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新型双功能融合蛋白GAD的PEG-马来酰亚胺修饰及其性质研究

Modification of mPEGylated novel bifunctional fusion protein GAD and its propenty

  • 摘要: 为了解决新型双功能融合蛋白GAD复性困难、具有免疫原性等问题,采用PEG-马来酰亚胺修饰变性后的GAD包涵体。通过圆二色谱、色氨酸荧光分析等方法对比修饰前后GAD构象变化,并采用OGTT、脂清除实验验证其生物学活性。研究结果表明,PEG-马来酰亚胺能够完成变性条件下GAD的定点修饰,提高其复性效率,且修饰产物二、三级结构与修饰前一致;PEG-GAD的免疫原性降低(P<0.01),具有显著的降糖降脂作用(P<0.001),且体内药效时间延长。此方法可为强疏水性多肽类药物的开发提供有效策略。

     

    Abstract: In order to solve the difficucties of renaturation and immunogenicity of new bifunctional fusion protein GAD, inclusion bodies of GAD were modified by PEG-maleimide. Conformational changes of the modified GAD were compared by circular dichroism and tryptophan fluorescence spectroscopy. The biological activity was verified by oral glucose tolerance test and lipid scavenging. The results showed that PEG-maleimide completed the specific-point modification of GAD, and improved its refolding efficiency. The secondary and tertiary structures of mPEGylated GAD were consistent with that of GAD. PEG-GAD has significant hypoglycemic and lipid-lowering effects(P<0. 001)with longer half life in vivo and lower immunogenicity(P<0. 01). This study provides effective strategies for the development of strongly hydrophobic peptide drugs.

     

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