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。
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-32 a 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 1 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 FliC 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.
作者
田文静
廖成水
王晓利
郁川
余祖华
张梦珂
李婧
张春杰
李银聚
程相朝
TIAN Wen-jing LIAO Cheng-shui WANG Xiao-li YU Chun YU Zu-hua ZHANG Meng-ke LI Jing ZHANG Chun-jie LI Yin-ju CHENG Xiang-chao(Luoyang Key Laboratory of Live Carrier Biomaterial and Anirnal Disease Prevention and Control~College ofA nimal Science and Technology,Henan University of Science and Technalogy,Luoyang 471001 ,China Medical College ,Henan University of Science and Technology ,Luoyang 471001, China Luoyang Vocational and Technical College ,Luoyang 471003, China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2017年第7期810-816,共7页
Chinese Veterinary Science
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)
