人FGF12融合蛋白在大肠杆菌中的高效表达

目的：在原核表达系统中获得高效表达的人成纤维生长因子12融合蛋白。方法：采用逆转录PCR方法获得hFGF12 cDNA，将其连接到pET 28a原核表达载体，转化入BL21(DE3)菌中表达，经金属螯合亲和层析法和肝素亲和层析法纯化，以刺激NIH 3T3成纤维细胞增殖实验；测定纯化的hFGFl2融合蛋白活性。结果：表达产物经SDS-PAGE电泳，在Mr31000附近新增一条明显区带，与预期的相对分子质量相符。重组融合蛋白能刺激NIH3T3细胞增殖。结论：在大肠杆菌中表达了具有促有丝分裂活性的融合蛋白。

Study on high-level expression of human fibroblast growth factor 12 fusion protein in Escherichia coli

Objective To express recombinant human fibroblast growth factor 12 fusion protein in E.coli.Methods hFGF12 cDNA obtained by using RT-PCR was cloned into pET28a expression vector. The recombinant hFGF12 fusion protein was expressed in BL21 (DE3) E.coli cells and the product was purified by metal chelate affinity chromatograph and heparin affinity chromatograph. The activity of fusion protein was assayed by proliferation of NIH 3T3 cells.Results Recombinant hFGF12 fusion protein was expressed in BL21 (DE3) cells under the induction of IPTG and was observed as a band of M r 31 000 on a SDS-PAGE gel. The recombinant fusion protein was also found to be able to stimulate proliferation of NIH3T3 cells.Conclusion Recombinant hFGF12 fusion protein was expressed in E.coli and will be used as an immunogen to prepare specific antibodies.