鼠伤寒沙门菌鞭毛蛋白FliC的表达纯化及其生物学特性的研究

发布时间：2018-12-31 19:33

【摘要】：为研究鼠伤寒沙门菌鞭毛蛋白FliC的生物学特性,利用PCR方法扩增鼠伤寒沙门菌鞭毛蛋白FliC基因,构建重组表达质粒p ET-32a-FliC,经大肠杆菌Rosetta(DE3)表达、镍离子亲和层析法纯化后用SDS-PAGE和Western-blot方法分析。重组蛋白与巨噬细胞RAW264.7共孵育,ELISA和CCK-8法分别检测巨噬细胞细胞因子和巨噬细胞增殖情况。重组蛋白免疫小鼠后不同时间点检测血清抗体效价和细胞因子水平。结果显示,鼠伤寒沙门菌鞭毛蛋白FliC在大肠杆菌中呈可溶性表达,且可获得纯度较高的重组鞭毛蛋白。50 ng/m L和500 ng/m L重组鞭毛蛋白可刺激巨噬细胞增殖,同时促进巨噬细胞分泌IL-1、IL-4、IL-6、IL-8、IFN-γ和TNF-α。重组蛋白免疫小鼠后效价在第3周达到1∶128 000,同时小鼠血清细胞因子的分泌量呈倒V型变化。研究结果为后续进一步研究FliC蛋白在细菌感染、侵袭和免疫方面的作用研究提供了参考数据。
[Abstract]:In order to study the biological characteristics of flagellin FliC of Salmonella typhimurium, the FliC gene of flagellin of Salmonella typhimurium was amplified by PCR, and the recombinant expression plasmid p ET-32a-FliC, was constructed and expressed by Rosetta (DE3). Nickel ion affinity chromatography was used to purify and analyze by SDS-PAGE and Western-blot. The recombinant protein was co-incubated with macrophage RAW264.7. Macrophage cytokines and macrophage proliferation were detected by ELISA and CCK-8. Serum antibody titers and cytokines levels were measured at different time points after immunization with recombinant protein. The results showed that the flagellin FliC of Salmonella typhimurium was soluble expressed in Escherichia coli, and the purified recombinant flagellin could be obtained. 50 ng/m L and 500 ng/m L of recombinant flagellin could stimulate the proliferation of macrophages. At the same time, the macrophages secreted IL-1,IL-4,IL-6,IL-8,IFN- 纬 and TNF- 伪. The titer of recombinant protein reached 1: 128 000 in the third week after immunization, and the secretion of cytokines in serum of mice showed reverse V type change. The results provide reference data for further study on the role of FliC protein in bacterial infection, invasion and immunity.
【作者单位】：河南科技大学动物科技学院/洛阳市活载体生物材料与动物疫病防控重点实验室;河南科技大学医学院;洛阳职业技术学院;
【基金】：国家自然科学基金项目(31302059、31572489) 河南省科技攻关项目(152102110078) 河南科技大学博士启动基金项目(13480071);河南科技大学青年科学基金资助项目(2015QN033);河南科技大学省部级科技创新平台培育项目(2015SPT004)
【分类号】：S852.61

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