Abstract:

Abstract:Objective
To express and purify the fusion protein of rhaFGF-tat with biological activity,and provide a foundation for development of an genetically engineered drug. Methods The aFGF-tat gene fragment through digesting the pMD18-T-aFGF-tat was obtained,then the recombinant plasmid was transformed into E.coil BL21 (DE3) and induced by IPTG. The protein was purified by CM-Sepharose FF cation exchange and heparin-Sepharose FF affinity. The activity of aFGF-tat was detected with NIH 3T3 proliferation assay. Results Acquired gene fragments of aFGF-tat  identified by digestion and DNA sequencing were coincident with the human aFGF-tat reported in GenBank. The recombinant vector of pET3c-aFGF-tat was constructed successfully. SDS-PAGE result proved that aFGF-tat fusion protein with a relative molecular mass of about 19 102 was expressed. The purity of fusion protein was more than 95%,Western blotting results showed that the expressed products had specific reaction with anti-human aFGF polyclonal antibody and was able to promote the proliferation of NIH 3T3 cells. Conclusion　aFGF-tat fusion gene is expressed in E.coil and purified successfully,and the aFGF-tat fusion protein can promote the proliferation of cells.

Key words: acidic fibroblast growth factor；transcriptional activator protein；expression；purification；activity assay