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| Author(s): |
Ana Lia Pradella Puglia
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2014-04-28 |
| Examining board members: |
Renato Mancini Astray;
Ronaldo Zucatelli Mendonça;
Jaime Henrique Amorim Santos
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| Advisor: | Renato Mancini Astray |
| Abstract | |
The recombinant Semliki Forest Virus (rSFV) has been considered as one of the most promising viral vectors, both for the expression of proteins of biotechnological interest or as a vector for immunizations. In this study, an rSFV carrying the gene of the green fluorescent protein (GFP) was obtained (rSFV-GFP) and used for the standardization of rSFV production and of a novel titration methodology. Through the analysis of the amounts of GFP expressed in BHK-21 cells infected with virus stocks obtained by transfection with a lipid reagent or electroporation, we established the best conditions for rSFV production based on the production of this recombinant protein. In addition, a standardized method of absolute quantitative RT-PCR (qRT-PCR), based in a standard curve and applicable to virtually all rSFV particles, regardless of the heterologous gene cloned, was validated. The direct analysis of the quantity of infected cells, by flow cytometry and fluorescence microscopy, confirmed that the rSFV-GFP was an appropriated tool to support ours studies of viral titration by qRT-PCR. It was demonstrated the association between the amount of copies of viral RNA used for culture infection and the amount of GFP expression. This approach led to the development of technical and technological competence (construction, evaluation and titration) in the laboratory, that shall help the establishment of other rSFV of interest. We concluded that electroporation is the best methodology to obtain rSFV particles and the viral titre, as determined by qRT-PCR, can be used to calculate the volume of an rSFV stock necessary to obtain the desired multiplicity of infection. (AU) | |
| FAPESP's process: | 11/15269-0 - Construction and utilization of SFV vector expressing GFP and RVGP |
| Grantee: | Ana Lia Pradella Puglia |
| Support Opportunities: | Scholarships in Brazil - Master |