Exendin-4在大肠杆菌中的串联表达、纯化及活性鉴定  

Tandem Expression,Purification and Activity Analysis of Exendin-4 in E.coli

作　　者：刘延杰[1] 林鲁霞[1] 宋长征[1] 季虹[1] 荣海钦[1] 

机构地区：[1]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

出　　处：《生物技术》2011年第5期40-44,共5页Biotechnology

基　　金：山东省科技攻关项目("重组肠促胰岛素样肽Exendin-4的研制";编号:2009GG20002046"重组胰高血糖素样肽1(GLP-1)类似物的研制";编号:2009GG20002049)资助

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。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 were analyzed by SDS-PAGE.Ni-NTA affinity chromatography,enterokinase cleavage,high performance liquid chromatography,and quadrupole time of flight LC/MS were used to perform purification and identification.The insulin releasing activity of Exendin-4 was detected by INS-1 cell.Result：Prepared expression plasmids pET28a-（Exendin-4）n（n=2,4,6）expressed soluble protein,but the express level does not conform to the copy numbers.The synthesized Exendin-4 was identified with a purity of 98%,and has a similar glucose-stimulated insulin secretion activity compared to standard sample in vitro.Conclusion： These results showed that we successfully prepared the Exendin-4 protein,and validated its biological activity preliminary,which will have an influential research into the therapy of type 2 diabetes mellitus.

关 键 词：EXENDIN-4 串联表达 纯化 胰岛素释放试验 2型糖尿病 

分 类 号：Q786[生物学—分子生物学]