Fusion expression and bioactivity comparison of porcine beta-defensin-2 and porcine interferon-gamma in Pichia pastoris.
- Author:
Dingyong ZHANG
1
;
Lei SUN
;
Limin YANG
;
Wenjun LIU
Author Information
1. Key Laboratory of Pathogenic Microbiology and Immunology, Center for Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Genetic Vectors;
genetics;
Interferon-gamma;
biosynthesis;
genetics;
Pichia;
genetics;
metabolism;
Recombinant Fusion Proteins;
biosynthesis;
chemistry;
genetics;
Swine;
beta-Defensins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2010;26(12):1652-1659
- CountryChina
- Language:Chinese
-
Abstract:
In order to study PBD-2 and PoIFNgamma, the chimeric gene PBD-2-PoIFNgamma was synthesized by overlap extension PCR, and amplified PoIFNgamma on the basis of this sequence, then cloned into yeast expression vector pPICZalphaA separately to get the recombinant plasmid pPICZalphaA-PBD-2-PoINFgamma and pPICZalphaA-PoINFgamma. The recombinant plasmid was digested by Sac I and introduced into Pichia pastoris X33 cells by electroporation. Positive clones were screened and cultivated in BMMY medium containing 0.5% methanol for 72 h. SDS-PAGE and Western blotting analysis showed that the screened recombinant could secrete PBD-2-PoINFgamma and PoINFgamma separately. The activity of fusion protein was not detected by cytopathic effect inhibition assay and agar diffusion assay, but detected obvious antiviral activity of PoINFgamma. The helix and random coil contents was showed vary greatly between PoIFNgamma and PBD-2-PoLNFgamma by circular dichroism analysis. It was speculated that the fusion protein was not correctly folded and may affect the activity of PBD-2-PoINFgamma.
