大肠不耐热肠毒素B亚单位作为佐剂的分子机理研究

发布时间：2018-04-19 21:06

本文选题：LTB + RAW　；参考：《重庆医科大学》2016年硕士论文

【摘要】：大肠杆菌不耐热肠毒素B亚单位是一种非常有效的粘膜免疫佐剂,它能刺激机体产生强烈的粘膜和系统免疫应答。LTB通过与细胞表面的GM1结合,不仅能增强机体对抗原的免疫应答效应,还能够改变粘膜免疫应答的类型。LTB的免疫调节作用包括促进Th1和Th2型细胞反应的细胞因子的表达,CD8+T细胞的凋亡和CD4+T细胞的增殖;使B细胞活化的MHC‖、ICAM-1、CD25、CD40分子表达增加,影响B细胞和巨噬细胞的抗原处理和递呈过程。但在巨噬细胞的具体免疫机制尚不清楚,本文将在这方面进行研究。目的:从LTB处理的RAW264.7细胞中,筛选与LTB有相互作用的蛋白质分子,并通过生物信息学方法预测其与LTB佐剂活性的相关性;并利用分子生物学技术对它们的相互作用进行验证,最终探索LTB作为免疫佐剂的分子机理。方法:(1)在大肠杆菌中诱导表达含His-tag的LTB融合蛋白,用镍磁珠纯化。(2)复性后的LTB融合蛋白去除内毒素,然后处理RAW264.7细胞。(3)提取LTB处理后RAW264.7细胞总蛋白,用pull down的方法,分离与LTB有相互作用的蛋白分子。(4)PAGE检测分离得到的蛋白分子,并收集该蛋白质进行质谱鉴定。(5)运用生物信息软件从质谱鉴定结果中筛选出可能的相互作用蛋白分子,并对其进行功能性分析和信号通路分析,构建出这些蛋白分子相互作用的网络图,筛选出有意义蛋白分子。(6)免疫荧光验证这些分子在细胞内与LTB相互作用。(7)Western blot检测下游分子的表达。结果:(1)通过pull down捕获到与LTB有相互作用的蛋白分子,并通过质谱鉴定了这些蛋白分子。(2)通过对这些蛋白分子进行生物学分析后,筛选出junction plakoglobin、heat shock protein 1、carbamoyl-phosphate synthetase 1、vimentin等25种有统计学意义的蛋白分子。(3)通过对25种蛋白分子进行功能及其信号通路分析(3)免疫荧光结果显示,Vimentin在生理状态下不能与LTB相互作用,而GM130在细胞内与LTB有相互作用,表明LTB在细胞内是以内吞小泡的形式运输的。(4)Western blot检测显示,LTB处理RAW 264.7细胞12 h后,β-actin表达上调,Hspd1表达无变化,表明LTB与Actb确实存在相互作用。结论:通过质谱鉴定结果和免疫荧光实验,我们可以推测LTB通过与免疫细胞表面GM1结合,在Keratin家族和Actb等细胞骨架相关蛋白分子的协助下内吞,并以小泡的形式到达高尔基体,通过高尔基体的加工、转运到Jup处或以抗原的形式直接递呈给T细胞,通过Jup激活TCF/LEF,促进T细胞的活化、增殖与分化,以及相关细胞因子的分泌,达到促进免疫应答的作用。
[Abstract]:E. coli heat-labile enterotoxin B subunit is a very effective mucosal immune adjuvant that stimulates the production of strong mucosal and systemic immune responses. LTB binds to GM1 on the cell surface. It can not only enhance the immune response to antigen, but also change the type of mucosal immune response. LTB can promote the expression of cytokines of Th1 and Th2 type reaction and the apoptosis of CD8 T cells and the proliferation of CD4 T cells. The expression of ICAM-1 CD25 + CD40 in B cells was increased, which affected the process of antigen treatment and presentation of B cells and macrophages. However, the specific immune mechanism of macrophages is not clear, this paper will study this aspect. Objective: to screen protein molecules interacting with LTB from RAW264.7 cells treated with LTB, and to predict their correlation with LTB adjuvant activity by bioinformatics, and to verify their interaction by molecular biological techniques. Finally, the molecular mechanism of LTB as an immune adjuvant was explored. Methods the LTB fusion protein containing His-tag was induced and expressed in E. coli. The LTB fusion protein was purified by nickel beads. The endotoxin was removed by refolding LTB fusion protein. The total protein of RAW264.7 cells treated with LTB was extracted from RAW264.7 cells. The total protein of RAW264.7 cells was extracted by the method of pull down. Isolation of protein molecules with interaction with LTB. Page was used to detect the protein molecules, and the protein was collected for mass spectrometry identification. 5) the possible interacting protein molecules were screened by bioinformatics software from the results of mass spectrometry identification. Functional analysis and signal pathway analysis were performed to construct the network diagram of the interaction of these proteins. The significant protein molecules were screened out and immunofluorescence was used to detect the expression of the downstream molecules by LTB interaction. Western blot was used to detect the expression of these molecules. Results the protein molecules interacting with LTB were captured by pull down and identified by mass spectrometry. 25 protein molecules, such as junction plakglobin shock protein 1 carbamoyl-phosphate synthetase 1 vimentin and so on, were screened. The immunofluorescence results showed that Vimentin could not interact with LTB in physiological state by analyzing the function and signal pathway of 25 protein molecules. However, GM130 interacted with LTB in cells, indicating that LTB was transported in the form of endocytosis vesicles. Western blot analysis showed that 尾 -actin expression was up-regulated in RAW 264.7 cells after 12 h treatment, indicating that there was indeed interaction between LTB and Actb. Conclusion: based on the results of mass spectrometry and immunofluorescence assay, we can speculate that LTB can be ingested with the help of Keratin family and cytoskeleton related protein molecules such as Actb, and reach Golgi body in the form of vesicles by binding to GM1 on the surface of immune cells. Through the processing of Golgi body, it was transported to Jup or presented directly to T cells in the form of antigen. TCFR / LEF was activated by Jup, which promoted the activation, proliferation and differentiation of T cells, and the secretion of related cytokines, so as to promote the immune response.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R392

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