發(fā)布時間：2018-01-15 22:19

  本文關(guān)鍵詞：細菌納米磁小體與神經(jīng)節(jié)苷脂及凝集素偶聯(lián)復合物的制備與應(yīng)用　出處：《江南大學》2015年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 磁小體 神經(jīng)節(jié)苷脂 凝集素 糖生物學

【摘要】：磁小體(Magnetosome)是一種由趨磁細菌(Magnetotactic bacteria)生產(chǎn)的磁性納米顆粒,具有生物相容性好、大小均一及超順磁性等特點,已在免疫檢測、載藥體系、固定化酶等多領(lǐng)域得到廣泛應(yīng)用,但磁小體在糖生物學研究中的應(yīng)用還較少。神經(jīng)節(jié)苷脂(Ganglioside)及凝集素(Lectin)作為兩種重要的功能性分子,在糖生物學研究中有著重要的作用。神經(jīng)節(jié)苷脂是一種攜帶一至多個唾液酸的糖鞘脂(Glycosphingolipid)。其可作為細胞表面的識別分子,在細胞識別及細胞與細胞的相互識別中扮演重要角色。人們常利用外源添加神經(jīng)節(jié)苷脂的方法來研究其功能與作用,然而神經(jīng)節(jié)苷脂在水溶液中易形成脂質(zhì)球,從而抑制了細胞對神經(jīng)節(jié)苷脂的吸收。因此尋找一種好的固相載體來負載神經(jīng)節(jié)苷脂,以提高細胞對神經(jīng)節(jié)苷脂的利用率,是十分必要的。凝集素是一種可以與糖鏈特異性結(jié)合的蛋白,將其與固相載體偶聯(lián),可用于糖復合物的分離,從而在糖組學中發(fā)揮重要作用。首先,本研究對神經(jīng)節(jié)苷脂及磁小體進行提取。實驗對豬腦組織及狗血中的神經(jīng)節(jié)苷脂進行分離純化,最終獲得43.26 mg GM1及53.26 mg GM3,經(jīng)斑點印跡(Dot-blot)、薄層色譜(TLC)分析,鑒定所提取的神經(jīng)節(jié)苷脂GM1及GM3的純度分別為96.7%和98.2%。接著,實驗利用趨磁細菌M.gryphiswaldense MSR-1菌株進行發(fā)酵,共獲得磁小體213.8 mg。經(jīng)紅外傅立葉紅外光譜(FTIR)、電鏡觀察(TEM),所提取磁小體擁有完整的膜結(jié)構(gòu),其表面存在氨基,為功能性分子的偶聯(lián)提供了條件。而后,本研究以GM1、GM3為例制備了神經(jīng)節(jié)苷脂-磁小體復合物,實驗首先利用十二烷基硫酸鈉(SDS)將磁小體膜蛋白去除,再將其與神經(jīng)節(jié)苷脂超聲孵育,從而制備神經(jīng)節(jié)苷脂-磁小體復合物。所制備的GM1-磁小體復合物可以有效地促進細胞對神經(jīng)節(jié)苷脂GM1的吸收,在外加磁場幫助下,促進作用更為明顯。此外,通過體外實驗證明,所制備的GM3-磁小體復合物可以與表皮生長因子受體(EGFR)特異性結(jié)合,并能有效的抑制細胞內(nèi)EGFR的磷酸化,在外加磁場幫助下其抑制作用進一步加強。最后,本研究利用磷脂酰乙醇胺(PE)修飾磁小體,以雙(硫代琥珀酰亞胺基)辛二酸酯鈉鹽(BS3)為連接臂,制備凝集素-PE-磁小體復合物,用于糖蛋白的分離。實驗結(jié)果表明,改造后的PE-磁小體分散性更好,每毫克PE-磁小體可偶聯(lián)294.64μg麥胚凝集素(WGA),較修飾前磁小體偶聯(lián)量提高1.6倍。在外加磁場作用下,所制備的WGA-PE-磁小體可以特異性的從雞卵清蛋白(OVA)及伴刀豆凝集素(Con A)混合液中分離OVA。因此,制備的凝集素-PE-磁小體復合物能很好的應(yīng)用于糖蛋白分離純化,從而在糖組學研究中發(fā)揮重要作用。
[Abstract]:Magnetosome is a kind of magnetic nanoparticles produced by Magnetotactic bacterium (Magnetotactic bacterium) with good biocompatibility. The characteristics of uniform size and superparamagnetism have been widely used in many fields, such as immunological detection, drug loading system, immobilized enzyme and so on. Ganglioside (Ganglioside) and Lectinin (Ganglioside) are two important functional molecules. Ganglioside is a kind of Glycosphingolipid carrying one or more sialic acids. It can be used as a recognition molecule on cell surface. It plays an important role in cell recognition and the mutual recognition between cells and cells. People often use exogenous methods to study the function and function of ganglioside. However ganglioside is easy to form lipid spheres in aqueous solution. It can inhibit the absorption of ganglioside, so we seek a good solid phase carrier to load ganglioside, in order to improve the cell utilization of ganglioside. Lectin is a kind of protein that can specifically bind to sugar chain. It can be coupled with solid phase carrier and can be used in the separation of sugar complex, which plays an important role in glycometry. In this study, ganglioside and magnetic bodies were extracted and purified from pig brain and dog blood. Finally, 43.26 mg GM1 and 53.26 mg GM3 were obtained and analyzed by dot blotting and TLC. The purity of ganglioside GM1 and GM3 were 96.7% and 98.2, respectively. The magnetic body 213.8 mg / g was obtained by fermentation with M. gryphiswaldense MSR-1 strain. The extracted magnetic bodies have a complete membrane structure and have amino groups on their surface, which provide the conditions for the coupling of functional molecules. Then, GM1 was used in this study. GM3 was used as an example to prepare ganglioside magnetic body complex. Firstly, magnetic body membrane protein was removed by sodium 12 alkyl sulfate, and then incubated with ganglioside ultrasonic. The GM1-magnetosome complex can effectively promote the absorption of ganglioside GM1, with the help of external magnetic field. In addition, the GM3-magnetosome complex can specifically bind to EGFR, an epidermal growth factor receptor (EGFR). And can effectively inhibit the phosphorylation of EGFR in cells, with the help of external magnetic field to further enhance its inhibitory effect. Finally, the use of phosphatidylethanolamine (PE) modified magnetic bodies. The lectin-PE-magnetic body complex was prepared with BS3 (thiosuccinyl imidyl) sodium octanedioate (BS3) as the connecting arm. The results showed that the lectin-PE-magnetic body complex could be used for the separation of glycoproteins. The modified PE-magnetosome has better dispersibility and can be coupled with 294.64 渭 g wheat germ agglutinin (WGA) per mg PE-magnetic body. The coupling amount of magnetic bodies was 1.6 times higher than that of premodified magnetic bodies. The prepared WGA-PE-magnetosome can specifically separate OVA from the mixture of ovalbumin (OVA) and concanavan agglutinin (Con A). The lectin-PE-magnetosome complex can be used in the separation and purification of glycoproteins and play an important role in the study of glycometry.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：TB383.1;Q93

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本文編號：1430317