Identification and characterization of IL-1 receptor-associated kinase-4 (IRAK-4) in half-smooth tongue sole Cynoglossus semilaevis

As a crucial component in TLR/IL-1R signaling pathways, IRAK-4 plays a central role in innate and adaptive immunity. In the present study, the cDNA of IRAK-4 was cloned for the first time from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of csIRAK-4 was 2149 bp and contained a 168 bp 5′ UTR, a 580 bp 3′ UTR and a 1401 bp CDS. The predicted protein sequence of csIRAK-4 had two typical domains, a death domain (DD) at the N terminus and a serine/threonine/tyrosine protein kinase domain (STYKc) at the C terminus. RT-PCR showed that csIRAK-4 mRNA was detected in all tested tissues, especially in immune-related organs, gonads and brain. After injected with inactivated Vibrio anguillarum, the expressions of csIRAK-4 were up-regulated significantly (P < 0.05) in spleen and head kidney. During development, csIRAK-4 was expressed at all selected stages and low-level expression was detected at metamorphosis. Taken together, the present study indicated that csIRAK-4 played a crucial role in immune responses and might be involved in the process of development.     

Gene cloning and expression analysis of IRF1 in half-smooth tongue sole (Cynoglossus semilaevis)

Interferon regulatory factor 1 (IRF1) was known to play key roles in antiviral defense in several species, and some other important biological processes. In this report, full length cDNA of IRF1 from Cynoglossus semilaevis (CsIRF1) was identified. It was of 1,455 bp, containing a 5′ UTR of 104 bp, a 3′ UTR of 541 bp with a poly (A) tail and an ORF of 810 bp encoding a putative protein of 269 amino acids. The putative CsIRF1protein contained one conserved IRF domain (1–113aa), and two low complexity regions (140–158aa and 230–242aa, respectively). Phylogenetic analysis showed that CsIRF1 was conserved in the teleost evolutionary branch, which was independent of mammalian, birds and amphibians. Additionally, CsIRF1 had the 96 % homology with marine fishes, while 66 % with freshwater fishes. The expression profiles of CsIRF1was analyzed by quantitative real-time PCR in healthy tissues and in immune tissues challenged with different pathogens [Vibrio anguillarum and Lymphocystis disease virus (LCDV)], respectively. CsIRF1 was widely expressed in healthy tissues of Cynoglossus semilaevis and with the highest expression in blood, as much as 19 times of that in liver. V. anguillarum and LCDV both induced the CsIRF1 gene expression distinctly in liver, with the peak value reached to 98-fold at 6 h and 25-fold at 24 h, respectively. The bacteria induced CsIRF1 suddenly up-expression in each detected tissues. However, at the initial stage of the challenge of virus LCDV, the CsIRF1 expression in blood and spleen were up regulated; on the contrary, its expression in liver and head kidney were down regulated, 0.3 and 0.4-fold 6 h post virus injection, respectively. These results suggested that CsIRF1 gene might involve in not only antiviral activity but also antibacterial procedure, indicating its vital role in Cynoglossus semilaevis innate defense system.     

Molecular characterization and expression analysis of the IκB gene from pearl oyster Pinctada fucata

Inhibitor of NF-κB (IκB) is one important member of NF-κB signal pathway and plays a pivotal role in regulating the innate immune response of invertebrate. Herein, we described the isolation and characterization of pearl oyster Pinctada fucata IκB gene (designated as poIκB). The poIκB cDNA was 1975 bp long and consisted of a 5′ untranslated region (UTR) of 73 bp, a 3′ UTR of 807 bp with three RNA instability motifs (ATTTA) and a polyadenylation signal (AATAAA) at 13 nucleotides upstream of the poly (A) tail, and an open reading frame (ORF) of 1095 bp encoding a polypeptide of 364 amino acids with an estimated molecular mass of 40.11 kDa and theoretical isoelectric point of 4.61. A conserved degradation motif (DS₃₅GFSS₃₉) and six ankyrin repeats were identified in the poIκB by SMART analysis. Homology analysis of the deduced amino acid sequence of the poIκB with other known IκB sequences by MatGAT software revealed that the poIκB shared 23.5–63.3% similarities with other known IκB isoforms. The poIκB mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the haemocyte. The poIκB mRNA was up-regulated and increased 4.13- and 5.28-fold after LPS and Vibrio alginolyticus stimulation, respectively. These results suggested that the poIκB was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of pearl oyster.     

