本文選題：α干擾素　切入點：聚乙二醇修飾　出處：《重慶理工大學(xué)》2014年碩士論文

【摘要】：研究背景：干擾素是一類廣譜抗病毒、影響細(xì)胞生長、調(diào)節(jié)細(xì)胞免疫等多功能蛋白質(zhì)。干擾素在臨床應(yīng)用方面十分廣泛，尤其針對病毒性疾病有很好的療效。普通干擾素的缺陷在于體內(nèi)半衰期短，難以持久地抑制病毒。使得長效干擾素的研發(fā)有著重要的意義。人類干擾素的研究取得了長足的進步，聚乙二醇人干擾素已經(jīng)實現(xiàn)了商品化，如佩樂能和派羅欣。而聚乙二醇化豬干擾素研究進展較為緩慢，目前國內(nèi)尚沒有相關(guān)藥品上市和有效專利。 研究目的：建立一種簡便的制備聚乙二醇化重組豬干擾素方法，獲得生物活性保留較好的單聚乙二醇干擾素。 研究方法：在重組豬干擾素融合蛋白可溶性表達的基礎(chǔ)上，使用EK酶將融合蛋白酶切，鎳柱純化后，制成干擾素原液。根據(jù)重組豬α干擾素的氨基酸序列特點，分別采用了相對分子質(zhì)量為20kDa的直鏈型mPEG-ALD（單甲氧基聚乙二醇丙醛）和mPEG-SPA（單甲氧基聚乙二醇琥珀酰亞胺丙酸酯）修飾重組豬α干擾素。優(yōu)化重組豬干擾素的聚乙二醇化條件，使用mPEG-ALD大量修飾rpoIFNα，采用SP陽離子交換層析法純化單PEG修飾偶聯(lián)物，SDS-PAGE檢測修飾產(chǎn)物，運用細(xì)胞病變抑制法對其生物活性進行了初步研究。 研究成果：（1）制備重組豬α干擾素的純度在90%以上，達到初步實驗研究的要求。（2）通過優(yōu)化PEG修飾干擾素的條件，篩選出兩種PEG修飾劑的最優(yōu)修飾反應(yīng)條件：①PEG-ALD修飾的最優(yōu)條件：50mmol/L PB，pH=6.0、4℃、（PEG/protein）摩爾比為3：1，，反應(yīng)24h。②mPEG-SPA修飾的最優(yōu)條件：50mmol/L PB中，pH=9.0、4℃、（PEG/protein）摩爾比為5:1，反應(yīng)2h。（3）PEG-ALD對rpoIFNα的單修飾率達到60%以上，單聚乙二醇化干擾素的活性達到未修飾干擾素的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 偽 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 偽 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 偽 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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