|Budget Amount *help
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
In 1997, in my laboratory, a novel unenveloped virus with a small, covalently closed circular genome of single-stranded DNA of 3.9 kilobases was discovered from the serum of a patient with transfusion-related acute hepatitis of unknown etiology, and named as TT virus (TTV) after the initial of the index patient. The aims of the present study were to obtain recombinant TTV proteins by gene expression and to develop assay systems of TTV-related antigens and antibodies. In the first fiscal year, parts of TTV ORF1 and ORF2 proteins were expressed in E.coli, and purified. Using the expressed proteins as immunogens, two distinct murine monoclonal antibodies (mAbs) were raised against ORF1 protein (anti-ORF1 mAb) and ORF2 protein (anti-ORF2 mAb). In the second fiscal year, antigenic epitopes to which anti-ORF1 mAb and anti-ORF2 mAb are directed, were determined using multipin peptides. These two mAbs were successfully applicable to Western blotting for the detection of TTV proteins. Then, for the purpose of large-scale preparation of TTV proteins, four ORFs (ORF1-ORF4) of TTV were expressed separately or in combinations in silkworms using recombinant baculovirus vectors, and tandem dimers of TTV genome were introduced into cultured hepatoma cell line (HuH7). Unexpectedly, TTV proteins were not yielded in both expression experiments, enough for the establishment of TTV antibody detection system. However, in this study, I could first develop two mAbs against TTV ORF1 and ORF2, both of which are useful for specifically detecting TTV-related antigens.