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Protein & Cell ; (12): 317-327, 2014.
Article in English | WPRIM | ID: wpr-757497


Cytotoxic T cells (CTLs) play a key role in the control of Hepatitis B virus (HBV) infection and viral clearance. However, most of identified CTL epitopes are derived from HBV of genotypes A and D, and few have been defined in virus of genotypes B and C which are more prevalent in Asia. As HBV core protein (HBc) is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool covering HBc to screen and identify specific CTL epitopes. An unconventional HLA-A2-restricted epitope HBc141-149 was discovered and structurally characterized by crystallization analysis. The immunogenicity and anti-HBV activity were further determined in HBV and HLA-A2 transgenic mice. Finally, we show that mutations in HBc141-149 epitope are associated with viral parameters and disease progression in HBV infected patients. Our data therefore provide insights into the structure characteristics of this unconventional epitope binding to MHC-I molecules, as well as epitope specific CTL activity that orchestrate T cell response and immune evasion in HBV infected patients.

Adult , Amino Acid Sequence , Animals , Binding Sites , Epitopes , Chemistry , Allergy and Immunology , Metabolism , Female , Genotype , HEK293 Cells , HLA-A2 Antigen , Metabolism , Hepatitis B Core Antigens , Chemistry , Allergy and Immunology , Metabolism , Hepatitis B virus , Genetics , Metabolism , Humans , Hydrogen Bonding , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Middle Aged , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Structure, Tertiary , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Metabolism
Chinese Journal of Biotechnology ; (12): 595-604, 2014.
Article in Chinese | WPRIM | ID: wpr-279480


Secretory anti-gp96 scFv fragment was expressed in Pichia pastoris to obtain a small molecule antibody that specifically recognizes heat shock protein gp96. The gp96-scFv fragment gene was synthesized and cloned to Pichia pastoris expression plasmid pPICZa-A. Pichia pastoris X33 was electroporated with the linearized recombinant expression vector, and expression of gp96-scFv fragment was induced by methanol. The His-tagged recombinant protein was then purified by affinity chromatography and analyzed with SDS-PAGE and Western blotting assays. The biological activities of recombinant gp96-scFv fragment were determined by Western blotting, Immunofluorescence, ELISA and FACS assays. The gp96-scFv fragment was expressed successfully in Pichia pastoris. About 50 mg of recombinant protein could be purified from 1 liter of the Pichia pastoris culture supernatant. Its molecular weight was about 15 kDa. The gp96-scFv fragment could specifically bind to gp96 protein by Western blotting, immunofluorescence, ELISA and FACS analyses. Pichia pastoris-expressed gp96-scFv fragment specifically recognizes gp96 protein, which could be used for Western blotting, Immunofluorescence, ELISA and FACS analyses.

Blotting, Western , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Membrane Glycoproteins , Allergy and Immunology , Pichia , Metabolism , Plasmids , Recombinant Proteins , Single-Chain Antibodies