Artificial mosaic proteins as new immunodiagnostic reagents: the hepatitis E virus experience.
BACKGROUND
Naturally occurring viral proteins derived from cell culture and recombinant proteins expressed in procaryotic systems have been used extensively as target proteins in the development of immunoassay methods for the detection of antibodies. However, immunoassays utilizing these proteins often yield false-positive reactions suggesting that it may be possible to identify and remove regions responsible for these non-specific reactions.


OBJECTIVE
In this paper we describe a new strategy for the construction of immunoreactive recombinant proteins designed to improve immunoassay specificity.


METHODS
A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligonucleotides by the polymerase chain reaction (PCR). The polypeptide comprises a mosaic of three antigenically 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 strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione-S-transferase or beta-galactosidase.


RESULTS
Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by immunoblot analysis and by enzyme immunoassay (EIA) the presence and accessibility of all HEV-specific antigenic epitopes designed into the mosaic protein. Both hybrid proteins were shown by immunoblot analysis using a panel of human anti-HEV-positive and -negative sera to be HEV-specific. A sensitive and specific EIA was developed to detect IgG anti-HEV activity in human sera. A neutralization test using individual synthetic peptides corresponding to the epitopes designed into the mosaic protein was also developed to confirm IgG anti-HEV activity by absorbing the specimen before retesting by EIA.


CONCLUSIONS
An artificial mosaic protein composed of short linear HEV-specific antigenic epitopes was constructed from synthetic oligonucleotides by PCR and used to develop a sensitive and specific EIA for the detection of anti-HEV activity in human sera.
Fields
H A
HA
Hepatitis Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, 1600 Clifton Rd., Atlanta, GA 30333, USA.
Khudyakov
Y E
YE
Favorov
M O
MO
Khudyakova
N S
NS
Cong
M E
ME
Holloway
B F
BF
Lambert
S B
SB
Jue
D L
DL