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1.
Transpl Immunol ; 51: 1-5, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30081185

RESUMO

BACKGROUND AND OBJECTIVES: This work aims to present the expert system EpAssistant, a platform algorithm designed to accurately and automatically identify the EPLETS specificities of anti-HLA (Human Leukocyte Antigen) antibodies in the sera of transplantation candidates. MATERIALS AND METHODS: RESULTS: As preliminary results, we present the development and establishment of the EpAssistant platform. EpAssistant analyses can be performed for Class I (-A, B and C) and Class II (-DR, -DQ and -DP) HLA molecules. CONCLUSIONS: EpAssistant automates the EPLETS reactivity analysis process and drastically reduces the time required to produce final results, enabling large-scale data analyses in a simple, inexpensive and rapid manner and facilitating the allocation of donated organs via the EPLETS Virtual Crossmatch (EVxM) system.


Assuntos
Epitopos de Linfócito B/isolamento & purificação , Epitopos/isolamento & purificação , Rejeição de Enxerto/imunologia , Antígenos HLA/imunologia , Teste de Histocompatibilidade/métodos , Transplante de Rim , Transplantados , Algoritmos , Processamento Eletrônico de Dados , Rejeição de Enxerto/prevenção & controle , Humanos , Imunização , Isoanticorpos/metabolismo , Software
2.
Mol Med Rep ; 16(3): 2851-2855, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28656246

RESUMO

Platanus acerifolia (P. acerifolia) is an important cause of pollinosis in cities. The use of allergen extracts on patients with allergic diseases is the most commonly applied method to attempt to treat pollinosis. Pla a 3, a non­specific lipid transfer protein, is a major allergen present in P. acerifolia pollen extracts. In the present study, the Pla a 3 gene was sub­cloned into a pSUMO­Mut vector using Stu I and Xho I sites and transformed into the Arctic Express™ (DE3) RP E. coli host strain. The purified Pla a 3 allergen was analyzed by western blotting and the results revealed that the Pla a 3 allergen has the ability to bind IgE in the P. acerifolia pollen of allergic patients' sera. Moreover, the authors predicted the potential B cell epitopes of the Pla a 3 allergen using the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides system and the BepiPred 1.0 server. In addition, the T cell epitopes were predicted by the SYFPEITHI database and the NetMHCII­2.2 server. As a result, two B cell epitopes (35­45 and 81­86) and four potential T cell epitopes including 2­15, 45­50, 55­61 and 67­73 were predicted in the present study. The current results can be used to contribute to allergen immunotherapies and useful in peptide­based vaccine designs of pollen allergy.


Assuntos
Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Imunoglobulina E/imunologia , Magnoliopsida/imunologia , Rinite Alérgica Sazonal/imunologia , Adolescente , Adulto , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Clonagem Molecular , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/isolamento & purificação , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/isolamento & purificação , Escherichia coli/genética , Feminino , Humanos , Imunoglobulina E/sangue , Magnoliopsida/química , Magnoliopsida/genética , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Pólen/química , Pólen/genética , Pólen/imunologia , Conformação Proteica , Rinite Alérgica Sazonal/sangue , Adulto Jovem
3.
Viruses ; 9(5)2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28509875

RESUMO

Hepatitis B virus (HBV) infection has persisted as a major public health problem due to the lack of an effective treatment for those chronically infected. Therapeutic vaccination holds promise, and targeting HBV polymerase is pivotal for viral eradication. In this research, a computational approach was employed to predict suitable HBV polymerase targeting multi-peptides for vaccine candidate selection. We then performed in-depth computational analysis to evaluate the predicted epitopes' immunogenicity, conservation, population coverage, and toxicity. Lastly, molecular docking and MHC-peptide complex stabilization assay were utilized to determine the binding energy and affinity of epitopes to the HLA-A0201 molecule. Criteria-based analysis provided four predicted epitopes, RVTGGVFLV, VSIPWTHKV, YMDDVVLGA and HLYSHPIIL. Assay results indicated the lowest binding energy and high affinity to the HLA-A0201 molecule for epitopes VSIPWTHKV and YMDDVVLGA and epitopes RVTGGVFLV and VSIPWTHKV, respectively. Regions 307 to 320 and 377 to 387 were considered to have the highest probability to be involved in B cell epitopes. The T cell and B cell epitopes identified in this study are promising targets for an epitope-focused, peptide-based HBV vaccine, and provide insight into HBV-induced immune response.


