發(fā)布時間：2018-03-22 02:07

  本文選題：克氏原螯蝦　切入點：L型凝集素　出處：《華中農(nóng)業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：甲殼動物缺乏適應性免疫系統(tǒng),主要依賴先天性免疫系統(tǒng)抵御病原感染。凝集素(lectin)是一類可以結(jié)合特異性糖分子的蛋白,在免疫識別和免疫應答中發(fā)揮重要的作用。C-型凝集素、L-型凝集素屬于動物型凝集素。甲殼動物凝集素的研究,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)及纖維膠凝樣蛋白(Ficolin-like proteins,FLPs)凝集素的研究比較少。本課題以克氏原螯蝦為模型,研究了LTL和FLPs在抵御病原感染中的功能,結(jié)果如下:1、從克氏原螯蝦體內(nèi)鑒定了一種新的LTL,并命名為PcL-lectin。生物信息學分析表明PcL-lectin同內(nèi)質(zhì)網(wǎng)-高爾基體交換蛋白-53 kDa(endoplasmic reticulum Golgi intermediate compartment-53 kDa,ERGIC-53)有較高的相似性。在白斑綜合征病毒(WSSV)刺激后,PcL-lectin表達水平顯著下降。進一步分析表明,體內(nèi)注射PcL-lectin的重組蛋白(rPcL-lectin)可顯著促進WSSV在克氏原螯蝦體內(nèi)的復制水平。而體內(nèi)敲降PcL-lectin的表達水平能有效抑制WSSV在克氏原螯蝦體內(nèi)的復制能力。免疫細胞定位分析表明,Pc L-lectin可定位在細胞膜上。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表現(xiàn)出結(jié)合活性。這些結(jié)果表明WSSV的感染過程需要PcL-lectin的參與,為抗WSSV感染研究提供了新的依據(jù)。2、從克氏原螯蝦體內(nèi)鑒定了一種新的FLP,命名為PcFLP1。PcFLP1在血細胞、心、肝胰腺、鰓、胃和腸等組織中均有分布。在血細胞、肝胰腺和腸道中,PcFLP1被副溶血弧菌(Vibrio parahaemolyticus)誘導上調(diào)表達。PcFLP1的重組蛋白(rPcFLP1)能有效抑制副溶血弧菌(弧菌)對肝胰腺造成的損傷。細菌清除實驗表明,rPcFLP1可有效增強克氏原螯蝦對細菌的清除能力。在體內(nèi)對PcFLP1進行敲降后,克氏原螯蝦對細菌的清除能力明顯減弱。PcFLP1抑菌活性結(jié)果表明PcFLP1表現(xiàn)出廣譜的抑菌能力。這些結(jié)果表明PcFLP1在克氏原螯蝦抗弧菌免疫中發(fā)揮了關(guān)鍵作用。
[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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S945.4

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