Issued: May 12, 1998
Inventor: Gary W. Blissard and Scott C. Monsma, Ithaca, NY
Assignee: Boyce Thompson Institute for
Plant Research, Inc., Ithaca, NY
Contact: Christopher Michaels, at Brown & Michaels, Ithaca (see below for address and phone number) - firstname.lastname@example.org
Disclosed herein is a novel baculovirus cloning system. The new cloning system is a marker-rescue system, using an essential gene, e.g. gp64. In this system, a gene essential for viral replication, growth, or propagation in cell culture is removed from or inactivated in the viral genome. Once a null baculovirus is created, it is propagated in a host cell that expresses the essential protein or a functional homolog.
For cloning into the baculovirus containing the null-mutation, the virus is used to infect wild type host cells and the same cells are transfected with a plasmid that contains the essential gene, or a functional homolog, linked to a foreign gene under the control of a selected promoter. The baculovirus is "rescued" by the rescue gene linked to the foreign gene and is able to propagate normally and express the foreign gene.
The recombinant "rescued" baculovirus can be used for gene expression, biological control or presentation of a foreign protein on the surface of the virus for vaccines and antibody production. As an example of this new cloning system, disclosed herein are recombinant baculoviruses that contain an insertionally inactivated or deleted gp64 efp gene, a gene that encodes a protein essential for viral infectivity and propagation in cell culture and in animals. To generate the virus the GP64 EFP protein was supplied in trans, from a stably transfected cell line. Homologous recombination was the used to generate inactivated gp64 efp genes in the context of otherwise wild type AcMNPV baculoviruses.
For generating the stably transfected cell line, we selected a heterologous gp64 efp gene (derived from a different baculovirus, OpMNPV). A lacZ marker gene was fused in-frame with the wt AcMNPV gp64 efp gene, and the "loss-of-function" phenotype of the recombinant virus was analyzed by detection of the b -galactosidase marker.
A virus containing a complete deletion of the gp64 ORF was also generated by this method. To demonstrate rescue by the gp64 gene, a plasmid containing the native AcMNPV gp64 gene was transfected into wild type Sf9 cells that were infected with a gp64-null baculovirus. The gp64-null virus was rescued and a viable baculovirus was obtained after transfection and standard virus isolation.
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