新型法氏囊活性肽调节免疫反应和B细胞分化的作用机制研究

Bursal of Fabricius (BF) is the only acknowledged central humoral immune organ, which plays the vital roles on the B cell differentiation and antibody production. Bursin (Lys-His-Gly) is the first reported bursal-derived active peptide which enhances the humoral immune response. However, expect for Bursin, the other novel bursal-derived active peptides and the mechanism on immune regulation and B cell development is little reported. In our work, various novel immunomodulatory peptides were isolated from BF. To investigate the immunomodulatory mechanism of the novel bursal peptides on immune responses, firstly, the different animal immunization models will be established to detect the antibody response, cytokines and T cell subtype. Furthermore, in view of the important role of BF in the immune system, in this project, hybridoma cells will be established, and ELISA and gene microarray will be performed to detect the regulatory mechanisms of the novel bursal peptides on the antibody production. It has been reported that BF plays the vital roles on B cell differentiation. To investigate the mechanism of the novel bursal peptides on B cell differentiation, the bone marrow cells and the immature B cells models will be established, and 2D gel electrophoresis, MADIL-TOF-MS and gene microarray will be performed to detect the molecular basis at the protein and molecular level of the novel bursal peptides on B cell development. Also, the proteome analysis and genome analysis will be performed to detect the regulation of the novel bursal peptides on antioxidant effect and the target molecule. In this project, these results on will elucidate the the immunomodulatory mechanism of bursal-derived peptides on the immune responses and B cell differentiation, and will play the important foundation for the development of novel vaccine adjuvant, which will have the important guiding significance on the current clinical disease prevention.

法氏囊是目前世界上唯一认可的中枢体液免疫器官，在B细胞分化、抗体生成中发挥及其重要的作用。囊素三肽（Lys-His-Gly）是第一个法氏囊来源的促进体液免疫反应的活性分子。然而除囊素三肽外，其他法氏囊来源的活性肽及其免疫调节机制鲜见报道。本项目以本课题组新发现的法氏囊活性肽为试验材料，首先以动物免疫为模型，通过抗体、细胞因子、T细胞亚型等方面研究新法氏囊多肽的免疫调节功能；然后以杂交瘤细胞为模型模型，采用ELISA、基因芯片等技术，研究新法氏囊多肽调节抗体生成的作用机理；最后以骨髓干细胞、未成熟B细胞为模型，采用2-D电泳、质谱、基因芯片等技术，在蛋白和分子水平上研究新法氏囊多肽调节B细胞分化的机理及其抗氧化机制。本项目中，研究结果有助于阐明新型法氏囊多肽的免疫调节机制，并为疫苗新型佐剂的开发奠定基础，对当前临床疫病防治具有重要的指导意义。

法氏囊是目前世界上认可的中枢体液免疫器官，在B细胞分化、抗体生成中发挥极其重要的作用。本项目以4-6周龄BALB/C小鼠为实验动物，新法氏囊活性肽分别与禽流感病毒（H9N2）灭活疫苗和灭活抗原充分混合，以腹腔注射免疫小鼠，证实了新法氏囊活性肽BPP-I、BP4等促进AIV疫苗免疫小鼠的免疫反应，如抗体及亚型和细胞因子水平升高、T淋巴细胞等。为了研究新法氏囊活性肽在本体动物（鸡）体内的免疫调节活性，新法氏囊多肽分别与AIV疫苗或抗原混合，以颈背皮下注射的方式免疫，证实了新型法氏囊活性肽能够调节鸡机体的免疫反应，为其临床应用提供了重要参考依据。以分泌JEV抗体的杂交瘤细胞为成熟B细胞模型，通过抗体生成等证实了BSP-II促进抗体生成，并通过基因芯片、定量PCR等技术证实了新法氏囊活性肽调节抗体生成的分子基础，进而证实了BSP-II调节体液免疫的作用机制。以C57/BL6小鼠为实验动物，分离骨髓干细胞，活性肽刺激后，采用荧光标记的单克隆抗体B220、CD43、IgM、IgD，通过流式细胞术分析，证实了新法氏囊活性肽对小鼠骨髓干细胞的分化调节作用。以未成熟B细胞（WEHI-231细胞）为模型，通过流式细胞术、细胞增殖、双相电泳、MALDI-TOF-MS等技术，证实了新法氏囊活性肽调节B细胞分化的作用机制进行研究，并发现与细胞免疫系统相关联的作用机制。检测WEHI-231 B细胞内ROS水平，证实了法氏囊活性肽BP5对胞内活性氧情况的调节作用。采用双相电泳和MALDI-TOF质谱相结合的方法，证实了BP5调节B细胞内抗氧化生物学过程中的靶蛋白及重要通路，并通过RNA干扰等技术证实了BP5对G6pdx活性的调节作用，从而揭示其抗氧化作用机制。本项目中，研究结果有助于阐明新型法氏囊多肽的免疫调节机制，并为疫苗新型佐剂的开发奠定基础，对当前临床疫病防治具有重要的指导意义。