FGFR2c-L-Fc的可溶性表达、纯化及初步活性研究

objective: To improve stablity and the half-life of FGFR2c for better efficacy , a new construction of recombinant FGFR2c was genereated by fusion of FGFR2c and Fc frangment of human IgG with flexible Linker. Methods: Gene of FGFR2c-L-Fc were amplified by PCR and overlap PCR. FGFR2c-L-Fc gene was cloned into pET43.1 expression vector. Fusion protein was expressed in E.coli. Gel chromatographic separated the target protein. FGFR2c-L-Fc was removed by cleaving the NusA fusion protein with enterokinase. The recombinant protein was purified by heparin affinity chromatography. Activity of FGFR2c-L-Fc was studied by Western Blot and CCK-8 assay. Results: Double restriction enzyme digestion and sequencing results showed that FGFR2c-L-Fc-pET43.1 recombinant plasmid was correct. SDS-PAGE results showed that a single band in 110 kd by heparin affinity chromatography. Western Blot results showed that the target protein could recognize FGFR. The CCK-8 assay results showed that FGFR2c-L-Fc inhibited the proliferation of DU145 at 320ng/ml, and the inhibitory ratios of DU145 was about 25%. Conclusions: FGFR2c-L-Fc was expressed in E.coli，can be combined with FGFR，and significantly inhibited the proliferation of DU145.