表皮生长因子介导的基因转移载体蛋白的制备

Expression and Purification of Gene Transfer Vehicle Mediated by Epidermal Growth Factor Receptor

目的制备一种表皮生长因子(EGF)受体介导的、完全非病毒基因转移载体。方法通过PCR分别扩增出人类组蛋白第一功能域(H1)基因的编码序列和EGF的受体结合结构域(C环)基因的编码序列:将它们以EcoRⅠ位点连接后作为模板,PCR扩增出融合蛋白H1EGFc的基因。在对此融合基因进行克隆、测序之后,构建原核表达载体PLYI-H1EGFc,并转化大肠杆菌DH5α进行诱导表达。将表达出的融合蛋白利用SDS-PAGE电泳凝胶切割法进行纯化。结果表达出的目的蛋白与设计的融合蛋白分子量相符;经SDS-PAGE扫描,纯化后的目的蛋白纯度达94.02%。结论表达并纯化了融合蛋白H1EGFc。

Objective To create a gene transfer vehicle for targeting gene therapy of cancer with epidermal growth factor receptor overexpressing.Methods Encoding sequences of the first domain of histone gene (H1)and EGF C-loop (EGFc)were obtained by PCR amplification.These two DNA fragments were ligated by EcoRⅠsite,and cloned and sequenced.E.coli expression vector of the fusion gene was then constructed.The fusion protein H1EGFc was purified by specific band isolation from SDS-PAGE.Results The molecular weight of purified protein was consistent with the designed request.Its purity reached94.02%.Conclusion A fusion protein H1EGFc was expressed and purified.