Assuntos
Epitopos/imunologia , Produtos do Gene pol/efeitos dos fármacos , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Hepatite B/prevenção & controle , Vacinas Virais/imunologia , Sequência de Aminoácidos , Linhagem Celular , Simulação por Computador , Epitopos/química , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/isolamento & purificação , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/química , Antígeno HLA-A2/imunologia , Humanos , Imunogenicidade da Vacina/imunologia , Simulação de Acoplamento Molecular , Peptídeos/síntese química , Peptídeos/farmacologia , Vacinas Virais/farmacologia
4.
Parasitol Int ; 66(3): 324-330, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28110081

RESUMO

Humoral and cellular immune responses play an important role during Giardia lamblia infection. Several Giardia proteins have been identified as immunogenic antigens based on their elicited humoral immune response. Poorly is known about Giardia antigens that stimulate a cellular immune response. The main purpose of this study was to isolate and partial characterize an immunogenic antigen (5G8) of G. lamblia. The 5G8 protein was isolated from G. lamblia trophozoite lysates by affinity chromatography using moAb 5G8-coupled CNBr-Sepharose. The isolated protein was analysed by electrospray tandem mass spectrometry (ESI-MS/MS), and by diverse bioinformatics tools (GiardiaDB, BLASTn, BLASTp and ExPASy). Additionally, several biochemical and immunological characteristics of the isolated protein were analysed. By ESI-MS/MS the amino acidic 5G8 sequence was deduced. The 5G8 antigen belongs to the VSP family proteins of G. lamblia. This protein is composed by one polypeptide chain (±71kDa). Using the algorithm SYFPHEITI, we identified candidate CD4+ T-cell epitopes from the 5G8 antigen, which can elicit cell-mediated immune responses. In this study, we have identified a G. lamblia protein that induces a strong immune response in infected mice. The biochemical and immunological characterization of the immunogenic 5G8 antigen may contribute to the rational design of a Giardia vaccine.


Assuntos
Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Giardia lamblia/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/genética , Antígenos de Protozoários/isolamento & purificação , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/isolamento & purificação , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/isolamento & purificação , Imunidade Celular , Imunidade Humoral , Proteínas de Membrana/genética , Camundongos , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Espectrometria de Massas em Tandem , Trofozoítos/imunologia
5.
Biotechnol Appl Biochem ; 64(1): 117-125, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26549515

RESUMO

Glucose-regulated protein 78 (GRP78) is a typical endoplasmic reticulum luminal chaperone having a main role in the activation of the unfolded protein response. Because of hypoxia and nutrient deprivation in the tumor microenvironment, expression of GRP78 in these cells becomes higher than the native cells, which makes it a suitable candidate for cancer targeting. Suppression of survival signals by antibody production against C-terminal domain of GR78 (CGRP) can induce apoptosis of cancer cells. The aim of this study was in silico analysis, recombinant production, and characterization of CGRP in Escherichia coli. Structural prediction of CGRP by bioinformatics tools was done and the construct containing optimized sequence was transferred to E. coli T7 shuffle. Expression was induced by isopropyl-ß-d-thiogalactoside, and recombinant protein was purified by Ni-NTA agarose resin. The content of secondary structures was obtained by circular dichroism (CD) spectrum. CGRP immunogenicity was evaluated from the immunized mouse sera. SDS-PAGE analysis showed CGRP expression in E. coli. CD spectrum also confirmed prediction of structures by bioinformatics tools. The enzyme-linked immunosorbent assay using sera from immunized mice revealed CGRP as a good immunogen. The results obtained in this study showed that the structure of truncated CGRP is very similar to its structure in the whole protein context. This protein can be used in cancer researches.


Assuntos
Epitopos de Linfócito B , Expressão Gênica , Proteínas de Choque Térmico , Animais , Epitopos de Linfócito B/biossíntese , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/isolamento & purificação , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/imunologia , Proteínas de Choque Térmico/isolamento & purificação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
6.
Hum Immunol ; 77(10): 824-831, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27374949

RESUMO

The concept that HLA antibodies recognize epitopes is leading to new approaches of HLA matching at the epitope level. HLA-DQ plays an important role and many studies have identified structurally defined DQ epitopes specifically recognized by antibodies; they have been recorded in the International HLA Epitope Registry http://www.epregistry.com.br but the list is still incomplete. Pregnancy offers an attractive model to study antibody responses to HLA epitopes. The current analysis was done on 42 DQ-reactive post-pregnancy sera tested in binding assays with a panel of DQ heterodimers. The reactivity of 29 sera corresponded fully to the presence of antibody-verified DQA and DQB epitopes recorded in the Registry. Analysis of the remaining 13 sera led to the identification of additional antibody-defined DQB and DQA epitopes. We have designed the first version of an eplet map for DQ alleles which includes antibody-defined DQA and DQB epitopes and shows sequence positions with polymorphic residues which can be used in HLA epitology studies to identify new antibody-defined DQ epitopes.


