JPH08205893A - Method for measuring activity of hcv protease - Google Patents

Abstract

PURPOSE: To measure the subject enzymic activity required for developing an antihepatitis C virus (HCV) agent by expressing a gene in a specific region of an HCV precursor polyprotein in Escherichia coli and reacting the resultant HCV protease with a synthetic substrate. CONSTITUTION: An HCV protease obtained by integrating a gene capable of coding an NS3 region and NS4A region and, as necessary, a region on the downstream side thereof in a nonstructural protein of an HCV precursor protein into a vector, then transducing the resultant vector into Escherichia coli and expressing the gene therein is reacted with a synthetic substrate peptide labeled with biotin or an antigenic substance or an antibody substance [label (a)] and a labeled peptide which is a cleavage product thereof is further labeled with an antigenic substance [label (b)] to join the antigenic substance labeled in the peptide to an enzymically labeled antibody against the antigen. The enzymic activity thereof is then measured to measure the HCV protease activity useful for the screening, etc., of an HCV protease inhibitor with good operating efficiency and quantitative determinability.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an assay system indispensable for developing anti-HCV agents. More specifically, the invention relates to methods for measuring HCV protease activity.

[0002]

2. Description of the Related Art It is estimated that there are 1.2 million chronic non-A non-B hepatitis patients in Japan, 60% of which are attributed to hepatitis C virus (HCV). .
Chronic hepatitis C is highly likely to be transferred to cirrhosis and hepatocellular carcinoma. Moreover, HCV carriers occupy 1 to 2% of the population in Japan, and although the ratio decreases as the younger age group, these carriers carry the risk of developing hepatitis in the future. Therefore, it is necessary to eliminate the virus early in the onset of non-A non-B hepatitis. Treatment with interferon is the most common treatment, but its cure rate is only 20-40%,
It also has serious side effects and expensive costs. Therefore, development of more effective anti-HCV agents is desired.

Since HCV revealed the nucleotide sequence of a part of the viral gene in 1988 by Chiron (USA) ((Q.-L. Choo et al., Science, 244, 359, (1989),
G. Quo, et al. Science, 244, 362 (1989))), and molecular elucidation by molecular biology techniques. As a result H
CV is an RNA similar to flavivirus and pestivirus
It became clear that it was a virus. In these viruses, a long continuous protein is translated in a host cell by using single-stranded RNA that is a gene as mRNA. Viral proteins are divided into structural proteins that make up viral particles and nonstructural proteins (NS) that perform the necessary functions in the viral life cycle. Structural proteins are cleaved by the endopeptidase of the host and are nonstructural. The protein is cleaved by a protease translated from a viral gene to become a functional protein. Virus-derived proteases include other viruses such as retroviruses such as HIV (human immunodeficiency virus), HAV (A
Its presence has been confirmed in piconaviruses such as hepatitis C virus (H.-G. Krausslich and E. Wimmer, Ann.
Rev. Biochem. 57, 701, (1988)). Yellow fever virus belonging to HIV or flavivirus (TJChambers
et al., Proc. Natl. Acad. Sci. USA, 87, 8898 (199
In 0)), it was revealed that viral infectivity was lost by inhibition of viral protease. In HCV, the presence of protease was predicted by the gene analysis as described above, but the fact that the protease activity was confirmed was confirmed by cell-free transcription / translation experiments of viral gene DNA or animal cells (M.Hijikata et al., J. Virol., 67,
4665 (1993), A. Grakoui et al., J. Virol., 67, 2832
(1993)) or in a transient expression system in E. coli.

On the other hand, an enzyme reaction system for measuring protease activity using an isolated and purified enzyme is expected to have good operability and quantification. Japanese Patent Publication No. 5-507612 suggests the possibility of an assay using an isolated and purified enzyme, but the content is preliminary and does not meet the requirements required as a new enzyme. That is, the characteristics of the enzyme as a substance, the specification of the amino acid sequence, the specification of the cleavage site as the action of the enzyme, the method for measuring the titer, etc. are not shown.

The present inventors have previously applied for a patent (Japanese Patent Application No. 5-1
8854), the possibility of the assay using the isolated and purified NS3 enzyme was shown, and the enzyme characteristics and the cleavage site were clarified. Here, the NS3 enzyme refers to a protease existing in the NS3 region of the nonstructural protein region of HCV (see FIG. 1). However, the enzyme due to the expression of only NS3 is unstable and has low activity, and a more stable enzyme with high cleavage activity is desired.

[0006] On the other hand, as a possibility of other proteins contributing to the activity of the enzyme, Failla et al. Expressed that not only NS3 but also NS4A in Hela cells was NS3 / NS4A,
It is required for cleavage of NS4B / 5A, and it has been reported that expression of NS4A also accelerates cleavage of NS4A / 4B, NS4B / 5A (J. Virol., 68, 3753-376).
0 (1994)). However, such an animal cell expression system has various problems in terms of operability and quantification as described above.
That is, the protease inhibitor assay in the cell culture system has the following drawbacks in terms of operability as compared with the enzyme reaction system: To carry out the assay by the method of Failla et al., I) pre-incubate Hela cells up to 4 x 10 ⁵ cells / plate (1 to 7 days) ii) adsorb vaccinia virus to Hela cells (~ 2
time). iii) Transfection of expression vector (~ 1
Time) iv) Labeling with ³⁵ S-methionine (up to 1 hour) v) Recovering cells after 3 hours vi) Immunoprecipitation of the protein is required, and the above procedure requires at least 2 days. 2. Consumables are expensive, including cell culture media, serum and disposables. 3. Operations such as aseptic operation are complicated and require skill. 4. A clean bench is required for cell manipulation, and a facility suitable for recombination experiments is required.

In addition, it has the following drawbacks in terms of sensitivity and quantitativeness: In order to detect a small amount of protein expressed in cells, a detection method having a radioisotope or equivalent high sensitivity must be used. 2. The results are highly dependent on the state of the cells and the values vary between assays. 3. It is difficult to control the enzymatic reaction quantitatively and temporally. That is, since the enzyme and the substrate are biosynthesized in the cell and the enzymatic reaction occurs successively, and the biosynthesis and the enzymatic reaction occur almost in parallel, it is difficult to determine the amounts of the enzyme and the substrate and the starting point of the enzymatic reaction. 4. Numerous control experiments are needed to rule out the involvement of unknown factors in cells.

