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【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Gen Bank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。
【Objective】 The objective of this study was to construct a gene deletion mutant and to express and purify the gene SlyD of Salinylidene prolyl cis-trans isomerase, which laid the foundation for studying its role in pathogenicity and stress of Salmonella enteritidis. 【Method】 Based on the genome sequence of Salmonella enteritidis infected by Gen Bank, we designed a specific primer for slyD knockout and prokaryotic expression. The suicide plasmid-mediated homologous recombination technique was used to knock out the SlyD gene of Salmonella enteritidis C50041 to construct C50041ΔslyD The SlyD protein was expressed in prokaryotic cells and its PPIase activity was measured by α-chymotrypsin coupling method. The amino acid sequence and functional domain of SlyD protein were analyzed by bioinformatics software. 【Result】 The results of PCR and sequencing proved that Salmonella enteritidis C50041ΔslyD deletion mutant was successfully constructed and its growth characteristics were basically the same as those of wild type. SDS-PAGE and PPIase activity analysis showed that the soluble SlyD protein with bioactivity was obtained. Bioinformatics The analysis showed that the SlyD protein consists of three functional domains, FKBP-like prolyl cis-trans isomerase domain, chaperone domain and metal-binding domain. 【Conclusion】 Salmonella enteritidis C50041ΔslyD deletion mutant and recombinant SlyD protein with PPIase activity were successfully obtained.