Baculovirus-directed high level expression of the hepatitis delta antigen in Spodoptera frugiperda cells.
The hepatitis delta antigen (HDAg) is a multifunctional protein. It forms the core-like structure of the hepatitis delta virus (HDV) but also enhances replication of HDV in the nucleus of the hepatocyte. A cDNA fragment encoding HDAg was inserted adjacent to the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus present in the baculovirus transfer vector pVL941. After transfection of Spodoptera frugiperda (Sf9) cells a recombinant baculovirus Ac delta 1 was isolated and purified using filter hybridization techniques. Sf9 cells infected with Ac delta 1 express the HDAg as a non-fused, non-glycosylated protein with an abundance of up to 25% of the total cellular protein mass. Immunoblot analysis using a human polyclonal anti-HD conjugate identified a 22K and a 24K protein in the nucleus of Ac delta 1-infected Sf9 cells. Electron microscopic studies using immunogold labelling showed that the recombinant HDAg (recHDAg) was associated with the hetero-chromatin of the Sf9 cells. The recHDAg produced by Sf9 cells elicited anti-HD antibodies in chimpanzees when injected intramuscularly.
Kos
T
T
Department of Chronic and Infectious Diseases, Primate Center, ITRI-TNO, Rijswijk, The Netherlands.
Molijn
A
A
Blauw
B
B
Schellekens
H
H
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005814:Genes, Viral; D005822:Genetic Vectors; D003698:Hepatitis Delta Virus / Q000235:genetics* / Q000276:immunology / Q000648:ultrastructure; D035923:Hepatitis delta Antigens; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009036:Moths; D002679:Pan troglodytes; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D014162:Transfection
D000956:Antigens, Viral; D004279:DNA, Viral; D035923:Hepatitis delta Antigens; D012367:RNA, Viral; D011994:Recombinant Proteins; C442244:hepatitis delta virus large antigen