Expression of immunoreactive forms of the hepatitis C NS5A protein in E. coli and their use for diagnostic assays

Abstract

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.

Production of Herpes B Virus Recombinant Glycoproteins and Evaluation of Their Diagnostic Potential

“…Recombinant-based serological assays have been developed for the diagnosis of many viral infections, including human cytomegalovirus (11), hepatitis C virus (27), hepatitis E virus (46), human papillomavirus (49), Ebola virus (45), and many others. Several recombinant glycoprotein G (gG)-based immu-noassays for HSV type-specific serodiagnosis are commercially available (19,44).…”

Calcium-Dependent Calpain Proteases Are Implicated in Processing of the Hepatitis C Virus NS5A Protein

“…The pA-EUA2 vector has been derived from the pA-SK amplicon plasmid and carries the green fluorescent protein-coding sequence and the BGH poly(A)-taken from pIRES-EGFP (Clonetech)-under the HSV-1 IE4 (␣22/␣47) promoter (A. L. Epstein and V. Revol-Guyot, unpublished data). Plasmid pHPI 1406 encodes an N-terminal deletion (Ϫ162 amino acids) fragment of NS5A and was constructed following amplification of the corresponding sequence from pHPI 611 (31). The sense primer was 5Ј-CCAAGCTTGCCATGGCGCCCCCTTGC-3Ј (HindIII and NcoI restrictions sites are underlined; the translation initiation codon is indicated in bold), and the antisense primer was as described above.…”

“…The MBP-NS5A.1 protein was used to generate a polyclonal antibody against NS5A. The protein was produced in Escherichia coli harboring the pHPI 662 plasmid and purified by affinity chromatography, as previously described (31). Rabbits were immunized against the NS5A protein following three injections, each containing approximately 300 g of the purified fusion protein, at intervals of about 3 weeks.…”

Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays

“…12 In this report we mapped the antigenic sites in Core and E2 genes of HCV-3a and cloned in pET-28a expression vector for the production of truncated Core and E2 recombinant antigens, which were then studied for their potential assessment as diagnostic antigens for the identification of anti-HCV antibodies in HCV infected sera. The production of truncated recombinant Core and E2 antigens was confirmed by western blotting, and their serological reactivity was examined by immunoblotting and ELISA.…”

“…11 Several efforts have been made to develop efficient serological tests for the screening of HCV infection. 12 The ELISA assay or immunoblot assays uses HCV recombinant viral antigens corresponding to multiple polypeptides from various viral fragments, including structural proteins and non-structural proteins. 13 These serological screening assays combine improved sensitivity and specificity, however still fail to diagnose HCV infection more accurately.…”