Expression of the nucleoprotein of rabies virus in Escherichia coli and mapping of antigenic sites.
Investigations were performed to delineate the antigenic sites I and IV of rabies virus nucleoprotein (N), the former of which is well conserved among the rabies and rabies-related viruses. The N cDNA of the RC-HL strain was inserted into an expression vector pET3a, with which the E. coli BL21(DE3) was transformed. Upon induction with isopropyl-1-thio-beta-D-galactoside, the transformants produced a protein with a size (56 k-Da) almost identical to that of the authentic N protein. The protein also reacted with a panel of our N protein-specific monoclonal antibodies (N-MAbs) including the antibodies against the antigenic sites I and IV. By using the cDNA, various deletion mutants were generated and expressed in E. coli to examine the reactivity of mutant proteins with N-MAbs by Western blot analysis. Deletion of the C-terminal 67 amino acid residues did not abolish their reactivity with any of the N-MAbs specific for the sites I and IV. When 91 residues or more were deleted from the C-terminus, however, the protein lost the reactivity, indicating that the antigenic sites I and IV are mapped to a small region which is comprised of at most 24 amino acid residues from positions 360 to 383. Comparison of the 24-amino acid sequence with the corresponding region of N protein of several other Lyssavirus strains suggests that the antigenic site I is mapped to positions 360 to 369.
Goto
H
H
Department of Veterinary Public Health, Faculty of Agriculture, Gifu University, Japan.
Minamoto
N
N
Ito
H
H
Luo
T R
TR
Sugiyama
M
M
Kinjo
T
T
Kawai
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D002213:Capsid / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D017931:DNA Primers; D018604:Epitope Mapping; D004926:Escherichia coli; D015967:Gene Expression Regulation, Viral; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D011820:Rabies virus / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014758:Viral Core Proteins / Q000235:genetics* / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D017931:DNA Primers; D011994:Recombinant Proteins; D014758:Viral Core Proteins