重组葡激酶二聚体的聚乙二醇氮末端定点修饰的研究
文献类型：学位论文
| 作者 | 刘如艳 |
| 学位类别 | 硕士 |
| 答辩日期 | 2011-05-26 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 胡涛 |
| 关键词 | PEG化 二聚体化 葡激酶（SAK） 体外活性 亲和力 |
| 其他题名 | Research on N-terminal monoPEGylation of recombinant Staphylokinase dimers |
| 学位专业 | 生物化工 |
| 中文摘要 | 聚乙二醇化（PEG）可以通过提高蛋白质药物在体内的循环半衰期以及降低免疫原性和抗原性来提高蛋白质药物的药效。然而，PEG修饰会造成蛋白质药物出现明显的生物活性的损失，部分原因可能是由于PEG链对蛋白质造成的空间位阻效应。蛋白质的二聚体化可以通过二价反应增强其与胞内受体的亲和力，并且通过使分子量成倍增加而延长其体内的循环半衰期。因此，二聚体化可以有效地提高PEG修饰蛋白质的生物活性，延长其循环半衰期。 本论文以葡激酶（SAK）为模型蛋白，它具有很好的溶栓效应，但其体内循环半衰期很短。本论文的主要成果在于：首先，提出了一个很新颖的制备N-末端PEG定点修饰葡激酶二聚体的方法；其次，通过与PEG-SAK相比较，发现PEG-dSAK具有更好的结构特征以及生物活性和亲和力。具体如下： 首先，通过基因工程方法在SAK的C-末端引入半胱氨酸以获得自由巯基，然后用交联剂1,4-二马来酰亚胺丁烷(1,4-bismaleimidobutane)与半胱氨酸反应制备C-末端交联的SAK二聚体（dSAK）。使PEG-醛定点修饰到dSAK中一个SAK的N-末端，从而保证其与另外一个SAK具有较远的空间距离。 其次，用圆二色光谱、内源荧光谱和外源荧光光谱对PEG-dSAK的结构进行表征。结果发现与SAK相比，PEG-dSAK的二级结构没有发生变化，而其三级结构只受到了轻微的影响。与PEG-SAK相比，PEG-dSAK受PEG空间位阻效应影响较小。与SAK相比，dSAK与受体纤溶酶原的结合力和体外活性基本上没有发生改变，而PEG-SAK与受体纤溶酶原的结合力和体外活性分别下降了46.5倍和2.6倍。然而，PEG-dSAK的活性是PEG-SAK的1.5倍，并且亲和力是PEG-SAK的4.5倍。因此，PEG-dSAK在改良SAK药学性质方面具有更大的优势。本论文的修饰策略可对其它蛋白质和多肽类药物的药学性质改善提供借鉴。 |
| 英文摘要 | PEGylation can improve the therapeutic efficacy of proteins by increasing serum half-life and reducing immunogenicity and antigenicity. However, such PEGylated molecules at times show a substantial loss of the functional biological activity, partially due to steric hindrance of the polyethylene glycol (PEG) chain. Dimerization of the proteins can increase the binding affinity to their cellular receptors through divalent interactions and improve the serum half-life by doubling the molecular weight. Therefore, dimerization is a potential strategy to improve the biological activity and the serum half-life of the PEGylated protein. In the present study, staphylokinase (SAK), a protein with therapeutic potential for coronary thrombolysis, was chosen as the model protein for its relatively short plasma half-life. The principal findings of the present study are (i) successful N-terminal monoPEGylation of C-terminally linked dSAK and (ii) improved structural property, biological activity and binding affinity of the PEG-dSAK as compared to the PEG-SAK. The details were as following: (i) The C-terminally linked SAK dimers (dSAK) was prepared by engineering free cysteine residue at the C-terminus of SAK and subsequently intermolecular crosslink of the cysteine residue with 1,4-bismaleimidobutane. PEG aldehyde chemistry was used for specifically N-terminal monoPEGylation of adjacent SAK in dSAK, where the distal SAK is far from the PEG. (ii) Structural characterization of the PEG-dSAK was carried out using circular dichroism spectra, intrinsic and extrinsic fluorescence spectra. The present study suggested that the secondary structure of the PEG-dSAK was not altered as compared to that of SAK; the tertiary structure of the PEG-dSAK was only slightly perturbed as compared to that of SAK; the PEG-dSAK showed lower steric hindrance effect of PEG as compared to the PEG-SAK. Compared to SAK, the in vitro biological activity of dSAK and its binding affinity to the receptor, plasminogen, were fully retained, whereas those of the PEG-SAK were 2.6-fold and 46.5-fold lower, respectively. However, the PEG-dSAK showed a 1.5-fold biological activity and 4.5-fold binding affinity higher than that of the PEG-SAK. Therefore, the PEG-dSAK would be a better strategy to improve the therapeutic efficacy of SAK. The present strategy may rationally optimize and tailor the pharmacological properties of peptides and proteins. |
| 公开日期 | 2013-09-24 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1763]  |
| 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 | 刘如艳. 重组葡激酶二聚体的聚乙二醇氮末端定点修饰的研究[D]. 北京. 中国科学院研究生院. 2011. |
入库方式： OAI收割
来源：过程工程研究所
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