Tagged epitope protein transposable element

Abstract

A transposable element is provided that has a 3′ and a 5′ end. The transposable element includes a 5′ recombining site 5′ of a nucleic acid sequence encoding a selectable marker, a 3′ recombining site 3′ of the nucleic acid sequence encoding a selectable marker, a nucleic acid sequence encoding an MHC epitope 5′ to the 5′ recombining site or 3′ to the 3′ recombining site, and an insertion end comprising an inverted repeat sequence sufficient for integration of the transposable element at the 5′ and the 3′ end of the transposable element. In one embodiment, a transposable element is provided that has a 5′ and a 3′ end. The transposable element includes a 5′ loxP sequence 5′ of a nucleic acid encoding a selectable marker, a 3′ loxP sequence 3′ of a nucleic acid encoding the selectable marker, an MHC epitope 5′ to the 5′ loxP sequences or 3′ of the 3′ loxP sequence, an insertion end at the 5′ end of the transposable element, and an insertion end at the 3′ of the transposable element. A method is provided for detecting an antigenic epitope of a pathogen by infecting a pathogenic cell with a transposable element of the invention, wherein the infection results in the integration of the transposable element in a nucleic acid sequence of the bacterial cell; transforming the pathogenic cell with a vector comprising a transposase; contacting a eukaryotic cell that can internalize the pathogenic cell with the pathogenic cell infected with the transposable element; contacting the eukaryotic cell with a specific binding partner that recognizes the MHC epitope; identifying the labeled eukaryotic cells and externalizing the bacteria cell. The externalized bacterial cell may be grown to produce a population of bacterial cells, and the nucleic acid sequence of the bacterial cell that has the integrated transposable element is identified. This nucleic acid sequence encodes the antigenic element of the pathogen. A method is also provided for generating a carrier vaccine by infecting a bacterial cell with the transposable element of the invention, wherein the transposable further comprises an antigen associated with a disease operably linked to the MHC epitope of the transposable element. The infection of the bacteria results in the integration of the transposable element in a nucleic acid sequence of the bacterial cell. The pathogenic cell is then internalized into a eukaryotic cell and the eukaryotic cell is exposed to a specific binding agent that recognizes the MHC epitope, identifying labeled eukaryotic cells are identified and lysed to externalize the bacteria cell, which is cultured to produce a population of bacterial cells. The nucleic acid sequence of the bacterial cell that has the integrated transposable element is identified, wherein the nucleic acid sequence encodes the antigenic element of the pathogen, The growing bacterial cell identified, and may be used as the carrier vaccine.

1 - 36 . (canceled)  
     
     
         37 . A method for identifying a protein secreted by an intracellular pathogen having access to an MHC class II pathway of a eukaryotic cell infected with the intracellular pathogen, comprising: 
 (i) transfecting an intracellular pathogen with a transposable element, wherein the transposable element has a 3′ and a 5′ end and comprises a 5′ recombining site 5′ of a nucleic acid sequence encoding a selectable marker, a 3′ recombining site 3′ of the nucleic acid sequence encoding a selectable marker, a nucleic acid sequence encoding an MHC class II epitope 5′ to the 5′ recombining site or 3′ to the 3′ recombining site, and an insertion end comprising an inverted repeat sequence sufficient for integration of the transposable element at the 5′ and the 3′ end of the transposable element, and wherein the transfection results in the integration of the transposable element in a nucleic acid sequence of the intracellular pathogen;    (ii) transforming the intracellular pathogen with a vector comprising a transposase;    (iii) contacting a eukaryotic cell that can internalize the pathogenic cell with the pathogen transfected with the transposable element, wherein an MHC class II haplotype of the eukaryotic cell is matched to the MHC II epitope;    (iv) contacting the eukaryotic cell with a labeled antibody that recognizes the MHC class II epitope, thereby generating a labeled eukaryotic cell;    (v) identifying the labeled eukaryotic cell;    (vi) lysing the labeled eukaryotic cell to externalize the intracellular pathogen;    (vii) growing the externalized intracellular pathogen to produce a population of intracellular pathogen; and    (viii) identifying the nucleic acid sequence of the intracellular pathogen that has the integrated transposable element, wherein the nucleic acid sequence encodes the secreted protein having access to an MHC class II pathway of a eukaryotic cells infected with the intracellular pathogen.    
     
