Characterization of a Trichoplusia ni hexamerin-derived promoter in the AcMNPV baculovirus vector

•We have tested a Trichoplusia ni insect-derived hexamerin promoter, denominated pB2, in the AcMNPV baculovirus vector.•This promoter activity occurred earlier in baculovirus-infected cells than that achieved by a conventional polyhedrin promoter.•A combination of pB2 with the conventional promoter...

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Bibliographic Details
Published in:Journal of biotechnology 2013-06, Vol.165 (3-4), p.201-208
Main Authors: López-Vidal, Javier, Gómez-Sebastián, Silvia, Sánchez-Ramos, Ismael, Escribano, José M.
Format: Article
Language:English
Subjects:
AcMNPV
Animals
Autographa californica multiple nucleopolyhedrovirus
Baculovirus expression vector system
Base Sequence
Basic juvenile hormone-suppressible protein 2
Biotechnology
chimerism
DNA
genes
Genetic Vectors - genetics
Green Fluorescent Proteins
hexamerins
Insect Proteins - genetics
Insect Proteins - metabolism
insects
Larva - genetics
Larva - metabolism
Molecular Sequence Data
Moths - genetics
Nucleopolyhedrovirus - genetics
Promoter
promoter regions
Promoter Regions, Genetic - genetics
protein synthesis
Recombinant protein expression
recombinant proteins
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Sf9 Cells
transcription (genetics)
Trichoplusia ni
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Summary:•We have tested a Trichoplusia ni insect-derived hexamerin promoter, denominated pB2, in the AcMNPV baculovirus vector.•This promoter activity occurred earlier in baculovirus-infected cells than that achieved by a conventional polyhedrin promoter.•A combination of pB2 with the conventional promoter p10 increased expression levels at early times with respect to polh promoter.•Promoter pB2 is the first isolated from the Lepidoptera T. ni to be assayed in a baculovirus expression vector. 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.
ISSN:0168-1656
1873-4863
DOI:10.1016/j.jbiotec.2013.03.012