RESUMO
In fish, interleukin (IL)-2, IL-21 and IL-15 genes have recently been identified by in-silico cloning. Fish IL-15 gene is similar to counterparts from mammals and other vertebrates. A zebrafish genomic database-search initiated to find IL-2 and IL-21 genes from zebrafish (Danio rerio) led to the identification of an IL-15 like gene (IL-15L). This gene was cloned by prediction and the transcripts were subsequently cloned by PCR. The predicted translation yielded a 162 amino acid protein with a 42 amino acid-long signal peptide. This protein shared identities of 28.4% to 31.5% with other mammalian and vertebrate IL-2, IL-15 and IL-21 genes. The gene occupies 7.7 kb of the genomic DNA and the coding region spans into four exons and is interrupted by three introns, which is similar to the genomic structure of IL-2 gene. The chromosomal synteny and phylogenetic analyses support our view that this IL-15L gene is specific to teleosts. Furthermore, two alternatively spliced forms have been identified with differential exon usage translating for proteins of 108 and 120 amino acids in length. The analysis of the alternative splicing suggests it may play an important role in regulating the function of this novel gene. Analyses by RT-PCR and in situ hybridization show gene expression in lymphoid tissues like intestine, gills, spleen, pancreas and kidney suggestive of a role in immunity of fish.
Assuntos
Interleucina-15/biossíntese , Interleucina-15/genética , Peixe-Zebra/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/química , Primers do DNA/química , Perfilação da Expressão Gênica/veterinária , Ordem dos Genes , Hibridização In Situ/veterinária , Interleucina-15/análise , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA/veterinária , Análise de Sequência de Proteína/veterinária , Homologia de Sequência do Ácido Nucleico , Peixe-Zebra/imunologiaRESUMO
Cytokines are one of the major signaling molecules involved in immunity. Many of these cytokines have been isolated in vertebrates and found to play a significant role in host defense mechanism. Interleukin-17 (IL-17) family of genes are known to have pro-inflammatory actions and associated with specific disease conditions, these genes are conserved across vertebrate evolution. In this study, computational screening for the zebrafish (Danio rerio) genome resulted in identification of five contigs harboring IL-17 genes. Zebrafish cDNA encoding five IL-17 genes exhibited percentage identities of 19.3%-61.9% with that of human homologs. The molecules show conservation of cysteines, important for disulphide bonds for IL-17 molecules. The structural composition of these genes shows two introns and three exons except for IL-17D gene that has only one intron and two exons. Phylogenetic analysis using maximum parsimony algorithm showed that zebrafish IL-17 genes clustered well with other IL-17 homologs further proving the structural similarity with IL-17 genes from other organisms. Expression analysis by RT-PCR revealed expression of IL-17 genes in normal and stimulated tissues of kidney, spleen, gills and intestine. The expression of IL-17 in un-stimulated tissues indicates that these genes may play important roles in normal conditions as well.
Assuntos
Proteínas de Peixes/genética , Interleucina-17/genética , Peixe-Zebra/fisiologia , Animais , Primers do DNA/química , Proteínas de Peixes/biossíntese , Proteínas de Peixes/química , Proteínas de Peixes/efeitos dos fármacos , Ordem dos Genes , Interleucina-17/biossíntese , Interleucina-17/química , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/métodos , Alinhamento de Sequência/veterinária , Homologia de Sequência de Aminoácidos , Peixe-Zebra/genética , Peixe-Zebra/imunologiaRESUMO
A reverse transcription loop-mediated isothermal amplification (RT-LAMP) protocol was developed for detection of infectious hematopoietic necrosis virus (IHNV) RNA in rainbow trout (Oncorhynchus mykiss). A set of four primers, two outer and two inner primers for the RT-LAMP and the LAMP assay, were designed based on the sequence of G-protein of IHNV. Time and temperature conditions were optimized for 60 min at 63 degrees C for both RT-LAMP and LAMP protocols. The detection limit was found to be similar for both RT-LAMP and LAMP. When the sensitivity of RT-LAMP and LAMP were compared with conventional nested PCR, a10-fold higher sensitivity was seen for the LAMP protocols.
Assuntos
Doenças dos Peixes/virologia , Vírus da Necrose Hematopoética Infecciosa/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Oncorhynchus mykiss/virologia , Infecções por Rhabdoviridae/veterinária , Animais , Primers do DNA , DNA Complementar/metabolismo , Vírus da Necrose Hematopoética Infecciosa/genética , Reação em Cadeia da Polimerase/métodos , RNA Viral/análise , RNA Viral/isolamento & purificação , Infecções por Rhabdoviridae/virologia , Sensibilidade e EspecificidadeRESUMO
Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a protocol for koi herpes virus (KHV) detection in common carp was designed. A set of four primers, two inner and two outer, were designed based on the sequence of the thymidine kinase (tk) gene of KHV. Time and temperature conditions for detection of KHV were optimized for 60 min at 65 degrees C. The detection limit using LAMP was found to be similar to that by polymerase chain reaction. In this study, we have developed a highly sensitive and rapid diagnostic procedure for detection of KHV infection in common carp.
