JPH0454197A - Synthetic peptide antigen for detection of hepatitis c virus antibody, composition thereof and use thereof - Google Patents

Abstract

PURPOSE:To provide the subject synthetic peptide antigen corresponding to a partial protein in a protein region coded by HCV gene and capable of executing a high-sensitive HCV infection diagnosis with very excellent specificity. CONSTITUTION:One or more antigenic peptides selected from peptides synthesized by a peptide synthesizer based on cDNA sequence informations of HCV (causative virus of heptatisis C) and having amino acid sequences of formulae I-V or a part of the above-mentioned peptides. The above-mentioned peptides are useful for a high-reliable and specific diagnosis of an HCV-infected HC patient or for a high reliable and specific HCV infection screening of blood or blood derivatives respectively remarkably low in wrong diagnosis. A combination between C825 group of formulae I-III and C100J group of formula IV or V, especially a combination between a peptide of formula I and a peptide of formula IV is preferably used.

Description

[Detailed Description of the Invention] No. 1 The present invention provides a synthetic peptide antigen related to the virus (HcV) that causes serum hepatitis that is neither hepatitis A nor hepatitis B, particularly hepatitis C, and a diagnostic reagent for detecting antibodies against HCV. Regarding.

Several types of viruses that cause viral hepatitis have been identified.

Hepatitis A virus is a 27n-diameter RNA virus of the Picornaviridae family that causes epidemics through oral infection (Fi
neston, S, M, et al, 5cie
nce, 1J3j: Pl 026, l 973
> Hepatitis B virus is a DNA virus with a diameter of 42 nm that belongs to the Hepadnaviridae family and is mainly transmitted through blood (Dane, 0.5., et al.,
Lancet, I: p, 241.1974),
Six definitive methods have been established for these viruses, and preventive measures have also been established.

Hepatitis viruses that do not belong to any of these were called non-A, non-B hepatitis viruses. Recently, it has become clear that there are two types of non-A, non-B hepatitis viruses. One is a calicivirus-like hepatitis virus with a diameter of 27 to 32 ns that is prevalent through oral infection in India, Myanmar, Afghanistan, North Africa, etc. (Khuroo, M, S^m, J, Med, 1
)Jl: p, 818-824.1980), This WiJ reply is epidemic or c++ter
Hepatitis E virus by taking the E of tc
It is called (HEV).

The other is a virus that causes most cases of post-transfusion hepatitis.
It is a virus called V). The existence of this virus has been confirmed for a long time (Tabor, E.
, et al Lancet, I:p, 463.19
78>, despite the efforts of many researchers around the world,
The virus itself is still not fully understood.
Under these circumstances, in 1989, Choo of the United States,
Q. succeeded in cloning HCV cDNA,
We reported that a detection system for anti-HCV antibodies was established using some of the expressed proteins (Choo, Q. et al.
,, 5science, 214. P.

359-362.1989; Kuo, Get a
t,, 5science, 'IJJp, 362-3
64.19891゜The present inventors, apart from Choo et al.,
They independently succeeded in cloning HCV cDNA using high PT plasma and have already filed patent applications (Japanese Patent Application No. 1-238848, Japanese Patent Application No. 2-22549).

Choo, Qratsuku O-ning Shiri HCV f) c
cD cloned by DNA and the inventors
N A (C825, C100J) has 78% and 74% homology at the nucleic acid level, suggesting that they are different strains, and there is a difference in the degree of poliovirus type 1 and type 2 between these strains. The 9 strains that exist have some different immunoserological properties, so when using the same diagnostic method, the difference between the strains has a large impact on the detection sensitivity or specificity.
It is expected that the cDNA of the present inventors will be extremely useful in the detection of.

Anti-HCV antibody detection ELISA prepared by Kuo, G et al.
The system uses superoxide dismutase (Superθ
A fusion protein with 1de dismutase (5OD) expressed in yeast is used as an antigen. This method can specifically and sensitively detect viral antibodies possessed by HCV-infected people around the world, and is highly valuable as a screening method for blood transfusions that can transmit hepatitis C after transfusion. found. However, the following problems still remain in detection systems that use fusion protein virus antigens produced by recombinant DNA technology.

l) There is a risk that the desired expressed protein may be degraded by protease derived from the cultured recombinant microorganism.

2) Cultured recombinant microorganism, cultured cell upper groove, bacterial body,
When purifying a target antigen protein from cells, the starting material for purification contains a large amount of protein derived from recombinant organisms, cultured cells, and the medium used, and purification generally requires many steps. It is a complicated process that involves

3) Non-specific reactions caused by the fusion protein and the recombinant microorganism used for expression may occur.

