CAJ | 학술논문

为探讨Nramp基因在草鱼(Ctenopharyngodon idellus)免疫应答中的作用,采用实时荧光定量PCR技术,对比研究了Nramp基因在草鱼不同发育阶段、不同组织及嗜水气单胞菌(Aeromonas hydrophila)攻毒感染后4个组织不同时间的差异表达,并利用原核表达技术对草鱼Nramp基因 ORF区642 bp的目的片段进行蛋白表达,获得40 kD的融合蛋白。实时荧光定量PCR分析结果表明, Nramp在草鱼胚胎发育过程中的表达量随着发育时间的增加而逐渐增强,在幼鱼期的表达量达到最大。成年健康草鱼不同组织中的表达量差异显著(P<0.05),在肝中的表达量最高,其次是在脾、头肾、肠中的表达量较高,而在眼和鳔中的表达量最低。草鱼感染嗜水气单胞菌后,在头肾、肝、脾、肠中Nramp的表达量都显著上升(P<0.05),在头肾中感染12 h后显著上升,48 h达到最大,在72 h和96 h时又显著下调,在7 d时表达量继续下调而逐渐回归到初始水平；在肝中Nramp基因的表达量在4 h时出现下调,在12 h又显著上调,随后在24 h出现下调,在48 h又上调,72 h达到最大并保持较高水平到7 d；在肠中mRNA表达量在12 h显著上调,48 h达到最大,在72 h和96 h显著下调,在7 d时表达量基本回归到初始水平；在脾中mRNA表达量在4h开始上升,12h达到最大,在24h和48h表达水平显著下调,在7d时表达量回归到初始水平。草鱼感染致病菌可引起免疫相关组织中Nramp的表达量上升,说明Nramp在硬骨鱼类的天然免疫反应过程中发挥着重要作用。
위탐토Nramp기인재초어(Ctenopharyngodon idellus)면역응답중적작용,채용실시형광정량PCR기술,대비연구료Nramp기인재초어불동발육계단、불동조직급기수기단포균(Aeromonas hydrophila)공독감염후4개조직불동시간적차이표체,병이용원핵표체기술대초어Nramp기인 ORF구642 bp적목적편단진행단백표체,획득40 kD적융합단백。실시형광정량PCR분석결과표명, Nramp재초어배태발육과정중적표체량수착발육시간적증가이축점증강,재유어기적표체량체도최대。성년건강초어불동조직중적표체량차이현저(P<0.05),재간중적표체량최고,기차시재비、두신、장중적표체량교고,이재안화표중적표체량최저。초어감염기수기단포균후,재두신、간、비、장중Nramp적표체량도현저상승(P<0.05),재두신중감염12 h후현저상승,48 h체도최대,재72 h화96 h시우현저하조,재7 d시표체량계속하조이축점회귀도초시수평；재간중Nramp기인적표체량재4 h시출현하조,재12 h우현저상조,수후재24 h출현하조,재48 h우상조,72 h체도최대병보지교고수평도7 d；재장중mRNA표체량재12 h현저상조,48 h체도최대,재72 h화96 h현저하조,재7 d시표체량기본회귀도초시수평；재비중mRNA표체량재4h개시상승,12h체도최대,재24h화48h표체수평현저하조,재7d시표체량회귀도초시수평。초어감염치병균가인기면역상관조직중Nramp적표체량상승,설명Nramp재경골어류적천연면역반응과정중발휘착중요작용。
To explore the function of Nramp in the grass carp (Ctenopharyngodon idellus) immune system, gene expression was analyzed in different developmental stages and tissues. Four tissues of grass carp at different times after challenge with the pathogenic bacterium Aeromonas hydrophila were analyzed by real-time quantitative RT-PCR. The 642 bp Open Reading Frame (ORF) region of grass carp Nramp was expressed using prokaryotic expression technology. The fusion protein had a relative molecular mass of 40 kD. Real-time quantitative RT-PCR analysis indicated that the Nramp mRNA level in embryos gradually increased during embryogenesis from cleav-age to fry stages after fertilization, with the highest level in the fry stage. In adult fish, the Nramp mRNA level in different tissues significantly differed; Nramp transcripts were found to be highly abundant in liver; moderately abundant in the head kidney, spleen, and intestine;and least abundant in the eye and bladder. After challenge with A. hydrophil, with uninjected groups as the control, the Nramp mRNA level in the head kidney, liver, spleen, and intestine significantly increased (P<0.05). There was significant up-regulation in head kidney at 12 h post-treatment, with a maximum level at 48 h post-infection;significant down-regulation at 72 h and 96 h;and the expression regressed to the initial level by 7 d. Nramp expression in the liver was significantly increased at 12 h post-treatment;followed by a decrease at 24 h post-treatment;another increase at 48 h, with a maximum level at 72 h post-infection;and the high expression level was maintained at 96 h and 7 d post-treatment. There was sig-nificant up-regulation in the intestine at 12 h post-treatment, with a maximum level at 48 h post-treatment; sig-nificant down-regulation at 72 h and 96 h; and the expression regressed to the initial level by 7 d. In the spleen, Nramp mRNA level increased at 4 h post-treatment, with a maximum level at 12 h; then, there was significant down-regulation at 24 h and 48 h;and the expression regressed to the initial level by 7 d. Bacterial infection can increase Nramp expression in immune-related organs, which may indicate that Nramp plays an important role in the innate immune response of grass carp.