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Production of leptospiral LipL32 antigen in Pichia pastoris and its use in an enzyme-linked immunosorbent assay BABT
Monte,Leonardo Garcia; Leal,Fernanda Munhoz Dos Anjos; Hartwig,Daiane Drawanz; Vasconcellos,Sílvio Arruda; Brihuega,Bibiana; Dellagostin,Odir Antonio; Hartleben,Cláudia Pinho.
The production of recombinant LipL32 protein using Escherichia coli has been used extensively for the development of vaccines and diagnostic tests for leptospirosis. However, E. coli has demonstrated limitations, including low yield and lack of post-translational modifications. In this study, rLipL32 was produced in eukaryotic expression system (Pichia pastoris) and evaluated the antigen by enzyme-linked immunosorbent assay (ELISA). The yield obtained from the culture supernatant reached 270 mg/L and ELISA showed an accuracy of 95.34%. In summary, the production of rLipL32 using P. pastoris did not impair the antigenic characteristics of this antigen and ensured its use for detecting the leptospiral antibodies in swine sera.
Tipo: Info:eu-repo/semantics/article Palavras-chave: Pichia pastoris; LipL32; ELISA; Leptospirosis.
Ano: 2014 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132014000300007
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Proporção macho: fêmea de embriões bovinos cultivados na presença ou ausência de glicose após FIV com espermatozóides selecionados por Swim-up ou Gradiente de Percoll Braz. J. Vet. Res. Anim. Sci.
Rheingantz,Maria Gabriela Tavares; Pegoraro,Ligia Margareth Cantarelli; Dellagostin,Odir Antonio; Pimentel,Anita Mylius; Bernardi,Mari Lourdes; Deschamps,João Carlos.
No sistema de PIV em bovinos, tem sido obtida uma elevada porcentagem de embriões machos. Este experimento foi realizado para determinar se a presença de glicose no meio de cultivo afeta a proporção macho:fêmea (M:F) dos embriões bovinos PIV a partir da FIV com espermatozóides preparados pelos métodos do swim-up (S) ou do gradiente de Percoll (P). Após a MIV, os COCs foram divididos em dois grupos e inseminados com espermatozóides preparados por um dos métodos. Os zigotos foram cultivados em meio com ou sem 5,56mM de glicose, totalizando 4 tratamentos: S-Gli, S+Gli, P-Gli e P+Gli e 48h após a inseminação, os embriões de cada tratamento foram submetidos à sexagem por PCR (n=845). O efeito da glicose no meio de cultivo sobre a proporção M:F dos embriões PIV...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Fecundação in vitro; Proporção macho:fêmea; Glicose; Gradiente de Percoll; Swim-up.
Ano: 2004 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1413-95962004000100006
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Effect of the Kozak sequence on seroconversion of mice immunized with a DNA vaccine against swine colibacilosis BJM
Michelon,André; Michelon,Marcelo; Simionatto,Simone; Lagranha,Valeska Lizzi; Conceição,Fabrício Rochedo; Vaz,Eliana Knackfus; Cerqueira,Gustavo Maia; Dellagostin,Odir Antonio.
The neonatal diarrhea in swine caused by enterotoxigenic Escherichia coli (ETEC) is responsible for high mortality and low growth rate in pigs and it is mainly dependent on the capacity of E. coli to attach to the surface of the small intestine, a property mediated by fimbria. In this study the faeC gene, which codes for the minor fimbrial subunit of E. coli K88ab, was cloned in the eukaryotic expression vector pcDNA3, associated or not to the Kozak sequence. Plasmid DNA of the two versions of the vaccine candidate was inoculated in mice by the intramuscular route, in two doses, at 0 and 21 days. The animals that received the DNA vaccine containing faeC associated to the Kozak sequence presented seroconversion significantly higher (P<0.05) than the one...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Swine colibacilosis; DNA vaccine; FaeC; Kozak sequence.
Ano: 2003 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822003000500029
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LTB-R1: an alternative to swine mycoplasmal pneumoniae control BJM
Conceição,Fabrício Rochedo; Michelon,André; Michelon,Marcelo; Cerqueira,Gustavo Maia de; Dellagostin,Odir Antonio.
