本文選題：Pyrobaculum　切入點：neutrophilum　出處：《吉林大學》2017年碩士論文　論文類型：學位論文

【摘要】：絲氨酸蛋白酶抑制劑(serine protease inhibitors,serpins)廣泛存在于動物、植物及微生物體內(nèi),是絲氨酸蛋白酶的天然抑制劑。參與生物體內(nèi)許多重要的生命過程,如凝血、補體激活、炎癥反應(yīng)、細胞遷移、細胞基質(zhì)重建以及腫瘤抑制等。目前對于serpins缺陷相關(guān)疾病主要采用增補療法,而臨床上應(yīng)用的主要是人血漿純化獲得的蛋白制劑,其成本較高,不利于廣泛應(yīng)用。自然界中微生物的多樣性為人們開發(fā)新藥提供了不竭源泉,而嗜熱微生物及其來源的蛋白質(zhì)具有較高的穩(wěn)定性,可開發(fā)更有價值的生物活性物質(zhì)。本論文對嗜熱微生物Pyrobaculum neutrophilum來源的絲氨酸蛋白酶抑制劑Pnserpin的理化性質(zhì)及抗炎活性進行研究,以期為serpins缺陷和炎癥相關(guān)疾病的治療提供新的備選藥物分子。一、Pnserpin的表達、純化及性質(zhì)表征將Pnserpin全長基因,克隆到pET-28a表達載體中,并轉(zhuǎn)化大腸桿菌BL21誘導表達,利用鎳親和柱層析純化獲得高純度Pnserpin蛋白。以絲氨酸蛋白酶和半胱氨酸蛋白酶為底物,測定Pnserpin基本動力學常數(shù)及其抑制酶活力的最適反應(yīng)溫度、pH和熱穩(wěn)定性等理化性質(zhì)。研究結(jié)果表明Pnserpin是具有高度熱穩(wěn)定性的絲氨酸蛋白酶抑制劑。二、Pnserpin抗炎活性研究通過二甲苯致小鼠耳腫脹的急性炎癥模型和脂多糖(Lipopolysaccharide,LPS)刺激小鼠巨噬細胞RAW264.7的炎癥細胞模型,研究了不同濃度Pnserpin對小鼠的抗炎作用。實驗結(jié)果顯示,Pnserpin對二甲苯引起的小鼠耳腫脹具有明顯抑制作用;而對LPS誘導的RAW264.7細胞中IL-6、IL-1β、TNF-α和NO的表達也具有明顯抑制作用。這表明Pnserpin具有較好的抗炎活性,可進一步開發(fā)和應(yīng)用。
[Abstract]:Serine protease inhibitors (serine inhibitors) exist widely in animals, plants and microbes, and are natural inhibitors of serine protease. They are involved in many important biological processes, such as coagulation, complement activation, inflammation. Cell migration, cell matrix reconstruction and tumor suppression. At present, supplementation therapy is mainly used for serpins defection-related diseases, and the clinical application is mainly human plasma purified protein preparation, which has a high cost. The diversity of microorganisms in nature provides an inexhaustible source for the development of new drugs, while thermophilic microbes and proteins derived from them have high stability. In this paper, the physicochemical properties and anti-inflammatory activity of Pnserpin, a serine protease inhibitor derived from thermophilic microorganism Pyrobaculum neutrophilum, were studied. In order to provide a new alternative drug molecule for the treatment of serpins defects and inflammatory related diseases. The expression, purification and characterization of Pnserpin full-length gene were cloned into pET-28a expression vector and transformed into Escherichia coli BL21 induced expression. High purity Pnserpin protein was purified by nickel affinity chromatography. Serine protease and cysteine protease were used as substrates. The basic kinetic constants of Pnserpin and its optimum reaction temperature, pH and thermal stability were determined. The results show that Pnserpin is a serine protease inhibitor with high thermal stability. The acute inflammatory model of mouse ear swelling induced by xylene and the inflammatory cell model of murine macrophage RAW264.7 stimulated by lipopolysaccharide and lipopolysaccharide were studied. The anti-inflammatory effects of different concentrations of Pnserpin on mice were studied. The results showed that Pnserpin could inhibit ear swelling induced by xylene in mice. It also inhibited the expression of IL-6, IL-1 尾, TNF- 偽 and no in RAW264.7 cells induced by LPS, which indicated that Pnserpin had good anti-inflammatory activity and could be further developed and applied.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R915

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