JPH0998788A - Judging method for effectiveness of therapy to hepatitis c virus of xeno-type 1b and peptide therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for judging an effectiveness of an interferon therapy to a hepatitis C virus(HCV) by examining whether the ISD region of the HCV belonging to a xeno-type 1b in a specimen has specific amino acid sequences or not by an immunological measuring method. SOLUTION: In this method for judging an effectiveness of an interferon therapy to a HCV by examining whether the amino acid sequences of the ISD region consisting of amino acids from 2209th to 2248th of the HCV belonging to a xeno-type 1b in a specimen matches with the amino acid sequences expressed in the formula or not, the examination is conducted by an immunological measurement method utilizing an antigen antibody reaction by using an antibody to an antigen of the ISD region or its partial polypeptide and the ISD region of the HCV in the specimen as the antigen, and the administration of the interferon is judged as the more effective as the amino acid sequences of the ISD region in the specimen becomes the more different from the amino acid sequences of the region expressed by the formula.

Description

Detailed Description of the Invention [Industrial applications]

The present invention relates to a method for determining the effectiveness of treatment against hepatitis C virus belonging to genotype 1b (hereinafter sometimes referred to as "HCV-1b") and a peptide used therefor.

[0002]

BACKGROUND OF THE INVENTION Interferon is one root treatment for chronic hepatitis C. However, even among HCVs belonging to the same genotype 1b, there are effective cases that are completely cured by interferon administration and ineffective cases that are completely ineffective, and intermediate forms thereof exist. Heretofore, as the factors that regulate the susceptibility of HCVs belonging to the same genotype to interferon, the amount of HCV in blood, the degree of histological progression, and the diversity of HCV genes have been suggested, but these are behind these. The underlying cause is unknown.

[0003]

If the susceptibility of HCV-1b to treatment such as interferon administration can be diagnosed, whether or not interferon should be administered can be known, and interferon does not work. This is advantageous because it avoids the wasteful treatment of administering interferon.

Therefore, it is an object of the present invention to provide a method of determining the efficacy of treatment for HCV-1b.

[0005]

As a result of earnest research, the inventors of the present application have found that the 2209th to 22nd HCV-1b are present.
It was found that the sequence of the 48th amino acid, especially the sequence of the 2217th to 2220th amino acids, is closely related to the susceptibility to interferon, and based on this, the susceptibility to interferon is determined by an immunological method. A method was developed and the present invention was completed.

That is, according to the present invention, the amino acid sequence of the ISD region consisting of the 2209th to 2248th amino acids of the hepatitis C virus belonging to genotype 1b in the sample is represented by SEQ ID NO: 1 in the sequence listing. Genotype 1b, consisting of checking for sequence identity
A method for determining the effectiveness of treatment against hepatitis C virus, which comprises using the above-mentioned ISD region or a part of the polypeptide as an antigen and utilizing an antigen-antibody reaction with an antibody against the antigen in a sample. To provide a determination method by the method. The present invention also relates to genotype 1 in the sample.
2209th to 2nd hepatitis C virus belonging to group b
C of genotype 1b, which comprises examining whether or not the amino acid sequence of the ISD region consisting of the 248th amino acid is the same as the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.
A method for determining the effectiveness of treatment against hepatitis B virus, which is carried out by an immunoassay using an antigen-antibody reaction using the ISD region in a sample as an antigen, using an antibody that reacts with the ISD region or a part thereof. Provide a judgment method.
Furthermore, the present invention provides a peptide used in the above-mentioned determination method of the present invention.

The present invention will be described in detail below.

The interferon for which the effectiveness of treatment for HCV-1b is determined by the determination method of the present invention is not particularly limited, and examples thereof include interferon α2a, interferon α2b, interferon beta and natural interferon α. But,
Other drugs (such as antiviral drugs) are also used in the treatment of hepatitis C, and the present invention is not limited to interferon.

The inventors of the present invention determined the amino acid sequence of HCV-1b in serum samples of patients with chronic hepatitis C caused by a large number of HCV-1b, and carefully compared and examined the relationship between these and the effectiveness of interferon administration. As a result, HCV-1b
Amino acids 2209 to 2248 (in this specification, this region is referred to as "ISDR" (InterferonSe
nsitivity Determining Region) or “ISD region”), especially the sequence of the 2217th to 2220th amino acids (herein, this region is referred to as “ISD core region”) is closely related to interferon sensitivity. Therefore, it was found that the efficacy of interferon administration can be determined by examining this area. The amino acid sequence of HCV-1b and its numbering are shown in Kato, N, et al., Proc. Natl. Acad. Sci., 1
990; 87 9524-8).

