Artificial mosaic protein containing antigenic epitopes of hepatitis E virus

Abstract

A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or I-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and ,-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and-negative sera. The data

Locally acquired hepatitis E virus infection, El Paso, Texas

“…IgM and IgG anti-HEV were detected by enzyme immunoassay (EIA) using as the target antigen a mixture comprised of a recombinant mosaic protein (MPr) containing antigenic determinants of HEV ORF-2 and ORF-3 [Khudyakov et al, 1994] and a complete ORF-2 baculovirus expressed protein (55 k) [Tsarev et al, 1993]. The target antigen mixture (MPr55k mixture), containing 15 ng MPr and 20 ng 55 k protein in PBS, was adsorbed to flat-bottomed wells (Immulon 2HB Styrene Immunoassay Plates, Dynex Technologies, Chantilly, VA) at room temperature for 16-18 hr.…”

Hepatitis E: current status

“…Recently, some studies reveal the role of ORF3 protein in virus egress and the presence of this protein on the virion surface possibly in association with lipids . All ORFs are expressed during viral infection because antibodies against these regions have been detected in naturally infected humans and in experimentally infected monkeys .…”

Hepatitis E: Clinical Presentation in Disease-Endemic Areas and Diagnosis

“…The available anti-HEV assays have used (1) recombinant proteins expressed in Escherichia coli, 61,62 baculovirus-infected insect cells, [63][64][65] animal cells, 66 plant cells, 67,68 or insect larvae 69,70 (2) short peptides; or (3) artificial "mosaic" proteins that contain several short segments of HEV proteins with antigenic epitopes. 71,72 The recombinant proteins generally perform better because humoral epitopes of HEV are known to be conformational in nature. 62,73,74 Importantly, sensitivity, specificity, and duration of detectable immune response against a diagnostic reagent may vary between infection with different HEV genotypes.…”