Abstract: Objective To explore the reasons for the low intracellular transduction efficiency of a previously constructed His-TAT-Flag recombinant protein and establish a more efficient transduction system.Methods The Flag tag of pET14b-His-Tat-Flag vector was deleted with PCR mutant kit,and enhanced green fluorescent protein(EGFP)coding sequence was inserted into the new pET14b-His-TAT recombinant vector.Enzyme digestion and DNA sequencing were performed for identification of pET14b-His-TAT-EGFP vector,which was then transformed into E.coli BL21(DE3).After IPTG induction,the recombinant protein of His-TAT-EGFP was isolated and analyzed with SDS-PAGE.Purified His-TAT-EGFP recombinant protein was added to ECV304 cells and the fluorescence was observed to evaluate the transduction efficiency.Results pET14b-His-TAT vector and pET14b-His-TAT-EGFP vector were successfully constructed,which was identified with enzyme digestion and DNA sequencing.His-TAT-EGFP fusion protein was expressed and purified successfully and showed cellular transduction activity.Conclusion The prokaryotic expression vector has been successfully constructed by modifying pET14b-His-TAT-Flag,and the expressed and purified recombinant protein of His-TAT-EGFP possesses high efficiency of intracellular transduction activity.