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1.
Microb Pathog ; 148: 104459, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32835775

RESUMEN

Coronavirus disease (COVID-19) is an infectious disease caused by a newly discovered coronavirus that it disease spreads in over the world. Coronaviruses are single-stranded, positive-sense RNA viruses with a genome of approximately 30 KD, the largest genome among RNA viruses. Most people infected with the COVID-19 virus will experience mild to moderate respiratory illness and recover without requiring special treatment. Older people and those with underlying medical problems like cardiovascular disease, diabetes, chronic respiratory disease, and cancer are more likely to develop serious illness. At this time, there are no specific vaccines or treatments for COVID-19. So, there is an emergency need for vaccines and antiviral strategies. The spike protein is the major surface protein that it uses to bind to a receptor of another protein that acts as a doorway into a human cell. The putative antigenic epitopes may prove effective as novel vaccines for eradication and combating of COV19 infection. A combination of available bioinformatics tools are used to synthesis of such peptides that are important for the development of a vaccine. In conclusion, amino acids 250-800 were selected as effective B cell epitopes, T cell epitopes, and functional exposed amino acids in order to a recombinant vaccine against coronavirus.


Asunto(s)
Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Sitios de Unión , COVID-19/inmunología , COVID-19/virología , Diseño de Fármacos , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/aislamiento & purificación , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/aislamiento & purificación , Humanos , Modelos Moleculares , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Glicoproteína de la Espiga del Coronavirus/química
2.
Transpl Immunol ; 51: 1-5, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30081185

RESUMEN

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.


Asunto(s)
Epítopos de Linfocito B/aislamiento & purificación , Epítopos/aislamiento & purificación , Rechazo de Injerto/inmunología , Antígenos HLA/inmunología , Prueba de Histocompatibilidad/métodos , Trasplante de Riñón , Receptores de Trasplantes , Algoritmos , Procesamiento Automatizado de Datos , Rechazo de Injerto/prevención & control , Humanos , Inmunización , Isoanticuerpos/metabolismo , Programas Informáticos
3.
Mol Med Rep ; 16(3): 2851-2855, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28656246

RESUMEN

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.


Asunto(s)
Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Inmunoglobulina E/inmunología , Magnoliopsida/inmunología , Rinitis Alérgica Estacional/inmunología , Adolescente , Adulto , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/inmunología , Antígenos de Plantas/aislamiento & purificación , Clonación Molecular , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/aislamiento & purificación , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/aislamiento & purificación , Escherichia coli/genética , Femenino , Humanos , Inmunoglobulina E/sangre , Magnoliopsida/química , Magnoliopsida/genética , Masculino , Persona de Mediana Edad , Modelos Moleculares , Polen/química , Polen/genética , Polen/inmunología , Conformación Proteica , Rinitis Alérgica Estacional/sangre , Adulto Joven
4.
Viruses ; 9(5)2017 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-28509875

RESUMEN

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.


Asunto(s)
Epítopos/inmunología , Productos del Gen pol/efectos de los fármacos , Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Hepatitis B/prevención & control , Vacunas Virales/inmunología , Secuencia de Aminoácidos , Línea Celular , Simulación por Computador , Epítopos/química , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/aislamiento & purificación , Epítopos de Linfocito T/inmunología , Antígeno HLA-A2/química , Antígeno HLA-A2/inmunología , Humanos , Inmunogenicidad Vacunal/inmunología , Simulación del Acoplamiento Molecular , Péptidos/síntesis química , Péptidos/farmacología , Vacunas Virales/farmacología
5.
Parasitol Int ; 66(3): 324-330, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28110081

RESUMEN

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.


Asunto(s)
Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Giardia lamblia/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/administración & dosificación , Antígenos de Protozoos/genética , Antígenos de Protozoos/aislamiento & purificación , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/aislamiento & purificación , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/aislamiento & purificación , Inmunidad Celular , Inmunidad Humoral , Proteínas de la Membrana/genética , Ratones , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Espectrometría de Masas en Tándem , Trofozoítos/inmunología
6.
Biotechnol Appl Biochem ; 64(1): 117-125, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26549515

RESUMEN

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.


Asunto(s)
Epítopos de Linfocito B , Expresión Génica , Proteínas de Choque Térmico , Animales , Chaperón BiP del Retículo Endoplásmico , Epítopos de Linfocito B/biosíntesis , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/aislamiento & purificación , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/inmunología , Proteínas de Choque Térmico/aislamiento & purificación , Humanos , Ratones , Ratones Endogámicos BALB C , Dominios Proteicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación
7.
Hum Immunol ; 77(10): 824-831, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27374949

RESUMEN

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.


