【作者】 窦文芳；

【导师】 金坚；

【作者基本信息】 江南大学 ， 发酵工程， 2008， 博士

【摘要】 胰高血糖素样多肽-1（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。体内外研究显示GLP-1具有增强葡萄糖依赖性的胰岛素分泌、减少食物的摄取、减慢胃的排空、以及抑制胰高血糖素的分泌、刺激胰β细胞增殖、促进胰岛再生、以及抑制胰β细胞的凋亡等功能,且无胰岛素和磺脲类降糖药物的低血糖危险。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。但在体内其极易被二肽酰基肽酶Ⅳ降解,限制了GLP-1药物化的进展,本论文主要围绕GLP-1的长效性研究展开,主要结论如下:利用PCR的方法克隆获得GLP-1及其突变体GLP-1_A2G的基因,并构建了大肠杆菌表达菌株BL21 （pGEX-4T-1-GLP-1）和BL21 (pGEX-4T-1-GLP-1_A2G)。SDS-PAGE显示阳性克隆在分子量29.0 kDa处有明显的表达条带,与预测的分子量大小相近。凝血酶酶切后,进一步利用GST亲和层析与分子筛G75纯化获得了目标蛋白GLP-1和GLP-1_A2G。小鼠糖耐量实验证明,纯化获得的GLP-1和GLP-1_A2G在小鼠体内均有良好的控制血糖的生物活性,并且生物活性基本没有差异。利用生物信息学软件InsightII、ICM pro、Amber计算得出GLP-1的突变体GLP-1_A2G与其突变体的串联体(GLP-1_A2G)2的合理构象,并完成了其与GLP-1受体的分子对接,主要结合氨基酸残基为V30、L32、V36、Y69和P90,大部分是脂肪族和芳杂环类氨基酸,提示四个配体与nGLP-1R间的相互作用主要为疏水作用,并且计算得出GLP-1和突变体GLP-1_A2G的与受体结合能相差不大,而(GLP-1_A2G)2与受体结合能大于GLP-1和突变体GLP-1_A2G,说明(GLP-1_A2G)2较GLP-1_A2G更适合与白蛋白融合表达。利用重叠PCR技术,在体外成功拼接获得了(GLP-1_A2G)2和HSA的融合基因（GGH）,构建重组表达质粒pPIC9K-GGH,并将其通过电转化法转入毕赤酵母表达系统,经G418抗性筛选得到高表达菌株KM71（pPIC9K-GGH）,在甲醇诱导下其表达量为162 mg/L, western-blot结果表明诱导表达的融合蛋白为GLP-1突变体和HSA的杂合分子。对高表达菌株的发酵过程进行了优化,确定了毕赤酵母KM71（pPIC9K-GGH）的最佳生长条件:温度30℃、pH 6.0、装液量50-60 mL、甘油初始浓度4.0%、蛋白胨浓度2.0%。最佳表达条件:温度30℃、pH 6.0、装液量50-60 mL、诱导甲醇浓度2.0 %。在最优的条件下,毕赤酵母KM71（pPIC9K-GGH）菌体浓度最高达到21.0 g/L、最大比生长速率达到1.70 g/L·h、目标蛋白的最高产量达到245 mg/L、最大比产物生成速率达到3.38 mg/L,并且连续5个批次发酵水平稳定,批次之间的相对误差≤5%。确定了融合蛋白GGH的分离纯化的主要步骤,即10000 r/min离心10 min,分子量10 kDa的biomax超滤膜浓缩20倍,大孔树脂DA101脱色,Q FF离子交换,Sephacryl S-200凝胶层析,整个纯化过程的回收率为50.1%。经SDS-PAGE和HPLC检测纯度大于95%,等电聚集测定等电点为4.6,符合下一步生物活性和药代动力学实验的要求。融合蛋白GGH在体外对胰岛原代细胞生长有较好的刺激作用,在浓度45 nmoL/L时增殖率为35.4%,与单体的GLP-1相差不大。在糖耐量实验中,融合蛋白GGH可以较好的控制小鼠的血糖水平,并且在给药72 h后中、高剂量组仍然有控制血糖的生物活性,而单体的GLP-1在给药4 h后就检测不到生物活性。药代动力学研究表明,融合蛋白GGH在腹腔皮下单次给药8 h左右,血药浓度达到最高,分布容积为53.4 mg/L·h,体内吸收半衰期为26.6 h,体内消除半衰期约为57.8 h。大鼠皮下注射该药后,主要脏器均于6 h达峰,小鼠的胃、肾、肺和肌肉等组织器官中单位重量放射性积聚较高,其中以胃为最高;在肠、胰腺、肝、生殖器和脂肪中单位重量的放射性活度次之,而在心和脑中的分布最少。300 h尿中平均累积排泄率为80.1%,说明肾脏是消除同位素的主要途径,间接反映出该药（包括代谢产物）主要通过肾排泄。300 h粪便中平均累积排泄率为16.0%,说明该药可部分通过粪便排泄。更多还原

