【摘要】 目的构建人胰岛素样生长因子-1(hIGF-1)基因原核表达载体,并检测表达的重组人IGF-1(rhIGF-1)蛋白的生物学活性。方法以pUC57-hIGF-1质粒为模板,PCR扩增hIGF-1基因,克隆入pTEtrans载体,进行DNA序列分析后,亚克隆入表达载体pTE,转化大肠杆菌W3110,进行诱导表达,表达产物经阳离子交换层析和凝胶过滤层析纯化,并进行Tricine-SDS-PAGE和Western blot分析。MTT法检测rhIGF-1促MCF-7细胞的增殖作用。结果转化质粒pTE-hIGF-1的大肠杆菌经诱导后,以可溶性形式表达相对分子质量约为7700的目的蛋白,表达量约占菌体总蛋白的8%。Western blot证实该蛋白具有良好的反应原性,Tricine-SDS-PAGE证实纯度达98%以上,MTT证实具有良好的促MCF-7细胞增殖的作用。结论已成功构建了含hIGF-1基因的原核表达载体,并获得具有生物学活性的高纯度rhIGF-1。更多还原

【Abstract】 Objective To construct a prokaryotic expression vector for human insulin-like growth factor-1 (hIGF-1) gene and determine the biological activity of expressed protein. Methods Amplify hIGF-1 gene by PCR using plasmid pUC57-hIGF-1 as template and clone into vector pTEtrans. The constructed recombinant plasmid pTEtrans-hIGF-1 was identified by DNA sequencing, then subcloned into expression vector pTE. Transform the constructed recombinant plasmid pTE-hIGF-1 to E. coli W3110 for expression. The expressed product was purified by cation exchange and gel filtration chromatography, identified by Tricine-SDS-PAGE and Western blot and determined for promoting effect on proliferation of MCF-7 cells by MTT method. Results The target protein with a relative molecular mass of 7 700 was expressed in a soluble form in E. coli transformed with recombinant plasmid pTE-hIGF-1, contained about 8% of total somatic protein and showed good reactogenicity as proved by Western blot. Tricine-SDS-PAGE proved that the purity of expressed rhIGF-1 reached more than 98%. MTT method showed significantly promoting effect of rhIGF-1 on proliferation of MCF-7 cells. Conclusion The prokaryotic expression vector for hIGF-1 gene was successfully constructed, and rhIGF-1 with high purity and biological activity was expressed.更多还原