[0008] Therefore, there is a demand for a method of assaying HCV protease activity which is superior in quantification with a simpler operation.

[0009]

It is considered that viral protein processing is essential for the production of infectious HCV. Therefore, inhibition of the viral protease essential for processing of HCV nonstructural proteins is
It is believed to block infections and thus has the potential to be an effective anti-HCV agent with few side effects. To develop such inhibitors, it is essential to establish an efficient method for measuring HCV protease activity.

[0010]

As a result of intensive studies by the present inventors to complete an efficient method for measuring HCV protease activity by overcoming the above-mentioned drawbacks of the prior art, HC
N, which was previously considered necessary and sufficient as a V protease
By expressing not only the S3 region but also the downstream region from NS3 in Escherichia coli at the same time and isolating it by a simple purification method, it was possible to obtain a large amount of an isolated enzyme that was stable and had high cleavage activity. In addition, by using a synthetic peptide in the vicinity of the cleavage site of HCV protease as a substrate and combining it with the above enzyme, we succeeded in completing a measurement method excellent in operability and quantification. The method for measuring HCV protease activity of the present invention is also useful for screening protease inhibitors.

That is, the present invention expresses in Escherichia coli the genes encoding the NS3 region and NS4A region in the nonstructural protein of the hepatitis C virus (HCV) precursor polyprotein, and optionally the downstream region thereof, Provided is a method for measuring HCV protease activity, which comprises reacting an HCV protease obtained by separation and purification with a synthetic substrate peptide.

Further, the present invention provides the hepatitis C virus (H
CV) N in the nonstructural protein of the precursor polyprotein
Expressing in S. coli the genes encoding the S3 and NS4A regions, and optionally the downstream region thereof,
In a system in which an HCV protease obtained by isolation and purification is reacted with a synthetic substrate peptide, a test compound is added to the reaction system to allow the cleavage reaction of the synthetic substrate peptide to proceed.
Provided is a screening method for HCV protease inhibitors, which comprises comparing with and without addition of a test compound.

The assay method and screening method of the present invention do not use a cell culture system, but isolate and purify H in vitro.
CV protease and synthetic substrate peptide are used.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, HC to be expressed
The V protease region may be any region as long as it is a region containing NS3 and NS4A. Therefore,
The HCV protease region to be expressed includes i) a region containing the NS3 region and NS4A region, ii) a region containing the NS3 region, NS4A region and NS4B region, iii) a region containing the NS3 region, NS4A region, NS4B region and NS5A region, Or iv) NS3 region, NS4A region, NS4B region, NS5
A region including the A region and the NS5B region is included. Although several subtypes of HCV are known, the amino acid sequence of any subtype may be used.

Corresponds to the above HCV nonstructural region gene
The construction and construction of the cDNA fragment are described in the specification of the previous patent application (Japanese Patent Application No. 5-18854) and J. Virol., 67, 4665 (19).
93), or Proc. Natl. Acad. Sci. USA, 90, 10773 (1
It can be carried out by the method described in 993).

HCV protease uses the above gene,
It can be expressed in Escherichia coli, animal cells, insect cells, rabbit reticulocyte hemolysate and the like.

The expression vector for expressing these HCV proteases may be any one as long as it can efficiently express the fusion protein. For example, pTZ18, pT
Z19, pUC18, pUC19, Bluescript
t KS, SK, pHSG398, pRSET, pGE
Examples include X-2T and pRIT2T. In order to increase the stability of the expressed protease and facilitate purification, pMAL-c or the like which is expressed in the state of being fused with maltose binding protein (MBP) on the amino acid terminal side of the protease is preferable. As the Escherichia coli strain used for expression, HB101, TB1, JM105 and the like can be used, but recA ⁻ strain JM109, which is unlikely to cause mutation in the recombinant plasmid, is preferable.

The synthetic peptide used as a substrate is HCV.
NS3 / NS4 identified as a protease cleavage site
A, NS4A / NA4B, NS4B / NS5A, NS5A / NS5B amino acid sequences near each cleavage site may be used, and one or more amino acids other than these consensus amino acids may be substituted or deleted. Alternatively, it may be added. Consensus amino acids NS3 / NS4A, NS4A / NA4B, NS4B / NS5A, NS5A
/ NS5B is an amino acid conserved in the amino acid sequence near each breakpoint. That is, the underlined amino acids in Table I below.

Table I HCV Subtype Sequence HCV Site H-FDA CMSA D LEVV T S TWVLVGGVL NS3 / NS4A H-AP CMSA D LEVV T S TWVLVGGVL HCV-1 CMSA D LEVV T S TWVLVGGVL HCV-J CMSA D LEVV T S TWVLVGGVL HCV-BK CMSA D LEVV T S TWVLVGGVL HC-J6 CMQA D LEVM T S TWVLAGGVL HCV-T CMSA D LEVV T S TWVLVGGVL HC-J8 CMQA D LEIM T S SWVLAGGVL HCV-JT, JT 'CMSA D LEVV T S TWVLVGGVL H-FDA YQEF D EMEE C S QHLPYIEQG NS4A / NS4B H-AP YQEF D EMEE C S QHLPYIEQG HCV-1 YREF D EMEE C S QHLPYIEQG HCV-J YQEF D EMEE C A SHLPYIEQG HCV-BK YQEF D EMEE C A SHLPYIEQG HC- J1, J4 YEAF D EMEE C A SRAALIEEG HCV-T YQEF D EMEE C A SHLPYIEQG HC-J8 YQAF D EMEE C A SKAALIEEG HCV-JT, JT 'YREF D EMEE C A SHLPYIEQG H-FDA WISS E CTTP C S GSWLRDIWD NSB NS5A H-AP WISS E CTTP C S GSWLRDIWD HCV-1 WISS E CTTP C S GSWLRDIWD HCV-J WINE D CSTP C S GSWLKDVWD HCV-BK WINE D CSTP C S GSWLRDVWD HC-J1, J4 WITE D CPIP C S GSWLRDVWD HCV- T WINE D CSTP C S GSWLRDVWD HC-J8 WITE D CPVP C S GSWLQDIWD HCV-JT WINE D CSTP C S GSWLKDVWD HCV- JT 'WINE D CSTP C S GSWLRDVWD H-FDA GADT E DVVC C S MSYTWTGAL NA5A / NS5B H-AP GADT E DVVC C S MSYSWTGAL HCV-1 EANA E DVVC C S MSYSWTGAL HCV-J GEAG E DVVC C S MSYTWTGAL HCV-BK EEAS E DVVC C S MSYTWTGAL HC-J6 SEED D SVVC C S MSYSWTGAL HCV-T EEDG E GVIC C S MSYTWTGAL HC-J8 SDQE D SVIC C S MSYSWTGAL HCV-JT, JT 'GEAS D DIVC C S MSYTWTGAL Consensus DCSETA (Grakoui, Grakoui, et al., J. Virol., 67, 2832 (1993))

