[Expression purification and verification of HBscFv-IFNgamma in Pichia pastoris x33].
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.
Zhou
Shishui
S
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510640, China. hgzhouss@scut.edu.cn
Wang
Xiaoning
X
D002846:Chromatography, Affinity; D005818:Genetic Engineering; D005822:Genetic Vectors; D006510:Hepatitis B Antibodies / Q000096:biosynthesis / Q000235:genetics*; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics*; D030781:Organisms, Genetically Modified; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D007371:Interferon-gamma