Expression of equine herpesvirus type 1 glycoprotein gp14 in Escherichia coli and in insect cells: a comparative study on protein processing and humoral immune responses.
The extracellular portion (amino acids 1 to 844) of the equine herpesvirus type 1 (EHV-1) glycoprotein gp14, the homologue of gB of herpes simplex virus, was expressed in Escherichia coli and in insect cells using a recombinant baculovirus. Immunoblot analysis revealed that the recombinant E. coli expressed a fusion protein of M(r) 135K which was composed of the truncated gp14 and the maltose-binding protein (MBP) provided by the vector and a 90K protein lacking the MBP moiety. Both proteins were sequestered within the cells in form of inclusion bodies. Infection of insect cells with the recombinant baculovirus resulted in the production of a 115K to 118K glycoprotein which was cleaved intracellularly into two subunits of M(r) 55K and 63K to 65K. The cleaved subunits were secreted into the cell culture supernatant and formed disulphide-linked dimers of M(r) 120K to 122K. The recombinant proteins produced in E. coli and in insect cells elicited EHV-1-specific antibodies in goats as demonstrated by Western blot analysis. The gp14 expressed in insect cells induced antibodies with virus-neutralizing activity. In contrast, the truncated gp14 expressed by E. coli failed to elicit neutralizing antibodies. The results suggest that post-translational modification of the EHV-1 gp14 may be important for the expression of epitopes necessary for the induction of neutralizing antibodies.
Osterrieder
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N
Institute for Medical Microbiology, Infectious and Epidemic Diseases, Ludwig-Maximilians-University Munich, Germany.
Wagner
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R
Pfeffer
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M
Kaaden
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OR
D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics; D004861:Herpesvirus 1, Equid; D007313:Insecta / Q000166:cytology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D013045:Species Specificity; D014759:Viral Envelope Proteins
D000914:Antibodies, Viral; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C063677:gp14 protein, equine herpesvirus 1