Expression in insect cells and immune reactivity of a 28K tegument protein of human cytomegalovirus.
The gene encoding the highly antigenic 28K (pp28) tegument phosphoprotein of human cytomegalovirus (HCMV) was expressed in insect cells utilizing a recombinant baculovirus. The mature intracellular form of the recombinant-derived pp28 had mobility on SDS-polyacrylamide gels similar to that of native pp28 from HCMV strain Towne-infected human foreskin fibroblasts (HFFs). In vitro labelling of recombinant Autographa california nuclear polyhedrosis virus-infected Spodoptera frugiperda cells or of HCMV-infected HFFs with [32P]orthophosphate followed by immunoprecipitation showed that both the insect cell-derived and HCMV strain Towne-infected fibroblast-derived pp28 were phosphorylated. The mobility of pp28 derived from these two sources as well as from extracellular HCMV virions indicated the existence of multiple charged forms of the protein, and a difference in the relative amounts of these forms expressed in HCMV-infected HFFs and recombinant baculovirus-infected insect cells. The recombinant pp28 expressed in insect cells was readily and specifically recognized by antibodies to native pp28, including HCMV-seropositive human serum, and was used in an ELISA to screen human sera for seropositivity.
Giugni
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TD
Division of Pediatrics, City of Hope National Medical Center, Duarte, California 91010.
Churchill
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MA
Pande
H
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Campo
K
K
Guha
M
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Zaia
J A
JA
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology*; D000917:Antibody Formation; D000937:Antigen-Antibody Reactions; D000956:Antigens, Viral / Q000276:immunology*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D009036:Moths; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000276:immunology*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis; D014764:Viral Proteins / Q000096:biosynthesis* / Q000276:immunology*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C070746:human cytomegalovirus protein pp28