That is, it was found that in wild-type HCV-1b, the amino acid sequence of the ISDR is the sequence shown in SEQ ID NO: 1 in the sequence listing, and in this case, interferon administration is ineffective (0% efficiency). It was In addition, ISDR
The probability that the interferon administration will be significantly effective as the amino acid sequence of
When 4 or more amino acid residues differed, in the following example in which 70 cases were investigated, the efficiency of interferon treatment reached 100%. When the number of different amino acid residues was 1 to 2 (there was not 3), the efficiency was 13% in the following examples. In another example in which 20 cases were investigated, the number of different amino acid residues was 3 or more, the efficiency was 100%, the efficiency was about 13% in the cases of 1 and 2, and the ineffective rate was 100% in the wild type (the efficiency was 0%). )
It became. The aspect of the difference in amino acid residues referred to here includes not only substitution but also insertion, deletion and termination codon.

Therefore, the ISDR of HCV-1b in the sample
Whether or not the interferon treatment may be effective can be known by examining whether or not the amino acid sequence of is the same as the sequence shown in SEQ ID NO: 1.

Further, an example of a partial sequence of ISDR has been clarified when interferon treatment was significantly effective. These sequences are shown in SEQ ID NOs: 2 to 14 in the sequence listing. Therefore, if the amino acid sequence of ISDR of HCV-1b in the sample is the same as any of the sequences of SEQ ID NOs: 2 to 14, it is determined that the interferon treatment is likely to be significantly effective.

Among the above ISDRs, 4 amino acids from the 2217th position to the 2220th position (this region is referred to as "ISD core region" for convenience in the present specification)
The sequence of was found to be particularly important. When the ISD core region is wild type, that is, in the case of Thr His His Asp (SEQ ID NO: 15), interferon treatment is ineffective, and when the amino acid sequence of the ISD core region is different from this sequence, interferon treatment is It may be effective. Then, as the number of amino acid residues in which the amino acid sequence of the ISD core region differs from that of the wild-type sequence increases, the possibility that interferon treatment will be more effective increases. 5 in total
In the following reference example in which 6 cases were examined, the ineffective rate of interferon treatment was 0% (remarkable efficiency 100%) when 3 or more of the 4 amino acids in the ISD core region differed, and 16 in the case of 2 amino acids. 0.7%, 62.5% in the case of one, and 100% in the case of the wild type.

Therefore, whether interferon treatment may be effective by examining whether or not the amino acid sequence of the ISD core region of HCV-1b in the sample is the same as the wild type sequence shown in SEQ ID NO: 15. You can know whether or not.

In addition to the above ISDR (existing in NS5A region) of the amino acid sequence of HCV-1b, the present inventors have found amino acid mutations in each case in the hypervariable region (HVR). I was observed. HVR is HCV
It is a region where the sequence is known to frequently differ depending on the strain of -1b. However, HVR is silent
H with different interferon susceptibility even if there is only mutation (that is, the encoded amino acid sequence is the same)
The presence of CV-1b and the lack of a relationship between the sequence and interferon susceptibility suggest that it is unlikely that HVR directly determines interferon susceptibility. Therefore, interferon sensitivity is
It became clear that it was determined by R.

The determination method of the present invention utilizes an immunoassay technique. That is, for example, a polypeptide having the amino acid sequence of wild type ISDR shown in SEQ ID NO: 1 or a part thereof is synthesized. This polypeptide can be easily produced using a commercially available peptide synthesizer or by a well-known genetic engineering technique. By carrying out an immunoassay using this polypeptide as an antigen, it can be known whether or not an antibody specifically reacting with the polypeptide is present in a sample. If such an antibody is present, it can be seen that wild-type HCV-1b is present in the patient, and that interferon treatment is ineffective. The immunoassay method itself is well known, and any of the well-known immunoassay methods can be applied. That is,
If classified according to the measurement format, for example, sandwich method, competition method, agglutination method, etc. are applicable, and if classified according to label, for example, enzyme immunoassay, radioimmunoassay, fluorescent immunoassay, biotin immunoassay. Etc. are applicable. Details of the immunoassay method are well known and are also specifically described in Examples below.

Alternatively, an animal is immunized with a polypeptide having the amino acid sequence of wild-type ISDR or a part thereof, and an antibody against the animal is recovered. You can know if it exists. The antibody may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred. The monoclonal antibody can be easily obtained based on the well-known Koehler and Milstein method. The immunoassay method is well known as described above, and any known immunoassay method can be applied. According to this method, it is possible to know whether or not wild-type ISDR is present in the sample.

The above method examines the difference between the amino acid sequence shown in SEQ ID NO: 1 and, as described above, if it is the same as the sequence shown in SEQ ID NO: 2-14, the interferon treatment is markedly effective. Therefore, it is also possible to use the sequences of SEQ ID NOs: 2 to 14 in place of the sequence of SEQ ID NO: 1 in the above immunoassay method.