The consensus amino acid D or E,
C or T, as well as S or A can be freely combined at each position. That is, not only the above combination of D, C, S, E, T, A, but also D, T, S, E,
It may be a combination of C and S.

The length of the synthetic peptide is preferably 5 to 25 amino acids, but preferably 10 to 20 amino acids. For example, YQEFDEMEEC ASHLPYIEQG, WINEDCSTPC SGSWLKDVWD,
GEAGDDIVCC SMSY TWTGAL etc. are mentioned. Most preferred are GEAGDDIVPC SMSYTWTGAL, GEAGDDIVPC SMSYTW
T, DDIVPC SMSYTWT, and DDIVPC SMSYT, which have almost the amino acid sequence containing the cleavage point of NS5A / NS5B in which the cleavage reaction proceeds most rapidly in E. coli. Moreover, in the original amino acid sequence GEAGDDIVCC SMSYTWTGAL, the disadvantage that two adjacent cysteines rapidly form a disulfide bond and become a compound that does not undergo an enzymatic reaction is improved by converting P2 into proline. Proline at the P2 position is present at the natural NS4B / NS5A cleavage point, and not only does it hinder the recognition of the natural cleavage point of HCV protease, but also enhances the solubility of the peptide.

The N-terminal or C-terminal of these peptides can be labeled with a fluorescent, luminescent, chromogenic group, an affinity ligand, an antigen or the like. For example, if the N-terminal is labeled with a dansyl group (Dns-) or FITC group of a fluorescent reagent, the signal of the peptide can be detected with high sensitivity by HPLC detection without interference of contaminants. Biotin, which is an affinity ligand, is labeled at the N-terminal or C-terminal and immobilized on an avidin-coated microtiter plate, and detection can be performed by ELISA. Alternatively, it can be detected by the ABC method. In addition, the antigen digoxigenin
If the N terminal or C terminal is labeled with (Dig), it can be detected with an anti-Dig antibody.

The enzyme reaction can be carried out at a pH of 5 to 10, preferably pH 7 to 8. Calcium, magnesium and manganese can be added as divalent ions. The reaction temperature may be 0 to 90 ° C, preferably 25 to 60 ° C.

The reaction can be detected by HPLC, TLC or ELISA.

The reaction proceeds with time, and includes i) a region containing the NS3 region and NS4A region, ii) a region containing the NS3 region, NS4A region and NS4B region, and iii) an NS3 region, NS4A region, NS4B region and NS5A region. And iv) NS3 region, NS4A region, NS4B region, NS5
A region including the A region and the NS5B region is expressed by any of 2
Over 90% of the substrate was cleaved after time. On the other hand, when the HCV protease expressing only the NS3 region was used, the progress of the reaction was significantly slower than that of other proteases (see FIG. 3 and Table 2).

Further, protease inhibitors can be screened by using the HCV protease activity assay method of the present invention. In a system for reacting the above-mentioned HCV protease with a synthetic substrate peptide, a test compound that is a candidate for an inhibitor is added to the reaction system, and the progress of the cleavage reaction of the synthetic substrate peptide is not added to that with the test compound added. Screening can be done by comparison with the ones. In the screening method of the present invention, as shown in Example 4, a test compound was added to the enzyme reaction solution and preincubated at 37 ° C. for 15 minutes,
Then, a peptide substrate with fluorescently labeled N-terminal was added to
Incubate for 10 minutes at 7 ° C. After that, the reaction can be stopped by heating at 90 ° C. for 5 minutes, and stored on ice for measurement. Therefore, 1
The measurement can be completed within the time, and the measurement can be performed in an extremely short time as compared with the above-mentioned cell culture system.
Further, the facility to be used may be simple without complicated operations such as aseptic operation. Furthermore, since no cells are used,
Compared to cell culture systems, there is no variation between measurements and reliable results are obtained. It is also an advantage of the present invention that it is not necessary to use a detection method for radioactive isotopes.

In another aspect of the present invention, the present invention provides a method for measuring HCV protease activity, which comprises the following steps: (1) In a non-structural protein of hepatitis C virus (HCV) precursor polyprotein HC obtained by expressing genes encoding NS3 region and NS4A region, and optionally downstream region thereof in Escherichia coli, isolating and purifying
V protease is reacted with biotin or a synthetic substrate peptide labeled (labeled a) with an antigenic substance or an antibody substance; (2) the labeled peptide that is the cleavage product of step (1) is further labeled with an antigenic substance (labeled b) (3) bind an enzyme-labeled antibody against the antigen to the antigen substance labeled with a peptide; and (4) measure the activity of the enzyme in step (3); provided that steps (2) and ( 3) or before step (4), the label a
The labeled peptide is immobilized by utilizing its affinity for.

This method enhances the versatility of the ELISA method, which has a high processing capacity, when the activity of a large number of samples has to be measured, such as screening of HCV protease inhibitors, and it is easy to change the substrate. It is useful in that it can deal with. This method is
This is a post-labeling method in which an antigenic substance is bound to the product after the enzymatic reaction prior to the ISA method.

The synthetic substrate peptide used in the above method is
In order to specifically perform the labeling reaction with respect to the amino group generated by protease cleavage, there should be no reactive amino group other than this amino group. Further, in order to immobilize the reaction product obtained by the labeling reaction, it is necessary to previously label the C-terminal side or the N-terminal side of the cleavage site of the synthetic substrate peptide with biotin or an antigen substance or an antibody substance ( This sign is called sign a). As long as such conditions are satisfied, the amino acid sequence of the substrate peptide can be freely designed within the substrate specificity of the enzyme.

