Baculovirus-based expression system

Details Baculovirus-based expression system Baculovirus-based expression system

2002-01-16

2004-11-09

Guzo, David (Department: 1636)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C424S093100, C424S093600, C536S023100, C536S023200, C536S024100, C435S320100, C435S235100, C435S005000, C435S006120, C435S069100, C435S029000, C435S455000, C435S456000, C435S325000, C435S348000, C435S366000, C435S252300

Reexamination Certificate

active

06814963

ABSTRACT:

BACKGROUND
The baculoviruses are a family (Baculoviridae) of DNA viruses which primarily infect insects of the order
Lepidoptera
in nature. Baculoviruses have been used to express exogenous genes in insect cells for some time. One of the most studied baculoviruses is the
Autographa californica
multiple nuclear polyhedrosis virus (AcMNPV).
Baculoviruses can express large quantities of exogenous (non-baculovirus) proteins driven by the endogenous polyhedrin and p10 promoters, both of which are strongly active during the late stage of the natural life cycle of baculoviruses. Although the natural host spectrum of baculovirus is often defined in terms of the ability of the virus to replicate in a given cell, baculovirus can penetrate and express genetic materials in non-natural (non-permissive) host cells, e.g., mammalian cells (see, e.g., Hofmann et al., 1995, Proc. Natl. Acad. Sci. USA 92, 10099-10103; Boyce et al., 1996, Proc. Natl. Acad. Sci. USA 93, 12348-2352; Shoji et al., 1997, J. Gen. Virol. 78, 2657-2664; and Yap et al., 1997, Virology 231, 192-200) and
Drosophila
cells (Pennock et al., 1984, Mol. Cell Biol. 4, 399-406).
Conventional baculovirus expression systems offer the advantages that a) baculoviruses are not normally pathogenic to humans; b) the viruses can be propagated in serum-free media and grown to a titer of over 10
8
pfu/ml; and c) the genome of the virus (approximately 80 to 230 kb) accepts large exogenous DNA molecules. The conventional baculovirus expression system is limited in that a) protein expression can result in cell-lysis; and b) when one baculovirus infects its host for a period of time, the entrance of another baculovirus into the same host (successive infection) becomes non-permissive. Because successive infection (superinfection) is not permissible, it is impossible to utilize a conventional baculovirus to express two or more genes in its host cells at different times.
SUMMARY
The present invention is based on the development of a recombinant virus-based vector, e.g., a baculovirus-based vector, that allows the expression of an exogenous target protein in non-permissive cells (e.g., non-permissive insect cells or mammalian cells) in the absence of expression of a detectable selection marker. The baculovirus-based vector includes a nucleic acid sequence encoding a detectable selection marker which is controlled by a promoter that is active in host cells (e.g., Sf21 or Sf9 cells) used to screen for recombinant virus but is silent in the non-permissive cells (e.g.
Drosophila
cells and mammalian cells) used for expression of the exogenous target protein. The vector also includes an exogenous nucleic acid sequence encoding a target protein under the control of a promoter that is active in the non-permissive cell but inactive in host cells. This system allows the selection marker to be expressed during viral plaque screening, but not while the target protein is being produced. Further, a recombinant virus according to the invention is able to successively infect a non-permissive insect host cell, permitting the expression of a plurality of target proteins in the same non-permissive insect cell.
Accordingly, in one aspect, the invention features a recombinant virus, e.g., a baculovirus, which includes a first nucleic acid sequence encoding a detectable marker operably linked to a first promoter that is active in a host cell culture and is inactive in a non-permissive cell; and also includes a second nucleic acid sequence encoding an exogenous protein operably linked to a second promoter, which second promoter is active in the non-permissive cell.
In another aspect, the invention features a method for selecting a viral plaque, e.g., for infection of a non-permissive cell. The method includes: providing a recombinant virus, e.g., a baculovirus, capable of infecting the non-permissive cell, which virus includes a first nucleic acid sequence encoding a detectable marker operably linked to a promoter that is active in a host cell but is silent in the non-permissive cell; and a second nucleic acid sequence comprising an exogenous nucleic acid sequence operably linked to a second promoter, wherein the second promoter is active in the non-permissive cell; infecting a host cell culture with the recombinant baculovirus; and identifying a viral plaque by detecting expression of the detectable marker.
In another aspect, the invention features a method for producing a protein in a non-permissive cell. The method includes: providing a recombinant virus, e.g., a virus described herein, e.g., a baculovirus described herein; infecting a host cell culture with the recombinant virus; selecting a viral plaque by identifying expression of the detectable marker; amplifying the virus from the selected viral plaque; and infecting a non-permissive cell with the amplified virus. The non-permissive cell thereby produces the protein encoded by the exogenous nucleic acid sequence and does not express the detectable marker.
In a preferred embodiment, the non-permissive cell is infected in vitro. In another preferred embodiment, the non-permissive cell is infected in vivo. Preferably, the method further includes the step of re-infecting a non-permissive cell, e.g., a non-permissive insect cell, with a recombinant virus, e.g., the same or a different virus described herein. This method provides a superinfection system that allows the expression of a plurality of proteins in the same host cell. This system is thereby useful in a two-hybrid screening assay.
In each of the aspects of the invention, a preferred recombinant virus described herein is a baculovirus.
In preferred embodiments, a non-permissive cell described herein is an insect cell, e.g., a
Drosophila
cell (e.g., an S2 cell or Kc cell) or a mosquito cell (e.g., a C6/36 cell); or a mammalian cell (preferably a human cell, e.g., a human primary cell or an established cell line). Preferred promoters that are active in a host cell and inactive in a non-permissive cell include, e.g., the viral polyhedrin promoter or the P10 viral promoter. Preferred promoters that are active in a non-permissive cell, e.g., a mammalian cell, can include, e.g., a CMV promoter, a RSV promoter, or a SV40 promoter. Other preferred promoters that are active in a non-permissive cell, e.g., an insect cell, include a heat shock protein promoter (e.g., hsp7O), a
Oravia pseudotsuciata
nuclear polyhedrosis virus immediate early promoter, an OP1E2 promoter, a MT promoter, or an actin 5C promoter. The detectable marker is preferably a fluorescent protein, e.g., a green fluorescent protein (GFP) or an enhanced GFP (EGFP).
As used herein, the term “exogenous nucleic acid sequence” refers to any nucleic acid sequence that is not part of the virus genome in nature. Such a sequence can include a nucleic acid sequence that is normally present in the non-permissive cell to be infected. Also included is a sequence that is not normally present in the non-permissive cell to be infected (e.g., related and/or unrelated genes of other cells and of other species). The term “non-permissive” when referring to a cell means that the cell does not permit replication of a virus described herein and does not permit the virus to enter the lytic phase within the cell. Such non-permissive cells can nonetheless be infected with a virus described herein and can produce a protein encoded by a nucleic acid sequence of the virus. By “operably linked” is meant a functional linkage between a promoter and a second nucleic acid sequence, wherein the promoter sequence can initiate and/or mediate transcription of the DNA sequence corresponding to the second sequence. Operably linked sequences can be contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. A “protein” or a “target protein” refers to a peptide of any length.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the

Affiliated with

Also associated with

Rating

Baculovirus-based expression system does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Baculovirus-based expression system, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Baculovirus-based expression system will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3280874