Expression of an immunogenic region of HIV by a filamentous bacteriophage vector.
Vectors derived from the Escherichia coli filamentous phage, fd-tet, expressing parts of the human immunodeficiency virus (HIV) gag genes were constructed and analyzed. The immunoreactive domain of HIV Gag antigens was produced in the form of a fusion protein, with a phage minor coat protein, called protein III, playing an important role in phage infectivity. A micropanning procedure, utilizing the strong affinity of biotinylated antibody to streptavidin, was applied for the selection of clones. A simple preparation procedure consisting of polyethyleneglycol precipitation of the recombinant phage from the E. coli supernatant allowed us to detect HIV antigens by enzyme-linked immunosorbent assay (ELISA). Cloned FUSE-gag, as isolated using anti-Gag RL4.72.1 monoclonal antibody (mAb), contained a nucleotide sequence coding for 91 amino acids of HIV Gag p24. It specifically reacted with the mAb in the ELISA. Construction of the mAb-selectable phages permitted localization of epitopes for mAb. Infectivity of the phage clone was specifically neutralized by the anti-HIV mAb. Immunoelectroblotting analysis of recombinant phages revealed the presence of an about 65-kDa band reacting with anti-HIV mAb. This Mr corresponded to the size of the fused form of the FUSE 1 protein III. Human sera from HIV-infected and uninfected individuals reacted with recombinant protein III, as well as the original form of protein III.
Unité de Recherches sur les Rétrovirus et Maladies Associées, U322 de l'INSERM, Marseille, France.
Y
Y
Tsunetsugu-Yokota
M
M
Tatsumi
V
V
Robert
C
C
Devaux
B
B
Spire
J C
JC
Chermann
I
I
Hirsch
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D015683:Gene Products, gag / Q000096:biosynthesis / Q000276:immunology*; D015750:Genes, gag; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D014760:Viral Fusion Proteins / Q000096:biosynthesis / Q000276:immunology*
D015683:Gene Products, gag; D014760:Viral Fusion Proteins