麦胚系统真核表达小鼠伯氏疟原虫TIP样蛋白的实验研究

目的体外真核表达小鼠伯氏疟原虫TIP样蛋白（PbTIP）胞外域重组蛋白。方法合成PbTIP胞外域DNA片段，克隆入真核表达载体pEU-E01-His-TEV—MCS，采用CFS真核无细胞蛋白表达系统（麦胚系统），在体外表达可溶性PbTIP胞外域重组蛋白，SDS-PAGE凝胶电泳和Western-blot检测并鉴定目的蛋白表达情况。结果获得了可溶性表达的PbTIP胞外域重组蛋白，分子量约70000Mr，与预期相符，目的蛋白浓度约为0．1mg／ml。结论采用麦胚系统在体外获得了较高浓度的可溶性PbTIP胞外域重组蛋白，不但为后续的PbTIP胞外域功能研究打下了良好的基础，也为众多疟原虫未知蛋白的功能研究提供了新的方法。

Eukaryotic expression of PbTIP with wheat germ system in vitro

Objective To express the extracellular domain fragment of TIP like protein of Plasmodium berghei （PbTIP） in vitro. Methods DNA fragment of PbTIP extracellular domain was synthesized, and cloned into the eukaryotic expression vector pEU-E01-His-TEV-MCS. The soluble recombinant protein was expressed using the CFS eukaryotic cell free protein expression system （wheat germ system） in vitro, and then the target protein was identified by SDS-PAGE and western blot assay. Results A 70 000 Mr, soluble extracellular domain fragment of the PbTIP which consistent with expectation was obtained by the wheat germ expression system, the concentration of target protein was about 0.1 mg/ml. Conclusion The soluble extracellular domain fragment of the PbTIP can be successfully expressed in the wheat germ system in vitro. The present study laid a foundation for the further functional clarification of PbT1P extracellular domain, and also for the development of a new approach for the functional study of numbers of unknown P. berghei proteins.