Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents |
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| Authors: | R L Shoeman D Young R Pottathil J Victor R R Conroy R M Crowl T Coleman E Heimer C Y Lai K Ganguly |
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| Institution: | 1. Roche Institute of Molecular Biology, Roche Research Center, Hoffmann-LaRoche, Inc., Nutley, New Jersey 07110 USA;2. Department of Molecular Genetics, Roche Research Center, Hoffmann-LaRoche, Inc., Nutley, New Jersey 07110 USA;3. Department of Diagnostic Research, Roche Research Center, Hoffmann-LaRoche, Inc., Nutley, New Jersey 07110 USA;4. Department of Peptide Research, Roche Research Center, Hoffmann-LaRoche, Inc., Nutley, New Jersey 07110 USA;5. Department of Protein Chemistry, Roche Research Center, Hoffmann-LaRoche, Inc., Nutley, New Jersey 07110 USA;6. Department of Bioprocess Development, Roche Research Center, Hoffmann-LaRoche, Inc., Nutley, New Jersey 07110 USA;1. Department of Biochemistry, Faculty of Chemistry—University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia;2. Department of Organic Chemistry, Faculty of Chemistry—University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia;3. Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, Knez Mihailova 35, 11000 Belgrade, Serbia;1. Sorbonne Université, Institut de Biologie Paris-Seine (IBPS), CNRS UMR 8256, Inserm ERL U1164, Biological Adaptation and Ageing, 75005, Paris, France;2. Université de Paris, Unit of Functional and Adaptive Biology, CNRS UMR 8251, 75013, Paris, France;1. Institute of Applied Computer Science, Lodz University of Technology, Stefanowskiego 18/22, 90-924 Lodz, Poland;2. Department of Plant Physiology and Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland |
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| Abstract: | Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting. |
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