Scielo RSS <![CDATA[Electronic Journal of Biotechnology]]> vol. 15 num. 5 lang. es <![CDATA[SciELO Logo]]> <![CDATA[<b>Isolation and analysis of differentially expressed genes from peanut in response to challenge with <i>Ralstonia solanacearum</i></b>]]> Background: Bacterial wilt caused by Ralstonia solanacearum is the most devastating disease in peanut. Planting resistant peanut cultivars is deemed as the sole economically viable means for effective control of the disease. To understand the molecular mechanism underlying resistance and facilitate breeding process, differences in gene expression between seeds of Rihua 1 (a Virginia type peanut variety resistant to bacterial wilt) inoculated with the bacterial pathogen suspension (10(9) cfu ml-1) and seeds of the same cultivar treated with water (control), were studied using the GenefishingTM technology. Results: A total of 25 differentially expressed genes were isolated. Expression of genes encoding cyclophilin and ADP-ribosylation factor, respectively, were further studied by real time RT-PCR, and full length cDNAs of both genes were obtained by rapid amplification of cDNA ends. Conclusions: The study provided candidate genes potentially useful for breeding peanut cultivars with both high yield and bacterial wilt resistance, although confirmation of their functions through transgenic studies is still needed. <![CDATA[<b>Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis</b>]]> Background: Altering target cell apoptosis is one of the challenging ideas of biotechnological applications. There are several applications of over expressing Bcl-xL anti-apoptotic protein from recombinant protein production to DNA vaccination strategies. The aim of the present study is to evaluate the anti-apoptotic efficacy of Bcl-xL expressing dual promoter plasmid system as a candidate to be used for recombinant protein production and DNA vaccination approaches. For this purpose, Bcl-xL anti-apoptotic protein gene was inserted in a dual expressing vector system in frame with EGFP (enhanced green fluorescence protein) after IRES (internal ribosomal site). The plasmid has a multiple cloning site after CMV (cytomegalovirus promoter) left empty to be inserted a biopharmaceutical protein gene region or DNA vaccine antigens. Results: In order to determine the anti-apoptotic efficacy of Bcl-xL inserted dual expressing vector, BHK-21 cells were transfected both with this plasmid and empty vector as control. Apoptosis was stimulated by several apoptosis inducing agents and serum deprivation in the transfected cells for 48 hrs. Cells expressing Bcl-xL protein in frame with EGFP were determined by flow cytometry as an indicator of cell viability. Additionally, apoptosis were determined by intracellular cleaved Casp 3 staining in Bcl-xL expressing EGFP positive cells. The dual expression plasmid bearing Bcl-xL anti-apoptotic protein prolonged the cell survival rate and protected cells from apoptosis upon apoptosis induction by doxorubicin and camptothecin in which the anti-apoptotic efficacies are inhibited through over expressing of Bcl-xL. pIRES2EGFP/Bcl-xL transfected cell ratio was significantly higher compared to empty vector transfected cells (P < 0.001). In contrast, apoptotic cell ratio was significantly lower in pIRES2EGFP/Bcl-xL transfected cell population compared to empty vector transfected cells (P < 0.001). Conclusion: In conclusion, it was shown that in vitro transient expression of Bcl-xL efficiently inhibited apoptosis induced by serum deprivation, doxorubicin and camptothecin. Thus, the dual expression plasmid bearing Bcl-xL anti-apoptotic protein could be a good candidate for recombinant protein production and DNA vaccination applications. <![CDATA[<b>Stimulation of <i>trans</i>-resveratrol biosynthesis in <i>Vitis vinifera</i> cv. Kyoho cell suspension cultures by 2, 3-dihydroxypropyl jasmonate elicitation</b>]]> Background: Plant cell suspension culture of Vitis vinifera is a promising technology for investigating different factors that are able to induce and/or modify stilbenes biosynthesis. Jasmonates have been reported to play an important role in a signal transduction pathway that regulates defence responses as well as the production of secondary metabolites. In this study, 2, 3-dihydroxypropyl jasmonate (DHPJA) was used to investigate its effect on stimulating trans-resveratrol (t-R) accumulation and the plant defence responses in Vitis vinifera cv. Kyoho cell suspension cultures for the first time. Results: It demonstrated that DHPJA had superior effects on stilbenoids accumulation over methyl jasmonate (MeJA). The optimal condition was 150 μM DHPJA added on day 15 of cultivation period, with the highest level of t-R accumulation which was increased 1.8-fold and 1.3-fold compared with the control and 150 μM MeJA respectively. DHPJA induced stronger plant defence responses, including oxidative burst and activation of L-phenylalanine ammonia lyase (PAL) than MeJA. H2O2 generation induced by DHPJA played a significant role in enhancing t-R accumulation. Adding a specific inhibitor of H2O2 signalling pathway inhibited DHPJA-induced t-R accumulation, but had no effects on DHPJA-induced other metabolites accumulation, which resulted in regulations of product diversity. Conclusions: This study demonstrated that DHPJA was an efficient elicitor to enhance t-R accumulation by activating stronger oxidative burst, and H2O2 signalling pathway could regulate product diversity in DHPJA-induced V. vinifera cv. Kyoho cell suspension cultures. <![CDATA[<b>Characterization of a thermostable extracellular tannase produced under submerged fermentation by <i>Aspergillus ochraceus</i></b>]]> Background: Tannases are enzymes that may be used in different industrial sectors as, for example, food and pharmaceutical. They are obtained mainly from microorganisms, as filamentous fungi. However, the diversity of fungi stays poorly explored for tannase production. In this article, Aspergillus ochraceus is presented as a new source of tannase with interesting features for biotechnological applications. Results: Extracellular tannase production was induced when the fungus was cultured in Khanna medium with tannic acid as carbon source. The extracellular tannase was purified 9-fold with 2% recovery and a single band corresponding to 85 kDa was observed in SDS-PAGE. The native apparent molecular mass was estimated as 112 kDa. Optima of temperature and pH were 40ºC and 5.0, respectively. The enzyme was fully stable from 40ºC to 60ºC during 1 hr. The activity was enhanced by Mn2+ (33-39%) and NH4+ (15%). The purified tannase hydrolyzed tannic acid and methyl gallate with Km of 0.76 mM and 0.72 mM, respectively, and Vmax of 0.92 U/mg protein and 0.68 U/mg protein, respectively. The analysis of a partial sequence of the tannase encoding gene showed an open read frame of 567 bp and a sequence of 199 amino acids were predicted. TLC analysis revealed the presence of gallic acid as a tannic acid hydrolysis product. Conclusion: The extracellular tannase produced by A. ochraceus showed distinctive characteristics such as monomeric structure and activation by Mn2+, suggesting a new kind of fungal tannases with biotechnological potential. Further, it was the first time that a partial gene sequence for A. ochraceus tannase was described. <![CDATA[<b>Complement dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling</b>]]> Oligosaccharides are implicated in the development of the immune response notably in complement activation. Anti-tumoural immunotherapy by monoclonal antibodies (mAbs) offers some advantages to chemotherapy including cell targeting but some of them are inefficient to generate cytotoxicity dependent complement (CDC) known to be important in the antibody’s efficacy. The aim of this study is to give a CDC activity of mAb by linkage of a complement activating oligosaccharide to this antibody via a hetero-bifunctional linker allowing control of the conjugation reaction. We worked on non Hodgkin Burkitt’s lymphoma as cancer source, Fab fragments of rituximab devoid of complement activity as mAb and the trisaccharide Galα(1→3)Galβ(1→4)GlcNAc as immunogenic glycan. The bioconjugate Fab-Gal was characterized by biochemical methods and we demonstrated that the α-Gal epitope was recognized by seric immunoglobulins. After checking the recognition capacity of the Fab-Gal conjugate for the CD20 epitope, in vitro assays were performed to evaluate the activation of the complement cascade by the Fab-Gal conjugate. The effect of this bioconjugate was confirmed by the evaluation of the proliferation response of Burkitt’s cell line. The relative facility realization of this strategy represents new approaches to increase activities of mAbs. <![CDATA[<b>Induction of the expression of defence genes in <i>Carica papaya</i> fruit by methyl jasmonate and low temperature treatments</b>]]> The defence mechanisms that are activated by methyl jasmonate (MJ) in fruits are not well understood. In this work, we studied the expression of defence