抗菌肽分子多样性及抗菌功能的分子结构基序研究

Antimicrobial peptides were the main effector molecules that involved in the clearance process of bacteria and fungi in invertebrate innate immune system. There were a lot of signal pathways which were involved in the antimicrobial peptides producing processes. At present, in the process of antimicrobial peptides exerting their antibacterial activities, the research results about the synergistic effect mechanism between antimicrobial function and other functions of the molecules themselves, and the research results about the relationship between antibacterial activity and antibacterial peptides molecular structure were very limited. In this project, the gene expression regulation and molecules function of antimicrobial peptides were systematically studied in Procambarus clarkii. The research contents included gene cloning, tissue distribution, the gene expression profile under the pathogens stimulation, and so on. The antiserum preparation, function research in vivo and vitro were done, after recombinant expression of antibacterial peptides genes was achieved. At the same time, the structural motif which could decide the antimicrobial activity was studied, using the site-directed mutagenesis technique. And the crystal structures of natural antimicrobial peptides and mutational molecules were analyzed to explore the relationship between molecular structure motif and function. In addition, the relationship between the antimicrobial peptides and antiviral immune response under viral stimulation was centrally studied. The results of this project has a great significance for further improvement of the invertebrate innate immunity system basal theory. And there are very important theoretical promotion and practical guiding significance to the immune prevention and control strategies for aquatic economical animal.

抗菌肽是无脊椎动物先天免疫系统中清除细菌、真菌的主要免疫效应分子，其产生过程受到多种信号通路的调节。目前，在抗菌肽发挥抗菌活性过程，关于抗菌功能与分子本身其他功能的协同作用机制的研究结果，以及抗菌肽分子抗菌活性与分子结构基序之间关联的研究结果，十分有限。因此，本课题进行淡水小龙虾抗菌肽基因的表达调控以及功能的系统研究，包括基因克隆、组织分布、病原刺激下的表达模式。在重组表达抗菌肽基因后，制备抗血清，从体内、体外研究基因的多种功能。同时，利用基因定点突变技术，研究决定抗菌肽分子抗菌活性的分子结构基序；并且，解析天然抗菌肽分子与突变分子的晶体结构，来探索分子结构基序与抗菌功能之间的关系。此外，重点研究在病毒刺激下，抗菌肽分子与抗病毒免疫反应之间的关联。本课题对于进一步完善无脊椎动物先天免疫系统基础理论，具有较大促进意义，对于水产类经济动物免疫防治策略的制定具有重要的理论促进和实践指导意义。

无脊椎动物先天免疫系统中，抗菌肽分子是发挥清除细菌、真菌以及病毒的主要免疫效应分子；同时，抗菌肽的表达过程也受到多种信号通路的调节。深入研究抗菌肽分子的多样性以及发挥免疫功能的分子机制可以进一步完善低等生物先天免疫的基础理论。本课题以淡水小龙虾抗菌肽作为研究对象，进行了表达调控以及功能方面的系统研究，包括基因克隆、组织分布以及表达模式与生物活性的研究，实验结果初步确定了富含Gly与Pro结构基序的抗菌肽分子很大程度上会具有抗菌功能。在小龙虾抗病毒先天免疫机制的研究方面，转录组测序的结果显示：小龙虾淋巴器官、鳃、小肠组织参与抗病毒免疫的差异表达基因具有多样性的特点；同时，预测细胞凋亡信号途径、黑化反应途径、Toll样受体信号途径、Jak-STAT signaling pathway、nsulin signaling pathway、Wnt signaling pathway很可能是小龙虾参与抗病毒的主要途径。microRNA测序结果预测出20个新的microRNA参与到了小龙虾的抗病毒先天免疫过程。在小龙虾血细胞抗病毒免疫机制的研究方面，研究结果初步揭示Bax与Bax inhibitor 1分子在参与小龙虾血细胞抗病毒先天免疫方面具有重要作用，Bax可以通过促进受侵染血细胞的凋亡，来起到抑制病毒在小龙虾体内增殖的作用。在小龙虾黑化反应重要分子的研究方面，对小龙虾神经系统重要调节分子多巴脱羧酶（DDC）的先天免疫功能进行了深入研究，实验结果显示小龙虾DDC不仅是神经系统重要的调节分子，它在小龙虾对抗细菌侵染方面发挥着重要作用。此外，研究过程还发现一些重要的免疫相关分子，包括小龙虾丝氨酸蛋白酶抑制因子（Serpin）基因、神经肽（NP）基因、过氧素（PX）基因、抑制蛋白（PB）基因、白介素增强子结合因子（IEBF）基因、M型凝集素（M-Lectin）基因以及肿瘤坏死因子受体相关的因子（TRAF）基因。本课题的研究结果对于进一步完善无脊椎动物先天免疫系统基础理论，具有较大促进意义，对于水产类经济动物免疫防治策略的制定具有重要的理论促进和实践指导意义。