Assuntos
Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/isolamento & purificação , Antígenos HLA-DQ/isolamento & purificação , Gravidez/imunologia , Software , Alelos , Anticorpos/sangue , Simulação por Computador , Epitopos de Linfócito B/metabolismo , Feminino , Genótipo , Antígenos HLA-DQ/metabolismo , Teste de Histocompatibilidade , Humanos , Imunidade Humoral , Conformação Proteica , Sistema de Registros
7.
Protein Expr Purif ; 102: 38-44, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24996028

RESUMO

Beta toxin (btx) is the prime virulence factor for the pathogenesis of Clostridium perfringens type C strain, known to cause necrotic enteritis and enterotoxaemia in mammalian species. The existing vaccines targeting btx are formaldehyde inactivated culture filtrates of Clostridium. These filtrates raise antigenic load in the host leading to nonspecific and poor responses. The present study aimed to overcome these drawbacks and generate a chimeric protein carrying in silico identified B-cell epitope of btx fused with a carrier protein as a vaccine candidate. Using bioinformatic tools, three stretches of amino acids were predicted as putative B-cell epitopes. One of the epitopes spanning 140-156 amino acid residues was genetically conjugated with B-subunit of heat labile enterotoxin (LTB) of Escherichia coli and expressed as a translational fusion in Vibrio cholerae secretory expression system. High level expression of the recombinant fusion protein rLTB-Btx140-156 was obtained and the protein was successfully purified. The recombinant protein retained the native LTB property to pentamerize and bind to GM1 ganglioside receptor of LTB. The antigenicity of both the epitope and the carrier protein was maintained in fusion protein as indicated by immunoblotting against anti-LTB and anti-btx antibody. The rLTB-Btx140-156 fusion protein therefore can be evaluated as a potential vaccine candidate against C. perfringens.


Assuntos
Toxinas Bacterianas/genética , Clostridium perfringens/genética , Enterotoxinas/genética , Epitopos de Linfócito B/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Vibrio cholerae/genética , Toxinas Bacterianas/isolamento & purificação , Vacinas Bacterianas/genética , Vacinas Bacterianas/isolamento & purificação , Sequência de Bases , Enterotoxinas/isolamento & purificação , Epitopos de Linfócito B/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação
8.
Exp Parasitol ; 135(3): 606-10, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24090568

RESUMO

Newly synthesized epitopes are one of the most promising antigens for the development of diagnostic kits and peptide vaccines. Very little is known about the B cell epitopes on GRA1 of Toxoplasma gondii, which are recognized by the humoral immune response in pigs. In this study, epitopes derived from GRA1 of T. gondii were identified using synthetic peptide techniques and bioinformatics. Three (PG10, PG13 and PG18) out of the eighteen peptides tested were recognized by all pig sera from different time points after infection, and the other peptides were recognized by select sera from various time points after infection. Our data indicate that many regions of GRA1, and in particular, the regions represented by the peptides PG10, PG13 and PG18, are involved in the pig antibody response. The identification of specific epitopes targeted by the host antibody response is important both for understanding the natural response to infection and for the development of epitope-based marker vaccines and diagnostic tools for toxoplasmosis.


Assuntos
Antígenos de Protozoários/imunologia , Epitopos de Linfócito B/isolamento & purificação , Doenças dos Suínos/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Epitopos de Linfócito B/imunologia , Epitopos Imunodominantes/imunologia , Epitopos Imunodominantes/isolamento & purificação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Vacinas Protozoárias , Kit de Reagentes para Diagnóstico/veterinária , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/parasitologia , Toxoplasma/genética , Toxoplasmose Animal/diagnóstico , Vacinas de Subunidades
9.
Allergol Int ; 62(4): 435-45, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23963475