Molecular cloning and characterization of a C-type lectin in roughskin sculpin (Trachidermus fasciatus)

C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5′ untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3′ UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys⁶¹-Cys¹⁵⁸, Cys¹³⁴-Cys¹⁵⁰) and a Ca²⁺/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca²⁺-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca²⁺-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR.     

Sexually dimorphic expression and regulatory sequence of dnali1 in the olive flounder Paralichthys olivaceus

Dynein axonemal light intermediate chain 1 (dnali1) is an important part of axonemal dyneins and plays an important role in the growth and development of animals. However, there is little information about dnali1 in fish. Herein, we cloned dnali1 gene from the genome of olive flounder (Paralichthys olivaceus), a commercially important maricultured fish in China, Japan, and Korea, and analyzed its expression patterns in different gender fish. The flounder dnali1 DNA sequence contained a 771 bp open reading frame (ORF), two different sizes of 5′ untranslated region (5′UTR), and a 1499 bp 3′ untranslated region (3′UTR). Two duplicated 922 nt fragments were found in dnali1 mRNA. The first fragment contained the downstream coding region and the front portion of 3′UTR, and the second fragment was entirely located in 3′UTR. Multiple alignments indicated that the flounder Dnali1 protein contained the putative conserved coiled-coil domain. Its expression showed sexually dimorphic with predominant expression in the flounder testis, and lower expression in other tissues. The gene with the longer 5′UTR was specifically expressed in the testis. The highest expression level in the testis was detected at stages IV and V. Transient expression analysis showed that the 922 bp repeated sequence 3′UTR of dnali1 down-regulated the expression of GFP at the early stage in zebrafish. The flounder dnali1 might play an important role in the testis, especially in the period of spermatogenesis, and the 5′UTR and the repetitive sequences in 3′UTR might contain some regulatory elements for the cilia.

     

斜带石斑鱼TLR3基因的克隆及其在刺激隐核虫感染时的表达分析

TLR3(Toll like receptor 3)是Toll样受体家族的重要成员,通过识别病原相关分子模式,诱导宿主天然免疫应答。研究从斜带石斑鱼(Epinephelus coioides)中克隆得到TLR3 cDNA序列,全长为2937 bp,包括107bp的5′非编码区、100 bp的3′非编码区和编码909个氨基酸的2730 bp的开放阅读框。TLR3全长氨基酸序列包含1个信号肽、18个富含亮氨酸的重复序列(Leucine-rich Repeat LRR)、1个跨膜结构域和1个胞内TIR结构域(IL-R1 homologous region)。同源比对显示,斜带石斑鱼TLR3与其他已报道硬骨鱼类的TLR3具有较高的同源性(52%—67%)。组织表达分析显示,TLR3在健康斜带石斑鱼的组织中具有较广的表达分布,其中在前脑、体肾和脾脏中表达量较高。刺激隐核虫(Cryptocaryon irritans)感染斜带石斑鱼后:在皮肤中TLR3的表达量呈现先降低后升高的趋势,从感染后第7天开始上调,并在第10天达到高峰;而在脾脏中,TLR3的表达量在感染6h时就显著上调并达到峰值。结果表明斜带石斑鱼TLR3在抗刺激隐核虫的免疫应答过程中可能发挥重要作用。     

Cloning and expression of HSP70 gene of sipuncula Phascolosoma esculenta

In this study the gene encoding HSP70 was isolated from Phascoloma esculenta by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length of cDNA (2520 bp) consists of a 5′-terminal untranslated region (UTR) (125 bp), a 3′-terminal UTR (421 bp) with a canonical polyadenylation signal sequence (AATAAA), a poly (A) tail, and an open reading frame (ORF) (1974 bp). The predicted molecular mass and isoelectric point for HSP70 is 71.6 kDa and 5.15, respectively. BLAST analysis showed that P. esculenta HSP70 gene shared high similarity. Classical HSP signature motifs, ATP/GTP-Binding Site Motif A, Bipartite Nuclear Targeting Sequence, the cytosolic HSP70 could be expressed in Escherichia coli BL21. After purification, the recombinant pET-HSP70 protein was used to produce the polyclonal antibody in mice and the specificity of the antibody was confirmed by Western blot analysis. Fluorescent real-time quantitative PCR analysis showed that expression of Hsp70 in sipuncula was increased significantly after exposure to 10 mM Zn for12 h, Cd for 24 h, Cu for 48 h, and was exposure to 37 °C for 24 h sea water.     

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.