title: Characterization of a Trichoplusia ni hexamerin-derived promoter in the AcMNPV baculovirus vector.
abstract: The promoter sequences of the encoding genes for the three most abundant hexamerins of the Lepidoptera Trichoplusia ni were isolated and cloned into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-derived baculovirus expression vector. From the sequences analyzed, the DNA region driving the expression of the Basic juvenile hormone-suppressible protein 2 (BJHSP-2), denominated pB2, presented the highest promoter strength in the context of the baculovirus vector in Sf21 insect cells. This promoter activity occurred earlier in baculovirus-infected cells than that achieved by a conventional polyhedrin promoter (polh), but surprisingly stopped at 48h post-infection. A mapping of pB2 essential promoter elements determined that a region of about 400bp, denominated pB29, retained and even increased the transcriptional activity with respect to the parental full-length sequence. Finally, several chimeric combinations of the insect-derived pB2 with the virus-derived conventional polh or p10 promoters were constructed and incorporated into an AcMNPV baculovirus vector. The pB2-p10 combination showed increased recombinant protein expression at early times post-infection and similar expression levels at very late times post-infection in Sf21 cells with respect to conventional late promoters. To the best of our knowledge, pB2 is the first promoter isolated from the Lepidoptera T. ni, the natural host of AcMNPV, to be assayed in a baculovirus expression vector.
authors.affiliation: Alternative Gene Expression S.L. (ALGENEX), Centro Empresarial, Parque Científico y Tecnológico de la Universidad Politécnica de Madrid, Campus de Montegancedo, 28223 Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: López-Vidal
authors.forename: Silvia
authors.initials: S
authors.lastname: Gómez-Sebastián
authors.forename: Ismael
authors.initials: I
authors.lastname: Sánchez-Ramos
authors.forename: José M
authors.initials: JM
authors.lastname: Escribano
mesh_terms: D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D007814:Larva / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells
chemical_list: D019476:Insect Proteins; D011994:Recombinant Proteins; C083993:basic juvenile-hormone suppressible protein-2, insect; C041137:storage proteins, Insecta; D049452:Green Fluorescent Proteins