RESUMO

BACKGROUND: In Japan, hydrolyzed wheat proteins (HWP) have been reported to cause wheat-dependent exercise-induced anaphylaxis (WDEIA) by transcutaneous sensitization using HWP-containing soap. Patients develop allergic reactions not only with soap use, but also with exercise after the intake of wheat protein (WP). ω5-Gliadin and HMW-glutenin were identified as major allergens in conventional WP-WDEIA patients. However, the allergens in HWP-WDEIA have yet to be elucidated. METHODS: Sera were obtained from 22 patients with HWP-sensitized WDEIA. The allergenic activities of HWP and six recombinant wheat gluten proteins, including α/ß-, γ-, ω1,2- and ω5-gliadin and low- and high molecular weight (HMW)-glutenins, were characterized by immunoblot analysis and histamine releasing test. IgE-binding epitopes were identified using arrays of overlapping peptides synthesized on SPOTs membrane. RESULTS: Immunoblot analysis showed that IgE antibodies (Abs) from HWP-WDEIA bound to α/ß-, γ- and ω1,2-gliadin. Recombinant γ-gliadin induced significant histamine release from basophils in eight of 11 patients with HWP-WDEIA. An IgE-binding epitope "QPQQPFPQ" was identified within the primary sequence of γ-gliadin, and the deamidated peptide containing the "PEEPFP" sequence bound with IgE Abs more strongly compared to the native epitope-peptide. The epitope-peptide inhibited IgE-binding to HWP, indicating that the specific IgE to HWP cross-reacts with γ-gliadin. CONCLUSIONS: HWP-WDEIA patients could be sensitized to HWP containing a PEEPFP sequence, and WDEIA symptoms after WP ingestion could partly be induced by γ-gliadin. These findings could be useful to help develop tools for diagnosis and desensitization therapy for HWP-WDEIA.


Assuntos
Anafilaxia/imunologia , Exercício Físico , Gliadina/imunologia , Sabões/efeitos adversos , Triticum/imunologia , Hipersensibilidade a Trigo/imunologia , Adolescente , Adulto , Idoso , Alérgenos/química , Alérgenos/imunologia , Antígenos de Plantas/imunologia , Basófilos/imunologia , Células Cultivadas , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/isolamento & purificação , Feminino , Gliadina/química , Glutens/imunologia , Histamina/metabolismo , Humanos , Hidrólise , Imunização , Imunoglobulina E/metabolismo , Japão , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Sabões/química , Triticum/química , Adulto Jovem
10.
Hum Immunol ; 74(9): 1141-52, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23756161

RESUMO

Donor-specific antibodies (DSA) in sera of sensitized transplant patients are often produced against the specific epitopes on mismatched HLA antigens. In this study, we selected sera from 30 kidney transplant patients with DSA and AMR to define DQ epitopes. Using adsorption and elution assays, we identified 18 antibody reaction patterns to define 6 new epitopes and to confirm 12 previously defined epitopes. In one patient case, one mismatched antigen produced 3 different antibodies and, in another, antibodies were produced against the alpha and beta chains of the same antigen. For some sera, a single epitope can explain reactions for 27 of the 29 DQ beads in the single antigen panel. Several studies highlighted the prevalence of anti-DQ antibodies. In 2011, Almeshari et al. observed DQ DSA in 34/46 (74%) of rejection episodes - 44 patients had DSA and 20 lost their graft due to AMR. Other studies have shown a high prevalence of anti-DQ antibodies and an association with adverse effects on the graft. We conclude that analysis of the epitopes of the DQ antibodies using Adsorption/Elution and testing on single antigen DQ beads helps to better understand the specificities and cross-reactions of DQ antibodies in transplant patients.


Assuntos
Epitopos de Linfócito B/imunologia , Rejeição de Enxerto/imunologia , Cadeias alfa de HLA-DQ/imunologia , Cadeias beta de HLA-DQ/imunologia , Transplante de Rim , Complicações Pós-Operatórias/imunologia , Citotoxicidade Celular Dependente de Anticorpos/genética , Apresentação do Antígeno , Sítios de Ligação de Anticorpos/genética , Linhagem Celular , Mapeamento de Epitopos , Epitopos de Linfócito B/isolamento & purificação , Cadeias alfa de HLA-DQ/genética , Cadeias beta de HLA-DQ/genética , Histocompatibilidade , Humanos , Isoanticorpos/sangue , Isoanticorpos/imunologia , Ligação Proteica , Transgenes/genética
11.
Vet Microbiol ; 164(1-2): 18-26, 2013 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-23434015

