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

Anais da Academia Brasileira de Ciências

Sabrina R.a. Queiroz¹  Andréa N.m.r. Silva¹  Jefferson J.s. Santos¹  Ernesto T.a. Marques Jr¹  Giovani R. Bertani¹  Laura H.v.g. Gil¹ 

关键词: cloning technique;    homologous recombination;    replicon;    reporter gene;    yellow fever virus;    técnica de clonagem;    recombinação homóloga;    replicon;    gene repórter;    vírus da febre amarela;   

DOI  :  10.1590/S0001-37652013005000008

来源: SciELO

【 摘 要 】

RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potencial 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.

【 授权许可】

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