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脂筏是细胞膜上富含胆固醇、鞘脂类和糖基磷脂酰肌醇锚着蛋白的去垢剂不溶性微结构域,被认为是多种细胞膜孔毒素在细胞表面形成寡聚体的平台。为研究脂筏与Bt毒素在细胞膜上形成寡聚体膜孔的关系,本文对棉铃虫Helicoverpa armigera幼虫中肠脂筏的制备与鉴定方法进行了研究。根据脂筏在低温(4℃)下不溶于去垢剂的特性,采用Triton X-100处理棉铃虫幼虫中肠刷状缘膜囊泡,溶解非脂质筏成分,以OptiPrep为介质进行密度梯度离心,分离去垢剂不溶组分,成功地得到了棉铃虫幼虫中肠上皮细胞的脂筏。再以脂筏的特有化学成分神经节苷脂GM1作为脂筏的标志分子,利用霍乱毒素β亚基能与GM1特异性结合的特性,以辣根过氧化物酶标记的霍乱毒素β亚基用点印迹法化学发光检测神经节苷脂的分布,从而对脂筏进行定性鉴定。结果表明我们建立的脂筏制备方法简便、易行,比传统的蔗糖梯度离心法大大缩短了制备所需时间。
Lipid rafts are detergent insoluble microstructures that are rich in cholesterol, sphingolipids and glycosylphosphatidylinositol anchored proteins on the cell membrane and are thought to be platforms for the formation of oligomers on the cell surface by a variety of cell membrane pore toxins. In order to study the relationship between lipids rafts and the formation of oligomeric membrane pores on lipid membranes in Bt toxins, a method for the preparation and identification of midgut lipid rafts in Helicoverpa armigera larvae was studied. According to the characteristics of lipid raft insolubilized in detergent at low temperature (4 ℃), the brush border membrane vesicles of midgut of H. armigera larvae were treated with Triton X-100 to dissolve the non-lipid raft components and the density gradient of OptiPrep Centrifugation, separation of detergent insoluble components, successfully obtained cotton bollworm larval midgut epithelial cells of lipid rafts. Then the ganglioside GM1, a peculiar chemical component of lipid raft, was used as a marker molecule for lipid rafts. Based on the characteristic that cholera toxin β subunit can specifically bind to GM1, the horse radish peroxidase-labeled cholera toxin β subunit Dot blot chemiluminescence detection of ganglioside distribution, lipid raft qualitative identification. The results showed that the lipid raft preparation method we established was simple and easy to implement, which greatly shortened the preparation time than the traditional sucrose gradient centrifugation.