genes in papaya fruit that are induced by the exposure to MJ and/or low temperatures. The papaya fruits ‘Maradol’ were randomly divided into two groups: one group was the untreated control and the other was treated with 10-4 M of MJ. Half of the fruits from each of the two groups were stored after treatment for 5 days at 5ºC and 2 days at 20ºC. We studied the expression levels of the pdf1.1 and pdf1.2 genes by amplification from expression libraries created from the pulp and skin tissues of the papaya fruit. As a reference, the mRNA level of the 18s ribosomal gene was used. In the skin tissue, the expression levels of the pdf1.1 and pdf1.2 genes were higher immediately after MJ treatment compared to the control. Furthermore, the expression of pdf1.2 remained high after MJ treatment and subsequent storage compared to the control. It was therefore concluded that the activation of the pdf1.1 and pdf1.2 genes forms part of the molecular defence mechanism in fruits that is activated by exposure to MJ. To our knowledge, this is the first study that analyzes the gene expression in papaya fruit that is induced by the exogenous application of methyl jasmonate and cold treatment. <![CDATA[<b>A molecular marker approach using intron flanking EST-PCR to map candidate genes in peach (<i>Prunus persica</i>)</b>]]> In Peach (Prunus persica) several physiological changes, such as woolliness, triggered by chilling injury are involved in major production losses due to cold storage of the fruits during shipping. Additionally, the low level of polymorphisms among peach varieties is an important limitation in the search for new molecular markers that could be associated with economically important traits. Therefore, a functional approach was employed to associate candidate genes with an informative marker in peach. The data was obtained from the results of an in silico analysis of four different cold peach treatments. Thirty two candidate genes were selected that were aligned against Arabidopsis thaliana genomic sequences to design intron-flanking EST-PCR markers. These markers were used to position the candidate genes on the Prunus genetic reference map. In the physiological response to chilling injury, cell wall integrity, carbohydrate metabolism and stress response pathways could be involved, therefore candidate genes associated by Gene Ontology annotation to these pathways were included in the analysis. The designed markers were positioned to the Texas X Earlygold (TxE) genetic reference map through selective mapping methodology (Bin mapping). 72% of these new markers showed polymorphism in the TxE Binset population and 31% of them were successfully mapped to a genetic position on the Prunus reference map. The bioinformatic methodology used in this work includes a first approach in search for functional molecular markers associated to differentially expressed genes under certain physiological condition which in addition to the Bin mapping approach allows addressing a genetically anchored position to these new markers. <![CDATA[<b>Analysis of genetic variability by ISSR markers in <i>Calibrachoa caesia</i></b>]]> Background: Calibrachoa Cerv. (ex La Llave & Lexarza) is a genus of the Solanaceae family (La Llave and Lexarza, 1825). This genus has a high ornamental and economic value due to its intrinsic variability and multiplicity of flower colours. In Argentina there are eight native species, and one of them is Calibrachoa caesia. The genetic diversity among 35 accessions of C. caesia, from five departments in the province of Misiones, was analyzed using ISSR markers. Results: Thirteen ISSR primers yielded a reproducible banding pattern, with 701 amplified loci and 98% of polymorphism. The ISSR primers 5’CT, 5’CA, 5’GA, 5’GACA, 3’CAC, 3’TG and 3’TC generated 100% polymorphic patterns. The Rp values ranged from 23.20 to 10.29 for 5’GACA and 3’AG primers, respectively, while the average values for MI and PIC were 0.367 and 0.231, respectively. The more informative primers were 5’GACA and 5’GA, and the less informative was 3’AC. Simple matching coefficient of similarity varied from 0.8875 to 0.6659, indicating high levels of genetic similarity among the genotypes studied. The UPGMA cluster analysis indicated three distinct clusters; one comprised genotypes of the five departments, while the second included individuals from Guaraní and Oberá regions and the third cluster included the San Pedro individuals. The overall grouping pattern is in agreement with principal coordinate analysis (PCoA). Conclusions: The Bayesian cluster analysis revealed structuring of the C. caesia population and two clusters were identified, which correspond to UPGMA major clades. The AMOVA test for all populations showed highest genetic variation within populations (90%), meanwhile the Fst coefficient was 0.098, indicating a medium differentiation between populations. These results showed a great intrapopulation genetic diversity but no significant difference was detected among populations. In this work the use of thirteen ISSR markers, allowed the characterization of every individual examined. Therefore, the possibility of using molecular data for varietal identification is a promising tool for application in future breeding programs in the genus Calibrachoa. <![CDATA[<b>Molecular cloning, expression pattern, and putative cis-acting elements of a 4-coumarate:CoA ligase gene in bamboo (<i>Neosinocalamus affinis</i>)</b>]]> Background: 4-coumarate:CoA ligase (4CL) plays an important role at the divergence point from general phenylpropanoid metabolism to several branch pathways. Although 4CL sin higher plants have been extensively studied, little has known about the 4CL gene of bamboo. Results: In current study, a Na4CL gene putative encoding 4-coumarate:CoA ligase (4CL) and its 5’-flanking region were isolated from bamboo (Neosinocalamus affinis) by RACE-PCR and genomic DNA walker, respectively. Na4CL encodes a predicted protein of 557 amino acids, with conserved motifs of adenylate-forming enzymes. Phylogenetic analysis showed that Na4CL shared 62~85% identity with other known plant 4CLs, and cluster closely with some known 4CLs in monocots. Sequence analysis revealed conserved cis-acting elements (Box A and AC-II element) present in the Na4CL promoter. Additionally, a Na4CL RNAi construct was transformed into tobacco. Transgenic tobaccos displayed significant down-expression of endogenesis 4CL and reduced lignin contents. Conclusion: These results contribute to the knowledge of the presence of Na4CL gene and its possible role in phenylpropanoid metabolism. <![CDATA[<b>Isolation of high quality RNA from <i>Polyporus umbellatus</i> (Pers.) Fries</b>]]> Background: The dried sclerotium of medicinal fungus Polyporus umbellatus (Pers.) Fries has many pharmacological functions such as diuretic and anticancer activity, in which high-content polysaccharides may play an important role. However, RNA isolation is difficult in filamentous fungi and lacking in P. umbellatus. Results: Five methods for RNA extraction from five strains collected from four provinces were assessed for their ability to recover a high-quality RNA applicable for sequence-related amplification polymorphism (SRAP) PCR and GDP-D-mannose pyrophosphorylase (GMP) gene expression profiles. Both A260/A280 and A260/A230 ratios of the best Trizol Plus + RNAiso-mate for Plant Tissue method are around 2 with a yield of 1122.00 ± 0.21 ng μl-1. The Trizol method also showed good quality with the yield 469.60 ng μl-1. The SRAP PCR amplified clear and polymorphic bands in all five cDNA samples transcribed from RNA by using primer Me4-Em4. GMP gene fragment (1251 bp) was successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. Conclusion: All these results showed that the total RNA isolated by this protocol is of sufficient quality for subsequent molecular applications. <![CDATA[<b>Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (<i>Oncorhynchus mykiss</i>)</b>]]> Background: Apoptosis is an active cell death process mediated by caspases activation, in which different extrinsic or intrinsic signalling pathways result in direct activation of effector caspases. Caspase-3 is considered to be the most important of the executioner caspases, which cause the morphological and biochemical changes detected in apoptotic cells. Different bacterial and virus pathogens have developed different strategies to survive inside the host and overcome natural protections, one of them is inducing apoptotic death in infected cells. We have demonstrated previously that Piscirickettsia salmonis activates this process in monocytes/macrophages from salmonid RTS11 cell line both by morphological and caspase detection assays; nevertheless, recognition of caspase activation by western blot was impossible since most of the commercially available antibodies for mammalian caspases are not cross-reacting. Results: We have generated a monospecific polyclonal antibody directed to an epitope region of salmonid caspase-3; the selected epitope present high homology