4) Cultivation of recombinant microorganisms is essential, and differences between lots are likely to occur.

The HCV antibody detection system described above is extremely significant in that it can quickly and easily detect antibodies against hepatitis C virus, which has not been possible in the past.
Furthermore, from the viewpoint of detection rate and suppression of non-specific reactions,
% accurate diagnosis is desired. There is also room for improvement in terms of quality control, and further development is underway to achieve this goal.

The inventors of the present invention have investigated non-specific reactions caused by the fusion protein, non-specific reactions caused by the transformed microorganism used for expression,
In order to avoid difficulties in quality control, we prepared a synthetic peptide of the region encoded by the uniquely cloned cDNA, and proceeded with the development of a system that utilizes this for several antibody detection methods, thereby completing the present invention. reached.

The object of the present invention is to provide several novel synthetic peptides corresponding to antigenic HCV proteins that can be used with excellent specificity, selectivity, and high sensitivity in HCV infection detection and diagnosis. The present invention aims to provide a method for detecting anti-HCV antibodies that directly uses the newly synthesized peptide or a portion thereof.

The inventors of the present invention cloned HCV Cr) c DNA from plasma with abnormal liver function values (GOT, GPT) of a Japanese person thought to be infected with non-A, non-B hepatitis. For details, see the previous patent applications filed by the inventors (Japanese Patent Application No. 1-238848, No. 2-22549).
It is written in.

The present inventors incorporated these cDNAs into plasmids and transformed microorganisms to express them.

The cDNA was expressed by incorporating it into culture cells, and the expression product was purified to investigate an anti-HCV antibody detection system.
We have synthesized a peptide by replacing the sea fence with amino acids, and have been investigating an anti-HCV antibody detection system using this peptide.

The peptide shown below was synthesized using a peptide synthesizer based on the sea fence information of the cDNA.

C825-37mer ^5p-Pro-5et Lys-＾rg-＾``g Ser-Leu-Ala Ser-Alt-Pr.

C825-34mer Thr-Thr-Arg His-1ie-Thr-^1a-Glu-Thr-^
1aLeu-^1a-^rg-Gly-3er-Pro
-Pr.

5er-5er-5er-^1a-3er-Gln-L
euSer-Leu-Lys-Ala 1(is-Asp-3er-Pro-Asp-Ala-
^spLeu-eye-Glu-^1a-^5n-Leu
-Leu-Trp-^"g-GlnGlu-)let-
Gly-Gly-Asn-1)e-Thr-^rg-'
Val-GluSer-Gln-^sn-Lys C825-30mar Glu-Net-Gly-GIY-^9n-1)e-T
hr-^rg-4al-GluSer-Glu-^5n
-Lys-Vat-Val-1)e-Leu-^5p-
5erPhe-^5p-Pro-Leu-backg-Ala
-Glu-Glu-^sp-GluClooJ-29m
er Glu-Phe-Asp-Glulet-Glu-Gl
u-Cys-^1a-5erHis-Leu-Pro-
Tyr-1) e-Glu-Gln-Gly-Met-G
inLeu-^1a-Glu-Gln-1'he-Ly
s-Gln-Lys-^1aC100J-37mer Pro-Asp-^rg-Glu-Val-Leu-T
yr-^rg-Glu-PheAsp-Glu-1)e
t-Glu-Glu-Cys-^1a-3er-His
-LeuPro-Tyr-Val-Glu-Gln-G
ly-Met-Gln-Leu-^1aGlu-Gln
-1'be-Lys-Gin-Lys-^1a A second object of the present invention is to at least one peptide selected from the above peptide group, preferably at least one peptide selected from the 0825 group, or Part and C
An antigenic peptide consisting of at least one or more peptides selected from the 100J group or a combination of a portion thereof,
contacting the sample under conditions such that an immunological conjugate is formed between the anti-HCV antibody present in the sample and the peptide;
Measuring the formation of said immunological conjugate to detect anti-HCV in a sample
This is accomplished by an anti-HCV antibody detection method that confirms the presence of antibodies.

In other words, the present invention provides a synthetic peptide antigen corresponding to a part of the fourth nonstructural region (NS4) of polyprotein encoded by the HCV gene, and a synthetic peptide antigen corresponding to a part of the protein of the fourth nonstructural region (NS4) of polyprotein encoded by the HCV gene. This was achieved through the discovery of synthetic peptide antigens corresponding to some proteins in region (1) 55>, and these peptides can be used to diagnose HC patients due to HCV infection and to detect HC in blood and blood products.
It is useful as a method for screening for V exposure with high reliability, high specificity, and extremely low erroneous results.

