中国兽医科学2017，Vol.47Issue(7)：810-816,7.DOI:10.16656/j.issn.1673-4696.2017.07.002
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
Expression and biological characteristics of FliC of Salmonella typhimurium
摘要
Abstract
In order to study the biological characteristics of flagellin FliC of Salmonella typhimurium,the FliC gene of Salmonella typhimurium was amplified using PCR and inserted into pET-32a vector to construct the recombinant plasmid pET-32a-FliC.The recombinant plasmid pET-32a-FliC was expressed in E.coli Rosetta(DE3) by induced with l mmol/L IPTG.After purified with Ni-NTA,the recombinant FliC protein was analyzed using SDS-PAGE and Western-blot.The level of cytokines and the proliferation of macrophages RAW264.7 were detected by ELISA and CCK-8 after in vitro incubation with the recombinant FIiC protein.Meanwhile,the level of antibody and cytokines in serum of mice immunized with the recombinant protein were measured at different time points.The results showed that the recombinant bacteria was constructed successfully,and the high purity recombinant protein was obtained.The recombinant protein (50 ng/mL and 500 ng/mL) stimulated macrophage proliferation and increased the secretion of IL-1,IL-4,IL-6,IL-8,IFN-γ,and TNF-α.The level of antibodies reached a maximum of 1∶128 000 in the third week.The level of cytokines revealed the inverse V-shape distribution in sera of mice immunized with the recombinant protein.These results will provide the basis for the study of the relationship between FliC and bacterial infection,invasion,and immunity.关键词
鼠伤寒沙门菌/鞭毛蛋白/FliC/巨噬细胞Key words
Salmonella typhimurium/flagellin/FliC/macrophage分类
农业科技引用本文复制引用
田文静,程相朝,廖成水,王晓利,郁川,余祖华,张梦珂,李婧,张春杰,李银聚..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[J].中国兽医科学,2017,47(7):810-816,7.基金项目
同家自然科学基金项目(31302059、31572489) （31302059、31572489）
河南省科技攻关项目(152102110078) （152102110078）
河南科技大学博士启动基金项目(13480071) （13480071）
河南科技大学青年科学基金资助项目(2015QN033) （2015QN033）
河南科技大学省部级科技创新平台培育项目(2015SPT004) （2015SPT004）
