發(fā)布時間：2018-04-17 09:58

  本文選題：先天免疫 + 模式識別受體��； 參考：《山東大學》2017年博士論文

【摘要】：無脊椎動物主要依靠先天免疫系統(tǒng)抵御病原體的感染,在此過程中模式識別受體對病原微生物的識別是啟動先天免疫反應的關鍵步驟。在對蝦中,已發(fā)現(xiàn)的模式識別受體有十多種,但對C型清道夫受體及其功能了解的還不多。我們以日本囊對蝦Marsupenaeus japonicus為研究對象,發(fā)現(xiàn)了 C型清道夫受體可以作為一種重要的模式識別受體促進對蝦血細胞對病毒和多種細菌的吞噬作用以及調節(jié)抗菌肽基因的表達。(1)C型清道夫受體通過增強對蝦血細胞的吞噬作用提高宿主抗病毒的能力至今已在多種無脊椎動物中發(fā)現(xiàn)有C型清道夫受體(SRC)的存在,然而SRC在抗病毒方面的研究報道還很少。在本研究中,我們首次從日本囊對蝦,Mjaponicus,中克隆到了一種SRC基因,并將其命名為MjSRC。當對蝦受到白斑綜合征病毒(WSSV)的刺激以后,MjSRC在mRNA以及蛋白水平呈顯著的上調表達。敲低MjSRC導致WSSV病毒的含量顯著增加。同時,過表達MjSRC會有效地提高血細胞的吞噬率,從而降低對蝦體內WSSV的含量。Pull-down實驗結果表明MjSRC可以與WSSV的囊膜蛋白VP19相互作用。透射電鏡的觀察結果顯示膠體金標記的MjSRC胞外域可以附著在WSSV的囊膜表面。在受到WSSV刺激后,MjSRC可以形成三聚體并伴隨病毒的內吞而進入細胞質,這類內化過程依賴Mjβ-arrestin2的存在。進一步的實驗結果表明MjSRC的胞內域可以與Mjβ-arrestin2相互作用,進而介導WSSV以網(wǎng)格蛋白依賴的方式進入細胞質。進入細胞質的病毒與溶酶體融合,當注射溶酶體抑制劑(氯喹)以后,病毒的拷貝數(shù)顯著增加。本研究結果顯示MjSRC通過其胞外域識別并結合WSSV,啟動血細胞的吞噬作用,從而清除對蝦體內的病毒。本文首次報道了 SRC作為一種模式識別受體參與對病毒吞噬和清除。(2)MjSRC在對蝦抗細菌感染中的功能研究我們進一步研究了 MjSRC作為模式識別受體參與細菌的的識別和清除的分子機制。當對蝦受到弧菌(Vibrio anguiarum)或金黃色葡萄球菌(Staphylococcusaureus)刺激以后,MjSRC的mRNA水平在血細胞中顯著上調表達。MjSRC的胞外域(MAM和CCP結構域)具有體外結合不同細菌以及細菌來源多糖的功能。在敲低MjSRC表達的情況下,對蝦血細胞的細菌清除能力和吞噬率與對照組相比顯著下降。此外,MjSRC具有調節(jié)抗菌肽(AMPs)基因表達的功能。綜上所述,MjSRC通過增強對蝦的細胞免疫和體液免疫有效地保護對蝦免受細菌的侵染。
[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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S945.4

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本文編號：1763120