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Mol Biotechnol ; 56(11): 963-70, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24939577

RESUMEN

Recombinant FMDV empty capsids have been produced in insect cells and larvae using the baculovirus expression system, although protein yield and efficiency of capsid assembly have been highly variable. In this work, two strategies were compared for the expression of FMDV A/Arg/01 empty capsids: infection with a dual-promoter baculovirus vector coding for the capsid precursor (P12A) and the protease 3C under the control of the polyhedrin and p10 promoters, respectively (BacP12A-3C), or a single-promoter vector coding the P12A3C cassette (BacP12A3C). Expression levels and assembly into empty capsids were analyzed in insect cells and larvae. We observed that the use of the single-promoter vector allowed higher levels of expression both in insect cells and larvae. Recombinant capsid proteins produced by both vectors were recognized by monoclonal antibodies (mAbs) directed against conformational epitopes of FMDV A/Arg/01 and proved to self-assemble into empty capsids (75S) and pentamers (12S) when analyzed by sucrose gradient centrifugation.


Asunto(s)
Biotecnología/métodos , Proteínas de la Cápside/genética , Cisteína Endopeptidasas/genética , Virus de la Fiebre Aftosa/fisiología , Mariposas Nocturnas/virología , Proteínas Recombinantes/genética , Proteínas Virales/genética , Proteasas Virales 3C , Animales , Baculoviridae/genética , Proteínas de la Cápside/inmunología , Cisteína Endopeptidasas/inmunología , Virus de la Fiebre Aftosa/inmunología , Humanos , Mariposas Nocturnas/embriología , Regiones Promotoras Genéticas , Proteínas Recombinantes/inmunología , Células Sf9 , Spodoptera , Proteínas Virales/inmunología
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