| Exendin -4在大肠杆菌中的串联表达、纯化及活性鉴定 |
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| 引用本文： | 刘延杰,林鲁霞,宋长征,季虹,荣海钦.Exendin -4在大肠杆菌中的串联表达、纯化及活性鉴定[J].生物技术,2011,21(5). |
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| 作者姓名： | 刘延杰 林鲁霞 宋长征 季虹 荣海钦 |
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| 作者单位： | 山东省内分泌与代谢病研究所山东省医学科学院,山东济南,250062 |
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| 基金项目： | 山东省科技攻关项目，重组胰高血糖素样肽1(GLP-1)类似物的研制项目 |
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| 摘 要： | 目的:完成钝尾毒蜥Exendin -4基因在大肠杆菌中的串联高效表达.方法:按照大肠杆菌偏爱密码子设计Exendin -4基因片段,利用同尾酶构建多拷贝串联的表达载体,于大肠杆菌BL21( DE3)中诱导表达,SDS - PAGE鉴定结果,表达产物Ni柱纯化后肠激酶切割,高效液相色谱及四级杆-飞行时间质谱纯化、鉴定,确定目标产物,作用胰岛瘤细胞INS -1检测胰岛素释放活性.结果:成功构建重组载体pET28a -(Exendin -4)n(n=2,4,6),且均获得可溶性表达产物,重组表达蛋白经切割、纯化、鉴定和制备,最终得到纯度98%的Exendin -4,其具有与标准品相似的促葡萄糖刺激的胰岛素释放活性.结论:试验成功利用串联表达方式制备有活性的Exendin -4,为下一步2型糖尿病治疗药物的研究奠定了基础.
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| 关 键 词： | Exendin -4:串联表达 纯化 胰岛素释放试验 2型糖尿病 |
Tandem Expression, Purification and Activity Analysis of Exendin -4 in E.coli |
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| LIU Yan-jie,LIN Lu-xia,SONG Chang-zheng,JI Hong,RONG Hai-qin.Tandem Expression, Purification and Activity Analysis of Exendin -4 in E.coli[J].Biotechnology,2011,21(5). |
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| Authors: | LIU Yan-jie LIN Lu-xia SONG Chang-zheng JI Hong RONG Hai-qin |
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| Institution: | LIU Yan-jie,LIN Lu-xia,SONG Chang-zheng,JI Hong,RONG Hai-qin(Shandong Institute of Endocrine and Metabolic Diseases,Shandong Academy of Medical Science,Jinan 250062,China) |
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| Abstract: | Objective: The sake of a high expression of heloderma suspectum Exendin-4 in E.coli.Method: The original Exendin-4 gene in light of its amino acid sequence and the code bias of E.coli was modified.The expression plasmids pET28a-(Exendin-4)n(n=2,4,6)were constructed containing a multicopy of tandem oligonucleotide which were joined by a passage of DNA coding enterokinase.The positive clone was identified by a restriction enzyme and further confirmed by sequencing,and expressed in E.coli BL21,and the products... |
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| Keywords: | Exendin-4 tandem expression purification insulin releasing test type 2 diabetes mellitus |
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