| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2003: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
We first cloned human CD81 gene that had been reported to bind to the envelope protein of hepatitis C virus (HCV) by PCR. Then, we subcloned the gene into a stable expression vector and selected COS7 cell clones highly expressing CD81 with G418.
We made 36 large E.coli agar plates, each representing 1x10^4 different clones, and made DNA preparation after overnight culture. After that, we transfected CD81-expressing COS7 cells with library DNA preparations by lipofection. Twenty-four hours later, we added HCV containing patient serum to the culture media. After 4 days, cells were harvested and total RNA was extracted. We examined negative strand RNA of HCV, meaning viral replication, by strand-specific multi-step RT-PCR. There were six positive pools among 36 at the first round screening. We divided the pool by 600 genes into subpools, and continued the screening in the same way. Finally, we obtained 8 positive clones in the final round. However, as far as we checked, all clones seemed false-positive. We now re-try the screening by using retrovirus-based expression library and by more specific RT-PCR with Tth polymerase.
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