Identification And Characterization Of Immune Relevant Genes From Large Yellow Croaker (Pseudosciaena Crocea)

Large yellow croaker (Pseudosciaena crocea), is a species of jewfish and is found mainly in the coast in the temperate zone. Large yellow croaker is an economically important marine fish species in China, and also represents the largest yield for a single species in Chinese marine net-cage farming. In recent years, with the rapid development of large yellow croaker culture industry, the infectious diseases caused by viruses, bacteria, and parasites are becoming more and more severe, resulting in great economical losses. At present little is known about the genetic and immunological basis of this fish. This lack of knowledge may represent a major obstacle that hinders the establishment of effective measures in disease control and genetic improvement.To better understand the molecular mechanism of the immune system of large yellow croaker and increase genomic resources in cultured fish, we constructed a cDNA library from mRNA isolated from the spleens of large yellow croaker stimulated with a viral micmic,polyinosinic polycytidynic acid (poly I: C). 1039 ESTs from the library were sequenced and compared with sequences in GenBank, and 252 genes were identified by EST analysis, of which 46 genes may be implicated in the immune functions, including complement system components, immunoglobulins, antigen processing and presentation proteins, interferon system proteins, cytokines, and some innate defense molecules. The expression analysis of selected genes during polyI:C induction was performed by reverse transcription-PCR (RT-PCR), including Mx protein, beta2-microglobulin (β2m), CD2 binding protein 1(CD2BP1), placenta-specific 8 genes, MHC class II associated invariant chain (li) and Cytochrome b-245 alpha peptide (Cyba). The results revealed that expression levels of Mx protein,β2m, placenta-specific 8 genes, and Cyba were significantly upregulated at 30h after induction with poly I:C, and the CD2BP1expression was also induced by polyI:C, suggesting that these genes may be involved in an immune response induced by poly I:C in large yellow croaker. In this study we also report two novel immune-related genes (CD2BP and placenta-specific 8 gene) and the presence of polymorphism at the maturation site of hepcidin.Based on the work of large yellow croaker EST project, we further clone and analyze interferon-γ-inducible-lysosomal thiol reductase (GILT) gene of large yellow croaker. In mammals, GILT has been demonstrated to play a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. Here, we reported the cloning of a GILT gene homologue from the spleen of large yellow croaker(LycGILT). The full length cDNA of LycGILT gene is 1033 nucleotides (nt) encoding a protein of 256 amino acids (aa),with a putative molecular weight of 28.9 kDa. The deduced protein is highly homologous to that of mammalian and zebrafish GILTs and shares 54.1% sequence identity to that of zebrafish and 43.2%-39.2% sequence identity to that of various mammals. The deduced LycGILT possesses the typical structural feature of mammalian GILT, including an active-site CXXC motif, a GILT signature sequence CQHGX2ECX2NX4C, and other six cysteines responsible for the formation of disulfide bonds in the C-terminus. Genomic analysis revealed that LycGILT gene, spanning a 3159 nt fragment, contained seven exons interrupted by six introns and exhibited a similar exon-intron organization to human and mouse GILT genes except for a slightly more compact intron arrangement. The LycGILT expression is obviously up-regulated in spleen and kidney after immunization with inactivated trivalent bacterial vaccine consisting of Vibrio alginolyticus, Vibrio paraphaemolyticus, and Aeromonas hydrophila although it is also constitutively expressed in liver, gills, brain, and heart, suggesting that LycGILT may be involved in the immune response to bacterial challenge in large yellow croker. A search of NCBI sequence data with LycGILT cDNA identified a pufferfish (fugu rubrides) GILT homologue cDNA and its genomic DNA sequence, where two putative interferon-γactivation sites (GAS) were found within the promoter region. This provided evidence that a fish GILT homologue like mammalian GILT, may also be regulated by interferon-γ(IFN-γ) through the JAK-STAT signal pathway. These results indicate that the bony fish GLIT is a functional homologue of mammalian GILT.Finally we clone and characterize G-type lysozyme from large yellow croaker (lyc-g-lys). the full length of lyc-g-lys is 716bp encoding a protein of 193 aa, with a putative molecular weight of 21kd.The deduced protein show 40~85% homologous to other speice. The deduced amino acid squence possesses the typical structural feature of other G-type lysozyme including catalytical residues (E71,D84,D101) and substrate binding site(L97,L121,L128,G152). Genomic analysis revealed that lyc-g-lys gene has similar exon-intron organization to other bony fish G-lysozyme gene.Expression analysis by relative quantification real time PCR shown that g-lys was unregulated about 8~10 fold in instine, spleen and kidney after the trivalent bacterial, vaccine while the expression of G-lys in other organ was not change obviously, these results indicating complexity of G-lysozyme regulation mechanism and the important role of G-lysozyme in the innate immunity. The recombinant large yellow croaker g-lysozyme expressed by yeast shown lytic activity against M.lysodeikticus,A.sobria,V. alginolyticus,V. parahae molyticus,V.vulnficus.