鼠表皮生长因子受体胞外段原核表达、纯化与复性

用多聚酶链反应(PCR)方法从既往构建质粒扩增鼠表皮生长因子受体(EGFR)胞外段基因及适宜酶切位点，进而构建原核表达载体。克服质粒丢失的不足，在大肠杆菌中表达目的蛋白，并在变性条件下通过。Ni^2 柱亲和层析纯化表达蛋白，聚丙烯酰胺凝胶电泳(SDS—PAGE)示纯度在95％以上。去除组氨酸标记的纯化蛋白在梯度尿素中透析复性。复性蛋白在表皮生长因子的作用下可以同源二聚化而在非还原SDS—PAGE及蛋白印迹分析中呈二聚体状态，其所产生抗体可以识别复性蛋白及表达于肺癌细胞上的EGFR，提示复性后大肠杆菌表达鼠EGFR胞外段重组蛋白已获得一定空间结构，并具有免疫原性，可以进一步用于EGFR功能及免疫学研究。

Expression, Purification and Refolding of Extracellular Domain of Mouse Epidermal Growth Factor Receptor

A DNA fragment encoding extracellular domain of mouse epidermal growth factor receptor (EGFR) was obtained by PCR from a previous recombinant plasmid. The DNA fragment was then ligated into prokaryotic expression vector, and expressed in Escherichia Coli. The recombinant protein was purified under denature conditions by affinity chromatography, and refolded with gradient dialysis. The recombinant protein could produce antibodies to recognize extracellular domain and full length of mouse EGFR, and form homodimer in the presence of EGF detected by western blot analysis. These findings provide evidence that the renatured recombinant extracellular domain of mouse epidermal growth factor receptor is immunogeneic and may be important for further application of this protein in functional and immunological research.