RESUMO

A major immunogenic region of equine herpesvirus (EHV)-1 glycoprotein E (gE) was identified. Firstly, the various fragments of EHV-1 gE were expressed as fusion proteins with glutathione S-transferase (GST) in Escherichia coli and their antigenicities were compared by immunoblot analysis using sera from horses experimentally infected with EHV-1. Thirty-three amino acids of gE (a.a. 169-201) specifically and sensitively reacted with the antibodies induced by EHV-1 but not EHV-4 infection. The corresponding region of EHV-4 gE (a.a. 169-199) did not react with antibodies to EHV-1, indicating that this region is specific for each virus. In addition, when the antigenicities of three 20-mer synthetic peptides of EHV-1 gE, gE1(169-188), gE1(176-195) and gE1(182-201) were compared by enzyme-linked immunosorbent assay (ELISA), gE1(169-188) was found to contain a major B-cell epitope. ELISA using two synthetic peptides, gE1(169-188) and gG4(319-330), previously identified as the major EHV-4-specific epitope in gG, was developed and could specifically detect antibodies to EHV-1 and EHV-4, respectively. In Japan, the EHV-1 deleted in gE gene (EHV-1 ΔgE) virus is expected to be introduced in the field as a new modified live vaccine. This ELISA did not react with antibodies induced by inoculation with EHV-1 ΔgE, indicating that it is a useful method to differentiate between EHV-1 infection and EHV-1 ΔgE inoculation. In conclusion, the ELISA described herein, using synthetic peptides, is a simple method to distinguish between EHV-1 and EHV-4 infections and will be suitable as a vaccine marker after introduction of EHV-1 ΔgE into field horses.


Assuntos
Epitopos de Linfócito B/isolamento & purificação , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1 , Doenças dos Cavalos/diagnóstico , Proteínas do Envelope Viral/isolamento & purificação , Animais , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Herpesviridae/diagnóstico , Herpesvirus Equídeo 4 , Cavalos
12.
Protein Expr Purif ; 88(1): 7-12, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23220378

RESUMO

Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides.


Assuntos
Epitopos de Linfócito B/genética , Epitopos de Linfócito T/isolamento & purificação , Receptores ErbB/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Epitopos de Linfócito B/biossíntese , Epitopos de Linfócito B/isolamento & purificação , Epitopos de Linfócito T/biossíntese , Epitopos de Linfócito T/genética , Receptores ErbB/biossíntese , Receptores ErbB/genética , Escherichia coli , Expressão Gênica , Humanos , Masculino , Vírus do Sarampo/química , Vírus do Sarampo/genética , Camundongos , Peptídeos/genética , Peptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Virais de Fusão/biossíntese , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/isolamento & purificação
13.
Mol Biol (Mosk) ; 46(2): 251-9, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22670521

RESUMO

Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polyprotein that is cleaved into 10 proteins. Recently, a novel, so called "ARFP/F", or "core+1", protein, which is expressed through a ribosomal frame shift within the capsid-coding sequence, has been described. Herein, to produce and characterize a recombinant form of this protein, the DNA sequence corresponding to the ARFP/F protein (amino acid 11-161) was amplified using a frame-shifted forward primer exploiting the capsid sequence of the 1b-subtype as a template. The amplicon was cloned into the pET-24a vector and expressed in different Escherichia coli strains. The expressed protein (mostly as insoluble inclusion bodies) was purified under denaturing conditions on a nickel-nitrilotriacetic acid (Ni-NTA) affinity column in a single step with a yield of 5 mg/L of culture media. After refolding steps, characterization of expressed ARFP/F was performed by SDS-PAGE and Western blot assay using specific antibodies. Antigenic properties of the protein were verified by ELISA using HCV-infected human sera and by its ability for a strong and specific interaction with sera of mice immunized with the peptide encoding a dominant ARFP/F B-cell epitope. The antigenicity plot revealed 3 major antigenic domains in the first half of the ARFP/F sequence. Immunization of BALB/c mice with the ARFP/F protein elicited high titers of IgG indicating the relevance of produced protein for induction of a humoral response. In conclusion, possibility of ARFP/F expression with a high yield and immunogenic potency of this protein in a mouse model have been demonstrated.


