mRNA PCR-Based Epitope Chase Method
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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.
