适用于双向电泳的米曲霉孢子破壁方法研究

为探究米曲霉(Aspergillus oryzae)孢子适用于双向电泳的最佳破壁方法,采用5种不同的破壁方法对米曲霉孢子进行破壁,用血球计数板进行破壁率计算,Bradford方法测定释出的可溶性蛋白含量,并进行双向电泳可行性验证。结果表明,在普通光学显微镜下,破壁后的米曲霉孢子多为碎片,极少数为孢壁内空圆球。5种破壁方法中石英砂研磨+超声、液氮研磨、MP·Fast-prep均质器法在孢子浓度较低(107个/m L)时破壁效果较佳,但是随着孢子浓度的不断提升(109个/m L),只有均质器法能保证较高的破壁率,破壁率高达90%,且适用于双向电泳的蛋白质提取。

Spore Wall Breaking Method of Aspergillus oryzae Suitable for Two-D Electrophoresis

In order to explore the optimum method of spore wall breaking suitable for two-D electrophoresis in Aspergillus oryzae, five different methods were used to break the spore wall of A.oryzae and the wall breaking rates were calculated using the blood count plate, then the concentration of soluble protein delivered from the spores was determined by the Bradford method and the quality of protein sample was tested by two-D electrophoresis. The results showed that after breaking the spore wall, the spores of A.oryzae were mostly fragments, and a few were hollow spheres under ordinary optical microscope. Three of the five methods, quartz sand grinding plus ultrasonic treatment method, liquefied nitrogen grinding method, and MP·Fast-prep homogenizer method, worked pretty well when the spores in a low concentration (10⁷ spores per mL). However, as the concentration increase to 10⁹ spores per mL, only the MP·Fast-prep homogenizer method worked well, and the wall breaking rate kept as high as 90%, and it was suitable for protein extraction for two-D electrophoresis.