人EGF受体胞外域在大肠杆菌中表达、复性及抗原性鉴定  

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

作　　者：徐丽慧[1] 洪岸[2] 何贤辉[1] 

机构地区：[1]暨南大学生命科学技术学院组织移植与免疫实验中心 [2]暨南大学生命科学技术学院生物工程研究所,广州510632

出　　处：《免疫学杂志》2006年第4期362-365,共4页Immunological Journal

基　　金：国家自然科学基金(30230350和30371651);国家"十五"重大专项基金(2002AA2Z3344);广东省"十五"重大专项基金(2001A1090208)资助项目

摘　　要：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(EGFRLSL)的DNA片段,并在其3′端加入编码His6标签的序列,与pET3c连接构建EGFRLSL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(EGFRL2)制备抗血清,以此抗血清鉴定表达产物。结果EGFRLSL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EGFRLSL蛋白;免疫印迹分析表明,EGFRLSL与小鼠抗EGFRL2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EGFRLSL蛋白。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 antisenan against EGFR-L2 domain. Methods DNA fragment, encoding the extracellular domains L1, S1, and L2 of EGFR （EGFR-LSL）, containing a His6-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. Antisenan 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-His6 antibody. Soluble EGFR-LSL was purified through on-colunm renaturing of denatured EGFR-LSL bound to Ni26＋ -NTA. Furthermore, EGFR-LSL had highly spe- cific reactivity with mouse anti-EGFR-L2 antiserum. Conclusion The soluble EGFR-LSL obtained from E. coli possesses specific antigenicity.

关 键 词：表皮生长因子受体 胞外域 原核表达 包涵体 抗原性 

分 类 号：Q78[生物学—分子生物学] R392[医药卫生—免疫学]