肺炎链球菌蛋白质组双向电泳技术的建立及优化

Establishment and Optimization of Technique of Two-dimensional Electrophoresis for Proteome of Streptococcus pneumoniae

为了建立适合肺炎链球菌(Streptococcus pneumoniae)蛋白质组的双向电泳技术,对蛋白样品提取、裂解、上样量、IEF和银染等关键步骤进行了优化,探索出一套有效的肺炎链球菌蛋白分析的双向电泳方法,即以裂解液[15 mmol/L Tris-HCl、7 mol/L尿素、2 mol/L硫脲、4%(m/v)CHAPS,使用前现加2%(v/v)IPG缓冲液、1%(m/v)二硫苏糖醇(DTT)和1%(m/v)蛋白酶抑制剂混合物]结合反复冻融和超声裂解菌体的方法来提取蛋白质样品,按80μg蛋白上样量,并结合适宜的双向电泳参数,可获得蛋白分辨率高、重复性较好的双向电泳图谱。

In order to establish an eftieient two - dimensional gel eleetrophoresis （2 - DE） protocol for proteomic study of Strepto- coccus pneumoniae, the processes of cell lysis, protein extraction, protein loading, IEF and silver staining were optimized. It was found that the following treatments could obtain 2 - DE protein map with high resolving rate and good repeatability ： cells were treated consecutively with a lysis buffer containing 15 mmol/L Tris -HC1, 7 moL/L urea, 2 mol/L thiourea, 1% DTF, 4% CHAPS, 2% （v/ v） IPG buffer with pH -value 4 -7 and 1% PMSF, frozen -thaw for three times and then sonicated for 10 inin; 80 μg protein was subjected to 2 - DE separation with suitable electrophoresis parameters.