Site-specific Listeria integration vectors and methods for their use are provided. The subject vectors include a bacteriophage integrase gene and a bacteriophage attachment site, where in many embodiments the bacteriophage that is the source of these elements is a listeriophage. In certain embodiments, the subject vectors further include a multiple cloning site, where the multiple cloning site may further include a polypeptide coding sequence, e.g., for a heterologous antigen. The subject vectors and methods find use in a variety of different applications, including the study of Listeria species and the preparation of Listeria vaccines.
1. A recombinant integration vector capable of integrase mediated site-specific Listeria genome integration, wherein said vector is a plasmid, said vector comprising;
a listeriophage integrase attachment (attPP) site and a cloning site containing a heterologous coding sequence, wherein said attPP site is capable of integrase mediated recombination with a listeriophage integrase bacterial attachment (attBB) site; and
a nucleic acid sequence encoding a listeriophage integrase capable of catalyzing attPP′-mediated integration of the vector at an attBB′ site.
2. The integration vector according to claim 1 , wherein said attachment site provides for integration at an integration site selected from the group consisting of: a comK integration site and a tRNA Arg integration site.
3. The integration vector according to claim 1 , wherein said integration vector further includes a multiple cloning site.
4. The integration vector according to claim 3 , wherein said integration vector further includes a coding sequence.
5. The integration vector according to claim 1 , wherein said coding sequence encodes a polypeptide.
6. The integration vector according to claim 5 , wherein said polypeptide is an antigen.
7. A method of transforming a Listeria, said method comprising: contacting said Listeria with an integration vector according to claim 1 under conditions sufficient for said integration vector to integrate into said Listeria's genome.
8. A Listeria transformed with a vector according to claim 1 .
9. A strain of Listeria cells according to claim 8 , wherein said heterologous coding sequence encodes a heterologous antigen and said Listeria cells express said heterologous antigen encoded by said coding sequence.
10. The strain according to claim 9 , wherein said Listeria cells are attenuated.
11. A recombinant culture of Listeria cells according to claim 8 .
12. The recombinant culture according to claim 11 , wherein said Listeria cells are attenuated.
13. A kit comprising:
a vector according to claim 3 ; and
at least one nuclease that cuts said vector at a multiple cloning site.
14. The kit according to claim 13 , wherein said kit further comprises a host cell.
15. A kit comprising:
a vector according to claim 3 ;
at least one nuclease that cuts said vector at a multiple cloning site; and
a Listeria cell.
16. A system comprising:
a vector according to claim 3 ;
at least one nuclease that cuts said vector at a multiple cloning site;
said heterologous coding sequence; and
Listeria cells.
17. The integration vector according to claim 1 , wherein said listeriophage integrase attachment site is a PSA bacteriophage integrase attachment site.
18. The integration vector according to claim 17 , wherein said attachment site is at least 17 nucleotides in length.
19. The integration vector according to claim 1 , wherein said listeriophage integrase attachment site is selected from a U153 attPP attachment site and a PSA attPP attachment site.
20. A recombinant plasmid comprising:
a listeriophage integrase U153 attachment site (U153 attPP′ site), wherein said U153 attPP′ site is capable of integrase mediated integration with a comK listerial genome attachment site (comK attBB′ site) and a nucleic acid sequence encoding a U153 integrase; or
a listeriophage integrase PSA attachment site (PSA attPP′ site), wherein said PSA attPP′ site is capable of integrase mediated integration with a tRNA Arg listerial genome attachment site (attBB′ site) and a nucleic acid sequence encoding a PSA integrase,
wherein said plasmid further comprises a cloning site for a nucleic acid encoding a heterologous coding sequence.
21. The plasmid of claim 20 , wherein the plasmid comprises a U153 attPP′ site and a nucleic acid encoding U153 integrase, or a PSA attPP′ site and nucleic acid encoding PSA integrase, wherein the nucleic acid encoding the integrase is operably linked with a listerial promoter.
22. The plasmid of claim 21 , wherein the nucleic acid encoding the integrase is operably linked with a listerial promoter from p60, actA, plcA, mpl, plcB, or inlA.
23. The plasmid of claim 20 , wherein the plasmid comprises a non-listerial bacterial origin of replication.
24. The plasmid of claim 20 , further comprising a nucleic acid encoding an origin of transfer.
25. The plasmid of claim 20 , wherein the cloning site is a multiple cloning site.
26. The plasmid according to claim 20 , wherein said plasmid further comprises a nucleic acid encoding a heterologous coding sequence.
27. The plasmid of claim 26 , wherein the heterologous coding sequence encodes a polypeptide.
28. The plasmid of claim 26 , wherein the heterologous coding sequence encodes an antigen.
29. The plasmid of claim 26 , wherein the antigen is a tumor antigen.
30. A Listeria bacterium comprising the plasmid of claim 20 .
31. The Listeria bacterium of claim 30 , wherein the plasmid is genomically integrated.
32. The Listeria bacterium of claim 30 , wherein the Listeria is attenuated.