[0019]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Reference Example 1 (1) Purification of RNA 1 serum each of 70 patients with chronic hepatitis C due to HCV-1b
RNAzolB (trade name, manufactured by BIOTEX LAB) in 00 μl
900 μl and 150 μl of chloroform were added, and the mixture was stirred in a 2 ml Eppendorf tube for 15 seconds and left at 4 ° C. for 5 minutes. After centrifuging at 12000 rpm for 15 minutes at 4 ° C., the supernatant was transferred to a 1.5 ml Eppendorf tube, and 650 μl of isopropanol was added thereto, and the mixture was stirred 1
After standing for 5 minutes, it was centrifuged at 12000 rpm, 4 ° C. for 20 minutes to obtain a precipitate. Further, 1 ml of 75% ethanol was added, and after stirring, the mixture was centrifuged at 12000 rpm and 4 ° C. for 5 minutes, and the supernatant was discarded. After repeating this operation two more times, the obtained precipitate was dried.

(2) cDNA synthesis Purified RNA was added to 2 μl of 5 × RT buffer, 0.1M
DTT 1 μl, 10 mM dNTP 1 μl, random primer (absorbance at 260 nm 5, below,
In such a case, it is described as “5 OD 260 nm” 1
μl and 2 μl of DEPC-treated water were added, and after stirring, the mixture was heated at 37 ° C. for 45 minutes for cDNA synthesis.

(3) First PCR PCR was performed by the hot start method. That is,
Lower mix (primer HCV-9 (SEQ ID NO: 16) and primer HCV-10 into a 0.6 ml PCR tube)
(SEQ ID NO: 17) 2.5 O.V. D. 260 nm to 0.
5 μl each, 10 mM dNTP 1 μl, 25 mM M
gCl _{2 (} 3 μl, sterilized MQ water 5 μl) (10 μl), ampliwax 100 (trade name: Perkin Elmer Co., Ltd.), 1 grain, heated at 90 ° C. for 1 minute, and then at 25 ° C.
It was left for 3 minutes to form a film. Upper mix on this membrane
(10x PCR buffer (100 mM Tris-H
Cl, 5 mM of 500 mM KCl (pH 8.3), 35 μl of sterilized MQ water, 0.2 μl of Taq polymerase (trade name, manufactured by Promega) and 1 μl of the above cDNA
PCR was performed according to the following program. When using Thermal Circular PJ9600 (trade name, manufactured by Perkin Elmer): 94 ° C. 20 seconds (denaturation) 55 ° C. 20 seconds (annealing) 77 ° C. 20 seconds (extension) 35 cycles Thermal Circular PJ2000 or 480 (trade name, Perkin) When using Elmer Co., Ltd .: 94 ° C. 60 seconds (denaturation) 55 ° C. 60 seconds (annealing) 77 ° C. 60 seconds (extension) 35 cycles

(4) Second PCR PCR was performed by the hot start method. That is,
Lower mix (primer HCV-ES (SEQ ID NO: 18) and primer HCV-into a 0.6 ml PCR tube.
E-AS (SEQ ID NO: 19) 2.5 each. D. 260n
m 0.5 μl each, 10 mM dNTP 1 μl, 25
mM MgCl ₂ 3 μl, sterile MQ water 5 μl) 10
After taking 1 μl, ampliwax 100 (trade name: manufactured by Perkin Elmer) was added and heated at 90 ° C. for 1 minute.
A film was formed by leaving it at 25 ° C. for 3 minutes. Upper on this membrane
mix (10 x PCR buffer 5 μl, sterile MQ water 35
μl, Taq polymerase (trade name, manufactured by Promega) 0.2 μl) and 1 μl of the first PCR product
PCR was performed using the same program as the first PCR.

(5) Purification of PCR product The second PCR product was purified using Quick Spin Column G50 (TE) (trade name, manufactured by Boehringer Mannheim).

(6) Nucleotide sequence determination The nucleotide sequence was determined using the dye terminator method with a 373A autosequencer (trade name, manufactured by Perkin Elmer). For the reaction, a PRISM kit (trade name, manufactured by Perkin Elmer) was used, and the reaction mixture was 9.5 μl, SEQ ID NO: 3
PCR was carried out in the following program in a 0.2 ml PCR tube containing 5.0 μl of the purified second PCR product S (3.2 μmol), the primer S for determining the nucleotide sequence having the nucleotide sequence shown in 1 above, and 4.5 μl of MQ water. It was That is, using a thermal circular PJ9600 (trade name, manufactured by Perkin Elmer), 96 ° C for 15 seconds, 50 ° C
After 25 cycles of 1 second at 60 ° C. for 4 minutes, the plate was left at 4 ° C. until the next operation. 20 μl of the above reaction solution was purified by Quick Spin Column G50 (TE), dried, and then 373
6 with A Auto Sequencer (Perkin Elmer)
Electrophoresis using% acrylamide (19: 1)
The nucleotide sequence of the fragment containing ISDR was determined. After converting the obtained base sequence into amino acids, 40 amino acids of ISDR were compared with the wild-type ISDR shown in SEQ ID NO: 1.

