皱纹盘鲍丝氨酸蛋白酶的基因克

发布时间：2018-03-31 04:34

本文选题：皱纹盘鲍　切入点：丝氨酸蛋白酶　出处：《集美大学》2017年硕士论文

【摘要】：鲍鱼作为中国传统名贵海产品,一直受到人们的喜爱。随着我国经济快速增长以及人们生活水平的提高,鲍鱼的需求量不断增加,同时,也带动了鲍鱼养殖业的蓬勃发展。但是,海水污染、高密度养殖等因素导致鲍鱼疾病时常发生,造成巨大的经济损失。鲍鱼病害的防治已成为鲍鱼养殖业中亟待解决的问题,其中,副溶血弧菌一直是鲍鱼养殖的主要病原。先天免疫是鲍鱼抵抗病原侵袭的主要途径。因此,深入探索参与鲍鱼先天性免疫反应的关键基因和蛋白的功能对于解决鲍鱼病害具有特别重要的意义。前期研究提示,丝氨酸蛋白酶在鲍鱼先天免疫中扮演重要的角色。本研究根据其他软体动物丝氨酸蛋白酶基因的保守序列设计特异性引物,采用巢式PCR和RACE技术从皱纹盘鲍的肝脏组织克隆获得了丝氨酸蛋白酶(Haliotis discus hanai serine proteinase,Hdh-SP)基因c DNA全长序列,序列分析结果显示Hdh-SP c DNA全长共1107 bp,包含5’UTR 111 bp,3’UTR 39 bp,ORF 957 bp,编码319个氨基酸残基。通过序列比对结果显示,该氨基酸序列与太平洋牡蛎(Crassostrea gigas)、栉孔扇贝(Azumapecten farreri)、缢蛏(Sinonovacula constricta)和马氏珠母贝(Pinctada fucata)中丝氨酸蛋白酶的同源性分别为54%、50%、47%和40%。Hdh-SP基因编码蛋白的预测相对分子质量为34.0 k Da,理论等电点为6.89,属于亲水蛋白,预测其N-末端为信号肽。该蛋白的二级结构以延伸链和无规卷曲为主,其中α-螺旋占15.72%,无规卷曲占50.63%,延伸链占33.65%。根据系统发育关系分析Hdh-SP与太平洋牡蛎丝氨酸蛋白酶具有较高的保守性并且亲缘关系最近。构建了原核重组表达载体p ET28a-SP,使用原核表达系统表达并纯化出重组Hdh-SP,用于制备兔抗鲍鱼丝氨酸蛋白酶的多克隆抗体。构建昆虫细胞重组表达载体,利用昆虫-杆菌表达系统表达出可溶的Hdh-SP,SDS-PAGE显示其分子量为30 k Da,与预测的分子量大小相吻合,Western blot结果显示兔抗鲍鱼丝氨酸蛋白酶的多克隆抗体与Hdh-SP有很好的的免疫杂交反应,且大部分位于胞浆内。使用荧光定量PCR与Western blot技术检测Hdh-SP在鲍鱼血淋巴、肝胰腺、肌肉、性腺、鳃、外套膜等组织的基因和蛋白水平的表达情况。结果显示,肝胰腺组织中Hdh-SP基因水平和蛋白水平的表达量明显高于其他组织。用副溶血弧菌侵染鲍鱼之后,其血淋巴细胞内Hdh-SP基因水平与蛋白水平都有不同程度上调,因此推断Hdh-SP参与鲍鱼免疫反应。同时,用副溶血弧菌刺激鲍鱼之后,发现与鲍鱼免疫密切相关的四个免疫因子细胞凋亡酶-8(cysteinyl aspartate specific proteinase 8,Caspase 8)、异体移植炎症因子(Allograft inflammatory factor,ALIn Fa)、巨噬细胞表达蛋白(macrophage expressed protein,MEP)、转录因子(Nuclear factorκB,Rel/NF-κB)在血淋巴细胞内不同程度明显上调,说明这四个免疫因子参与到副溶血弧菌侵染后的免疫反应中。为了进一步了解Hdh-SP在免疫调节方面的作用,采用RNAi技术沉默Hdh-SP基因。结果显示,Hdh-SP的表达被抑制后,Rel/NF-κB、MEP、ALIn Fa和Caspase 8这四个免疫因子基因的相对表达量都会不同程度下降,提示在鲍鱼免疫反应中,Hdh-SP可能是通过调控免疫因子的表达来参与免疫调节。鲍鱼血淋巴细胞凋亡检测结果表明当Hdh-SP基因表达受到抑制后,血淋巴细胞的凋亡比例明显下降,说明Hdh-SP通过调控Caspase 8基因的表达而参与调控鲍鱼血淋巴细胞凋亡。本论文围绕Hdh-SP在鲍鱼先天性免疫反应中的作用展开研究。结果显示,副溶血弧菌侵染皱纹盘鲍后,Hdh-SP基因与蛋白水平显著上调表达。Hdh-SP通过促进Rel/NF-κB基因表达,启动下游MEP、ALIn Fa和Caspase 8基因的转录从而调节免疫反应来抵御致病菌入侵。本文的研究结果对于理解鲍鱼的先天性免疫反应具有重要意义,也为鲍鱼疾病的防治策略提供了理论参考。
[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.

【学位授予单位】：集美大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S943

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