可解离的人胰高血糖素样肽1与人血清白蛋白融合蛋白的构建表达及其初步药代动力学和药效动力学评价

发布时间：2018-09-12 05:21

【摘要】：目的构建4种在体内以不同速率解离的人胰高血糖素样肽1(GLP-1)与人血清白蛋白融合蛋白,探索各种融合蛋白的疗效与其解离速率之间的关系,获得在药代动力学和药效动力学之间保持平衡的最佳融合蛋白。方法通过重叠延伸PCR扩增出具有不同多肽接头的融合蛋白基因,将其克隆到p PIC9载体,电击转化毕赤酵母GS115,经甲醇诱导,分泌表达各种融合蛋白,将各种融合蛋白分离纯化后进行初步药代学和药效学研究。结果融合蛋白与弗林蛋白酶作用后,不可解离的Gly2-GLP-1-GGGGG-HSA融合蛋白未发生解离,而可解离的融合蛋白Gly2-GLP-1-VTR-HSA为慢解离,Gly2-GLP-1-SARSVRA-HSA为中等解离,Gly2-GLP-1-GRSRVTRSV-HSA为快解离。体外生物学活性表明,各种融合蛋白均具有促进MIN6细胞胰岛素分泌的功能。体内药代学检测结果表明,各种融合蛋白在体内的半衰期大小依次为Gly2-GLP-1-GGGGG-HSA、Gly2-GLP-1-VTR-HSA、Gly2-GLP-1-SARSVRA-HSA、Gly2-GLP-1-GRSRVTRSV-HSA。体内药效学检测结果表明,各种融合蛋白均表现出降糖活性,且其活性高低依次为Gly2-GLP-1-VTR-HSA、Gly2-GLP-1-SARSVRA-HSA、Gly2-GLP-1-GRSRVTRSV-HSA、Gly2-GLP-1-GGGGG-HSA。结论引入蛋白酶切割位点后,GLP-1可在体内从融合蛋白中解离,使其恢复生物学活性。只有控制融合蛋白在体内适当的解离速率才能达到最佳疗效,从而在药代动力学和药效动力学性质之间达到平衡。
[Abstract]:Objective to construct four fusion proteins of human glucagon like peptide-1 (GLP-1) and human serum albumin (HSA) at different rates of dissociation in vivo, and to explore the relationship between the efficacy of these fusion proteins and their dissociation rate. The optimal fusion protein was obtained to maintain the balance between pharmacokinetics and pharmacokinetics. Methods the fusion protein gene with different polypeptide junctions was amplified by overlapping extension PCR and cloned into p PIC9 vector. The fusion protein was secreted and expressed by electroporation of Pichia pastoris GS115, induced by methanol. The fusion proteins were isolated and purified for preliminary pharmacokinetic and pharmacodynamic studies. Results after the fusion protein was treated with Flynn protease, there was no dissociation of the insoluble Gly2-GLP-1-GGGGG-HSA fusion protein, while the dissociable fusion protein Gly2-GLP-1-VTR-HSA was Gly2-GLP-1-SARSVRA-HSA, which was the medium dissociated gly2-GLP-1-GRSRVTRSV-HSA. In vitro biological activity showed that all fusion proteins could promote insulin secretion of MIN6 cells. The results of in vivo pharmacokinetics showed that the half-life of the fusion proteins in vivo was Gly2-GLP-1-GGGGG-HSA,Gly2-GLP-1-VTR-HSA,Gly2-GLP-1-SARSVRA-HSA,Gly2-GLP-1-GRSRVTRSV-HSA.. The results of in vivo pharmacodynamics test showed that all fusion proteins showed hypoglycemic activity, and the order of their activity was Gly2-GLP-1-VTR-HSA,Gly2-GLP-1-SARSVRA-HSA,Gly2-GLP-1-GRSRVTRSV-HSA,Gly2-GLP-1-GGGGG-HSA.. Conclusion GLP-1 can be dissociated from the fusion protein in vivo after the introduction of protease cleavage site to restore its biological activity. Only by controlling the proper dissociation rate of the fusion protein in vivo can the optimal efficacy be achieved, thus achieving a balance between pharmacokinetics and pharmacokinetic properties.
【作者单位】：安徽大学生命科学学院;军事医学科学院生物工程研究所微生物工程研究室;山西康宝生物制品股份有限公司;
【基金】：国家自然科学基金面上资助项目(81172968)
【分类号】：R96

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