On the other hand, each patient was given interferon α 6
The total dose was 516-880 MU administered over a period of months. The presence or absence of blood HCV-RNA and the GPT value (UV method) were measured for 6 months after the treatment. Blood HCV-R
NA concentration was determined by branched DNA probe assay (Lancet, 1993, 3
41: 1501-1504). The GPT value is J
The measurement was performed at a measurement temperature of 37 ° C. using an SCC-compliant prescription reagent. For 6 months after the treatment, those showing negative blood HCV-RNA and normal GPT were markedly effective, and the others were invalid.

The results are shown in FIGS. 1 and 2. 1 and 2
As is clear from the figure, the wild type ISDR has a marked efficiency of 0%, and the wild type having a difference of 1 or 2 amino acids has a marked efficiency of 13%, which is different by 4 or more. The product had a remarkable efficiency of 100%. Age, sex, total dose of tissue-type interferon, GP before treatment
There was no significant difference in T value. Therefore, ISD
By examining whether or not the sequence of R is the same as the wild type, it is possible to know whether or not the interferon treatment is ineffective.
In addition, by examining the number of amino acid residues different from the wild type, the probability that interferon treatment is significantly effective can be estimated.

Further, I of the excellent cases found by the above examination
The amino acid sequences of 29 amino acid residues from the N terminus of the amino acid sequence of SDR are shown in SEQ ID NOs: 2 to 14. HC in the sample
When the amino acid sequence of ISDR of V-1b is the same as any of these sequences, it is considered that interferon treatment is highly effective with a high probability.

Reference Example 2 77 patients with HCV-1b-induced chronic hepatitis C were examined in the same manner as in Reference Example 1 (the total dose of interferon is 324 MU or more). Among the determined amino acid sequences, the amino acid sequence of the ISD core region is the sequence shown in SEQ ID NO: 15, namely Thr His His
The number of mutated amino acid residues was examined by comparison with Asp. The results are shown in Tables 2 and 3. In Table 2, “mutant type” means that the sequence of the ISD core region differs from the wild type by at least one amino acid.

[0030]

[Table 2]

[0031]

[Table 3]

From Tables 2 and 3, it can be seen that interferon treatment is ineffective when the amino acid sequence of the ISD core region is wild type. Further, it can be seen that the ineffective rate of interferon treatment decreases as the number of amino acids different from the wild type increases, and the ineffective rate becomes 0% when the number of amino acids differing by 3 or more.

Reference Example 3 The same test as in Reference Example 1 was performed except that the primer used in the second PCR was changed. For the second PCR, HCV-ISDR-S (SEQ ID NO: 20) and HCV
-ISDR-AS (SEQ ID NO: 21) was used. Reference example 1
Similarly to, the fragment containing ISDR was amplified and its nucleotide sequence was determined.

Reference Example 4 The same test as in Reference Example 1 was carried out except that the primers used for the second PCR and nucleotide sequence determination were changed.
HCV-ISDR-T7 (SEQ ID NO: 22) and HCV-ISDR-R22 (SEQ ID NO: 2) were included in the second PCR.
3) was used. T720 (SEQ ID NO: 32) and R22 (SEQ ID NO: 33) were used for nucleotide sequence determination. Reference example 1
Similarly to, the fragment containing ISDR was amplified and its nucleotide sequence was determined.

Example 1 AB is a peptide M0 having the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing and peptides M1 and M2 having the amino acid sequences shown in SEQ ID NOS: 24 and 25, respectively.
Synthesized using a 430A peptide synthesizer manufactured by Company I
~ 0.4ml is filtered with a 0.45μm filter, HP
After LC purification, it was freeze-dried.

The synthesized peptides M0, M1 and M2 were dissolved in 10 mM phosphate buffer (pH 7.0) to prepare 1
It was adjusted to μl / ml. 96-well ELIS each
A plate (MaxSorb, trade name, manufactured by nunc)
50 μl / well. It was left at room temperature for 2 hours. Wells were washed twice with 100 μl PBS. 5% B
250 μl of PBS containing SA or 0.05% gelatin was added to each well and left for 16 hours.

Next, 100 μl of pretreated sample serum was added to each well and left for 1 hour. It was washed twice with PBS. PB containing 5% BSA or 0.05% gelatin
Add 50 μl per well of peroxidase-labeled rabbit anti-human immunoglobulin antibody diluted with S, and add 3
The reaction was carried out for 0 to 60 minutes. 100 μl P per well
Washed 3 times with BS. Ortho-phenylenediamine (OD
E) Solution (citric acid-phosphate buffer (citric acid, 0.5
g, disodium hydrogen phosphate 1.84 g, distilled water 10
(0 ml, pH 5.0) 40 mg of ODE was dissolved in 100 ml, and 30 μl of 30% hydrogen peroxide solution was further added).
Was added to each well at 100 μl. After reacting for 10 to 30 minutes, 50 μl / well of 2 M sulfuric acid was added to stop the reaction. The absorbance was measured with an ELISA reader (OD 492 nm). cut off is O. D. 492nm
= 0.5, and all of M0, M1, and M2 were negative in the five HCV antibody-negative patients. HCV-1b type (interferon α ineffective case) positive in 5 cases, HCV-1b
Negative was determined in 5 cases of type (interferon excellent response cases).

Example 2 From the serum of a patient with chronic hepatitis C, HCV was prepared in the same manner as in Reference Example 1.
-RNA was extracted, cDNA synthesis and first PCR was performed. Next, 100 ng or less of each primer MM
Using F1 (SEQ ID NO: 26) and MMF2 (SEQ ID NO: 27), 1.25 units of Taq DNA polymerase, 1
0 mM Tris-HCl (pH 8.3), 50 mMK
1 μl of the first PCR product in 100 μl of a solution of Cl, 1.5 mM MgCl ₂ , 0.01% gelatin
In addition, 25 cycles of PCR were performed at 95 ° C for 1 minute, 55 ° C for 20 seconds, and 72 ° C for 20 seconds. Restriction enzyme E
5 units of coRI, treated with restriction enzyme, EcoRI
The ends were made. 5 μl of this and the expression vector pGEX
-1 Lambda T EcoRI / BAP Glutathione-S-
Transferase (GTS) fusion vector (pGEX manufactured by Pharmacia Biotech Co., Ltd. with lambda T phage and G
Vector incorporating a fusion protein of TS) 100n
g, 66 mM Tris-HCl (pH 7.6), A
TP 1 mM, spermidine 1 mM, MgCl ₂ 10
A solution of mM, DDT 15 mM and T4 ligase 300 units (total 10 μl) was reacted at 22 ° C. for 3 hours. Transformed into E. coli JM109, cultured in culture agar medium, and confirmed insert by colony hybridization. Furthermore, the nucleotide sequence was determined, and it was confirmed that the gene encoding the ISDR sequence was incorporated into the expression vector. In addition, E. The expression vector obtained in E. coli was transformed to prepare an ISDR sequence expression vector strain. The expressed fusion protein of ISDR and GTS was purified by affinity chromatography using Glutathion Sepharose 4B (trade name, Pharmacia Biotech Co., Ltd.), and a site-specific protease (thrombin) located immediately upstream of the cloning site.
It was separated by utilizing the recognition site of 1 mM and prepared in 10 mM phosphate buffer (pH 7.0) so as to be 1 μg / ml.
50 μl / well of each well was placed in a 96-well ELISA plate (MaxSorb, manufactured by Nunc). It was left at room temperature for 2 hours. 2 wells with 100 μl PBS
Washed times. 250 μl of PBS containing 5% BSA or 0.05% gelatin was added to each well and left for 16 hours. Thereafter, an ELISA was performed in the same manner as in Example 6. As a result, the HCV antibody-negative patients were negative. HC
V-1b type (interferon α ineffective case) positive in 5 cases, HCV-1b type (interferon markedly effective case)
Negative was judged in 5 cases.

Example 3 16 mg of keyhole limpet hemocyanin (KL
H) in 1 ml of sodium phosphate buffer (pH 7.2)
, And 9.3 μl of m- with stirring with a stirrer.
A maleimidobenzoyl-N-hydrosuccinimide ester (MB) solution (a solution of MB in dimethylformamide at 0.3 mg / μl) was added,
Stirred at room temperature for 30 minutes. The mixture was transferred to a 1.5 ml Eppendorf tube and centrifuged at 4 ° C. and 16000 rpm for 5 minutes. The supernatant was applied to Sephadex G-25 (trade name, manufactured by Pharmacia) using a glass column having an inner diameter of 1 cm and a length of 30 cm (50 mM sodium phosphate buffer,
pH 6.0 was used) 1 ml aliquots were collected, and among them, fractions containing the KLH-MB conjugate were found by Coomassie Brilliant Blue (CBB) staining and stored together at -80 ° C. The CBB staining was performed as follows.
The PVDV membrane was wet with methanol and then replaced with distilled water. 1 μl of each fraction was spotted and decolorization solution (50
% Methanol, 5% acetic acid). Further 0.1%
It was immersed in methanol containing CBB for 10 seconds and transferred to the decolorizing solution. The KLH-MB fraction was stained blue.

