Nucleic acid and gene derived from novel HCV strain and replicon-replicating cell using said gene
View Patent ↗The present invention relates to a gene derived from a novel fulminant hepatitis C virus strain, an HCV replicon RNA with a high replication efficiency obtained using the gene, and an HCV replicon-replicating cell transfected with the replicon RNA. When the HCV replicon RNA and the HCV replicon-replicating cell of the present invention are used, HCV proteins can be continuously produced in a large amount.
1. An isolated nucleic acid comprising:
(a) the nucleotide sequences shown by the 5′ untranslated region of SEQ ID NO:3, the nucleotide sequence encoding NS3, NS4A, NS4B, NS5A and NS5B proteins from nucleotides 1-6012 of SEQ ID NO:1 and the 3′ untranslated region of SEQ ID NO:4; or
(b) an entire nucleotide sequence according to (a) one or a plurality of nucleotide substitutions which functions as an RNA replicon, wherein said nucleotide substitutions reflect degeneracy in the genetic code;
provided that, if the nucleic acid is a DNA, the nucleotide symbol “u” in the sequence listings shall be replaced with “t”.
2. A replicon RNA comprising, in order, the nucleotide sequences shown by the 5′untranslated region of SEQ ID NO:3, the nucleotide sequence encoding NS3, NS4A, NS4B, NS5A and NS5B proteins from nucleotides 1-6012 of SEQ ID NO:1 and the 3′ untranslated region of SEQ ID NO:4.
3. The replicon RNA according to claim 2 , further comprising an IRES sequence.
4. The replicon RNA according to claim 3 , further comprising a selection marker gene or a reporter gene.
5. A replicon RNA consisting of (a) or (b):
(a) the nucleotide sequence shown by SEQ ID NO:7; or
(b) an entire nucleotide sequence according to (a) one or a plurality of nucleotide substitutions which has an autonomous replication ability, wherein said nucleotide substitutions reflect degeneracy in the genetic code.
6. A replicon-replicating cell created by introducing the replicon RNA according to any one of claims 2 to 5 into isolated cells.
7. The replicon-replicating cell according to claim 6 , wherein the isolated cells are human liver-derived cells, human cervix-derived cells, or human embryonic kidney-derived cells.
8. A method for screening a substance to stimulate or suppress replication of hepatitis C virus, comprising culturing the replicon-replicating cell according to claim 6 in the presence of a test substance and detecting replication of a replicon RNA in the resulting culture.
9. The replicon RNA according to claim 3 , further comprising a selection marker gene.