Patent ID: 6846622
Filing Date: 2005-01-25
Classification: A61K,C07K,C12N,C12Q,Y02A

Abstract:
1. A method for identifying a protein secreted by an intracellular pathogen and having access to an MHC class I pathway of a eukaryotic cell infected with the intracellular pathogen, comprising: (i) transfecting 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 I 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 intracellular pathogen with the pathogen transfected with the transposable element, wherein an MHC class I haplotype of the eukaryotic cell is matched to the MHC I epitope; (iv) contacting the eukaryotic cell with a labeled antibody that recognizes the MHC class I 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 I pathway of eukaryotic cell infected with the intracellular pathogen.