However, as shown in the following Examples, when cysteine is contained in the substrate peptide, cysteine thiol (S
The H) group may also be labeled with an antigenic substance. When such a substrate peptide is used, the thiol group of cysteine must be modified to prevent the thiol group from being labeled with the antigenic substance, prior to the labeling reaction with the amino group. The modifier for the thiol group is not particularly limited as long as it does not inhibit the subsequent labeling reaction with the amino group and does not affect the subsequent treatment. Preferred thiol group modifiers are N-methylsuccinide, N-
Includes N-alkylsuccimides such as ethylsuccinide, and iodoacetic acid, sodium iodoacetate. Iodoacetic acid and sodium iodoacetate are particularly preferable because of their high solubility in water. These modifiers include water or buffers,
It is used as a solution of alcohol, dimethyl sulfoxide, dimethylformamide or the like. The reaction conditions are not particularly limited, but a reaction temperature of room temperature to 50 ° C. and a reaction time of 30 minutes to 60 minutes are sufficient. The amount of the thiol group modifying agent is not particularly limited as long as the modification reaction with respect to the thiol group contained in the sample completely proceeds under the above reaction conditions.

Next, the labeled peptide which is the cleavage product in the above reaction is further labeled with an antigenic substance (this label is referred to as label b). For example, when the label a is attached to the N-terminal side of the cleavage site of the substrate peptide, the label b is applied to the carboxyl group.

The type of the antigenic substance is not limited as long as it is a substance capable of producing an antibody against it. This antibody corresponds to the secondary antibody in a normal ELISA, and it must be labeled with the enzyme used in the detection reaction in actual use. Therefore, an enzyme-labeled antibody is commercially available because it is easily available. Digoxigenin, which has been used, is suitable. However, since digoxigenin itself does not react with an amino group, in order to label the amino group with digoxigenin, digoxigenin must be converted to hydroxysuccimidation which is a modifier of the amino group. That is, the labeling reaction for the amino group is digoxigenin-3.
-O-methylcarbonyl-ε-aminocaproic acid-N-
Hydroxysuccinimide ester (hereinafter referred to as DIG-NHS
) And other digoxigenin labeling reagents.
As a solvent for DIG-NHS, it is completely miscible with water, D
There is no particular limitation as long as it does not react with IG-NHS itself. Dimethyl sulfoxide and dimethylformamide are preferred. The reaction temperature is not particularly limited, but may be room temperature to
A range of 50 ° C is sufficient. The reaction time is also not particularly limited, but 30 minutes to 60 minutes is sufficient. DIG-N
The concentration of HS is not particularly limited, but it can be used in the range of 0.01 to 1 mg / ml depending on the reaction temperature, the reaction time, and the amount used.

An excess amount of D was added to the labeling reaction of digoxigenin.
Since IG-NHS is used, unreacted DIG-N is used so as not to hinder the subsequent steps (immobilization and enzyme reaction).
Inactivate HS. The inactivation treatment can be carried out by adding a substance having an excess amount of amino groups and reacting with DIG-NHS. Such a substance is not particularly limited as long as it has a primary amino group and is a water-soluble substance. Preferred examples include amino acids and their derivatives. The pH of the aqueous solution is slightly alkaline,
The pH is preferably in the range of 7.5 to 10. The concentration and the amount used can be changed according to the concentration and the amount used of DIG-NHS, and it is not particularly limited as long as it completely reacts with unreacted DIG-NHS. The reaction conditions are not particularly limited, but may be 30 minutes to 60 at a temperature in the range of room temperature to 50 ° C.
Minutes are enough.

The above treatment enables selective labeling of digoxigenin with respect to the terminal amino group of the fragment generated by protease cleavage.

Next, the obtained digoxigenin-labeled cleavage fragment is immobilized. However, as described in the claims, the time of immobilization may be at this stage, before the above-mentioned labeling b, or immediately before the measurement of the enzyme activity.

To perform immobilization, the affinity for the label a is used. For example, when the label a is biotin, avidin or streptavidin is used, when the label a is an antigen, an antibody against the antigen is used, and when the label a is an antibody, the antigen against the antibody is used as a carrier. Immobilize. As the carrier, a solid carrier is preferable, and for example, in addition to a polymer carrier such as styrene or polystyrene molded into an arbitrary size and shape, an inner wall of a reaction container molded with these materials, specifically, with respect to the label a It is carried out using a tube coated with a substance showing affinity (streptavidin, antibody, antigen), microtiter plate, beads, column carrier and the like. Immobilization can be performed using the whole amount or a part of the digoxigenin-labeled reaction liquid depending on the detection sensitivity, the mass of the substrate and the amount of the reaction liquid. At that time, it is also possible to dilute with a buffer solution containing a surfactant. As the surfactant, Nonidet P-
40, Tween 20, Triton
A nonionic surfactant such as ton) X-100 is preferable, and its concentration of 0.001 to 1% is sufficient. The temperature and time for immobilization are not particularly limited, but the temperature is 4
C. (in the refrigerator) to 37.degree. C., and the time can be 1 to 24 hours.

After immobilizing the unreacted substrate peptide and the labeled cleavage product fragment, washing is performed to remove contaminants such as enzymes in the reaction solution. In addition to the diluent used for immobilization, TBS (Tris Buffered Salin), which is widely used in biochemical experiments, is used as the washing solution.
e) and PBS (Phosphate Buffered)
A buffer such as Saline) can be used.

Subsequent operations can be performed in the same manner as in a normal ELISA. That is, after washing, the peptide-labeled antigenic substance is subjected to enzyme-labeled anti-digoxigenin antibody (hereinafter referred to as enzyme-labeled antibody) against the antigen.
Combine. Prior to the binding step, a treatment with a blocking reagent is preferably performed in order to prevent nonspecific adsorption on the surface of the device used for immobilization. Skim milk used in Western blotting as a blocking reagent,
Casein etc. can be used. The blocking treatment can be performed at a temperature of 4 ° C to 37 ° C for 1 to 24 hours.

