猪链球菌细胞壁蛋白SSU05_0272的表达、纯化及生物活性分析

摘要

In order to prepare recombinant Streptococcus suis, China cell zvall protein SSL/05_0272 and assess its functions on the regulation of cylokines, SSL/05_0272 gene was amplified by using the genome of S. Suis 05ZYH33 and cloned to the expression plasmid pET28a. The recombinant protein was induced with 1 mM IPTG to express in E. Coli BL21 and purified by nickel affinity chromatography and HiTrap Q HP chromatography in turn. SDS-PAGE analysis showed that the purify of the recombinant protein was up to 90%. The purified protein was then treated with Triton X-114 to remove the contamination of endotoxin. Then, the human brain microvascular endothelial cell hCMEC/D3 was treated by SSU05_0272 for different time and tested the expression of cytokines by real-time PCR. Real-time PCR assay indicated SSU05_0272 could up-regulate the expression of several cytokine such as IL-6, IL-8, TNF-α, IL-1β and down-regulate the expression of IL-12P40. As an important cell wall protein of S. Suis, SSU05_0272 might contribute to the inflammation responses induced by S. Suis.%目的 制备获得高纯度的重组SSU05_0272蛋白,并研究其对细胞因子的调控.方法 以05ZYH33基因组为模版,PCR扩增SSU05_0272基因,构建表达质粒,转化至大肠杆菌BL21(DE3)中并诱导表达.上清表达蛋白先后经过镍亲和层析和离子交换层析方法 进行纯化.实时定量PCR方法 检测纯化后的SSU05_0272蛋白与人脑微血管内皮细胞作用后其细胞因子的转录水平变化.结果 制备的SSU05_0272蛋白具有较高的纯度,RT-PCR结果 显示,SSU05_0272蛋白与人脑微血管内皮细胞作用后能上调IL-6、IL-8、TNF-α、IL-1β的表达,下调IL-12P40的表达.结论 SSU05_0272蛋白能够调控促炎症细胞因子和趋化因子转录水平变化,提示其在猪链球菌致炎症反应中起重要作用.