JP2000078985A - Hcv-derived rna polymerase gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new RNA polymerase gene derived from the hepatitis C virus (HCV), having a specific amino acid sequence, encoding a protein having RNA polymerase activity and useful for screening substances inhibiting the RNA polymerase. SOLUTION: This new RNA polymerase gene derived from the hepatitis C virus encodes a protein having an amino acid sequence of the formula, or a protein allowed to include alterations selected from the group consisting of at least one amino acid deletion, substitution or addition and having RNA polymerase activity. The RNA polymerase is an enzyme playing the important role of the multiplication of HCV, can easily screen substances inhibiting the RNA polymerase and can contribute the development of remedy for HCV. The gene is obtained by extracting RNA from blood of non-A, non-B hepatitis patients, by synthesizing cDNA using the RNA, by conducting PCR using the cDNA as a template with primers comprising partial sequences.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an RNA polymerase gene derived from hepatitis C virus (hereinafter referred to as "HCV"), a screening method using the gene or the RNA polymerase protein, and an RNA polymerase gene isolated by the screening method. Substances.

[0002]

2. Description of the Related Art As viral hepatitis, hepatitis A, which is mainly transmitted orally, and hepatitis B, which is transmitted through blood, are generally known, but non-A which is transmitted via blood transfusion separately from these. There is also hepatitis called non-B hepatitis. Non-A
Non-hepatitis B is one of the diseases for which it is urgent to establish a reliable treatment because most infected people become chronic and the rate of transition to liver cirrhosis and liver cancer is high.

[0003] The causative factor of non-A, non-B hepatitis has been unknown for a long time, but the causative virus was isolated by M. Houghton et al. (Japanese Patent Publication No. 2-500880) and named HCV. HCV
Is a single-stranded RNA virus belonging to the Flaviviridae family, and its genomic RNA is about 9.4 kb in total length. Genomic RNA
Is divided into seven regions: core, E1, E2 / NS1, NS2, NS3, NS4, and NS5, and genes involved in virus growth and the like are mainly contained in the region downstream from NS3.

[0004] HCV RNA polymerase is an enzyme that is involved in the transcription and replication of genomic RNA and plays an important role in the growth of HCV. The gene encoding it is thought to be included in the NS5 region described above. (ZHYuan et al., Biochemica
l and Biophysical Research Communications 232, 231
-235 (1997), SBHwang et al., Virology 227, 439-446 (19
97), SEBehrens et al., The EMBO Journal 15 12-22 (199
6)).

[0005]

If the gene encoding HCV RNA polymerase can be isolated, it can be used to
Inhibitors to RNA polymerase can be easily screened, which can greatly contribute to the development of HCV therapeutic agents. At present, although a partial nucleotide sequence of the NS5 region has been elucidated (JP-A-6-225770) No.),
The entire nucleotide sequence of the RNA polymerase gene has not yet been elucidated.

[0006] It is an object of the present invention to isolate a gene encoding the full length of HCV-derived RNA polymerase, determine its nucleotide sequence, and establish an expression system. It is a further object of the present invention to provide a method for screening a substance that inhibits the activity of the gene or the protein, using the gene or the RNA polymerase protein.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems, and as a result, succeeded in isolating the gene encoding the full length of HCV-derived RNA polymerase, and completed the present invention. Reached. That is, the present invention provides the following (1)
To (3). (1) A gene encoding the following protein (a) or (b): (a) a protein comprising the amino acid sequence of SEQ ID NO: 2; (b) a protein comprising the amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids have been deleted, substituted or added; Protein.

(2) A method for screening a substance that inhibits the activity of the gene described in (1) or the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, wherein (a)
(B) contacting a test sample with the gene or the protein comprising the amino acid sequence of SEQ ID NO: 2 or a partial peptide thereof, (b) the gene of (1) or the amino acid of SEQ ID NO: 2; Selecting a substance that inhibits the activity of a protein consisting of a sequence or a partial peptide thereof. (3) (1) which can be isolated by the method described in (2).
A substance that inhibits the activity of the gene consisting of the gene described in (1) or the amino acid sequence represented by SEQ ID NO: 2.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The gene of the present invention comprises (a) a protein having the amino acid sequence of SEQ ID NO: 2, or (b) an amino acid having one or more amino acids deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 Consists of an array, RN
Encodes a protein with A polymerase activity.

