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

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.