| 题名 | 干扰素α-2b的聚乙二醇修饰研究 |
| 作者 | 吴影新 |
| 学位类别 | 硕士 |
| 答辩日期 | 2008-06-02 |
| 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 | 干扰素α-2b 聚乙二醇 化学修饰 复染 |
| 其他题名 | Study on Modification of Interferon α-2b with Polyethylene Glycol |
| 学位专业 | 生物化工 |
| 中文摘要 | 干扰素α-2b(Interferon α-2b)是干扰素的一种，是机体感染病毒时，白细胞通过抗病毒应答反应而产生的一种低分子量的糖蛋白，具有抗病毒、抗肿瘤、免疫调节等生物学活性。干扰素α-2b目前被广泛用于肝炎和肿瘤的治疗，但在临床应用中，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、需要多次给药等问题，给患者的日常生活带来很大的不便。聚乙二醇(PEG)化学修饰能够有效延长药物半衰期，是解决上述问题的一种重要的有效途径。 针对目前已上市聚乙二醇干扰素α-2b水溶液稳定性不好、循环半衰期仍然不够长的缺点，并且为了制备具有高创新性的修饰产物，进一步提高疗效，提升我国基因药物的国际竞争力，本研究采用分子量更大，反应温和的聚乙二醇丙醛(20kD)修饰剂，并通过控制修饰反应条件，以期望获得半衰期更长、更稳定的、具有自主知识产权的PEG化干扰素α-2b产物。 本论文以高单修饰产物活性保留和高修饰转化率为目标，对修饰反应各影响因素，如离子强度、pH、温度、反应时间、修饰剂/蛋白质配比等分别进行了考察，优化出最佳修饰反应条件，建立了高效、低成本干扰素α-2b的聚乙二醇修饰工艺。 建立了单修饰聚乙二醇-干扰素α-2b的分离纯化工艺。采用常规分离介质阳离子交换色谱梯度洗脱、一步纯化即达到对产物的高效精制，既减少了分离步骤、经济简便，又提高了蛋白收率、降低活性损失，产品纯度可达97.0％以上。 对自制的聚乙二醇-干扰素α-2b的稳定性和药代动力学进行了研究。实验结果表明，该制备得到的聚乙二醇-干扰素α-2b抗酶解能力以及动物体内的药代动力学性质与未修饰干扰素α-2b相比都得到了较大提高，体内循环半衰期延长了20倍左右，比已上市的聚乙二醇干扰素α-2b(PEG-intron)提高2倍。 此外，针对聚乙二醇修饰蛋白体系，提出了两种SDS-PAGE复染方法并对其进行了比较。通过复染，实现了在同一凝胶上先后或同时观察到聚乙二醇修饰蛋白体系的所有组分，简化了实验操作，方便了实验结果的分析对比；并且考察了聚乙二醇分子量对复染灵敏度的影响。 |
| 英文摘要 | Interferon α-2b is one of the widely applied protein drugs used as anti-virus and anti-tumor agent. Similar with the other protein drugs, however, the use of interferonα-2b has been limited by its short half-lives, easy clearance by enzyme, antigen-antibody reaction and so forth. So far, the protein PEGylation technology has become one of the important methods for overcoming these problems. The PEG-IFNα-2b developed by a foreign company has come into the market, but it’s not stable in aqueous and its half life is not long enough. Therefore, PEG-ALD with molar weight of 20kD was chosen to modify IFN α-2b to obtain a new type of PEG-IFNα-2b in this study. Compared with the commercial PEG-IFNα-2b, the conjugate of PEG-ALD and IFN α-2b is more stable, and the half-life should be larger because of the larger molar weight. The process optimization of bioconjugate chemistry and the separation and purification of PEGylated IFNα-2b were studied. The reaction conditions were optimized for obtaining more mono PEG-IFNα-2b with higher bioactivity, and the effect of each reaction condition on bioactivity of mono PEG-IFNα-2b and the pegylation degree was studied. The separation and purification process of PEGylated IFNα-2b was investigated. Ion-exchange chromatography was used to separate mono PEGylated IFN α-2b from the reaction mixture by using SP Sepharose Fast Flow. The method was simple and convenient, and could increase the protein recovery and the bioactivity. By stability studies, it was found that PEG-IFNα-2b was much more stable than the native IFNα-2b, and its cleasrance was much slower after injection to rats. It prolonged the serum half-lives 20 times compared with native IFN α-2b, while the commercial PEG-IFN α-2b is only 10 times longer. Two new restaining methods of SDS-PAGE were developed and compared. All of the components of the mixture after PEGylation could be seen on one gel in succession or at the same time. It could reduce half of the load and the cost of the gels, and make the observation of the results more convenient. |
| 语种 | 中文 |
| 公开日期 | 2013-09-13 |
| 页码 | 87 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1229]  |
| 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 | 吴影新. 干扰素α-2b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. 2008. |
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