A portion of mutant p53 cDNA from human lung tumor was amplified from cDNA by PCR and cloned into a eukaryotic expression vectors. One of these vectors contained an E3 signal peptide from adenovirus which causes proteins to be quickly translocated to the endoplasmic reticulum for MHC class I presentation to T cells.
Plasmid DNAs (vectors) were fixed to micron-sized gold particles. Gold is used because it is chemically inert and very dense which allows the DNA to be propelled through the cell membrane into the cell (particle bombardment).
The beads were loaded into a macrocarrier disk, and then the disk was loaded into a helium-driven gene gun which shot the beads into the ears of anesthetized mice.
The gold beads enter the cells, the plasmid replicates, and the mutant p53 mini-gene is transcribed and translated. Mutant p53 is produced, and the peptides that are translated from the vector in the endoplasmic reticulum bind to the cleft of the MHC class I (a protein which presents antigenic peptide to T cells) where it is then translocated to the surface.
The advantage of the E3 signal peptide is that it directs the mutant p53 to the endoplasmic reticulum.
Since mutant p53 is "non-self" (nonmutated p53 would be "self") the immune system sees mutated p53 as foreign. Therefore, the T cells recognize the foreign p53 presented by MHC and becomes stimulated. The stimulated T cells then divide and look for other cells bearing the same antigenic peptide.
