草鱼免疫相关基因Prkrip1的克隆和表达分析

随着草鱼养殖规模的扩大, 草鱼的病毒性疾病极大地影响着草鱼的产量。开展鱼类病毒免疫反应相关功能基因的研究意义重大。研究首先通过同源克隆的方法从草鱼中克隆到了一段Prkrip1基因的EST序列, 进一步通过RACE、长片段PCR和Genome walking的方法获得了该基因的全长cDNA序列、基因组DNA序列和启动子区序列。氨基酸序列分析显示, Prkrip1含有3个核定位信号和一个双链RNA结合区, 并具有与PKR结合的保守N端区; 荧光报告基因的表达证实我们所克隆到的启动子区是有活性的, 可用于后续该基因的转录调控分析; Real-time PCR分析发现, Prkrip1 基因在草鱼的肝和血中表达量最高, GCRV感染后在大部分免疫组织中均上调表达, 说明该基因确实与病毒感染相关。研究结果为Prkrip1基因在硬骨鱼类的功能研究提供了线索, 也为鱼类天然免疫反应中调控PKR信号通路的系统研究提供了理论依据。       

草鱼CD81基因的克隆及功能分析

为研究白细胞表面分化抗原81(CD81)的功能, 对草鱼CD81进行了克隆, CD81全长共1376 bp, 其中5'非翻译区87 bp, 3'非翻译区581 bp, 开放阅读框为708 bp, 包括8个外显子, 7个内含子, 编码235个氨基酸。实验采用实时荧光定量PCR的方法检测了CD81在健康草鱼不同组织中的表达情况及草鱼出血病病毒(GCRV)攻毒前后的表达变化情况。结果显示草鱼CD81在所有被检测组织中均有表达, 在头肾中表达量最高。在GCRV攻毒前后草鱼鳃、脾、肝、肠及头肾5个组织中的CD81表达量均有明显变化。同时, 采用绿色荧光蛋白(GFP)来示踪CD81的亚细胞表达部位, 激光共聚焦显微镜显示, 同人类一样, 草鱼CD81定位于细胞膜上。       

Structure,organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3′ and 5′ RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5′-terminal untranslated region (UTR), a 355 bp 3′-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74–96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1-k long genomic DNA of carp NKEF-B containing six exons and five introns. Real-time RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity.     

草鱼钠磷协同转运载体Slc34a2基因的克隆及组织表达分析

为了揭示草鱼对磷的吸收机制,运用RT-PCR和快速扩增cDNA末端方法,从草鱼（Ctenopharyngodon idella）肠中克隆获得钠磷协同转运载体基因Slc34a2,该基因全长为2446 bp,包含了1938 bp的开放阅读框,47 bp的5非编码区（Untranslated region,UTR）和461 bp的3UTR,编码645个氨基酸。草鱼SLC34A2蛋白的分子式为C3215H5125N801O902S30,分子量大小为70.39 kD,等电点为5.68,总平均疏水指数为0.458。对草鱼SLC34A2蛋白结构和功能预测分析,发现SLC34A2蛋白有11个跨膜域,1个半胱氨酸富集区,且N-端在胞外而C-端在胞内,也在第二个细胞外环中发现4个N-糖基化位点。用邻接法构建系统进化树,发现草鱼Slc34a2基因与硬骨鱼类聚类为一支,且草鱼SLC34A2蛋白与鲤（Cyprinus carpio）和斑马鱼（Danio rerio）SLC34A2的相似性最高,分别为90.3%和87.0%。实验采用了实时荧光定量PCR对草鱼Slc34a2 mRNA进行组织表达分析,结果表明Slc34a2 mRNA在组织中广谱表达,且在肠中表达最高,其次是肝脏、鳃、肾脏、脾脏、皮肤、肌肉、脑和头肾。实验为以后研究提高鱼对磷的利用和减少磷的排放奠定分子基础。     

Cloning, characterization and expression analysis of a novel CXC chemokine from turbot (Scophthalmus maximus)

Chemokines represent a superfamily of chemotactic cytokines playing an important role in leucocyte chemotaxis. Here we report a novel turbot CXC chemokine screened from a turbot spleen cDNA library. The complete cDNA of the turbot CXC chemokine contains an 81bp 5' UTR, a 414bp open reading frame (ORF) encoding 137 amino acids and a 449bp 3' UTR. Four exons and three introns are identified in the turbot CXC chemokine gene. Phylogenetic analysis showed that the turbot CXC chemokine clustered apart from all other CXC chemokines. RT-PCR demonstrated that turbot CXC chemokine was expressed highly in spleen and head kidney. During the early stages of embryo development after fertilization, it appears that low expression level of turbot CXC chemokine was firstly observed at somites stage. Interestingly, the turbot chemokine was highly and rapidly (5h) induced in liver, spleen and head kidney of turbot after challenge with Vibrio anguillarum. Furthermore, the expression of CXC chemokine was also dramatically increased after challenge in turbot embryonic cells (TECs). These results indicated that the turbot CXC chemokine played an important role in turbot immune response.     

