重组人表皮生长因子的构建与表达

Construction and expression of recombinant human epidermal growth factor

目的应用原核表达系统研究提高重组表皮生长因子(rhEGF)产量的方法。方法用RT-PCR方法克隆hEGF基因并插入表达载体pGEX-4T-1,获得重组的表达质粒pGEX-4T-1-hEGF转化大肠杆菌BL21并筛选得到工程菌株,以异丙基-β-D-硫代半乳糖苷诱导目的蛋白表达,亲和层析纯化rhEGF,MTT法测定活性。结果成功扩增获得hEGF基因,部分表达产物为可溶性的、部分形成包涵体。菌体裂解上清液经亲和层析纯化后纯度达98%,复性后的rhEGF具有促细胞生长活性。结论成功构建并原核表达了重组人表皮生长因子基因,其表达产物具有很好的促细胞增殖活性。

Objective To research the method for increasing the yield and activity of rhEGF by prokaryotic expression vector. Methods The human EGF gene was amplified by RT-PCR, and was inserted into prokaryotic expression vector pGEX-4T-1. After identified by restriction mapping and sequencing, the recombinant plasmid pGEX-4T-1-hEGF was transformed into E. coli BL21 cell. Recombinant was purified with affinity chromatography and its bioactivity was determined with MTT assay. Results The human EGF gene was amplified successfully. The expression product was partially soluble and partially inthe form of inclusion body. After purified with afinity chromatography, the soluble rhEGF in the supernatant of cell lysate reached a purity of 98%. The refolded rhEGF exhibited proliferation-stimulating activity on human embryo kidney cells （HEK293）. Conclusion The rhEGF gene is successfully constructed and expressed, and the expression product has the biological activity in promoting cell proliferation.