RESUMEN
A host range expanded recombinant Autographa californica multiple-nucleocapsid nucleopolyhedrosis virus AcMNPV/r2 was obtained by cotransfection of the bacmid DNA from Escherichia coli DH10Bac along with a plasmid pBmH-M containing HindIII M fragment of Bombyx mori nuclear polyhedrosis virus (BmNPV) genomic DNA. A recombinant transposon vector carrying a mutant green fluorescent protein gene (GFP) and a polyhedrin gene was constructed. Transposition was carried out in both E. coli DH10Bac and E. coli DH10BmH, which contains AcMNPV/r2 and a helper plasmid. Recombinant DNAs were transfected into Sf-9 cells to generate recombinant virus AcMNPV/r3 and AcMNPV/r4 respectively. Viral stock of AcMNPV/r4 was then infected into Bombyx mori cells (BmN) and Bombyx mori larvae (silkworm). Analysis shows that GFP was highly expressed in Bombyx mori larvae. This expression system, is practicable therefore for mass production of foreign gene products.
Asunto(s)
Elementos Transponibles de ADN , Expresión Génica , Vectores Genéticos , Mutagénesis Insercional , Nucleopoliedrovirus/genética , Recombinación Genética , Animales , Secuencia de Bases , Bombyx/genética , Bombyx/virología , Línea Celular , ADN Recombinante , Escherichia coli/genética , Proteínas Fluorescentes Verdes , Larva/virología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Nucleocápside , Proteínas de la Matriz de Cuerpos de Oclusión , Plásmidos/genética , Proteínas Recombinantes/biosíntesis , Spodoptera , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Estructurales Virales , Replicación ViralRESUMEN
Recombinant transposing plasmids pFH24 and pFH41 were constructed by cloning the human immunodeficiency virus 1 (HIV-1) p24 and gp41 genes, respectively, into the transposing vector pFastBacHTa. Recombinant bacmids rBH24 and rBH41 were obtained by transposing pPolh/p24 and pPolh/gp41 expression cassettes from recombinant plasmids pFH24 and pFH41, respectively. Recombinant viruses rAcH24 and rAcH41 were generated by transfection of the Spodoptera frugiperda (Sf9) cells with the DNAs of plasmids rBH24 and rBH41, respectively. Analysis of the expressed p24 or gp41 proteins with an antiserum to HIV-1 (HIV-1 antiserum) by an enzyme-linked immunosorbent assay (ELISA) and dot blot assay showed high biological activity of these proteins; p24 was more active than gp41. Also a Western blot analysis showed stronger bands for p24 than for gp41. The high reactivities of p24 and gp41 with the HIV-1 antiserum suggest that these proteins could also be used as specific standard antigens in HIV-1 diagnostics.