肺炎支原体双向电泳条件的探索
发布时间：2018-09-19 14:07
【摘要】：目的建立适用于肺炎支原体(mycoplasma pneumoniae,MP)的双向电泳技术。方法对双向电泳实验中的三个关键因素:全蛋白提取量、固相PH梯度胶条范围、蛋白上样量进行摸索,比较和分析标准株FH电泳图谱,利用液相色谱串联质谱技术(LC-MS/MS)鉴定部分蛋白斑点。结果采用菌液离心结合超声破碎的方法提取全蛋白,上样量为2mg,固相PH梯度胶条选取PH4-7,可以得到蛋白点分布均匀的双向电泳图谱,蛋白点数为60±2。利用液相色谱串联质谱(LC-MS/MS)技术成功获得了分子伴侣蛋白(Chaperone protein DnaK),鉴定成功率为100%。结论成功建立了适用于肺炎支原体蛋白质组分析的双向电泳技术条件,为肺炎支原体蛋白质组学的研究提供一个可靠的技术平台。
[Abstract]:OBJECTIVE To establish a two-dimensional electrophoresis technique for Mycoplasma pneumoniae (MP). Methods Three key factors in the two-dimensional electrophoresis experiment were explored, including the total protein extraction, the range of solid phase PH gradient strips and the sample size on the protein. The electrophoretic patterns of FH of the standard strain were compared and analyzed, and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results The whole protein was extracted by centrifugation and ultrasonic fragmentation. The sample size was 2 mg, and the solid phase PH gradient strip was PH4-7. The two-dimensional electrophoresis pattern with uniform distribution of protein spots was obtained. The number of protein spots was 60. The chaperone protein (Chaper) was successfully obtained by liquid chromatography-mass spectrometry (LC-MS/MS). CONCLUSION A two-dimensional electrophoresis technique suitable for the proteomic analysis of Mycoplasma pneumoniae has been successfully established, which provides a reliable technical platform for the study of Mycoplasma pneumoniae proteomics.
【作者单位】： 军事医学科学院附属医院呼吸与危重症医学科;军事医学科学院疾病预防控制所;
【基金】：首都特色基金(Z141107002514182) 国家自然科学基金(81400009)
【分类号】：R375.2
[Abstract]:OBJECTIVE To establish a two-dimensional electrophoresis technique for Mycoplasma pneumoniae (MP). Methods Three key factors in the two-dimensional electrophoresis experiment were explored, including the total protein extraction, the range of solid phase PH gradient strips and the sample size on the protein. The electrophoretic patterns of FH of the standard strain were compared and analyzed, and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results The whole protein was extracted by centrifugation and ultrasonic fragmentation. The sample size was 2 mg, and the solid phase PH gradient strip was PH4-7. The two-dimensional electrophoresis pattern with uniform distribution of protein spots was obtained. The number of protein spots was 60. The chaperone protein (Chaper) was successfully obtained by liquid chromatography-mass spectrometry (LC-MS/MS). CONCLUSION A two-dimensional electrophoresis technique suitable for the proteomic analysis of Mycoplasma pneumoniae has been successfully established, which provides a reliable technical platform for the study of Mycoplasma pneumoniae proteomics.
【作者单位】： 军事医学科学院附属医院呼吸与危重症医学科;军事医学科学院疾病预防控制所;
【基金】：首都特色基金(Z141107002514182) 国家自然科学基金(81400009)
【分类号】：R375.2
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