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  • Title: High efficient production of Pr55(gag) virus-like particles expressing multiple HIV-1 epitopes, including a gp120 protein derived from an Ugandan HIV-1 isolate of subtype A.
    Author: Buonaguro L, Buonaguro FM, Tornesello ML, Mantas D, Beth-Giraldo E, Wagner R, Michelson S, Prevost MC, Wolf H, Giraldo G.
    Journal: Antiviral Res; 2001 Jan; 49(1):35-47. PubMed ID: 11166859.
    Abstract:
    The main goal of this study was to investigate a novel approach for an efficient and reproducible production of Virus-Like Particles (VLPs) expressing multiple HIV-1 epitopes. The HIV-1 Pr55(gag)-based VLPs have been produced in a Baculovirus expression system, using a transfer vector able to support the independent expression of different open reading frames (ORFs). In this regard, the gp120 derived from 94UG018 HIV-1(A) isolate, previously studied in our laboratory, has been packaged into the VLPs together with nef and pol ORFs. In particular, the gp120(UG) sequence shows a 90% homology in the V3 region compared to African HIV-1 strains of the A-clade. This novel approach is extremely effective for the production of VLPs expressing all the epitopes, as confirmed by Western Blot characterization. Furthermore, the resulting HIV-VLP(A)s show the expected density (1.14--1.18 g/ml) on a 10--60% sucrose gradient and the morphology of an immature virion at standard transmission electron microscopy. Our results demonstrate that this strategy is highly efficient for expressing a balanced amount of multiple epitopes and their packaging in VLP structures, without affecting the Pr55(gag) autoassembling capacities. Furthermore, the genetic transposition performed in a modified E. coli represents a methodological improvement, allowing a faster and more reproducible identification of recombinant Baculovirus DNA molecules.
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