JP2000249704A - Preparation of virus antigen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a virus antigen with high antigen activity by reacting peptidyl prolyl cis-trans isomerase (PPI ase) with the virus antigen. SOLUTION: A hepatitis B virus surface antigen (HBS antigen) or a hepatitis B virus core antigen (HBC antigen) is used for a virus antigen, for example, and it is preferably manifested by the gene recombination method with bacteria such as colibacillus and yeast used as a host. PPI ase of one or more kinds of cyclophilin (binding protein to cyclosporin A which is an immunosuppressive agent) type and FKBP (binding protein to FK 506 which is an immunosuppressive agent) type is used, and the PPI ase is prepared from an eukaryotic organism, eubacteria or old bacteria, or is manifested from DNA prepared from the eukaryotic organism, eubacteria or old bacteria. A phosphoric acid buffer solution, for example, is used as a diluting liquid in the process for reacting the PPI ase with virus antigen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preparing a virus antigen capable of obtaining a virus antigen having enhanced antigen activity.

[0002]

2. Description of the Related Art In recent years, with the diversification of viral infections, the importance of detecting viral infections has increased.
As one of such virus detection methods, there is an immunodiagnosis method in which the presence or absence of infection is determined by measuring whether or not a specific antibody against the virus has been produced in the body.

For example, in the case of hepatitis B virus (HBV), Dane having a diameter of 42 nm is contained in the serum of a patient at the time of infection.
In addition to double-stranded DNA viruses called particles,
nm spherical particles or 50-700 nm in length with the same outer diameter
Are known to exist. These HB
V has a surface of HBV, a core (cor)
e) surface antigen (HBs antigen), nuclear antigen (HBs antigen)
c antigen).

[0004] Detection of such antibodies against each antigen is performed by
Important for clinical diagnosis. In particular, since the HBs antibody is a protective antibody against HBV, the HBs antibody is detected in the blood when HBV infection has been eliminated in the past and has already been eliminated or after HBV vaccination. Therefore, HB
Positive s antibody indicates resistance to HBV and no risk of reinfection.

[0005] In such a method for detecting an HBs antibody, an HBs antigen labeled with an enzyme or adsorbed and immobilized on an insoluble carrier is used as a reagent. Therefore H
Although a large-scale preparation system for Bs antigens is required, such HBs antigens are currently used mainly as small spherical particles extracted from serum of patients who are HBs antigen positive and HBV is not detected. And the supply amount of serum is limited.

[0006] In recent years, with the development of genetic recombination technology,
Production of proteins has been put to practical use, and production of antigen proteins using HBs antigens by genetic recombination techniques has been attempted. However, when Escherichia coli is used as a host, although low-molecular-weight proteins are produced, they do not aggregate and do not become particulate, so that the antigen activity was extremely weak (Pumpen et al, Ge).
ne, 30, 201 (1984)). In addition, when yeast is used as a host, there is a problem that secretion of antigen protein from cells does not occur and purification is difficult (Miyanoha).
ra, A et al, Proc. Nat. Acad. S
ci. , 80, 1 (1983)).

[0007] Therefore, a system using cultured cells such as CHO cells, Vero cells, and L cells instead of Escherichia coli or yeast as a host has been devised and studied, and production of an antigen protein with enhanced antigen activity has been reported (particularly). 63-2306
No. 39). However, such cultured cells are
Compared to Escherichia coli, yeast, etc., it takes much more days to culture and requires a skillful operation, so that it is complicated and unsuitable for mass production. Therefore, the development of a system that uses Escherichia coli or yeast, which has a short culture period and simple culturing operation, as a host and produces a virus antigen having high antigen activity is desired.

[0008]

SUMMARY OF THE INVENTION In view of the above circumstances, an object of the present invention is to provide a method for preparing a virus antigen capable of obtaining a virus antigen having enhanced antigen activity.

[0009]

Means for Solving the Problems The present invention is a method for preparing a virus antigen comprising a step of reacting a virus antigen with peptidyl prolyl cis trans isomerase (hereinafter, also referred to as "PPIase").

The above-mentioned virus antigens are not particularly limited, and include, for example, hepatitis B virus surface antigen (HBs antigen), hepatitis B virus core antigen (HBc antigen), hepatitis C virus antigen, HIV antigen and the like.

The above-mentioned virus antigens may be those directly extracted from the serum of a positive patient or the like, bacteria such as Escherichia coli, and yeasts;
Cells expressed by genetic recombination using cultured cells such as cells, Vero cells, and L cells as hosts can be mentioned, but from the viewpoint of short culture days, simple culturing operations, and large-scale preparation. And those expressed by a gene recombination method using bacteria such as Escherichia coli and yeast as hosts.

For example, in a system using Escherichia coli, proteins produced in the cells are formed as granules, and these granulated proteins are often insolubilized and do not show antigen activity. Even in the absence of granule formation, virus antigens often do not show antigen activity unless the antigen protein is in a specific aggregated state, ie, spherical particles. In the present invention, from the point that it is possible to prepare a particulate antigen having enhanced antigen activity from a virus antigen having low antigen activity without being granulated and insolubilized or spherical particles, the gene using bacteria and yeast as hosts is also considered. It can be suitably applied to those expressed by a recombinant method.

The method for preparing a viral antigen of the present invention comprises a step of reacting the viral antigen with PPIase. By including the step of reacting the viral antigen with PPIase, the three-dimensional structure of the viral antigen is stabilized, and the structural change is quickly repaired, thereby preventing a decrease in antigen activity.

[0014] The PPIase used in the present invention is an enzyme having an action of promoting the rotation of the peptide bond on the N-terminal side of a proline residue. The N-terminal peptide bond of the proline residue, which is a constituent amino acid in the protein, cannot rotate freely, and spontaneous cis-trans isomerization hardly occurs.
As a result, when a structural change occurs, the conversion of the protein chain to a normal structure is limited by such a cis-trans isomerization reaction of proline, and the spontaneous regeneration is inhibited.
PPIase that promotes this isomerization reaction is an enzyme that promotes the regeneration of higher-order structures of proteins and can regenerate denatured proteins.

The PPIase used in the present invention includes cyclophilin (protein binding to cyclosporin A which is an immunosuppressant) type and FKBP (protein binding to FK506 which is an immunosuppressant) type. At least one P
PIase can be used.

