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

目的构建弗氏2a志贺菌301株赖氨酸营养缺陷型突变株,为将细胞培养条件下稳定同位素标记(SILAC)技术应用于志贺菌蛋白质组学研究奠定基础。方法采用λ-Red重组系统对弗氏2a志贺菌301株lysA基因进行缺失,对野生株和突变株进行基本的表型测评和毒力评价,并利用双向电泳技术比较二者在全菌蛋白表达谱上的差异,最后将突变株分别在含有12C6-赖氨酸和13C6-赖氨酸的培养基中培养,根据双向电泳胶图取对应蛋白点进行胶内酶切及MALDI-TOF/TOF分析。结果成功构建了301 lysA基因缺失突变株,蛋白鉴定结果显示所取对应蛋白点为同一蛋白,在质谱图上轻重同位素峰位移为6 amu。结论本研究所构建的赖氨酸营养缺陷型突变株适合进行SILAC分析。

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 k-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 eleetrophoresis （2-DE）. Finally, the protein samples of the mutant strain grown on the media contai- ning 12C6-1ysine and 13C6-1ysine 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 S1LAC analysis.