人表皮生长因子-天花粉蛋白融合蛋白的基因克隆、表达及纯化

目的 构建人表皮生长因子(EGF)-linker-天花粉蛋白(TCS)融合蛋白的表达载体,在大肠杆菌中表达并纯化EGF-linker-TCS融合蛋白.方法 用PCR方法扩增EGF及柔性连接肽Linker基因片段,插入质粒PQE30-TCS中,转化至大肠杆菌M15中,表达该融合蛋白(EGF-linker-TCS).表达产物经Ni-NTA Agrose亲和层析柱纯化.结果 PQE30-EGF-linker-TCS蛋白在大肠杆菌M15中获得稳定、高效的融合表达.目的蛋白占细胞总蛋白的40%左右,以可溶性形式存在,表达上清柱纯化后纯度达95%以上.结论 应用基因工程技术,成功地构建了融合蛋白EGF-linker-TCS的表达载体,表达并纯化了该融合蛋白,为进一步研究其结构功能关系和肿瘤临床应用奠定了基础.

Gene cloning,expression and purification of fusion protein epidermal growth factor-linker-trichosanthin

Objective To construct a recombinant expression vector of the fusion protein epidermal growth factor (EGF)- Linker-trichosanthin (TCS) and achieve its expression in E.coli to obtain purified EGF-linker-TCS fusion protein. Methods The gene fragments of EGF-linker were amplified by PCR and inserted into the expression plasmid PQE30-TCS, followed by transformation of the recombinant plasmid into E.coli M15 for expression of the fusion protein. Ni-FF column chromatography was utilized for purification of the expressed product. Results The recombinant plasmid PQE30-EGF- linker-TCS was stably and highly expressed in E. coli M15. The expressed product existed in the form of soluble protein accounting for about 40% of total cellular protein and reached a purity of above 95% after purification with Ni-FF column chromatography. Conclusion The recombinant plasmid PQE30/EGF-linker-TCS has been successfully constructed, which provides a basis for further structural and functional study of EGF and TCS and their potential clinical application for cancer therapy.