with caspase-3 from others teleost species and includes the active site of the enzyme. The peptide was designed using bioinformatics tools and was chemically synthesized using the Fmoc strategy, analysed by RP-HPLC, its molecular weight confirmed by mass spectrometry and its structure analyzed by circular dichroism. The synthetic peptide was immunized and antibodies from ascitic fluid were enriched for immunoglobulins using caprylic acid and then purified by activated affinity columns. The anti-peptide activity of purified antibodies was verified by ELISA, and the ability of the anti-peptide to recognize salmonid caspase-3 activation was demonstrated with the molecule in P. salmonis RTS11 infected cells by western blotting, ELISA and immunocytochemistry. Conclusions: This is the first antibody available for a fish caspase, specifically for trout caspase-3, whose applications were validated by different immunological assays. <![CDATA[<b>Marine genome resource sustainability in Central America</b>]]> In an effort to raise awareness of the major environmental challenges facing the region’s coastal and marine ecosystems, and to highlight the potential research and socioeconomic benefits of this program, the authors provide summaries of key lectures and conclusions presented in the international conference on Central American Marine Biodiversity and Genomics, in April of 2012, dedicated to “Genomic Archiving and Coastal Marine Biodiversity Exploration, Conservation and Sustainable Development”, and describe the main components of the initiative for the benefit of other actors, stakeholders and donors in the field of marine biodiversity. <![CDATA[<b>Vaccines for finfish aquaculture</b>: <b>What do we need to know to make them work?</b>]]> Aquaculture still faces serious economic impacts due to the loss of animals to disease. A conservative estimate of 5% losses due to disease means that the finfish aquaculture industry loses over $1 billion annually on a global scale. One proven way to prevent costly disease outbreaks is to vaccinate fish against common or known pathogens. Current vaccination schemes still result in losses, however, and this may be due in part to vaccine design. Vaccines are currently designed using state of the art knowledge of immune responses, which is based primarily on mammalian studies. Just how applicable is this information to fish immunity and vaccine design, however? This review discusses what is currently known for teleost fish about two key processes that drive immune response: antigen presentation and cytokine regulation. In both cases many of the genes known to be involved have been identified; in the case of cytokines recent genome projects have added to the total rapidly in recent years. Most functional studies to date in these areas have focused on gene expression and mRNA levels, due to a lack of available antibodies that are required for studies at the protein level. These studies are confounded by the fact that in many cases the teleost equivalents of single copy mammalian genes are duplicated and are regulated in very different ways. This suggests that vaccines designed around mammalian immunological principles will not be as efficient as they could be. Future research goals for fish immunologists should be to develop the antibodies required for protein level functional studies in order to provide the true understanding of fish immunity that is required for the design of finfish aquaculture vaccines that are truly effective. <![CDATA[<b>A microbial community analysis of the octocoral <i>Eunicea fusca</i></b>]]> While there is a significant and growing body of knowledge describing the microbial communities of marine invertebrates such as sponges, there are very few such studies focused on octocorals. The octocoral Eunicea fusca is common on reefs in various regions of the Caribbean and has been the subject of natural product investigations. As part of an effort to describe the microbial community associated with octocorals, a culture-independent analysis of the bacterial community of E. fusca was conducted. Specifically, a 16S rDNA clone library analysis was performed to provide baseline data. A total of 40 bacteria members from 11 groups were found. In general, Proteobacteria were the dominant group with a total of 24 species and α-Proteobacteria represented the highest percentage of bacteria associated with E. fusca (27.5%). Other prominent groups observed were Acidobacteria, Actinobacteria, Cyanobacteria, Planctomycetes, δ-Proteobacteria, Lentisphaerae