[Analysis of monoclonal antibody binding sites in ovine prion protein].
Binding sites of five monoclonal antibodies were obtained by reinforceable method of overlapping recombinant prion protein and synthetic peptide. Overlapping peptides of PrP core were expressed in Escherichia coli by insertion of serial PCR amplicons of ovine PrP gene fragments into pET32a. The expressed fusion peptides were then tested for the binding activity to PrP monoclonal antibodies in Western blotting. The binding sites of 5 monoclonal antibodies of ovine PrP were located respectively as follows: 2H3 in 199 aa-213 aa, 4C6, 5F11 and 7F11 in 139 aa-168 aa and 7F1 in 214 aa-227 aa. There oligo peptides were synthesized and used in ELISA test for more accurate localization of the binding sites. The binding sites of 4C6, 5F11 and 7F11 were further confirmed to be in 149 aa-158 aa. This conclusion may contribute to the research for pathogenesis and diagnostic method of scrapie and bovine transmissible spongiform encephalopathy.
Zhang
Yongqiang
Y
National Diagnostic Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center, Qingdao 266032, China.
Wu
Xiaodong
X
Zhao
Yonggang
Y
Bao
Endong
E
Wang
Qinghua
Q
Zhang
Wei
W
Liu
Yutian
Y
Wang
Zhiliang
Z
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000276:immunology*; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017096:Prion Diseases / Q000175:diagnosis; D011328:Prions / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012608:Scrapie / Q000175:diagnosis; D012756:Sheep
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011328:Prions; D011993:Recombinant Fusion Proteins