Use of the recombinant chimera proteins, LacZ-Env and Gag-Env, for immunological studies on HIV-1 infection.
To use Env proteins as antigens for detection of the human immunodeficiency virus type-1 (HIV-1) antibodies, we attempted to overexpress the Env proteins in Escherichia coli. To study the epitopes in the Env proteins recognized by the sera of HIV carriers, various regions of the proviral DNA encoding the Env region were fused to the 3' end of the lacZ gene. The immunoblotting analysis of the LacZ-Env(512-611) and LacZ-Env(721-826) proteins with the 41 positive sera revealed that the former and the latter immunologically reacted with 100 and 78% of the sera, respectively. To avoid rare false-positive reactions due to the LacZ moiety of the fusion protein, we attempted to express the Env(512-611) alone or Gag-Env(512-611) under the control of bacteriophage T7 promoter. Although we could express only a low level of the Env(512-611) peptide in E. coli, we succeeded in producing large amounts of the Gag(121-406)-Env(512-611) and Gag(308-406)-Env(512-611) proteins as chimeric proteins. Both of these chimera proteins strongly reacted with the 41 positive sera. We purified these proteins and analyzed the immunological reactivity by dot blot with the 60 positive sera and the 84 normal sera. As little as 20 ng of the dotted proteins was enough for the reaction with the positive sera, whereas as much as 320 ng of them did not show false-positive reactions with the normal sera. We obtained highly purified Gag-Env proteins with highly specific seroreactivity, which should be useful for diagnosis and prognosis.
Morimoto
M
M
Osaka Prefectural Institute of Public Health, Japan.
Saitoh
A
A
Ueba
N
N
Nakata
A
A
Shinagawa
H
H
D000386:AIDS-Related Complex / Q000175:diagnosis / Q000276:immunology; D000163:Acquired Immunodeficiency Syndrome / Q000175:diagnosis / Q000276:immunology; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D000939:Epitopes / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D005189:False Positive Reactions; D015683:Gene Products, gag / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D015483:HIV Antibodies / Q000097:blood*; D015700:HIV Envelope Protein gp41 / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D015658:HIV Infections / Q000175:diagnosis* / Q000276:immunology*; D015497:HIV-1 / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
D000939:Epitopes; D015683:Gene Products, gag; D015483:HIV Antibodies; D015700:HIV Envelope Protein gp41; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase