RESUMEN
Insertion of foreign oligopeptide sequences (40-50 amino acids in length) into the Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids in Escherichia coli cells. These capsids are morphologically and immunologically similar to native HBcAg, but expose the inserted oligopeptides on their outer surface and exhibit antigenic and immunogenic characteristics of the latter. As a source of model antigenic determinants, the appropriate DNA copies excised from cloned viral genes such as the pre-S region of hepatitis B virus, the transmembrane protein gp41 of human immunodeficiency virus 1 and the envelope protein gp51 of bovine leukemia virus have been used. The localization of the inserted antigenic determinants on the surface of chimeric capsids does not depend on the presence or absence of the arginine-rich, 39 amino acid-long C terminus of HBcAg.
Asunto(s)
Antígenos Virales/genética , Antígenos de la Hepatitis B/genética , Antígenos del Núcleo de la Hepatitis B/inmunología , Cápside/inmunología , ADN Recombinante , Epítopos , Genes Virales , Vectores Genéticos , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/inmunología , Antígenos del Núcleo de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/ultraestructura , Inmunohistoquímica , Virus de la Leucemia Bovina/genética , Virus de la Leucemia Bovina/inmunología , Datos de Secuencia Molecular , Vacunas Sintéticas , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Expression of the coat protein gene of RNA bacteriophage fr in Escherichia coli cells leads to the formation of capsid-like structures of ca. 25 nm in diameter, which are immunologically indistinguishable from the native phage fr capsids. The modification strategy of the coat protein gene by gene engineering technique was developed in order to localize coat protein regions, which are exposed on the capsid surface and are capable to include foreign amino acid inserts without an appreciable effect on the capsid self-assembly. The oligonucleotide linkers, coding short amino acid sequences and bearing also convenient restriction sites, were synthesized and inserted into different regions of the coat protein gene. The mutant proteins, containing insertions of 2-12 amino acids in potentially exposed regions, were obtained. It was shown that N- and C-terminal insertions, as well as the insertion into codon 51 in the RNA-binding region, do not prevent the self-assembly. The regions (codons 96 and 112) were also revealed, insertions in them decreased drastically the protein yield as a consequence of a block in the self-assembly.
Asunto(s)
Genes Virales , Ingeniería Genética , Mutación , Fagos ARN/genética , Proteínas del Envoltorio Viral/genética , Secuencia de Bases , Epítopos/análisis , Escherichia coli/genética , Inmunodifusión , Datos de Secuencia Molecular , Plásmidos , Fagos ARN/inmunología , Fagos ARN/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestructura , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/ultraestructuraRESUMEN
Insertion of 48 amino acid long sequence of envelope protein gp51 of bovine leukemia virus (BLV), located from position 56 till 103 of mature protein, into Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids. These capsids preserve morphology of intact HBcAg but expose on their outer surface BLV epitopes which are localised in the inserted gp51 fragment and responsible for the recognition of chimeras by monoclonal anti-gp51 antibodies MAK14. The anti-genicity of gp51 epitopes within chimeric capsids is not disturbed after shortening of C terminal part of inserted gp51 fragment by deletion of amino acids 73-103. The resulting chimeras show the same capsid-forming ability as well as HBcAg and gp51 antigenic properties.