截短型胰高血糖素样肽-1的融合构建与表达

为了延长截短型胰高血糖素样肽1（shorted glucagon like peptide1，sGLP1）在血浆中的半衰期，将sGLP1的C端与人血清白蛋白（human albumin，HSA）的N端通过6个氨基酸的柔性连接子融合在一起，构建了融合表达菌株Pichia pastoris GS115/sGLP1HSA。以4g/L G418筛选出来的阳性工程菌株，经5L发酵罐培养，甲醇诱导48h，表达量可达0.8g/L。培养液经超滤、葡聚糖G25除盐、蓝色凝胶亲和层析纯化后，经HPLC分析产物纯度达95%，回收率达60%。正常小鼠糖耐量实验和对GKⅡ型糖尿病模型鼠的治疗证明sGLP1/HSA具有明显的降糖效果，为获得大量sGLP1/HSA融合蛋白进行临床实验研究奠定了基础。

Construction and Expression of sGLP-1-HSA Fusion Protein

To reduce the clearance of sGLP-1, sGLP-1-HSA fusion protein was constructed by the fusion of the C-terminus of sGLP-1 to the N-terminus of HSA via an 6 amino acids linker and expressed in the pichia pastoris.Screen the strain with resistsnce of 4g/L G418. After fermentation in a 5L bioreactor，the expression of sGLP-1- HSA was induced by methanol.After 48h, the expression level reached 0.8g/L.The sGLP-1- HSA was abtained following a series of purification steps such as ultrafiltration, sephadex G-25 and blue- sepharose. The purity of the fermentation product could reach 95% by means of HPLC，and the total recovery yield could reach 60%.The biological activity of sGLP-1- HSA in GKⅡ diabetes rat and healthy rat suggested that the plasma glucose level was significantly lower after subcutaneous administration of sGLP-1- HSA..Expression of sGLP-1- HSA fusion protein can provide foudation for abtaining a larger quantity of recombinant sGLP-1- HSA for experimented and clinic studies.