军事医学2012，Vol.36Issue(5)：345-350,6.
弗氏志贺菌ΔlysA突变株的构建及SILAC技术的应用尝试
Construction of a lysA deletion mutant of Shigella flexneri and pre-experiment of SILAC
摘要
Abstract
Objective To construct the lysine auxotrophic mutant of Shigella flexneri 2a strain 301 to facilitate pro-teomic studies by stable isotope labeling with amino acids in cell culture ( SILAC ). Methods Gene lysA was knocked out from Sh.flexneri 2a strain 301 using λ-Red recombination system. The biochemical phenotypes and the virulence of the mu-tant strain were tested. Then, the protein expression profiles of the mutant strain and the wild-type strain were analyzed by two-dimensional gel electrophoresis ( 2-DE ). Finally, the protein samples of the mutant strain grown on the media contai-ning 12C6-lysine and 13C6-lysine respectively were prepared and separated by 2-DE. The corresponding spots were cut off from the gels, digested by trypsin and analyzed by MALDI-TOF/TOF. Results The lysA deletion mutant of Sh. flexneri 2a strain 301 was constructed. The corresponding spots were identified to be the same protein, and the 6 Dalton shift of the mass peaks was observed in the mass spectra. Conclusion Lysine auxotrophic mutant of Sh. flexneri 2a strain 301 is appro-priate for SILAC analysis.关键词
志贺菌,弗氏/赖氨酸营养缺陷型突变株/基因缺失/λ-Red重组系统/双向电泳/SILACKey words
Shigella flexneri/ lysine auxotrophic mutant/ gene deletion/ \-Red recombination system/ two-dimensional gel electrophoresis/ SILAC分类
医药卫生引用本文复制引用
李月玥 ,刘先凯,冯尔玲,王恒樑,陈福生,朱力..弗氏志贺菌ΔlysA突变株的构建及SILAC技术的应用尝试[J].军事医学,2012,36(5):345-350,6.基金项目
国家自然科学基金资助项目(81071324,81171531) （81071324,81171531）
国家973计划资助项目(2011CB504901) （2011CB504901）
