Construction, expression and activity evaluation of recombinant NuBCP-9/ Tumstatin(74-98) fusion polypeptide
10.3321/j.issn:1000-5048.2009.04.015
- VernacularTitle:重组NuBCP-9/Tumstatin(74-98)融合多肽的构建、表达和活性研究
- Author:
Jiasen YANG
;
Jianing ZOU
;
Wei FANG
;
Yingying XING
;
Tao XI
- Publication Type:Journal Article
- Keywords:
NuBCP-9;
tumstatin;
fusion polypeptide;
antitumor;
prokaryotic expression
- From:
Journal of China Pharmaceutical University
2009;40(4):364-369
- CountryChina
- Language:Chinese
-
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 chromatogra-phy, 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.