Deletion of one or more amino acids, etc.
Techniques commonly used at the time of filing the present application, such as site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1
982). The gene of the present invention can be obtained from RNA in the blood of a non-A non-B hepatitis patient as described in Examples, but Escherichia coli introduced with a vector (pCALN / HCV RBZ) containing the gene of the present invention Since it has been deposited at the National Institute of Advanced Industrial Science and Technology,
RM BP-6763, date of deposit: October 31, 1997), and can also be obtained from the strain.

[0011] The present invention also provides a method for screening a substance that inhibits the activity of the gene or the protein using the RNA polymerase protein comprising the gene of the present invention or the amino acid sequence of SEQ ID NO: 2,
And a substance that can be isolated by the screening method. 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, substances that inhibit this enzyme are considered to prevent the growth of HCV, and are therefore promising as therapeutic agents for non-A non-B hepatitis. By utilizing the gene of the present invention,
HCV-derived RNA polymerase can be produced easily and in large quantities, and accordingly, the search for inhibitors can be facilitated.

The protein of the present invention used for screening may be any of a recombinant type, a natural type and a partial peptide. Further, it may be a purified protein or a partial peptide thereof. One embodiment of this screening method comprises: (a) a step of contacting a test sample with a gene of the present invention or a protein consisting of the amino acid sequence of SEQ ID NO: 2 or a partial peptide thereof; (b) a gene or sequence of the present invention. Selecting a substance that inhibits the activity of the protein consisting of the amino acid sequence of No. 2 or a partial peptide thereof. The test sample used in this screening method is not particularly limited, and includes, for example, a cell extract, a cell culture supernatant, a protein, a peptide, and a synthetic low-molecular compound.

[0013]

Example (1) Isolation of HCV RNA from Hepatitis Patient Blood and Amplification of the Isolated RNA by PCR RNA was extracted from the blood of a non-A non-B hepatitis patient by the guanidine thiocyanate and phenol / chloroform methods. RT-PCR according to the method described in JP-225770-cD
I got NA. Using this cDNA as a template, PCR was performed using primers described in JP-A-6-225770 (Science 230:
1350 (1985)), and the four amplified fragments (C6-62 region, C
6-66, C6-79, C6-82). These amplified fragments were cloned using the cloning vector pBM, and Sanger dideoxy chain termination method (Science, 214, 120
5 (1981)), the nucleotide sequence of the amplified fragment was determined. In addition,
The cloning vector pBM is a vector constructed in consideration of the nature of the HCV gene into which mutations are liable to be introduced during replication and cloning so that mutations are unlikely to occur (Japanese Patent Application Laid-Open No. 6-225770). Determine the position of each amplified fragment by comparing the homology between the amplified fragment in the clone obtained by the above method and the previously reported non-A non-B hepatitis virus gene,
Each was ligated by PCR, and the desired DNA fragment (amplified DNA) was cloned into a vector. Using this cloning vector, pCALN / HCV RBZ was prepared.

(2) Determination of the nucleotide sequence of the fragment encoding NS5B Using pCALN / HCV RBZ as a template, the fragment encoding NS5B was amplified by PCR using the following primers. PCR primer NS5B1 5'-ATC CCT CGA GAT GTC CTA CAC ATG GAC
AGG-3 (SEQ ID NO: 3) NS5B2 5'-TAT GGA TCC AAG CTT C
AC CGG TTG GGG AGC AGG T-3 '(SEQ ID NO: 4)

