Expression of immunoreactive forms of the hepatitis C NS5A protein in E. coli and their use for diagnostic assays.
In this study different forms of the hepatitis C virus (HCV) NS5A protein, including a nearly full-length, an amino-terminal and a carboxy-terminal truncated form were produced in E. coli as fusion proteins with the MBP or the GST protein. The chimeric proteins were tested for their reactivity with sera from HCV infected patients by immunoblot and ELISA assays. A panel of 110 sera specimens, including 39 HCV-positive sera, 27 sera from patients with non-HCV-associated liver disease and 44 healthy individuals were analyzed for the presence of antibodies to NS5A. Twenty four (61 %) out of the 39 HCV positive sera, showed reactivity against the nearly full length NS5A, 21 (54 %) against the amino-terminal part of NS5A and 20 (51 %) against the carboxy-terminal part of the NS5A protein in immunoblot assays, suggesting that immunoreactive epitopes are present both at the carboxy- and the amino- terminal part of the protein. None of the 71 HCV-negative serum samples showed any reactivity against the NS5A antigens. With the exception of one patient, similar data were obtained with an ELISA assay based on the use of the nearly full-length NS5A antigen. The data indicate that new forms of NS5A may be potentially valuable antigens for the development of serological assays for HCV.
Kalamvoki
M
M
Molecular Virology Laboratory, Hellenic Pasteur Institute, Athens, Greece.
Miriagou
V
V
Hadziyannis
A
A
Georgopoulou
U
U
Varaklioti
A
A
Hadziyannis
S
S
Mavromara
P
P
D000293:Adolescent; D000328:Adult; D000368:Aged; D000914:Antibodies, Viral / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006526:Hepatitis C / Q000175:diagnosis*; D006801:Humans; D015151:Immunoblotting; D008297:Male; D008875:Middle Aged; D011994:Recombinant Proteins / Q000276:immunology; D012698:Serologic Tests; D017361:Viral Nonstructural Proteins / Q000276:immunology*; D012324:RNA-Dependent RNA Polymerase
D000914:Antibodies, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase