PMID: 7537750Feb 1, 1995Paper

A simple procedure to generate chimeric Pr55gag virus-like particles expressing the principal neutralization domain of human immunodeficiency virus type 1

Journal of Virological Methods
D BrandF Barin

Abstract

The Pr55gag human immunodeficiency virus type 1 (HIV-1) precursor protein that is capable of auto-assembling was used as a carrier for a consensus sequence of the principal neutralization domain (PND) of the HIV-1 envelope. For this purpose, a modified HIV-1 gag gene with deletion of the sequence encoding a previously described p24 epitope (amino acids 196-228 of Pr55gag) was first obtained using PCR with degenerate primers, and then cloned. This deleted gag gene allowed in a second time the insertion of a synthetic oligonucleotide cassette encoding the North American/European consensus PND precisely in place of the p24 epitope. The chimeric gene was then inserted into a baculovirus transfer vector and expressed in insect cells. The construct formed 100-140 nm virus-like particles that were released into the extracellular medium. The use of a serum-free medium that supports growth of insect cells facilitated the downstream purification of the extracellular particles. The chimeric particles were recognized by monoclonal antibodies directed to V3 by Western blot but not by immune electron microscopy, suggesting that, although the inserted sequence was still antigenic it was not exposed at the surface of the particles. The results...Continue Reading

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