本文關(guān)鍵詞： 降糖藥 利拉魯肽 融合表達(dá) 純化 蛋白修飾　出處：《遵義醫(yī)學(xué)院》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：利拉魯肽是一種長效GLP-1類似物，在人體內(nèi)能模仿GLP-1發(fā)揮顯著降低血糖、促進(jìn)胰島素分泌、修復(fù)胰島β細(xì)胞等生理功能。它的半衰期約為11~13h，每天只需注射一次就能很好地控制血糖濃度，并降低患者的體重，是治療T2DM的理想藥物。 目的：利用大腸桿菌蛋白融合表達(dá)系統(tǒng)、腸激酶裂解蛋白技術(shù)、色譜技術(shù)分離純化蛋白及蛋白修飾技術(shù)等生物工程技術(shù)，實(shí)現(xiàn)利拉魯肽的高效表達(dá)及制備，并通過小鼠實(shí)驗(yàn)分析利拉魯肽的降糖活性，為日后國內(nèi)進(jìn)一步研發(fā)利拉魯肽的制備工藝提供參考。 方法：PCR法擴(kuò)增獲得在Lys26Arg34GLP-1(7-37)基因的N端緊鄰牛腸激酶位點(diǎn)、C端帶有終止密碼子的目的基因LG1，然后連接到pET31b(+)載體中，構(gòu)建成表達(dá)KSI-Arg34GLP-1(7-37)的重組融合表達(dá)載體pET31b(+)-LG1，并轉(zhuǎn)化至（Escherichiacoli）BL21(DE3)構(gòu)建表達(dá)工程菌株。利用IPTG誘導(dǎo)發(fā)酵菌體表達(dá)目的蛋白，發(fā)酵菌體經(jīng)超聲破碎、洗滌后得到包涵體蛋白。包涵體蛋白變性、復(fù)性處理后，經(jīng)過強(qiáng)陰離子交換色譜技術(shù)初步純化獲得純度較高的融合蛋白。融合蛋白經(jīng)過腸激酶進(jìn)一步裂解，并利用反相色譜技術(shù)純化獲得利拉魯肽前體分子Arg34GLP-1(7-37)。最后在Arg34GLP-1(7-37)分子的Lys26上連接一條棕櫚脂肪酸側(cè)鏈，得到利拉魯肽分子，反相色譜技術(shù)進(jìn)一步純化后制成制劑，并通過小鼠體內(nèi)活性實(shí)驗(yàn)測試它的降糖活性。 結(jié)果：菌體蛋白以包涵體形式表達(dá)，表達(dá)量約為2.98g/L，且KSI-Arg34GLP-1(7-37)占總蛋白的35%以上。離子交換色譜純化重組融合蛋白后，經(jīng)SDS-PAGE檢測它的純度達(dá)85%以上。腸激酶可裂解重組融合蛋白獲得大小與理論值相符的Arg34GLP-1(7-37)單體，并通過在它的Lys26上連接脂肪酸側(cè)鏈后得到的利拉魯肽分子，其分子量與理論值（3750Da）相符，反相色譜純化后，經(jīng)高效液相檢測其純度在98%以上，小鼠體內(nèi)活性實(shí)驗(yàn)表明它具有顯著的體內(nèi)降糖活性。 結(jié)論：本研究在實(shí)驗(yàn)室階段初步建立了一種制備大腸桿菌利拉魯肽的方法，為下一步研究利拉魯肽的生產(chǎn)工藝奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943

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本文編號(hào)：1459620