肠炎沙门菌肽脯氨酰顺反异构酶SlyD的基因敲除、表达及酶学特征

Deletion-mutant construction, prokaryotic expression and characterization of peptidal-prolyl cis-trans isomerase SlyD from Salmonella enteritidis

摘要:

[目的] 以肠炎沙门菌肽脯氨酰顺反异构酶SlyD为对象，构建基因缺失株及表达纯化该蛋白，为研究其在肠炎沙门菌致病性与应激等方面的作用奠定基础。[方法] 参考GenBank登录的肠炎沙门菌基因组序列设计用于slyD基因敲除及原核表达的特异引物，运用自杀质粒介导的同源重组技术对肠炎沙门菌C50041 slyD基因进行敲除，构建C50041ΔslyD缺失株；原核表达SlyD蛋白，通过α-糜蛋白酶耦联法对其PPIase活性进行测定；利用生物信息学相关软件，分析SlyD蛋白的氨基酸序列及功能域。[结果] PCR鉴定与测序结果证明成功构建了肠炎沙门菌C50041ΔslyD缺失株，其生长特性与野生株基本一致；SDS-PAGE及PPIase活性分析表明，获得了具有生物活性的可溶性SlyD蛋白；生物信息学分析显示SlyD蛋白由FKBP样肽脯氨酰顺反异构酶结构域、分子伴侣功能域和金属结合区域3个功能区域组成。[结论] 成功获得了肠炎沙门菌C50041ΔslyD缺失株和具有PPIase活性的重组SlyD蛋白。

Abstract:

[Objective] Salmonella enterica serovar enteritidis is an important food-borne pathogen of human and animal. To further study the function of SlyD associated with virulence and regulation in stress responses of Salmonella Enteritidis, we constructed slyD gene-deletion mutant,, expressed it in E. coli, and characterized the PPIase enzyme obtained.[Methods] The slyD gene-deletion mutant of Salmonella enteritidis C50041 was constructed by suicide plasmid mediated homologous recombination. Salmonella enteritidis slyD prokaryotic expression vector was carried out in E. coli, and PPIase activity of recombination SlyD was measured in protease-coupling assay with chymotrypsin. For amino acids conservation studies, functional domain searches and secondary structure predictions, the BLAST, SMART, TMHMM, SignalP, PHD and SWISS MODEL were used.[Results] Salmonella enteritidis C50041 ΔslyD mutant strain was successfully constructed. The growth rate of slyD-deleted strain was identified consistent with its parent strain C50041. A soluble recombinant SlyD protein was expressed in Escherichia coli BL21(DE3) cells and confirmed by SDS-PAGE. Catalytic activity confirmed that the SlyD protein was biologically active. Bioinformatic analysis showed that Salmonella Enteritidis SlyD as a multifaceted protein including three separated domains, the FKBP type peptidal-prolyl cis-trans isomerase domain, the IF chaperone domain and the metal-binding domain.[Conclusion] Salmonella enteritidis C50041 ΔslyD mutant strain and soluble SlyD protein was obtained, and the present study may provide a basis for further study of the role of SlyD in Salmonella enteritidis.