嗜热丝氨酸蛋白酶抑制剂Pnserpin的克隆表达及抗炎活性研究

发布时间：2018-03-05 17:40

本文选题：Pyrobaculum　切入点：neutrophilum　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：丝氨酸蛋白酶抑制剂(serine protease inhibitors,serpins)广泛存在于动物、植物及微生物体内,是丝氨酸蛋白酶的天然抑制剂。参与生物体内许多重要的生命过程,如凝血、补体激活、炎症反应、细胞迁移、细胞基质重建以及肿瘤抑制等。目前对于serpins缺陷相关疾病主要采用增补疗法,而临床上应用的主要是人血浆纯化获得的蛋白制剂,其成本较高,不利于广泛应用。自然界中微生物的多样性为人们开发新药提供了不竭源泉,而嗜热微生物及其来源的蛋白质具有较高的稳定性,可开发更有价值的生物活性物质。本论文对嗜热微生物Pyrobaculum neutrophilum来源的丝氨酸蛋白酶抑制剂Pnserpin的理化性质及抗炎活性进行研究,以期为serpins缺陷和炎症相关疾病的治疗提供新的备选药物分子。一、Pnserpin的表达、纯化及性质表征将Pnserpin全长基因,克隆到pET-28a表达载体中,并转化大肠杆菌BL21诱导表达,利用镍亲和柱层析纯化获得高纯度Pnserpin蛋白。以丝氨酸蛋白酶和半胱氨酸蛋白酶为底物,测定Pnserpin基本动力学常数及其抑制酶活力的最适反应温度、pH和热稳定性等理化性质。研究结果表明Pnserpin是具有高度热稳定性的丝氨酸蛋白酶抑制剂。二、Pnserpin抗炎活性研究通过二甲苯致小鼠耳肿胀的急性炎症模型和脂多糖(Lipopolysaccharide,LPS)刺激小鼠巨噬细胞RAW264.7的炎症细胞模型,研究了不同浓度Pnserpin对小鼠的抗炎作用。实验结果显示,Pnserpin对二甲苯引起的小鼠耳肿胀具有明显抑制作用;而对LPS诱导的RAW264.7细胞中IL-6、IL-1β、TNF-α和NO的表达也具有明显抑制作用。这表明Pnserpin具有较好的抗炎活性,可进一步开发和应用。
[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- 伪 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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

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