Conjugative plasmids and methods of use thereof

Abstract

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).