本文選題：膜聯(lián)蛋白　切入點(diǎn)：囊尾蚴　出處：《第二軍醫(yī)大學(xué)》2005年博士論文

【摘要】：囊尾蚴引起的囊蟲(chóng)病對(duì)人畜的危害是很大的,且囊蟲(chóng)病的預(yù)防仍然是薄弱環(huán)節(jié)。囊尾蚴能夠在人體中存活幾年的時(shí)間,而在存活的囊尾蚴周?chē)挥泻茌p微的炎癥反應(yīng)。這個(gè)現(xiàn)象表明存活的囊尾蚴可能通過(guò)某種機(jī)制維持了宿主和寄生蟲(chóng)之間的平衡。 Annexin B1是孫樹(shù)漢等從豬囊尾蚴cDNA文庫(kù)中篩選得到的一個(gè)新基因,現(xiàn)有研究證實(shí)其屬于annexin家族。Annexin蛋白家族是一群結(jié)構(gòu)相似的Ca~(2+)依賴(lài)性磷脂結(jié)合蛋白,廣泛存在于動(dòng)植物的各個(gè)組織器官,并且含量豐富。但人們對(duì)annexin的在體功能仍然所知甚少。Annexin B1是扁形動(dòng)物門(mén)(platyhelminth)中首次被鑒定的一個(gè)annexin成員,研究它的生理功能,不僅可以了解annexin B1對(duì)于豬囊尾蚴的寄生、移行、生長(zhǎng)、發(fā)育等生命活動(dòng)中的重要意義,為治療和預(yù)防該類(lèi)疾病提供新的藥物靶點(diǎn),而且可以研究annexin在不同生物中的生理功能以及分子進(jìn)化。 本研究具體結(jié)果如下: 1.首先我們構(gòu)建了annexin B1的非融合表達(dá)載體,在大腸桿菌中實(shí)現(xiàn)了高表達(dá),利用膜聯(lián)蛋白的鈣依賴(lài)性磷脂結(jié)合活性,采用鈣離子誘導(dǎo)的沉淀反應(yīng)來(lái)純化重組蛋白,建立了一種新的膜聯(lián)蛋白純化方法。 2.以制備的蛋白為抗原,采用脾內(nèi)包埋法免疫小鼠,無(wú)菌取出脾細(xì)胞與骨髓瘤細(xì)胞SP2/0進(jìn)行融合,隨后以Elisa方法和有限稀釋法進(jìn)行3次篩選和克隆化,獲得一株雜交瘤細(xì)胞株2B10H5,能穩(wěn)定地分泌高滴度的針對(duì)annexin B1的特異單抗,為下一步功能研究打下了良好的基礎(chǔ)。 3.采用常規(guī)H-E染色和免疫組化結(jié)合的方法,發(fā)現(xiàn)annexin B1主要位于囊尾蚴的體表,另外在炎性囊壁有不同程度的分布,炎細(xì)胞層和肌纖維母細(xì)胞呈特異性的陽(yáng)性。提示annexin B1有可能作為一種分泌蛋白,被囊尾蚴階段特異性地分泌,在寄生蟲(chóng)與宿主的相互作用中起著抗炎和抗纖維化的作用。 4.以annexin B1為融合標(biāo)簽構(gòu)建表達(dá)載體,能夠用來(lái)純化重組蛋白。而且在標(biāo)簽和目的蛋白之間引入了蛋白內(nèi)含肽,在合適的條件下誘發(fā)自切割,釋放出完整的目的蛋白。該系統(tǒng)大大降低了蛋白純化的工作量,簡(jiǎn)便快速,易于操作,成本低廉,處理量大,有可能對(duì)生物工程制品下游的中試和生產(chǎn)提供一定的幫助。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：Q51

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 陳江濤;豬源膜聯(lián)蛋白A2的克隆、表達(dá)及其多克隆抗體的制備[D];中國(guó)農(nóng)業(yè)科學(xué)院;2010年

，

本文編號(hào)：1659301