BACULOVIRAL EXPRESSION SYSTEM

Claims

1. A recombinant bacmid containing: bacterial replicon; selectable marker sequence; a first reporter gene comprising a first preferred target site for transposon insertion; a second reporter gene functionally linked to a baculovirus-inducible promoter and a second preferred target site capable of mediating a site-specific recombinant event.

2. The recombinant bacmid of claim 1, wherein the bacterial replicon is a low copy number replicon, wherein, optionally, the low copy number replicon is a mini-F replicon.

3. The recombinant bacmid of claim 1, wherein the selectable marker sequence comprises an antibiotic resistance gene, wherein, optionally, the antibiotic resistance gene is a kanamycin resistance gene.

4. The recombinant bacmid of claim 1, wherein the first preferred target site for insertion of the transposon does not disrupt the reading frame of the first reporter gene.

5. The recombinant bacmid of claim 1, wherein the second preferred target site comprises a LoxP site or a variant thereof, and wherein the site-specific recombinant event is mediated by Cre recombinase.

6. The recombinant bacmid according to claim 1, further comprising: a heterologous sequence inserted into the first reporter gene, where the inserted heterologous sequence disrupts the reading frame of the first reporter gene.

7. The recombinant bacmid of claim 6, wherein the protein is a Rep protein isolated from the genome of a member of the Parvoviridae family of viruses, wherein, optionally, the Rep protein is selected from the group consisting of B19 Rep, AAV2 Rep, HBoV1 Rep, and GPV Rep.

8. A recombinant bacmid according to claim 6, comprising: a heterologous sequence inserted into the mini-attTn7 site, where the heterologous sequence encodes a Rep, and where the inserted Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; and LoxP website or its variant.

9. A recombinant bacmid according to claim 8, comprising: bacterial replicon; the first antibiotic resistance gene; a heterologous sequence inserted into the mini-attTn7 site, where the heterologous sequence encodes B19 Rep, and where the inserted B19 Rep disrupts the reading frame of the LacZα gene or a functional portion thereof, a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter and LoxP website or its variant.

10. The recombinant bacmid according to claim 8, comprising: bacterial replicon; the first antibiotic resistance gene; a heterologous sequence inserted into the mini-attTn7 site, where the heterologous sequence encodes a GPV Rep, and where the inserted GPV Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter and LoxP website or its variant.

11. A recombinant bacmid according to claim 8, comprising: bacterial replicon; the first antibiotic resistance gene; a heterologous sequence inserted into the mini-attTn7 site, where the heterologous sequence encodes an AAV2 Rep, and where the inserted AAV2 Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter and LoxP website or its variant.

12. A recombinant bacmid containing: a first heterologous sequence inserted into a first reporter gene, wherein the inserted heterologous sequence disrupts the reading frame of the first reporter gene; the first preferred target site capable of mediating a site-specific recombinant event; a multiple cloning site containing a second heterologous sequence; and a second preferred target site capable of mediating a site-specific recombinant event.

13. The recombinant bacmid according to claim 12, comprising: a sequence encoding Rep, where the introduced Rep sequence disrupts the reading frame of the LacZα gene or a functional portion thereof, and a heterologous sequence containing, from 5' to 3': an inverted 5'-terminal repeat, wild-type or truncated, derived from the first genome of a member of the Parvoviridae family of viruses; protein coding sequence; one or more expression control sequences operably linked to the protein coding sequence, and an inverted 3'-terminal repeat, wild-type or truncated, derived from the second genome of a member of the Parvoviridae family of viruses.

14. The recombinant bacmid according to claim 12, comprising: a sequence encoding the Rep protein, where Rep disrupts the reading frame of a reporter gene or its functional portion, and a heterologous sequence comprising the nucleotide sequence of SEQ ID NO:

20.

15. The recombinant bacmid of claim 12, wherein the heterologous sequence comprises the nucleotide sequence of SEQ ID NO:

19.

16. A recombinant bacmid according to claim 12, comprising: a sequence encoding the Rep protein, where Rep disrupts the reading frame of a reporter gene or its functional portion, and a heterologous sequence comprising the nucleotide sequence of SEQ ID NO:

29.

17. A host cell containing the recombinant bacmid of claim 1.

18. A host cell containing the recombinant bacmid of claim 12.

19. A method for producing a recombinant baculovirus, comprising transfecting the recombinant bacmid of claim 12 into an insect cell under suitable conditions.

20. A method for creating a closed-end DNA molecule (ceDNA), comprising: infecting an insect cell with a recombinant baculovirus containing a recombinant bacmid according to claim 1.

21. A method for creating a closed-end DNA molecule (ceDNA), comprising: transfecting the recombinant bacmid of claim 13 into an insect cell under conditions suitable for producing a recombinant baculovirus, and infection of a second insect cell with a recombinant baculovirus under conditions suitable for the creation of the ceDNA molecule.

22. A stable cell line containing a nucleic acid sequence according to claim 21, wherein the nucleic acid sequence is stably integrated into the genome of the stable cell line.

23. A set of recombinant bacmids comprising a first bacmid and a second bacmid, wherein the first bacmid contains a sequence encoding a Rep inserted into the mini-attTn7 site, wherein the inserted Rep disrupts the reading frame of the reporter gene or its functional portion, and wherein the second bacmid comprises a heterologous sequence, wherein the heterologous sequence comprises, from 5' to 3': an inverted 5'-terminal repeat (ITR), wild-type or truncated, derived from the first genome of a member of the Parvoviridae family of viruses; protein coding sequence; one or more expression control sequences operably linked to the protein coding sequence, and an inverted 3'-terminal repeat (ITR), wild-type or truncated, derived from the second genome of a member of the Parvoviridae family of viruses.

24. A method for creating a closed-end DNA molecule (ceDNA), comprising: infecting an insect cell with a recombinant baculovirus containing a set of recombinant bacmids according to claim 23.

25. A method for creating a closed-end DNA molecule (ceDNA), comprising: transfecting a set of recombinant bacmids according to claim 23 into an insect cell under conditions suitable for producing a recombinant baculovirus, and infection of a second insect cell with a recombinant baculovirus under conditions suitable for the creation of the ceDNA molecule.

26. The recombinant bacmid of claim 11, comprising: bacterial replicon; the first antibiotic resistance gene; a sequence encoding HBoV1 Rep, where the inserted HBoV1 Rep sequence disrupts the reading frame of the LacZα gene or a functional portion thereof, a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter and LoxP website or its variant.