Expression purification and verification of HBscFv-IFNgamma in Pichia pastoris x33 / 生物工程学报
Chinese Journal of Biotechnology
; (12): 423-429, 2008.
Article
in Zh
| WPRIM
| ID: wpr-276105
Responsible library:
WPRO
ABSTRACT
In order to effectively cure hepatitis B virus (HBV), we studied on fusion protein HBscFv-IFNgamma, which was connected with single-chain Fv against HBV surface antigen(HBscFv) and gamma-interferon(IFNgamma) of being used in clinic against HBV. Adopting overlap PCR, the hbscfv and the ifngamma were connected into hbscfv-ifngamma. Then the pPICZalphaA/(hbscfv-ifngamma)(1,2,4) of multi-copy recombinant plasmid were constructed and transformed into Pichia pastoris x33. The engineering strain x4 was screened from transformed x33 and could secretively express HBscFv-IFNgamma. The preliminary verification indicates that HBscFv-IFNgamma has the bioactivity of HBscFv and IFNgamma by SDS-PAGE, Western blotting and ELISA. The supernatant of culturing X4 was purified by 14F7 affinity chromatography to HBscFv-IFNgamma with purity of 95%-98%. The HBscFv-IFNgamma is able to bind 27.9% HBV surface antigen (HBsAg) in the serum of HBV transgenic mice, which shows the antibody of HBscFv-IFNgamma has bioactivity in vivo. Therefore HBscFv-IFNgamma can shed light on the development of a new promising HBV-targeted drug.
Full text:
1
Index:
WPRIM
Main subject:
Pichia
/
Recombinant Fusion Proteins
/
Immunoglobulin Variable Region
/
Immunoglobulin Fragments
/
Genetic Engineering
/
Chromatography, Affinity
/
Interferon-gamma
/
Organisms, Genetically Modified
/
Allergy and Immunology
/
Genetic Vectors
Language:
Zh
Journal:
Chinese Journal of Biotechnology
Year:
2008
Type:
Article