As the PPIase, those prepared from eukaryotes, eubacteria or archaebacteria, or those expressed from DNA prepared from eukaryotes, eubacteria or archaebacteria can be used.

The above-mentioned eukaryotic PPIase is not particularly restricted but includes, for example, pig-derived 19K PPIase, Drosophila-derived ninaA product, and cyclophilin (Cy) derived from higher plants such as tomato and corn.
clofilin), cyclophilin derived from Neurospora crassa (Cyclophilin), Nc-FKBP, cyclophilin derived from yeast (Cyclophilin),
CRG protein, Sc-FKBP and the like. For example, Cyclophilin-type PPIase (derived from calf thymus) and FKBP-type PPIase (derived from human), which are commercially available from Sigma, can be easily obtained.

The above-mentioned PPIase derived from eubacteria is not particularly limited. For example, PPIase derived from Escherichia coli is used.
α, PPIase-β and the like (Protein nucleic acid enzyme Vol. 36, No. 11 (1991)).

The archaea include, for example, the genus Sulfolobus, the genus Desulfurococcus, the genus Pyrodictium, the genus Thermofilum, and the thermoproteus (T).
genus thermoproteus, Pyrobaculum (Py)
robaculum, Pyrococ (Pyrococ)
cus), Methanobacterium (Methanobaba)
cterium), Methanococcus (Methano)
coccus), Methanopyrus (Methanopy)
rus), Methanotherther
mus), Archaeoblobus (Archaeoglo)
bus, halobacterium (Halobacter)
ium), Methanopranus
us) genus, Methanospiram
illum), Methanosarcia (Methanosa)
rcina) can be used. Among the archaea, in particular, those prepared from Methanococcus archaea, or DNAs prepared from Methanococcus archaea
Are preferably expressed.

The above-mentioned Methanococcus genus belongs to the marine archaeal methanogen and is a group of spheroid bacteria characterized by producing methane from hydrogen, carbon dioxide and formic acid.
The Methanococcus spp. Are classified into mesophilic and thermophilic bacteria based on their optimal growth temperature, and include Methanococcus jannasii (Methanococcus jannas).
chii: optimal temperature of 85 ° C.), Methanococcus thermolystroficus (Methanococcus th)
thermolytropicus: ATCC35
097, optimal temperature 65 ° C). Optimal temperature is 2
Mesophilic bacteria at 0 to 40 ° C include Methanococcus voltae (ATC).
C33273), Methanococcus deltae (Meth
nanococuus deltae: ATCC3529
4), Methanococcus frischus (Methanoc)
ocus frisius: ATCC43340),
Methanococcus maripaldis
cusus maripaludidis: ATCC4300
0), Methanococcus bannieri (Methanoc)
ocus vannieliei: ATCC3508
9) and the like.

The above-mentioned bacterium belonging to the genus Methanococcus has 7 to 8 types of PPIases, unlike Escherichia coli, and each of them does not act in a complicated manner.
Since there are only types, versatility for proteins is high. The PPIase used in the present invention can be obtained by directly purifying from the above-mentioned bacterium belonging to the genus Methanococcus. However, by cloning the DNA base sequence encoding the PPIase of these organisms, a rapidly growing organism such as Escherichia coli or yeast can be used. It is preferable to prepare it by incorporating it into a product because mass production is possible.

The method for directly purifying the above-mentioned PPIase is not particularly limited, and for example, it can be obtained by the following method. The bacteria are cultured, for example, in a seawater-based medium (yeast extract 2 g, bactotryptone 2 g, sodium acetate 1 g, PIPES 1.7 g, DAB vitamin solution 10 mL / L seawater pH 6.8) under anaerobic conditions and hydrogen / carbon dioxide gas. 37 under aeration of a (4: 1) gas mixture.
Cultivate all day and night at ℃ to collect the cells. 25 collected cells
The cells were crushed by osmotic shock by suspending them in an mM phosphate buffer (pH 7.0), and the supernatant was collected by centrifugation.
Sepharose column: manufactured by Pharmacia, etc.), gel filtration column chromatography (Superrose)
A 12 HR column: manufactured by Pharmacia and the like, and an anion exchange column chromatography (MonoQ: manufactured by Pharmacia) can collect a PPIase-active fraction.

The obtained fraction can be confirmed to be a single fraction by SDS polyacrylamide gel electrophoresis. In the above purification, the PPIase activity can be confirmed by a coupling assay with an α-chymotrypsin reaction. For example, HEPE
In S buffer (pH 7.8), N-suc-Ala-Ala-Ala-Phe-pNA in which the N-terminal is modified with an N-succinyl group and the C-terminal is modified with p-nitroanilide, respectively.
Or N-suc-Ala-Ala-Leu-Phe-p
After injecting the sample into the cell using the peptide of NA (final concentration: 17 μM) as a substrate, the mixture is incubated at 25 ° C., and the reaction is started by adding a chymotrypsin solution. The enzyme activity can be measured by monitoring the increase in absorption at 390 nm due to the release of pNA under stirring throughout. Examples of the peptide serving as the substrate include “N-suc-Ala-Ala-Pro-Phe-p” (trade name).
A commercially available product of “-nitroanilide” (manufactured by Sigma) can also be used.

D prepared from the above Methanococcus bacterium
As a method for expressing from NA, a conventional method such as a gene experiment protocol described in “Guide to Neoplastic Chemistry Experiment” (Chemical Doujinsha) or the like can be applied.

The DNA sequence of the PPIase of the bacterium belonging to the genus Methanococcus and the method for confirming the enzyme are described in, for example,
No. 91, and Japanese Patent Application No. 9-235999, which can be used. As an example, the DNA sequence of PPIase derived from Methanococcus thermolystrophycus is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 2. As a method for expressing from the DNA, for example, a primer for PCR is prepared by synthesizing a part of the base sequence so as to include an appropriate restriction enzyme site. Then, the genomic DNA of the genus Methanococcus
Is used as a template for amplification by PCR. The PPIase gene obtained from the PCR product is inserted into a plasmid vector containing an appropriate resistance marker, and the resulting plasmid is used to transform fast-growing organisms such as Escherichia coli and yeast.
The transformant is cultured and purified from the cultured cells by a conventional method such as hydrophobic chromatography or ion exchange chromatography to obtain PPIase.