【Abstract】 Glucagon-like peptide-1 （GLP-1） is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. GLP-1 plays an important role in stimulating insulin secretion in a glucose induced manner, regulation of feeding, inhibiting glucagon secretion , increasingβcell proliferation, inhibitingβcell apoptosis and without low blood glucose dangerous with insulin and sulfonylureas hypoglycemic drugs. GLP-1 hypoglycemic unique mechanism is unparalleled to the existing anti-diabetes drugs. While, GLP-1 could easily degradated by DPPⅣin vitro, the progress to make GLP-1 as a drug was restricted. This study is mainly focused on long-term nature of GLP-1. The main conclusions are as follows:PCR technology was employed to amplify the GLP-1and GLP-1_A2G, and then the express strains BL21 （pGEX-4T-1-GLP-1） and BL21 (pGEX-4T-1-GLP-1_A2G) were constructed. The SDS-PAGE analysis showed the molecular weight of fusion protein GST- GLP-1 and GST- GLP-1_A2G was about 29.0KDa. The target protein of GLP-1 and GLP-1_A2G was obtained by GST affinity chromatography and Superdex G75 after digested with thrombin. The protein of GLP-1 and GLP-1_A2G show a good biological activity.Bioinformatics softwares were used to calculated the rational conformations of GLP-1_A2G mutant and its concatenate, we also completed the two docking with the GLP-1 receptor molecule. The main binding amino acids were V30, L32, V36, Y69 and P90, most of which are aliphatic and heterocyclic amino acids, suggesting that the interaction between the four ligands and nGLP-1R is hydrophobic interaction. According calculating, there is no significated of acceptor binding energy between GLP-1 and GLP-1_A2G, while the acceptor binding energy of (GLP-1_A2G)₂ is larger than GLP-1 and GLP-1_A2G, indicating that (GLP-1_A2G)₂ is more fit for expression with albumin.Overlap PCR technology was employed to amplify the fusion gene GGH, the construction of recombinant expression plasmid pPIC9K-GGH was electrotransformated to Pichia expression system. The high expression strain KM71（pPIC9K-GGH） was selected under G418 resistance, which production was 162 mg/L under the inducing with methanol. Using western-blot technology to identify the fusion protein is a GLP-1 and the HSA hybrid elements.The fermentation process of high expression strain has been optimized, and the best growth condition of KM71（pPIC9K-GGH） has been determined: temperature 30℃, pH 6.0, liquid volume 50-60 mL in 500 mL flask,the initial concentration of glycerol 4.0%, the concentration of peptone 2.0%. The optimized expression condition consisted temperature 30℃, pH 6.0, liquid volume 50-60 mL in 500 mL flask, the methanol induced concentration 2.0 %. On the optimal condition, the highest cell concentration was 21.0 g/L, the largest specific growth rate reached 1.70 g/L·h, the highest production of target protein was up to 245 mg/L, and the best specific production formation rate was 3.38 mg/L·h. The result has been proved to be stable after five batches of fermentation, and the error of the five batches is no more than 5%.The major steps of fusion protein separation and purification have been determined, first centrifuge at 10000 r/min 10 min, and then use biomax membrane （10 kDa mochular weight） concentration 20 times, to decolored with resin DA101 and use Q FF to ion exchange, gel chromatography was used by Sephacryl S-200. The recovery rate of purification in entire process is 50.1%. The purity detection through SDS-PAGE and HPLC was upper than 95%, this can be in line with pharmacokinetics request.The fusion protein GGH can stimulate the growth of islet cell in vitro. When the concentration of GGH is 45 nmoL, the growth rate is 35.4%. The result was similar as GLP-1. In the glucose tolerance test, GGH fusion protein in mice can better control the blood sugar levels. After 72 h administration, biological activity of high-dose group still can be detected. However, there was no biological activity after 4 h administration GLP-1.that the, indirectly reflects 300 hours in was 16.0 percent, that part of the drug through fecal excretion.Pharmacokinetics research showed that the maximum plasma concentration reached the highest after 8 h administration the fusion protein GGH by abdominal subcutaneous, the volume of distribution was 53.4 mg/L·h, the half-life of absorbtion in vivo was 26.6 h, the elimination half-life in vivo was about 57.8 h. Subcutaneous injection of the fusion protein GGH in mice, the main organs were all reached peak in 6 h, There were higher accumulations of radioactive by the unit weight in mice stomach, kidney, lung and muscle, especially it was highest in stomach, intestine, pancreas, liver, fat and genital were followed by. What’more, there had the least distribution in heart and brain.The average cumulative fecal excretion rate in urine after 300 h was 80.1%. It is suggested that kidney was the main way to eliminate isotope, and also reflected that the drug （including metabolites） mainly egested through kidney. The average cumulative fecal excretion rate in manure after 300 h was 16.0%. It is suggested that this drug can egest by manure.更多还原