人EGFR胞外段原核表达及免疫原性研究

目的：研究人EGFR胞外段在原核本质的表达，纯化条件以及对小鼠的免疫原性。方法：以既往构建包含人EGFR胞外段基因的表达质粒为基础，采用基因工程方法构建pET原核表达载体。在大肠杆菌中表达目的蛋白，表达蛋白在变性条件下，通过Ni^2 柱亲和层析纯化以包涵体形式存在的His-Tag融合蛋白，SDS－PAGE检测其纯度。去余His-Tag的纯化蛋白在梯度尿素中透析复性。以复性蛋白免疫小鼠获得抗血清，western blotting及流式细胞仪器检测特异抗体的存在。结论：通过限制酶切分析及测序鉴定表明获得了人EGFR胞外段基因的pET原核表达载体，SDS－PAGE分析表明在IPTG诱导下可以表达90Kd目的蛋白其纯度大于95％，所产生抗体可以识别复性蛋白及表达于A549细胞上的EGFR。提示在大肠杆菌中表达的人EGFR胞外段重组蛋白具有免疫原性，可以进一步用于EGFR免疫研究。

Prokaryotic Expression of Extracellular Domain of Human EGFR and It's Immunogenity

The object is to investigate the end its immunogenity in mouse.A DNA fragment encoding extracellular domain of human EGFR was obtained from a previoused constructed plasmid.The DNA fragment was then ligated into prokaryotic expression vector,and was expressed in Escherichia Coli harboring the constructs.The recombinant protein within insoluble inclusion bodies was purified under denatured conditions by affinity chromatography,and the refolding of the recombinant protein was performed with gradient dialysis.The recombinant protein could induce immune response in mouse producing antibody to recognize the recombinant protein and EGFR expressed at A549 tumor cells by western blot analysis and flow cytometric analysis.These findings provided evidence of the renatured recombinant extracellular domain of human EGFR is immunogenity in mouse may be of important for the further exploration of the application of this protein in immunological and immunotherapy research.