利拉鲁肽在大肠杆菌中的表达、制备工艺研究及活性分析

发布时间：2018-01-24 08:36

本文关键词： 降糖药利拉鲁肽融合表达纯化蛋白修饰　出处：《遵义医学院》2014年硕士论文　论文类型：学位论文

【摘要】：利拉鲁肽是一种长效GLP-1类似物，在人体内能模仿GLP-1发挥显著降低血糖、促进胰岛素分泌、修复胰岛β细胞等生理功能。它的半衰期约为11~13h，每天只需注射一次就能很好地控制血糖浓度，并降低患者的体重，是治疗T2DM的理想药物。目的：利用大肠杆菌蛋白融合表达系统、肠激酶裂解蛋白技术、色谱技术分离纯化蛋白及蛋白修饰技术等生物工程技术，实现利拉鲁肽的高效表达及制备，并通过小鼠实验分析利拉鲁肽的降糖活性，为日后国内进一步研发利拉鲁肽的制备工艺提供参考。方法：PCR法扩增获得在Lys26Arg34GLP-1(7-37)基因的N端紧邻牛肠激酶位点、C端带有终止密码子的目的基因LG1，然后连接到pET31b(+)载体中，构建成表达KSI-Arg34GLP-1(7-37)的重组融合表达载体pET31b(+)-LG1，并转化至（Escherichiacoli）BL21(DE3)构建表达工程菌株。利用IPTG诱导发酵菌体表达目的蛋白，发酵菌体经超声破碎、洗涤后得到包涵体蛋白。包涵体蛋白变性、复性处理后，经过强阴离子交换色谱技术初步纯化获得纯度较高的融合蛋白。融合蛋白经过肠激酶进一步裂解，并利用反相色谱技术纯化获得利拉鲁肽前体分子Arg34GLP-1(7-37)。最后在Arg34GLP-1(7-37)分子的Lys26上连接一条棕榈脂肪酸侧链，得到利拉鲁肽分子，反相色谱技术进一步纯化后制成制剂，并通过小鼠体内活性实验测试它的降糖活性。结果：菌体蛋白以包涵体形式表达，表达量约为2.98g/L，且KSI-Arg34GLP-1(7-37)占总蛋白的35%以上。离子交换色谱纯化重组融合蛋白后，经SDS-PAGE检测它的纯度达85%以上。肠激酶可裂解重组融合蛋白获得大小与理论值相符的Arg34GLP-1(7-37)单体，并通过在它的Lys26上连接脂肪酸侧链后得到的利拉鲁肽分子，其分子量与理论值（3750Da）相符，反相色谱纯化后，经高效液相检测其纯度在98%以上，小鼠体内活性实验表明它具有显著的体内降糖活性。结论：本研究在实验室阶段初步建立了一种制备大肠杆菌利拉鲁肽的方法，为下一步研究利拉鲁肽的生产工艺奠定基础。
[Abstract]:Lilaru peptide is a long-acting GLP-1 analogue that mimics GLP-1 to play a significant role in reducing blood glucose and promoting insulin secretion in the human body. Repair of islet 尾 cells and other physiological functions, its half-life is about 113h, only once a day to control the concentration of blood sugar, and reduce the weight of patients, it is an ideal drug for the treatment of T2DM. Objective: to achieve the efficient expression and preparation of Lilaru peptide by using the fusion expression system of Escherichia coli protein, enterokinolysis protein technology, separation and purification of protein by chromatography and protein modification technology. The hypoglycemic activity of Lilaru peptide was analyzed by mouse experiment, which provided a reference for further research and development of the preparation process of Lilaru peptide in China. Methods the target gene of Lys26Arg34GLP-1H7-37) gene was amplified by 1: 10% PCR. The target gene of Lys26Arg34GLP-1 7-37) gene was obtained, which was adjacent to the bovine enterokinase site (BKK) and had a terminating codon. Then the recombinant fusion expression vector pET31b (pET31b) expressing KSI-Arg34GLP-1m7-37 was constructed by ligating into pET31b () vector. The recombinant strain was transformed into Escherichia coli BL21 (DE3). IPTG was used to induce the expression of the target protein in the fermentation cell, and the fermentation cell was broken up by ultrasound. After washing, inclusion body protein was obtained. After renaturation, the fusion protein was purified by strong anion exchange chromatography. The fusion protein was further cleavage by enterokinase. The precursor molecule Arg34GLP-1 was purified by reverse phase chromatography (RP-HPLC), and the precursor molecule Arg34GLP-1 was obtained from Arg34GLP-1 7-37. A palm fatty acid side chain is attached to the Lys26 of the molecule. The preparation was further purified by reverse phase chromatography and its hypoglycemic activity was tested by mouse bioassay in vivo. Results: the bacterial protein was expressed in the form of inclusion body, and the expression amount was about 2.98 g / L. KSI-Arg34GLP-17-37) accounted for more than 35% of the total protein. The recombinant fusion protein was purified by ion exchange chromatography. The purity of the fusion protein was more than 85% by SDS-PAGE. The recombinant fusion protein could be lysed by enterokinase to obtain Arg34GLP-1 7-37) monomer which was in accordance with the theoretical value. The molecular weight of the Lilaru peptide molecule obtained by ligating the side chain of fatty acid on its Lys26 was in accordance with the theoretical value of 3750Da. after purification by reversed-phase chromatography. Its purity was more than 98% by high performance liquid chromatography, and the activity of mice in vivo showed that it had remarkable antidiabetic activity in vivo. Conclusion: in the laboratory stage, a method was established for the preparation of Lilaru peptide of Escherichia coli, which laid a foundation for the further study of the production process of Lilaru peptide.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

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