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

【摘要】：重組人白介素-15具有廣泛的應(yīng)用前景,本論文研究了它的制備工藝。目前人們認(rèn)為hIL-15至少可以有以下三個(gè)用途:作為疫苗的佐劑;治療血小板減少;治療實(shí)體瘤。研究制備工藝意味著要在提高產(chǎn)率的同時(shí)降低成本。這里主要從原核表達(dá)和蛋白純化兩個(gè)方面進(jìn)行了工藝的改進(jìn)。 本論文的研究主要包括工程菌的構(gòu)建,蛋白的表達(dá)和純化。從外周血單核細(xì)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(dá)的工程菌株,構(gòu)建了四種類型的表達(dá)質(zhì)粒,從而比較他們的表達(dá)效率。通過(guò)優(yōu)化表達(dá)條件,提高了蛋白的表達(dá)量。然后通過(guò)三步純化,經(jīng)過(guò)包涵體的處理,離子交換層析,凝膠過(guò)濾層析,使蛋白的純度達(dá)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(fù)性條件和保存方法對(duì)蛋白活性的影響。 通過(guò)本論文的實(shí)驗(yàn),找到了一種可行的制備工藝。使蛋白的純度可以達(dá)到95.3%,比活能達(dá)到1.1×107IU/mg。實(shí)驗(yàn)結(jié)果達(dá)到了預(yù)期的目標(biāo),為以后工藝放大打下了堅(jiān)實(shí)的基礎(chǔ)。
[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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392;Q78

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本文編號(hào)：1551201