Abstract
Major Histocompatibility Complex class I molecules present antigens to the immune system. A mouse MHC class I gene was modified by linking individual subunits together with a peptide antigen. A disulfide bond, termed the disulfide trap, was added to stabilize the preloaded antigen. The original antigen peptide, SIINFEKL, was modified to test the disulfide trap for stabilization of MHC class I with a modified antigen. The SIINFEKL antigen was modified to SIINHEKL and SIINYEKL. DNA primers were designed to mutate the phenylalanine (F) amino acid to the new histidine (H) or tyrosine (Y). This was done through site directed mutagenesis, transformation, and transfection into tissue culture cells. Modifications of the peptide were tested using cytotoxic T cells that specifically recognized MHC I-SIINFEKL. Cells expressing the disulfide trap proved to be very stable and presented a recognizable antigen to the T cells even when the original peptide had been modified. For the SIINHEKL modification, the disulfide trap model was recognized by the T cells. However, the SIINHEKL without the disulfide trap model was weakly presented to the T cells. Flow cytometry of the tissue cultures showed very similar results. Flow cytometry uses antibodies specific for the MHC I molecule and the antigens being presented. This allows for proper folding of the antigen peptide on the MHC I to be detected. Molecules lacking the disulfide trap were not as stable and did not fold or present peptides well. The SIINYEKL molecule responded in much the same way. This serves as proof of the concept that peptides of our design can be presented efficiently to the immune system with disulfide trap stabilization. Further application of this research may allow DNA vaccines, expressing custom antigens, to be generated.