Baculovirus expression of the respiratory syncytial virus fusion protein using Trichoplusia ni insect cells.
Respiratory syncytial virus (RSV) is a major viral pathogen responsible for severe respiratory tract infections in infants, young children, and the elderly. The RSV fusion (F) protein is highly conserved among RSV subgroups A and B and is the major protective immunogen. A genetically-engineered version of the RSV F protein was produced in insect cells using the baculovirus expression system. To express a secreted form of this protein, the transmembrane domain was eliminated by removing the region of the gene encoding 48 amino acids at the C-terminus. Production of the truncated RSV F protein (RSV-Fs) was compared in two different insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five). The yield of RSV-Fs secreted from High Five insect cells was over 7-fold higher than that from Sf9 insect cells. Processing of the RSV-Fs protein was also different in the two insect cell lines. N-terminal sequencing demonstrated that while most of the RSV-Fs protein secreted by High Five cells was correctly processed at the F2-F1 proteolytic cleavage site, most of the RSV-Fs protein secreted by Sf9 cells was unprocessed or incorrectly processed. Antigenicity of the major RSV F neutralization epitopes was maintained in the RSV-Fs protein secreted from High Five cells. The RSV-specific neutralizing antibody titres in the sera of cotton rats immunized with the RSV-Fs protein were equivalent to those in the sera of animals intranasally inoculated with live RSV. Animals immunized with either live RSV or the immunoaffinity purified RSV-Fs protein from High Five cells were completely protected against live virus challenge.
Parrington
M
M
Connaught Centre for Biotechnology Research, Connaught Laboratories Ltd., North York, Ontario, Canada.
Cockle
S
S
Wyde
P
P
Du
R P
RP
Snell
E
E
Yan
W Y
WY
Wang
Q
Q
Gisonni
L
L
Sanhueza
S
S
Ewasyshyn
M
M
Klein
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000097:blood; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007114:Immunization; D008297:Male; D008969:Molecular Sequence Data; D009036:Moths; D009500:Neutralization Tests; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018357:Respiratory Syncytial Virus Infections / Q000276:immunology / Q000517:prevention & control; D012136:Respiratory Syncytial Viruses / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D016205:Sigmodontinae; D018411:Spodoptera; D014760:Viral Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D004279:DNA, Viral; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins