Expression of the F glycoprotein gene from human respiratory syncytial virus in Escherichia coli: mapping of a fusion inhibiting epitope.
A cDNA copy of the gene encoding the entire amino acid sequence of the fusion (F) protein of human respiratory syncytial virus (strain A2) was inserted into a bacterial expression vector containing the lambda PR promoter. Upon heat induction, Escherichia coli cells harboring the vector produced a 45-kDa peptide which reacted with rabbit polyclonal antiserum to the native F protein. Expression of the F gene resulted in severe inhibition of bacterial growth, which was overcome by deletion of the DNA sequences encoding the F signal peptide. The region of the F protein which reacted with a virus-neutralizing and fusion-inhibiting monoclonal antibody was probed by expressing cDNA fragments encoding different protein domains in E. coli and testing antibody reactivity by Western blot analysis. Analysis of six fragments yielded an overlapping antibody-reactive region between amino acids 253 and 298. Analysis of reactivity with a cassette of synthetic peptides confirmed that the virus-neutralizing epitope mapped between residues 289 and 298 defined by the amino acid sequence M-S-I-I-K-E-E-V-L-A.
Martin-Gallardo
A
A
Department of Virology Research, Praxis Biologics, Inc., Rochester, New York 14623.
Fien
K A
KA
Hu
B T
BT
Farley
J F
JF
Seid
R
R
Collins
P L
PL
Hildreth
S W
SW
Paradiso
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D000939:Epitopes / Q000032:analysis*; D005814:Genes, Viral; D005822:Genetic Vectors; D015707:HN Protein; D007106:Immune Sera; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010957:Plasmids; D011817:Rabbits / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D012136:Respiratory Syncytial Viruses / Q000235:genetics*; D015183:Restriction Mapping; D014759:Viral Envelope Proteins; D014764:Viral Proteins
D000956:Antigens, Viral; D000939:Epitopes; D015707:HN Protein; D007106:Immune Sera; D015345:Oligonucleotide Probes; D010455:Peptides; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116204:attachment protein G