Baculoviral DNA elements for the expression of recombinant proteins in a host insect
Inventors
Gomez Sebastian, Silvia • López Vidal, Javier • Martinez Escribano, José Angel
Assignees
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Publication Number
US-9701983-B2
Publication Date
2017-07-11
Expiration Date
Abstract
Reagents and methods are provided that allow for an improved expression of a recombinant protein in an insect, More specifically, the introduction of recombinant DNA elements into an insect larva allows for the increased expression of a recombinant protein, an improvement of the correct folding of said protein and an increase in the survival rate after infection of the insect These recombinant DNA elements can be introduced, for example, into insect larvae via a recombinant baculovirus, which has incorporated said elements. The recombinant DNA elements include nucleic acids encoding transcriptional regulators, such as IE-0 and IE-1, transcriptional, enhancer elements, such as the homologous region (hr) and promoters.
Core Innovation
The disclosed invention relates to improved baculovirus expression cassettes for recombinant protein production in insect larvae, using baculovirus transcriptional regulators IE-1/IE-0. The system overexpresses an extra copy of the Ac-ie-01 gene introduced into an insect by recombinant baculovirus, and the extra copy is expressed at levels greater than levels achieved by infecting with a corresponding control baculovirus that comprises one copy of the Ac-ie-01 gene in its genome and does not comprise the extra nucleic acid sequence.
The introduced first nucleic acid sequence includes nucleic acid sequences selected from defined SEQ ID NOS for Ac-ie-01 gene variants or amino-acid-encoding sequences, and further comprises at least one recombinant homologous region (hr) from a baculovirus as an enhancer region. The hr is operably linked to a promoter that drives expression of a recombinant heterologous protein, where the promoter is a nucleic acid sequence selected from defined SEQ ID NOS.
The described host insects include Lepidoptera, notably Trichoplusia ni and Spodoptera frugiperda, infected using recombinant baculovirus comprising the extra Ac-ie-01 sequence. The reported improvements include increased recombinant GFP expression compared to a conventional polhGFP approach, along with higher infectious-dose infection outcomes with increased larval survival and insect biomass recovery.
Claims Coverage
The partial claim set provided contains one independent claim, supported by dependent claims that narrow the enhancer sequence selection, the Ac-ie-01 nucleic-acid options, and the recombinant protein expression and production workflow. The independent claim contains inventive features centered on extra-copy Ac-ie-01 expression in Trichoplusia ni or Spodoptera frugiperda via an AcMNPV recombinant baculovirus, with expression enhanced by an hr enhancer operably linked to a selected promoter.
Extra-copy Ac-ie-01 gene expression in insect via AcMNPV
An insect of Trichoplusia ni or Spodoptera frugiperda comprising a first nucleic acid sequence introduced by a recombinant baculovirus, where the recombinant baculovirus is Autographa californica multinuclear polyhedrosis virus (AcMNPV) comprising one copy of an Ac-ie-01 gene and said first nucleic acid sequence; wherein said first nucleic acid sequence comprises an extra copy of the Ac-ie-01 gene under control of a promoter and directs expression of immediate early protein-1 (IE-1) or immediate early protein-0 (IE-0) in the insect at levels greater than levels of IE-1 or IE-0 achieved by infecting with a corresponding control baculovirus that comprises one copy of the Ac-ie-01 gene and does not comprise said first nucleic acid sequence.
Ac-ie-01 sequence selection for the first nucleic acid sequence
The first nucleic acid sequence comprises a nucleic acid sequence selected from the group consisting of the nucleic acid sequence of any of SEQ ID NOS:2-5 and the nucleic acid sequence encoding any of the amino acid sequences of SEQ ID NOS:6-9.
hr enhancer operably linked to a selected promoter for heterologous protein expression
The first nucleic acid further comprises at least one recombinant homologous region (hr) from a baculovirus as enhancer region, wherein the recombinant homologous region (hr) is operably linked to a promoter that drives expression of a recombinant heterologous protein, wherein the promoter is a nucleic acid sequence comprising any of SEQ ID NO:10-16.
Overall, the independent claim requires an insect (Trichoplusia ni or Spodoptera frugiperda) infected with an AcMNPV recombinant baculovirus that introduces an extra-copy Ac-ie-01 gene expressed at higher levels of IE-1/IE-0 than a corresponding control, where the regulator sequence is selected from defined SEQ ID options and is further coupled to at least one baculovirus hr enhancer operably linked to a promoter selected from defined SEQ ID NOs for heterologous protein expression.
Stated Advantages
Increased recombinant GFP expression compared to a conventional polhGFP approach.
Higher infectious-dose infection with increased larval survival.
Improved insect biomass recovery.
Improved recombinant protein post-translational processing/folding integrity, including expected GFP molecular weight and less degradation.
Preserved tubulin/cell integrity (cell integrity/morphology).
Documented Applications
Recombinant protein production in insect larvae in vivo using recombinant baculovirus expression cassettes (with GFP described as an example reporter protein).
Production of recombinant heterologous proteins in Lepidoptera hosts including Trichoplusia ni and Spodoptera frugiperda.
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