细菌纳米磁小体与神经节苷脂及凝集素偶联复合物的制备与应用

发布时间：2018-01-15 22:19

本文关键词：细菌纳米磁小体与神经节苷脂及凝集素偶联复合物的制备与应用　出处：《江南大学》2015年硕士论文　论文类型：学位论文

【摘要】：磁小体(Magnetosome)是一种由趋磁细菌(Magnetotactic bacteria)生产的磁性纳米颗粒,具有生物相容性好、大小均一及超顺磁性等特点,已在免疫检测、载药体系、固定化酶等多领域得到广泛应用,但磁小体在糖生物学研究中的应用还较少。神经节苷脂(Ganglioside)及凝集素(Lectin)作为两种重要的功能性分子,在糖生物学研究中有着重要的作用。神经节苷脂是一种携带一至多个唾液酸的糖鞘脂(Glycosphingolipid)。其可作为细胞表面的识别分子,在细胞识别及细胞与细胞的相互识别中扮演重要角色。人们常利用外源添加神经节苷脂的方法来研究其功能与作用,然而神经节苷脂在水溶液中易形成脂质球,从而抑制了细胞对神经节苷脂的吸收。因此寻找一种好的固相载体来负载神经节苷脂,以提高细胞对神经节苷脂的利用率,是十分必要的。凝集素是一种可以与糖链特异性结合的蛋白,将其与固相载体偶联,可用于糖复合物的分离,从而在糖组学中发挥重要作用。首先,本研究对神经节苷脂及磁小体进行提取。实验对猪脑组织及狗血中的神经节苷脂进行分离纯化,最终获得43.26 mg GM1及53.26 mg GM3,经斑点印迹(Dot-blot)、薄层色谱(TLC)分析,鉴定所提取的神经节苷脂GM1及GM3的纯度分别为96.7%和98.2%。接着,实验利用趋磁细菌M.gryphiswaldense MSR-1菌株进行发酵,共获得磁小体213.8 mg。经红外傅立叶红外光谱(FTIR)、电镜观察(TEM),所提取磁小体拥有完整的膜结构,其表面存在氨基,为功能性分子的偶联提供了条件。而后,本研究以GM1、GM3为例制备了神经节苷脂-磁小体复合物,实验首先利用十二烷基硫酸钠(SDS)将磁小体膜蛋白去除,再将其与神经节苷脂超声孵育,从而制备神经节苷脂-磁小体复合物。所制备的GM1-磁小体复合物可以有效地促进细胞对神经节苷脂GM1的吸收,在外加磁场帮助下,促进作用更为明显。此外,通过体外实验证明,所制备的GM3-磁小体复合物可以与表皮生长因子受体(EGFR)特异性结合,并能有效的抑制细胞内EGFR的磷酸化,在外加磁场帮助下其抑制作用进一步加强。最后,本研究利用磷脂酰乙醇胺(PE)修饰磁小体,以双(硫代琥珀酰亚胺基)辛二酸酯钠盐(BS3)为连接臂,制备凝集素-PE-磁小体复合物,用于糖蛋白的分离。实验结果表明,改造后的PE-磁小体分散性更好,每毫克PE-磁小体可偶联294.64μg麦胚凝集素(WGA),较修饰前磁小体偶联量提高1.6倍。在外加磁场作用下,所制备的WGA-PE-磁小体可以特异性的从鸡卵清蛋白(OVA)及伴刀豆凝集素(Con A)混合液中分离OVA。因此,制备的凝集素-PE-磁小体复合物能很好的应用于糖蛋白分离纯化,从而在糖组学研究中发挥重要作用。
[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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB383.1;Q93

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