As the genomic DNA, Japanese Patent Application No. 9-3
It can be prepared according to the method described in No. 50623. For example, when using Methanococcus thermolystrophycus DSM2095 strain, 65 ° C., anaerobic conditions, H ₂ / CO ₂ (4: 1) mixed gas ventilation (200 m
(L / min), and the above strain was cultured.
0 mL of TNE buffer (20 mM Tris-HCl (pH
8.0), 100 mM NaCl, 20 mM EDTA), the temperature was adjusted to 50 ° C., and 0.05 mL of proteinase K solution (20 mg / mL) was added thereto.
Perform shaking culture for hours. After performing phenol treatment and chloroform treatment on this culture solution, 0.05 mL of RNase was used.
Add solution A (0.5 mg / mL) and leave at 37 ° C. for 1 hour. Add 1 mL of 10% SDS to the solution after standing
Solution, 0.05 mL of proteinase K solution (20 mg
/ ML) and left again at 50 ° C. for 70 minutes. Furthermore, after performing phenol treatment and chloroform treatment (the solution volume at this time is 10 mL), 1 mL of 3 M sodium acetate solution (pH 5.2) and 25 mL of ethanol are added, and the mixture is left at -20 ° C for 2 hours. . The solution after standing is centrifuged with a high-speed centrifuge to precipitate DNA, and the precipitate is 3 mL.
, And dried with a centrifugal evaporator, and then dissolved in 0.5 mL of TNE buffer.
By this operation, about 2 mg of genomic DNA can be obtained.

Among the above-mentioned Methanococcus genus, Methanococcus thermolystroficus (Methanococcus)
PPIase obtained from P. cus thermolytropicus has excellent thermostability,
Even when left under high temperature conditions such as 0 ° C. and 100 ° C., it is hardly deactivated. PPIases that have been marketed so far include 6
Since it was almost inactivated at 5 ° C., it could not be used under high temperature conditions. However, the above Methanococcus thermolystroficus (Methanococcus)
The PPIase obtained from P. us themolithrophicus can be used not only at a medium temperature of 37 to 65 ° C but also at a high temperature of 80 to 90 ° C. Furthermore, a feature of these thermostable PPIases is that their activity is preserved for long periods.

The above-mentioned Methanococcus thermolystroficus (Methanococcus themolith)
PPIase from P. otropicus) can be
Since PIase has not only the peptidylproline cis-trans isomerization effect and the polypeptide chain folding effect but also the irreversible aggregation-inhibiting effect of chaperonin-like proteins, the effect of stabilizing the HBs antigen is large.

Among the mesophilic bacteria of the genus Methanococcus, in particular, Methanococcus voltae (Methanococcus)
cus voltae),
Methanococcus thermolystroficus (Metha
nococcus themolithotrophi
cus), it has not only the peptidylprolyl cis-trans isomerization effect and the folding effect of the posopeptide chain, but also the effect of inhibiting the irreversible aggregation of the chaperonin-like protein, thus stabilizing the HBs antigen. large.

The above Methanococcus voltae (Meth
(Anococcus voltae) is a mesophilic bacterium, so that PPIase derived from the bacterium has an effect of stabilizing the HBs antigen in a wide range of 4 to 40 ° C.

The present invention relates to the above-mentioned virus antigen,
The step of reacting ase. The dilution of the virus antigen is not particularly limited, and examples thereof include a phosphate buffer, a Tris buffer, a Good buffer, a glycine buffer, and a Tris-HCl buffer. The ionic strength of the diluent is not particularly limited as long as it is suitable for a virus antigen, but preferably contains 0.05 to 1 M of NaCl. The pH of the diluent is not particularly limited.
H4.5 to 9.0. An appropriate amount of a virus antigen is added to this to make a virus antigen solution. The virus antigen concentration is not particularly limited, and is usually 0.001 to 1
0 mg / mL.

The solution for dissolving the PPIase and the pH thereof are not particularly limited, and include, for example, those exemplified as the virus antigen diluent, and the concentration of the buffer is preferably 0.05 to 1M. The ionic strength in the diluent is not particularly limited as long as it is suitable for the PPIase reaction. For example, NaCl, KCl, MgC
It is preferable to contain 0.01 to ₂ M of l ₂ , (NH ₄ ) ₂ SO _{4 and the} like. The solution in which the PPIase is dissolved to perform the reaction is also referred to as a particle-constituting reaction solution in this specification. PPIase is dissolved and used in such a particle-constituting reaction solution, and its concentration is usually 0.001 to 10
mg / mL.

In the reaction between the virus antigen and the PPIase, PPIase is mixed and acted so that the molar ratio of the PPIase is about 0.01 to 200 times the virus antigen. If it is less than 0.01 times, a sufficient reaction is not performed, and
If it exceeds 0 times, the effect does not change and there is a high possibility that side reactions will occur as impurities. Preferably it is about 0.05 to 50 times.

The reaction conditions for the PPIase include, for example, incubation at 4 to 60 ° C. for 30 minutes to 72 hours. If desired, longer incubations may be used.

The above-mentioned particle-constituting reaction solution contains protein disulfide isomerase (PDI), thioredoxin, oxidized glutathione, reduced glutathione, etc. having an SS-bonding reaction promoting effect for the purpose of promoting disulfide (-SS-) recombination. May be added.

The present invention 2 relates to a method for preparing a virus antigen, comprising:
A method for preparing a virus antigen, comprising a step of treating with at least one selected from the group consisting of a reducing agent and a protein denaturant, and a step of reacting the treated virus antigen with peptidyl prolyl cis trans isomerase.

The present invention 2 includes a step of treating a virus antigen with at least one selected from the group consisting of a surfactant, a reducing agent and a protein denaturant. Examples of the above virus antigens include those exemplified in the present invention 1.

The surfactant is not particularly limited as long as it can remove lipids. For example, sodium dodecyl sulfate (SDS), sodium dodecyl sulfone sulfate, sodium cholate, sodium deoxycholate,
[(Cholamidopropyl) dimethylammonio] -1
-Propanesulfonic acid (CHAPS), octyl glucoside (OG), octyl thioglucoside, nonanoyl-
N-methylglucamide (MEGA-9), polyoxyethylene dodecyl ether (Briji), polyoxyethylene i-octyl phenyl ether (Triton
X), polyoxyethylene nonylphenyl ether (N
onidet P-40), polyoxyethylene fatty acid ester (Span), polyoxyethylene sorbitol ester (Tween) and the like. The concentration of the surfactant used depends on the concentration of the virus antigen to be treated, but is preferably in the range of 0.01 to 20%.

