Current drug delivery · 2020
Production of Recombinant HIV-1 p24-Nef Protein in Two Forms as Potential Candidate Vaccines in Three Vehicles.
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Larijani Mona Sadat, Pouriayevali Mohammad Hassan, Sadat Seyed Mehdi, Ramezani Amitis
Аннотация
BACKGROUND: Different approaches have been investigated to develop a preventive or therapeutic vaccine, although none of them has been fully practical. Therapeutic vaccines against HIV-1 have been studied with the aim of eliminating the virus from reservoir cells with or without HAART (Highly Active Antiretroviral Therapy). Fusion proteins with the most immunogenic features among conserved regions can facilitate this achievement in such a variable virus. To achieve the most immunogenic and also conserved regions, bioinformatics tools are widely used to predict antigens' features before applying them.
OBJECTIVE: This study aimed at the in vitro evaluation of p24 -Nef fusion protein based on the previous in silico design to achieve a potential therapeutic subunit vaccine against HIV-1.
METHODS: The truncated form of p24-Nef using AAY flexible linker and the full protein were expressed and evaluated in the prokaryotic system and confirmed by western blotting. We also used pcDNA3.1 to transfect Lenti-X 293T cells. Moreover, lentiviral vectors were applied to produce recombinant virions harboring the genes of interest and cell transduction.
RESULTS: Both fusion proteins in a truncated and a full form were expressed and confirmed by Anti Nef polyclonal antibody in western blotting. Recombinant virions were generated and transduced Lenti-X 293T cells confirming by immunofluorescence microscope and p24 ELISA assay kit. Transduced cells were analyzed by SDS-PAGE and western blotting, which resulted in approved protein expression.
CONCLUSION: Fusion protein of p24 and Nef is well expressed in eukaryotic cell lines according to its pre-evaluated features by bioinformatics tools.
OBJECTIVE: This study aimed at the in vitro evaluation of p24 -Nef fusion protein based on the previous in silico design to achieve a potential therapeutic subunit vaccine against HIV-1.
METHODS: The truncated form of p24-Nef using AAY flexible linker and the full protein were expressed and evaluated in the prokaryotic system and confirmed by western blotting. We also used pcDNA3.1 to transfect Lenti-X 293T cells. Moreover, lentiviral vectors were applied to produce recombinant virions harboring the genes of interest and cell transduction.
RESULTS: Both fusion proteins in a truncated and a full form were expressed and confirmed by Anti Nef polyclonal antibody in western blotting. Recombinant virions were generated and transduced Lenti-X 293T cells confirming by immunofluorescence microscope and p24 ELISA assay kit. Transduced cells were analyzed by SDS-PAGE and western blotting, which resulted in approved protein expression.
CONCLUSION: Fusion protein of p24 and Nef is well expressed in eukaryotic cell lines according to its pre-evaluated features by bioinformatics tools.