棉铃虫幼虫中肠细胞脂筏的制备及鉴定

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。

Isolation and identification of lipid rafts from the larval midgut of Helicoverpa armigera (Hiibner) ( Lepidoptera : Noctuidae)

Lipid raft is a detergent-resistant microdomain that enriches in cholesterol, sphingolipids and glycosylphosphatidylinositol-anchored proteins. It is considered as the platform that pore-forming toxins aggregate and form oligomer on the surface of cell membrane. In order to investigate the relationship between lipid raft and the formation of membrane pore by Bt β-endotoxins, the preparation and identification of lipid raft from the larval midgut of Helicoverpa armigera (Hübner) was performed. According to the insolubility of lipid raft in detergent at 4℃, brush border membrane vesicle （BBMV） of the larval midgut of H. armigera was treated with Triton X-100 to dissolve non-raft membrane fraction, and Triton insoluble fraction (lipid raft) was then isolated by OptiPrep gradient centrifugation. GM1 gangliosidoses, the specific component in lipid raft, was used as the hallmark of lipid raft. The distribution and expression of GM1 were probed by horse radish peroxidase (CTB-HRP) coupled to cholera β-subunit, and revealed by chemiluminiscent HRP substrates. The results indicate that the isolation method established in this study is simple and easy to operate, and also time saving compared with traditional sucrose density centrifugation.