重组人成纤维细胞生长因子8b原核表达载体的构建和纯化研究

成纤维细胞生长因子8b(fibroblast growth factor, FGF8b)在生长因子中与内分泌癌的发生和发展相关联,是一个有潜在临床应用价值的候选分子,为了满足重组人FGF8b的研发需要,论文采用分子克隆的方法构建了以包涵体形式表达FGF8b的重组大肠杆菌,并初步摸索出包涵体蛋白的纯化和复性工艺条件,通过Western blot和MTT法鉴定了FGF8b的生物化学特征和促增殖活性,表明利用包涵体复性技术初步得到了具有活性的蛋白,为后续研发进程奠定了基础。

The Constructing and Purification of Recombinant Human Fibroblast Growth Factor 8b Expressed Vector

FGF8b, which belongs to the fibroblast growth factor family, has been found to be associated with the regulation of growth and progression of hormonal cancers. Thus, it is believed that FGF8b could be a potential target in the treatment of hormonal cancers. To improve the production of recombinant hFGF8b to meet the increasing demand in basic research and clinical applications, an artificial gene encoding its mature peptide sequence was constructed and cloned into vector pET-3a. Then the recombinant proteins were expressed and presented in form of inclusion bodies in Escherichia coli BL21(DE3)pLysS. Expression, purification and renaturation conditions of the recombinant proteins were optimized. The preliminary biochemical characterization of FGF8b was further confirmed by Western blotting, and its mitogen activity was tested through MTT assay. In summary, this study has successfully acquired active recombinant FGF8b protein, which can be used for the further clinical development of embryology and the basic hormonal cancer researches.