【摘要】 目的构建含表皮生长因子(EGF)受体Ⅲ型突变体胞外区基因(vⅢECD)的重组质粒并进行表达和鉴定。方法应用PCR方法扩增EGFRvⅢ ECD片段,将其T-A克隆和测序,再将目的基因插入GST融合表达载体pGEX-4T-1中进行表达。采用双酶切鉴定插入序列的正确性,用SDS-PAGE及Western blotting分析融合蛋白的表达。结果测序结果证实插入DNA序列与EGFR胞外区序列完全一致;双酶切鉴定表明,EGFRvⅢ ECD序列已经正确克隆到GST融合表达载体中。SDS-PAGE电泳显示融合蛋白在E.coli BL21(DE3)中以包涵体形式表达,重组融合蛋白GST-vⅢECD的表达量占菌体总蛋白的15%以上。Western blotting分析证实重组融合蛋白可以被EGFR特异性抗体所识别。结论成功构建了融合表达载体(pGEX-4T-1/vⅢECD),并进行了融合蛋白的诱导表达和鉴定,可进一步用于EGFRvⅢ功能及免疫学研究。更多还原

【Abstract】 Objective To clone the extracellular domain (ECD) of the typeⅢvariant of human epidermal growth factor receptor (EGFRvⅢ) and construct the recombinant expression plasmid. Methods A DNA fragment (vⅢ ECD) encoding the extracellular domain of human EGFRvⅢ was obtained by PCR, and its T-A was cloned and sequenced. The DNA fragment was then ligated into the GST fusion expression vector to construct the recombination plasmid. After identification with restriction digestion and DNA sequencing, the recombinant plasmid was transformed into E. coli BL21 (DE3) for expression of the recombinant protein. The target protein was identified by SDS-PAGE and Western blotting. Results The results of restriction digestion and DNA sequencing confirmed the successful construction of the recombinant plasmid. SDS-PAGE showed that the fusion protein was expressed as inclusion bodies in E. coli BL21 (DE3), and the amount of the fusion protein expressed in the bacteria, after induction for 4 h, accounted for up to 15% of the total bacterial proteins. Western blotting demonstrated that the fusion protein could be recognized by the specific anti-EGFR antibody. Conclusion We have successfully constructed the recombinant expression vector of vⅢ ECD and induced the expression of the fusion protein, which may facilitate functional and immunological studies of EGFRvⅢ.更多还原