KLH-MB ISDR sequence peptide (M0), dissolved in 1.0 ml and 0.5 ml distilled water, 0.
0.5 ml of 2M sodium phosphate buffer was mixed. The reaction solution was blown with argon gas and shaken on a rotary shaker at room temperature for 3 hours. Dispense 200 μl of each,
The temperature was kept at 20 ° C.

200 ml of MO-K was mixed with 1.5 ml of Freund's complete adjuvant, and subcutaneously injected into rabbits (New Zealand white, female, around 2.5 kg, 5 animals) at about 20 sites. 5 second injections 3 weeks later
0 μl was performed, and thereafter, the injection was repeated every 10 days for 3 months.
The antibody detection system described in Example 6 was used for checking the anti-ISDR antibody. The obtained anti-ISDR sequence antibody is PBS
Dilute with 0.05% gelatin and add 96 wells of E
LISA plate (MaxSorb, trade name, nunc
50 μl / well). It was left at room temperature for 2 hours. Wells were washed twice with 100 μl PBS. 5
250 μl of PBS containing% BSA or 0.05% gelatin was added to each well and left for 16 hours. Thereafter, an ELISA was performed in the same manner as in Example 6. As a result, it was negative in two HCV antibody negative patients. HCV-1b type (interferon α ineffective case) was positive in 2 cases, HCV-
Negative was determined in 3 cases of type 1b (interferon markedly effective case).

Example 4 Peptides M0, M1 and M2 above and SEQ ID NO: 28,
Peptides M3, M4 and M5 having the sequences shown by 29 and 30, respectively, were synthesized using ABI 430A type peptide synthesizer, and 0.2-0.4 ml was 0.45 μm.
It was filtered with a filter, purified by HPLC, and then lyophilized.

Synthesized peptides M0, M1, M2, M
3, M4, and M5 are each 10 mM phosphate buffer (pH
It was dissolved in 7.0) and adjusted to 1 μg / ml. 96-well ELISA plate (Maxisorp;
50 μl / well was put in each of the product names (manufactured by nunc). It was left at room temperature for 2 hours. The peptide solution was removed. The well was washed twice with 100 μl of PBS. 5%
Weld BSA or PBS containing 0.05% gelatin each
250 μl was added to 1 and left for 16 hours.

Next, sample serum was added to 100 wells per well.
μl was added and left for 1 hour. It was washed twice with PBS. 5% B
SA (trade name; bovine serum albumin, Boehringer Mannheim) or peroxidase-labeled rabbit (or goat) anti-mouse immunoglobulin antibody diluted with PBS containing 0.05% gelatin was added in an amount of 50 μl per well and reacted at room temperature for 30 to 60 minutes. Each well was washed 3 times with 100 μl of PBS. Ortho-phenylenediamine (OD
E) Solution (citric acid-phosphate buffer (citric acid, 0.5
g, disodium hydrogen phosphate 1.84 g, distilled water 100
40 mg of ODE was dissolved in 100 ml of 100 ml of pH 5.0), and 30 μl of 30% hydrogen peroxide solution was added), and 100 μl was added to each well. After reacting for 10 to 30 minutes, 50 μl / well of 2M sulfuric acid was added to stop the reaction. The absorbance was measured with an ELISA reader (OD 492 nm). cut off, O. D. 492n
With m = 0.5, M0, M1, M2, M4, and M5 are
HCV antibody negative 5 patients negative, HCV-1b type (IFN alpha ineffective case) 5 positive, HCV-1b
Negative was judged in 5 cases of type (IFN excellent response cases).

[0045]

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to judge whether or not the interferon treatment for HCV-1b is effective. The present invention eliminates the waste of performing interferon treatment for patients who are not responding to interferon treatment. Therefore, the present invention is expected to contribute to the treatment of chronic hepatitis C with HCV-1b.

