胰高血糖素样肽-1的聚乙二醇修饰研究
文献类型：学位论文
| 作者 | 张磊 |
| 学位类别 | 硕士 |
| 答辩日期 | 2009-06-02 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 过程工程研究所 |
| 导师 | 马光辉 |
| 关键词 | 胰高血糖素样肽-1 聚乙二醇 定点化学修饰 |
| 其他题名 | Studies on PEGylation of Glucagon-like Peptide 1 and Interferon α-2b |
| 学位专业 | 生物技术与医学★ |
| 中文摘要 | 胰高血糖素样肽-1(Glucagon-like peptide-1，GLP-1)一种具有30个氨基酸的多肽，可以促进胰岛素分泌从而起到降低血糖的作用。但是，GLP-1存在体内循环半衰期短、具有免疫原性和抗原性、易被循环系统清除等问题，这大大限制了其在临床上的应用。本文采用聚乙二醇（PEG）化学修饰的方法，试图解决以上问题。 GLP-1的修饰位点主要His7、Lys26、Lys34等三个，其中Lys34位点的修饰产物活性最高。本研究在GLP-1的合成过程中，利用马来酸酐对Lys26先进行保护，然后采用分子量5 KD的单甲氧基聚乙二醇琥珀酰亚胺碳酸酯修饰剂对GLP-1进行修饰，并结合修饰反应过程控制对GLP-1进行Lys34的定点修饰，围绕如何获得高的单修饰产率和高活性的mono-PEG5K-GLP-1产物进行了研究。考察了各个反应条件对单修饰多肽的活性以及转化率的影响。在各个修饰反应条件中，pH值对单修饰产物活性影响不是很大，在pH 7.5时，单修饰产物活性保留相对较高；pH、多肽浓度、修饰剂与多肽摩尔配比以及反应时间对单修饰转化率影响较大。随着多肽浓度增加，单修饰转化率逐渐增加；而随着修饰剂与多肽摩尔配比的增加，单修饰转化率先增大后减小，在2:1时存在最大值；单修饰转化率随反应时间的延长而逐渐增大，1 h后逐渐趋于平缓。最终优化得到了最佳修饰反应条件：即4 ℃，在pH 7.5，50 mmol/l的磷酸缓冲溶液体系中，多肽浓度为1 mg/ml，修饰剂/多肽配比为2:1的时候反应1 h。此修饰工艺高效、简便、成本低，单修饰产率达到了50%以上。建立了一步法分离纯化mono-PEG-GLP-1的工艺。采用反相高效液相色谱梯度洗脱分离、一步纯化即达到对产物的高效精制，既减少了分离步骤，又保持了产物的高收率、降低了活性损失，而且得到了定点修饰的单一修饰产物，产物纯度可达96%以上，活性保留86%以上。此外，对获得的mono-PEG-GLP-1进行了质量鉴定，包括分子量、纯度、体外活性等，并且利用MALDI-TOF对修饰位点进行了检测，进一步验证了得到的mono-PEG-GLP-1的修饰位点为Lys34。研究了mono-PEG-GLP-1的药代动力学和稳定性，结果表明，与未修饰的GLP-1相比，本研究获得的mono-PEG5K-GLP-1在动物体内的循环半衰期（皮下注射）达到了60 min左右，是未修饰多肽的20倍左右。而文献报道的mono-PEG2K-GLP-1的半衰期只有33 min，是未修饰GLP-1的10倍左右。此外，mono-PEG5K-GLP-1还表现出了比原肽更好的长期稳定性。这说明本研究中采用分子量更大的聚乙二醇修饰剂，并对远离活性中心的Lys34采取的定点修饰策略，对延长胰高血糖素样肽-1在动物体内的半衰期起到了明显的效果。 |
| 英文摘要 | Glucagon-like peptide-1 (GLP-1) is a peptide with 30 amines. It can stimulate the secretion of insulin via glucose-dependent mechanism which was thought to be a potential advantage over conventional antidiabetic agents that secret insulin via glucose-independent mechanism. However, GLP-1 has a short half life(about 3min) in vivo and is easy to be cleared by circulatory system. These limitations pose great challenges in the development of GLP-1 as an antidiabetic pharmaceutical agent. The pegylation of GLP-1 is an efficient way to deal with the above limitations. In GLP-1，three amino acid sites(His7, Lys26, Lys34) can be used for pegylation，and the activity in vitro of pegylated conjugate in Lys34 is the highest. In this research Lys26 was protected with maleic anhydride during the synthesis of GLP-1. PEG-SC with molar weight of 5 KD was chosen to modify GLP-1. The effect of reaction conditions on the acitivty and production yield of mono-PEG5K-GLP-1 were investigated. It was found that pH did not influence the activity of mono-PEG5K-GLP-1 much, but at pH 7.5, the retaining activity of mono-PEG5K-GLP-1 was relatively high. Besides, pH, the concentration of GLP-1, molar ratio between PEG and GLP-1 and reaction time were the key factors that influence the production yield of mono-PEG5K-GLP-1. Increasing the concentration of GLP-1 increased the production yield of mono-PEG5K-GLP-1; with the increasing of the molar ratio between PEG and GLP-1, the production yield increased at first and decreased later, and got the highest yield at 2:1; the production yield also increased with the increasing of the reaction time, but stayed unchanged after 1 h. The optimized reaction conditions were as follows: the concentration of GLP-1 was 1 mg/ml, the molar ratio between PEG and GLP-1 was 2:1, and the reaction was processed in 50 mmol/l phosphate buffer at 4℃ for 1 h. This reaction process was effient, convenient and low-cost. The production yield of the mono-PEG5K-GLP-1 could reach 50% or above. The separation and purification process of pegylated GLP-1 were investigated. Reverse phase high performance liquid chromatogram was used to separate mono-PEG-GLP-1 from the reaction mixture. The method was simple and convenient, could maintain the bioactivity in vitro of the peptide at a high level(86%), and the purity of the mono-PEG5K-GLP-1 was up to 96%. Besides, the pegylation site was examined with MALDI-TOF, and the results showed that the pegylation site was Lys34. It was also found that mono-PEG5K-GLP-1 was much more stable than the native GLP-1 in the long time stability studies, and its half life was more than 60mins, about 20 times of the native GLP-1. Compared with the non-site-specific PEG2K-GLP-1 with half life of 33mins as reported in the preference, the site-specific PEG5K-GLP-1 strategy was proved to be more effective in prolonging the half life of the GLP-1. |
| 语种 | 中文 |
| 公开日期 | 2013-09-16 |
| 页码 | 66 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1298]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 张磊. 胰高血糖素样肽-1的聚乙二醇修饰研究[D]. 过程工程研究所. 中国科学院过程工程研究所. 2009. |
入库方式： OAI收割
来源：过程工程研究所
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