Objective:We sought to construct the recombinant expression vector pED-Tat-PFKMVV to express and purify the recombinant peptide Tat-PFKMVV. Methods: The gene fragment encoding a recombinant peptide Tat-PFKMVV was constructed by PCR and ligated into expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The recombinant plasmid vector was transduced into E.coli. The expressed recombinant peptide was partially purified by the wash solution of Tris-Cl/TritonX-100,alcohol precipitation,acid hydrolysis and isoelectric point precipitation. Results:The correct recombinant DNA coding fragment was validated by DNA sequencing. The 17 000 AsnB-C-Tat-PFKMVV fusion protein was highly expressed in E.coli BL21 transduced by pED-Tat-PFKMVV. The analysis of the purified Tat-PFKMVV by mass spectrometry indicated that its molecular weight was 2 659. GST-pull down experiments showed that Tat-PFKMVV in vitro disturbed the coupling of nNOS and PFK-M. Conclusion:The experiment constructed the recombinant expression vector of AnsB-C-Tat-PFKMVV,and expressed and purified the peptide Tat-PFKMVV. The novel preparation method is a potentially useful strategy for the large-scale preparations of bioactive peptides due to its high yield and simple steps.