两种凝集素参与克氏原螯虾先天性免疫反应的分子机制研究

发布时间：2018-03-22 02:07

本文选题：克氏原螯虾　切入点：L型凝集素　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：甲壳动物缺乏适应性免疫系统,主要依赖先天性免疫系统抵御病原感染。凝集素(lectin)是一类可以结合特异性糖分子的蛋白,在免疫识别和免疫应答中发挥重要的作用。C-型凝集素、L-型凝集素属于动物型凝集素。甲壳动物凝集素的研究,主要集中在C型凝集素(C-type lectin,CTL),然而L型凝集素(L-type lectin,LTL)及纤维胶凝样蛋白(Ficolin-like proteins,FLPs)凝集素的研究比较少。本课题以克氏原螯虾为模型,研究了LTL和FLPs在抵御病原感染中的功能,结果如下:1、从克氏原螯虾体内鉴定了一种新的LTL,并命名为PcL-lectin。生物信息学分析表明PcL-lectin同内质网-高尔基体交换蛋白-53 kDa(endoplasmic reticulum Golgi intermediate compartment-53 kDa,ERGIC-53)有较高的相似性。在白斑综合征病毒(WSSV)刺激后,PcL-lectin表达水平显著下降。进一步分析表明,体内注射PcL-lectin的重组蛋白(rPcL-lectin)可显著促进WSSV在克氏原螯虾体内的复制水平。而体内敲降PcL-lectin的表达水平能有效抑制WSSV在克氏原螯虾体内的复制能力。免疫细胞定位分析表明,Pc L-lectin可定位在细胞膜上。Pull-down和免疫共沉淀(Co-immunoprecipitation,Co-IP)研究表明,PcL-lectin同WSSV囊膜蛋白VP24表现出结合活性。这些结果表明WSSV的感染过程需要PcL-lectin的参与,为抗WSSV感染研究提供了新的依据。2、从克氏原螯虾体内鉴定了一种新的FLP,命名为PcFLP1。PcFLP1在血细胞、心、肝胰腺、鳃、胃和肠等组织中均有分布。在血细胞、肝胰腺和肠道中,PcFLP1被副溶血弧菌(Vibrio parahaemolyticus)诱导上调表达。PcFLP1的重组蛋白(rPcFLP1)能有效抑制副溶血弧菌(弧菌)对肝胰腺造成的损伤。细菌清除实验表明,rPcFLP1可有效增强克氏原螯虾对细菌的清除能力。在体内对PcFLP1进行敲降后,克氏原螯虾对细菌的清除能力明显减弱。PcFLP1抑菌活性结果表明PcFLP1表现出广谱的抑菌能力。这些结果表明PcFLP1在克氏原螯虾抗弧菌免疫中发挥了关键作用。
[Abstract]:Crustaceans lack an adaptive immune system and rely primarily on the innate immune system to fight off pathogenic infections. Lectin is a class of proteins that bind to specific sugar molecules. Plays an important role in immune recognition and immune response. C- lectin L- lectin belongs to animal lectin. However, the studies of L-type lectin (L-type lectin LTL) and Ficolin-like protein (Ficolin-like proteins-FLPs) lectin were few. The purpose of this study was to study the function of LTL and FLPs in resisting pathogen infection. The results are as follows: 1. A new species of LTL was identified from protochelfish, named PcL-lectin.Bioinformatics analysis showed that PcL-lectin was similar to endoplasmic reticular-Golgi exchange protein -53 kDa(endoplasmic reticulum Golgi intermediate compartment-53 kDaerGIC-53. In leukoplakia syndrome, a new type of LTL was identified as PcL-lectin.There was a high similarity between PcL-lectin and endoplasmic reticulum Golgi exchange protein (-53 kDa(endoplasmic Golgi intermediate compartment-53 kDaGIC-53). The expression of PcL-lectin was significantly decreased after WSSV stimulation. RPcL-lectin, a recombinant protein injected with PcL-lectin in vivo, could significantly promote the replication of WSSV in crayfish, and knock down the expression level of PcL-lectin in vivo could effectively inhibit the replication ability of WSSV in the body of prochelate crayfish. The localization of immunocytes was analyzed. These results suggest that Pc L-lectin can be localized on the membrane of the cell membrane. Pull-down and co-immunoprecipitation Co-IPs have shown that PcL-lectin binds to WSSV envelope protein VP24. These results suggest that PcL-lectin is necessary for the process of WSSV infection. In order to provide a new basis for the study of WSSV infection, a new PcFLP1.PcFLP1 was identified from the body of P. claricarp, named PcFLP1.PcFLP1, which was distributed in blood cells, heart, hepatopancreas, gills, stomach and intestine. In hepatopancreas and intestines, PcFLP1 was induced by Vibrio parahaemolyticus (Vibrio parahaemolyticus) to up-regulate the expression of rPcFLP1 (rPcFLP1), which can effectively inhibit the damage caused by Vibrio parahaemolyticus (Vibrio parahaemolyticus) to hepatopancreas. The ability of crayfish to remove bacteria. After knocking down PcFLP1 in vivo, The bacteriostatic activity of PcFLP1 showed that PcFLP1 showed broad-spectrum bacteriostasis. These results indicated that PcFLP1 played a key role in the immunity of P. claridinensis against Vibrio claroides.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S945.4

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