[0015] The reaction solution was placed in a 0.5 ml tube with 10X PCR buffe.
r II (500 mM KCl, 100 mM Tris-HCl pH 8.3, 15 mM MgCl ₂ )
10 μl, 1.25 mM dNTP 16 μl, 20 μM 2 primers
(NS5B1, NS5B2) 5 μl each, 1 unit / μl AmpliTaq DNA
Add 0.5 μl of Polymerase (PERKIN ELMER) and add 10
It was adjusted to 0 μl. Temperature conditions were as follows: first, heating at 95 ° C for 5 minutes, followed by 25 cycles of denaturation at 95 ° C for 1 minute, annealing at 55 ° C for 1 minute, and extension at 72 ° C for 3 minutes.
It was set to hold for minutes. A part of the reaction completed solution was subjected to agarose gel electrophoresis to confirm a specifically amplified DNA fragment.

After purifying this DNA fragment according to the method of Gene Clean (Biolol), it was digested with XhoI and then with BamHI. The digestion reaction end solution is subjected to agarose gel electrophoresis,
The target DNA fragment was extracted from the gel, and the concentration of DNA in the extract was measured. The cloning vector pET-15b (Novagen) was similarly digested with XhoI and BamHI and purified. The digestion reaction end solution was subjected to agarose gel electrophoresis, the target DNA fragment was extracted from the gel, and the concentration of DNA in the extract was measured.

For the above two types of gel-extracted DNA fragments, DN
A ligation reaction was performed according to the method of A ligation Kit ver. 2 (Takara), and Escherichia coli was transformed using a part of the reaction solution. The resulting transformant was plated on an LB-Amp plate (1%
(Bactotripton, 0.5% yeast extract, 1% sodium chloride, 1.4% agar, ampicillin 100μg / ml) overnight, then colonies appeared on the plate 2m each
l LB-Amp medium (1% bactotryptone, 0.5% yeast extract, 1
% Sodium chloride and ampicillin (100 μg / ml) in a tube (37 ° C., 16 hours), and the culture was centrifuged to collect the bacteria. The plasmid DNA was mini-prepared to prepare a DNA solution. This was digested with XhoI and BamHI, and the enzyme reaction completed solution was subjected to agarose gel electrophoresis to obtain a clone into which the target DNA fragment was inserted. Transfer this clone to pET-15b HCV po
Named l.

The clone pET-15b HCV pol obtained above
Was digested with XbaI and BamHI, and the resulting enzyme reaction solution was subjected to agarose gel electrophoresis to extract a target DNA fragment. Similarly, the baculovirus transfer vector pVL1392 (Ph
armigen) was digested with XbaI and BamHI, and the resulting enzyme reaction solution was subjected to agarose gel electrophoresis to extract a target DNA fragment.

By performing a ligation reaction on the above two types of gel-extracted DNA fragments, the NS5B gene in which 6 × His was tagged at the N-terminal was inserted into pVL1392. Escherichia coli was transformed using a part of the ligation reaction solution. The resulting transformant was cultured overnight on an LB-Amp plate (1% bactotryptone, 0.5% yeast extract, 1% sodium chloride, 1.4% agar, 100 μg / ml ampicillin), and then the colonies that appeared on the plate Each in 2 ml LB-Amp medium (1%
(Bactotryptone, 0.5% yeast extract, 1% sodium chloride, ampicillin 100μg / ml)
(37 ° C., 16 hours), the culture was centrifuged to collect bacteria, and the plasmid DNA was mini-prepared to prepare a DNA solution. This was digested with XbaI and BamHI, and the enzyme-finished solution was subjected to agarose gel electrophoresis to obtain a clone into which the target DNA fragment was inserted. This clone was named pVL1392 HisHCV pol. Baculo gold using pVL1392 His HCV pol
(Pharmigen) recombinant baculovirus (BacHisHCVp
ol). Baculovirus production is Baculovirus
Expression Vector System: Procedures and Methods
Performed according to the description of Manual (Pharmigen).

