人EGF受体胞外域在大肠杆菌中表达、复性及抗原性鉴定

目的 构建人EGF受体（EGFR）胞外域的原核表达载体并在大肠杆菌中表达，制备EGFR特异性抗体，对表达产物进行鉴定。方法 以PCR方法从克隆的EGFR胞外区cDNA中扩增编码胞外结构域L1、S1、L2（EGFR-LSL）的DNA片段，并在其3′端加入编码His6标签的序列，与pET-3c连接构建EGFR-LSL原核表达载体，在大肠杆菌中进行表达；同时以纯化的EGFRL2结构域蛋白（EGFR-L2）制备抗血清，以此抗血清鉴定表达产物。结果 EGFR-LSL在大肠杆菌BL21（DE3）中获得高效表达，抗His6抗体免疫印迹分析表明，表达产物全部以包涵体形式存在，通过Ni2^＋．NTA柱上复性获得纯化的可溶性EGFR-LSL蛋白；免疫印迹分析表明，EGFR-LSL与小鼠抗EGFR-L2抗血清具有高特异性反应。结论 从大肠杆菌中获得具有特异抗原性的可溶性EGFR-LSL蛋白。

Renaturing and antigenic identification of human EGF receptor extracellular domain inclusion body overexpressed in Escherichia coli

Objective To construct a prokaryotic expression vector for the extracellular domain of EGFR and to identify the its expression product in Escherichia coli (E. coli) with specific antiserum against EGFR-L2 domain. Methods DNA fragment, encoding the extracellular domains L1, S1, and L2 of EGFR (EGFR-LSL), containing a His_6-tag at the carboxyl terminus, was amplified by PCR from the cDNA of EGFR extracellular region, and then was inserted into pET-3c to construct the prokaryotic expression vector for EGFR-LSL. Antiserum was prepared with the purified EGFR L2 domain (EGFR-L2) and was used to identify the EGFR-LSL. Results EGFR-LSL was highly expressed in E. coli BL21(DE3) strain and was only present in the inclusion body as revealed by immunoblotting analysis with anti-His_6 antibody. Soluble EGFR-LSL was purified through on-column renaturing of denatured EGFR-LSL bound to Ni~ 2+-NTA. Furthermore, EGFR-LSL had highly specific reactivity with mouse anti-EGFR-L2 antiserum. Conclusion The soluble EGFR-LSL obtained from E. coli possesses specific antigenicity.