Assuntos
Epitopos de Linfócito B , Proteínas do Core Viral , Animais , Anticorpos Antivirais/imunologia , Epitopos de Linfócito B/biossíntese , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/isolamento & purificação , Epitopos de Linfócito B/farmacologia , Escherichia coli , Expressão Gênica , Hepacivirus/genética , Hepacivirus/imunologia , Humanos , Imunização , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genética , Proteínas do Core Viral/isolamento & purificação , Proteínas do Core Viral/metabolismo , Proteínas do Core Viral/farmacologia
14.
J Immunoassay Immunochem ; 31(3): 181-92, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20623405

RESUMO

Epitope mapping of ModD of Mycobacterium paratuberculosis was performed using overlapping peptides. In total, 80 overlapping peptides, covering the entire mature ModD, were commercially synthesized. Each peptide spanned 14 amino acids with an offset of 4 amino acids, i.e., with an overlap of 10 amino acids. Synthetic peptide antigenicity was evaluated by enzyme-linked immunosorbent assay (ELISA) using rabbit antisera to culture filtrate (CF) of M. avium or M. paratuberculosis or recombinant ModD (rModD). The peptides o f ModD reacting most strongly (ELISA OD > 1.0) were clustered near the N- and C-terminal ends. The peptides around the C-terminal end only showed the greatest specificity for M. paratuberculosis, yielding high ELISA OD values with rabbit anti-M. paratuberculosis CF serum and low ELISA OD values with rabbit anti-M. avium CF serum. Sera from naturally M. paratuberculosis-infected cattle, however, bound poorly to the short, 14-amino-acid peptides. Thus, two longer peptides covering amino acids 100 to 125 and 328 to 353 were synthesized based on their broad reactivity to rabbit serum against M. paratuberculosis CF. The peptide covering amino acids 328 to 353 showed the highest level of specific bovine antibody binding.


Assuntos
Especificidade de Anticorpos , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Epitopos de Linfócito B/imunologia , Mycobacterium avium subsp. paratuberculosis/química , Animais , Reações Antígeno-Anticorpo , Antígenos de Bactérias/isolamento & purificação , Bovinos , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos de Linfócito B/isolamento & purificação , Mycobacterium avium subsp. paratuberculosis/imunologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/imunologia , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Coelhos , Sensibilidade e Especificidade
15.
Vet Microbiol ; 142(3-4): 293-302, 2010 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-19913364

RESUMO

Enzootic pneumonia (EP) in pigs caused by Mycoplasma hyopneumoniae is a highly prevalent, chronic respiratory disease, which causes considerable economic losses in the swine industry. Most herds are vaccinated, but classical bacterin vaccines do not prevent colonization and it is not possible to detect flourishing M. hyopneumoniae infections in vaccinated herds since commonly used commercial ELISAs cannot differentiate infected from vaccinated animals. To solve this problem, new immunogenic proteins, up-regulated or solely expressed during infection, need to be identified. For this purpose a peptide-spot array was constructed which presents 105 potential linear B-cell epitopes identified by in silico analysis in 35 putative lipoproteins encoded on the genome of M. hyopneumoniae type strain 232. Subjecting this array to immunoblotting using porcine convalescent serum revealed a single strongly immunoreactive epitope on the Mhp366 protein which did not react with serum from bacterin-immunized pigs. In addition, it was not possible to detect Mhp366 in total cell lysates of in vitro grown M. hyopneumoniae strains, using a polyclonal rabbit serum raised against a recombinant GST-Mhp366 fusion protein. To investigate the possibility of using an Mhp366-based ELISA in the field for differentiating vaccinated herds with and without a flourishing infection it was shown that (i) homologues of the corresponding mhp366 gene were present in all 17 M. hyopneumoniae strains and porcine lung samples tested from different geographic origins and (ii) an ELISA based on epitope-specific synthetic peptides as solid phase antigen allowed a classification of field samples. Therefore, Mhp366 might be the first antigen identified which facilitates the detection of flourishing M. hyopneumoniae infections even in vaccinated herds.


