Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2001-12-21
2003-10-28
Housel, James (Department: 1648)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S005000, C435S069100, C536S023100, C536S023200, C536S023400, C536S023720
Reexamination Certificate
active
06639053
ABSTRACT:
This application is a 371 of PCT/JP99/03381, filed Jun. 24, 1999.
TECHNICAL FIELD
The present invention relates to an RNA polymerase gene derived from hepatitis C virus (referred to as “HCV” herein), a method of screening using this gene or this RNA polymerase protein, and a substance able to be isolated by this screening method.
PRIOR ART
Generally known viral hepatitis includes hepatitis A which is mainly orally transmitted, and hepatitis B transmitted by means of the blood. Moreover, apart from these hepatitis, there is hepatitis called non-A, non-B hepatitis which is transmitted by means of blood transfusion. Since most of these infected with non-A, non-B hepatitis become chronic, and the incidence of development into cirrhosis and hepatoma is high, this is one disease for which the establishment of a certain means of treatment is urgently sought.
Through the causative agent of non-A, non-B hepatitis had been unclear for a long time, recently the causative virus was isolated by M. Houghton et al. (Japanese Patent Application Laid-Open (Kohyo) No. 2-500880), and was termed “HCV”. HCV is a single-stranded RNA virus belonging to the Flavivirdae, the length of its whole genomic RNA is about 9.4 kb. The genomic RNA is divided into 7 regions; core, E1, E2/NS1, NS2, NS3, NS4, and NS5; and the genes related to virus growth, etc. are primarily included in downstream regions from NS3.
HCV RNA polymerase is related to the transcription and replication of genomic RNA, and plays an important role in the reproduction of HCV. The gene encoding this polymerase is thought to be included in the above-mentioned NS5 region (Z. H. Yuan et al., Biochemical and Biophysical Research Communications 232, 231-235(1997), S. B. Hwang et al., Virology 227, 439-446(1997), S. E. Behrens et al., The EMBO Journal 15 12-22(1996)).
The Problem to Be Solved by the Invention
If the gene encoding HCV RNA polymerase can be isolated, it will become possible using this gene to easily screen for substances inhibiting RNA polymerase, and contribute greatly to the development of drugs for treating HCV. However, at present, although the nucleotide sequence of a portion of the NS5 region has been clarified (Japanese Patent Application Laid-Open (Kokai) No. 6-225770), the entire nucleotide sequence of the RNA polymerase gene has yet to be clarified.
The object of the present invention is to isolate the gene encoding the full length of HCV-derived RNA polymerase, to determine its nucleotide sequence, as well as to establish its expression system.
A further object of the present invention is to provide a screening method for a substance which inhibits the activity of this gene or this protein employing this gene or this RNA polymerase protein.
Means for Solving the Problem
In order to solve the above problem, the present inventors, as result of deliberate and focused research have succeeded in isolating the gene encoding the full-length of HCV-derived RNA polymerase, thereby completing the present invention.
That is to say, the present invention relates to the following (1) to (3).
(1) A gene encoding the following protein (a) or (b):
(a) a protein consisting of the amino acid sequence represented in SEQ ID NO:2;
(b) a protein consisting of an amino acid sequence derived from the amino acid sequence represented in SEQ ID NO:2 by deletion, substitution or addition of one or several amino acid(s), and which has RNA polymerase activity.
(2) A method of screening a substance which inhibits the activity of the gene of (1) above, or of the protein consisting of the amino acid sequence represented in SEQ ID NO: 2, wherein this method comprises the following steps:
(a) a step of contacting the gene of (1) above or the protein consisting of the amino acid sequence represented in SEQ ID NO: 2, or a fragment of this protein, with a test sample; and,
(b) a step of selecting a substance which inhibits the activity of the gene of (1) above, or of the protein or the partial peptide fraction consisting of the amino acid sequence represented in SEQ ID NO: 2.
(3) A substance able to be isolated by the method of (2) above, wherein this substance inhibits the activity of the gene of (1) above or of the protein consisting of the amino acid sequence represented in SEQ ID NO: 2.
The descriptions contained in the specification of Japanese Patent Application No. 10-177817, which forms the basis of the right of priority of the present application, are incorporated herein in their entirety.
DISCLOSURE OF THE INVENTION
Below, the present invention will be explained in detail.
The gene of the present invention encodes (a) a protein consisting of the amino acid sequence represented in SEQ ID NO: 2; or, (b) a protein consisting of an amino acid sequence derived from the amino acid sequence represented in SEQ ID NO:2 by deletion, substitution, or addition of one or several amino acid(s) and having RNA polymerase activity.
The deletion, etc. of one or several amino acid can be performed by techniques in common use at the time of filing this application, such as, for example, site-specific mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1982).
The gene of the present invention is able to be obtained from the blood of non-A, non-B hepatitis patients as described in the examples, or from the strain of
E. coli
into which a vector (pCALN/HCV RBZ) comprising the gene of the present invention was introduced, has been deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-1-3, Higashi, Tsukuba-shi, Ibaraki-ken, Japan) (Accession No. FERM BP-6763) on Oct. 31, 1997).
Further, the present invention relates to a screening method for a substance which inhibits the activity of this gene or this protein, employing the gene of the present invention or an RNA polymerase protein consisting of the amino acid sequence represented in SEQ ID NO: 2; and, to a substance able to be isolated by this screening method employing this gene or this RNA polymerase protein.
The RNA polymerase encoded by the gene of the present invention is an enzyme involved in the transcription and replication of HCV genomic RNA. Therefore, a substance which inhibits this enzyme is thought to be able to prevent the reproduction of HCV, and is promising as a drug for treating non-A, non-B hepatitis. By using the gene of the present invention it will be possible to produce HCV-derived RNA polymerase easily and in great quantities, and as a result of this, the screening of inhibitory substances for the RNA polymerase will become simpler.
The protein of the present invention that can be used for screening can be either a recombinant type, a wild type, or a partial peptide. Further it can be a purified peptide or a partial peptide thereof.
One embodiment of this method of screening comprises the steps of (a) contacting the gene of the present invention or the protein consisting of the amino acid sequence represented by SEQ ID NO: 2, or a fragment of this protein, with a test sample; and, (b) selected a substance which inhibits the activity of the gene of the present invention or the protein consisting of the amino acid sequence represented by SEQ ID NO: 2. There is no particular limitation on what can be used as a test material in this screening method but for example, a cell extract, a cell culture supernatant, a protein, a peptide, or synthetic low molecular weight compound can be used.
REFERENCES:
patent: 6-225770 (1994-08-01), None
patent: 10-507370 (1998-07-01), None
patent: 11-514862 (1999-12-01), None
patent: WO 96/37619 (1996-11-01), None
Sequence comparison sheet with SEQID No:2 of WO 96/37619.*
Behrens, et al.; “Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus” The Embo Journal; vol. 15, No. 1, 1996; pp. 12-22.
Hwang, et al.; “Hepatitis C virus NS5B protein is a membrane-associated phosphoprotein with a predominantly perinuclear localization” Virology; vol. 227, No. 2, 1997; pp. 439-446.
Al, R.H. et al., “Expression of Recombinant Hepatitis C Virus Non-Structural Protein 5B inEscherichia coli”
Higashi Kazuhiro
Kohara Kyoko
Kohara Michinori
Toyoda Tetsuya
Tsuchiya Masayuki
Davidson, Davidson & Kappel
Hill Myron G.
Housel James
Toyoda Tetsuya
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