The reducing agent is not particularly limited as long as it can dissociate the disulfide bond of the protein. For example, β-mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), dithioerythritol (DTE) , Sodium borohydride, monothiophosphoric acid, and the like. The use concentration of the reducing agent varies depending on the concentration of the virus antigen to be treated, but can usually be used so that the concentration in the solution is 0.01 to 200 mM.

The above-mentioned protein denaturing agent is not particularly limited as long as it loosens the higher-order structure of the protein and dissociates hydrogen bonds and hydrophobic interactions between polypeptide side chains. For example, guanidine hydrochloride (Gdm-HCl) ), Urea and the like. The concentration of the protein denaturant varies depending on the concentration of the virus antigen to be treated, but is preferably in the range of 0.1 to 8M.

The above reducing agents, surfactants and protein denaturants may be used alone or as a mixture. Buffer for dissolving the reducing agent, surfactant and protein denaturant, and its ionic strength and p
H includes, for example, those exemplified as the virus antigen diluent in the present invention 1. In this specification,
A solution obtained by dissolving an appropriate amount of at least one treating agent selected from the group consisting of a reducing agent, a surfactant, and a protein denaturant in the buffer solution is converted into a solution for lowering the molecular weight, and further the solution for lowering the molecular weight. Treating the virus antigen with a low molecular weight treatment.

In the step of treating a virus antigen with at least one selected from the group consisting of a surfactant, a reducing agent and a protein denaturing agent, the virus antigen diluent and the depolymerizing solution are treated with And incubating at 4 to 50 ° C. for about 30 minutes to 48 hours. If necessary, a stirring operation or ultrasonic treatment may be performed. The concentration of the virus antigen in the solution to be treated is not particularly limited, and is usually 0.001 to 10 mg / mL.

In the method for preparing a virus antigen according to the second aspect of the present invention, a step of reacting the treated virus antigen with PPIase is then performed. In the present invention 2, the surfactant, the three-dimensional structure of the virus antigen damaged by the depolymerization treatment by at least one selected from the group consisting of a reducing agent and a protein denaturing agent,
By rapidly repairing structural changes, it is possible to prevent a decrease in viral antigen activity. For example, a PPIase is reacted with the antigen-protein that has been granulated and insolubilized in the cells of genetically modified Escherichia coli to reduce the molecular weight, thereby preparing a particulate antigen with enhanced antigen activity or extracting from a positive patient serum. After performing the molecular weight reduction treatment using the obtained natural-type particulate antigen protein, a PPIase is allowed to act on the molecule to reconstitute it into a particulate form, thereby preparing an antigen having higher activity than before the treatment.

The PPIase and its reaction conditions include those exemplified in the present invention 1.

In the present invention 1 and the present invention 2, the above PP
Iase may be used not only in a solution but also as a bioreactor by immobilizing it on an insoluble carrier. in this case,
The insoluble carrier to be used is not particularly limited as long as it is a usual enzyme-immobilizing carrier.For example, cellulose, agarose, dextran, chitin, collagen, amino acid polymer, polystyrene, polyacrylamide, ion-exchange tree, vinyl polymer, glass beads, Ceramics, photocrosslinkable resins, polyurethanes and the like can be mentioned. A carrier obtained by activating cellulose or agarose with cyanogen bromide or a carrier obtained by introducing a functional group into a vinyl polymer is commercially available as a protein binding carrier, and these may be used.

The method of bonding the insoluble carrier to the PPIase is not particularly limited as long as it is a method used for immobilizing proteins, and examples thereof include a covalent bonding method, a physical adsorption method, an ionic bonding method, and a crosslinking method. Method, a comprehensive lattice type, a microcapsule lattice type, and the like. PP to the above insoluble carrier
The amount of Iase adsorbed is not particularly limited. For example, the protein amount is 0.1 to 1000 per cm ^{3 of} the insoluble carrier.
mg is preferred.

The reaction operation using the insoluble carrier to which the PPIase is bound is not particularly limited.
(1) An insoluble carrier to which PPIase has been bound by mixing and reacting the insoluble carrier to which PPIase has been bound, the virus antigen diluent after the completion of the low molecular weight treatment, and the reaction solution for forming particles, and then centrifuging the mixture. (2) Packing a column with an insoluble carrier to which PPIase is bound, and adding a virus antigen diluent and a particle composing reaction solution after completion of the lowering operation Expand
A method of recovering the eluted virus antigen after the reaction, (3) mixing and reacting the virus antigen diluent and the particle-constituting reaction solution after the completion of the lowering operation with the PPIase-binding membrane to react the virus antigen. There is a method of collecting.
The insoluble carrier to which the PPIase has been bound can be used repeatedly.

The virus particles prepared in the present invention 1 and the present invention 2 can be used as an antigen for immunodiagnostic agents, vaccines and the like. The immunodiagnostic agent can perform quantitative measurement and the like of a virus antibody in a sample such as serum,
An immunoassay utilizing an antigen-antibody reaction can be used. In the above immunoassay, (1) a hemagglutination method or a latex agglutination method in which the virus antigen prepared according to the present invention is carried on an insoluble carrier such as blood cells or latex, (2)
An enzyme immunoassay (EIA) using an enzyme-labeled antigen obtained by labeling a virus antigen prepared according to the present invention with an enzyme may be mentioned.

In the hemagglutination method or latex agglutination method, the virus antigen prepared according to the present invention is carried on latex, blood cells, etc., and mixed with a virus antibody-containing specimen. As a result of the antigen-antibody reaction, latex and blood cells are cross-linked and agglutinated by the antibody, and the virus antibody in the sample can be quantified by detecting the agglutination.

In the enzyme immunoassay described above, for example,
In the case of the one-step sandwich method, the virus antigen prepared according to the present invention is immobilized on beads or an insoluble carrier such as the inner wall of a well of a microtiter plate. A virus antibody-containing specimen and an enzyme-labeled virus antigen are added to the mixture to generate an immobilized virus antigen-virus antibody-enzyme-labeled virus antigen immune complex. After the washing operation, an enzyme substrate is added, and a product produced by the enzyme reaction is detected, whereby the amount of the virus antibody in the sample can be quantified.

[0051]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Reference Example 1 Preparation of PPIase (Derived from Methanococcus thermolystrophicus) (1) Construction of Expression Plasmid and Transformation into Escherichia coli A fragment of the gene encoding PPIase of Methanococcus thermolysosophycus was used. The plasmid pUFKC1 deposited as FERM P-15622 with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology was used as a template and amplified by a PCR method using the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 in the sequence listing. . 3 to 8 from the 5 'end of the sequence shown in SEQ ID NO: 3
The second base sequence is a recognition site for NcoI. Also,
The 3rd to 8th base sequence from the 5 'end of the sequence shown in SEQ ID NO: 4 is a BamHI recognition site. The fragment amplified and recovered by the PCR method using the above primers was digested with BamHI and NcoI, and then ligated to an expression vector pET-11d (manufactured by Stratigen) which had been treated with the same restriction enzymes. Thereafter, this was transformed into BL21 (DE3) strain (manufactured by Stratigen), and ampicillin 100 μg / m
After culturing on an LB medium plate containing L (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl / 1 L (pH 7.0)), colonies were picked up and used as glycerol stock.

(2) Preparation of PPIase from recombinant Escherichia coli 200 m of LB medium containing 100 μg / mL of ampicillin
L, BL21 (DE carrying the above expression plasmid)
3) One platinum loop of the strain (recombinant E. coli) was inoculated and precultured at 37 ° C. for 16 hours. This culture solution was inoculated into 6 liters of LB medium, and main culture was performed using a jar fermenter. Three hours after the start of the main culture, IPTG (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a concentration of 1 mM, and the culture was further continued for 4 hours.
Continued for hours. After completion of the culture, collect the bacteria by centrifugation,
It was stored frozen at -30 ° C. Next, the recovered recombinant Escherichia coli was suspended in a 50 mM phosphate buffer (pH 7.5) and disrupted by sonication. The supernatant was collected by centrifugation, and heat-treated in a water bath at 80 ° C. for 30 minutes to denature and aggregate the contaminating proteins, and removed by centrifugation. Further, ammonium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the supernatant so as to be 80% saturated, and salting out was performed. 3
After 0 minute, the precipitate collected by centrifugation was placed in a 50 mM phosphate buffer (pH 8.0) containing 1.8 M ammonium sulfate.
7.8).

This solution was applied to a TSK-Gel Ether-5PW column (7.5 mm × 7.5 cm) equilibrated with 50 mM phosphate buffer (pH 7.8) containing 1.8 M ammonium sulfate (Tosoh Corporation). 5)
Minutes later, PPIase was eluted with a linear gradient of solution B: 50 mM phosphate buffer (pH 7.8) for 60 minutes. PPIase appeared in the fraction eluted at around 0.5M ammonium sulfate concentration. This fraction was desalted by dialysis and used in the following Examples.

Reference Example 2 Preparation of PPIase (from Methanococcus voltae) (1) Construction of expression plasmid and transformation into Escherichia coli Genome DNA of Methanococcus voltae prepared according to the method described in Japanese Patent Application No. 9-350623. Was used as a template to perform a polymerase chain reaction (PCR) using the primers of SEQ ID NOs: 5 and 6 in the sequence listing. PC
The R product was converted to pT7 blue vector (Novag
after the ligation to E. coli JM1
Transform to 09, 100μg / mL
Was cultured at 37 ° C. overnight in an LB medium plate containing ampicillin to prepare a library of PCR products. This was transferred to Hybond-N ⁺ membrane (Amersham). DIG-DNA using the digoxigenin-labeled gene used in the PPIase gene screening described in JP-A-10-91 as a probe
Using a detection kit (Boehringer Mannheim), M.P. The VPI-derived PPIase gene was screened. The plasmid was recovered from the obtained positive clone, the inserted sequence was cut out with NcoI and BamHI, and recovered by electrophoresis.

This was ligated to an expression vector pET-11d (manufactured by Stratigen) which had been treated with the same restriction enzymes. After that, this is BL21 (DE
3) The strain was transformed into a strain (manufactured by Stratigen) and cultured on an LB medium plate containing 100 μg / mL of ampicillin. Thereafter, colonies were picked up and used as a glycerol stock.

(2) Preparation of PPIase from recombinant Escherichia coli 200 m of LB medium containing 100 μg / mL of ampicillin
L, BL21 (DE carrying the above expression plasmid)
3) One platinum loop of the strain (recombinant E. coli) was inoculated and precultured at 37 ° C. for 16 hours. This culture solution was inoculated into 6 liters of LB medium, and main culture was performed using a jar fermenter. Three hours after the start of the main culture, IPTG (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a concentration of 1 mM, and the culture was further continued for 4 hours.
Continued for hours. After completion of the culture, collect the bacteria by centrifugation,
It was stored frozen at -30 ° C. Next, the recovered recombinant Escherichia coli was suspended in a 50 mM phosphate buffer (pH 7.5) and disrupted by sonication. The supernatant was collected by centrifugation, and ammonium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) was
% Saturation was carried out. After 30 minutes, the precipitate collected by centrifugation was dissolved in a 50 mM phosphate buffer (pH 7.8) containing 1.8 M ammonium sulfate.

This solution was subjected to separation and purification of PPIase using high performance liquid chromatography, and the eluate containing the enzyme was desalted by dialysis and used in the following Examples.

Reference Example 3 Preparation of PPIase Immobilized Carrier PPIase derived from Methanococcus thermolystrophicus prepared in Reference Example 1 and C as an insoluble carrier
NBr-activated Sepharose 4B gel (Pharmacia) was used. 1 g of this gel was weighed and 1 mM H
It was washed and swollen with Cl. 0.5 M NaCl.
PPI in 10 mL of 1 M NaHCO ₃ solution (pH 8.3)
The swollen gel was mixed with a protein solution in which 35 mg of ase was dissolved, and shaken at room temperature for 2 hours. After removing the supernatant, 10 mL of 0.2 M glycine (pH 8.0) was added, and the mixture was shaken at room temperature for 2 hours. This reaction blocked excess active groups. Next, it was washed with 20 mL of a 0.1 M NaHCO ₃ solution (pH 8.3) containing 0.5 M NaCl, and then washed with 20 mL of a 0.1 M, pH 4 acetate buffer containing 0.5 M NaCl. After washing again with 20 mL of the same solution, this gel was used as a PPIase-bound carrier in the following Examples.

