草鱼MHC相关多肽与肿瘤坏死因子受体相关因子的克隆及其表达分析
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摘要
草鱼(Ctenopharyngodon idellus)是我国重要的淡水养殖鱼类。在草鱼养殖中,疾病是限制其养殖生产的一个重要因子,免疫防治是控制疾病的重要而有效的途径。本文对草鱼三个免疫因子进行了克隆鉴定以及表达分析,这三个基因涉及到糖基化修饰、肿瘤免疫以及细胞凋亡三个方面。
通过RACE技术获得草鱼SIMP以及两个凋亡相关基因的cDNA全长,并采用PCR和Genome Walking的方法克隆了SIMP启动子区域,对SIMP进行了基因组结构分析。
草鱼免疫优势MHC相关多肽(gcSIMP) cDNA序列全长为4384 bp。其中5′、3′非编码区分别为1117 bp、849 bp,开放阅读框包含2418 bp,编码805个氨基酸。获得的gcSIMP基因组全长约为24212bp,整个基因组结构跨越16个外显子和15个内含子。启动子区域缺乏典型的TATA或CAAT结构,但存在GCN4, YY1, Sp1, Palpha, TBP, Hb,C / EBPalp,C / EBPbeta,Oct-1和AP-1等潜在的转录因子结合位点。
草鱼肿瘤坏死因子受体相关因子1(gcTRAF1)基因的cDNA序列全长为2235 bp。其中5′、3′非编码区分别为250 bp、326 bp,开放阅读框包含1659 bp,编码552个氨基酸。
草鱼肿瘤坏死因子受体相关因子2(gcTRAF2)全长为3162 bp。其中5′、3′非编码区分别为60 bp、1491 bp,开放阅读框包含1611 bp,编码536个氨基酸。
对三个基因转录和表达产物的器官组织分布进行了详细的分析。Real time PCR表明这三个基因在草鱼组织中呈组成型表达,且三者具有不同的组织分布模式。分别构建gcSIMP、gcTRAF1和gcTRAF2基因的原核表达质粒,通过原核表达、表达蛋白纯化、并制备多克隆抗体,对脑、鳃、心脏、头肾、中肾、肝脏、肌肉、脾脏、和胸腺等器官中的目的基因蛋白进行免疫印迹分析。Western blotting结果显示gcTRAF1和gcTRAF2蛋白在各个组织中广泛表达。两种分析方法均显示了在心脏中目的基因gcTRAF1和gcTRAF2的表达量最高,在中肾中的表达量最低。
The grass carp (Ctenopharyngoden idellus) is an important species in aquaculture industry in China. The culture of grass carp has been threatened seriously by many kinds of diseases. It is thus important and necessary to develop some effective methods for the control of diseases. The present study aimed to clone some immune genes for the grass carp, with their expression analysis. The three immune genes obtained are concerned with glycosylation modification, tumor immune and cell apoptosis.
The SIMP cDNA full sequence of grass carp has been cloned using RACE-PCR and PCR. The genomic structure including the promoter region has been obtained by PCR and Genome Walking method. In addition, two apoptosis related genes were cloned for the first time in the grass carp, i.e. TNFR-associated factor 1 (gcTRAF1) and TNFR-associated factor 2 (gcTRAF2).
The gcSIMP full length cDNA is 4384 bp, including a 1117 bp 5′UTR (untranslated region), a 2418 bp open reading frame, and a 849 bp 3′UTR. The putative protein of gcSIMP is 805 aa. The gcSIMP spans over more than 24,212 bp in length, containing 16 exons and 15 introns. The gcSIMP promoter contains many putative transcription factor binding sites, such as Oct-1, GCN4, YY1, Sp1, Palpha, TBP, GATA-1, C / EBPbeta, and five C/ EBPalp binding sites. The gcTRAF1 full length cDNA is 2235 bp, including a 250 bp 5′UTR (untranslated region), a 1659 bp open reading frame, and a 326 bp 3′UTR. The putative protein of gcTRAF1 is 552 aa. The gcTRAF2 full length cDNA is 3162 bp, including a 60 bp 5′UTR (untranslated region), a 1611 bp open reading frame, and a 1491 bp 3′UTR. The putative protein of gcTRAF2 is 536 aa.
