本文選題：皺紋盤鮑　切入點：絲氨酸蛋白酶　出處：《集美大學》2017年碩士論文

【摘要】：鮑魚作為中國傳統(tǒng)名貴海產品,一直受到人們的喜愛。隨著我國經濟快速增長以及人們生活水平的提高,鮑魚的需求量不斷增加,同時,也帶動了鮑魚養(yǎng)殖業(yè)的蓬勃發(fā)展。但是,海水污染、高密度養(yǎng)殖等因素導致鮑魚疾病時常發(fā)生,造成巨大的經濟損失。鮑魚病害的防治已成為鮑魚養(yǎng)殖業(yè)中亟待解決的問題,其中,副溶血弧菌一直是鮑魚養(yǎng)殖的主要病原。先天免疫是鮑魚抵抗病原侵襲的主要途徑。因此,深入探索參與鮑魚先天性免疫反應的關鍵基因和蛋白的功能對于解決鮑魚病害具有特別重要的意義。前期研究提示,絲氨酸蛋白酶在鮑魚先天免疫中扮演重要的角色。本研究根據(jù)其他軟體動物絲氨酸蛋白酶基因的保守序列設計特異性引物,采用巢式PCR和RACE技術從皺紋盤鮑的肝臟組織克隆獲得了絲氨酸蛋白酶(Haliotis discus hanai serine proteinase,Hdh-SP)基因c DNA全長序列,序列分析結果顯示Hdh-SP c DNA全長共1107 bp,包含5’UTR 111 bp,3’UTR 39 bp,ORF 957 bp,編碼319個氨基酸殘基。通過序列比對結果顯示,該氨基酸序列與太平洋牡蠣(Crassostrea gigas)、櫛孔扇貝(Azumapecten farreri)、縊蟶(Sinonovacula constricta)和馬氏珠母貝(Pinctada fucata)中絲氨酸蛋白酶的同源性分別為54%、50%、47%和40%。Hdh-SP基因編碼蛋白的預測相對分子質量為34.0 k Da,理論等電點為6.89,屬于親水蛋白,預測其N-末端為信號肽。該蛋白的二級結構以延伸鏈和無規(guī)卷曲為主,其中α-螺旋占15.72%,無規(guī)卷曲占50.63%,延伸鏈占33.65%。根據(jù)系統(tǒng)發(fā)育關系分析Hdh-SP與太平洋牡蠣絲氨酸蛋白酶具有較高的保守性并且親緣關系最近。構建了原核重組表達載體p ET28a-SP,使用原核表達系統(tǒng)表達并純化出重組Hdh-SP,用于制備兔抗鮑魚絲氨酸蛋白酶的多克隆抗體。構建昆蟲細胞重組表達載體,利用昆蟲-桿菌表達系統(tǒng)表達出可溶的Hdh-SP,SDS-PAGE顯示其分子量為30 k Da,與預測的分子量大小相吻合,Western blot結果顯示兔抗鮑魚絲氨酸蛋白酶的多克隆抗體與Hdh-SP有很好的的免疫雜交反應,且大部分位于胞漿內。使用熒光定量PCR與Western blot技術檢測Hdh-SP在鮑魚血淋巴、肝胰腺、肌肉、性腺、鰓、外套膜等組織的基因和蛋白水平的表達情況。結果顯示,肝胰腺組織中Hdh-SP基因水平和蛋白水平的表達量明顯高于其他組織。用副溶血弧菌侵染鮑魚之后,其血淋巴細胞內Hdh-SP基因水平與蛋白水平都有不同程度上調,因此推斷Hdh-SP參與鮑魚免疫反應。同時,用副溶血弧菌刺激鮑魚之后,發(fā)現(xiàn)與鮑魚免疫密切相關的四個免疫因子細胞凋亡酶-8(cysteinyl aspartate specific proteinase 8,Caspase 8)、異體移植炎癥因子(Allograft inflammatory factor,ALIn Fa)、巨噬細胞表達蛋白(macrophage expressed protein,MEP)、轉錄因子(Nuclear factorκB,Rel/NF-κB)在血淋巴細胞內不同程度明顯上調,說明這四個免疫因子參與到副溶血弧菌侵染后的免疫反應中。為了進一步了解Hdh-SP在免疫調節(jié)方面的作用,采用RNAi技術沉默Hdh-SP基因。結果顯示,Hdh-SP的表達被抑制后,Rel/NF-κB、MEP、ALIn Fa和Caspase 8這四個免疫因子基因的相對表達量都會不同程度下降,提示在鮑魚免疫反應中,Hdh-SP可能是通過調控免疫因子的表達來參與免疫調節(jié)。鮑魚血淋巴細胞凋亡檢測結果表明當Hdh-SP基因表達受到抑制后,血淋巴細胞的凋亡比例明顯下降,說明Hdh-SP通過調控Caspase 8基因的表達而參與調控鮑魚血淋巴細胞凋亡。本論文圍繞Hdh-SP在鮑魚先天性免疫反應中的作用展開研究。結果顯示,副溶血弧菌侵染皺紋盤鮑后,Hdh-SP基因與蛋白水平顯著上調表達。Hdh-SP通過促進Rel/NF-κB基因表達,啟動下游MEP、ALIn Fa和Caspase 8基因的轉錄從而調節(jié)免疫反應來抵御致病菌入侵。本文的研究結果對于理解鮑魚的先天性免疫反應具有重要意義,也為鮑魚疾病的防治策略提供了理論參考。
[Abstract]:As a famous traditional Chinese abalone seafood, has been loved by the people. With the rapid growth of China's economy and the improvement of people's living standard, the demand of abalone is increasing, at the same time, also led to the rapid development of abalone aquaculture. However, water pollution, high density culture and other factors lead to diseases of abalone occurred frequently, causing huge economic loss. Prevention of abalone diseases has become an urgent problem, which in abalone aquaculture, Vibrio parahaemolyticus has been the main pathogen of abalone breeding. Innate immunity is the main way to resist pathogen invasion of abalone. Therefore, exploring the key genes and proteins involved in the innate immune response of abalone function is of special significance for the to solve the abalone diseases. Previous studies have suggested that serine proteases play an important role in the innate immunity of abalone. This research is based on other software Conserved sequence specific primers were designed animal serine protease gene, the serine protease obtained from liver tissue cloned abalone by nested PCR and RACE (Haliotis discus Hanai serine proteinase, Hdh-SP) C sequence DNA, sequence analysis results showed that Hdh-SP C DNA contained 1107 BP, including 5 'UTR 111 BP 3, UTR 39 BP, ORF 957 BP, encoding 319 amino acid residues. The sequence alignment showed that the amino acid sequence of the Pacific Oyster (Crassostrea gigas), Chlamys Scallop in Shell (Azumapecten farreri), sinonovaculaconstricta (Sinonovacula constricta) and pinctadamartensii (Pinctada fucata) in serine protease homology respectively 54%, 50%, 47% and 40%.Hdh-SP genes encoding predicted protein