Molecular Cloning And Expression Of Cytokine Genes From Lateolabrax Japonicus And Acanthopagrus Schlegeli

Based on the technicques of homology cloning and RACE, several full lengthcDNA of fish cytokenses genes such as interleukin 1a (IL-1a), tumor necrosis factorá (TNFá) and translationally tumor protein (TCTP) from Japanese sea perch(Lateolabrax japonicus) or Cobia (Acanthopagrus schlegeli) were isolated, and theirtemporal expressions patterns were investigated by semi-quantitative RT-PCR. Themain results are reported as follow.1. Molecular cloning and expression of IL-1a from Japanese sea perch and Cobia The IL-1 gene were cloned from Japanese sea perch and Cobia by using thetechniques of homology cloning and anchored PCR. The full length cDNA of seaperch IL-1 contained a 5' untranslated region (UTR) of 136 bp, an open readingframe ORF of 774 bp encoding a polypeptide of 258 amino acids with an estimatedmolecular mass of 29.31 kDa and a 3' UTR of 430bp. The full length cDNA of CobiaIL-1 was of 1104 bp, containing a 5' UTR of 108bp, an ORF of 738 bp encoding apolypeptide of 246 amino acids with an estimated molecular mass of 27.68 kDa, and a3' UTR of 255bp. The searches for nucleotides and protein sequence similarities withBLAST analysis indicated that the deduced amino acid sequence of sea perch andcobia IL-1 were homological to the IL-1 in other fish species and even themammalian. Conserved signature sequences of IL-1 gene family were found in thesea perch and cobia IL-1 deduced amino acid sequence. Just as other knownnonmammalian IL-1 genes, the ea perch and cobia IL-1 all lacked an aspartic acidin the cut region of mammalian IL-1 s which was required for cleavage by ICE a邱丽华 花鲈和军曹鱼部分细胞因子基因的克隆及表达特征研究 博士学位论文(interleukin-1 converting enzyme). The temporal expressions of IL-1 gene in theLPS or iridovirus challenged and the healthy sea perch or cobia were measured bysemi-quantitative RT-PCR. In tne controlled fish, the mRNA expression of IL-1gene could be dectected in most tissues. After stimulated by LPS or iridovirus, theIL-1 expressions in most of the examined tissues were up-regulated. The resultindicated that sea perch and cobia IL-1 were constitutive and inducible expressedand could play a critical role in the host-pathogen interaction.2 Cloning and expression analysis of TNF from Japanese sea perch The techniques of homology cloning and anchored PCR were used to clone theTNFá gene from Japanese sea perch. The full length cDNA of TNFá contained a 5'UTR of 72 bp, followed by an ORF of 990 bp, and a 3' UTR of 192 bp. The ORF wascapable of encoding a polypeptide of 241 amino acids with an estimated molecularmass of 26 kDa. Conserved signature sequences of TNFá gene family were found inthe sea perch TNFá deduced amino acid sequence. The temporalexpressions of TNFágene in sea perch challenged by LPS or iridovirus were measured bysemi-quantitative RT-PCR. High constitutive expression was detected in the tissues ofhead-kidney and spleen. The TNFá gene expressionin sea perch was up-regulated bythe stimulation of LPS or iridovirus, but the expression varied in different tissues.After challenged by LPS, the expression of TNFá was found to be higher inhead-kidney and spleen; while challenged by iridovirus, the expression of TNFá inliver was found to be higher than that in head-kidney and spleen. The result indicatedthat sea perch TNFá was a constitutive and inducible acute-phase protein that couldplay a critical role in the host-pathogen interaction.3 cDNA Cloning and mRNA expression of the TCTPgene from Japanese sea perch A homologue of the lower vertebrates TCTP was cloned from the marine fishJapanese sea perch by the technique of homology cloning and anchored PCR. The fulllength cDNA sequence of the sea perch TCTP gene contained a 5' UTR of 47 bp, a 3'UTR of 433 bp, and a putative ORF of 510 bp encoding a polypeptide of 170 aminoacids. Sequence a...