Mycoplasmal pneumoniae is the main respiratory disease in swine. The most efficient way to control it is through the use of vaccines (bacterins), whose production cost is high. The objective of this work was to develop a new alternative for controlling Swine Mycoplasmal Pneumoniae, based on a recombinant subunit vaccine containing the R1 region of P97 adhesin of Mycoplasma hyopneumoniae fused to the B subunit of the heat-labile enterotoxin of Escherichia coli (rLTB-R1). In this work we report the amplification of the genes, genetic fusion between LTB and R1 coding sequences, cloning, construction of the expression vector, as well as expression and purification of rLTB-R1 in E. coli.
Tipo: Info:eu-repo/semantics/article Palavras-chave: Swine mycoplasmal pneumoniae; P97; LTB.
Ano: 2003 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822003000500027
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Isolation and characterization of Leptospira interrogans from pigs slaughtered in São Paulo State, Brazil BJM
Miraglia,Fabiana; Moreno,Andréa Mike; Gomes,Cleise Ribeiro; Paixão,Renata; Liuson,Esequiel; Morais,Zenaide Maria; Maiorka,Paulo; Seixas,Fabiana Kömmling; Dellagostin,Odir Antonio; Vasconcellos,Silvio Arruda.
With the aim of isolating Leptospira spp., blood serum, kidney, liver and genital tract of 137 female swine (40 sows and 97 gilts) and also urine samples from 22 sows were collected in a slaughterhouse in the State of São Paulo, from April 2003 to August 2004. Four isolates were obtained from animals that presented microagglutination test (MAT) titers > 100 for the serovar Pomona and one was obtained from an animal negative by MAT in which Leptospira was isolated from the liver and reproductive tract. The presence of leptospiral DNA was investigated by PCR, and positive results were found in kidneys of 11 females, liver of two, genital tract of two and urine of one of them. Nephrosis, interstitial multifocal nephritis, moderate to severe changing,...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Pomona; Swine; VNTR; Culture; PCR; Genital Tract.
Ano: 2008 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822008000300017
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Leptospirosis diagnosis using Nested-PCR BJM
Nassi,Fernanda; Seixas,Fabiana Kömmling; Jouglard,Sandra Denize Dorneles; Simionatto,Simone; Silva,Everton Fagonde; Seyffert,Núbia; Brod,Claudiomar Soares; Dellagostin,Odir Antonio.
Leptospirosis is a worldwide sanitary problem. Its clinical signs resemble that of other diseases like Dengue and Flu, and it is difficult to distinguish between them. Currently available diagnostic methods shown low sensitivity and specificity. Efforts have been made to develop simpler, faster and more efficient diagnostic methods. The aim of this work was to evaluate and optimize a Nested-PCR method for diagnosis of leptospirosis. Primers were designed to amplify a 264 bp region within the LipL32 gene. The sensitivity and specificity of the assay was evaluated using seven saprophytic serovars and 35 pathogenic serovars. This technique showed to be very specific for pathogenic serovars, however it lacked sensitivity. In order to enhance the sensitivity,...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Leptospirosis; PCR.
Ano: 2003 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822003000500031
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PRODUCTION OF PCB01, A PLASMID FOR DNA IMMUNIZATION AGAINST THE ADHESIN OF ESCHERICHIA COLI K88AB BJM
Gil-Turnes,Carlos; Conceição,Fabrício Rochedo; Dellagostin,Odir Antonio.
Growth characteristics and plasmid yields of Escherichia coli JM109 transformed with pCB01, a plasmid that encodes genes for the fimbrial adhesin of E. coli K88ab and for ampicillin resistance, grown in two culture media in agitated flasks and in fermentor, are reported. The rate of plasmid loss during growth was estimated by the differential counts in media with and without ampicillin. Plasmid yields of cultures grown in flasks varied from 0.9 to 67 µg/ml of medium, while those grown in fermentor attained 62 µg/ml of medium after 8 hours of culture. Plasmid bearing cells were outgrown by plasmid free cells in proportions varying from 5 to 1.2 non-transformed for each transformed cell during growth. Generation times of total population and plasmid bearing...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Plasmid production; Genetic immunization; Fae G; Escherichia coli K 88ab.
