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目的建立重组人胰高血糖素样肽前药(prodrug of recom binant human GLPs,Pro-rhGLPs)的表达、纯化方法,研究Pro-rhGLPs对体内血糖水平及血清胰岛素水平的影响。方法采用异丙基-β-D-硫代半乳糖苷(IPTG,0.01mmol·L-1)诱导原核表达系统E.coliBL21(DE3)/pET32a(+)-hGLPs表达Pro-rhGLPs,12%SDS-PAGE检测蛋白表达量,Ni-NTA亲和层析纯化Pro-rhGLPs,应用Westernblot在体外观察前体药物的活性分子释放过程。在C57BL/6小鼠上进行葡萄糖耐量实验检测其生物学活性,不同时间间隔取血测定血糖及血清胰岛素水平。在糖尿病db/db小鼠上观察其降糖作用。结果所构建的Pro-rhGLPs原核表达系统中,Pro-rhGLPs表达量约为细菌总蛋白的50%。纯化得到的蛋白纯度达到95.43%,并且可以在特异性酶作用下缓慢降解,释放出多个胰高血糖素样肽1(GLP-1)分子。纯化的Pro-rhGLPs呈剂量依赖性降低血糖浓度,同时升高血清胰岛素水平。等剂量Pro-rhGLPs的降糖作用较GLP-1作用更强。结论建立了Pro-rhGLPs高效表达、纯化方法,获得了高纯度Pro-rhGLPs,对糖尿病小鼠具有明显的降血糖作用。
Objective To establish the expression and purification methods of prodrug of recombinant human GLPs (Pro-rhGLPs) and to study the effect of Pro-rhGLPs on in vivo blood glucose and serum insulin levels. Methods Pro-rhGLPs and 12% SDS were induced by E.coli BL21 (DE3) / pET32a (+) - hGLPs induced by IPTG (0.01mmol·L-1) -PAGE protein expression, Ni-NTA affinity chromatography purification of Pro-rhGLPs, the use of Westernblot observed in vitro prodrug active molecule release process. The C57BL / 6 mice were subjected to glucose tolerance test to detect the biological activity, blood was taken at different time intervals to measure the blood glucose and serum insulin levels. Diabetic db / db mice observed its hypoglycemic effect. Results Pro-rhGLPs prokaryotic expression system constructed, Pro-rhGLPs expression of about 50% of total bacterial protein. The purity of the purified protein reached 95.43%, and it could be slowly degraded under the action of specific enzymes to release a number of glucagon-like peptide 1 (GLP-1) molecules. Purified Pro-rhGLPs reduced blood glucose levels in a dose-dependent manner, while elevating serum insulin levels. Equal doses of Pro-rhGLPs hypoglycemic effect than GLP-1 stronger. Conclusion Pro-rhGLPs were highly expressed and purified, and high-purity Pro-rhGLPs were obtained, which showed obvious hypoglycemic effect on diabetic mice.