黄芪多糖对大黄鱼头肾巨噬细胞免疫调节作用的机制研究

Large yellow croaker (Larimichthys crocea) possess an important position in aquaculture industry of China, however, diseases take place frequently during the cultivation. The use of immunopotentiator is an effective way to prevent and treat the diseases. In our previous research, Astragalus polysaccharide showed immunoregulation effects on macrophages from head kidney of L. crocea. In this project, the activation role of APS on macrophages will be further studied by detecting the influence of APS on cell phagocytic ability, respiratory burst activity and transcriptional level of cytokines. In addition, receptors possibly participate in recognition of APS will be screened by qPCR. The extracellular domain of the candidate receptor will be expressed in Escherichia Coli, and antibody will be prepared with the purified recombined receptor. The receptor will be pre-screened by observing the binding of macrophages and fluorescence labeled APS with or without antibody blocking. APS receptor will be revealed after verification of the interaction between APS and the receptor with Surface Plasmon Resonance Technique. Downstream adaptor protein will be screened by Yeast Two-Hybrid and Co-Immunoprecipitation Assay. Antibody Blocking Assay will be used in order to test the influence of APS on downstream signal and effector molecules, so the the receptor of APS and the signal path to activate macrophages will be clarified. These expected results are of great theoretical significance in understanding the regulatory mechanism of APS on macrophages from head kidney of L. crocea. And the results will also provide basis for application of APS as an immunopotentiator for preventing and treating the diseases during L. crocea cultivation.

鉴于大黄鱼在我国养殖产业中的重要地位和病害频发的现状，应用免疫增强剂来防治疾病是一种有效手段。本项目拟在前期发现黄芪多糖（APS）对大黄鱼头肾巨噬细胞具有免疫调节作用的基础上，通过研究APS对巨噬细胞吞噬活性、呼吸爆发活性及细胞因子转录水平的影响，明确APS对巨噬细胞的激活作用；采用qPCR筛选可能参与APS识别的受体，将候选受体胞外段原核表达并制备抗体，分析抗体阻断前后荧光标记APS与细胞的结合情况，初步筛选与APS结合的受体，再通过表面等离子共振技术验证APS与受体的互作，确定APS的受体；利用酵母双杂交和免疫共沉淀鉴定受体的下游接头蛋白，通过抗体阻断试验研究APS对受体途径下游信号和效应分子基因表达的影响，阐明APS的受体及其诱导巨噬细胞活化的信号通路。预期结果对于认识APS对大黄鱼头肾巨噬细胞的调控机制具有重要理论意义，也将为APS作为免疫增强剂应用于大黄鱼病害防治提供依据。

黄芪多糖是鱼类养殖过程中广泛应用的免疫增强剂，然而其对鱼类的免疫调节作用机制尚不清楚。本项目明确了黄芪多糖对大黄鱼头肾巨噬细胞的激活作用；制备了黄芪多糖脂质体，通过体内外试验证实了脂质体包被后黄芪多糖的免疫调节和抗氧化功能得到显著提高，为黄芪多糖给药方式的改进提供了思路；从黄芪多糖处理后的大黄鱼头肾组织转录组及离体头肾巨噬细胞转录组数据库中筛选并鉴定了大黄鱼凝集素受体基因CD209，原核表达了该受体的胞外段蛋白并进行了凝集活性和糖结合特异性分析；用生物素标记黄芪多糖，Pull-down试验证实了受体与黄芪多糖的结合；进一步，筛选到对LcCD209干扰效果较好的siRNA，成功制备了CD209受体胞外段的多克隆抗体，通过RNA干扰和抗体封闭试验证实了黄芪多糖通过该受体对大黄鱼头肾巨噬细胞发挥免疫调节作用。本研究结果为进一步揭示黄芪多糖对大黄鱼头肾巨噬细胞免疫调控的分子机制奠定了基础，也为黄芪多糖在鱼类养殖业中的推广应用提供了理论依据。