Construction and utilization of SFV vector expressing GFP and RVGP

• Grant number: 11/15269-0
• Support Opportunities: Scholarships in Brazil - Master
• Start date: March 01, 2012
• End date: August 31, 2013
• Field of knowledge: Biological Sciences - Immunology
• Principal Investigator: Renato Mancini Astray
• Grantee: Ana Lia Pradella Puglia
• Host 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. (11/15269-0, 10/08742-8)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

PUGLIA, Ana Lia Pradella. Production and utilization of an SFV vector expressing GFP.. 2014. Master's Dissertation - Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) São Paulo.