mRNA PCR-based epitope chase method

Abstract

The identification of specific viral and tumor antigen epitopes recognized by CD4(+) or CD8(+) T lymphocytes remains a challenge. Unfortunately, epitope mapping methods are generally costly and time-consuming. This chapter details a polymerase chain reaction (PCR)-based mRNA epitope identification method called mPEC, which is designed to rapidly and precisely identify relevant T cell epitopes recognized by previously isolated CD8(+) or CD4(+) T lymphocytes.This method is based on the use of mRNA fragments synthesized from PCR-amplified cDNA with a variety of 3'end iterative deletions. mRNA fragments are electroporated into autologous antigen-presenting cells to map the epitope in a given protein antigen. Considering mRNA's sensitivity to degradation, we also insert a control define epitope at the mRNA's 3'end to control for electroporated mRNA's integrity and capacity to be translated.