Grass carp (Ctenopharyngodon idella) TRAF6 and TAK1: Molecular cloning and expression analysis after Ichthyophthirius multifiliis infection

Ichthyophthirius multifiliis, a pathogenic ciliate parasite, infects almost all freshwater fish species and causes significant economic losses. Tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (TAK1) are two important signaling molecules involved in toll-like receptor (TLR) signal transduction. To date, the roles of TRAF6 and TAK1 in host defense against fish parasites are still poorly understood. In the present study, TRAF6 (CiTRAF6) and TAK1 (CiTAK1) were identified from grass carp (Ctenopharyngodon idella). The full-length cDNA sequence of CiTRAF6 (2250 bp) includes an open reading frame (ORF) of 1629 bp, which shows a high similarity to that of Cyprinus carpio TRAF6 and encodes a putative protein of 542 amino acids containing one RING domain, two zinc fingers, one coiled-coil region, and one MATH domain. The full-length CiTAK1 cDNA sequence is 2768 bp and includes an ORF of 1626 bp that encodes a putative protein of 541 amino acids containing a conserved serine/threonine protein kinase catalytic domain and a coiled-coil region. Phylogenetic analysis showed that CiTRAF6 and CiTAK1 were clustered with TRAF6 and TAK1 of other teleosts, respectively. CiTRAF6 and CiTAK1 were both constitutively expressed in all examined tissues but with varied expression levels. The highest expressions of CiTRAF6 and CiTAK1 were in the head kidney and spleen, respectively. The expression profiles of CiTRAF6 and CiTAK1 were detected in grass carp after I. multifiliis infection. Expressions of both genes were significantly up-regulated in the skin, gill, head kidney, and spleen at most time points after infection, indicating that CiTRAF6 and CiTAK1 may play essential roles in grass carp defense against I. multifiliis.     

二龄草鱼脾脏、肝脏组织高表达甘露糖结合凝集素mRNA

Innate immunity is expected to be very important in fish. Mannose-bingding lectin (MBL) participates in the innate immune system as an activator of the complement system and as an opsonin after binding to certain carbohydrate structures on microorganisms. In this experiment, total mRNA was isolated from spleen, liver, gills, thymus, head kidney and kidney of adult and immature grass carp Ctenopharygodon idllus. The cDNA of MBL was obtained by RT-PCR using total mRNA from the spleen of carp as template. Such cDNA was labled with ^32p and used as probe for Northern analysis, and autoradiographic signals were quantified by densitometry analysis. The results showed that MBL was high expressed in the spleen and liver and low in gills, thymus, head kidney and kidney of adult grass carp, and MBL was much lower expressed in spleen and liver of immature grass carp than those of adult grass carp. The results might partially explain why immature grass carp are vulnerable to grass carp hemorrhage virus (GCHV) whereas adult grass carp are not.This suggested that MBL mav be an imoortant anti-GCHV factor [Acta Zoologica Sinica 50 (1): 137 - 140. 2004].     

Cloning, characterization and expression analysis of interleukin-10 from the common carp, Cyprinus carpio L.

Interleukin (IL)-10 was cloned from the common carp (Cyprinus carpio L.) using IL-10 primers from carp head kidney following stimulation with concanavalin A and lipopolysaccharide. The cDNA consisted of a 1096 bp sequence containing a 55 bp 5' untranslated region and a 498 bp 3' untranslated region. An open reading frame of 543 bp encoded a putative 180 amino acid protein with a putative signal peptide of 22 amino acids. The signature motif of IL-10 is conserved in carp sequence. A 2083 bp genomic sequence of carp IL-10 was found to contain five exons interrupted by four introns. With the exception of much more compact introns, the genomic structure was similar to that of mammalian IL-10. By homology, phylogeny and genomic analyses, the carp gene cloned was designated as IL-10. Carp IL-10 was expressed in head, kidney, liver, spleen and intestine during the resting phase. The gene was also expressed in head kidney and liver following in vitro stimulation with lipopolysaccharide.     

