重组人胰高血糖素样肽－1的表达及生物学活性

将人工合成的人胰高血糖素样肽-1（human glucagon like peptide-1, hGLP-1）基因插入质粒载体pET-32a(+)中，构建成rhGLP-1与硫氧还蛋白（thioredox）及六聚组氨酸（hexahistidine）的融合表达载体pET32-GLP-1，转化大肠杆菌BL21（DE3）获得表达菌株，经IPTG诱导发酵的菌体超声破碎后，裂解液用Ni离子亲和层析纯化得到融合蛋白，经肠激酶裂解，再次Ni离子亲和层析，得到rhGLP-1样品。经SDS PAGE 和等电聚焦检测，样品纯度大于90％, 等电点介于pH5.2～pH5.85之间。质谱测定rhGLP-1分子量为3 355.0kDa，肽图分析得到2 097.7kDa和1 005.5kDa两个胰蛋白酶酶解片断，均与理论分析结果一致。动物实验表明重组蛋白具有明显的降血糖活性和促胰岛素分泌作用。

Expression and Bioactivites of Recombinant Glucagon-like Peptide 1

The synthesized full length hGLP-1 gene was cloned into pET-32a(+) to get the recombinant plasmid pET32-GLP-1, which could express a fusion protein containing thioredox， hexahistidine， and rhGLP-1 consecutively. The recombinant plasmid containing hGLP-1 was transformed into E.coli BL21 (DE3) and expressed by IPTG induction. The fusion protein was purified from lysates with Ni·IDA His·Bind affinity chromatography. rhGLP 1 with the purity of 90% was achieved after enterokinase digestion, Ni·IDA His·Bind affinity chromatography again, then was concentrated by ultrafiltration. The purified rhGLP 1 showed a single band on IEF gel with an isoelectric point between pH5.2 and pH5.85. ESI mass spectrometry showed that the molecular weight was 3355.0kDa as expected. rhGLP-1 was digested with trypsin followed by mass analysis and the peptide mapping produced two expected fragments with the molecular weights of 2097.7kDa and 1005.5kDa, respectively. The purified rhGLP-1 also showed obvious biological activity for both lowering plasma glucose and stimulating insulin secretion in mice.