【作者】 杨涛；

【导师】 许正宏；

【作者基本信息】 江南大学 ， 微生物与生化药学， 2009， 硕士

【摘要】 GLP-1是由肠道L细胞合成和分泌的由30个氨基酸组成的多肽,它不仅能促进胰岛素合成,而且能够促进胰β细胞增殖和抑制胰β细胞凋亡。GLP-1独特的降血糖作用机制,是现有抗糖尿病药物无可比拟的,但由于其在体内的半衰期非常短,大大限制了其应用。为克服GLP-1半衰期短的缺点,实现其在体内的长效作用,本实验室成功构建了胰高糖素样多肽-1(GLP-1)突变串联体与人血清白蛋白的表达质粒pPIC9K/(GLP-1A2G)2-HSA,并且初步实现了在毕赤酵母KM71中的表达。在此基础上,本文主要围绕提高融合蛋白GGH的产量和分离纯化进行研究,主要结论如下:(1)统计分析了融合基因GGH在不同毕赤酵母宿主(KM71、GS115、SMD1168)中的表达,并通过荧光定量PCR研究了三株产量差异较大的GS115重组子基因剂量与融合蛋白GGH表达量的关系,实验结果表明:宿主GS115更有利于融合蛋白GGH的表达,融合蛋白的表达量与目的基因的剂量成正相关,最终确定高一株产菌株GS115/F2,在摇甁中的稳定表达量为328 mg/L。(2)研究了高表达菌株的发酵过程,确定了其在5 L发酵罐上的发酵工艺。实验结果表明有机氮源培养基更有利于融合蛋白GGH高密度表达及控制目的蛋白的降解;在有机氮源培养基中,0.5%的甲醇脉冲式流加方式和30℃诱导温度为最佳诱导条件,最终融合蛋白GGH的产量达到642 mg/L。(3)初步建立了融合蛋白GGH的分离纯化路线,即发酵液8000 r/min离心5 min, 10 kDa截留量的超滤膜超滤浓缩20倍, Blue Sepharose亲和吸附、Sephadex G25脱盐、Q Sepharose FF离子交换层析,整个纯化过程回收率为33.4%,经SDS-PAGE和HPLC检测纯度大于95%,符合下一步生物活性和药代动力学实验和药剂学试验的要求。更多还原

【Abstract】 Glucagon-like peptide-1(GLP-1) is 30-residual peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. It plays an important role in simulating insulin secretion, inducing pancreatic beta cells proliferation and inhibiting pancreatic beta cells apoptosis. The unique hypoglycemic mechanism of GLP-1 is unparalleled to the existing anti-diabetes drugs. Consequence to its natural function as locally secreted short-term messenger, its half-life in circulation is very short and provided mainly by fast renal clearance due to its low molecular weight and proteolysis susceptibility, resulting in low clinical utility. In our lab, A GLP-1 fusion protein named GGH comprising double tandem GLP-1 and human serum albumin (HSA) was constructed to prolong its half-life. The GLP-1 fusion protein was expressed in pichia pastoris KM71 transformed with vector pPIC9K/(GLP-1A2G)2-HSA. In this study, improving the expression level and purification of fusion protein GGH were focused on. The main conclusions are as follows:The fusion protein GGH expression level in different host strains (KM71, GS115 and SMD1168) was determined and analyzed by statistical methods. The relevance of gene dosage and fusion protein GGH expression level was investigated by RT-PCR in three GS115 recombinants with different expression level. The results showed that the host strain GS115 was more suitable for expressing fusion protein GGH. It was a positive correlation between gene dosage and fusion protein GGH expression level. A high yield recombinant strain GS115/F2 with expression level of 328 mg/L in shake flask culture was selected out for further study.After investigating the fermentation process of high yield recombinant strain, the process was confirmed. The results demonstrated that organic nitrogen sources were more conducive to high density culture of recombinant strain and inhibiting proteolysis. A pulse feeding strategy of 0.5% methanol each time was the optimal mode for inducing fusion protein expression. 30℃was the optimal temperature for inducing. The yield of fusion protein GGH in the fermentation process of recombinant strain reached to 642 mg/L。A preliminary purification process was established. The process with a total recovery of 33.4% included five steps: centrifugation, filtration, Blue sepharose, Sephadex G25 and Q sepharose chromatography. The fusion protein GGH was purified to apparent homogeneity with purity greater than 95% analyzed by SDS-PAGE and HPLC, meeting the requirement of the study of pharmacodynamics, pharmacokinetics and pharmacy.更多还原