Asunto(s)
Mapeo Epitopo/métodos , Epítopos de Linfocito B/aislamiento & purificación , Antígenos HLA-DQ/aislamiento & purificación , Embarazo/inmunología , Programas Informáticos , Alelos , Anticuerpos/sangre , Simulación por Computador , Epítopos de Linfocito B/metabolismo , Femenino , Genotipo , Antígenos HLA-DQ/metabolismo , Prueba de Histocompatibilidad , Humanos , Inmunidad Humoral , Conformación Proteica , Sistema de Registros
8.
Protein Expr Purif ; 102: 38-44, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24996028

RESUMEN

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.


Asunto(s)
Toxinas Bacterianas/genética , Clostridium perfringens/genética , Enterotoxinas/genética , Epítopos de Linfocito B/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Vibrio cholerae/genética , Toxinas Bacterianas/aislamiento & purificación , Vacunas Bacterianas/genética , Vacunas Bacterianas/aislamiento & purificación , Secuencia de Bases , Enterotoxinas/aislamiento & purificación , Epítopos de Linfocito B/aislamiento & purificación , Proteínas de Escherichia coli/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación
9.
Exp Parasitol ; 135(3): 606-10, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24090568

RESUMEN

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.


Asunto(s)
Antígenos de Protozoos/inmunología , Epítopos de Linfocito B/aislamiento & purificación , Enfermedades de los Porcinos/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Epítopos de Linfocito B/inmunología , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/aislamiento & purificación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Vacunas Antiprotozoos , Juego de Reactivos para Diagnóstico/veterinaria , Porcinos , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/parasitología , Toxoplasma/genética , Toxoplasmosis Animal/diagnóstico , Vacunas de Subunidad
10.
Allergol Int ; 62(4): 435-45, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23963475

RESUMEN

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.


Asunto(s)
Anafilaxia/inmunología , Ejercicio Físico , Gliadina/inmunología , Jabones/efectos adversos , Triticum/inmunología , Hipersensibilidad al Trigo/inmunología , Adolescente , Adulto , Anciano , Alérgenos/química , Alérgenos/inmunología , Antígenos de Plantas/inmunología , Basófilos/inmunología , Células Cultivadas , Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/aislamiento & purificación , Femenino , Gliadina/química , Glútenes/inmunología , Histamina/metabolismo , Humanos , Hidrólisis , Inmunización , Inmunoglobulina E/metabolismo , Japón , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Jabones/química , Triticum/química , Adulto Joven
11.
Hum Immunol ; 74(9): 1141-52, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23756161

RESUMEN

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.


Asunto(s)
Epítopos de Linfocito B/inmunología , Rechazo de Injerto/inmunología , Cadenas alfa de HLA-DQ/inmunología , Cadenas beta de HLA-DQ/inmunología , Trasplante de Riñón , Complicaciones Posoperatorias/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Presentación de Antígeno , Sitios de Unión de Anticuerpos/genética , Línea Celular , Mapeo Epitopo , Epítopos de Linfocito B/aislamiento & purificación , Cadenas alfa de HLA-DQ/genética , Cadenas beta de HLA-DQ/genética , Histocompatibilidad , Humanos , Isoanticuerpos/sangre , Isoanticuerpos/inmunología , Unión Proteica , Transgenes/genética
12.
Vet Microbiol ; 164(1-2): 18-26, 2013 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-23434015

RESUMEN

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.


Asunto(s)
Epítopos de Linfocito B/aislamiento & purificación , Infecciones por Herpesviridae/veterinaria , Herpesvirus Équido 1 , Enfermedades de los Caballos/diagnóstico , Proteínas del Envoltorio Viral/aislamiento & purificación , Animales , Ensayo de Inmunoadsorción Enzimática/veterinaria , Infecciones por Herpesviridae/diagnóstico , Herpesvirus Équido 4 , Caballos
13.
Protein Expr Purif ; 88(1): 7-12, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23220378

RESUMEN

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.


Asunto(s)
Epítopos de Linfocito B/genética , Epítopos de Linfocito T/aislamiento & purificación , Receptores ErbB/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Animales , Epítopos de Linfocito B/biosíntesis , Epítopos de Linfocito B/aislamiento & purificación , Epítopos de Linfocito T/biosíntesis , Epítopos de Linfocito T/genética , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Escherichia coli , Expresión Génica , Humanos , Masculino , Virus del Sarampión/química , Virus del Sarampión/genética , Ratones , Péptidos/genética , Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Virales de Fusión/biosíntesis , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/aislamiento & purificación
14.
Mol Biol (Mosk) ; 46(2): 251-9, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22670521

RESUMEN

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.