(3) Expression and Purification of RNA Polymerase BacHisHCVpol was used to infect sf21 AE cells with moi = 1 or 2 and the cells were recovered 3 days (72 hours after) infection with 10 mM Tris / HCl
pH 7.9, 0.5 M NaCl, 1.5 mM MgCl ₂ , 7 mM 2-ME, 0.1% Tri
ton X-100, 25% glycerol, 1 mM PMSF, 10 μg / ml leupep
Extracted with a buffer containing tin. First, extract the whole cell extract with N
Pass through an i-NTA column (QIAGEN, 60 mM imidazole), then
Purification was performed by PLC (MonoQ column, manufactured by Pharmacia). For MonoQ columns, RNA polymerase is 0.1-1.0 M NaCl gradien
It was eluted with a 0.5-0.6M NaCl fraction over time. (4) Confirmation of RNA polymerase activity Prior to measurement of HCV RNA polymerase activity, HCV-specific RNA was prepared as a template for the polymerase reaction to determine the optimal concentration of magnesium acetate and KCl.

HCV cD downstream from the PvuII site (9240) of HCV
NA was cloned into Bluescript KSII (+) and this vector was named HCV RNA-9610. The prepared cloning vector Bluescript KSII (+) HCV RNA-9610 was converted to DraI, Pst
After digesting with I, NheI, etc., and purifying, it was subjected to an in vitro transcription reaction using T7 RNA polymerase. Synthesized RNA
Have different lengths due to the different restriction enzymes used, such as DraI, PstI, and NheI (377 for DraI).
nts, 340 nts for PstI and 305 nts for NheI). Each RNA was converted to HCV RNA-9610, HCV RNA-9576, H
It was named CV RNA-9541. HCV RNA-9610 has two extra uracils at the 3 'end.

Using the above HCV RNA as a template, the optimum concentration of magnesium acetate in the HCV RNA polymerase activity measuring system was determined. 5 μl of HCV RNA polymerase (MonoQ column fraction) is buffered (20 mM HEPES / KOH pH7.6, 50 mM KC
l, 1 mM DTT, 25 μg / ml actinomycin D, 0.5 mM ATP, CTP,
GTP, 50 μM UTP, 5 μCi [α- ³² P] UTP (15 TBq / mmol, Amer
sham), 50 μl of 10 pmole HCV RNA-9541, 400 U / ml RNase inhibitor (TOYOBO), and the magnesium acetate concentration in the solution was set in 11 steps within a range of 0-10 mM, 29 ° C., and 1.
Incubated for 5 hours. Labels synthesized at each concentration of magnesium acetate were electrophoresed on 4% PAGE / 6M urea, and PSL of the synthesized product was measured with BAS. As a result, the optimal concentration of magnesium acetate was 3-4 mM.

Similarly, the HCV RNA polymerase activity measurement system
The optimal KCl concentration was determined. Here, HCV R
PolyA was used instead of NA. 10 μl HCV RNA polymerase
Buffer fraction (20 mM HEPES / KOH pH7.6, 5 mM acetic acid
Magnesium, 1 mM DTT, 25 μg / ml actinomycin D, 10
μMUTP, 2.5 μCi [α-³²P] UTP (15 TBq / mmol, Amersha
m), 10 μg / ml poly A (Pharmacia), 100 μM UpU (Sigm
a), added to 50 μl of 400 U / ml RNase inhibitor (Takara)
The KCl concentration is set in 10 steps within the range of 20-200 mM, and
The UMP uptake in degrees was measured. The measurement itself is
First, [α-^{Three Two}P] UMP precipitated in 10% TCA
And collect it on a glass filter (GF / C, Whatman).
It can be measured with a chelation counter (Aloka)
It is. Using polyA as template and in the presence of UpU primer
The optimal KCl salt concentration in the system where the polymerase activity was observed was 100
mM.

Further, the optimal concentration of KCl in the HCV RNA polymerase activity measuring system when HCV RNA-9541 was used as a template was determined. The reaction volume was 5 μl of HCV RNA polymerase.
And the magnesium acetate concentration to 3.5 mM, 10 pmole HC
Except that V RNA-9541 was used as a template, it was the same as the experimental system in which the optimal concentration of magnesium acetate was determined. The magnesium acetate concentration is set in 9 steps within the range of 50-200 mM. As a result of the measurement, the optimum KCl concentration was 50 mM. Here, it was inferred that the difference in the optimum KCl concentration was due to the secondary structure of the template.

