Cloning and expression of human rotavirus spike protein, VP8*, in Escherichia coli.
A system for the expression and purification of soluble VP8*, part of the human rotavirus (HRV) spike protein, was established by expressing VP8* as a fusion protein with glutathione S-transferase (GST). VP8 cDNA, from the Wa strain of HRV, was prepared by RT-PCR, cloned into a pUC18 plasmid, and inserted into a pGEX-4T-2 GST fusion vector. The GST-VP8* fusion protein was expressed in Escherichia coli, and the VP8* was purified by Glutathione Sepharose 4B affinity chromatography, yielding 1.8 mg VP8*/L culture. The purified VP8* was used to vaccinate chickens, eliciting antibodies which displayed high neutralization activity against the Wa strain of HRV, suggesting its use for the induction of specific neutralizing antibodies for potential immunotherapeutic applications for the prevention of HRV infection.
Kovacs-Nolan
J
J
Department of Food Science, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
Sasaki
E
E
Yoo
D
D
Mine
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000302:isolation & purification; D001483:Base Sequence; D002213:Capsid / Q000235:genetics / Q000276:immunology; D036022:Capsid Proteins; D002460:Cell Line; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D000882:Haplorhini; D007136:Immunoglobulins / Q000097:blood / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D016133:Polymerase Chain Reaction; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012401:Rotavirus / Q000235:genetics* / Q000276:immunology; D012698:Serologic Tests; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D010948:Viral Plaque Assay
D000914:Antibodies, Viral; D036022:Capsid Proteins; C016512:IgY; D007136:Immunoglobulins; D010446:Peptide Fragments; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C060339:VP4 protein, Rotavirus; D017361:Viral Nonstructural Proteins; C079234:NS35 protein, rotavirus; D005982:Glutathione Transferase