Baculovirus expression system
AssigneeBioverativ Therapeutics Inc.
InventorsTongyao Liu, Ajay Maghodia, Philip Zakas
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Abstract
Provided herein is a baculovirus expression vector system for the production of a desired protein. In one aspect, the desired protein is a closed-ended DNA (ceDNA) molecule comprising wild-type and/or truncated inverted terminal repeats derived from a genome of a member of the viral family Parvoviridae.
Claims41
A recombinant bacmid comprising:
- a bacterial replicon;
- a selectable marker sequence;
- a first reporter gene comprising a first preferential target site for the insertion of a transposon;
- a second reporter gene operably linked to a baculovirus-inducible promoter;
- and
- a second preferential target site capable of mediating a site-specific recombination event.
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5-8. (canceled)
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The recombinant bacmid of claim 20, wherein the protein is a Rep protein isolated from a genome of a member of the viral family Parvoviridae, optionally wherein the Rep protein is selected from the group consisting of a B19 Rep, an AAV2 Rep, a HBoV1 Rep, and a GPV Rep.
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45-94. (canceled)
A recombinant bacmid comprising:
- a first heterologous sequence inserted into a first reporter gene, wherein the inserted heterologous sequence disrupts the reading frame of the first reporter gene;
- a first preferential target site capable of mediating a site-specific recombination event;
- a multiple cloning site comprising a second heterologous sequence;
- and
- a second preferential target site capable of mediating a site-specific recombination event.
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159-160. (canceled)
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A stable cell line comprising the nucleic acid sequence of claim 170, wherein the nucleic acid sequence is stably integrated in the genome of the stable cell line.
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A set of recombinant bacmids comprising a first bacmid and a second bacmid,
- wherein the first bacmid comprises a sequence encoding a Rep inserted into a mini-attTn7 site, wherein the inserted Rep disrupts the reading frame of a reporter gene or functional portion thereof;
- and
- wherein the second bacmid comprises a heterologous sequence, wherein the heterologous sequence comprises from 5′ to 3′:
- a wild-type or truncated 5′ inverted terminal repeat (ITR) derived from a first genome of a member of the viral family Parvoviridae;
- a sequence encoding a protein;
- one or more expression control sequences operably linked to the sequence encoding a protein;
- and
- a wild-type or truncated 3′ inverted terminal repeat (ITR) derived from a second genome of a member of the viral family Parvoviridae.
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Dependencies are inferred from the claim text.