猪链球菌 2 型不同温度条件下分泌蛋白的差异研究 2 实验材料 ....................................................................................................................23 2.1 菌株及培养基 .....................................................................................................23 2.2 主要实验仪器 .....................................................................................................23 2.3 主要化学试剂 .....................................................................................................23 2.4 主要溶液配方 .....................................................................................................24 3 方法 ............................................................................................................................26 3.1 PCR 鉴定菌株毒力因子.....................................................................................26 3.1.1 引物的设计与合成.......................................................................................26 3.1.2 PCR 反应程序..............................................................................................27 3.2 SC-21 菌株的培养条件......................................................................................27 3.3 分泌蛋白样品的制备 .........................................................................................27 3.4 THB 培养基蛋白样品的制备 ............................................................................27 3.5 蛋白定量 .............................................................................................................28 3.5.1 精确定量：Quantity Kit 定量....................................................................28 3.5.2 粗略定量：Bradford 法...............................................................................28 3.6 1D SDS-PAGE 电泳............................................................................................28 3.7 双向聚丙烯酰胺凝胶电泳(2D-PAGE) ..............................................................28 3.7.1 IEF ................................................................................................................28 3.7.2 IPG 胶条平衡...............................................................................................29 3.7.3 灌制凝胶 ......................................................................................................30 3.7.4 胶条的转移 ..................................................................................................30 3.7.5 打开循环水浴 ..............................................................................................30 3.7.6 参数设置 ......................................................................................................30 3.7.7 凝胶染色（银染） ......................................................................................30 3.8 图像扫描及分析 .................................................................................................31 3.9 差异蛋白质点的鉴定 .........................................................................................31 3.9.1 切取蛋白点 ..................................................................................................31 3.9.2 胶内酶解 ......................................................................................................31 3.9.3 LC/ESI-MS/MS 质谱分析及数据库搜索...................................................32 3.10 实时荧光定量 PCR...........................................................................................32 3.10.1 基本原理 ....................................................................................................32 3.10.2 总 RNA 的提取..........................................................................................33 3.10.3 cDNA 的合成.............................................................................................34 3.10.4 引物的合成 ................................................................................................34 3.10.5 实时荧光定量 PCR 检测...........................................................................35 4 结果 ............................................................................................................................36 II