(5) Inhibition of UMP Uptake of HCV RNA Polymerase by Rabbit Anti-HCV RNA Polymerase Antibody NS5B (HCV RNA polymerase) in pET-15b HCV pol was expressed in Escherichia coli and purified with a Ni-NTA (QIAGEN) column. Using this protein as an antigen, rabbits are immunized and antibodies (anti-HCVpo
l) was prepared. First, 20 μl of HCV RNA polymerase purified by Ni-NTA was incubated with a rabbit anti-HCV RNA polymerase antibody IgG at a final concentration of 3.1, 6.3, 12.5, 25, 50 μg / ml or a normal rabbit antibody IgG as a control at room temperature for 30 minutes. . The composition of the buffer used at that time was 20 mM HEPES / KOH pH7.6, 100 mM KCl.

After the incubation, the HCV RNA polymerase solution contained 3 mM magnesium acetate, 1 mM DTT, 25 μg
/ ml actinomycin D, 10 μM UTP (Pharmacia), 2.5 μCi
[α- ³² P] UTP (15 TBq / mmol, Amersham), 10 μg / ml poly
A (Pharmacia), 100 μM UpU (Sigma), 400 U / ml Prime RNa
It was re-prepared to have the composition of se inhibitor (5'-3 'Inc.) and incubated at 29 ° C for 1.5 hours. The incorporated [α- ³² P] UMP was precipitated in 10% TCA, and the glass filte
r (GF / C, Whatman) and measured on a liquid scintillation counter (Aloka). As a result, rabbit anti-HCV R
Inhibition of UMP uptake of HCV RNA polymerase by NA polymerase antibody was observed.

[0027]

Industrial Applicability The present invention provides a novel RNA polymerase gene derived from HCV, and a screening method using the gene or the RNA polymerase protein. By using this gene, it is possible to easily search for an inhibitor against RNA polymerase.

[0028]

