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Construction and utilization of SFV vector expressing GFP and RVGP

Grant number: 11/15269-0
Support type:Scholarships in Brazil - Master
Effective date (Start): March 01, 2012
Effective date (End): August 31, 2013
Field of knowledge:Biological Sciences - Immunology
Principal Investigator:Renato Mancini Astray
Grantee:Ana Lia Pradella Puglia
Home Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Using the Semliki Forest Virus (SFV) for production of recombinant proteins in mammalian cells or to therapeutic and vaccine purposes has been increasingly studied. Obtaining and using this viral vector require the execution of various stages of molecular biology and cell culture. In order to establish and standardize these different methodologies we intend to use a vector able to express the green fluorescent protein gene (GFP). This SFV-RVGP will be utilized for the development and standardization of a quantitative RT-PCR as a novel method for viral titration, by the correlation of RNA amounts with the infection ratios measured by flow cytometry. Parameters will be established to study the best relationship between the quantities of virus/cell (multiplicity of infection - MOI) and the time and temperature of infection more suitable for recombinant expression. The relationship between viral titer and recombinant protein expression will be determined under standard conditions, comparing SFV-GFP and SFV carrying the glycoprotein gene of rabies virus (RVGP). Characterized and quantified SFV-RVGP and the RVGP produced in BHK-21 will be utilized for mice immunization. The immune responses to the same protein produced in vivo by SFV-RVGP or in vitro by SFV-RVGP infected BHK-21 will be studied.

Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
PUGLIA, ANA L. P.; REZENDE, ALEXANDRE G.; JORGE, SORAIA A. C.; WAGNER, RENAUD; PEREIRA, CARLOS A.; ASTRAY, RENATO M. Quantitative RT-PCR for titration of replication-defective recombinant Semliki Forest virus. Journal of Virological Methods, v. 193, n. 2, p. 647-652, NOV 2013. Web of Science Citations: 7.

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