表皮生长因子受体胞外区的原核表达和鉴定

目的 克隆表皮生长因子受体(EGFR)胞外区段基因序列(EGFR ECD),构建重组融合表达载体.方法 采用常规PCR方法 扩增EGFR的胞外区DNA序列,并将扩增产物克隆入GST融合表达载体pGEX-4T-1中,构建重组质粒pGEX-4T-1/EGFRECD,转化BL21(DE3)宿主菌;采用Sal Ⅰ和Not Ⅰ双酶切和序列分析鉴定插入序列的正确性:并用IPTG诱导工程菌,SDS-PAGE及Western blotting分析融合蛋白的表达.结果 双酶切鉴定表明,EGFR胞外区序列已经正确克隆到GST融合表达载体中,测序结果证实插入DNA序列与EGFR胞外区序列完全一致.SDS-PAGE电泳显示,融合蛋白在BL21(DE3)中以包涵体形式表达,重组融合蛋白GST-EGFRECD的表达量占菌体总蛋白的12%左右.经Western blotting分析证实,重组融合蛋白可以被EGFR特异性抗体所识别.结论 本试验成功克隆了EGFR胞外区DNA序列,构建了融合表达载体,并进行了融合蛋白的诱导表达和鉴定,可进一步用于EGFR功能及免疫学研究.

Expression and identification of the extracellular domain of human epidermal growth factor receptor

Objective To clone the extracellular domain(ECD) of human epidermal growth factor receptor(EGFR) to and construct the recombinant expression plasmid.Methods A DNA fragment encoding extracellular domain of human EGFR was obtained by PCR,and the DNA fragment was then ligated into GST fusion expression vector to construct the recombination plasmid(pGEX-4T-1/EGFR ECD).After identification by restriction digestion and DNA sequencing,the recombinant plasmid was transformed into E.coli BL21(DE3) and expressed the recombinant proteins after induction with IPTG.The target proteins were identified by SDS-PAGE and Western blotting.Results The restriction digestion and DNA sequencing could prove that the recombinant plasmid was correct.SDS-PAGE showed that the fusion protein was expressed as inclusion body formation in E.coli BL21 and the amount of fusion protein was about 12% in the whole protein of E.coli.Western blotting showed that the fusion protein could be detected by the specific anti-EGFR antibody.Conclusion We successfully constructed the recombinantexpression vector of EGFR ECD and the inducing fusion protein mayprovide the foundation for further application of this protein in the functional and immunological study of EGFR.