关键词: 棉铃虫, 脂筏, 中肠, 刷状缘膜囊泡, OptiPrep

Abstract: 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.

Key words: Helicoverpa armigera, lipid raft, midgut, brush border membrane vesicle （BBMV）, OptiPrep