草鱼TRIM32基因克隆表达及功能研究

发布时间：2018-03-16 02:13

本文选题：TRIM　切入点：克隆　出处：《水生生物学报》2017年04期 　论文类型：期刊论文

【摘要】：TRIM(Tripartite-motif protein)家族是机体先天性免疫的重要成员,参与细胞增殖分化、细胞凋亡、肿瘤抑制、抗病毒等过程。为了进一步探究TRIM蛋白在鱼类免疫中的作用,实验利用PCR方法克隆了草鱼TRIM32基因的编码区全长,共1980 bp,编码660个氨基酸。草鱼TRIM32具有TRIM家族典型的3个结构域,与斑马鱼TRIM32的核苷酸序列同源性较高,遗传进化分析也聚为一支。间接免疫荧光、Western-blotting检测结果显示草鱼TRIM32在EPC细胞中成功表达,主要以点状分布在细胞质中,细胞核中表达量较少;组织分布分析表明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;不同胚胎发育时期的表达分析进一步表明,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。此外,双荧光素酶检测系统显示TRIM32能够激活NF-κB信号通路,诱导下游的炎症反应。结果显示,草鱼TRIM32广泛分布于鱼体内,并参与机体的天然免疫反应,对于抵抗病原体的入侵具有重要作用。
[Abstract]:TRIM(Tripartite-motif protein family is an important member of innate immune system. It is involved in cell proliferation and differentiation, apoptosis, tumor inhibition, antivirus and so on. In order to further explore the role of TRIM protein in fish immunity, The coding region of the TRIM32 gene of grass carp was cloned by PCR method, encoding 660 amino acids. The TRIM32 of grass carp has three typical domains of TRIM family, and has high homology with TRIM32 nucleotide sequence of zebrafish. The results of indirect immunofluorescence Western-blotting analysis showed that grass carp TRIM32 was successfully expressed in EPC cells, mainly in the cytoplasm with a small amount of expression in the nucleus. Tissue distribution analysis showed that the expression of TRIM32 in 11 tissues was significantly higher than that in other tissues, and the expression of TRIM32 in fertilized eggs was further demonstrated at different embryonic stages. The expression level in cleavage stage and blastocyst stage was significantly higher than that in other stages, and then decreased, and then increased again after membrane exudation. In addition, the double luciferase detection system showed that TRIM32 could activate NF- 魏 B signaling pathway. The results showed that grass carp TRIM32 was widely distributed in fish and participated in the innate immune response, which played an important role in resisting the invasion of pathogens.
【作者单位】：华中农业大学水产学院水生动物医学系;中国水产科学研究院长江水产研究所;淡水水产健康养殖湖北省协同创新中心;池塘健康养殖湖北省工程实验室;
【基金】：湖北省科技支撑计划项目(2015BBA234)资助~~
【分类号】：S917.4

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