Localization of antigenic sites at the amino-terminus of rinderpest virus N protein using deleted N mutants and monoclonal antibody.
The nucleocapsid (N) protein of rinderpest virus (RPV) is highly conserved, immunogenic, and abundantly expressed during infection. Six antigenic sites (sites A, B, C, D, E and F), defined previously by a competitive binding assay using corresponding monoclonal antibodies (Mabs), have been further localized by immunoassays using deleted N mutants. Five different forms of RPV N protein, containing residues aa 1-79, aa 1-149, aa 1-421, aa 414-525 and aa 1-525, were expressed as glutathione S transferase (GST) fusion proteins (designated as GST-N1-79, GST-N1-149, GST-N1-421, GST-N414-525, and GST-N1-525, respectively) in E.coli BL21 cells. In ELISA using deleted N mutants, Mabs recognizing sites A, B, C, D and E reacted with 3 GST fusion proteins (GST-N1-149, GST-N1-421 and GST-N1-525), indicating that they are located at aa 80-149. Mab recognizing site F reacted with 4 GST fusion proteins (GST-N1-79, GST-N1-149, GST-N1-421 and GST-N1-525), indicating that site F is located at aa 1-79. Identification of the amino-terminal antigenic sites of the N protein would provide antigen basis for developing sensitive and specific diagnostic reagents for RPV, although it remains to be further investigated antigenic sites at the carboxyl-terminus.
Choi
Kang-seuk
KS
National Veterinary Research and Quarantine service, Ministry of Agriculture and Forestry, 480 Anyang, Gyounggi 430-824, Korea. choiks@nvrqs.go.kr
Nah
Jin-ju
JJ
Ko
Young-joon
YJ
Kang
Shien-young
SY
Joo
Yi-seok
YS
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D012302:Rinderpest virus / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014709:Vero Cells; D014764:Viral Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C481383:nucleocapsid protein, Rinderpest virus