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目的研究HMGN2对大肠埃希氏菌TA系统作用。方法用5%高氯酸萃取兔胸腺组织,通过反相高效液相色谱进一步分离纯化蛋白,用Tricine-SDS-PAGE、AU-PAGE和Dot-blot进行鉴定。采用凝胶迁移阻滞试验(EMSA)检测HMGN2对大肠埃希氏菌TA系统的结合作用;平板菌落记数法检测TA系统激活或非激活条件下,菌体过表达重组HMGN2和对照组对细菌存活百分率的影响。结果HMGN2可阻滞mazEF基因的泳动速率,且随HMGN2浓度增加,阻滞作用增强。利福平激活TA系统,菌体过表达重组HMGN2后,与对照组比较细菌存活量减少约30%,有统计学差异。而头孢哌酮不能激活TA系统,过表达重组HMGN2组和空质粒组CFU%分别为70.2%、68.9%,两者无明显差异。结论HMGN2可加强mazEF系统介导的细菌程序性死亡;HMGN2对TA系统的作用也可能是其抗大肠埃希氏菌作用的机制之一。
Objective To study the effect of HMGN2 on Escherichia coli TA system. Methods Rabbit thymus tissue was extracted with 5% perchloric acid and further purified by reversed-phase high performance liquid chromatography. The protein was identified by Tricine-SDS-PAGE, AU-PAGE and Dot-blot. The binding of HMGN2 to Escherichia coli TA system was measured by gel permeation retardation assay (EMSA). The number of bacteria in the over-expressed recombinant HMGN2 and the control group were detected by plate colonies counting method under the activation or non-activation of TA system. The impact of survival percentage. Results HMGN2 could block the mazEF gene migration rate, and with the increase of HMGN2 concentration, the blocking effect was enhanced. Rifampin activated TA system, bacterial cells over-expression of recombinant HMGN2, compared with the control group, bacterial viability decreased by about 30%, with statistical differences. However, cefoperazone failed to activate TA system. The CFU% of over-expressed recombinant HMGN2 group and empty plasmid group were 70.2% and 68.9% respectively. There was no significant difference between the two groups. Conclusion HMGN2 can enhance the mazEF system-mediated bacterial programmed death. The effect of HMGN2 on TA system may also be one of the mechanisms of its anti-escherichia coli effect.