Mapping unprocessed epitopes using deletion mutagenesis of gene fusions.
To locate the antigenic determinant recognized by a monoclonal antibody directed against a baculovirus capsid protein, a series of overlapping deletions of a fusion protein were immunologically screened with the monoclonal antibody. The immunoreactive fusion protein was derived from a restriction fragment which contained a large portion of a baculovirus capsid protein open reading frame fused in-frame with a truncated trpE gene in a bacterial (pATH3) expression system. To map the epitope, nested sets of 5' and 3' deletion mutants were generated. Mutants were characterized by the DNA insert size or by the size of the expressed fusion protein. Selected N- and C-termini truncated fusion proteins were Western blotted and incubated with the monoclonal antibody to identify mutants which retained the epitope. Plasmid DNA from mutants which flank the 5' and 3' junction of the antigenic determinant were sequenced to determine the epitope junction. By screening forty 3' deletions and sixty-four 5' deletions, the antigenic determinant was localized to a region of seven amino acids.
Oregon State University.
C H
CH
Gross
G F
GF
Rohrmann
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002213:Capsid / Q000235:genetics; D002872:Chromosome Deletion; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D000939:Epitopes / Q000235:genetics*; D005822:Genetic Vectors; D008722:Methods; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology
D000939:Epitopes; D011993:Recombinant Fusion Proteins