The use of the polymerase chain reaction to map CD4+ T cell epitopes.
CD4+ T cells recognize processed exogenous antigen in the form of peptides bound to syngeneic major histocompatibility complex class II molecules on antigen-presenting cells. We have developed a novel and convenient method to synthesize and map CD4+ T cell epitopes of cloned antigens using polymerase chain reaction (PCR)-directed construction of genes expressing recombinant protein fragments. Unique restriction sites incorporated into the PCR primers were employed for the unidirectional cloning of gene fragments into a bacterial expression vector that can be induced to high-level expression. The bacterial lysate could be used directly in T cell proliferation assays. Overlapping recombinant fragments spanning the entire protein were generated and tested. The length of the sequence containing the epitope was further reduced by utilizing PCR to generate 3' truncations. Finally, a small number of overlapping peptides spanning a sequence of 39 amino acids were synthesized to identify a thirteen-amino acid peptide epitope within chicken transferrin that stimulates the T helper cell clone D10.G4.1. PCR-directed construction of fragments of antigen allows for optimal design of strategies for the mapping and analysis of CD4+ T cell epitopes.
Nakagawa
T Y
TY
Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510.
Von Grafenstein
H
H
Sears
J E
JE
Williams
J
J
Janeway
C A
CA
Flavell
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003207:Conalbumin / Q000276:immunology; D000939:Epitopes; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010455:Peptides / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000276:immunology; D014168:Transferrin / Q000276:immunology
D000939:Epitopes; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D003207:Conalbumin