摘要
以人胰高糖素样肽-1(human glucagons-like peptide-1,GLP-1))为研究对象,以克隆载体pPblu2SKP/(GLP-1A2G)2基因为模板,利用PCR扩增获得GLP-1与其突变体GLP-1A2G的编码基因,并将其插入原核表达质粒pGEX-4T-1中,利用氯化钙法将其转入表达宿主大肠杆菌(Escherichia coli)BL21(DE3)中诱导表达,经IPTG诱导后,收集菌体。菌体经超声破碎后,裂解液用GST亲和层析纯化得到融合蛋白,经凝血酶酶解,用Superdex G-75凝胶层析,得到GLP-1及其突变体GLP-1A2G的样品,经SDS-PAGE电泳测定,样品纯度>90%。小鼠糖耐量试验表明,相对于空白对照组而言,GLP-1及其突变体GLP-1A2G可以明显地控制血糖的水平,两者的生物活性并无明显差异。由于突变体GLP-1A2G较GLP-1可以更加有效的抵制DPPⅣ的降解,提示突变体GLP-1A2G较GLP-1在白蛋白融合或PEG修饰等方面具有更大的优势。
Glucagon-like peptide-1（GLP-1） is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion.This article focused on the expression purification and bio-activity of GLP-1 and GLP-1A2G.These two genes were amplified by PCR and constructed in plasmid of pGEX-4T-1.The recombinant plasmids of pGEX-4T-1/GLP-1 were transformed into Escherichia coli BL21（DE3） and expression continued as induced by IPTG.The fusion proteins were purified from lysates with GST affinity chromatography.The GLP-1 and GLP-1A2G was obtained following two purification steps including cutting with thrombin and Superdex G75.The Tricine-SDS-PAGE showed that the molecular weight of the GLP-1 and GLP-1A2G was 3.1 kD and the purity of the GLP-1 and GLP-1A2G was more than 90%.The Glucose tolerance tests in mice model suggested that plasma glucose level were significantly lower after administration of GLP-1 or GLP-1A2G in combination with glucose,comparing with glucose alone.Thus the GLP-1A2G Seems to be more suits for protein fusion with HSA or modification by PEG than GLP-1.
出处
《生物技术通报》
CAS
CSCD
2008年第6期139-143,共5页
Biotechnology Bulletin
基金
863探索类项目(2006AA10Z449)
江苏省六大人才高峰计划
