异型双功能连接剂辅助PEG修饰核糖核酸酶A
文献类型：学位论文
| 作者 | 刘慎翔 |
| 学位类别 | 硕士 |
| 答辩日期 | 2012-05-30 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 胡涛 |
| 关键词 | PEG修饰 聚乙二醇 PEG单修饰 核糖核酸酶A 巯基 |
| 其他题名 | Mono-PEGylation of ribonuclease A by thiolation with small molecular weight reagent |
| 学位专业 | 化学工程 |
| 中文摘要 | 聚乙二醇（PEG）修饰技术可以提高蛋白质和多肽类药物在体内的循环半衰期、降低免疫原性和抗原性，从而改善药物的药代动力学和药效。然而，常用的PEG修饰方法多是针对蛋白质上?-氨基的随机修饰，修饰产物不均一，反应可控性差，存在大量的二修饰和多修饰产物，而且产物的活性损失较大。蛋白质的PEG单修饰（即蛋白质分子连接1个PEG分子）有利于蛋白质药物的工业生产、分离纯化以及产物鉴定。本论文提出异型双功能连接剂辅助PEG修饰蛋白质，通过两种异型双功能连接剂SATA和IT在蛋白质分子上引入巯基，随后马来酰亚胺-PEG与引入的巯基共价连接，完成蛋白质的PEG修饰。本论文选用具有抗肿瘤活性的牛胰核糖核酸酶A（RNase A）为模型蛋白。通过上述方法，RNase A的PEG单修饰产物产率达到了60%以上，而二修饰和多修饰产物基本为零。这主要因为引入的巯基减少了PEG修饰位点，增加了PEG与蛋白质共价连接的难度，从而减少PEG的二修饰和多修饰反应。本论文用圆二色光谱、内源荧光谱和外源荧光光谱、动态光散射以及分析超离心等方法对RNase A的PEG单修饰产物进行结构表征。与未修饰的RNase A相比，本方法制备的PEG单修饰产物的二级结构和三级结构未有明显变化，表面疏水性轻微增大、流体力学半径增大、修饰产物的不对称性增加、沉降系数降低。体外活性实验和细胞毒性实验结果表明，由于PEG的空间位阻作用，RNase A的酶活降低，细胞毒性增强。 |
| 英文摘要 | PEGylation can improve the both pharmacokinetic and the pharmacodynamic properties of proteins by increasing serum half-life and reducing immunogenicity and antigenicity. However, the common PEGylation that targets at the ?-amino groups of proteins can lead to imprecise control of the stoichiometry of the protein-PEG conjugate (i.e., mono-, di- and multi-PEGylated protein) and an apparent loss of the bioactivity of proteins. To prepare a PEGylated therapeutic protein, it is desirable that the protein is mono-PEGylated for industrial production, convenient purification and analytical characterization. Here, N-hydroxysuccinimide esters of S-acetylthioacetic acid (SATA) and 2-iminothiolane (IT) were used to introduce thiol groups on proteins, followed by maleimide chemistry based PEGylation of the thiolated proteins. In the present study, bovine pancreatic ribonuclease A (RNase A) was chosen as the model protein for its clear structure and functions. PEGylation can improve the therapeutic potential of RNase A, as a cancer chemotherapeutic agent. We found that the yield of mono-PEGylated RNase A was higher than 60%, and di- or multi-PEGylated RNase A were absent in the PEGylated product. Presumably, the limited number and low solvent accessibility of the introduced thiol group favored mono-PEGylation of RNase A. In the present study, structural characterization of the mono-PEGylated RNase A was carried out using circular dichroism spectra, intrinsic and extrinsic fluorescence spectra, dynamic light scattering and sedimentation velocity analytical ultracentrifugation. As compared to the unmodified RNase A, the mono-PEGylated RNase A showed unchanged structural properties, slightly increased hydrophobility, larger hydrodynamic radius, significantly decreased sedimentation coefficient and symmetry. Due to the steric hindrance of PEG to RNase A， in vitro enzymatic activity of RNase A was slightly decreased and anti-proliferative ability of RNase A was increased. Our study is expected to control the PEGylation process and optimize the industrial pharmaceutical production of PEGylated proteins. |
| 语种 | 中文 |
| 公开日期 | 2013-09-25 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1810]  |
| 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 | 刘慎翔. 异型双功能连接剂辅助PEG修饰核糖核酸酶A[D]. 中国科学院研究生院. 2012. |
入库方式： OAI收割
来源：过程工程研究所
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