囊泡的提取和纯化 10使用液氮研磨5 g稻瘟病菌菌丝至粉末状，加入15 ml 0.1 M醋酸钠（含有0.07%巯基乙 醇），4°C匀速搅拌2小时6500 rpm, 4°C,离心30 min,收取上清至新的离心管中待用 沉淀再次加入15 ml 0.1 M醋酸钠（含有0.07%巯基乙醇）,搅拌均匀后，6500 rpm, 4C, 离心30 min,收取上清，并与上一步上清合并合并后的上清，7500 rpm, 4C,离心30 min, 进一步除去没有沉淀干净的菌丝碎片上清转入Beckman超速离心机专用离心管中，选择 70Ti转头，50000 rpm离心90 min倒去上清，沉淀使用1 ml TMD缓冲液（50 mM Tris pH 7.5, 10 mM MgCI2, 5 mM DTT）溶解 30 min8000 rpm, 4°C,离心 3 min,取上清加 入终浓度为15%的甘油，-80C保存待用（建议6个月内使用）囊泡裂解和蛋白质纯化取400 口1保存的囊泡样品，加入200 口1膜蛋白裂解液（5 M尿素,2 M硫脲,2% CHAPS, 2% SB3-10, 40 mM Tris, 5 mM巯基乙醇，0.17%蛋白酶抑制剂），充分混匀后，加入2-3倍体 积的预冷丙酮（含10%三氯醋酸，0.07%巯基乙醇）,-20C沉淀至少30 min。
13000 rpm, 4°C,离心30 min,沉淀使用预冷丙酮（含0.07%巯基乙醇）清洗3次后，自然晾干，加 入200 口1膜蛋白质裂解液，充分溶解后，4C最大转速离心，除去不溶杂质，使用GE公司 的 2D-Clean up 试剂盒进一步纯化About 5 g mycelia were ground in liquid nitrogen with a mortar and pestle and the mycelia powder was suspended in 25 ml of 0.1 M sodium acetate containing 0.07% 0-mercaptoethanol, then stirred at 4C for 2 h. The mixture was centrifuged at 6500 rpm for 30min and moved the supernatant to a new centrifuge tube. The precipitation was resuspe nded in 25 ml 0.1 M sodium acetate containing 0.07% p-mercaptoetha nol, after well-distributed, the mixture was centrifuged at 6500 rpm for 30min, the supernatant was mixed with the last one. In order to remove the debris of mycelia , the mixed supernatant was centrifuged at 7500rpm for 30 min at 4C. The supernatant was ultracentrifuged at 50000 rpm for 90 min using ?. After ultracentrifuged, removed the supernatant and dissolved the precipitation with 1 ml of TMD buffer （50 mM Tris pH 7.5, 10 mM MgCl2, 5 mM DTT） for 30 min. The mixture was centrifuged at 8000 rpm for 3 min at 4C and then added glycerol to final concentration was 15% to the supernatant, and stored at -80C for standby. For protein extraction, 400 ul vesicle sample were dissolved with 200 ul of lysis buffer （5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3- 10, 40 mM Tris, 0.07% p-mercaptoethanol, 1 mM PMSF） and mixed uniform. The proteins were precipitated by addition of two volumes of pre-cold acetone containing 10% TCA and 0.07% p-mercaptoethanol and kept at - 20C for 30 min at least and centrifuged at 13000 rpm, 4C for 3o min. The pellet was washed with pre-cold acetone for 3 times and dry naturally,. The protein was re-dissolved in 200 ul of lysis buffer and centrifuged at 13000rpm to get rid of the insoluble impurity. For further purity using the 2D Clean-Up kit （GE Healthcare）.。