The binding of the enzyme-labeled antibody to digoxigenin selectively labeled on the cleaved fragment of the substrate peptide depends on the stability of the labeled enzyme (labeled enzyme), but
It can be carried out at a temperature of from 370C to 37 ° C for 1 to several hours. Usually, one hour at room temperature is sufficient. Alkaline phosphatase and peroxidase are often used as the labeling enzyme, but the labeling enzyme is not particularly limited.

To remove excess enzyme-labeled antibody that has not bound, wash with a buffer such as TBS. Then, a substrate solution for the labeling enzyme is added to carry out an enzymatic reaction. After stopping the enzymatic reaction with acid or alkali, the absorbance or fluorescence intensity of the product generated by the enzymatic reaction is measured.

When this method according to the present invention is used, the reaction product can be labeled with an antigenic substance such as digoxigenin, and the measurement can be carried out in the same manner as in the conventional ELISA method.
Further, the conventional ELISA method requires an antibody (primary antibody) to the substance to be measured, but the method of the present invention does not require an antibody specific to the substance to be measured, and thus can be used in a wide range.

In Examples 7 to 12 below, as a HCV protease, a protease (345P derived from a region containing the NS3 region, NS4A region and NS4B region of HCV) was used.
s), biotinylated (ε-amino group of C-terminal lysine) and acetylated (N-terminal amino group) as a substrate peptide, Ac-GEAGDDIVPCSMSYTWTK [Bi
However, the method of the present invention can be widely used without being limited to this example.

The method for measuring HCV protease activity and the screening for protease inhibitors of the present invention will be explained in detail by the following examples, but the scope of the present invention is not limited thereto.

[0044]

【Example】

Example 1: Construction of enzyme expression vector The construction and construction of a cDNA fragment corresponding to the HCV nonstructural region gene was described in the specification of the previous patent application (Japanese Patent Application No. 5-18854).
No.), and J. Virol., 67, 4665 (1993), or Proc.
Natl. Acad. Sci. USA, 90, 10773 (1993).

PMANS2d3X : As described in Japanese Patent Application No. 5-18854, a vector pMal-c is obtained by inserting a cDNA fragment corresponding to amino acids 985-1325 on the ORF (open reading frame) of HCV.

PMANS34NsH : HCZ ORF on pTZ18 722-
PN722 containing a cDNA fragment corresponding to the 1647th amino acid
-1647 was digested with Sac II-Hind III and inserted into pMANS2d3X digested with the same restriction enzymes to obtain pMANS34Ns. This was cleaved with EcoT22-HindIII and a synthetic DNA fragment was inserted to obtain pMANS34NsH.

PMANS34Sm : pTZ18 on HCV ORF 722-1
PN722 into which a cDNA fragment corresponding to amino acid 908 was inserted
The fragment obtained by cutting 1908 with Sac II-Hind III was replaced with the fragment cut with the same restriction enzymes of pMANS2d3X to obtain pMANS34Sm. p MANS345C : H on pTZ18
PN729-3010 into which a cDNA fragment corresponding to amino acids 729-3010 on the ORF of CV was inserted was cleaved with Sac II-Sal I to give p
Insert into MANS2d3X digested with the same restriction enzyme and insert into pMAN
I got S345C.

PMANS345Bs : HCZ ORF on pTZ18 722-
PN722 containing a cDNA fragment corresponding to the 2472nd amino acid
The fragment obtained by digesting-2472 with Pst I-Hind III was replaced with the fragment digested with the same restriction enzyme of pMANS345C to obtain PMANS345Bs.

PMANS345Ps : pMANS345C was cut with Pst I and re-enzymatically ligated to delete a small fragment resulting in pMANS345Ps.

Example 2 Expression and Purification of Enzymes E. coli strain JM109 competent cells are transformed with the above expression vector. The transformed E. coli was pre-incubated overnight at 37 ° C with L Broth containing 50 μg / μl ampicillin, and 50 μg /
Dilute with L Broth containing 20 μl ampicillin (20-30 times),
The cells were cultured at 37 ° C for about 2 hours. When the turbidity reached OD ₆₀₀ = 0.5 to 0.7, isopropyl beta thiogalactoside at a final concentration of 0.5 mM was added, the mixture was further cultured at 20 ° C. for 3 hours, and then the culture solution was centrifuged to collect bacterial cells. The cells were stored in a freezer at -80 ° C.

The preserved cells were buffered with 1/10 volume of the buffer A (10 mM Na-phosphate buffer pH 7.2, 30 mM NaCl,
The cells were suspended in 10 mM beta-mercaptoethanol) and disrupted with an ultrasonic disruptor for 10 minutes under ice cooling. The supernatant was obtained by centrifugation, and ammonium sulfate was added to the supernatant to 70% saturation to precipitate the protein. The precipitate was collected by centrifugation, dissolved in 1/2 volume of the supernatant, Buffer A, and applied to an amylose resin column. After washing the column with buffer A, buffer B (10 mM Na-phosphate buffer pH7.2, 30
mM NaCl, 10 mM beta mercaptoethanol, 0.2%
Tween 20), buffer C (10 mM Na-phosphate buffer pH
7.2, 500 mM NaCl, 10 mM beta-mercaptoethanol). The protein bound to the column was eluted with buffer A containing 10 mM maltose. The eluted protein was concentrated by ultrafiltration, glycerol was added to a final concentration of 50%, and the protein was stored in a freezer at -20 ° C. The resulting enzyme was analyzed. That is, the obtained enzyme preparation was dissolved in a sample buffer, heated at 100 ° C for 5 minutes, and then SDS-
After applying to polyacrylamide gel and electrophoresis,
Stained with Macy Brilliant Blue. Alternatively, after electrophoresis, the developed protein is transferred to a PVDF membrane to
Western blot was performed with 3 antibody and anti-MBP antibody.