(Outline of Embodiments) The present invention relates to an antigenic peptide corresponding to a part of the protein region encoded by the HCV gene, and the present invention relates to an antigenic peptide corresponding to a part of the protein in the polyprotein NS4, NS5 region. Several peptides consisting of ~37 amino acids are provided. These novel peptides are HCV
The peptide contained in the present invention, which is useful for diagnosing the presence or absence of infection or virus exposure, is an oligopeptide having an amino acid sequence that includes a sequence constituting a sequential epitope corresponding to HCV antibodies. Consisting of

The peptides provided by the present invention thus include HC
Some peptides having immunoreactivity with V can be synthesized by known solid phase peptide synthesis methods. The peptide may be synthesized by adding one or two amino acids that are not in the sequence of the underlying protein to the amino or carboxyl terminus of the peptide, by such addition of amino acids.

The present peptide can be bound to a carrier protein or support. Amino acids useful for these purposes include:
Tyrosine, lysine, glutamic acid, aspartic acid,
Cysteine and derivatives thereof. Furthermore, conventional protein modification methods can be used, such as acetylation of the amino terminus,
It is also possible to add a means for binding the present peptide to other proteins, peptide molecules, or supports, such as by amidation of the carboxyl terminus.

The synthesized desired peptides can also be purified and recovered by conventional purification means.For example, these synthetic peptides are generally purified by high performance liquid chromatography (HPLC) using a synthetic peptide purification column, are dried using a rotary evaporator, and these synthetic peptides are recovered and subjected to the following measurement method.

These peptides can be used in methods for detecting antibodies against HCV antigens or HCV associated/bound antigens. In the method using this peptide to detect the presence of HCV-specific antibodies in a sample, the peptide antigen and HCV in the sample are
Under conditions where an immunological complex (antigen-antibody conjugate) can be formed with the antibody.

It is desirable to contact the present peptide with a sample, and if even a small amount of antigen-antibody conjugate is formed, it means that HCV antibodies are present in the sample;
This can be detected and measured by suitable means.

One such method is enzyme-linked immunoassay (ELI).
SA), radioimmunoassay (RIA), Western blotting, or methods using various carrier agglutination reactions. Among these assay methods, ELISA is particularly suitable for rapid, large-scale clinical screening of patient serum, blood for transfusion, blood products, and the like.

Hereinafter, specific embodiments will be outlined using ELISA as an example.

The above synthetic peptides are dissolved alone or in a mixture in a suitable buffer such as PBS, placed in each well of an ELISA plate, and left overnight at 4°C to allow the synthetic peptides to bind to the plate. 005%PB S Tween
After washing at 20°C, add 1% BSA to each well at 37°C.
Leave at ℃ for 1 hour for blocking, then 0.
Wash with a washing solution such as 0.05% PBS Tween 20, dilute the test sample, add to each well, and react at 37°C for 1 hour. Again 005%P B S Tw
After washing with een 20, anti-human IgG-HRP conjugate solution is added to each well and reacted at 37°C for one hour. After washing with 0.05% PBS Tween 20, add OPD substrate solution and allow color to develop in the dark at room temperature. 3
After 0 minutes, 2M sulfuric acid is added to stop the reaction, and the absorbance at -492 ns is measured.

When an ELISA system was created using synthetic peptides of C825-37mer and C10C100J-29, antibodies were detected in approximately 80% of non-A, non-B hepatitis patients, whereas antibodies were detected in 293 normal patients. It was revealed that the antibody was detected in only 3 cases, or about 1%, and that the specificity of non-A, non-B hepatitis was high, and the antibody detection rate in patients with non-A, non-B hepatitis was also very high. (Table 1) Table 1 NAIIBI ( 96/122 B 8/37 21.6 ＾utoi-une 1/4 25.0 tellltitis ＾1col ic 2/7 28.5 PBC 0/3 orma1 3/293 1 .02 PBC: Primary biliary cirrhosis Although the above-mentioned embodiments mainly describe the case where only chemically synthesized peptides are used, the present invention is not limited to this, and the synthetic peptides and genetically recombinant peptides are not limited to this. It is also possible to construct a suitable anti-HCV antibody detection system by appropriately combining antigen peptides prepared by techniques, or by using any part of the peptides.

The ELISA system using the polypeptide of the present invention has the following features compared to conventional commercially available ELISA methods using antigens expressed by recombinant microorganisms and culture II cells.