Molecular cloning, characterization and expression analysis of an IL-21 homologue from Tetraodon nigroviridis

Interleukin-21 (IL-21) is an important immune cytokine that was well characterized in human and mammals, but little is known in fish. In present study, an IL-21 homologue was cloned and well characterized from Tetraodon nigroviridis. The full-length Tetraodon IL-21 cDNA was 849bp in size, containing an open reading frame (ORF) of 438bp that translated a 145 amino-acid peptide, a 5' untranslated region (UTR) of 69bp, and a 3' UTR of 342bp. The deduced peptide shared identity of 20-49% with other known IL-21 sequences. The Tetraodon IL-21 gene had six exons while both human and Takifugu IL-21 gene contained only five exons. However, the level of synteny between human, Takifugu and Tetraodon genomes was well conserved during evolution. In vivo expression study showed that Tetraodon IL-21 mRNAs were constitutively expressed at a low level and only in limited tissues, including gut, gill and gonad in healthy fish, and stimulation with LPS increased the expression of IL-21 in these tissues and induced the expression of IL-21 in kidney, spleen and skin, indicating that IL-21 is an inflammatory stress inducible gene associated with the anti-bacterial defense in fish. Our study provided further evidence for the existence of IL-21 in fish, and gained further insight into the immunological functions of IL-21 gene in fish.     

赤眼鳟Toll样受体3基因cDNA全长克隆及表达分析

为探究赤眼鳟（Squaliobarbus curriculus）Toll样受体3（Toll-like receptor 3,ScTLR3）基因是否参与抗病毒免疫反应,实验运用RACE技术,克隆得到ScTLR3基因cDNA全长序列,并进行了生物信息学分析;通过Real-Time qPCR技术,检测了ScTLR3 mRNA在健康赤眼鳟10个组织中的分布以及感染草鱼呼肠孤病毒（GCRV）后肝脏、脾脏、体肾和头肾中的表达特征。结果表明:ScTLR3基因cDNA序列全长4043 bp,包括5-非编码区（UTR）216 bp,开放阅读框（ORF）2715 bp和3-UTR 1112 bp;ScTLR3共编码904个氨基酸残基,推导的蛋白分子量102.67 kD,理论等电点8.76;SMART结构域预测显示,ScTLR3由N端的信号肽（SP）、富亮氨酸结构域（LRRs）、跨膜结构域（TM）和C端的Toll/白介素-1受体结构域（TIR）组成。实时荧光定量结果显示,ScTLR3mRNA在检测的各组织中均有表达,肝脏中的相对表达量极显著高于其他组织（P0.01）;感染GCRV后,肝脏、脾脏、体肾和头肾组织中ScTLR3 mRNA均上调表达,肝脏、脾脏和体肾组织中的相对表达量在24h达到峰值,分别为对照组的5倍、7倍和6倍。研究表明,ScTLR3具有TLRs家族基因的典型结构特征,并能被GCRV诱导表达,推测其在赤眼鳟抗GCRV入侵免疫反应中发挥了重要作用。     

Cloning and analysis of non-specific cytotoxic cell receptor (NCCRP)-1 from common carp Cyprinus carpio L

Nonspecific cytotoxic cell receptor protein (NCCRP-1) provides an important function in target cell recognition and activation of cytotoxicity. NCCRP-1 has been cloned from common carp Cyprinus carpio L. from fish barbel by EST analysis. The isolated gene is composed of 945 bp with a 79 bp 5' UTR, 714 bp open reading frame and 152 bp 3' UTR. The predicted NCCRP-1 gene is composed of 237 amino acid residues and its predicted signal peptide is 19 amino acid residues in length. This gene has conservation of all the related domains characteristic to the NCCRP-1 gene in fish. Phylogenetic and genomic analyses showed that carp NCCRP-1 was similar to other fish orthologues. The expression of NCCRP-1 gene was constitutive in both lymphoid and non-lymphoid tissues. Furthermore, by semi-quantitative RT-PCR studies, we showed that NCCRP-1 gene expression is increased in anterior kidney challenged with Aeromonas hydrophila.     

Molecular cloning of interleukin 1beta from rainbow trout Oncorhynchus mykiss reveals no evidence of an ice cut site.

The complete coding sequence of rainbow trout IL-1beta has been obtained. The gene contains a short 5' UTR (97 bp), a 780 bp open reading frame and a 466 bp 3' UTR, which includes a polyadenylation signal, 7 ATTTA motifs and an 18 bp poly A tail. The predicted amino acid sequence (260 amino acids) contains 3 potential glycosylation sites, with a predicted molecular weight of 29 kDa, and shows between 49 and 56% amino acid similarity to mammalian IL-1betas and 57% similarity to carp IL-1beta. Greatest homology was apparent within the secondary structure of the gene, with few of the amino acids known to bind to the IL-1 receptor being conserved. No ICE cut site was apparent but multiple alignment with mammalian sequences allowed a putative mature peptide of 166 amino acids to be identified, in which Ala(95)would be the amino terminus. Northern blot analysis showed that whilst no IL-1beta expression was detectable in head kidney leukocytes immediately after isolation, expression could be induced by stimulation with LPS for 4 h in culture. Similarly, with isolated head kidney macrophages expression was significantly increased following stimulation with LPS.