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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 a lysine auxotrophic mutant of 301 strains of Shigella spp., Which lays a foundation for the application of SILAC technology in Shigella proteomics research. Methods The λ-Red recombination system was used to delete the lysA gene of 301 strains of Shigella flexneri. The phenotypes and virulence of wild-type and mutant strains were evaluated. Two-dimensional gel electrophoresis And then the mutants were respectively cultured in culture medium containing 12C6-lysine and 13C6-lysine. According to the two-dimensional electrophoresis gel map, the corresponding protein spots were subjected to gel digestion and MALDI-TOF / TOF analysis. Results The 301 lysA gene deletion mutant was successfully constructed. The protein identification results showed that the corresponding protein spots were the same protein, and the light and heavy isotopic peak shifts were 6 amu on the mass spectrum. Conclusion The lysine auxotrophic mutant constructed in this study is suitable for SILAC analysis.