First, there is no need for the construction of expression plasmids, production of recombinant microorganisms, cloning, or expression experiments, and there is no need to cultivate recombinant microorganisms. Furthermore, there is no fear that the expressed protein will be degraded by the protease of the cultured recombinant microorganism.

When purifying the target antigen protein from cultured recombinant microorganisms, cultured cell supernatants, facets, and cells, the recombinant microorganisms, cultured cells, and sources of the medium used are included in the purification starting materials. Due to the large amount of protein contamination, purification is generally a complex process involving many steps. On the other hand, the method of synthesizing synthetic peptides using a peptide synthesizer is mostly automated, and purification can be done by HP.
This can be easily carried out using LC. In addition, there is no risk of contamination with proteins that cause non-specific reactions derived from recombinant microorganisms, cultured cells, or culture media, or denaturation or inactivation of expressed proteins during purification. Since there is no difference between the samples, quality control is easy.

Furthermore, since the HCV antigen peptide of the present invention is prepared based on the cDNA of a major HCV strain in Japan, it has the great advantage of being able to complement parts that cannot be detected with conventional commercially available kits. Furthermore, the antibody detection system of the present invention can detect antibodies that appear earlier than conventional commercially available kits, further supporting the usefulness of the present invention.

Hereinafter, in order to clarify the characteristics of the present invention, the present invention will be described in detail along with examples, but these examples are for explaining various specific examples of the present invention, and the present invention is based on these examples. It is not limited to.

Fruit'1'L-↓ C825 clone and C isolated from concentrated Japanese plasma
100J clone sea fence information (patent application Hei l-2
38848, patent application No. 2-22549), C82
For the synthesis of 5-37mer and C1O[IJ-29mer peptides, ^pplied Biosystem was used.
Oboro S Company's 430＾ Peptide 5ynthe
Purification was performed using HPLC for purifying synthetic peptides using a sizer.
Column (＾quapore Prep-10, C-8,
300A pore 5ize, 20uw 5phe
rical 5ilica 1(1+m ID
x 250 likes, ^pplied Bio
system (S company) was used. When the purified synthetic peptide was subjected to compositional analysis according to a conventional method, it was confirmed that the desired peptide was synthesized.

C825-37mer P B S (0,1
M Phosphate buffered 5a
200 IA was added to each well of the immunoplate and reacted overnight at 4°C. PBS containing O1% Tween 80
(PBS-T) and 0.1% BSA (B
Post-coating was performed with 250 JA/well of PBS solution (Ovine Serum Albumin), PBS solution.

Thereafter, 20 rows of 0.1% BSA PBS-T were added to each well, 20 Δ of plasma samples were added thereto, and after stirring gently with a bra mixer, the mixture was reacted at 37° C. for 1 hour.

Next, after washing with PBS-T, anti-Human Ig
Add G-HRP conjugate at 200 d/well;
The reaction was carried out at 37°C for 1 hour.

After washing with P B S-T, T M B Z (3, 3,
5,5,-Tetramethylbenzidine
After adding 200 A of the substrate solution (hydrochloride) to each well and reacting at 37°C for 30 minutes, 50 A/well of 2M sulfuric acid was added to stop the reaction. Emaxprecisio this
n 5icroplate reader (Mole
450nm/
Measurements were performed at two wavelengths of 650 nm. (The above measurement system and operation are referred to as 37aer ELISA for convenience.) Similarly, C825-37mer and C,100J-29
Mix wr e r and dissolve in PBS to 1M each.
It was bound to an immunoplate as g/- and ELISA was performed. The 37+29ser ELISA (referred to as 37+29mer ELISA for convenience) is a commercially available HCV Ab EL manufactured by Ortho, which is widely used worldwide.
When 87 IS^Kit-negative normal human plasma samples were measured, the average value (^VG) was 0.0467, standard deviation (SD) 0.
Two ELIs of the present invention with a cutoff value (COV) of 0385 and AVG + 6SD = 0.214
When approximately 90 hepatitis patient plasma samples were measured using the SA system, the results shown in Table 2 were obtained. At the same time, the results measured using the ortho HCV^b ELISA kit for this patient group are shown. Ortho HCV Ab in PBC 37+29ser ELISA
Samples that could not be detected with the ELISA kit were detected, and this is thought to be due to differences in the third gene region encoding the antigenic peptide used in the ELISA system. 68,4%) (28,9%) (71,IJ) B BC Phage with C8251 gene inserted into λgtll,
λgt1)-C825 (patent application No. 1-238848) was cut with EC0RI to extract the C825 gene, and this was used as a plasmid for expressing beta-galactosidase fusion protein.
It was integrated into puEX 2 (manufactured by 7v>vmu Co., Ltd.) to produce a plasmid for expressing beta-galactosidase C825 (βgalC825) fusion protein.