[Sequence List] SEQUENCE LISTING <110> CHUGAI SEIYAKU KABUSHIKI KAISHA TOKYO METROPOLITAN ORGANIZATION FOR MEDICAL RESEARCH Tetsuya Toyoda International Reagents Corporation <120> An RNA polymerase gene derived from HCV <130> P99-0350 <160> 4 <170> Patent In version 2.0 <210> 1 <211> 1773 <212> DNA <213> HCV <220> <221> CDS <222> (1) ... (1773) <400> 1 tca atg tcc tac aca tgg aca ggc gcc ttg atc acg cca tgc gcc gcg 48 Ser Met Ser tyr Thr trp Thr Gly Ala Leu Ile Thr Pro Cys Ala Ala 1 5 10 15 gag gaa agc aag ttg ccc atc aac ccg ttg agc aac tct ttg ttg cgt 96 Glu Glu Ser Lys Leu Pro Ile Asn Pro Leu Ser Asn Ser Leu Leu Arg 20 25 30 cac cac aac atg gtc tat gct aca aca tcc cgc agc gca ggc cta cgg 144 His His Asn Met Val tyr Ala Thr Thr Ser Arg Ser Ala Gly Leu Arg 35 40 45 cag aag aag gtc acc ttt gac aga ctg caa gtc ctg gac gac cac tac 192 Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu Asp Asp His tyr 50 55 60 cgg gac gtg ctc aag gag atg aag gcg aag gcg tcc aca gtt gct 240 Arg Asp Val Leu Lys Glu Met Lys Ala Lys Ala Ser Thr Val Lys Ala 65 70 75 80 aaa ctc cta tcc ata gaa gaa gcc tgt aag ctg acg ccc cca cat tcg 288 Lys Leu Leu Ser Ile Glu Glu Ala Cys Lys Leu Thr Pro Pro His Ser 85 90 95 gcc aga tcc aaa ttt ggc tat ggg gca aag gac gtc cgg aac cta tcc 336 Ala Arg Ser Lys Phe Gly tyr Gly Ala Lys Asp Val Arg Asn Leu Ser 100 105 110 agc aag gcc gtt aac cac atc cgc tcc gtg tgg aag gac ttg ctg gaa 384 Ser Lys Ala Val Asn His Ile Arg Ser Val trp Lys Asp Leu Leu Glu 115 120 125 gac act gag aca cca att gac acc acc gtc atg gca aaa agt gag gtt 432 Asp Thr Glu Thr Pro Ile Asp Thr Thr Val Met Ala Lys Ser Glu Val 130 135 140 ttc tgc gtc caa cca gag aaa gga ggc cgc aag cca gct cgc ctt atc 480 Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile 145 150 155 160 gta ttc cca gac ttg ggg gtt cgt gta tgc gag aag atg gcc ctt tat 528 Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu tyr 165 170 175 gac gtg gtc tcc acc ctt cct cag gcc gtg atg ggc tcc tca tac gga Asp Val Val Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser tyr Gly 180 185 190 ttc cag tac tcc cct gga cag cgg gtc gag ttc ctg gtg aat gcc tgg 624 Phe Gln tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Asn Ala trp 195 200 205 aaa tca aag aaa tgc cct atg ggc ttt tca tat gac acc cgc tgt ttt 672 Lys Ser Lys Lys Cys Pro Met Gly Phe Ser tyr Asp Thrr Arg Cys Phe 210 215 220 gac tcg aca gtc act gag agt gac atc cgt gtt gag gag tca att tac 720 Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Val Glu Glu Ser Ile tyr 225 230 235 240 caa tgt tgt gac ttg gcc ccc gaa gcc aga cag gcc ata aag tcg ctc 768 Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala Ile Lys Ser Leu 245 250 255 aca gag cgg ctt tac att ggg ggt ccc ctg acc aat tca aaa ggg cag 816 Thr Glu Arg Leu tyr Ile Gly Gly Pro Leu Thr Asn Ser Lys Gly Gln 260 265 270 aac tgt ggc tat cgc cgg tgc cgc gcg agt ggc gtg ctg acg acc agc 864 Asn Cys Gly tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser 275 280 285 285 tgc ggt aat acc ctt aca tgt tac ttg aag gcc tct gca 912 Cys Gly Asn Thr Leu Thr Cys tyr Leu Lys Ala Ser Ala Ala Cys Arg 290 295 300 gct gca aag ctc cgg gac tgc acg atg ctc gtg aac gga gac gac ctc 960 Ala Ala Lys Leu Arg Asp Cys Thr Met Leu Val Asn Asp Asp Leu 305 310 315 320 gtc gtc atc tgt gag agt gcg gga acc caa gag gat gag gcg aac cta 1008 Val Val Ile Cys Glu Ser Ala Gly Thr Gln Glu Asp Glu Ala Asn Leu 325 330 335 cga gtc ttc acg gag gct act agg tat tct gcc ccc ccc ggg gac 1056 Arg Val Phe Thr Glu Ala Met Thr Arg tyr Ser Ala Pro Pro Gly Asp 340 345 350 ccg ccc cga cca gaa tac gac ttg gag cta ata aca tca tgt tcc tcc 1104 Pro Pro Arg Pro Glu tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser 355 360 365 aat gtg tcg gtc gcg cac gat gca tct ggc aaa agg gta tac tac ctc 1152 Asn Val Ser Val Ala His Asp Ala Ser Gly Lys Arg Val tyr tyr Leu 370 375 380 acc cgc gac ccc tcc acc ccc ctt gca cgg gct gcg tgg gag aca gct 1200 Thr Arg Asp Pro Ser Thr Pro Leu Ala Arg Ala Ala trp Glu Thr Ala 385 390 395 400 400 aga cac act cca gtt aat tcc tgg cta ggc aa c atc att atg tat gcg 1248 Arg His Thr Pro Val Asn Ser trp Leu Gly Asn Ile Ile Met tyr Ala 405 410 415 ccc acc tta tgg gca agg atg att ctg atg acc cat ttc ttc tcc atc 1296 Pro Thr Leu trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile 420 425 430 ctt cta gcc cag gag caa ctt gaa aaa gcc ctg gat tgc cag atc tac 1344 Leu Leu Ala Gln Glu Gln Leu Glu Lys Ala Leu Asp Cys Gln Ile tyr 435 440 440 ggg tgt tac tcc att gag