[0046]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 40 Sequence type: Amino acid sequence Pro Ser Leu Lys Ala Thr Cys Thr Thr His His Asp Ser Pro Asp Ala 1 5 10 15 Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn 20 25 30 Ile Thr Arg Val Glu Ser Glu Asn 35 40

SEQ ID NO: 2 Sequence length: 40 Sequence type: Amino acid sequence Pro Ser Leu Lys Ala Thr Cys Thr Thr Tyr His Gly Pro Pro Asp Ala 1 5 10 15 Asp Leu Ile Glu Ala Asn Leu Leu Trp Trp Gln Glu Met Gly Gly Asn 20 25 30 Ile Thr Arg Val Glu Ser Glu Asn 35 40

SEQ ID NO: 3 Sequence length: 40 Sequence type: Amino acid sequence Pro Ser Leu Lys Ala Thr Cys Thr Thr Asn His Asp Ser Leu Gly Val 1 5 10 15 Asp Leu Ile Glu Ala Asn Leu Leu Trp Gln Gln Glu Met Gly Gly Asn 20 25 30 Ile Thr Arg Val Glu Ser Glu Asn 35 40

SEQ ID NO: 4 Sequence length: 40 Sequence type: Amino acid sequence Pro Ser Leu Lys Ala Thr Cys Thr Thr Leu His Asp Ser Leu Gly Val 1 5 10 15 Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg His Glu Met Gly Gly Asn 20 25 30 Ile Thr Arg Val Glu Ser Glu Asn 35 40

SEQ ID NO: 5 Sequence length: 40 Sequence type: Amino acid sequence Pro Ser Leu Lys Thr Thr Phe Thr Ala Cys Pro Asn Ser Pro Asp Val 1 5 10 15 Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly His 20 25 30 Ile Thr Arg Val Glu Ser Glu Asn 35 40

SEQ ID NO: 6 Sequence length: 40 Sequence type: Amino acid sequence Pro Ser Leu Arg Ala Thr Cys Thr Ala Arg His Gly Ala Pro Asp Thr 1 5 10 15 Asp Leu Ile Asp Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asp 20 25 30 Ile Thr Arg Val Glu Ser Glu Asn 35 40

SEQ ID NO: 7 Sequence length: 40 Sequence type: Amino acid sequence Pro Ser Ser Arg Ala Thr Trp Ala Ala Tyr His Asp Ser Pro Asp Val 1 5 10 15 Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg His Glu Met Gly Gly Asp 20 25 30 Ile Thr Arg Val Glu Ser Glu Asn 35 40

SEQ ID NO: 8 Sequence length: 40 Sequence type: Amino acid sequence Ala Ser Leu Lys Ala Thr Cys Pro Thr Gln His Asp Ser Pro Asp Thr 1 5 10 15 Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn 20 25 30 Ile Thr Arg Val Glu Ser Glu Asn 35 40

SEQ ID NO: 9 Sequence length: 40 Sequence type: Amino acid sequence Leu Ser Leu Lys Ala Ala Cys Thr Ala His His Ala Pro Pro Asp Val 1 5 10 15 Asp Leu Ile Glu Ala Asn Leu Leu Trp Gln Gln Glu Met Gly Gly Asn 20 25 30 Ile Thr Arg Val Glu Ser Glu Asn 35 40

SEQ ID NO: 10 Sequence length: 40 Sequence type: Amino acid sequence Leu Ser Leu Lys Ala Ala Cys Thr Ala Pro His Gln Ala Pro Asp Val 1 5 10 15 Asp Leu Ile Glu Ala Asn Leu Leu Trp Gln Gln Glu Met Gly Gly Gly 20 25 30 Ile Thr Arg Val Glu Ser Glu Asn 35 40

SEQ ID NO: 11 Sequence length: 40 Sequence type: Amino acid sequence Leu Ser Leu Lys Ala Ala Cys Thr Gly Arg His Asp Ser Pro Asp Val 1 5 10 15 Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn 20 25 30 Ile Thr Arg Val Glu Ser Asn 35 40

SEQ ID NO: 12 Sequence length: 40 Sequence type: Amino acid sequence Val Ser Leu Lys Ala Ala Cys Thr Thr Arg His Asp Ser Pro Asp Ala 1 5 10 15 Asp Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Ser 20 25 30 Ile Thr Arg Val Glu Ser Glu Lys 35 40

SEQ ID NO: 13 Sequence length: 40 Sequence type: Amino acid sequence Val Ser Leu Lys Ala Ala Cys Thr Ala Arg His Gly Ser Pro Asp Ala 1 5 10 15 Asp Leu Ile Asp Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Thr 20 25 30 Ile Thr Arg Val Glu Ser Glu Asn 35 40

SEQ ID NO: 14 Sequence length: 48 Sequence type: Amino acid sequence Pro Ser Leu Lys Ala Thr Cys Thr Thr Arg Asn Asp Ser Ser Trp His 1 5 10 15 Asn Ser Pro Ala Ala Pro Asp Ala Asp Leu Ile Glu Ala Asn Leu Leu 20 25 30 Trp Arg Gln Glu Met Gly Gly Ser Ile Thr Arg Val Glu Ser Glu Asn 35 40 45

SEQ ID NO: 15 Sequence length: 4 Sequence type: Amino acid

SEQ ID NO: 16 Sequence length: 25 Sequence type: Nucleic acid sequence TCTTTCTCCG TGGAGGTGGT ATTGG 25

