| Backgroud and ObjectiveChronic hepatitis B is a common disease of a serious hazard to human health and about350million people infect chronic hepatitis B virus worldwide. Also, there are around180million people suffering chronic hepatitis C. The risk of these diseases mainly is its high chronicity rate and possibly serious complications, including hepatic cirrhosis and cancer. So, prevention and treatment of viral hepatitis is a global public health problem and it has attracted the attention of the world.Interferon (IFN) is widely recognized as an effective antiviral medicine, and so far has been used in clinic for over20years. However, as a small molecule protein, interferon is cleared fast from plasma, the clear phase half-life is about3h to8h after injection. IFN can not detected from the plasma24hour post-injection subcutaneously, so it need to be repeated to achieve therapeutic effect in clinical treatment, which result in some inconvenience of clinical applications. Therefore, prolonging the half-life of IFNa and reducing its side effects have great significance to the clinical application of IFNa. Currently, the methods of extending interferon half-life are polyethylene glycol (PEG) modification and the fusion protein. In recent years, antibody IgG Fc fragment-based fusion protein (IFN/Fc) gained attention for its biological activity and pharmacological effects. As a new strategy of genetically engineering medicine, it has been in cutting-edge research and pharmaceutical field.Based on the progress of protein expression system of recent years, we use the Bac-to-Bac baculovirus insect expression system, express the long-acting interferon IFN/Fc fusion protein. According to the characteristics of the IgG Fc and IFN, we explore its basic biological activity of IFN/Fc, expect to improve the interferon antiviral treatment, and provide new methods for long-acting interferon antiviral therapy.Methods1. Using molecular cloning technology to get human IFNa2b and IgG4Fc gene fragment, and constructing the baculovirus shuttle vector pFastBacl-IFN/Fc2. Transformation into DH10Bac competent strains to obtain the Bacmid-IFN/Fc3. Transfection of Bacmid-IFN/Fc into insect cells High Five to obtain recombinant baculovirus using liposomal transfection techniques4. The expression of IFN/Fc fusion protein was detected by ELISA and Western-Blot5. The biological activity were assessed by the cytopathic effect inhibition methodResults1. IFNa2b and IgG4Fc fragment were obtained and cloned into the baculovirus shuttle vector pFastBacl correctly, confirmed by PCR, restriction enzyme digestion and nucleotide sequencing analysis2. The recombinant Bacmid-IFN/Fc was gotten by screening in DH10Bac competent cells3. Cytopathic effect appeared in insect cells High five after transfection of Bacmid-IFN/Fc, indicating successful recombinant baculovirus packaging4. ELISA confirmed that rabbit anti-human IFNa antibody can identify the recombinant IFN/Fc fusion protein5. Western-Blot analysis showed a specific band at the45kD6. The in vitro antiviral activity showed580IU/ml using the cytopathic effect inhibition methodConclusionRecombinant human IFN/Fc fusion protein was expressed successfully using the baculovirus insect system, which provides the experimental basis for the development of new long-acting interferon and the treatment of chronic viral hepatitis. |
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