Assuntos
Epitopos de Linfócito B/isolamento & purificação , Lipoproteínas/isolamento & purificação , Mycoplasma hyopneumoniae/imunologia , Pneumonia Suína Micoplasmática/microbiologia , Análise Serial de Proteínas/veterinária , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Epitopos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Immunoblotting/veterinária , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/imunologia , Pneumonia Suína Micoplasmática/diagnóstico , Pneumonia Suína Micoplasmática/imunologia , Coelhos , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/imunologia
16.
Vet Microbiol ; 140(1-2): 25-33, 2010 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-19699594

RESUMO

The objective of this study was to screen and identify the B cell epitopes of structural proteins of foot-and-mouth disease virus (FMDV) serotype Asia1. The complete amino acid sequence of all the four structural proteins (P1 region) was analyzed using the DNAStar Protean system. Seventeen peptides were predicted and selected as potential B cell epitopes. The potential B cell epitope genes were cloned into the pGEX-6P-1 plasmid, then expressed and purified. The resulting 17 glutathione S-transferase (GST) fusion peptides were detected by Western blot and ELISA for evaluation of their antigenicity. Six of the 17 fusion peptides were identified successfully by sera from rabbits immunized with the purified P1 polyprotein of FMDV type Asia1. The six fusion proteins were epi1-1 (VP1:(1)TTTTGESADPVT(12)), epi1-2 (VP1:(17)NYGGETQTARRLH(29)), epi1-6 (VP1:(194)TTQDRRKQEIIAPEKQTL(211)), epi2-2 (VP2:(40)EDAVSGPNTSG(50)), epi3-1 (VP3:(26)YGKVSNPPRTSFPG(39)), and epi4-2 (VP4:(30)YQNSMDTQLGDN(41)). The results of this study lay a foundation for further study of the structure and function of the structural proteins and may aid in the design of an epitope vaccine against foot-and-mouth disease (FMD) type Asia1. This study has also shown that the bioinformatics method, in combination with molecular biology methods can be used to map the B cell epitopes on viral proteins.


Assuntos
Epitopos de Linfócito B/imunologia , Vírus da Febre Aftosa/imunologia , Proteínas Estruturais Virais/imunologia , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Sequência de Bases , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/isolamento & purificação , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , Dados de Sequência Molecular , Coelhos , Sorotipagem , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética
17.
BMC Microbiol ; 7: 113, 2007 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-18088426

RESUMO

BACKGROUND: The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. RESULTS: An antigenic sequence corresponding to amino acids 420-457 (epiA) of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. CONCLUSION: The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Epitopos de Linfócito B/imunologia , Epitopos Imunodominantes/imunologia , Serina Endopeptidases/imunologia , Streptococcus pneumoniae/imunologia , Anticorpos Antibacterianos/imunologia , Especificidade de Anticorpos , Epitopos de Linfócito B/isolamento & purificação , Humanos , Epitopos Imunodominantes/isolamento & purificação , Imunoglobulina A/metabolismo , Biblioteca de Peptídeos , Infecções Pneumocócicas/imunologia , Streptococcus/enzimologia , Streptococcus pneumoniae/enzimologia
18.
J Gen Virol ; 87(Pt 1): 217-23, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16361434

RESUMO

Avian hepatitis E virus (avian HEV) was recently discovered in chickens from the USA that had hepatitis-splenomegaly (HS) syndrome. The complete genomic sequence of avian HEV shares about 50 % nucleotide sequence identity with those of human and swine HEVs. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, but the B-cell epitopes in the avian HEV ORF2 protein have not been identified. Nine synthetic peptides from the predicted four antigenic domains of the avian HEV ORF2 protein were synthesized and corresponding rabbit anti-peptide antisera were generated. Using recombinant ORF2 proteins, convalescent pig and chicken antisera, peptides and anti-peptide rabbit sera, at least one epitope at the C terminus of domain II (possibly between aa 477-492) that is unique to avian HEV, one epitope in domain I (aa 389-410) that is common to avian, human and swine HEVs, and one or more epitopes in domain IV (aa 583-600) that are shared between avian and human HEVs were identified. Despite the sequence difference in ORF2 proteins between avian and mammalian HEVs and similar ORF2 sequence between human and swine HEV ORF2 proteins, rabbit antiserum against peptide 6 (aa 389-399) recognized only human HEV ORF2 protein, suggesting complexity of the ORF2 antigenicity. The identification of these B-cell epitopes in avian HEV ORF2 protein may be useful for vaccine design and may lead to future development of immunoassays for differential diagnosis of avian, swine and human HEV infections.