SEQ ID NO: 17 Sequence length: 25 Sequence type: Nucleic acid Sequence TGGATGGAGT GCGGTTGCAC AGGTA 25

SEQ ID NO: 18 Sequence length: 38 Sequence type: Nucleic acid sequence TGTAAAACGA CGGCCAGTCA GGTACGCTCC GGCGTGCA 38

SEQ ID NO: 19 Sequence length: 38 Sequence type: Nucleic acid sequence CAGGAAACAG CTATGACCGG GGCCTTGGTA GGTGGCAA 38

SEQ ID NO: 20 Sequence length: 42 Sequence type: Nucleic acid Sequence TGTAAAACGA CGGCCAGTAG CGTAGGCTGG CCAGGGGGTC TC 42

SEQ ID NO: 21 Sequence length: 42 Sequence type: Nucleic acid Sequence CAGGAAACAG CTATGACCAC GGATACTTCC CTCTCATCCT CC 42

SEQ ID NO: 22 Sequence length: 44 Sequence type: Nucleic acid sequence TAATACGACT CACTATAGGG AGCGTAGGCT GGCCAGGGGG TCTC 44

SEQ ID NO: 23 Sequence length: 46 Sequence type: Nucleic acid sequence TCACACAGGA AACAGCTATG ACACGGATAC TTCCCTCTCA TCCTCC 46

SEQ ID NO: 24 Sequence length: 20 Sequence type: Amino acid sequence Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys Thr Thr His His Asp Ser 1 5 10 15 Pro Asp Ala Asp 20

SEQ ID NO: 25 Sequence length: 12 Sequence type: Amino acid

SEQ ID NO: 26 Sequence length: 33 Sequence type: Nucleic acid Sequence TTTGAATTCC CTTCCTTGAA GGCGACATGC ACT 33

SEQ ID NO: 27 Sequence length: 33 Sequence type: Nucleic acid sequence TTTGAATTCA TTCTCTGACT CCACGCGAGT GAT 33

SEQ ID NO: 28 Sequence length: 12 Sequence type: Amino acid

SEQ ID NO: 29 Sequence length: 19 Sequence type: Amino acid sequence Asn Leu Lue Trp Arg Gln Glu Met Gly Gly Asn Ile Trp Arg Val Glu 1 5 10 15 Ser Glu Asn

SEQ ID NO: 30 Sequence length: 13 Sequence type: Amino acid sequence Gly Gly Asn Ile Tyr Arg Val Glu Ser Glu Asn Lys Val 1 5 10

SEQ ID NO: 31 Sequence length: 18 Sequence type: Nucleic acid Sequence TGTAAAACGA CGGCCAGT 18

SEQ ID NO: 32 Sequence length: 20 Sequence type: Nucleic acid sequence TAATACGACT CACTATAGGG 20

SEQ ID NO: 33 Sequence length: 22 Sequence type: Nucleic acid sequence TCACACAGGA AACAGCTATG AC 22

[Brief description of drawings]

FIG. 1 is a diagram showing the sequence of ISDR and the effect of interferon administration.

FIG. 2 is a diagram showing the sequence of ISDR and the effect of interferon administration.

Claims (9)

[Claims] 1. Whether the amino acid sequence of the ISD region consisting of amino acids 2209 to 2248 of hepatitis C virus belonging to genotype 1b in the sample is the same as the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing A method for determining the effectiveness of treatment of genotype 1b against hepatitis C virus, which comprises examining whether or not
A determination method, which uses a region or a part of the polypeptide as an antigen and performs an immunoassay using an antigen-antibody reaction with an antibody against the antigen in a sample. 2. Whether the amino acid sequence of the ISD region consisting of amino acids 2209 to 2248 of hepatitis C virus belonging to genotype 1b in the sample is the same as the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing A method for determining the effectiveness of treatment of genotype 1b against hepatitis C virus, which comprises examining whether or not
A determination method, which uses an antibody that reacts with a region or a part thereof, by an immunoassay utilizing an antigen-antibody reaction using the ISD region in a sample as an antigen. 3. The method according to claim 1, wherein the treatment is administration of interferon. 4. A peptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 5. A peptide having the amino acid sequence represented by SEQ ID NO: 24 in the sequence listing. 6. A peptide having an amino acid sequence represented by SEQ ID NO: 25 in the sequence listing. 7. A peptide having an amino acid sequence represented by SEQ ID NO: 28 in the sequence listing. 8. A peptide having an amino acid sequence represented by SEQ ID NO: 29 in the sequence listing. 9. A peptide having an amino acid sequence represented by SEQ ID NO: 30 in the sequence listing.