Asunto(s)
Epítopos de Linfocito B , Proteínas del Núcleo Viral , Animales , Anticuerpos Antivirales/inmunología , Epítopos de Linfocito B/biosíntesis , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/aislamiento & purificación , Epítopos de Linfocito B/farmacología , Escherichia coli , Expresión Génica , Hepacivirus/genética , Hepacivirus/inmunología , Humanos , Inmunización , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Proteínas del Núcleo Viral/biosíntesis , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/aislamiento & purificación , Proteínas del Núcleo Viral/metabolismo , Proteínas del Núcleo Viral/farmacología
15.
J Immunoassay Immunochem ; 31(3): 181-92, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20623405

RESUMEN

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.


Asunto(s)
Especificidad de Anticuerpos , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Epítopos de Linfocito B/inmunología , Mycobacterium avium subsp. paratuberculosis/química , Animales , Reacciones Antígeno-Anticuerpo , Antígenos Bacterianos/aislamiento & purificación , Bovinos , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos de Linfocito B/aislamiento & purificación , Mycobacterium avium subsp. paratuberculosis/inmunología , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Paratuberculosis/inmunología , Péptidos/síntesis química , Péptidos/química , Péptidos/inmunología , Conejos , Sensibilidad y Especificidad
16.
Vet Microbiol ; 140(1-2): 25-33, 2010 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-19699594

RESUMEN

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.


Asunto(s)
Epítopos de Linfocito B/inmunología , Virus de la Fiebre Aftosa/inmunología , Proteínas Estructurales Virales/inmunología , Animales , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Secuencia de Bases , Proteínas de la Cápside/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/aislamiento & purificación , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/genética , Datos de Secuencia Molecular , Conejos , Serotipificación , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/genética
17.
Vet Microbiol ; 142(3-4): 293-302, 2010 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-19913364

RESUMEN

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.


Asunto(s)
Epítopos de Linfocito B/aislamiento & purificación , Lipoproteínas/aislamiento & purificación , Mycoplasma hyopneumoniae/inmunología , Neumonía Porcina por Mycoplasma/microbiología , Análisis por Matrices de Proteínas/veterinaria , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Epítopos , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Immunoblotting/veterinaria , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/inmunología , Neumonía Porcina por Mycoplasma/diagnóstico , Neumonía Porcina por Mycoplasma/inmunología , Conejos , Proteínas Recombinantes de Fusión/inmunología , Alineación de Secuencia , Porcinos , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/inmunología
18.
BMC Microbiol ; 7: 113, 2007 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-18088426

RESUMEN

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.


Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Epítopos de Linfocito B/inmunología , Epítopos Inmunodominantes/inmunología , Serina Endopeptidasas/inmunología , Streptococcus pneumoniae/inmunología , Anticuerpos Antibacterianos/inmunología , Especificidad de Anticuerpos , Epítopos de Linfocito B/aislamiento & purificación , Humanos , Epítopos Inmunodominantes/aislamiento & purificación , Inmunoglobulina A/metabolismo , Biblioteca de Péptidos , Infecciones Neumocócicas/inmunología , Streptococcus/enzimología , Streptococcus pneumoniae/enzimología
19.
J Gen Virol ; 87(Pt 1): 217-223, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16361434

RESUMEN

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.


Asunto(s)
Antígenos Virales/inmunología , Cápside/inmunología , Epítopos de Linfocito B/aislamiento & purificación , Virus de la Hepatitis E/química , Animales , Antígenos Virales/genética , Enfermedades de las Aves/inmunología , Pollos , Reacciones Cruzadas , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Anticuerpos Antihepatitis/sangre , Hepatitis E/inmunología , Hepatitis E/veterinaria , Virus de la Hepatitis E/genética , Virus de la Hepatitis E/inmunología , Humanos , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/metabolismo , Porcinos
20.
Arch Virol ; 150(9): 1813-31, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15868095

RESUMEN

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.


Asunto(s)
Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Epítopos/aislamiento & purificación , Virus de la Bronquitis Infecciosa/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas de la Nucleocápside/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos , Pollos , Proteínas de la Nucleocápside de Coronavirus , Epítopos/inmunología , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/aislamiento & purificación , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/aislamiento & purificación , Hipersensibilidad Tardía , Inmunización , Epítopos Inmunodominantes/inmunología , Masculino , Glicoproteínas de Membrana/síntesis química , Glicoproteínas de Membrana/química , Proteínas de la Nucleocápside/síntesis química , Proteínas de la Nucleocápside/química , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/síntesis química , Proteínas del Envoltorio Viral/química
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