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Chinese Journal of Hepatology ; (12): 617-620, 2012.
Article in Chinese | WPRIM | ID: wpr-296838

ABSTRACT

To investigate a baculovirus insect cell system for expressing an interferon alpha 2b (IFNa2b)/immunoglobulin G-4 (IgG4) Fc fusion protein, which has long-acting antiviral effects. Human IFNa2b and IgG4 Fc cDNAs were generated by molecular cloning and inserted into a baculovirus shuttle vector, which was then transposed into the DH10 Bac strain to form recombinant Bacmid-IFN/Fc. The Bacmid-IFN/Fc was transfected into High five insect cells, and expression of the IFN/Fc fusion protein was detected by Western blotting and its biological activity was assessed by the cytopathic effect inhibition method. The IFNa2b and IgG4 Fc cDNA fragments were successfully amplified by RT-PCR using human peripheral lymphocytes. After cloning into the baculovirus shuttle vector, pFastBac1, and transforming into DH10 Bac competent cells, screening identified positive clones carrying the recombinant Bacmid-IFN/Fc. A Bacmid-IFN/Fc clone was successfully transfected into the High five insect cells and packaged into the baculovirus for expression of the IFN/Fc fusion protein. Western blotting revealed that the fusion protein expression was specific, and yielded a protein of 45 kD in size. The in vitro antiviral activity of the IFN/Fc fusion protein was 580 IU/mL. A novel IFN/Fc fusion protein was successfully generated using a baculovirus insect cell system, which may prove useful for providing future experimental data for development of a new long-acting interferon to treat chronic viral hepatitis.


Subject(s)
Animals , Antiviral Agents , Metabolism , Baculoviridae , Genetics , Cell Line , Cloning, Molecular , Gene Expression , Gene Fusion , Genetic Vectors , Humans , Immunoglobulin Fc Fragments , Genetics , Immunoglobulin G , Genetics , Insecta , Interferon-alpha , Genetics , Recombinant Fusion Proteins , Genetics , Recombinant Proteins , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection
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