and Nitrospirae. This is the first analysis of bacterial populations associated with the gorgonian E. fusca. <![CDATA[<b>Glucose and other hexoses transporters in marine invertebrates</b>: <b>A mini review</b>]]> Glucose and related hexoses are very important metabolic substrates. Their most important function is to provide quick fuel for most organisms in all three kingdoms because they are the first substrate for energy production in the form of ATP through glycolysis and the subsequent metabolic pathways. In this paper we review the current information about how glucose and related hexoses are transported across biological membranes to carry out their function either as a metabolic molecule or as energy store in marine invertebrate organisms. In these animals, there are two sugar transport systems that are mediated by the sodium/solute symporter family proteins (SGLT) and the major facilitative super-family proteins (GLUT). The most studied sugar transporters in marine invertebrates are involved with dietary sugar uptake, such as SGLT1, SGLT4, GLUT2 and GLUT5, however more studies need to be done to extend the knowledge about these and other sugar transporters involved in metabolic processes. <![CDATA[<b>The crustacean selenoproteome similarity to other arthropods homologs</b>: <b>A mini review</b>]]> Selenoproteins (Sels) are involved in oxidative stress regulation. Glutathione peroxidase (GPx) and thioredoxin reductase are among the most studied Sels in crustaceans. Since their expressions and activities are affected by pathogens, environmental and metabolic factors, their functions might be key factors to orchestrate the redox cellular balance. The most studied invertebrate selenoproteome is from Drosophila. In this fly, SelD and SelB are involved in selenoproteins synthesis, whereas SelBthD, SelH and SelK are associated with embryogenesis and animal viability. None of the Sels found in Drosophila have been identified in marine crustaceans yet, and their discovery and function identification is an interesting research challenge. SelM has been identified in crustaceans and is differentially expressed in tissues, while its function remains to be clarified. SelW and G-rich Sel were recently discovered in marine crustaceans and their functions are yet to be clearly defined. To fully understand the crustacean selenoproteome, it is still necessary to identify important Sels such as the SelD, SelBthD and SelB homologs. This knowledge can also be useful for marine crustacean industry to propose better culture strategies, enhanced health and improved profits. <![CDATA[<b>Bioprospection of cellulolytic and lipolytic South Atlantic deep-sea bacteria</b>]]> Background: Cellulases and lipases have broad industrial application, which calls for an urgent exploration of microorganisms from extreme environments as valuable source of commercial enzyme. In this context, the present work describes the bioprospection and identification of deep-sea bacteria that produce cellulases and lipases, as well their optimal temperature of activity. Results: The first step of this study was the screening of cellulolytic and lipolytic deep-sea bacteria from sediment and water column, which was conducted with substrates linked with 4-Methylumbelliferyl. Among the 161 strains evaluated, 40 were cellulolytic, 23 were lipolytic and 5 exhibited both activities. Cellulolytic and lipolytic bacteria are more common in sediment than at the water column. Based on the ability to produce cellulases and lipases three isolates were selected and identified (16S rRNA sequencing) as Bacillus stratosphericus, B. aerophilus and B. pumilus. Lipases of strain B. aerophilus LAMA 582 exhibited activity at a wide temperature range (4º to 37ºC) and include psychrophilic behaviour. Strain Bacillus stratosphericus LAMA 585 can growth in a rich (Luria Bertani) and minimal (Marine Minimal) medium, and does not need an inducer to produce its mesophilic cellulases and lipases. Conclusions: Deep-sea sediments have great potential for bioprospection of cellulase and lipase-producing bacteria. The strains LAMA 582 and LAMA 585 with their special features, exhibit a great potential to application at many biotechnology process. <![CDATA[<b>Selection of <i>Arthrospira platensis</i> strains with productivity in brackish water with high boron levels for commercial production in the Lluta Valley</b>]]> Adaptation and selection of Arthrospira platensis strains, for cultivation in brackish water with excess boron (B) in