Related Experiment Video
Updated: Jun 4, 2026
Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
Antigen-binding abilities of anti-nephrin antibody are prescribed by signal sequence of expression vector in genetic
Shokichi Naito1,2, Kouju Kamata3,4, Togo Aoyama1,2
1Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, Sagamihara, Kanagawa, 252-0374, Japan.
Background:
Nephrin is an essential protein for maintaining the normal structure of podocyte foot processes and the glomerular filtration barrier. To analyze the mechanism of proteinuria and nephrotic syndrome, we previously reported on a new method of producing polyclonal anti-nephrin antibody by genetic immunization. In the present paper, we investigate the effect of signal peptide sequence cDNA on the characteristics of polyclonal anti-nephrin antibodies induced by genetic immunization.
Methods:
Five fragments of nephrin cDNA with or without signal peptide sequence were inserted into the pTARGET™ vector. Rats were immunized with these vectors by the gene gun method. Sera obtained from rats at 14 weeks following immunization were analyzed by Western blotting, immunoprecipitation, flow cytometry and immunohistochemistry.
Results:
Four different antibodies induced by cDNA encoding nephrin protein fragments without signal peptide showed antigen site-specific binding to fragmented glycosylation-disturbed nephrin proteins. These antibodies also reacted to a glycosylation-disturbed full-length nephrin protein (inhibited by tunicamycin), but did not react to either a native nephrin protein or a fully glycosylated conformational nephrin protein. Four different antibodies induced by cDNA encoding nephrin fragments with signal peptide showed an antigen site-specific binding to glycosylation-disturbed nephrin protein fragments. In addition, these antibodies reacted to both a native nephrin protein and a full-length glycosylated conformational nephrin protein.
Conclusions:
The absence of signal peptide sequence cDNA in the expression vector produced antibodies specific for glycosylation-disturbed proteins, while its presence produced antibodies that bound to native or fully glycosylated conformational protein.
