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.
Tsunetsugu-Yokota
Y
Y
Unité de Recherches sur les Rétrovirus et Maladies Associées, U322 de l'INSERM, Marseille, France.
Tatsumi
M
M
Robert
V
V
Devaux
C
C
Spire
B
B
Chermann
J C
JC
Hirsch
I
I
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