the Lluta Valley can become an interesting alternative that would allow to extend these cultures to areas that possess the environmental conditions, but that lack the fresh water needed to do it. Strains TX98 and P88 were evaluated in laboratory conditions with three different media of brackish water and with the white medium, the Zarrouk modified medium (MZM). The growing media with brackish water with a B concentration present in the Lluta River of 20 mgL-1 (B20) and medium with 30 mgL-1 (B30), and 10 mg L-1 of B (B10). The effect of the different media on the growing parameters with a culture temperature of 25 ± 1ºC in the three treatments, strains TX98 and P88 triplicate, Arthrospira platensis, showed tolerance. It was statistically determined that in the growth, the two strains, the three treatments and in the interrelation of both there were significant differences (p < 0.05). The TX98 strain reached a concentration of 1.139 g L-1 (dry weight) in brackish water with medium B20. Therefore, the highest rate of specific growth (μmax) was obtained with the TX98 strain grown in the brackish medium B30 and the lowest duplication time (0.597 days). Cells grown in brackish water with B had a slightly biochemically modified composition with the white, in relation to the protein content, in the cases in which there are differences in the B content, specifically B30 treatment. For the culture with brackish water from the Lluta River, the TX98 strain is recommended with 10 mg of B using a laboratory to pilot scale. <![CDATA[<b>Anti-peptide antibodies</b>: <b>A tool for detecting IL-8 in salmonids</b>]]> Background: Interleukin 8 is a chemokine that is produced by several types of cells, like macrophages and has chemotactic activity in particular on neutrophils, playing a key role during the inflammatory process. It has been demonstrated at the molecular level that this molecule is present and conserved in several vertebrate groups, pointing its importance. Analysis of the amino acid sequence of IL-8, projected from cDNA of Salmo salar, presents homology with the sequences of mammals, poultry and lamprey, indicating the presence of a homologous molecule in higher fish. However, there is no information at protein level, which allows characterizing the regulatory role of this molecule during the immune response in fish. Results: In this work, we designed and synthesized an epitope peptide of 10 residues with a purity of 95% and mass of 1158.7 kDa, which showed a random coil structure. From this peptide it was able to generate a polyclonal mono-specific antibody which was capable of detecting the whole molecule of IL-8 in tissue and cellular model of salmonids. Conclusions: The resulting antibody is a versatile tool for detecting IL-8 by different immune techniques such as ELISA, dot blot, western blotting and immunocytofluorescence. Analysis of IL-8 at proteomic level is a useful method for characterizing immune properties of this molecule in fish. <![CDATA[<b>Immunological strategy for detecting the pro-inflammatory cytokine TNF-alpha in salmonids</b>]]> Background: Tumour necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine which exerts a variety of immunological functions in vertebrates. TNF-α has been identified and cloned in a number of teleost fish species; nevertheless, the functions displayed by this cytokine in fishes remain poorly understood, given that the low sequence identity compared to their mammalian counterpart, limit fish TNF-α detection using mammalian antibodies. Then, for fish immune response characterization is fundamental the production of specific fish anti-TNF-α antibody. Results: We have developed a monoespecific antibody against the pro-inflammatory molecule TNF-α of salmonid fish. TNF-α epitope region was identified and characterized using bioinformatic tools. The epitope sequence was chemically synthesized using Fmoc strategy, analyzed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was used to immunize mice and antibodies from ascitic fluid were purified. The resulting antibody was used for molecular and histochemical detection in gut samples from salmonid fishes treated with different food. By ELISA, we detected a differential expression of TNF-α, the western blot analysis shows recognition of the whole TNF molecule and by immunohistochemistry TNF-α positive cells were observed. Conclusions: We provide an immunological tool, validated through classical immunological assays, which can be a useful tool for characterizing fish TNF-α function.