Expression of avian leukaemia virus env-gp85 in Spodoptera frugiperda cells by use of a baculovirus expression vector.
We studied the genetic expression of gp85 of avian leukaemia virus (ALV) subgroup A in a baculovirus/insect cell system. 5'terminal sequences of the gag gene were added to precede the ALV gp85 sequence and a stop codon was introduced at the boundary of gp85 and gp37. The resulting construct was then cloned into the baculovirus transfer vector pAcYM1, which contains the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Cells of the insect Spodoptera frugiperda (Sf9) were cotransfected with the resulting recombinant transfer vector pAc85 and infectious AcNPV/E2 DNA. After cotransfection, recombinant baculovirus that lacked the polyhedrin gene and expressed gp85 was selected from the supernatant and used to infect Sf9 cells. The expression of the gp85 gene peaked 3 days after infection, but expression products were not released into the culture medium even though the signal peptide had been cleaved. Owing to incomplete N-glycosylation in the insect cells the largest gp85 product had an Mr of only 65,000. In immunofluorescence tests and immunoblots the recombinant gp85 products reacted with polyclonal and monoclonal antibodies directed against ALV gp85 of subgroup A. Chickens inoculated with crude lysates of Sf9 cells infected with gp85-expressing recombinant baculovirus developed antibodies directed against ALV gp85. These antibodies were not capable of neutralizing ALV.
Noteborn
M H
MH
Laboratory for Molecular Carcinogenesis, Sylvius Laboratory, University of Leiden, The Netherlands.
de Boer
G F
GF
Kant
A
A
Koch
G
G
Bos
J L
JL
Zantema
A
A
van der Eb
A J
AJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000096:biosynthesis; D001354:Avian Leukosis Virus / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D002645:Chickens; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000276:immunology*; D007700:Kinetics; D008970:Molecular Weight; D009036:Moths / Q000382:microbiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins