Study On Expression Of A Human Anti-HBsAg Single-chain Fv Antibody And Interferon Gamma In Pichia Pastoris

Hepatitis B virus infection is a worldwide public health problem affecting about 350 million people per year. Monoclonal antibodies or their fragments are only useful in prevention if they bind to and block surface regions of the virus. The fusion protein of a single-chain Fv antibody against HBsAg (HBscFv) and IFNγ which is cure well for virus in clinical practice can be used to prevent and treat HBV for directing to virus and to infected cell. The objective of this paper is to get single gene of HBscFv-IFNγby gene SOEing, and to express the fusion protein of HBscFv-IFNγin Pichia pastoris, and to purify and characterize the recombinant HBscFv-IFNγ.Construction of HBscFv-IFNγ and its vector in vivo. Gene of HBscFv and IFNγ was first amplified from vector separately by PCR. Then single gene of HBscFv-IFNγ was obtained overlap PCR. HBscFv-IFNywas sequenced and sub-cloned into expression vector pPICZaA. The recombinant plasmid, pPICZaA/HBscFv-IFNγ, was then transformed into TOP10. So, multi-copy pPICZaA/HBscFv-IFNywas then successfully construced, such as pPICZaA/(HBscFv-IFNγ)₂, pPICZaA/(HBscFv-IFNγ)₄, pPICZaA/(HBscFv-IFNγ)₆, and was successfully transformed into E. coli TOP10. So, the kinds of multi-copy recombinant vector can be extracted from cultivating transformed E. coli TOP10 with vector. This will be well foundation for transforming Pichia pastoris.Secretory expression of fusion protein of HBscFv-IFNywas produced by Pichia pastoris,GS115 and X33. pPICZaA/(HBscFv-IFNγ)₂, pPICZaA/(HBscFv-IFNγ)₄, pPICZaA/(HBscFv-IFNγ)₆ and pPICZaA/ HBscFv-IFNy linearized were transformed into Pichia pastoris GS115 or X33 by the methods of LiCl or electric transformation. It was found that 70% Pichia transformants had integrated target gene by analysis of PCR, and 4% transformants could growth in the medium containing 2000mg/L Zeocin. The transformants resistant to 100mg/L, 500mg/L , 1000mg/L 1500mg/L and 2000mg/L Zeocin were cultured and induced with methanol and HBscFv-IFNγ-like protein could be found in the culture supernatant. The 42kD protein in the culture supernatant was confirmed to be...