本文關鍵詞： 人白介素15 構(gòu)建 表達 純化 純度 活性　出處：《吉林大學》2006年碩士論文　論文類型：學位論文

【摘要】：重組人白介素-15具有廣泛的應用前景,本論文研究了它的制備工藝。目前人們認為hIL-15至少可以有以下三個用途:作為疫苗的佐劑;治療血小板減少;治療實體瘤。研究制備工藝意味著要在提高產(chǎn)率的同時降低成本。這里主要從原核表達和蛋白純化兩個方面進行了工藝的改進。 本論文的研究主要包括工程菌的構(gòu)建,蛋白的表達和純化。從外周血單核細胞提取總RNA,RT-PCR之后,獲得了人白介素-15的基因序列。為了篩選獲得一種高效表達的工程菌株,構(gòu)建了四種類型的表達質(zhì)粒,從而比較他們的表達效率。通過優(yōu)化表達條件,提高了蛋白的表達量。然后通過三步純化,經(jīng)過包涵體的處理,離子交換層析,凝膠過濾層析,使蛋白的純度達到要求,初步建立起了重組人白介素-15的純化工藝。也考慮了不同復性條件和保存方法對蛋白活性的影響。 通過本論文的實驗,找到了一種可行的制備工藝。使蛋白的純度可以達到95.3%,比活能達到1.1×107IU/mg。實驗結(jié)果達到了預期的目標,為以后工藝放大打下了堅實的基礎。
[Abstract]:Recombinant human interleukin-15 has a wide application prospect. In this paper, the preparation process of recombinant human interleukin-15 has been studied. At present, it is believed that hIL-15 can be used as adjuvant for vaccine, as adjuvant for treatment of thrombocytopenia; Treatment of solid tumor. The study of preparation technology means to increase the yield while reducing the cost. This paper mainly from the prokaryotic expression and protein purification two aspects of the improvement of the process. In this paper, the construction of engineering bacteria, the expression and purification of protein were studied. The gene sequence of human interleukin-15 was obtained after total RNA-RT-PCR was extracted from peripheral blood monocytes, and a highly expressed engineering strain was obtained. Four types of expression plasmids were constructed to compare their expression efficiency. By optimizing the expression conditions, the expression level of protein was improved. After three steps of purification, inclusion body treatment, ion exchange chromatography, gel filtration chromatography, The purification process of recombinant human interleukin-15 was preliminarily established, and the effects of different renaturation conditions and preservation methods on the protein activity were also considered. Through the experiments in this paper, a feasible preparation process was found. The purity of the protein can reach 95.3 and the specific activity can reach 1.1 脳 107 IU / mg. The experimental results have reached the expected goal and laid a solid foundation for the later process amplification.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392;Q78

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