Characterization of vaccinia surface antigen expressed by recombinant baculovirus.
The gene encoding the vaccinia surface antigen (S antigen) was inserted into a baculovirus transfer vector and a recombinant virus was isolated. The S antigen was expressed on the surface of Spodoptera frugiperda cells (Sf cells) infected with the recombinant baculovirus. Recombinant proteins were detected in immunoblotting with anti-vaccinia serum and have the apparent molecular weight of 40 and 50-kDa. The 50-kDa polypeptide was tunicamycin sensitive and was thus glycosylated. The glycosylated 50-kDa polypeptide was also secreted into culture supernatants. Antiserum directed against the expressed S antigen specifically reacted to the authentic S antigen that is located on the mammalian cells infected with vaccinia virus, but did not affect the production of infectious progeny virus. Antisera against both the terminal regions of the S antigen were prepared by immunizing rabbits with the recombinant fusion proteins produced in the bacterial expression vector. The S antigen on the cell surface reacted by immunofluorescence with anti-C-terminus serum but not or very weakly with anti N-terminus serum, indicating that the hydrophobic N-terminus functions both as a signal and a membrane anchor sequence. Interleukin (IL)-1 alpha failed to bind to the S antigen expressed on insect and mammalian cells, although the homology between IL-1 receptor and the S antigen has been reported by computer analysis of amino acid sequence.
Morikawa
S
S
Department of Virology I, National Institute of Health, Tokyo, Japan.
Ueda
Y
Y
D016475:3T3 Cells; D000818:Animals; D000954:Antigens, Surface / Q000032:analysis / Q000235:genetics*; D000956:Antigens, Viral / Q000032:analysis / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007375:Interleukin-1 / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009036:Moths; D011994:Recombinant Proteins / Q000032:analysis; D014616:Vaccinia virus / Q000235:genetics* / Q000254:growth & development; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics*
D000954:Antigens, Surface; D000956:Antigens, Viral; D007375:Interleukin-1; D011994:Recombinant Proteins; D014764:Viral Proteins; C080409:vaccinia surface antigen