质粒DNA混合,用于凝胶阻滞实验分析。结果:重组(KH)20-EGF的产量约为300μg/L。SDS-PAGE和Western blot表明该蛋白的凝胶迁移有些滞后;凝胶阻滞试验发现(KH)20-EGF对质粒DNA的迁移有阻滞作用。结论:成功表达非病毒载体(KH)-EGF并确认其DNA包装能力。Objective： To obtain a targeting cationic polymers （KH）20-EGF and examine its capacity for DNA condensation. Method： Prokaryotic expresssion plasmid pET28a-（ KH）20-EGF was constructed and transformed into BL21（ DE3 ）. The cationic polypeptide was induced by IPTG and purified using a Ni-NTA column. The purified polypeptide was determined by SDS-PAGE and Western blot analysis. The pDNA package ability of the resulted protein was examined by gel retardation assay. Results： The yield of （KH）20-EGF protein was about 300μg/L. The resulted protein was a little larger than expected when examined by SDS-PAGE and Western blot. Gel retardation assays indicated that （KH）20-EGF can retard the pDNA migration. Conclusion： The non-virus vector （KH）20-EGF was expressed successfully and its capability of DNA condensation was confirmed.

关 键 词：基因治疗 靶向性非病毒载体 阳离子多肽 原核表达纯化 DNA包装 

分 类 号：Q819[生物学—生物工程]