Expression, characterization, and immunoreactivities of a soluble hepatitis E virus putative capsid protein species expressed in insect cells.
The hepatitis E virus (HEV) open reading frame-2 (ORF-2) is predicted to encode a 71-kDa putative capsid protein involved in virus particle formation. When insect Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus containing the entire ORF-2 sequence, two types of recombinant proteins were produced; an insoluble protein of 73 kDa and a soluble protein of 62 kDa. The 62-kDa species was shown to be a proteolytic cleavage product of the 73-kDa protein. N-terminal sequence analysis of the 62-kDa protein indicated that it lacked the first 111 amino acids that are present in the full-length 73-kDa protein. A soluble 62-kDa protein was produced without the proteolytic processing by inserting the coding sequence of amino acids 112 to 660 of ORF-2 in a baculovirus expression vector and using the corresponding virus to infect Sf9 cells. The two recombinant 62-kDa proteins made by different mechanisms displayed immunoreactivities very compatible to each other. The 62-kDa proteins obtained by both proteolytic processing and reengineering demonstrated much higher sensitivities in detecting anti-HEV antibodies in human sera than the antigens made from bacteria, as measured by enzyme-linked immunosorbent assay. The data suggest that the soluble 62-kDa protein made from insect cells contains additional epitopes not present in recombinant proteins made from bacteria. Therefore, the 62-kDa protein may be useful for HEV diagnostic improvement and vaccine development. The reengineered construct allows for the consistent large-scale production of the soluble 62-kDa protein without proteolytic processing.
Zhang
Y
Y
Genelabs Technologies, Inc., Redwood City, California 94063, USA.
McAtee
P
P
Yarbough
P O
PO
Tam
A W
AW
Fuerst
T
T
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002213:Capsid / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002478:Cells, Cultured; D010450:Endopeptidases; D015967:Gene Expression Regulation, Viral / Q000276:immunology; D016752:Hepatitis E virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D007075:Immunoglobulin M / Q000276:immunology; D016366:Open Reading Frames / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D012995:Solubility; D018411:Spodoptera; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D007075:Immunoglobulin M; D011994:Recombinant Proteins; D014764:Viral Proteins; D010450:Endopeptidases