Abstract:
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.