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An Acad Bras Cienc. 2013 Mar;85(1):159-68. Epub 2013 Mar 1.

Construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique.

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1
Departamento de Virologia e Terapia Experimental, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Universidade Federal de Pernambuco, 50760-420 Recife, PE, Brasil.

Abstract

RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potential for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSC-repYFV-17D), previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP) and firefly luciferase (repYFV-17D-Luc) reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons.

PMID:
23460439
[Indexed for MEDLINE]
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