     
         38 . The method of  claim 37 , wherein the eukaryotic cell is a cell of the immune system.  
     
     
         39 . The method of  claim 38 , wherein the cell of the immune system is a macrophage.  
     
     
         40 . The method of  claim 37 , wherein the identification of the labeled eukaryotic cell is by fluorescence activated cell sorting.  
     
     
         41 . (canceled)  
     
     
         42 . (canceled)  
     
     
         43 . The method of  claim 37 , wherein the pathogen is a bacterial cell.  
     
     
         44 . The method of  claim 37 , wherein the pathogen is  Salmonella, Mycobacterium tuberculosis, Plasmodium , or  Listeria monocytogenes.    
     
     
         45 . A method for identifying a protein secreted by an intracellular pathogen and having access to an MHC class II pathway of a eukaryotic cell infected with the intracellular pathogen, comprising: 
 (i) transfecting an intracellular pathogen expressing a tranposase with a transposable element, wherein the transposable element has a 3′ and a 5′ end and comprises a 5′ recombining site 5′ of a nucleic acid sequence encoding a selectable marker, a 3′ recombining site 3′ of the nucleic acid sequence encoding a selectable marker, a nucleic acid sequence encoding an MHC class II epitope 5′ to the 5′ recombining site or 3′ to the 3′ recombining site, and an insertion end comprising an inverted repeat sequence sufficient for integration of the transposable element at the 5′ and the 3′ end of the transposable element, and wherein the transfection results in the integration of the transposable element in a nucleic acid sequence of the intracellular pathogen;    (ii) contacting a eukaryotic cell that can internalize the intracellular pathogen the pathogen transfected with the transposable element, wherein an MHC class II haplotype of the eukaryotic cell is matched to the MHC class II epitope;    (iiii) contacting the eukaryotic cell with a labeled antibody that recognizes the MHC class II epitope, thereby generating a labeled eukaryotic cell;    (iv) identifying the labeled eukaryotic cell;    (v) lysing the labeled eukaryotic cell to externalize the intracellular pathogen;    (vi) growing the externalized pathogen to produce a population of intracellular pathogen; and    (vii) identifying the nucleic acid sequence of the intracellular pathogen that has the integrated transposable element, wherein the nucleic acid sequence encodes the secreted protein having access to an MHC class II pathway of the intracellular pathogen.    
     
     
         46 . A method for identifying a secreted protein having access to an MHC class II pathway of an intracellualr pathogen, comprising: 
 (i) transfecting an intracellular pathogen with a transposable element, wherein the transposable element has a 3′ and a 5′ end and comprises a 5′ recombining site 5′ of a nucleic acid sequence encoding a selectable marker, a 3′ recombining site 3′ of the nucleic acid sequence encoding a selectable marker, a nucleic acid sequence encoding an MHC class II epitope 5′ to the 5′ recombining site or 3′ to the 3′ recombining site, an insertion end comprising an inverted repeat sequence sufficient for integration of the transposable element at the 5′ and the 3′ end of the transposable element, and a transposase, and wherein the transfection results in the integration of the transposable element in a nucleic acid sequence of the intracellular pathogen;    (ii) contacting a eukaryotic cell that can internalize the intracellular pathogen with the pathogen transfected with the transposable element, wherein an MHC class II haplotype of the eukaryotic cell is matched to the MHC class II epitope;    (iii) contacting the eukaryotic cell with a labeled antibody that recognizes the MHC class II epitope, thereby generating a labeled eukaryotic cell;    (iv) identifying the labeled eukaryotic cell;    (v) lysing the labeled eukaryotic cell to externalize the intracellular pathogen;    (vi) growing the externalized intracellular pathogen to produce a population of intracellular pathogen; and    (vii) identifying the nucleic acid sequence of the intracellular pathogen that has the integrated transposable element, wherein the nucleic acid sequence encodes the secreted protein having access to an MHC class II pathway of the intracellular pathogen.    
     