Example 1 Treatment of low molecular weight of natural HBs antigen and PPIase reaction (1) The HBs antigen was purified from human positive serum. The purification method is
The small spherical particle antigen was recovered by a cesium chloride solution and a sucrose density gradient method, and further purified by a gel filtration method. The obtained HBs antigen was added to 0.2 M NaCl / 50 mM
3 mg / mL in sodium phosphate buffer (pH 7.5)
Was dissolved to give an HBs antigen solution. The HBs antigen was converted to 4 M urea / 1 mM dithiothreitol (DTT) /
The mixture was incubated in a 0.5% octyl glucoside / 50 mM sodium phosphate buffer (pH 7.0) at 35 ° C. for 1 hour to perform a treatment for depolymerization and denaturation of the polypeptide. The treated HBs antigen was subjected to gel filtration using a TSKgel G4000SWXL column (Tosoh Corporation) using 50 mM sodium phosphate buffer (pH 7.0) /0.1% SDS as a developing solution. Chromatograms of the native form and the HBs antigen obtained by subjecting it to low molecular weight treatment are shown in FIGS. 1 and 2, respectively.

HBs treated by the above method to reduce the molecular weight
To 5 μL of the antigen (1.5 g / mL), 3 μL of the recombinant PPIase derived from Methanococcus thermolysorticus prepared in Reference Example 1 was added at 3 μL, 0.05 M
0.1 M sodium phosphate buffer containing NaCl (pH
7.8) was added and mixed, and the mixture was incubated at 35 ° C. for 3 hours. After completion of the reaction, E
The antigenicity was evaluated by IA, and the molecular weight distribution was measured by gel filtration. Gel filtration was performed using a TSKgel G4000SWXL column using 50 mM sodium phosphate buffer (pH 7.0) /0.1% SDS as a developing solution. The chromatogram is shown in FIG.

Evaluation method: Evaluation of antigenicity of HBs antigen by EIA Natural HBs antigen obtained in Example 1, low molecular weight treatment H
Each of the Bs antigen and the HBs antigen subjected to the PPIase reaction was 0.1% SDS / 50 mM sodium phosphate (p
H7.5), and diluted with human-managed serum containing 0.1% SDS, L-Consera N “Nissui” (Nissui Pharmaceutical) to 0.015 μg / m.
After diluting to L, antigenicity was evaluated using an EIA kit for measuring HBs antigen "Enzygnost HBsAgmonoclonal" (manufactured by Hoechst-Bering-Iguanostic). The results are shown in Table 1.

Example 2 Natural HBs Antigen Low Molecular Weight Treatment and PPIase Reaction (2) The HBs antigen purification and low molecular weight treatment were carried out in the same manner as in Example 1. 5 μL of the low molecular weight HBs antigen (1.5 g / mL) treated by the above method was added to the recombinant P derived from Methanococcus thermolystrophycus prepared in Reference Example 1.
3 μL of 1.6 mg / mL PIase, 2.5 mM oxidized glutathione, 0.25 mM reduced glutathione and bovine liver-derived protein disulphideiso
3 μL of a solution containing 1.0 mg / mL of melase (manufactured by Takara Shuzo Co., Ltd.) and 0.1 μL containing 0.05 M NaCl
195 μL of M sodium phosphate buffer (pH 7.8)
The procedure was performed in the same manner as in Example 1 except for adding and mixing. P
FIG. 4 shows a chromatogram of the HBs antigen subjected to PIase reaction.

Example 3 Treatment of Recombinant HBs Antigen Derived from Recombinant Escherichia coli and PPIase Reaction (1) HBs antigen S region gene cloned from a hepatitis B positive patient (serotype: adw) (Ono, Y. et a
l. , 1983, Nucleic Acids Res.
11, 1747; the DNA sequence is shown in SEQ ID NO: 7 in the sequence listing. ) Into which 2XY. T. Medium (10 g yeast extract, 20 g bactotryptone, 5 g NaCl,
After culturing with 100 mg / L of ampicillin (pH 7.8), when the OD 650 nm reached 0.8 to 1.2, IPTG was added to 1 mM and the cells were further cultured for 10 hours. After completion of the culture, the cells were collected, and 0.1 M Na
Cl / 50 mM sodium phosphate buffer (pH 7.5)
Ultrasonic crushing in. HBs gene expression product (rHBs
-Adw) was also produced in the soluble fraction, but was produced in large amounts in the precipitated fraction. 0.1 M NaC
After washing by centrifugation twice with 1/50 mM sodium phosphate buffer (pH 7.5), the resultant was dissolved in 50 mM sodium phosphate buffer (pH 7.0) / 4 M urea / 1 mM DTT, and treated at 35 ° C. for 1 hour. . The supernatant was recovered by centrifugation. 50 mM sodium phosphate buffer (pH 7.
0) / 4M urea / 1mM DTT as a developing solution and TSK
The low molecular weight recombinant HBs was purified using a gelG4000SWXL column. Purified low molecular weight rHBs-a
dw was concentrated to 1.5 mg / mL by ultrafiltration. A developing solution of the obtained low molecular weight rHBs-adw was used as a developing solution in a 50 mM sodium phosphate buffer (pH 7.0) / 0.
FIG. 5 shows a chromatogram of gel filtration using a TSKgel G4000SWXL column using 1% SDS.

The low molecular weight rHBs-adw obtained above
Using 5 μL (1.5 g / mL), 0.05 of Example 1 was used.
0.1 M sodium phosphate buffer containing MNaCl (p
H7.8) instead of 0.1M with 0.05M KCl
A PPIase reaction was carried out in the same manner as in Example 1, except that a sodium phosphate buffer (pH 7.8) was used. The chromatogram is shown in FIG.

Example 4 Low molecular weight treatment of recombinant HBs antigen derived from recombinant Escherichia coli and PPIase reaction (2) 50 mM sodium phosphate buffer (pH 7.0) of Example 3
0) / 4M urea / 1mM DTT instead of 50mM sodium phosphate buffer (pH 7.0) / 4M guanidium hydrochloride (Gdm-HCl) / 1mM DTT to depolymerize and TSKgelG4000SWXL
A low-molecular-weight rHBs-adw was prepared in the same manner as in Example 3, except that purification was performed using a column.