This was infected and transformed into Escherichia coli, pkk322,
Culture was performed. The cells were collected by centrifugation, and egg white lysozyme was added at a concentration of 20 (lμ9/-), and the cells were reacted at 4°C for 16 hours to digest the cells. The supernatant was removed by centrifugation, and the pellet was collected. This beret is 0.05
% Triton-X100 PBS solution,
The pellet was washed by centrifugation. This washing operation was repeated three times. Finally, the pellets were washed with PBS and collected.

1g of pellet was mixed with 8M urea, 20mM Tris-1
)c130 (1mM F1aCl, 28M D
Dissolve in T T solution 100- and centrifuge for 5 ephacr.
ylS-300 (5cmφ x 89.8cmh)
Column I/\ liquid was passed through. Fractionate the effluent and divide each fraction into 5
Checked with DSPAGE. β galc82
The purified fractions of 5 were pooled and dialyzed against PBS to recover 12.6 g of purified βgalc825.

Purified βgalc8251μg/d, synthetic peptide CI
Q(IJ29mer 1ttQ/d P B S solution was prepared, solidified in the same manner as in Example 1, and (βga1825
．． c100J-29++er) ELISA system was created. Using this ELISA system, 344 samples from normal subjects and 286 samples from patients with hepatitis were measured in the same manner as in Example 1.

The cut-off value was calculated as 0528 as a result of normal human sample measurement. Table 4 shows the measurement results of hepatitis patient samples.

Table 4 AIIB 2) 5%) IA ■R ^II (LC L Real IJL-Dan The above desired peptide was synthesized and purified, and C82530s
er 1 ttQ/d, C100J-29s+er
4 u9/- in PBS, assemble the same ELISA system (4 peptides) as in the example, measure normal human samples to calculate the cutoff value, and calculate the cutoff value.
A hepatitis patient specimen was measured using a LISA system. (Table 5) In this ELISA system, ortho-HCV Ab ELISA
It showed a higher detection rate for NANBH patients than the other kits.

ANBH A B ^IH LC Table 5 107 (86,5%) 0χ) Below margin tJJ! Non-specific reaction when using a fusion protein Using a plate immobilized with βgaic825 protein derived from genetic recombination technology, ELISA was performed on 50 samples from 14 chimpanzees uninfected with non-A, non-B hepatitis. However,
Chimpanzee testing positive (Ilo, 121No, 12
There were 3 serum samples of 4).

The three samples were mixed 1:1 with lysate of Escherichia coli HBIOI (which does not incorporate the expression plasmid), which is an expression host for βgal-C825 protein, reacted overnight at 4°C, and then subjected to the above ELISA. All samples turned negative, and the reaction was suppressed. Therefore, it was found that these chimpanzees possess antibodies against E. coli components, and that in ELISA using proteins expressed in microorganisms, contaminant proteins that cannot be removed during the culture purification process cause non-specific reactions. .

On the other hand, three synthetic peptides of the C825 region of the present invention (C
825-30, 34, 37mer) using EL
When ISA was prepared and the three samples mentioned above were measured, all showed negative results. The results are shown in Table 6.

βgal 00° Table 6 0v (30,34 D 30mer) Ov Margin below

Claims (5)

[Claims] (1) At least one type of antigenic peptide selected from peptides having the following amino acid sequences, or a part of the peptides: C825-37mer [Amino acid sequence is available] C825-34mer [Amino acid sequence is available] C825-30mer [ There is an amino acid sequence] (2) At least one type of antigenic peptide selected from peptides having the following amino acid sequences, or a part of the peptides: C100J-29mer [Amino acid sequence is available] C100J-37mer [Amino acid sequence is available] (3) The antigenic peptide described in claim (1) or a part of the peptide, and claim (2)
An immunological conjugate between the antigenic peptide described in item ) or a part of the peptide, and an anti-hepatitis C virus (HCV) antibody present in the sample and the peptide. A method for detecting an anti-HCV antibody, which comprises contacting a sample under conditions in which the above-mentioned immunological conjugate is formed, and measuring the formation of the immunological conjugate to confirm the presence of the anti-HCV antibody in the sample. (4) Claim No. 3 in which the antigenic peptide is a peptide represented by C825-37mer (has an amino acid sequence), C100J-29mer (has an amino acid sequence), or a mixture of a portion of each peptide. Anti-HCV described in section )
Antibody detection method. (5) Anti-HC according to claim (3) selected from ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), Western blotting, and agglutination method
V antibody detection method.