膜联蛋白B1的功能研究及其在蛋白分离和纯化中的应用

发布时间：2018-03-24 17:28

本文选题：膜联蛋白　切入点：囊尾蚴　出处：《第二军医大学》2005年博士论文

【摘要】：囊尾蚴引起的囊虫病对人畜的危害是很大的,且囊虫病的预防仍然是薄弱环节。囊尾蚴能够在人体中存活几年的时间,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是孙树汉等从猪囊尾蚴cDNA文库中筛选得到的一个新基因,现有研究证实其属于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,广泛存在于动植物的各个组织器官,并且含量丰富。但人们对annexin的在体功能仍然所知甚少。Annexin B1是扁形动物门(platyhelminth)中首次被鉴定的一个annexin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。本研究具体结果如下: 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,利用膜联蛋白的钙依赖性磷脂结合活性,采用钙离子诱导的沉淀反应来纯化重组蛋白,建立了一种新的膜联蛋白纯化方法。 2.以制备的蛋白为抗原,采用脾内包埋法免疫小鼠,无菌取出脾细胞与骨髓瘤细胞SP2/0进行融合,随后以Elisa方法和有限稀释法进行3次筛选和克隆化,获得一株杂交瘤细胞株2B10H5,能稳定地分泌高滴度的针对annexin B1的特异单抗,为下一步功能研究打下了良好的基础。 3.采用常规H-E染色和免疫组化结合的方法,发现annexin B1主要位于囊尾蚴的体表,另外在炎性囊壁有不同程度的分布,炎细胞层和肌纤维母细胞呈特异性的阳性。提示annexin B1有可能作为一种分泌蛋白,被囊尾蚴阶段特异性地分泌,在寄生虫与宿主的相互作用中起着抗炎和抗纤维化的作用。 4.以annexin B1为融合标签构建表达载体,能够用来纯化重组蛋白。而且在标签和目的蛋白之间引入了蛋白内含肽,在合适的条件下诱发自切割,释放出完整的目的蛋白。该系统大大降低了蛋白纯化的工作量,简便快速,易于操作,成本低廉,处理量大,有可能对生物工程制品下游的中试和生产提供一定的帮助。
[Abstract]:Cysticercosis caused by cysticercosis is very harmful to humans and animals, and the prevention of cysticercosis is still a weak link. But there is only a slight inflammatory response around the surviving cysticercaria, which suggests that the surviving cysticercus may have maintained a balance between the host and the parasite through some mechanism. Annexin B1 is a novel gene isolated from cDNA library of cysticercaria cellulosae. It has been confirmed that Annexin B1 belongs to the annexin family. Annexin protein family is a group of Ca~(2) -dependent phospholipid binding proteins with similar structure. It is widely found in various tissues and organs of animals and plants, and is rich in content. However, very little is known about the in vivo function of annexin. Annexin B1 is the first member of annexin identified in the phylum platyhelminthta to study its physiological function. Not only can we understand the importance of annexin B1 in the parasitism, migration, growth and development of cysticercus cellulosae, but also provide new drug targets for the treatment and prevention of these diseases. Moreover, the physiological function and molecular evolution of annexin in different organisms can be studied. The 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 recombinant protein was purified by Ca ~ (2 +) -induced precipitation reaction using the Ca ~ (2 +) -dependent phospholipid binding activity of annexin. A new method for purification of annexin was established. 2. Using the prepared protein as antigen, mice were immunized with intrasplenic entrapment method. Spleen cells were removed and fused with myeloma cells (SP2/0). Then three times of screening and cloning were carried out by Elisa method and finite dilution method. A hybridoma cell line 2B10H _ 5 was obtained, which could secrete a high titer specific monoclonal antibody against annexin B1, which laid a good foundation for further functional study. 3. Annexin B1 was found to be mainly located on the surface of cysticercus cysticercus, and distributed in different degree in inflammatory cyst wall by routine H-E staining and immunohistochemistry. The inflammatory cell layer and myofibroblast were specific positive, suggesting that annexin B1 may be a secretory protein secreted by cysticercaria, and play an anti-inflammatory and anti-fibrosis role in the interaction between parasite and host. 4. Using annexin B1 as fusion label, the expression vector can be used to purify recombinant protein. This system greatly reduces the workload of protein purification, is simple, fast, easy to operate, low cost and large amount of processing, which may be helpful to the pilot test and production of biological engineering products downstream.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：Q51

【引证文献】