CONJUGATIVE PLASMIDS AND METHODS OF USE THEREOF
The present invention provides novel C. botulinum conjugatively transmissible plasmids and methods of use thereof. Specifically, described herein are novel, conjugatively transmissible clostridial plasmids which are capable of being transferred among and between clostridial species. The novel plasmids of the present invention therefore permits the delivery of heterologous clostridial genes into a clostridial host, such as C. botulinum, and the expression of genes of interest in that host, including clostridial toxins and the nontoxigenic components of the toxin complex, toxin fragments, or antigenic portions thereof, in a way both that ensures abundant expression and facilitates purification. Furthermore, toxins with altered structures, chimeric, hybrid toxins, and other toxin derivatives valuable in medicine could be synthesized in this system.
1 . A conjugative transfer plasmid comprising:
an origin of replication effective in Clostridium species;
a protein coding sequence for a gene of interest operably joined to a promoter effective in Clostridium species; and
an origin of conjugative transfer capable of modulating the conjugative transfer of the plasmid into a recipient Clostridium species, wherein the gene of interest is expressed in the recipient Clostridium species.
2 . The conjugative transfer plasmid of claim 1 wherein the gene of interest is at least one gene for expressing clostridial toxins, toxin fragments, or antigenic portions thereof.
3 . The conjugative transfer plasmid of claim 1 wherein the plasmid is selected from C. botulinum serotypes A, B, C, D, E, F or G.
4 . The conjugative transfer plasmid of claim 1 wherein the plasmid is selected from C. botulinum strains Ba, Ab, Bf, Af or A(B).
5 . The conjugative transfer plasmid of claim 1 wherein the plasmid is selected from the same Clostridium species as the recipient Clostridium species.
6 . The conjugative transfer plasmid of claim 1 wherein the plasmid is selected from a different Clostridium species as the recipient Clostridium species.
7 . The conjugative transfer plasmid of claim 1 wherein the plasmid is selected from the group consisting of pBotCDC-A3 (SEQ ID NO: 1), pCLJ (SEQ ID NO: 2), pCLL (SEQ ID NO: 3), pBot81E-1133 and pCLD(SEQ IS NO: 4).
8 . The conjugative transfer plasmid of claim 1 wherein the promoter effective in Clostridium species is the NTNH promoter from Clostridium botulinum.
9 . The conjugative transfer plasmid of claim 1 wherein the recipient Clostridium species is toxic or nontoxic.
10 . The conjugative transfer plasmid of claim 1 wherein the recipient Clostridium species is proteolytic or nonproteolytic.
11 . The conjugative transfer plasmid of claim 1 wherein the recipient Clostridium species is LNT01 or Hall A-Hyper.
12 . The conjugative transfer plasmid of claim 1 further comprising an antibiotic resistant gene for conferring resistance to erythromycin, kanamycin, tetracycline, chloramphenicol, thiamphenicol, spectinomycin or streptomycin.
13 . A conjugative transfer plasmid comprising:
a BoNT-encoding plasmid having an origin of replication effective in Clostridium species, the BoNT encoded by the plasmid operably joined to a promoter effective in the Clostridium species; and
an origin of conjugative transfer capable of modulating the conjugative transfer of the BoNT-encoding plasmid into a recipient Clostridium species,
wherein the BoNT encoded by the plasmid is expressed in the recipient Clostridium species.
14 . The conjugative transfer plasmid of claim 13 wherein the BoNT-encoding plasmid is selected from the group consisting of pBotCDC-A3 (SEQ ID NO: 1), pCLJ (SEQ ID NO: 2), pCLL (SEQ ID NO: 3), pBot81E-1133 and pCLD(SEQ IS NO: 4).
15 . The conjugative transfer plasmid of claim 13 wherein the BoNT encoded by the plasmid is at least one C. botulinum neurotoxin gene for expressing C. botulinum toxins, toxin fragments, or antigenic portions thereof.
16 . The conjugative transfer plasmid of claim 13 wherein the BoNT-encoding plasmid is from the same Clostridium species as the recipient Clostridium species.
17 . The conjugative transfer plasmid of claim 13 wherein the BoNT-encoding plasmid is from a different Clostridium species as the recipient Clostridium species.
18 . The conjugative transfer plasmid of claim 13 further comprising an antibiotic resistant gene for conferring resistance to erythromycin, tetracycline, chloramphenicol or thiamphenicol.
19 . A method of conjugatively transferring a gene of interest into a recipient Clostridium species, the method comprising conjugatively transferring a plasmid comprising an origin of replication effective in Clostridium species, a protein coding sequence for a gene of interest operably joined to a promoter effective in Clostridium species, and an origin of conjugative transfer capable of modulating the conjugative transfer of the plasmid into the recipient Clostridium species, wherein the gene of interest encoded by the plasmid is expressed in the recipient Clostridium species.
20 . The method of claim 19 wherein the gene of interest is at least one gene for expressing clostridial toxins, toxin fragments, or antigenic portions thereof.
21 . The method of claim 19 wherein the plasmid is selected from the group consisting of pBotCDC-A3 (SEQ ID NO: 1), pCLJ (SEQ ID NO: 2), pCLL (SEQ ID NO: 3), pBot81E-1133 and pCLD(SEQ IS NO: 4).