Expression purification and verification of HBscFv-IFNgamma in Pichia pastoris x33.
- Author:
Shishui ZHOU
1
;
Xiaoning WANG
Author Information
1. School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510640, China. hgzhouss@scut.edu.cn
- Publication Type:Journal Article
- MeSH:
Chromatography, Affinity;
Genetic Engineering;
Genetic Vectors;
Hepatitis B Antibodies;
biosynthesis;
genetics;
Hepatitis B Surface Antigens;
immunology;
Immunoglobulin Fragments;
genetics;
Immunoglobulin Variable Region;
genetics;
Interferon-gamma;
biosynthesis;
genetics;
Organisms, Genetically Modified;
Pichia;
genetics;
metabolism;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
isolation & purification
- From:
Chinese Journal of Biotechnology
2008;24(3):423-429
- CountryChina
- Language:Chinese
-
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.
