The Cloning and Expression in Escherichia coli of Sequences Coding for p24, the Core Protein of Human Immunodeficiency Virus, and the Use of the Recombinant Protein in Characterizing a Panel of Monoclonal Antibodies against the Viral p24 Protein

Abstract

SUMMARYThe sequences encoding the p24 core protein of human immunodeficiency virus type 1 were identified in a cDNA library made from infected CEM cells. The nucleotide sequence of the DNA coding for p24 was shown to be very similar but not identical to the sequences of lymphadenopathy virus and human T-cell leukaemia virus type IIIb. These sequences were expressed in Escherichia coli at the amino terminus of fl-galactosidase and used to screen a panel of monoclonal antibodies raised against virus-expressed p24. Regions containing the epitopes of five of the monoclonal antibodies were located using a series of amino-and carboxy-terminal deletion mutants of the recombinant p24 protein.

Antisera raised against the second variable region of the external envelope glycoprotein of human immunodeficiency virus type 1 cross-neutralize and show an increased neutralization index when they act together with antisera to the V3 neutralization epitope

“…A similar assay was set up using a modified version of the gag p24 assay described by Moore et al (1990) and McKeating et al (1991) in which sheep polyclonal antisera raised against gag peptides (Aalto Bioreagents) are used as the solid phase and monoclonal antibody EH12EI (Spence et al, 1989) is used to detect captured gag p24 antigen. Bound monoclonal antibody is detected using 125I-radiolabelled anti-mouse IgG.…”

The Expression in Escherichia coli of Sequences Coding for the p18 Protein of Human Immunodeficiency Virus and the Use of the Recombinant Protein in Characterizing a Panel of Monoclonal Antibodies against the Viral p18 Protein

“…This change causes an amino acid substitution (arginine for leucine) when compared to both LAV and HTLV-IIIb (Sanchez-Pescador et al, 1985;Wain-Hobson et al, 1985). As the pDM614 plasmid contained 38 bp from the 3' end of the p 18 coding region at the 5' end of the p24 coding region (Spence et al, 1989) the 5' end of this expression fragment was also sequenced. A variation from the LAV sequence at position 714 was observed (CBL-1, T; HTLV-IIIb, T; LAV, C) which did not cause an amino acid substitution.…”

“…In this paper we describe the expression of p 18 sequences in Escherichia coli, the mapping of the epitopes of five p 18-reactive MAbs and fluorescence-activated cell sorter analysis (FACS) of HIV-infected cells using the MAbs. In the accompanying paper (Spence et al, 1989) we describe the cloning of the sequences encoding the p18 and p24 proteins from the CBL-1 isolate, the expression of p24 sequences and the epitope mapping of six anti-p24 MAbs.…”

“…BamHl linkers of 8 bp were ligated to the ends of the fragment which was then digested with both EcoRI and BamHI. The 340 bp product was cloned into the EcoRI-and BamHI-digested pXY460 expression vector (Spence et al, 1989). pDM602 was made from pB2 in a similar manner, except that because of methylation of the TaqI site at position 377 [nucleotide numbering from Wain-Hobson et al (1985)] only the TaqI site at position 232 was cleaved.…”

“…Bacterial cell lysates were run on 10 ~ or 10 to 25~ gradient SDS-PAGE gels (Laemmli, 1970) and processed as described (Spence et aL, 1989).…”