草鱼TRIM32基因克隆表达及其功能的初步研究

发布时间：2018-12-18 04:43

【摘要】：草鱼(Ctenopharyngodon idellus)是我国重要的淡水经济鱼类,在我国多个地区大规模养殖。但是随着草鱼养殖业的大力发展,草鱼的各类疾病频繁爆发,目前尚无健康有效的治疗方法,这成为草鱼养殖难以突破的瓶颈,严重阻碍了草鱼养殖业的可持续发展。在抵御各种病原体的入侵时,天然免疫系统发挥着至关重要的作用。TRIM(tripartite-motif protein)家族是机体先天性免疫的重要成员,在细胞的增殖、分化、肿瘤的抑制、病毒的抑制等方面发挥着重要的调控作用。TRIM家族蛋白,也称为RBCC家族蛋白,从N端到C端依次是RING结构域、B-box结构域、Coiled-coil结构域。在哺乳动物中TRIM32的RING结构域具有E3泛素连接酶的作用,可以调节信号通路的传导,参与机体的天然免疫反应。本论文以草鱼TRIM32为主要研究对象,旨在为草鱼疾病的防御与治疗方面提供进一步的理论依据。主要研究内容包括以下几个方面:1.利用PCR技术从草鱼的肝脏组织中克隆草鱼TRIM32基因的编码区全长,并翻译成对应的氨基酸序列。结果显示,草鱼TRIM32基因共1980 bp,编码660个氨基酸。2.对草鱼TRIM32进行结构域分析、核苷酸序列比对以及遗传进化树分析。结果显示,草鱼TRIM32具有TRIM家族3个典型的结构域,与斑马鱼TRIM32的核苷酸序列同源性较高,且遗传进化树分析也与斑马鱼TRIM32聚为一支。3.对草鱼TRIM32进行真核表达载体的构建,并通过间接免疫荧光和Western blotting分析草鱼TRIM32的表达与亚定位情况。结果显示草鱼TRIM32可以在EPC细胞中成功表达,主要以点状分布在细胞质中,而细胞核中的表达量较少。4.采用q PT-PCR检测草鱼TRIM32的组织分布,分析结果显示草鱼TRIM32在被检测的11个组织中均有表达,其中在头肾组织中的表达量明显高于其他组织。5.采用q PT-PCR检测草鱼TRIM32在不同胚胎发育时期的表达丰度,分析结果显示,草鱼TRIM32在受精卵时期、卵裂期和囊胚期的表达量显著高于其他时期(p0.05),随后表达量下降,直到出膜后表达量再次显著性升高。6.利用双荧光素酶报告系统检测草鱼TRIM32对NF-κB信号通路的影响。检测结果显示,实验组是对照组的1.66倍,说明草鱼TRIM32能够激活NF-κB信号通路,诱导下游的炎症反应。7.构建了草鱼TRIM32原核表达载体,并制备了高效价的抗草鱼TRIM32的多克隆抗体。
[Abstract]:Grass carp (Ctenopharyngodon idellus) is an important freshwater economic fish in China. However, with the vigorous development of grass carp culture, various diseases of grass carp frequently erupt, and there is no healthy and effective treatment, which has become the bottleneck of grass carp breeding, which seriously hinders the sustainable development of grass carp breeding. The innate immune system plays an important role in resisting the invasion of various pathogens. TRIM (tripartite-motif protein) family is an important member of innate immunity in the cell proliferation, differentiation, tumor suppression. TRIM family proteins, also known as RBCC family proteins, are RING domain, B-box domain and Coiled-coil domain from N-terminal to C-terminal. In mammals, the RING domain of TRIM32 has the function of E3 ubiquitin ligase, which can regulate the signaling pathway and participate in the innate immune response of the body. The purpose of this thesis is to provide further theoretical basis for the prevention and treatment of grass carp disease. The main research contents include the following aspects: 1. The coding region of TRIM32 gene of grass carp was cloned from liver tissue of grass carp by PCR technique and translated into corresponding amino acid sequence. The results showed that the TRIM32 gene of grass carp encodes 660amino acids with a total of 1980 bp,. Domain analysis, nucleotide alignment and genetic phylogenetic tree analysis of grass carp TRIM32 were carried out. The results showed that grass carp TRIM32 had three typical domains of TRIM family, and had high homology with zebrafish TRIM32, and the genetic evolutionary tree analysis was also associated with zebrafish TRIM32. The eukaryotic expression vector of grass carp TRIM32 was constructed, and the expression and sublocalization of TRIM32 in grass carp were analyzed by indirect immunofluorescence and Western blotting. The results showed that grass carp TRIM32 could be successfully expressed in EPC cells, mainly in the cytoplasm, but only in the nucleus. Q PT-PCR was used to detect the tissue distribution of grass carp TRIM32. The results showed that the expression of grass carp TRIM32 was detected in 11 tissues, and the expression level in head and kidney tissues was significantly higher than that in other tissues. Q PT-PCR was used to detect the abundance of TRIM32 in grass carp at different embryonic stages. The results showed that the expression of TRIM32 in fertilized egg, cleavage and blastocyst was significantly higher than that in other stages (p0.05), and then decreased. The expression level was significantly increased again until after membrane exudation. 6. 6%. The effects of grass carp TRIM32 on NF- 魏 B signaling pathway were detected by double luciferase reporting system. The results showed that the experimental group was 1. 66 times higher than the control group, indicating that grass carp TRIM32 could activate the NF- 魏 B signaling pathway and induce the downstream inflammatory reaction. The prokaryotic expression vector of grass carp TRIM32 was constructed and the polyclonal antibody against grass carp TRIM32 was prepared.
【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】：Q78;S917.4

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