     
         47 . (canceled)  
     
     
         48 . (canceled)  
     
     
         49 . (canceled)  
     
     
         50 . (canceled)  
     
     
         51 . (canceled)  
     
     
         52 . (canceled)  
     
     
         53 . (canceled)  
     
     
         54 . (canceled)  
     
     
         55 . The method of  claim 37 , wherein the 5′ recombining site or the 3′ recombining site is a loxP recombining site, a fit recombining site, a TN3 recombining site, a mariner recombining site, or a gamma/delta recombining site.  
     
     
         56 . The method of  claim 37 , wherein the 5′ recombining site or the 3′ recombining site is a loxP recombining site.  
     
     
         57 . The method of  claim 56 , wherein the loxP sequence comprises the sequence shown in SEQ ID NO: 11.  
     
     
         58 . The method of  claim 37 , wherein the MHC class II epitope is ASFEAQGALANIAVDKA (SEQ ID NO: 20) and the MHC class II haplotype of the eukaryotic cell is I-A b .  
     
     
         59 . The method of  claim 37 , wherein the selectable marker is a nucleic acid encoding antibiotic resistance.  
     
     
         60 . The method of  claim 59 , wherein the antibiotic resistance is ampicillin, kanamycin, zeomycin, hygromycin, tetracycline, puromycin or bleomycin resistance.  
     
     
         61 . The method of  claim 37 , wherein the selectable marker is detected by spectrophotometric properties.  
     
     
         62 . The method of  claim 37 , wherein the selectable marker is beta-galactosidase or green fluorescent protein.  
     
     
         63 . The method of  claim 37 , wherein the insertion end at the 5′ end of the transposable element is SEQ ID NO: 4 or SEQ ID NO: 5.  
     
     
         64 . The method of  claim 37 , wherein the insertion end at the 3′ end of the transposable element is SEQ ID NO: 3 or SEQ ID NO: 4.  
     
     
         65 . The method of  claim 63 , wherein the insertion end at the 5′ end of the transposable element comprises the sequence shown in SEQ ID NO: 5.  
     
     
         66 . The method of  claim 64 , wherein the insertion end at the 3′ end of the transposable element comprises the sequence shown in SEQ ID NO: 3.  
     
     
         67 . The method of  claim 37 , wherein the transposable element further comprises a nucleic acid sequence encoding a transposase.  
     
     
         68 . The method of  claim 67 , wherein the transposase is a Cre transposase.  
     
     
         69 . The method of  claim 37 , wherein the transposable element further comprises an affinity tag.  
     
     
         70 . The method of  claim 69 , wherein the affinity tag is 6× histidine, S-tag, glutathione-S-transferase, or streptavidin.  
     
     
         71 . The method of  claim 70 , wherein the affinity tag is 6× histidine.  
     
     
         72 . The method of  claim 69 , wherein the nucleic acid sequence encoding an affinity tag is 5′ of the 5′ recombining site.  
     
     
         73 . The method of  claim 69 , wherein the nucleic acid sequence encoding an affinity tag is 3′ of the 3′ recombining site.  
     
     
         74 . The method of  claim 45 , wherein the 5′ recombining site or the 3′ recombining site is a loxP recombining site, a fit recombining site, a TN3 recombining site, a mariner recombining site, or a gamma/delta recombining site.  
     
     
         75 . The method of  claim 74 , wherein the 5′ recombining site or the 3′ recombining site is a loxP recombining site.  
     
     
         76 . The method of  claim 75 , wherein the loxP sequence comprises the sequence shown in SEQ ID NO: 11.  
     
