| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
The inoculated serum which was used in our previous report contained heterogeneous clones of hepatitis C virus, so called quasi-species. However, we found that Limited species became predominant during the cultured Mr-2C cell system. In order to obtain such a predominant HCV species, we amplified 2 HCV genomic regions, the structure and the non-structure regions, by long RT-PCR for the RNA sample from HCV-infected MT-2C cells 8 days after inoculation. Then these PCR products were inserted into plasmid vector pBR322 leading to a plasmid library. Clones of full-length HCV genomic size were 196, of which 60 were available in the synthesis of HCV RNA of complete size and sufficient quantity. RNA in a set (3 to 5 cloned a set) was transfected into MT-2C cells .In three sets, HCV RNA was detected from media of transfected cells at both 7 and 14 days post-inoculation. Then we identified an individual clone which might have feasibility of infectious clone from three positive sets . Finally we confirmed three infectious clones by cell free transmission. However, the infectivities of these clones were faint.
Thus, in order to obtain a stronger infectivity, we reconstructed a new clone which has a consensus amino acid sequence among the last three clones. This clone was inserted into an expression vector under the CMV promoter. Consequently, we showed the expression of HCV protein, i.e., core, envelope 2, NS3, NS4A and NS5A. It is the first time to demonstrate the expression of HCV proteins using the clone originated from HCV-infected culture cells. By using this clone, we are on the process of performing RNA transfection to MT-2C cells and other cell lines and confirm a definite infectivity.
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