Construction，expression and activity evaluation of recombinant NuBCP-9/Tumstatin(74-98) fusion polypeptide

Aim ：To construct a prokaryotic expression vector carrying NuBCP-9-tumstatin(74-98) (abbreviated as NT) gene and to obtain the fusion peptide with antitumor activity. Methods ：Nucleotide sequences of antitumor peptides，NuBCP-9 and Tumstatin(74-98)，were connected via a linker(G_4S)₃ based on biased codons of E.coli the fused NT gene was reconstructed using SOE PCR，and inserted into pET32a(+) vector，and transformed in E.coli BL21(DE3).After expression，the novel fusion peptide was purified through nickel-affinity chromatography，Factor Xa digestion and ultrafiltration.Biological activity of the fusion peptide on ECV304 and A549 cells was evaluated by MTT assay. Results ：A prokaryotic expression system with NT gene was successfully constructed.The soluble fusion peptide was accounted for approximately 25% when induced by 0.5 mmol/L IPTG at 30 ℃ for 4 h.The purified fusion peptide could inhibit cell growth of ECV304 and A549 with inhibition rates of 60.8% and 65.2% at 20 μmol/L，respectively. Conclusion ：A novel fusion peptide with antitumor activity was cloned，expressed and purified.