Example 3: Synthesis of substrate peptides Peptides Dns-GEAGDDIVPC SMSYTWTGAL, Dns-GEAGDDIVAC
SMSYTWTGAL, Dns-GEAGDDIVPN SMSYTWTGAL, Dns-GEAGDDI
VP (D) C SMSYTWTGAL, Dns-GEAGDDIVPC SMSYTWT, Dns-GEA
GDDIVPC SMS, Dns-GEAGDDIVAC SMS, Dns-GEAGDDIVCC SM
S, Dns-DDIVPC SMSYT, Dns-DDIVPC SMS, Dns-DDIVPC SM
SYTWT, Dns-GEAGDDIVPC SMSYTWTK, Dns-GEAGDDIVSC SMS
YTWTGAL, Dns-WINEDCSTPC SGSWLKDVWP, Dns-YQEFDEMEEC
ASHLPYIEQG was performed by a solid phase method using a peptide synthesizer. That is, the α-amino group was protected with t-butyloxycarbonyl group, the side chain carboxyl group was protected with cyclohexyl group, the hydroxyl group was protected with benzyl group, the thiol group was protected with methylbenzyl group, and the ε-amino group was protected with benzyloxycarbonyl group. The two amino acids were condensed with dicyclohexylcarbodiimide to the first amino acid whose carboxyl group was bound to the resin and whose α-amino group was unprotected, and then the α-amino group was removed with trifluoroacetic acid. Similarly, the third and subsequent amino acids were sequentially synthesized from the C-terminal to the N-terminal. After the synthesis of the whole chain, the α-amino group was removed with trifluoroacetic acid, modified with dansyl chloride, and cleaved from the resin and deprotected with hydrogen fluoride to obtain the above peptides.

In Ac-GEAGDDIVPC SMSYTWTK-Bio, α-amino group was protected, ε-amino acid of lysine immobilized on resin was biotinylated, and the N-terminal was acetylated after solid phase synthesis using the protected amino acid. Later deprotected.

Dig-GEAGDDIVPC SMSYTWTK-Bio was prepared by the same solid-phase synthesis and deprotection to form a disulfide bond, and the N-terminal amino group was labeled with digoxigenin-3-O-succinyl [2- (N-maleimido)] ethyramide. (Digoxigenin-
3-O-succinyl-[2- (N -maleimido)]-ethylamid
It was possible to cleave the disulfide bond with DTT in step o).

Example 4: Enzymatic reaction A standard enzymatic reaction was carried out under the following conditions. 50 mM
Tris HCl pH 7.6, 30 mM NaCl, 10 mM DTT, enzyme protein 4
To 100 μl of enzyme reaction solution containing μg-8 μg, the final concentration of the test compound dissolved in DMSO or aqueous solution was 1-10 μg / ml.
And pre-incubate at 37 ℃ for 15 minutes. In HPLC method, the peptide substrate (DM
SO solution) A final concentration of 86 μM (final DMSO concentration: 2%) was added to start the reaction. After reacting at 37 ° C, the reaction was stopped by heating at 90 ° C for 5 minutes, and the mixture was stored on ice. In the ELISA method, a substrate whose N or C terminus was fluorescently labeled or affinity labeled was similarly reacted.

Example 5: Detection (1) HPLC method 5 μl of the reaction solution was taken and applied to reverse phase HPLC (φ 4.6 mm × 15 cm). Column is 50 mM ammonium acetate (pH 6.5)
22.5% → 60% acetonitrile linear concentration gradient (10
Min), unreacted peptide substrate and cleavage product N
Fluorescence signal of terminal fragment is excited: 340 nm, em
ission: detected at 510 nm. The cutting rate was calculated from the ratio of the areas of the two.

(2) ELISA method Dig-GEA substrate whose N-terminus is labeled with digoxigenin (Dig) and the amino group of the side chain of C-terminal lysine is labeled with biotin (Bio)
After enzymatic reaction of GDDIVPC SMSYTWTK-Bio, take 1 μl of the reaction mixture, dilute it 2000 times with phosphate buffer (pH 8.5) containing 2% 2-mercaptoethanol and 0.01% NP-40, and stir 10 μl. Added to microtiter plates coated with avidin. After standing at room temperature for 1 hour, the plate was washed and reacted with an alkaline phosphatase-labeled anti-Dig antibody at room temperature for 1 hour. After washing the plate, the alkaline phosphatase substrate 2,2'-azido-di- (3-ethylbenzthiazoline sulfate) (2,2'-Azido-di- (3-ethylbenzth
200 μl of iazoline sulfate)) solution was added and reacted at room temperature for 10 minutes, and 50 μl of 1 N NaOH was added to stop the reaction. The absorbance at 405 nm was measured to detect the amount of residual Dig.

Example 6: Results (1) Name of enzyme Expression plasmids pMANS34NsH, pMANS34Sm,
E. coli was infected with pMANS345C, pMANS345Bs, and pMANS345Ps, and the enzymes expressed as maltose-binding protein and fusion protein were purified with amylose resin to produce 34NsH, 34Sm, 345, respectively.
Called C, 345Bs, 345Ps. FIG. 1 shows the name of the expression plasmid and the inserted region of the HCV gene.

(2) Detection of enzymatic reaction 0.045 μg of enzyme 345Ps and synthetic substrate Dns-GEAGDDIVPC SMSYTWT
Fig. 2 shows a HPLC chromatogram obtained when the was reacted under the above conditions. The unreacted substrate was eluted at a retention time of 7 to 8 minutes, and the cleaved product was eluted at a retention time of 3 to 4 minutes. At this time, the cutting rate was 24%.

(3) Time reaction curve 0.08 μg / μl each of 34NsH, 34Sm, 345C, 345Bs and 345Ps was reacted with the synthetic substrate Dns-GEAGDDIVPC SMSYTWT under the above reaction conditions, and the time reaction curve is shown in FIG. The reaction proceeds with time, and after 2 hours, 34Sm, 345C, 345Bs, and 345Ps are
90% or more disconnected. However, the progress of the reaction was significantly slower with 34NsH than with other enzymes.

Then, the N of the peptide in the cleaved reaction solution was
The ends were analyzed with an amino acid sequencer. The uncleaved substrate and the N-terminal fragment of the cleavage product are protected at the amino terminus with a dansyl group and are not analyzed. The amino acid sequence from the N-terminus of the C-terminal fragment of the cleavage product can be analyzed, but the results are expected. It was the amino acid sequence of SMSYTWT as described above, and it was confirmed that the cleavage occurred at the correct cleavage site.

