C型清道夫受体在日本囊对虾先天免疫中的功能研究

发布时间：2018-04-17 09:58

本文选题：先天免疫 + 模式识别受体　；参考：《山东大学》2017年博士论文

【摘要】：无脊椎动物主要依靠先天免疫系统抵御病原体的感染,在此过程中模式识别受体对病原微生物的识别是启动先天免疫反应的关键步骤。在对虾中,已发现的模式识别受体有十多种,但对C型清道夫受体及其功能了解的还不多。我们以日本囊对虾Marsupenaeus japonicus为研究对象,发现了 C型清道夫受体可以作为一种重要的模式识别受体促进对虾血细胞对病毒和多种细菌的吞噬作用以及调节抗菌肽基因的表达。(1)C型清道夫受体通过增强对虾血细胞的吞噬作用提高宿主抗病毒的能力至今已在多种无脊椎动物中发现有C型清道夫受体(SRC)的存在,然而SRC在抗病毒方面的研究报道还很少。在本研究中,我们首次从日本囊对虾,Mjaponicus,中克隆到了一种SRC基因,并将其命名为MjSRC。当对虾受到白斑综合征病毒(WSSV)的刺激以后,MjSRC在mRNA以及蛋白水平呈显著的上调表达。敲低MjSRC导致WSSV病毒的含量显著增加。同时,过表达MjSRC会有效地提高血细胞的吞噬率,从而降低对虾体内WSSV的含量。Pull-down实验结果表明MjSRC可以与WSSV的囊膜蛋白VP19相互作用。透射电镜的观察结果显示胶体金标记的MjSRC胞外域可以附着在WSSV的囊膜表面。在受到WSSV刺激后,MjSRC可以形成三聚体并伴随病毒的内吞而进入细胞质,这类内化过程依赖Mjβ-arrestin2的存在。进一步的实验结果表明MjSRC的胞内域可以与Mjβ-arrestin2相互作用,进而介导WSSV以网格蛋白依赖的方式进入细胞质。进入细胞质的病毒与溶酶体融合,当注射溶酶体抑制剂(氯喹)以后,病毒的拷贝数显著增加。本研究结果显示MjSRC通过其胞外域识别并结合WSSV,启动血细胞的吞噬作用,从而清除对虾体内的病毒。本文首次报道了 SRC作为一种模式识别受体参与对病毒吞噬和清除。(2)MjSRC在对虾抗细菌感染中的功能研究我们进一步研究了 MjSRC作为模式识别受体参与细菌的的识别和清除的分子机制。当对虾受到弧菌(Vibrio anguiarum)或金黄色葡萄球菌(Staphylococcusaureus)刺激以后,MjSRC的mRNA水平在血细胞中显著上调表达。MjSRC的胞外域(MAM和CCP结构域)具有体外结合不同细菌以及细菌来源多糖的功能。在敲低MjSRC表达的情况下,对虾血细胞的细菌清除能力和吞噬率与对照组相比显著下降。此外,MjSRC具有调节抗菌肽(AMPs)基因表达的功能。综上所述,MjSRC通过增强对虾的细胞免疫和体液免疫有效地保护对虾免受细菌的侵染。
[Abstract]:Invertebrates mainly rely on the innate immune system to resist the infection of pathogens. The recognition of pathogenic microorganisms by pattern recognition receptors is the key step to initiate the innate immune response in this process.More than ten types of pattern recognition receptors have been found in prawns, but little is known about type C scavenger receptors and their functions.We used Marsupenaeus japonicus as the research object.It has been found that type C scavenger receptor can be used as an important pattern recognition receptor to promote phagocytosis of virus and a variety of bacteria in shrimp blood cells and to regulate the expression of antimicrobial peptide gene.The phagocytosis of shrimp blood cells enhances the ability of the host to resist virus. So far, the presence of type C scavenger receptor SRC has been found in many invertebrates.However, there are few reports of SRC in antiviral research.In this study, we first cloned a SRC gene from Penaeus japonicus and named it MjSRC.When the shrimp was stimulated by WSSV, the expression of MJSRC in mRNA and protein was significantly up-regulated.Knockout of MjSRC resulted in a significant increase in the content of WSSV virus.At the same time, overexpression of MjSRC could effectively increase the phagocytosis rate of blood cells, and thus reduce the content of WSSV in shrimp. Pull-down experiment showed that MjSRC could interact with the envelope protein VP19 of WSSV.The results of transmission electron microscopy showed that the MjSRC extracellular domain labeled by colloidal gold could be attached to the surface of the capsule of WSSV.After being stimulated by WSSV, MJSRC can form trimer and enter the cytoplasm with the endocytosis of the virus, which depends on the existence of Mj 尾 -arrestin2.The further results showed that the intracellular domain of MjSRC could interact with Mj 尾 -arrestin2, which mediated WSSV to enter the cytoplasm in a grid protein-dependent manner.When the lysosomal inhibitor (chloroquine) was injected into the cytoplasm, the copy number of the virus increased significantly.The results showed that MjSRC activated the phagocytosis of blood cells through its extracellular domain recognition and WSSV, thus clearing the virus in prawn.This paper reports for the first time that SRC, as a pattern recognition receptor, is involved in phagocytosis and clearance of viruses. We further studied the role of MjSRC as a pattern recognition receptor in the recognition of bacteria in Penaeus penaeus.And the molecular mechanisms of scavenging.When the shrimp was stimulated by Vibrio anguiarum) or Staphylococcus aureus (Staphylococcus aureus), the mRNA level of MjSRC was upregulated significantly in blood cells. The extracellular domain of MjSRC, MAM and CCP domain, had the function of binding different bacteria and polysaccharides from bacteria in vitro.When the expression of MjSRC was low, the bacterial clearance and phagocytic rate of blood cells of Penaeus penaeus decreased significantly compared with the control group.In addition, MjSRC has the function of regulating the gene expression of antimicrobial peptide (AMPs).In conclusion, MjSRC can effectively protect shrimp from bacterial infection by enhancing its cellular and humoral immunity.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S945.4

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