牛支原体蛋白质组学双向电泳技术的建立及初步分析

Establishment of two-dimensional gel electrophoresis for proteomic of Mycoplasma bovis

为了建立M.bovis蛋白质组学技术,试验采用M.bovis湖北分离株为实验材料,对样品处理、固相pH梯度胶条、上样量及染色等影响双向电泳图谱质量的关键因素与环节进行一系列的探索,2D Clean-up试剂盒处理的样品400μg在13 cm干胶条（pH 4~7）中进行双向凝胶电泳,考马斯亮蓝R350染色的方法,结果获得了能较好展示大部分蛋白点,分辨率及重复性均很好的双向电泳图谱,应用Image Master 2D Platinum version 6.0软件对图谱进行初步分析,不同重复图谱之间的平均匹配率达80.95%,随机取重复性好的20个蛋白点进行质谱分析,获得19蛋白质点的肽质量指纹图谱,鉴定成功率为95%。质谱鉴定结果与自建M.bovis蛋白库对比,蛋白点匹配率高于82分值限值（P〈0.05）的不同蛋白有12个,初步分析这些蛋白多为酶类。说明该技术平台能有效用于M.bovis蛋白质组学的后续研究之中。

To establish the two-dimensional electrophoresis（2-DE） proteomic analysis method of Mycoplasma bovis,we used M.bovis Hubei isolate as the experimental material to explore optimum conditions of 2-DE such as sample preparation,immobilized pH gradients,sample volume and staining.On 13 cm IPG strip（pH 4-7） for 400 μg M.bovis proteins extacted by 2D-Clean-up kit,we obtained 2-DE of high resolution and repeatability.The 2-DE could show the most protein spots that we were interested in,then these protein spots were analyzed using the software Image Master 2D Platinum version 6.0.The results revealed that more than 800 spots were detected,and the match rate of protein spots was 80.95%.20 proteins of high repeatability selected randomly were analyzed using mass spectrometry,and the peptide mass fingerprints of 19 proteins were successfully obtained with appraisal rate of 95%.Compared mass spectrum identification results with self-built M.bovis protein library,12 different proteins of matching rate higher than 82（P0.05） were obtained and they were mostly enzymes.In conclusion,this technology platform could be effectively used in the M.bovis proteomics investigation.