Obtención de clones recombinantes que expresan las proteínas prM-E-NS1 del virus dengue serotipo 2 / Obtaining recombinant clones expressing dengue virus serotype 2 prM-E-NS1 proteins

Objetivo: obtener clones recombinantes que expresen diferentes proteínas del virus dengue 2 en un vector de expresión en células eucariotas. Métodos: se realizó el clonaje de los genes prM, envoltura (E) y 65 aminoácidos (aa) de la proteína NS1 del virus dengue serotipo 2 (cepa Nueva Guinea C) en un vector de expresión en células eucariotas pcDNA (3.1). La obtención de los genes correspondientes a la zona prM/E/NS1 y prM/E truncada en 100 aa se realizó mediante reacción en cadena de la polimerasa (RCP). La detección de los posibles clones recombinantes se llevó a cabo mediante las técnicas de RCP, análisis de restricción enzimática y secuenciación nucleotídica. Se realizó la transfección de la línea celular CHO con cada plásmido recombinante. Para determinar la expresión transciente de los genes clonados se empleó la técnica de inmunofluorescencia indirecta (IFI) y trascripción reversa-RCP (TR-RCP). Resultados: se obtuvieron bandas de 2 202 y 1 600 pares de bases (pb), respectivamente. Se estudiaron 20 posibles colonias recombinantes, de las cuales, 7 resultaron positivas para prM-E-NS1 y 5 para prM/E truncada. Se obtuvieron células fluorescentes 48 h después de transfectadas, además una RCP positiva a ese mismo tiempo, lo que indicó la presencia de las proteínas en las células transfectadas. Conclusiones: el vector empleado fue eficiente para el clonaje y la expresión de las proteínas seleccionadas, por lo que las construcciones genéticas obtenidas pudieran ser evaluadas en animales como posibles candidatos vacunales para la obtención de una vacuna de ADN contra el dengue.

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.

Leishmania (Viannia) guyanensis: a new minicircle class exclusive to this specie isolated from a DNA cosmid library useful for taxonomic purposes.

A new minicircle class exclusive to this specie isolated from a DNA cosmid library useful for taxonomic purposes. Experimental Parasitology 94, 143-149. In this paper we describe a new minicircle class exclusive to Leishmania (Viannia) guyanensis. The minicircle class was obtained with the aid of a total DNA cosmid library. The library was screened with an EcoRI fragment isolated from L. (V.) guyanensis (M4147). After screening seven clones were selected which showed strong hybridisation. Clones were digested and hybridised with the same probe. After hybridisation only one clone containing the desired fragment was positive. The fragment sized around 1000 bp was subcloned into pBluescript for sequencing. Sequence analysis using the GCG programme showed no substantial homology with any sequences previously reported, apart from the expected homology with the conserved region of Leishmania kDNA sequences. The probe hybridised strongly only to L. (V.) guyanensis kDNA after medium stringency washing.

The four-way DNA junction: a fluorescence resonance energy transfer study.

Fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions have been carried out in order to analyze the global structure and its dependence on the concentration of several types of ions. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The results indicate that the four-way junction isomerizes from an unstacked extended square arrangement of the four duplex arms at low ion concentration to an antiparallel stacked X-structure as the salt is added. The ion-related conformational change progresses in a continuous non-cooperative manner as the ionic strength of the solution increases.