title: Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells.
abstract: Recombinant baculoviruses that produce a putative non-structural protein 1 (NS1) of hepatitis C virus (HCV), predicted to be the second envelope glycoprotein, were constructed. The recombinant NS1 protein (re-NS1) produced in infected insect cells was localized on the cell surface and was apparently glycosylated, because it was susceptible to treatment with both tunicamycin and N-glycanase. Furthermore, re-NS1 was effectively secreted into the culture supernatant when the putative NS1 signal peptide (SP) was replaced by the SP of rabies virus G protein, and the C-terminal hydrophobic region was eliminated. The secreted re-NS1 was tagged with six His residues at the C terminus and purified simply by native Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) affinity column chromatography. An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of HC using purified re-NS1. Anti-NS1 antibody (Ab) was detected in 55 of 60 patients (92%) with chronic HC liver diseases. Thus, this ELISA for Ab directed against HCV re-NS1 produced in insect cells is useful for the detection of chronic HC patients.
authors.affiliation: Chemo-sero Therapeutic Research Institute, Kikuchi Laboratories, Kumamoto, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Nishihara
authors.forename: C
authors.initials: C
authors.lastname: Nozaki
authors.forename: H
authors.initials: H
authors.lastname: Nakatake
authors.forename: K
authors.initials: K
authors.lastname: Hoshiko
authors.forename: M
authors.initials: M
authors.lastname: Esumi
authors.forename: N
authors.initials: N
authors.lastname: Hayashi
authors.forename: K
authors.initials: K
authors.lastname: Hino
authors.forename: F
authors.initials: F
authors.lastname: Hamada
authors.forename: K
authors.initials: K
authors.lastname: Mizuno
authors.forename: T
authors.initials: T
authors.lastname: Shikata
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis*; D016367:Baculoviridae; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D004274:DNA, Recombinant; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000096:biosynthesis; D016174:Hepacivirus / Q000737:chemistry*; D006508:Hepatitis Antibodies / Q000276:immunology; D006526:Hepatitis C / Q000175:diagnosis / Q000276:immunology; D018937:Hepatitis C Antibodies; D018936:Hepatitis C Antigens; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014759:Viral Envelope Proteins / Q000096:biosynthesis; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000956:Antigens, Viral; D004274:DNA, Recombinant; D006023:Glycoproteins; D006508:Hepatitis Antibodies; D018937:Hepatitis C Antibodies; D018936:Hepatitis C Antigens; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; C064951:glycoprotein G, Rabies virus