大肠不耐热肠毒素B亚单位佐剂活性相关蛋白的筛选

发布时间：2018-01-13 15:08

本文关键词：大肠不耐热肠毒素B亚单位佐剂活性相关蛋白的筛选　出处：《中国药理学通报》2016年12期 　论文类型：期刊论文

更多相关文章： LTB RAW . 相互作用蛋白 免疫佐剂内吞

【摘要】：目的通过对大肠杆菌不耐热肠毒素B亚单位相互作用蛋白的分析,探讨LTB佐剂活性的分子机制。方法纯化的LTB蛋白和生理盐水分别处理RAW 264.7细胞,pulldown实验分离与LTB有相互作用的蛋白分子,质谱鉴定这些相互作用蛋白;免疫荧光和Western blot分析验证与LTB相互作用蛋白在细胞内的相互作用。结果通过质谱鉴定了25种可能与LTB存在相互作用的蛋白分子,并构建了其相互作用网络图,筛选出可能与LTB免疫调节作用相关的蛋白分子;免疫荧光结果显示神经节苷脂能阻断LTB进入RAW 264.7细胞内,LTB在细胞内与GM130存在相互作用,而Vimentin在生理状态下与LTB不存在相互作用;LTB处理RAW 264.7细胞12 h后,β-actin表达上调,Hspd1表达无变化。结论 LTB佐剂活性的发挥是通过与免疫细胞表面GM1结合,引起内吞,并以小泡的形式到达高尔基体,通过高尔基体的加工。最终通过与Jup结合进而作用于TCF/LEF,引起Bcl-2、IL-6、Runx3等基因表达的变化,发挥促进T细胞和B细胞的增殖分化及活化、分泌细胞因子及免疫球蛋白的作用。
[Abstract]:Objective through the analysis of the Escherichia coli heat labile toxin B subunit interacting protein, to explore the molecular mechanism of LTB adjuvant activity. LTB protein and saline purification methods were treated RAW 264.7 cells, the protein molecules separation and LTB pulldown interaction, mass spectrometry identification of these interacting proteins; immunofluorescence and Western blot interaction analysis verify the interaction between LTB and protein in the cells. Results by mass spectrometry identified 25 possible protein interactions with LTB, and construct the interaction network graph, and to identify the possible immunomodulatory protein molecules related to LTB; immunofluorescence results showed that gangliosides can block LTB enter the RAW 264.7 cells, LTB interacts with GM130 in cells, and Vimentin in physiological condition and there is no LTB interaction; LTB RAW 264.7 cells 12 After h, upregulate the expression of beta -actin, Hspd1 showed no changes. Conclusion LTB play adjuvant activity is through a combination of GM1 and immune cells, caused by endocytosis, and in vesicles to the Golgi, through the Golgi processing. Eventually combined with Jup's influence on TCF/LEF, IL-6, Bcl-2 induced changes in expression Runx3 gene of T cells and promote the proliferation of B cell differentiation and activation, cytokine secretion and immune globulin.

【作者单位】：重庆医科大学分子医学与肿瘤研究中心生物化学与分子生物学教研室;
【基金】：国家自然科学基金资助项目(No 30972585)
【分类号】：R329-33
【正文快照】： R378.21;R977.6大肠杆菌不耐热肠毒素由产毒型大肠埃希菌(ETEC)产生,因其不耐热,在60℃处理30 min即被破坏,故称作不耐热肠毒素(LT)[1-2]。LT是一类六聚体大分子,由1个A亚基(LTA)和5个B亚基(LTB)组成,结构和功能与霍乱毒素相似[3]。LTB作为LT的非毒性亚基,能刺激机体产生强烈

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