RESUMO
The Q beta gene C has been proposed as a new carrier for the exposure of foreign peptide sequences. Contrary to well-known 'display vectors' on the basis of coat proteins of RNA phage group I, group III phage Q beta-based vectors suggested application of the 195-amino acid extension of coat protein (CP) within the so-called A1 protein for insertion of the appropriate immunological epitopes. 'Mosaic' capsids presenting model hepatitis B virus preS1 and HIV-1 gp120 epitopes and formed by Q beta CP together with A1-derived proteins were obtained as a result of (1) suppression of leaky UGA stop codon of the CP gene and (2) simultaneous expression of 'pure' CP and full-length A1-derived genes obtained after the changing of CP-terminating UGA to strong UAA stop codon or sense GGA codon, respectively.
Assuntos
Capsídeo/genética , Epitopos/genética , Vetores Genéticos , Fagos RNA/genética , Sequência de Aminoácidos , Códon de Terminação , Expressão Gênica , Proteína gp120 do Envelope de HIV/genética , Antígenos de Superfície da Hepatite B/genética , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fatores de Terminação de Peptídeos , Precursores de Proteínas/genéticaRESUMO
The Escherichia coli RNA phage Q beta coat protein-encoding gene (C) was amplified from native Q beta RNA using a reverse transcription-PCR technique. Gene C contains sequences coding for both the 133-amino acid (aa) Q beta coat protein (CP) and the 329-aa read-through protein (A1) consisting of CP and an additional 196-aa C-terminal sequence, separated from CP within the C gene by an opal (UGA) stop codon. Primers ensuring the natural environment for gene C, especially within the ribosome-binding site, and supplying C with unique restriction sites at both ends have been prepared. An amplified 1062-bp PCR fragment was positioned under the control of the strong E. coli trp promoter (Ptrp) within a pGEM-derived plasmid. The synthesis of gene C products was confirmed electrophoretically and immunologically. An immunodiffusion test with anti-Q beta phage antibodies and electron microscopy evaluation of the purified recombinant products showed that when expressed, the Q beta C gene was responsible for high-level synthesis and correct self-assembly of Q beta CP monomers into capsids indistinguishable morphologically and immunologically from Q beta phage particles, which we plan to use as surface display vectors.