(4) Reaction rate The reaction rates of 34NsH, 34Sm, 345C, 345Bs and 345Ps were compared. The reaction conditions were as follows: enzyme 0.08 μg / μl, standard solution (50 mM T
ris HCl (pH7.6), 30 mM NaCl, 5 mM CaCl ₂ , 10 mM DT
T) at 37 ° C for 30 min, then 0.05
μg / μl, 0.1 μg / μl, 0.2 μg / μl, 0.5 μg / μl, 1 μg
/ μl, 2 μg / μl of substrate was added and reacted at 37 ° C for 10 minutes. The reaction was stopped by heating at 70 ° C for 5 minutes. The results obtained are shown in the table below
Shown in II. As is clear from Table II, the cleavage activity is 345P.
s, 345Bs was the highest, followed by 345C, 34Sm. 34 Ns
H was significantly lower than these.

[0063]

[Table 1]

(5) Effect of Inhibitor Using the method described in Example 4, the effect of PMSF (phenylmethanesulfonyl fluoride), which is one of the serine protease inhibitors, on the protease activity was examined. Using 345Ps as the enzyme and Dns-GEAGDDIVPC SMSYTWT as the substrate, the effects of PMSF at concentrations of 1 mM and 10 mM were examined. Trypsin having protease activity was used as a control.

The obtained results are shown in FIG. Protease activity was inhibited by 10 mM PMSF as well as trypsin, but to a lesser extent than trypsin.

Example 7: Modification of thiol group The reaction solution (reaction time: 3 hours) obtained by the method described in Example 4 was used as a sample (in this reaction solution, H
The presence of unreacted substrate was not found by PLC analysis).

(1) Modification of thiol group 1 μl of the reaction solution was added to 5 μl of N-ethylmaleimide (100 mM, ethanol solution), and after stirring, at room temperature (23
The thiol group modification reaction was carried out for 30 minutes at (° C.). To this, 5 μl of DIG-NHS (Boehringer Mannheim: 0.4 mg / ml, dimethylsulfoxide solution) was added, stirred, and further reacted at room temperature for 30 minutes.
Then, 10 μl of glycine aqueous solution (500 mM, pH adjusted to 8.5 with sodium hydroxide) was added, and the reaction was further performed at room temperature for 90 minutes. 100 μl of a fixing diluent (50 mM, phosphate buffer (pH 8.5), 0.01% nonidet P-40) was added to the reaction solution thus obtained.
Was added and stirred.

(2) Immobilization on plate The whole amount of (1) was put into a streptavidin-coated microtiter plate (made by Boehringer Mannheim), stirred, and left at room temperature. After 1 hour, the contents were discarded, and 350 μl of the fixing diluent and TBS were sequentially washed. 300 μl of 50% Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd .: diluted with TBS) was added and left for 1 hour. The contents were discarded and washed twice with 300 μl TBS.

(3) Labeling with anti-digoxigenin antibody and alkaline phosphatase On the plate obtained in (2), 200 μl of the anti-digoxigenin antibody labeled with alkaline phosphatase (Boehringer Mannheim: diluted 1000 times with TBS: 0.7)
5 U / ml) was added and left for 1 hour. 300 μl of T
After washing twice with BS, 200 μl of a disodium p-nitrophenol phosphate solution (2.5 mg / ml: 100 mM sodium carbonate buffer (pH 9.8), 2 mM magnesium chloride) was used as a substrate for alkaline phosphatase.
Was added and reacted at room temperature (23 ° C.) for 15 minutes. 50μ
The reaction was stopped by adding 1 L of sodium hydroxide (1N), and the absorbance (405 nm) was measured. The same treatment was performed using an unreacted liquid (substrate solution) and TBS. Further, the same operation was performed even when the N-ethylmaleimide treatment for modifying the thiol group was not performed. The results obtained are shown in Table III.

Table III Effect of thiol group modification (absorbance) Modification reaction solution Unreacted solution TBS Yes 1.583 0.243 0.234 No 1.752 1.071 0.230

Example 8: Relation between amount of reaction product and absorbance (1) After the reaction solution and the unreacted solution used in Example 7 were diluted with an equal amount of dimethyl sulfoxide, the amount of biotin was kept constant. Both were mixed in various proportions. The same operation as in Example 7 was performed using 1 μl of the obtained mixed sample (room temperature: 23 ° C.). However, the N-ethylmaleimide treatment and the digoxigenin labeling reaction were performed at 37 ° C. The results obtained are shown in FIG.

Example 9: Relationship between the amount of reaction product and the absorbance (2) Each of the reaction solution and the unreacted solution used in Example 7 was 2 times.
After diluting with a double amount of dimethyl sulfoxide, both were mixed at various ratios so that the amount of biotin was constant. 2 μl of the mixed sample was put into a microtiter plate, and the same operation as in Example 7 was performed (room temperature: 23 ° C.). However,
Sodium iodoacetate (100 mM, dissolved in TBS) was used instead of N-ethylmaleimide, and DIG-NHS
The concentration was 0.2 mg / ml. The thiol group modification reaction and digoxigenin labeling reaction were carried out at 37 ° C. for 30 minutes, and the glycine treatment was carried out at 37 ° C. for 45 minutes. The color development time of alkaline phosphatase was set to 10 minutes. The obtained results are shown in FIG.

Example 10: Variation in measured values Each of the reaction solution and the unreacted solution was diluted with 4 times the volume of dimethyl sulfoxide, and then mixed at various ratios so that the amount of biotin was constant. 2 of the obtained mixed samples
The same operation as in Example 9 was performed using μl (room temperature:
23 ° C). However, sodium iodoacetate treatment and digoxigenin labeling reaction were carried out at 37 ° C. for 35 minutes, and glycine treatment was carried out at 37 ° C. for 40 minutes by adding 15 μl of a glycine solution. For immobilization on a streptavidin-coated microtiter plate, 100 μl of a diluting solution for immobilization was put, and 10 μl of a glycine-treated reaction solution was added thereto. The coloring time of alkaline phosphatase was set to 15 minutes. The results obtained are shown in Table IV.

[0074] *: Standard deviation / average value

Example 11: Case of Protease Inhibitor An enzyme reaction solution treated with PMSF (phenylmethanesulfonyl fluoride) was used as a protease inhibitor. To stop the reaction, 4-fold amount of dimethyl sulfoxide was added. The same operation as in Example 10 was performed using 1 μl of the stop reaction solution. The results obtained are shown in Table V.

