膜联蛋白B1的表达纯化与抗炎功能及细胞内定位研究

发布时间：2018-08-08 14:58

【摘要】：囊尾蚴引起的囊虫病是一种能造成很大危害的人畜共患疾病。囊尾蚴能够在宿主体中存活数年,而在存活的囊尾蚴周围只有很轻微的炎症反应。这个现象表明存活的囊尾蚴可能通过某种机制维持了宿主和寄生虫之间的平衡。 Annexin B1是孙树汉等用免疫筛选法从猪囊尾蚴cDNA文库中分离得到的一个新基因,已有研究证实其属于annexin家族。Annexin蛋白家族是一群结构相似的Ca~(2+)依赖性磷脂结合蛋白,存在于大多数真核细胞内,且含量丰富,具有广泛而重要的生理功能。Annexin B1是扁形动物门(platyhelminth)中首次被鉴定的一个annexin家族成员,研究它的生理功能,不仅可以了解annexin B1对于猪囊尾蚴的寄生、移行、生长、发育等生命活动中的重要意义,为治疗和预防该类疾病提供新的药物靶点,而且可以研究annexin在不同生物中的生理功能以及分子进化。所以接下来我构建了annexin B1的原核非融合表达载体,表达并纯化了该蛋白。由于在感染猪囊尾蚴的病猪肌肉的免疫组化切片发现:炎症反应强的组织中膜联蛋白B1含量明显比其他组织高。该结果提示膜联蛋白B1有抗炎活性并有可能以某种机制分泌到细胞外,因而我做了annexin B1抗炎活性和真核细胞内定位研究。本研究主要结果如下: 1.首先我们构建了annexin B1的非融合表达载体,在大肠杆菌中实现了高表达,利用annexin的钙依赖性磷脂结合活性优化了纯化方案,建立了简便有效并适宜于所有annexin家族成员的纯化方法。纯化得到了纯度大于95%的蛋白,为后续的功能研究打下了良好的基础。 2.为研究annexinB1的抗炎活性,我们检验它是否以界面竞争机制抑制cPLA2活性。我们运用自由脂肪酸探针ADIFAB来检测cPLA2活性,并以自行制备的脂质体为磷脂底物,来检测annexin B1对cPLA2活性的影响。结果显示annexinB1可抑制cPLA2活性,这种抑制作用依赖于适当的底物类型及钙离子浓度。cPLA2水解磷脂酰胆碱脂质体不能被annexinB1抑制,而水解磷脂酰丝氨酸脂质体可被annexin B1抑制,并且这种抑制作用是钙离子依赖的,钙离子在10~(-3)以下时这种抑制作用不明显,符合界面竞争机制。于是我们推论annexin B1在体外可以界面竞争机制抑制cPLA2活性,具有抗炎功能。
[Abstract]:Cysticercosis caused by cysticercosis is a zoonotic disease that can cause great harm. Cysticercaria can survive in the host for several years, but there is only a slight inflammatory response around the surviving cysticercus. This phenomenon suggests that survival cysticercaria may maintain the balance between host and parasite through some mechanism. Annexin B1 is isolated from pig sac tail by immune screening method by Sun Shuhan et al. A new gene isolated from cDNA library, It has been confirmed that it belongs to the annexin family. Annexin protein family is a group of similar structural Ca2 dependent phospholipid binding proteins, which exists in most eukaryotic cells and is rich in content. Annexin B1 is the first member of annexin family identified in (platyhelminth) of phylum oblate. Studying its physiological function can not only understand the parasitism, migration and growth of annexin B1 to cysticercus cellulosae. The significance of life activities such as development provides new drug targets for the treatment and prevention of these diseases and can be used to study the physiological functions and molecular evolution of annexin in different organisms. So then I constructed the prokaryotic expression vector of annexin B1, expressed and purified the protein. Because of the immunohistochemical section of the infected pig muscle, it was found that the content of integrin B1 in the tissue with strong inflammatory reaction was significantly higher than that in the other tissues. These results suggest that annexin B1 has anti-inflammatory activity and may be secreted out of cells by some mechanism, so I have done anti-inflammatory activity and eukaryotic localization of annexin B1. The main results of this study are as follows: 1. Firstly, we constructed a non-fusion expression vector of annexin B1, which was highly expressed in Escherichia coli. The purification scheme was optimized by using the calcium dependent phospholipid binding activity of annexin. A simple and effective purification method was established for all members of annexin family. The purity of the protein was more than 95%, which laid a good foundation for further functional study. 2. In order to study the anti-inflammatory activity of annexinB1, we examine whether it inhibits cPLA2 activity by interfacial competition mechanism. Free fatty acid probe (ADIFAB) was used to detect the activity of cPLA2 and liposome was used as phospholipid substrate to detect the effect of annexin B1 on the activity of cPLA2. The results showed that annexinB1 could inhibit the activity of cPLA2, which depended on the appropriate substrate type and calcium concentration. The hydrolysis of phosphatidylcholine liposomes could not be inhibited by annexinB1, but the hydrolysis of phosphatidylserine liposomes could be inhibited by annexin B1. The inhibitory effect is dependent on calcium ion. When calcium ion is below 10 ~ (-3), the inhibitory effect is not obvious, which accords with the mechanism of interfacial competition. Therefore, we infer that annexin B1 can inhibit the activity of cPLA2 by the mechanism of interfacial competition in vitro and has anti-inflammatory function.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363;Q78

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