Ano: 2001 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822001000300012
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Monoclonal antibodies against an outer membrane protein from pathogenic Leptospira BJM
Lüdtke,Charli Beatriz; Coutinho,Mariana Loner; Jouglard,Sandra Denise Dorneles; Moreira,Cecília Nunes; Fernandes,Cláudia Hartleben Pinho; Brod,Claudiomar Soares; Haake,David A.; Ko,Albert Icksang; Dellagostin,Odir Antonio; Aleixo,José Antonio Guimarães.
Two hybridomas secreting monoclonal antibodies (MAbs) that react with a lipoprotein (LipL32) of the outer membrane of pathogenic Leptospira were obtained. For hybridoma production, spleen cells from BALB/c mice imunized with recombinant LipL32 (rLipL32) were fused to SP2/O-Ag14 cells, selected in HAT medium and screened in an indirect ELISA. One MAb produced was of the IgG2b isotype and the other was an IgM. MAbs specificity was confirmed by indirect ELISA and immunoblotting using purified rLipL32 and whole-cell antigen preparations from Escherichia coli (E. coli) expressing LipL32 and from pathogenic and non-pathogenic serovars. Both Mabs reacted with most of the pathogenic serovars tested and none reacted with non-pathogenic Leptospira. The MAbs...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Leptospirosis; Monoclonal antibodies; LipL32.
Ano: 2003 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822003000500001
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Green turtles (Chelonia mydas) foraging at Arvoredo Island in Southern Brazil: genetic characterization and mixed stock analysis through mtDNA control region haplotypes Genet. Mol. Biol.
Proietti,Maíra Carneiro; Lara-Ruiz,Paula; Reisser,Júlia Wiener; Pinto,Luciano da Silva; Dellagostin,Odir Antonio; Marins,Luis Fernando.
We analyzed mtDNA control region sequences of green turtles (Chelonia mydas) from Arvoredo Island, a foraging ground in southern Brazil, and identified eight haplotypes. Of these, CM-A8 (64%) and CM-A5 (22%) were dominant, the remainder presenting low frequencies (< 5%). Haplotype (h) and nucleotide ( π) diversities were 0.5570 ± 0.0697 and 0.0021 ± 0.0016, respectively. Exact tests of differentiation and AMOVA ΦST pairwise values between the study area and eight other Atlantic foraging grounds revealed significant differences in most areas, except Ubatuba and Rocas/Noronha, in Brazil (p > 0.05). Mixed Stock Analysis, incorporating eleven Atlantic and one Mediterranean rookery as possible sources of individuals, indicated Ascension and Aves islands...
Tipo: Info:eu-repo/semantics/other Palavras-chave: Foraging grounds; Genetic diversity; Green turtle; MtDNA haplotypes; Natal origins.
Ano: 2009 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572009000300027
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Efeito do gene do estresse suíno sobre características de quantidade e qualidade de carcaça R. Bras. Zootec.
Bastos,Reginaldo Gaspar; Federizzi,Joreci; Deschamps,João Carlos; Cardellino,Ricardo Alberto; Dellagostin,Odir Antonio.
O gene do estresse suíno (gene hal), em homozigose recessiva (nn), está associado com a ocorrência da Síndrome do Estresse Porcino (PSS) e com a ocorrência da carne pálida, mole e exudativa (PSE). Em heterozigose (Nn), está relacionado com diminuição na qualidade da carne porém, com maior peso de carcaça. Neste estudo, foi caracterizado o genótipo do gene hal de 160 suínos por meio de análise do DNA extraído de um único folículo piloso. Estes animais foram abatidos e, após, mensuradas características de carcaça de cada um. Entre os 160 animais analisados, 82 (52,58%) foram caracterizados como NN, 67 (41,80%) como Nn e nove (5,62%) como nn. A variação das características de carcaça entre os genótipos foi avaliada por análise de variância, usando o SAS. Os...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Carcaça suína; Gene halotano; PCR; PSE; PSS.
Ano: 2001 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-35982001000100007
Registros recuperados: 10
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