Exendin-4类似物的克隆、表达与体内生物活性检测

为研究Exendin-4类似物的克隆,融合表达及在体内的生物活性,在pED载体融合伴侣序列中插入利于下游分离纯化的序列成为5#载体,将Exendin-4类似物基因与5#载体中的融合伴侣基因通过酸水解位点连接,转化至E.coli BL21中并诱导表达融合蛋白,酸水解将目的肽与融合伴侣分开后,经阴离子交换树脂分离得到目的肽。6周～8周正常雄性ICR小鼠皮下注射Exendin-4类似物后,口服糖耐量实验检测在不同时间段小鼠血液中葡萄糖及胰岛素含量的变化。结果表明:融合蛋白的表达量占菌体总蛋白的40%,Exendin-4类似物纯度达91.8%。Exendin-4类似物的活性与对照组相比,具有显著的降低血糖和显著促进胰岛素分泌的活性(P<0.01)。

Cloning, expressing of Exendin-4 analogue and bioactivity analysis in vivo

To construct,express and purify Exendin-4 analogue and detect its biological activity in vivo.Insert gene sequence into fusion partner of pED plasmid which is helped to purification,entitled the new recombinant plasmid 5#.Exendin-4 analogue polypeptide gene and fusion partner gene was linked by acid hydrolysis gene,transformed to E.coli BL21 and the fusion protein was induced by lactose.after acid hydrolysis,the Exendin-4 analogue polypeptide separated from fusion chaperon.Anion charge chromatography were used to further purification.6 to 8 week-old ICR mice were injected(s.c) with Exendin-4 analogue,blood glucose and plasma insulin level was detected in different period after oral glucose tolerance test.The results show that high expression of inclusion body was induced by lactose,which accounted for 40% of germ proteins,the Exendin-4 analogue was obtained with the purity of 91.8% after being purified by anion charge chromatography.Bioactivity assay showed that the level of blood glucose of mouse which treated with exendin-4 analogue was obviously decreased to normal(P<0.01),and the level of plasma insulin was increased obviously(P<0.01).