单甲氧基聚乙二醇修饰牛胰核糖核酸酶
文献类型：学位论文
| 作者 | 张颖 |
| 学位类别 | 硕士 |
| 答辩日期 | 2001-06 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 苏志国 |
| 关键词 | 单甲氧基聚乙二醇 活化 化学修饰 牛胰核糖核酸酶 |
| 其他题名 | Research of RNase A Chemical Modification by Monomethoxypoly (Ethylene Glycol) |
| 学位专业 | 生物化工 |
| 中文摘要 | 该文选取牛胰核糖核酸酶（RNaseA）为模型蛋白，对聚乙二醇修饰蛋白的技术以及修饰对蛋白性质的影响做了较为系统的研究.实验采用单甲氧基聚乙二醇（MPEG）为修饰剂.该文优化了MPEG的N-羟在琥珀酰亚胺活化法以及MPEG的1，1’-二咪唑代碳酸活化法.分别得到对蛋白质活性影响较小，修饰活性较高且较为稳定的活化产物MPEG-SS及MPEG-CDI.为了确定蛋白质的平均修饰度，该文采用TNBS法和毛细管电泳法两种方法对经MPEG-SS修饰的RNaseA进行了测定，得到了可以相互印证的结果，对RNaseA经过修饰后的酶学性质进行了探索.该文首次提出经过聚乙二醇修饰的RNaseA可能对癌细胞及受到病毒侵染的细胞具有细胞毒性，有可能发展成为一种抗癌及抗病毒的药物.MTT实验表明，在体外，5.8×10<'-6>mol/L的经过修饰的RNaseA对K562细胞系就表现出显著的细胞毒性. |
| 英文摘要 | Pharmaceutical research and application of proteins have attracted considerable research interest in recent years. Polypeptide, enzymes and cellular factors are used successfully in clinical therapy. However, there are still many problems remaining to be solved on the use of protein drugs, such as large dosage, immune response and short serum lifetime. Chemical modification with polyethylene glycol (PEG) may be an alternative way to circumvent these problems. It has been demonstrated that modifications of proteins with PEG could dimmish immune response. In addition, PEG attachment makes proteins much larger and thus reduces their rate of clearance through the kidney, resulting in enhanced serum lifetimes. Bovine pancreatic ribonuclease (RNase A) is an enzyme that catalyzes RNA depolymerization. Due to its virus resistance and tumor killing properties, RNase A has caused wide attention in recent years for its potential as a cancer killing reagent. In this dissertation, RNase A was modified with monomethoxypolyethylene glycol (MPEG) to increase its resistance to its inhibitor RI. The process of MPEG modification and its influence on the enzymatic characteristic were investigated. In order to solve the problem of the low chemical reaction activity of the hydroxyl group, MPEG was activated with two kinds of reagents, N-hydroxysuccinimide (NHS), and 1,1'-carbonyldiimidazole. The activation degree of MPEG was determined by different methods. When the activating reagents were NHS and 1,1'-carbonyldiimidazole, the activation degree was more than 80%, and the ester bonds existed in MPEG derivatives were verified by infrared spectrum. The MPEG derivatives activated by NHS and 1,l'-carbonyldiimidazole were used to modify RNase A. The effect of molar ratio of MPEG derivative to RNase A and reaction pH was investigated respectively by TNBS and capillary zone electrophoresis (CZE). The modification degree could be adjusted by changing the molar ratio of MPEG derivative to RNase A. When pH equals to 8.4, the fastest speed occurs. The enzymatic characteristic of the modified RNase A was investigated. The activity of RNase A with different modification degree was studied. With the molecular ratio between MPEG and RNase A increased, the activity of RNase A towards ribonucleic acid decreased, but the remaining activity was rather high. When the molecular ratio increased to 10:1, the remaining activity of RNase A towards ribonucleic acid was still as high as 64%. The thermostability was evaluated by enzymatic activity of the modified enzyme in comparison to the native one. According to the experimental results, modified RNase A adapts wider temperature range than the native one. The influences of the reaction buffer's pH and some cations on native and modified RNase A were studied. The modification changes the pH profile just slightly. And modified RNase A is more insensitive to cations than native RNase A. The influence of modification on the stability of RNase A was also assessed in the presence of proteolysis enzymes, including trypsin and pepsin. The results showed that, after modification, the stability of RNase A to proteolytic is increased. The enzyme kinetics of native and modified RNase A was studied, too. This study proposed, for the first time, that MPEG modified RNase A could have cytotoxicity towards cancer cells or virus-infected cells. Preliminary MTT assays demonstrated that the modified RNase A, with its concentration as low as 5.8 * 10~(-6) mol/L, was effectively cytotoxic to K562 human erythroleukemia cells in vitro. |
| 语种 | 中文 |
| 公开日期 | 2013-09-26 |
| 页码 | 82 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1901]  |
| 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 | 张颖. 单甲氧基聚乙二醇修饰牛胰核糖核酸酶[D]. 中国科学院研究生院. 2001. |
入库方式： OAI收割
来源：过程工程研究所
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