重组人胰高血糖素样肽前药的表达、纯化及生物活性分析

 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.coli BL21(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。

Expression,Purification and Bioactivity of Prodrug of Recombinant Human GLPs

OBJECTIVE To produce prodrug of recombinant human glucagon-like peptides (Pro-rhGLPs) and study its effects on regulations of blood glucose levels and insulin secretion in mice. METHODS The constructed E.coli BL21(DE3) /pET32a(+)-hGLPs was induced by 0.01 mmol·L^-1 Isopropgl-β-D-thiogalactopyranoside(IPTG). The expressed product was identified by 12% SDS-PAGE. The protein was purified by Ni-NTA purification system. Pro-rhGLPs was digested by thrombin in PBS buffer. The biological activity of Pro-rhGLPs was examined by sc injection in C57BL/6 mice or db/db mice. Blood glucose levels were measured using a blood glucose meter. Plasma insulin concentrations were determined by ELISA. RESULTS The expression output of Pro-rhGLPs was approximately 50% of the total bacterial proteins. The purity of recombinant Pro-rhGLPs was more than 95.43% by HPLC analysis.The protein was slowly digested by specific endoproteinase and bioactive glucagons peptide 1(GLP-1) was released gradually. The analysis of in vivo activity indicated that Pro-rhGLPs had the bioactivity of native GLP-1,and significantly decreased blood glucose and increased insulin secretion in dose-dependent manner. Pro-rhGLPs had a stronger effects than GLP-1. Moreover,basal random glycemia decreased rapidly and remained lower level for 10 h following a single injection. CONCLUSION A prokaryotic expression system of Pro-rhGLPs with high efficiency was successfully established and the protein was obtained. Preliminary studies showed a new potential for developing the long-acting GLP-1 analogs for clinical applications.