RESUMO
The hepatitis B core antigen (HBcAg) was used to present the HIV epitopes and mimics selected by phage display. The HIV epitopes were inserted into the el loop of HBcAg. The influence of insertions on the ability of chimeric HBcAg to assemble itself was studied. Special soft was made use of to detect the regularities between certain physical-and-chemical properties of amine-acid residua (belonging to an inserted alien peptide) and the presence or loss of the ability of HBcAg to assemble itself. Recommendations are provided of how to overcome difficulties related with the presentation of alien epitopes.
Assuntos
Epitopos/imunologia , HIV-1/imunologia , HIV-2/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Anticorpos Antivirais/biossíntese , Sistemas de Liberação de Medicamentos , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Humanos , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de Proteína , Vacinas Sintéticas , Vacinas ViraisRESUMO
Bacteriophages bearing peptides reacting with antihemagglutinating monoclonal antibodies (MAb) 10H10 to tick-borne encephalitis (TBE) virus protein E were selected from a phage-display peptide library by affinity selection and enzyme immunoassay. The library contained randomized peptides that are 6 amino acids long, fused with protein pIII and exposed on the surface of the bacteriophage. No significant homology between the sequences of selected peptides and TBE virus protein E was detected. Computer software was created to locate the conformation epitopes on the surface of protein E. Amino acids R73, C74, T76, M77, N103, C105, L107, and S112 were found to form a discontinuous epitope recognized by MAb 10H10. These amino acids are remote in the protein sequence but close in the tertiary structure and form a whole epitope located in the structural domain II of protein E. Presumably the localized amino acids bind to the cellular receptor for TBE virus.
Assuntos
Antígenos Virais/imunologia , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Hemaglutininas Virais/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Bacteriófagos/genética , Epitopos/análise , Epitopos/genética , Epitopos/imunologia , Técnicas Imunoenzimáticas , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/análise , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Alinhamento de Sequência , Proteínas do Envelope Viral/químicaRESUMO
Recombinant strains producing hepatitis B virus (HBcAg) core protein and chimeric core protein exposing on its surface the major immunogenic epitope of HBsAg (HBcAg-HBs) were constructed on the base of attenuated S. typhimurium SL 7202 strain. The resultant Salmonella strains produced proteins which were capable of self-assembly into virus-like particles and showed antigenic properties of both core and surface hepatitis B proteins. A single rectal immunization with recombinant S. typhimurium induced humoral and cellular immune response to HBcAg and HBsAg. Specific anti-HBcAg were detected in animal sera and intestinal tissues, which indicated the formation of specific mucosal immunity.
Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/genética , Salmonella typhimurium/genética , Animais , Epitopos/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/biossíntese , Antígenos de Superfície da Hepatite B/biossíntese , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Recombinação Genética , Salmonella typhimurium/imunologiaAssuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Viral da Expressão Gênica , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Recombinante , Vírus da Encefalite Equina Venezuelana/genética , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Plasmídeos , Proteínas Virais , alfa 1-Antitripsina/genética , beta-Galactosidase/genéticaRESUMO
Three new approaches to design effective immunogens are considered. At first, we derived an expression vector from bacteriophage M13 allowing the exposure of short peptides on the virion surface. EIA demonstrates that antibodies against a recombinant phage carrying the antigenic determinant of the HIV-1 gag protein reacted with the 17-kDa core protein of the virus and also with its polyprotein precursor p55 in immunoblotting. In another approach, we chose the hepatitis B core antigen (HBcAg) particle as a vehicle for the presentation of foreign antigenic determinants to the immune system. Chimerical particles of HBcAg containing epitope of the VEE virus were obtained. A vector system for insertion of foreign antigenic determinants and production of both hybrid and wild HBcAg proteins were also obtained. The third approach relies on construction of immunogens from different T- and B-cell epitopes of the HIV-1. We suggested to construct HIV-1 vaccines in a form of the TBI (T- and B-cell epitopes containing Immunogen) with a predetermined tertiary structure, namely, a four-alpha-helix bundle. The gene of the TBI protein consisting of nine HIV-1 epitopes was synthesized and expressed in Escherichia coli cells. Mice immunized with TBI showed humoral and cellular immune responses to HIV-1. Anti-TBI antibodies displayed HIV-1 neutralizing activity. These new approaches offer promise in the development of new effective vaccines.
Assuntos
Vacinas contra a AIDS , Antígenos Virais/imunologia , Vacinas Sintéticas , Vacinas Virais , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Antígenos Virais/genética , Bacteriófago M13 , Sequência de Bases , Primers do DNA , Desenho de Fármacos , Vírus da Encefalite Equina do Leste/genética , Vírus da Encefalite Equina do Leste/imunologia , Vírus da Encefalite Equina Venezuelana/genética , Epitopos/química , Epitopos/imunologia , Escherichia coli , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Genes gag , HIV-1/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/biossíntese , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Cavalos , Humanos , Camundongos , Modelos Estruturais , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Linfócitos T/imunologiaAssuntos
RNA Polimerases Dirigidas por DNA/genética , Regulação da Expressão Gênica , Vetores Genéticos , Fagos T/genética , Mapeamento Cromossômico , Escherichia coli/metabolismo , Cinética , Mapeamento por Restrição , Proteínas Ribossômicas/análise , Fagos T/enzimologia , beta-Galactosidase/biossínteseRESUMO
A hybrid beta-lactamase gene with a synthetic tuftsin-coding DNA fragment inserted at the Pst I-site of pBR322 plasmid has been obtained and its expression has been studied. Radioactive amino acids have been used to show that in E. coli chi 925 minicells up to 30% of newly synthesized chimeric protein is secreted into periplasm providing the tuftsin transport. After hybrid protein cleavage with CNBr, tuftsin has been isolated using ion-exchange and thin-layer chromatography.
