dapE gene of helicobacter pylori and dapE- mutant strains of helicobacter pylori

Abstract

The invention provides the dapE gene of Helicobacter pylori and H. pylon dapE − mutants and to methods of using the mutants to express foreign genes and immunize against foreign agents. The dapE gene can consist of the nucleotide sequence defined in SEQ ID NO:1. Nucleic acids of the gidA gene and ORF2 of H. pylori are provided. Examples of these nucleic acids can be found in SEQ ID NO:3 and SEQ ID NO:5, respectively. Having provided these nucleic acids, hybridizing nucleic acids in accord with the description of hybridizing nucleic acids of dapE are also provided.

What is claimed is:  
     
         1 . An isolated dapE gene of  Helicobacter pylori.    
     
     
         2 . The dapE gene of  claim 1 , consisting of the nucleotide sequence defined in SEQ ID NO:1.  
     
     
         3 . A  Helicobacter pylori -specific nucleic acid fragment of the gene of  claim 2 .  
     
     
         4 . A nucleic acid that encodes a naturally occurring DapE protein of  Helicobacter pylori  and hybridizes with the nucleic acid of SEQ ID NO:1 under the stringency conditions of about 16 hrs at about 65° C., about 5×SSC, about 0.1% SDS, about 2× Denhardt's solution, about 150 μg/ml salmon sperm DNA with washing at about 65° C., 30 min, 2×, in about 0.1×SSPE/0.1% SDS.  
     
     
         5 . A nucleic acid probe that hybridizes with the nucleic acid of SEQ ID NO:1 under the stringency conditions of about 16 hrs at about 65° C., about 5×SSC, about 0.1% SDS, about 2× Denhardt's solution, about 150 g/ml salmon sperm DNA with washing at about 65° C., 30 min, 2×, in about 0.1×SSPE/0.1% SDS.  
     
     
         6 . A nucleic acid primer that hybridizes with the nucleic acid of SEQ ID NO:1 under the stringency conditions of 35 cycles of 94° C. for 1 min, 50° C. for 2 min, and 72° C. for 2 min, with a terminal extension at 72° C. for 10 min.  
     
     
         7 . A purified mutant strain of  Helicobacter pylori  that does not express a functional DapE protein.  
     
     
         8 . The mutant strain of  claim 7  deposited with the American Type Culture Collection under accession no. ATCC 55897.  
     
     
         9 . The mutant  Helicobacter pylori  of  claim 7 , containing a plasmid comprising a nucleic acid encoding a functional DapE protein.  
     
     
         10 . The mutant strain of  claim 7 , having in its chromosome a nucleic acid encoding a foreign protein.  
     
     
         11 . The mutant strain of  claim 10 , wherein the foreign protein encoded is an immunogen of an infectious bacterium, virus, yeast, fungus or parasite selected from the group consisting of  Salmonella enteritidis , Shigella species, Yersinia, enterotoxigenic and enterohemorrhagic  E. coli, Mycobacterium tuberculosis, Streptococcus pyogenes, Bordatella pertussis, Bacillus anthracis, P. falciparum , human immunodeficiency virus, respiratory syncytial virus, influenza virus, histoplasma capsulatum.  
     
     
         12 . The mutant strain of  claim 10 , wherein the foreign protein encoded is a sperm immunogen.  
     
     
         13 . The mutant  Helicobacter pylori  of  claim 10 , containing a plasmid comprising a nucleic acid encoding a functional DapE protein.  
     
     
         14 . The mutant strain of  claim 7 , having a plasmid comprising a nucleic acid encoding a foreign protein.  
     
     
         15 . The mutant strain of  claim 14 , wherein the foreign protein encoded is an immunogen of an infectious bacterium, virus, yeast, fungus or parasite selected from the group consisting of  Salmonella enteritidis , Shigella species, Yersinia, enterotoxigenic and enterohemorrhagic  E. coli, Mycobacterium tuberculosis, Streptococcus pyogenes, Bordatella pertussis, Bacillus anthracis, P. falciparum , human immunodeficiency virus, respiratory syncytial virus, influenza virus,  histoplasma capsulatum.    
     
     
         16 . The mutant  Helicobacter pylori  of  claim 14 , containing a plasmid comprising a nucleic acid encoding a functional DapE protein.  
     
     
         17 . A method of maintaining long term expression of a foreign antigen in  Helicobacter pylori , comprising: 
 a. transforming a mutant  Helicobacter pylori  of  claim 7  with a plasmid comprising a nucleic acid encoding a functional DapE protein comprising a nucleic acid encoding the foreign protein; and    b. maintaining the mutant  Helicobacter pylori  from step a under conditions that permit expression of the foreign protein.    
     
     
         18 . A method of maintaining the expression of a foreign protein in  Helicobacter pylori , comprising: 
 a. transforming a mutant  Helicobacter pylori  of  claim 7  having in its chromosome a nucleic acid encoding the foreign protein with a plasmid comprising a nucleic acid encoding a functional DapE protein;    b. maintaining the mutant  Helicobacter pylori  from step a under conditions that permit expression of the foreign protein.    
     
     
         19 . A method of immunizing a subject against infection with  Helicobacter pylori , comprising: 
 a. administering to the subject the mutant strain of  claim 7;     b. supplementing the subject's diet with diaminopimelic acid to maintain the mutant strain in the subject at least long enough for the subject to mount an immune response to the strain; and    c. ceasing the supplementation of the subject's diet with diaminopimelic acid to kill the mutant strain in the immunized subject.    
     
     
         20 . A method of immunizing a subject against infection with a bacteria, virus, fungus or parasite, comprising: 
 a) administering to the subject the mutant strain of  claim 11;     b) supplementing the subject's diet with diaminopimelic acid to maintain the mutant strain in the subject at least long enough for the subject to mount an immune response to the foreign; and    c) ceasing the supplementation of the subject's diet with diaminopimelic acid to kill the mutant strain in the immunized subject.    
     
     
         21 . A method of immunizing a subject against infection with a bacteria, virus, fungus or parasite, comprising: 
 a) administering to the subject the mutant strain of  claim 15;     b) supplementing the subject's diet with diaminopimelic acid to maintain the mutant strain in the subject at least long enough for the subject to mount an immune response to the foreign protein; and    c) ceasing the supplementation of the subject's diet with diaminopimelic acid to kill the mutant strain in the immunized subject.