The low molecular weight rHBs-adw obtained above
In 5 μL (1.5 g / mL), recombinant P from Methanococcus thermolystrophycus prepared in Reference Example 1 was added.
3 μL of 1.6 mg / mL PIase, 3 μL of a solution containing 0.25 mM oxidized glutathione, 2.5 mM reduced glutathione, and 1.0 mg / mL recombinant Escherichia coli-derived thioredoxin (manufactured by Wako Pure Chemical Industries), and 0 .0
0.1 M sodium phosphate buffer containing 5KCl (pH
7.8) was added and mixed in the same manner as in Example 1 except that 195 μL of 7.8) was added and mixed. The chromatogram is shown in FIG.

Example 5 Recombinant HBs Antigen Derived from Recombinant Escherichia coli and PPIase Reaction (3) Escherichia coli transfected with HBs antigen S region gene cloned from a hepatitis B positive patient (serotype: adr) (L
oncarevic, I .; F. et al. , 199
0,18, Nucleic Acids Res. 1
8, 4940; the DNA sequence is shown in SEQ ID NO: 9 in the sequence listing. ) Was carried out in the same manner as in Example 3 except for using) to prepare a low-molecular-weight rHBs-adr. TSKgel using 50 mM sodium phosphate buffer (pH 7.0) /0.1% SDS as a developing solution
FIG. 8 shows a chromatogram of gel filtration using a G4000SWXL column.

The low molecular weight rHBs-adr obtained above
To 5 μL (1.5 g / mL) of the recombinant P derived from Methanococcus thermolystrophicus prepared in Reference Example 3
10 μL of PIase binding carrier and 0.05 M KCl
0.1 M sodium phosphate buffer (pH 7.8) containing
Was added and mixed, and incubated at 35 ° C. for 3 hours. After the reaction was completed, centrifugation was performed at 10,000 rpm for 10 minutes, and the supernatant was recovered. Using this supernatant, the antigenicity was evaluated by EIA and the molecular weight distribution was measured by gel filtration in the same manner as in Example 1. The chromatogram is shown in FIG.

Example 6 Recombinant E. coli recombinant HB
Small molecule treatment of s antigen and PPIase reaction (4) 50 mM sodium phosphate buffer of Example 5 (pH 7.0)
0) In place of 4 M urea / 1 mM DTT, a 50 M sodium phosphate buffer (pH 7.0) / 4 M guanidium hydrochloride / 1 mM DTT was used, and the molecular weight reduction treatment and purification using a TSKgel G4000 SWXL column were performed. In the same manner as in Example 5, low molecular weight rH
Bs-adw was prepared.

The low molecular weight rHBs-adr obtained above
In 5 μL (1.5 g / mL), the recombinant PPIase derived from Methanococcus voltae prepared in Reference Example 2 was prepared.
3 μL of 6 mg / mL, 2.5 mM oxidized glutathione, 0.25 mM reduced glutathione and bovine liver-derived p
protein disulphide isomeras
e (manufactured by Takara Shuzo Co., Ltd.)
L, and 195 μL of 0.1 M sodium phosphate buffer (pH 7.8) containing 0.05 M KCl were added and mixed, and the same procedure was performed as in Example 1. The chromatogram is shown in FIG.

[0072]

[Table 1]

Example 7 A double-stranded synthetic gene (697 bp, the amino acid sequence of which is shown in SEQ ID NO: 12) encoding the adr-type HBs antigen shown in SEQ ID NO: 11 in the sequence listing was digested with NdeI and BamHI, and the same procedure was followed. It was introduced into an expression vector carrying a T7 promoter which had been treated with a restriction enzyme, to construct a synthetic HBs gene expression vector pTSHBs. E. coli BL21
After transforming the (DE3) strain with pTSHBs, it was spread on an LB plate containing ampicillin (100 μg / ml) and cultured at 37 ° C. overnight.

The 10 colonies that had grown were inoculated into a 2XLB liquid medium containing ampicillin (100 μg / ml) and cultured at 35 ° C. OD650 of the culture solution is 0.8
When the number reached 5, 1 mM IPTG was added, and the cells were further cultured for 10 hours. After completion of the culture, the cells were collected by centrifugation. After disrupting the cells by sonication, the cells were separated into a precipitate fraction and a soluble fraction by centrifugation. As a result of confirming the expression of HBs protein in each fraction by SDS-polyacrylamide gel electrophoresis, 25 kDa
It was found that a large amount of a band corresponding to the HBs protein was present in the insoluble fraction.

The insoluble fraction was treated with 5 containing 0.15 M NaCl.
After thoroughly washing with 0 mM Na-phosphate buffer (pH 7.5), this was washed with 6 M guanidine hydrochloride / 1 mM DTT /
It was dissolved in 6 M guanidine hydrochloride / 50 mM Na-phosphate buffer (pH 7.8) and solubilized at 35 ° C. for 1 hour. This is treated with TSKgel using the same developing solution as the processing solution.
Purified by gel filtration on a G4000SWXL column. The purified fraction was concentrated to 10 mg / ml by ultrafiltration with a cut-off molecular weight of 5,000.

In 5 μl of the solubilized HBs, 3 μl of the recombinant PPIase derived from Methanococcus thermolystrophicus prepared in Reference Example 1 was added in an amount of 3 μl, 0.25 μl / ml.
3 μl of a solution containing mM oxidized glutathione, 2.5 mM reduced glutathione, and 1.0 mg / ml recombinant Escherichia coli-derived thioredoxin (Wako Pure Chemical Industries)
0.1 M Na-phosphate buffer containing 5 M KCl (pH
7.8) was added and mixed, and reacted at 35 ° C. for 2 hours. After completion of the reaction, the result of analyzing the regenerated HBs antigen by gel filtration using a TSKgel G4000 column is shown in FIG. The HBs antigen appeared as a peak at a retention time of 6.8 minutes, indicating that a uniform particle structure was formed.

