Construction and characterization of a minimized version of the HIV-1 pNL4-3 plasmid and its application for pseudotyping HIV-1 vectors.

The pUC-based pNL4-3 plasmid is the most widely used vector for in vitro manipulations of the HIV-1 proviral sequences. We have developed a minimal plasmid (pCHUS) based on pNL4-3, which may be useful to facilitate the design of HIV-based constructions. The strategy that has allowed us to construct pCHUS includes the following steps: (1) pNL4-3 digestion by using restriction sites contained within the long terminal repeats (LTRs), (2) recircularization of the fragment containing the pUC18 sequence, (3) amplification of the LTR region restored in the previous step, (4) double digestion of the products obtained in steps 2 and 3, (5) ligation of the fragment containing ColE1+Amp(R) with the LTR fragment, (6) linearization of the intermediate plasmid obtained, and (7) insertion of the fragment containing the proviral genome into the linearized vector. The pCHUS plasmid includes essential information for its replication and antibiotic selection in bacteria, but it lacks all the unnecessary sequences. Our results suggest that pCHUS may be more advantageous than pNL4-3 for in vitro manipulation of the HIV-1 proviral genome. In addition, we describe a potential application of this new vector for pseudotyping HIV-1 particles, using a single plasmid transfection, as a more helpful alternative to the traditionally used cotransfection method.

Inmunofluorescencia en el diagnóstico de la brucelosis método directo

Se procedió a seleccionar, purificar i aislar la inmunoglobulinas o anticuerpos específicos producidos por vacas portadoras de Brucellas abortus. Se marcó esta inmuglobulinas con isotiocianaton de fluorescencia, produciéndose el conjugado correspondiente. Se tituló el conjugado para determinar su óptima dilución de trabajo. Se procedió a colorear frotis de Brucellas abortus y otros gérmenes para observar el microscopio de fluorescencia la especificidad e inespecificidad de este método. Se realizan numerosas pruebas de control, inhibición y agotamiento para demostrar la positividad de este método(AU)