System for rapid generation of recombinant baculovirus-based expression vectors for silkworm larvae
First Claim
1. A recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV), which BmNPV has a genome comprising a restriction endonuclease site in a polyhedrin promoter region and a second restriction endonuclease site in an essential gene region located downstream of the polyhedrin promoter region, wherein the restriction endonuclease sites are not found outside of the segment of the genome delineated by the restriction endonuclease sites in the polyhedrin promoter region at the upstream end and the essential gene region in the downstream end, and wherein cutting of the genome by a restriction enzyme specific for the restriction site in the essential gene knocks out function of the essential gene.
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Abstract
Recombinant expression systems for the production of proteins, and particularly a system for rapidly generating recombinant silkworm baculoviruses. Bombyx mori nuclear polyhedrosis virus (BmNPV) with an efficiency approaching 100% has been developed. In a specific example, the vector of the invention was used to generate expression of a FLAG-epitope tagged HIV tat interacting protein of 30 kDa (f-TIP30) in BmN cells and silkworm larvae.
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32 Claims
- 1. A recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV), which BmNPV has a genome comprising a restriction endonuclease site in a polyhedrin promoter region and a second restriction endonuclease site in an essential gene region located downstream of the polyhedrin promoter region, wherein the restriction endonuclease sites are not found outside of the segment of the genome delineated by the restriction endonuclease sites in the polyhedrin promoter region at the upstream end and the essential gene region in the downstream end, and wherein cutting of the genome by a restriction enzyme specific for the restriction site in the essential gene knocks out function of the essential gene.
- 12. A linear BmNPV, which BmNPV has one end comprising a cut restriction endonuclease site in a polyhedrin promoter region and a second end comprising a second cut in a restriction endonuclease site in an essential gene region, wherein the essential gene is located downstream of the polyhedrin promoter region in an intact BmNPV genome.
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15. A method for preparing a recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV), which BmNPV has a genome comprising a restriction endonuclease site in a polyhedrin promoter region and a second restriction endonuclease site in an essential gene region located downstream of the polyhedrin promoter region, which method comprises:
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(a) introducing a restriction site into the polyhedrin promoter;
(b) introducing a restriction site into the essential gene; and
(c) selecting recombinant BmNPV that contain both restriction sites. - View Dependent Claims (16, 17)
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- 18. A transfer vector comprising a region of an BmNPV genome containing or upstream of a polyhedrin promoter, a cassette insertion site operably associated with the polyhedrin promoter or another promoter effective in silkworm cells, and a region of a BmNPV genome containing an essential gene, wherein the essential gene is located downstream of the polyhedrin promoter in a wildtype BmNPV genome and is oriented in the transfer vector the same way relative to the polyhedrin promoter as it is in wildtype BmNPV, and wherein the two regions are of sufficient size to permit homologous recombination with a BmNPV vector.
Specification