Assuntos
Antígenos Virais/imunologia , Capsídeo/imunologia , Epitopos de Linfócito B/isolamento & purificação , Vírus da Hepatite E/química , Animais , Antígenos Virais/genética , Doenças das Aves/imunologia , Galinhas , Reações Cruzadas , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Anticorpos Anti-Hepatite/sangue , Hepatite E/imunologia , Hepatite E/veterinária , Vírus da Hepatite E/genética , Vírus da Hepatite E/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Epitopos Imunodominantes/metabolismo , Suínos
19.
Arch Virol ; 150(9): 1813-31, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15868095

RESUMO

This paper describes mapping of antigenic and host-protective epitopes of infectious bronchitis virus proteins by assessing the ability of defined peptide regions within the S1, S2 and N proteins to elicit humoral, cell-mediated and protective immune responses. Peptides corresponding to six regions in the S1 (Sp1-Sp6), one in the S2 (Sp7) and four in the N protein (Np1-Np4) were synthesized and coupled to either diphtheria toxoid (dt) or biotin (bt). Bt-peptides were used to assess if selected regions were antigenic and contained B- or T-cell epitopes and dt-peptides if regions induced an antibody response and protection against virulent challenge. All S1 and S2 peptides were antigenic, being recognised by IBV immune sera and also induced an antibody response following inoculation into chicks. Three S1-and one S2-bt peptides also induced a delayed type hypersensitivity response indicating the presence of T-cell epitopes. The S2 peptide Sp7 (amino acid position 566-584) previously identified as an immundominant region, was the most antigenic of all peptides used in this study. Two S1 (Sp4 and Sp6) and one S2 peptide (Sp7), protected kidney tissue against virulent challenge. From four N peptides located in the amino-terminal part of the N protein, only one, Np2 (amino acid position 72-86), was antigenic and also induced a delayed type hypersensitivity response. None of the N peptides induced protection against virulent challenge. The results suggest that the S1 glycoprotein carries additional antigenic regions to those previously identified and that two regions located in the S1 and one in the S2 at amino acid positions 294-316 (Sp4), 532-537 (Sp6) and 566-584 (Sp7) may have a role in protection.


Assuntos
Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos/isolamento & purificação , Vírus da Bronquite Infecciosa/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas do Nucleocapsídeo/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Galinhas , Epitopos/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/isolamento & purificação , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/isolamento & purificação , Hipersensibilidade Tardia , Imunização , Epitopos Imunodominantes/imunologia , Masculino , Glicoproteínas de Membrana/síntese química , Glicoproteínas de Membrana/química , Proteínas do Nucleocapsídeo/síntese química , Proteínas do Nucleocapsídeo/química , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/síntese química , Proteínas do Envelope Viral/química
20.
Glycobiology ; 13(12): 955-60, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12966039

RESUMO

The type B-specific lectin from the mushroom Marasmius oreades was immobilized onto Sepharose 4B. The immobilized lectin bound murine laminin and bovine thyroglobulin, glycoproteins that contain the Galalpha1,3Galbeta1,4GlcNAc epitope. This epitope is responsible for hyperacute rejection of xenotransplants from lower mammals to humans, Old World monkeys, or apes. The immobilized lectin also bound a fraction of serum proteins from type B human serum but little or none from type A or O(H) serum. The major protein bound from human B serum was a portion of the alpha2-macroglobulin. Treatment of this fraction with N-glycosidase F resulted in decreased molecular weight of bands associated with alpha2-macroglobulin and loss of their M. oreades lectin reactivity, whereas on treatment with coffee bean alpha-galactosidase, this bound fraction also lost reactivity with M. oreades lectin but became reactive with Ulex europaeus I lectin, suggesting the presence of L-fucosyl-alpha1,2-terminated structures. The presence of blood group epitopes on alpha2-macroglobulin has been detected previously by immunological methods, but this is the first isolation and characterization of the specifically glycosylated fraction of this serum protein. The immobilized lectin also bound a number of proteins from pig, rabbit, and rat serum that were distinct in electrophoretic mobility from the human B-serum components and presumably contain the xenotransplantation epitope among their glycan structures. This report further demonstrates the utility of immobilized lectins in isolating and characterizing glycan structures of naturally occurring glycoproteins.


Assuntos
Aglutininas/química , Glicoproteínas/química , Lectinas/química , Sistema ABO de Grupos Sanguíneos , Aglutininas/imunologia , Aglutininas/metabolismo , Animais , Sítios de Ligação , Sequência de Carboidratos , Bovinos , Cromatografia em Gel , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/isolamento & purificação , Glicoproteínas/isolamento & purificação , Humanos , Lectinas/imunologia , Lectinas/metabolismo , Dados de Sequência Molecular , Coelhos , Ratos , Sefarose , Transplante Heterólogo
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