     
         77 . The method of  claim 45 , wherein the MHC class II epitope is ASFEAQGALANIAVDKA (SEQ ID NO: 20) and the MHC class II haplotype of the eukaryotic cell is I-A b .  
     
     
         78 . The method of  claim 45 , wherein the selectable marker is a nucleic acid encoding antibiotic resistance.  
     
     
         79 . The method of  claim 45 , wherein the selectable marker is detected by spectrophotometric properties.  
     
     
         80 . The method of  claim 45 , wherein the insertion end at the 5′ end of the transposable element is SEQ ID NO: 4 or SEQ ID NO: 5.  
     
     
         81 . The method of  claim 45 , wherein the insertion end at the 3′ end of the transposable element is SEQ ID NO: 3 or SEQ ID NO: 4.  
     
     
         82 . The method of  claim 45 , wherein the transposable element further comprises an affinity tag.  
     
     
         83 . The method of  claim 82 , wherein the affinity tag is 6× histidine, S-tag, glutathione-S-transferase, or streptavidin.  
     
     
         84 . The method of  claim 82 , wherein the nucleic acid sequence encoding an affinity tag is 5′ of the 5′ recombining site.  
     
     
         85 . The method of  claim 82 , wherein the nucleic acid sequence encoding an affinity tag is 3′ of the 3′ recombining site.  
     
     
         86 . The method of  claim 46 , wherein the 5′ recombining site or the 3′ recombining site is a loxP recombining site, a fit recombining site, a TN3 recombining site, a mariner recombining site, or a gamma/delta recombining site.  
     
     
         87 . The method of  claim 86 , wherein the 5′ recombining site or the 3′ recombining site is a loxP recombining site.  
     
     
         88 . The method of  claim 87 , wherein the loxP sequence comprises the sequence shown in SEQ ID NO: 11.  
     
     
         89 . The method of  claim 46 , wherein the MHC class II epitope is ASFEAQGALANIAVDKA (SEQ ID NO: 20) and the MHC class II haplotype of the eukaryotic cell is I-A b .  
     
     
         90 . The method of  claim 46 , wherein the selectable marker is a nucleic acid encoding antibiotic resistance.  
     
     
         91 . The method of  claim 46 , wherein the selectable marker is detected by spectrophotometric properties.  
     
     
         92 . The method of  claim 46 , wherein the insertion end at the 5′ end of the transposable element is SEQ ID NO: 4 or SEQ ID NO: 5.  
     
     
         93 . The method of  claim 46 , wherein the insertion end at the 3′ end of the transposable element is SEQ ID NO: 3 or SEQ ID NO: 4.  
     
     
         94 . The method of  claim 46 , wherein the transposable element further comprises an affinity tag.  
     
     
         95 . The method of  claim 94 , wherein the affinity tag is 6× histidine, S-tag, glutathione-S-transferase, or streptavidin.  
     
     
         96 . The method of  claim 94 , wherein the nucleic acid sequence encoding an affinity tag is 5′ of the 5′ recombining site.  
     
     
         97 . The method of  claim 94 , wherein the nucleic acid sequence encoding an affinity tag is 3′ of the 3′ recombining site.  
     
     
         98 . The method of  claim 37 , wherein the MHC class II epitope is anti-I-A k /Hen Egg Lysozyme (HEL 46-61 ) or anti-I-A k /Hen Egg Lysozyme (HEL 116-129 ), and the MHC class II haplotype of the eukaryotic cell is I-A b .  
     
     
         99 . The method of  claim 45 , wherein the MHC class II epitope is anti-I-A k /Hen Egg Lysozyme (HEL 46-61 ) or anti-I-A k /Hen Egg Lysozyme (HEL 116-129 ), and the MHC class II haplotype of the eukaryotic cell is I-Ab.  
     
     
         100 . The method of  claim 46 , wherein the MHC class II epitope is anti-I-A k /Hen Egg Lysozyme (HEL 46-61 ) or anti-I-A k /Hen Egg Lysozyme (HEL 116-129 ), and the MHC class II haplotype of the eukaryotic cell is I-A b .