Expression of herpes simplex virus 1 glycoprotein D in prokaryotic and eukaryotic cells.
Recombinant plasmids encoding either the full-length glycoprotein D (FLgD) or truncated gDs were constructed. The recombinant plasmids were expressed in Escherichia coli and BHK-21 cells. The strongest expression was obtained with the recombinant plasmid encoding a truncated gD which corresponded to the gD ectodomain. The cells transformed with this plasmid showed good exponential growth ensuring satisfactory yields of the expressed polypeptide in the form of the fusion protein. The fusion protein was biotinylated and efficiently purified. The shortest truncated gD, which contained the main continuous antigenic locus VII binding neutralization antibody and additional continuous antibody binding epitopes, still reacted with specific antibody as proven by immunoblot analysis. In addition, a shuttle vector for expression of FLgD in mammalian cells was constructed. This vector-transfected BHK-21 cells expressed gD for 40 days during 9 consecutive passages. The expression of gD began on day 2 and culminated at day 9 post transfection (p.t.).
Mosko
T
T
Institute of Microbiology and Immunology, Jessenius Faculty of Medicine, Comenius University, Martin, Slovak Republic.
Kosovský
J
J
Rezuchová
I
I
Durmanová
V
V
Kúdelová
M
M
Rajcáni
J
J
D000818:Animals; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D001710:Biotin / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D010957:Plasmids; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013194:Staining and Labeling; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1; D001710:Biotin