The organ distribution of the three genes was analyzed both in the transcription and protein levels. By real time PCR analysis, the transcription products of the cloned genes were detected in different tissues of healthy grass carp, and the three genes had different patterns of tissue distribution. Western blotting analyses revealed that both gcTRAF1 and gcTRAF2 protein existed broadly in examined tissues with the highest expression level in heart and lowest in trunk kidney.
通过RACE技术获得草鱼SIMP以及两个凋亡相关基因的cDNA全长,并采用PCR和Genome Walking的方法克隆了SIMP启动子区域,对SIMP进行了基因组结构分析。
草鱼免疫优势MHC相关多肽(gcSIMP) cDNA序列全长为4384 bp。其中5′、3′非编码区分别为1117 bp、849 bp,开放阅读框包含2418 bp,编码805个氨基酸。获得的gcSIMP基因组全长约为24212bp,整个基因组结构跨越16个外显子和15个内含子。启动子区域缺乏典型的TATA或CAAT结构,但存在GCN4, YY1, Sp1, Palpha, TBP, Hb,C / EBPalp,C / EBPbeta,Oct-1和AP-1等潜在的转录因子结合位点。
草鱼肿瘤坏死因子受体相关因子1(gcTRAF1)基因的cDNA序列全长为2235 bp。其中5′、3′非编码区分别为250 bp、326 bp,开放阅读框包含1659 bp,编码552个氨基酸。
草鱼肿瘤坏死因子受体相关因子2(gcTRAF2)全长为3162 bp。其中5′、3′非编码区分别为60 bp、1491 bp,开放阅读框包含1611 bp,编码536个氨基酸。
对三个基因转录和表达产物的器官组织分布进行了详细的分析。Real time PCR表明这三个基因在草鱼组织中呈组成型表达,且三者具有不同的组织分布模式。分别构建gcSIMP、gcTRAF1和gcTRAF2基因的原核表达质粒,通过原核表达、表达蛋白纯化、并制备多克隆抗体,对脑、鳃、心脏、头肾、中肾、肝脏、肌肉、脾脏、和胸腺等器官中的目的基因蛋白进行免疫印迹分析。Western blotting结果显示gcTRAF1和gcTRAF2蛋白在各个组织中广泛表达。两种分析方法均显示了在心脏中目的基因gcTRAF1和gcTRAF2的表达量最高,在中肾中的表达量最低。
The grass carp (Ctenopharyngoden idellus) is an important species in aquaculture industry in China. The culture of grass carp has been threatened seriously by many kinds of diseases. It is thus important and necessary to develop some effective methods for the control of diseases. The present study aimed to clone some immune genes for the grass carp, with their expression analysis. The three immune genes obtained are concerned with glycosylation modification, tumor immune and cell apoptosis.
The SIMP cDNA full sequence of grass carp has been cloned using RACE-PCR and PCR. The genomic structure including the promoter region has been obtained by PCR and Genome Walking method. In addition, two apoptosis related genes were cloned for the first time in the grass carp, i.e. TNFR-associated factor 1 (gcTRAF1) and TNFR-associated factor 2 (gcTRAF2).
The gcSIMP full length cDNA is 4384 bp, including a 1117 bp 5′UTR (untranslated region), a 2418 bp open reading frame, and a 849 bp 3′UTR. The putative protein of gcSIMP is 805 aa. The gcSIMP spans over more than 24,212 bp in length, containing 16 exons and 15 introns. The gcSIMP promoter contains many putative transcription factor binding sites, such as Oct-1, GCN4, YY1, Sp1, Palpha, TBP, GATA-1, C / EBPbeta, and five C/ EBPalp binding sites. The gcTRAF1 full length cDNA is 2235 bp, including a 250 bp 5′UTR (untranslated region), a 1659 bp open reading frame, and a 326 bp 3′UTR. The putative protein of gcTRAF1 is 552 aa. The gcTRAF2 full length cDNA is 3162 bp, including a 60 bp 5′UTR (untranslated region), a 1611 bp open reading frame, and a 1491 bp 3′UTR. The putative protein of gcTRAF2 is 536 aa.
The organ distribution of the three genes was analyzed both in the transcription and protein levels. By real time PCR analysis, the transcription products of the cloned genes were detected in different tissues of healthy grass carp, and the three genes had different patterns of tissue distribution. Western blotting analyses revealed that both gcTRAF1 and gcTRAF2 protein existed broadly in examined tissues with the highest expression level in heart and lowest in trunk kidney.
引文
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