cca ctt gac cta cct cag atc att gaa 1392 Gly Ala Cys tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile Glu 450 455 460 cga ctc cat ggt ctt agc gca ttt tca ctc cat agt tac tct cca g 1440 Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser tyr Ser Pro Gly 465 470 475 480 gag atc aat agg gtg gct tca tgc ctc agg aaa ctt ggg gta cca ccc 1488 Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val Pro Pro 485 490 495 ttg cga gtc tgg aga cat cgg gcc aga agt gtc cgc gct aag ctg ctg 1536 Leu Arg Val trp Arg His Arg Ala Arg Ser Val Arg Ala Lys Leu Leu 500 505 510 tcc cag ggg ggg agg gct gcc act tgt ggt aag tac ctc ttc aac tgg 1584 Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys tyr Leu Phe Asn trp 515 520 525 gca gta agg acc aag ctc aaa ctc act cca atc ccg gca gcg tcc Cag 1632 Ala Val Thr Lys Leu Lys Leu Thr Pro Ile Pro Ala Ala Ser Gln 530 535 540 ttg gac ttg tcc agc tgg ttc gtg gct ggt tac agc ggg gga gac ata 1680 Leu Asp Leu Ser Ser trp Phe Val Ala Gly tyr Ser Gly Gly Asp Ile 545 550 555 560 tat cac agc ctg tct cgt gcc cga ccc cgc tgg ttc atg ttg tgc cta 1728 tyr His Ser Leu Ser Arg Ala Arg Pro Arg trp Phe Met Leu Cys Leu 565 570 575 ctc cta ctt tca gta ggg gta gg gg ctc ccc aac cga 1773 Leu Leu Leu Ser Val Gly Val Gly Ile tyr Leu Leu Pro Asn Arg 580 585 590 <210> 2 <211> 591 <212> PRT <213> HCV <400> 2 Ser Met Ser tyr Thr trp Thr Gly Ala Leu Ile Thr Pro Cys Ala Ala 1 5 10 15 Glu Glu Ser Lys Leu Pro Ile Asn Pro Leu Ser Asn Ser Leu Leu Arg 20 25 30 His His Asn Met Val tyr Ala Thr Thr Ser Arg Ser Ala Gly Leu Arg 35 40 45 Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu Asp Asp His tyr 50 55 60 Arg Asp Val Leu Lys Glu Met Lys Ala Lys Ala Ser Thr Val Lys Ala 65 70 75 80 Lys Leu Leu Ser Ile Glu Glu Ala Cys Lys Leu Thr Pro Pro His Ser 85 90 95 Ala Arg Ser Lys Phe Gly tyr Gly Ala Lys Asp Val Arg Asn Leu Ser 100 105 110 Ser Lys Ala Val Asn His Ile Arg Ser Val trp Lys Asp Leu Leu Glu 115 120 125 Asp Thr Glu Thr Pro Ile Asp Thr Thr Val Met Ala Lys Ser Glu Val 130 135 140 Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile 145 150 155 160 Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu tyr 165 170 175 Asp Val Val Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser tyr Gly 180 185 190 Phe Gln tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Asn Ala trp 195 200 205 Lys Ser Lys Lys Cys Pro Met Gly Phe Ser tyr Asp Thr Arg Cys Phe 210 215 220 Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Val Glu Glu Ser Ile tyr 225 230 235 240 Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala Ile Lys Ser Leu 245 250 255 Thr Glu Arg Leu tyr Ile Gly Gly Pro Leu Thr A sn Ser Lys Gly Gln 260 265 270 Asn Cys Gly tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser 275 280 285 Cys Gly Asn Thr Leu Thr Cys tyr Leu Lys Ala Ser Ala Ala Cys Arg 290 295 300 Ala Ala Lys Leu Arg Asp Cys Thr Met Leu Val Asn Gly Asp Asp Leu 305 310 315 320 Val Val Ile Cys Glu Ser Ala Gly Thr Gln Glu Asp Glu Ala Asn Leu 325 330 335 Arg Val Phe Thr Glu Ala Met Thr Arg tyr Ser Ala Pro Pro Gly Asp 340 345 350 Pro Pro Arg Pro Glu tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser 355 360 365 Asn Val Ser Val Ala His Asp Ala Ser Gly Lys Arg Val tyr tyr Leu 370 375 380 Thr Arg Asp Pro Ser Thr Pro Leu Ala Arg Ala Ala trp Glu Thr Ala 385 390 395 400 Arg His Thr Pro Val Asn Ser trp Leu Gly Asn Ile Ile Met tyr Ala 405 410 415 Pro Thr Leu trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile 420 425 430 Leu Leu Ala Gln Glu Gln Leu Glu Lys Ala Leu Asp Cys Gln Ile tyr 435 440 445 Gly Ala Cys tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile Glu 450 455 460 Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser t yr Ser Pro Gly 465 470 475 480 480 Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val Pro Pro 485 490 495 Leu Arg Val trp Arg His Arg Ala Arg Ser Val Arg Ala Lys Leu Leu 500 505 510 Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys tyr Leu Phe Asn trp 515 520 525 Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Pro Ala Ala Ser Gln 530 535 540 Leu Asp Leu Ser Ser trp Phe Val Ala Gly tyr Ser Gly Gly Asp Ile 545 550 555 560 tyr His Ser Leu Ser Arg Ala Arg Pro Arg trp Phe Met Leu Cys Leu 565 570 575 Leu Leu Leu Ser Val Gly Val Gly Ile tyr Leu Leu Pro Asn Arg 580 585 590 <210> 3 <211> 30 < 212> DNA <213> Artificial Sequence <400> 3 atccctcgag atgtcctaca catggacagg 30 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <400> 4 tatggatcca agcttcaccg gttggggagc aggt 34

