Revista Cubana de Medicina Tropical
versión On-line ISSN 1561-3054
MUNE JIMENEZ, Mayra et al. Obtaining recombinant clones expressing dengue virus serotype 2 prM-E-NS1 proteins. Rev Cubana Med Trop [online]. 2008, vol.60, n.1, pp. 0-0. ISSN 1561-3054.
OBJECTIVE: To obtain recombinant clones expressing different dengue virus 2 proteins in an expression vector of eukaryote cells. METHODS: Cloning of prM genes, E envelope and 65 amino-acids (aa) of dengue virus serotype 2 NS1 proteins (Nueva Guinea strain) in an expression vector of pcDNA eukaryote cells (3.1). The prM/E/NS1 zone and the truncated prM/E zone at 100 aa genes were obtained by polymerase chain reaction (PCR). Possible recombinant clones were detected using PCR, enzyme restriction analysis and nucleotide sequencing. Transfection of the CHO cell line with each recombinant plasmid was performed. Indirect immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR) determined the transient expression of cloned genes. RESULTS: Bands of 2 202 and 1 600 base pairs (bp) respectively were obtained. Twenty possible recombinant colonies were studied, 7 of them were pr-E-NSI-positive and 5 truncated prM/E-positive. Fluorescent cells emerged 48 hours after being transfected in addition to positive PCR, all of which indicated that the studied proteins were present in transfected cells. CONCLUSIONS: The used vector proved to be efficient for cloning and expression of the selected proteins; therefore, the obtained genetic constructions could be evaluated in animals as likely vaccinal candidates for a dengue virus DNA vaccine.
Palabras llave : Cloning; genes; dengue 2; transfection; DNA vaccine.