Cloning, Expression And Purification Of RhaFGF-Tat Fusion Protein

Acidic fibroblast growth factor(aFGF) is a member of FGF family, with a wide range of physiological and pharmacological activities, it has extensive biological functions in organ and tissue development, angiogenesis, cell differentiation and apoptosis,tumorigenesis and wound healing. aFGF entering into cells as a function of biological macromolecules is achieved by activating its receptor. However, in the absence of its receptor, it is difficult for aFGF to enter into cells and play a role. It has great significance to express rhaFGF-tat fusion protein. This study used genetic engineering to fuse aFGF_19-154 and tat, after connecting with the expression vector into appropriate host cells to express highly aFGF_19-154-tat fusion protein, then by CM ion exchange chromatography and heparin affinity layer analysis of purified, to obtain a purified protein which can promote cell proliferation, in order to make the theoretical basis for the further research.Objective:The host bacterium is E. coli, using genetic engineering techniques to express recombinant human acidic fibroblast growth factor (aFGF)-penetrating peptide (tat) fusion protein, and establishment methodes of pilot-scale fermentation, purification and assay biological activity in vitro. And, to lay the foundation for development of innovative drugs and make the theoretical basis for the further developmentMethods:The total RNA was extracted from Human embryonic lung fibroblast cell, then aFGF_19-154-tat gene fragment was amplified by RT-PCR and aFGF_19-154-tat was cloned into the prokaryotic expression vector pET3c, and the constructed recombinant prokaryotic expression plasmid pET3c-aFGF_19-154-tat, the expression plasmid was transformed into E. coli BL21 (DE3) host bacteria and IPTG induction. Biomass fermentation and the fermentation tank with high-density fermentation, cell sonication method of breaking through the cation exchange (CM-Sepharose FF) chromatography, heparin affinity chromatography (Heparin-Sepharose CL-6B) and gel exclusion chromatography (Sephadex G-50) isolated and purified fusion protein aFGF_19-154-tat. Western blotting and HPLC analysis of purified aFGF-tat fusion protein, MTT method in vitro testing aFGF_19-154-tat fusion protein on NIH 3T3 cells, promoting proliferation.Results:We successfully constructed pET3c-aFGF_19-154-tat plasmid and expressed aFGF-tat fusion protein in BL21(DE3). The quantity can reach 15% when the concentration of IPTG was 0.5mM, the induced time was 4h and induced temperature was 37℃. We can get 90g thalli form 1L fermenting liquor, confirmed by SDS-PAGE, the relative molecular mass was consistent with the theoretical expected value, and the expression products was soluble. By cation exchange chromatography. heparin affinity chromatography and gel exclusion chromatography, the purity of protein was higher than 95%.Western blotting comfirmed that the expression products has specific binding ability with human aFGF polyclonal antibody. MTT confirmed the fusion protein can promote the proliferation of NIH 3T3 cells, which is the the same with the positive control group and significantly higher than the blank control group (P<0.01).Conclusion:pET3c-aFGF_19-154-tat gene was cloned, and the recombinant human aFGF_19-154-tat fusion protein engineering bacteria was obtained, the fusion gene was expressed in E.coil and purified successfully which can promote cell proliferation, which for the further research.