RESUMO
The hemagglutinin genes (HA1 subunit) from human and animal 2009 pandemic H1N1 virus isolates were expressed with a baculovirus vector. Recombinant HA1 (rHA1) protein-based ELISA was evaluated for detection of specific influenza A(H1N1)pdm09 antibodies in serum samples from vaccinated humans. It was found that rHA1 ELISA consistently differentiated between antibodies recognizing the seasonal influenza H1N1 and pdm09 viruses, with a concordance of 94% as compared to the hemagglutination inhibition test. This study suggests the utility of rHA1 ELISA in serosurveillance.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais , Técnicas de Laboratório Clínico/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/diagnóstico , Infecções por Orthomyxoviridae/diagnóstico , Animais , Antígenos Virais/genética , Baculoviridae/genética , Ensaio de Imunoadsorção Enzimática/métodos , Expressão Gênica , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia , Proteínas Recombinantes/genética , Sensibilidade e Especificidade , Soro/imunologiaRESUMO
Monoclonal antibodies (MAbs) against the E2 protein of classical swine fever virus (CSFV) are useful for diagnosis and strain characterization. A purified, baculovirus-expressed CSFV E2 protein from the Paderborn strain was formulated with a saponin adjuvant and successfully used to induce an antigen-specific immune response in mice. After cell fusion a panel, designated F92G, of 12 mouse hybridomas (5-2, 11-1, 14-1, 25-2, 28-2, 31-1, 34-1, 35-2, 37-3, 38-2, 39-1, 41-1) producing CSFV-E2 specific MAbs were selected based on their Ig subclass and secretion level (µg IgG/mL). Nine IgG 1/k, two IgG 2b/k, and one IgG 2a/k MAbs were further characterized using immunoperoxidase reactivity, ELISA, and Western blot analysis. Immunoglobulin concentration-dependent immunoperoxidase and ELISA reactivity was observed for some of the MAbs with certain antigens. In general there were several reactivity patterns exhibited by the MAbs, with CSFV strains representing different genetic subgroups (by immunoperoxidase staining) and recombinant antigens (by ELISA). It was interesting to note that in some cases the strain-specific reactivity of a MAb was dependent on the test, thereby providing a clue regarding the nature of the binding site.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Antígenos Virais/imunologia , Vírus da Febre Suína Clássica/imunologia , Imunoglobulina G/imunologia , Proteínas do Envelope Viral/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Baculoviridae/genética , Sítios de Ligação de Anticorpos , Western Blotting , Vírus da Febre Suína Clássica/química , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Hibridomas/imunologia , Imunização , Técnicas Imunoenzimáticas , Imunoglobulina G/biossíntese , Camundongos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Especificidade da Espécie , Suínos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Recently, we reported the expression and glycosylation of avian metapneumovirus attachment glycoprotein (AMPV/C G protein) in eukaryotic cell lines by a transient-expression method. In the present study, we investigated the biosynthesis and O-linked glycosylation of the AMPV/C G protein in a baculovirus expression system. The results showed that the insect cell-produced G protein migrated more rapidly in SDS-PAGE as compared to LLC-MK2 cell-derived G proteins owing to glycosylation differences. The fully processed, mature form of G protein migrated between 78 and 86 kDa, which is smaller than the 110 kDa mature form of G expressed in LLC-MK2 cells. In addition, several immature G gene products migrating at 40-48 and 60-70 kDa were also detected by SDS-PAGE and represented glycosylated intermediates. The addition of the antibiotic tunicamycin, which blocks early steps of glycosylation, to insect cell culture resulted in the disappearance of two glycosylated forms of the G protein and identified a 38 kDa unglycosylated precursor. The maturation of the G protein was completely blocked by monensin, suggesting that the O-linked glycosylation of G initiated in the trans-Golgi compartment. The presence of O-linked sugars on the mature protein was further confirmed by lectin Arachis hypogaea binding assay. Furthermore, antigenic features of the G protein expressed in insect cells were evaluated by ELISA.
Assuntos
Expressão Gênica , Metapneumovirus/genética , Processamento de Proteína Pós-Traducional , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Glicosilação , Lectinas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Spodoptera , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologiaRESUMO
Biosynthesis, glycosylation and cell surface expression of the AMPV/C G protein were examined in eukaryotic cell lines (LLC-MK2, CHO-K1, CHO-1d1D). Immature G gene products with a molecular mass of 42, 45 and 58-90 kilodaltons (kDa) were identified by SDS-PAGE and represented glycosylated intermediates. Tunicamycin treatment of transfected cells confirmed the presence of N-linked carbohydrate moieties on these intermediate species and identified a 38 kDa unglycosylated precursor. A fully processed, mature form of the protein migrated around 110 kDa. The presence of O-linked sugars on the mature G protein was confirmed by using the O-glycosylation deficient CHO-ldlD cell line supplemented with exogenous Gal and GalNAc. Binding of the lectin Arachis hypogaea (peanut agglutinin) confirmed the presence of O-linked sugars on the mature protein and its intracellular transport to the cell surface was demonstrated by indirect immunofluorescent staining.