Table V Inhibition by PMSF PMSF concentration Activity (% cleavage) 10 mM 71 1 mM 87 0 mM 100

Example 12: Scope of measurement As a sample, SMSYTWT of a cleaved fragment of a substrate peptide was used.
K [Bio] was used. 2 μl of the sample solution (dissolved in dimethyl sulfoxide) was treated with sodium iodoacetate (37 ° C., 30 minutes) as in Example 10.
Digoxigenin labeling (37 ° C., 30 minutes) and glycine treatment (37 ° C., 40 minutes) were performed, and 10 μl thereof was immobilized. The color reaction was carried out at room temperature (24.5 ° C) for 10 minutes. These operations were performed at various concentrations, and the results are shown in FIG.
Shown in

[0078]

INDUSTRIAL APPLICABILITY Since the method for measuring HCV protease activity of the present invention can be carried out in a test tube, it can be measured in an extremely short time as compared with a cell culture system. Further, the facility to be used may be simple without complicated operations such as aseptic operation. Further, since no cells are used, there is no variation between the measurements as compared with the cell culture system, and reliable results can be obtained. It is also an advantage of the present invention that it is not necessary to use a detection method for radioactive isotopes. In addition, the screening method for HCV protease inhibitors of the present invention enables extremely simple and reliable screening of inhibitors, which can be widely used in the development of anti-HCV agents.

Further, for the protease whose activity is difficult to measure by releasing the chromophoric atomic group, the HCV protease activity of the present invention is measured by the post-label ELISA method in which the antigen substance is bound to the product after the enzymatic reaction. The law is advantageous. This assay can easily adapt to changes in the substrate and has a wide range of applications.

[Brief description of drawings]

FIG. 1 shows the HCV precursor polyprotein structure, the name of the expression plasmid used in the present invention and the region of the HCV gene inserted in each. In the figure, C on the amino-terminal side represents a viral core protein, E1 and E2 represent envelope proteins 1 and 2, and these correspond to viral structural proteins. Products from the downstream region are nonstructural proteins (N
Equivalent to S).

FIG. 2 Enzyme 345Ps and synthetic substrate Dns-GEAGD
HPL when reacting DIVPC SMSYTWT
It is a chromatogram of C.

FIG. 3 Enzymes 34NsH, 34Sm, 345C, 345
Bs, 345Ps is a synthetic substrate Dns-GEAGDDIV
The time response curve at the time of making it react with PC SMSYTWT is shown.

FIG. 4: Enzyme 345Ps, synthetic substrate Dns-GEAGDDIVPC SM
The effect of adding PMSF as an inhibitor using SYTWT, and the substrate N-benzoyl FVR (Phe-Val-A) of trypsin in the presence of the same concentration of PMSF
The result of the rg) -p-nitroanilide cleavage reaction is shown.

FIG. 5 shows the relationship between the amount of reaction product and the absorbance (405 nm) when N-ethylmaleimide treatment was performed.

FIG. 6 shows the relationship between the amount of a reaction product and the absorbance (405 nm) when treated with sodium iodoacetate.

FIG. 7 shows a measurement applicable range when sodium iodoacetate treatment is performed.

Claims (11)

[Claims] 1. An NS3 region and N in a non-structural protein of hepatitis C virus (HCV) precursor polyprotein.
A method for measuring HCV protease activity, which comprises reacting an HCV protease obtained by isolating and purifying a gene encoding an S4A region, and optionally a downstream region thereof, with Escherichia coli and a synthetic substrate peptide. 2. The assay method according to claim 1, wherein the HCV protease is expressed using a gene encoding a region containing the NS3 region and the NS4A region. 3. The HCV protease comprises the NS3 region, N
The assay method according to claim 1, which is expressed using a gene encoding a region including the S4A region and the NS4B region. 4. The HCV protease comprises the NS3 region, N
Is expressed using a gene encoding a region including the S4A region, NS4B region and NS5A region,
The measuring method according to claim 1. 5. The HCV protease comprises NS3 region, N
S4A region, NS4B region, NS5A region and NS5
The assay method according to claim 1, which is expressed using a gene encoding a region including the B region. 6. The synthetic substrate peptide is NS3 / NS4A,
NS4A / NS4B, NS4B / NS5A or NS5
The assay method according to claim 1, which is a peptide in the vicinity of each cleavage site of A / NS5B, or a peptide in which one or more amino acids other than the consensus amino acid in these peptides are substituted, deleted or added. 7. The assay method according to claim 6, wherein the synthetic substrate peptide is 10 to 20 amino acids. 8. The synthetic substrate peptide is NS5A / NS5B
The assay method according to claim 6, which is a peptide near the cleavage site. 9. The NS3 region and N in a non-structural protein of hepatitis C virus (HCV) precursor polyprotein.
In a system in which a gene encoding the S4A region and optionally a downstream region thereof is expressed in Escherichia coli, and HCV protease obtained by isolation and purification is reacted with a synthetic substrate peptide, a test compound is added to the reaction system, HC which comprises comparing the progress of the cleavage reaction of the synthetic substrate peptide with and without the addition of the test compound.
Screening method for V protease inhibitors. 10. The method for measuring HCV protease activity according to claim 1, which comprises the following steps: (1) NS3 region and NS4A region in a nonstructural protein of hepatitis C virus (HCV) precursor polyprotein; HC obtained by optionally expressing a gene encoding a downstream region thereof in E. coli and isolating and purifying
V protease is reacted with biotin or a synthetic substrate peptide labeled (labeled a) with an antigenic substance or an antibody substance; (2) the labeled peptide that is the cleavage product of step (1) is further labeled with an antigenic substance (labeled b (3) bind an enzyme-labeled antibody against the antigen to the antigen substance labeled with a peptide; and (4) measure the activity of the enzyme in step (3), provided that steps (2) and ( 3) or before step (4), the label a
The labeled peptide is immobilized by utilizing its affinity for. 11. When the synthetic substrate peptide contains cysteine, the thiol group is modified prior to labeling with an antigen substance (label b) so that the thiol group of cysteine is not labeled with the antigen. Item 10. The measuring method according to Item 10.