草鱼TRIM32基因克隆表达及功能研究

THE PRELIMINARY STUDY ON CLONING, EXPRESSION PROFILES AND FUNCTION OF GRASS CARP(CTENOPHARYNGODON IDELLUS) TRIM32

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(P<0.05),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。

The TRIM family, an important member in natural immune system, regulates cell proliferation, differentia-tion, apoptosis, tumor suppression, antiviral activity and other processes. The TRIM32 full-length sequence of grasscarp （Ctenopharyngodon idellus） was cloned by using PCR method. It was 1980 bp in length encoding 660 aminoacids. The TRIM32 protein has three typical structural features of the TRIM family. Sequence analysis indicated thatTRIM32 of grass carp shared the highest identity with zebrafish, which belongs to the same branch on the phylogenetictrees. Immunofluorescence and Western-blotting showed that TRIM32 successfully expressed in cytoplasmic specklesof EPC cells. Tssue distribution analysis showed that TRIM32 was widely expressed, especially in the renal tissue.TRIM32 was significantly higher in zygote period, cleavage and blastula stage than that of the other stages during dif-ferent stages of embryonic development, and then decreased until hatching in which it increased significantly. In addi-tion, grass carp TRIM32 can activate the nuclear factor NF-κB based on dual luciferase assay and induce inflammation.