Produzione in pianta di “particelle simil-virali” per la formulazione di vaccini
Author(s)
Circelli, Patrizia
Date Issued
March 22, 2011
Type
Doctoral Thesis
Abstract
The aim of this thesis project is the production of chimeric plant virus-like particles (VLP), displaying immunogenic epitopes on the outer surface, derived from the Artichoke Mottled Crinkle Virus (AMCV), a member of genus Tombusvirus. The project was carried out following a combined approach consisting of computational and experimental virology.
The first step of the project was to verify the capability of AMCV coat protein (CP), expressed in plant tissues, to self-assemble into VLP. For this purpose the cp gene was expressed in Nicotiana benthamiana (N.benthamiana) leaves by an Agrobacterium mediated transient expression system boosted by the AMCV p19 silencing suppressor. The production of viral protein was evaluated by ELISA immunoassays and its ability to self-assemble into VLP was verified by electron microscopy.
Concerning the choice of immunogenic epitopes, we focused on highly conserved regions belong of glycoprotein (gp) 41 and gp120 of the human immunodeficiency virus type 1 (Human Immunodeficiency Virus, HIV-1) envelope. Two neutralising epitopes were chosen, the 2F5 of gp41 and the V3 loop of gp120. Thereafter, it was designed in silico model of the wild type VLP. This bioinformatics model has been of fundamental importance for the identification of useful sites for the insertion of heterologous sequences, coding for immunogenic peptides (epitopes) within the viral CP without interfering with the assembly of the VLP. After the characterization of the in silico wild type VLP, the bioinformatics models of chimeric VLP were performed. Following the computational analysis results, the expression cassettes containing the genes encoding chimeric CP were done. Expression and assembly of chimeric CP were evaluated by ELISA and electron microscopy.
2
In addition, part of the work was focused on the identification of the main factors responsible of the virion stability. In particular, we studied the variation of the interactions between CP molecules and between CP and RNA under different conditions of pH, ionic strength and different concentrations of divalent cations.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
File(s)![Thumbnail Image]()
Name
pcircelli_tesid.pdf
Size
2.82 MB
Format
Adobe PDF
Checksum (MD5)
47b7c426ca33e32ea2f5dc363dd5ef68