Continued on the front page (71) Applicant 000003311 Chugai Pharmaceutical Co., Ltd. 5-5-1 Ukima, Kita-ku, Tokyo (72) Inventor Ohara Michino 1308-19 Tsukazaki, Numaminamicho, Higashi-Katsushika-gun, Chiba Prefecture (72) Inventor Ohara Kyoko 1308-19, Tsukazaki, Numanamachi, Higashi-Katsushika-gun, Chiba Prefecture (72) Inventor Tetsuya Toyoda 1339-4, Tsufukuhoncho, Kurume-shi, Fukuoka (72) Inventor Kazuhiro Higashi 1-1-2 Muroya, Nishi-ku, Kobe-shi, Hyogo Pref. Research and Development Center Co., Ltd. (72) Inventor Masayuki Tsuchiya 1-135 Komamon, Gotemba, Shizuoka Prefecture

Claims (3)

[Claims] 1. A gene encoding the following protein (a) or (b): (a) a protein comprising the amino acid sequence of SEQ ID NO: 2; (b) a protein comprising the amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids have been deleted, substituted or added; Protein 2. A method for screening a substance that inhibits the activity of the gene according to claim 1 or the protein comprising the amino acid sequence according to SEQ ID NO: 2, wherein (a) the method according to claim 1 Contacting a test sample with a protein having the amino acid sequence according to the above or a partial peptide thereof, (b) inhibiting the activity of the gene having the gene according to claim 1 or the protein having the amino acid sequence of SEQ ID NO: 2 or a partial peptide thereof. Selecting a substance. 3. A substance that inhibits the activity of the protein comprising the gene of claim 1 or the amino acid sequence of SEQ ID NO: 2, which can be isolated by the method of claim 2.