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Cloning and Expression of IFN-α and Human LgG Fc Fusion Protein with Longer Serum Half-life

Author: LiJinJing
Tutor: XiaoWeiHua
School: University of Science and Technology of China
Course: Cell Biology
Keywords: IFN-α2b IgG fusion protein serum half-life Pichia yeast
CLC: R392.1
Type: Master's thesis
Year: 2011
Downloads: 108
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Abstract


The recombinant interferon such as IFN-α(including IFNα-2a, IFNα-2b, IFNα-1b and others) and its modifiers (e.g. multivalent IFN-α) have currently been used in clinic as an anti-virus or immuno-regulatory drug. Actually, natural IFN-αis a cytokine produced by some cells and has the effects of anti-tumor cell proliferation, anti-virus and immune response regulation. However, as a small protein, it rapidly degraded in vivo with a serum half-life less than half an hour, and this makes patients have to accept frequent dosing which increase the pain and economical burden. The peak plasma concentration will bring about side-effects, which is hard to achieve therapeutic targets. In order to extend the serum half-life of the IFN-α, we construct the recombinant gene encoding human interferon-alpha (rHuIFN-α) and immunoglobulin Fc fragment according to the characteristic of immunoglobulin with long-term half life in blood circulation in this study. This recombinant fusion protein was further modificated, including some site mutation in order to to reduce the side-effects (including ADCC and CDC activity of Fc fragment .The fusion protein not only maintain the natural N-terminal and bioactivity, but also has the charactristics of antibody, which may bind with Protein A through CH2 and CH3 regions and thus supply the advantage of protein purification. Moreover, the retained antibody hinge can make the two effective parts work independently.We also make two amino acid modifications to increase the affinity of the Fc fragment that binds with FcRn, which makes the fusion protein retain a longer half-life in serum. In addition, the fusion protein has successfully been expressed in a novel type of methanol-induced Pichia yeast and a standard protocol for protein purification has also been established. The purified protein with the purity more than 95% will be used for the biological activities, and should have great potential in the future clinical application.

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CLC: > Medicine, health > Basic Medical > Medical Immunology > Immunobiology
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