Expression Purification and Verification of HBscFv-IFNγ in Pichia pastoris x33

Abstract:

In order to effectively cure hepatitis B virus (HBV), we studied on fusion protein HBscFv-IFNγ, which was connected with single-chain Fv against HBV surface antigen(HBscFv) and γ-interferon(IFNγ) of being used in clinic against HBV. Adopting overlap PCR, the hbscfv and the ifnγ were connected into hbscfv-ifnγ. Then the pPICZαA/(hbscfv-ifnγ)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-IFNγ. The preliminary verification indicates that HBscFv-IFNγ has the bioactivity of HBscFv and IFNγ by SDS-PAGE, Western blotting and ELISA. The supernatant of culturing X4 was purified by 14F7 affinity chromatography to HBscFv-IFNγ with purity of 95%~98%. The HBscFv–IFNγ is able to bind 27.9% HBV surface antigen (HBsAg) in the serum of HBV transgenic mice, which shows the antibody of HBscFv-IFNγ has bioactivity in vivo. Therefore HBscFv-IFNγ can shed light on the development of a new promising HBV -targeted drug.