relative molecular mass of 34 K Da, isoelectric point was 6.89, belonging to the hydrophilic protein, predict its N- terminal signal peptide of the protein. The two stage structure to extended strand and random coil, the alpha helix random coil accounted for 15.72%, accounting for 50.63%, accounting for 33.65%. chain extension according to phylogenetic analysis of the Hdh-SP and the Pacific oyster highly conserved serine protease and the closest genetic relationship. Constructed recombinant prokaryotic expression vector p ET28a-SP, prokaryotic expression system to express and purify recombinant Hdh-SP for preparation of polyclonal antibody of Rabbit anti abalone serine protease. Construction of recombinant expression vector in insect cells, using insect - coli expression system to express soluble Hdh-SP, SDS-PAGE showed that the molecular weight of 30 K Da, consistent with the predicted molecular weight of Western, blot showed more polyclonal antibody and Hdh-SP Rabbit anti abalone serine protease has good hybridization reaction, most of which located in the cytoplasm. Using fluorescence quantitative PCR and Western blot Technology Detection of Hdh-SP in abalone hemolymph, hepatopancreas, muscle, gill, gonad, mantle tissue expression of gene and protein levels. The results showed that the expression of hepatic and pancreatic tissues in Hdh-SP gene and protein level was significantly higher than in other tissues. After infected with Vibrio parahaemolyticus abalone, its introlymphocytic Hdh-SP gene and the protein levels have different degrees of increase, so that Hdh-SP participate in the abalone immune response. At the same time, after stimulation with Vibrio parahaemolyticus found closely related with abalone, abalone immune four immune cell apoptosis factor -8 (cysteinyl aspartate specific enzyme proteinase 8, Caspase 8), Allograft inflammatory (allogeneic transplantation inflammatory factor factor, ALIn Fa), macrophage expressed protein (macrophage expressed protein MEP (Nuclear factor), transcription factor kappa B, Rel/NF- K B) in blood lymphocytes in Cheng Duming This shows that the immune response significantly up-regulated, four immune factors involved in Vibrio parahaemolyticus infection. In order to further understand the Hdh-SP regulatory role in immune, silencing the Hdh-SP gene by RNAi. The results showed that the expression of Hdh-SP was inhibited, Rel/NF- kappa B, MEP, Fa relative expression of ALIn and Caspase 8 four immune factor gene will be reduced to different degrees, suggesting that the immune response in the abalone, Hdh-SP may be through regulating the expression of immune factor in immune regulation. Abalone blood lymphocyte apoptosis test results showed that when Hdh-SP gene expression was inhibited, the apoptosis rate of lymphocytes decreased significantly, indicating that Hdh-SP regulate the expression of Caspase 8 gene participate in the apoptosis of lymphocytes in abalone blood control. This dissertation focuses on the role of Hdh-SP in abalone innate immune response. The results showed that Vibrio parahaemolyticus The infection of abalone, Hdh-SP gene and protein level of.Hdh-SP was significantly up-regulated by promoting Rel/NF- kappa B gene expression, promoter downstream MEP, transcription of ALIn Fa and Caspase 8 genes which regulate the immune response against pathogen invasion. The results of this study have important significance for understanding the innate immune responses of abalone, is also provided the theoretical reference for the prevention and control strategy of abalone diseases.

【學位授予單位】：集美大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S943

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