重组猪干扰素的PEG修饰及其活性分析

发布时间：2018-04-01 13:20

  本文选题：α干扰素　切入点：聚乙二醇修饰　出处：《重庆理工大学》2014年硕士论文

【摘要】：研究背景：干扰素是一类广谱抗病毒、影响细胞生长、调节细胞免疫等多功能蛋白质。干扰素在临床应用方面十分广泛，尤其针对病毒性疾病有很好的疗效。普通干扰素的缺陷在于体内半衰期短，难以持久地抑制病毒。使得长效干扰素的研发有着重要的意义。人类干扰素的研究取得了长足的进步，聚乙二醇人干扰素已经实现了商品化，如佩乐能和派罗欣。而聚乙二醇化猪干扰素研究进展较为缓慢，目前国内尚没有相关药品上市和有效专利。 研究目的：建立一种简便的制备聚乙二醇化重组猪干扰素方法，获得生物活性保留较好的单聚乙二醇干扰素。 研究方法：在重组猪干扰素融合蛋白可溶性表达的基础上，使用EK酶将融合蛋白酶切，镍柱纯化后，制成干扰素原液。根据重组猪α干扰素的氨基酸序列特点，分别采用了相对分子质量为20kDa的直链型mPEG-ALD（单甲氧基聚乙二醇丙醛）和mPEG-SPA（单甲氧基聚乙二醇琥珀酰亚胺丙酸酯）修饰重组猪α干扰素。优化重组猪干扰素的聚乙二醇化条件，使用mPEG-ALD大量修饰rpoIFNα，采用SP阳离子交换层析法纯化单PEG修饰偶联物，SDS-PAGE检测修饰产物，运用细胞病变抑制法对其生物活性进行了初步研究。 研究成果：（1）制备重组猪α干扰素的纯度在90%以上，达到初步实验研究的要求。（2）通过优化PEG修饰干扰素的条件，筛选出两种PEG修饰剂的最优修饰反应条件：①PEG-ALD修饰的最优条件：50mmol/L PB，pH=6.0、4℃、（PEG/protein）摩尔比为3：1，，反应24h。②mPEG-SPA修饰的最优条件：50mmol/L PB中，pH=9.0、4℃、（PEG/protein）摩尔比为5:1，反应2h。（3）PEG-ALD对rpoIFNα的单修饰率达到60%以上，单聚乙二醇化干扰素的活性达到未修饰干扰素的2.9%。
[Abstract]:Background: interferon is a class of broad-spectrum antiviral proteins that affect cell growth and regulate cellular immunity. Interferon is widely used in clinical applications. In particular, it has a good effect on viral diseases. The deficiency of common interferon is the short half-life of the body. It is difficult to suppress the virus permanently, which makes the research and development of long-acting interferon of great significance. Great progress has been made in the research of human interferon, and PEG-human interferon has been commercialized. For example, Perlenone and Perohin. The research progress of pegylated porcine interferon is slow, and there are no related drugs on the market and valid patents in China. Objective: to establish a simple method for preparation of recombinant porcine interferon with polyethylene glycol (PEG). Methods: on the basis of soluble expression of recombinant porcine interferon fusion protein, fusion protease was digested with EK enzyme and purified by nickel column. The recombinant porcine interferon 伪 was modified with straight chain mPEG-ALD (monomethoxypolyethylene glycol propionaldehyde) and mPEG-SPA (monomethoxypolyethylene glycol succinimide propionate) with relative molecular weight of 20kDa. The modified rpoIFN 伪 was modified by mPEG-ALD and purified by SP cation exchange chromatography. The modified product was detected by SDS-PAGE. The biological activity of the modified product was studied by cytopathic inhibition method. The purity of recombinant porcine interferon 伪 was more than 90%, which met the requirements of preliminary experimental study. 2) by optimizing the conditions of PEG modification of interferon, The optimum reaction conditions of two PEG modifiers: 1 PEG-ALD were selected. The optimum reaction conditions were: 1: 50 mmol / L, pH = 6.0 鈩

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