The recovered recombinant HBs antigen was
50 mM Na-phosphate buffer (pH 7.
0), and further diluted with human-managed serum L-Concera N “Nissui” (Nissui Pharmaceutical) at 0.015 μg /
Diluted to ml. EIA kit for measuring the antigenicity of an antigen diluent for HBs antigen, Enzygnost HBsAgm
Onoclonal (made by Hoechst Behring Diagnostics) was used. Table 2 shows the results. The same antigenicity evaluation was performed on the small spherical particles derived from the positive patient and the standard serum prepared in Example 1 as controls in the present example.

[0078]

[Table 2]

Example 8 A double-stranded synthetic gene (568 bp, coding for HBc (hepatitis B core antigen) antigen shown in SEQ ID NO: 13 in the sequence listing.
The amino acid sequence is shown in SEQ ID NO: 14) by NdeI and B
T7 digested with amHI and treated with the same restriction enzymes
Introduced into an expression vector holding a promoter,
A Bc gene expression vector pTHBc was constructed. After transforming Escherichia coli strain BL21 (DE3) with pTHBc,
The cells were spread on an LB plate containing ampicillin (100 μg / ml) and cultured at 37 ° C. overnight.

The 10 colonies that had grown were inoculated into a 2XLB liquid medium containing ampicillin (100 μg / ml) and cultured at 35 ° C. OD650 of the culture solution is 0.8
When the number reached 5, 1 mM IPTG was added, and the cells were further cultured for 10 hours. After completion of the culture, the cells were collected by centrifugation. After disrupting the cells by sonication, the cells were separated into a precipitate fraction and a soluble fraction by centrifugation. As a result of confirming the expression of HBc protein in each fraction by SDS-polyacrylamide gel electrophoresis, 21 kDa
It was found that a large amount of the band corresponding to the HBc protein was present in the insoluble fraction.

The insoluble fraction was treated with 5 containing 0.15 M NaCl.
After thoroughly washing with 0 mM Na-phosphate buffer (pH 7.5), this was washed with 6 M guanidine hydrochloride / 1 mM DTT /
It was dissolved in 0.5% octyl glucoside / 50 mM Na-phosphate buffer (pH 7.8) and solubilized at 35 ° C. for 1 hour. TSKg using the same developing solution as the processing solution
Purified by gel filtration on an elG4000SWXL column. The purified fraction was subjected to a molecular weight cutoff of 5000.
Was concentrated to 5.0 mg / ml by ultrafiltration.

In 5 μl of solubilized HBc, 3 μl of Reference Example 1
1.6 mg / ml and 0.0 mg of recombinant PPIase derived from Methanococcus thermolystrophicus prepared in
0.1 M Na-phosphate buffer containing 5 M KCl (pH
7.8) was added and mixed, and reacted at 35 ° C. for 2 hours. After completion of the reaction, the result of analyzing the regenerated HBc antigen by gel filtration using a TSKgel G4000 column is shown in FIG. The HBc antigen appeared as a peak at a retention time of 6.8 minutes, indicating that a uniform particle structure was formed.

[0083] The purified HBc particles were evaluated by ELISA. Anti-HB on 96-well micro-tarter plate
c coated with polyclonal antibody (manufactured by Quartet)
Next, after coating with bovine serum albumin, 50 mM Na-
After 10-fold dilution with a phosphate buffer (pH 7.0), an anti-HBc monoclonal antibody (manufactured by Chemicon) labeled with a peroxidase by a conventional method was allowed to act on the diluted solution. After the washing operation, ABTS (azinoethylbenzothiazolinesulfonic acid) was added as a color-developing substrate, and the color was developed.
The absorption at 05 nm was measured. Table 3 shows the results. As a control in this example, the same antigenicity evaluation was performed for the standard serum.

[0084]

[Table 3]

Example 9 A double-stranded synthetic gene (590 bp, whose amino acid sequence is shown in SEQ ID NO: 16) encoding the HCVc (hepatitis C core antigen) antigen shown in SEQ ID NO: 15 was digested with NdeI and BamHI, and It was introduced into an expression vector carrying a T7 promoter which had been treated with a restriction enzyme to construct a synthetic HCVc gene expression vector pTHCVc. E. coli BL2
After transforming strain 1 (DE3) with pTHCVc, it was spread on an LB plate containing ampicillin (100 μg / ml) and cultured at 37 ° C. overnight.

The 10 colonies that had grown were inoculated into a 2XLB liquid medium containing ampicillin (100 μg / ml) and cultured at 35 ° C. OD650 of the culture solution is 0.8
When the number reached 5, 1 mM IPTG was added, and the cells were further cultured for 10 hours. After completion of the culture, the cells were collected by centrifugation. After disrupting the cells by sonication, the cells were separated into a precipitate fraction and a soluble fraction by centrifugation. The expression of HCVc protein in each fraction was confirmed by SDS-polyacrylamide gel electrophoresis.
It was found that a band corresponding to the HCVc protein of a was present in a large amount in the insoluble fraction.

The insoluble fraction was made up of 5 containing 0.15 M NaCl.
After thoroughly washing with 0 mM Na-phosphate buffer (pH 7.5), this was washed with 6 M guanidine hydrochloride / 1 mM DTT /
Dissolve in 50 mM Na-phosphate buffer (pH 7.8)
The solution was solubilized at 35 ° C. for 1 hour. This was purified by gel filtration using a TSKgel G4000SWXL column using the same developing solution as the processing solution. The purified fraction was subjected to ultrafiltration with a cut-off molecular weight of 5000 to give 10 mg /
Concentrated to ml.

In 5 μl of the solubilized HCVc, 1.6 μg / ml of recombinant PPIase derived from Methanococcus thermolystrophycus prepared in Reference Example 1 and 3 μl of 0.1 μl were added.
0.1 M Na-phosphate buffer containing 05 M NaCl (p
H7.8) was added and mixed, and reacted at 35 ° C. for 2 hours. After completion of the reaction, the results of analyzing the regenerated HCVc antigen by gel filtration using a TSKgel G4000 column are shown in FIG. The HCVc antigen appeared as a peak at a retention time of 6.8 minutes, indicating that a uniform particle structure was formed.

After coating the purified HCVc core particles on a 96-well microtiter plate, blocking was performed with bovine serum albumin, and PBS-T buffer (10 mM
Na-phosphate buffer pH 7.5 / 0.8% sodium chloride / 0.05% Tween 20). Next, human positive or negative serum diluted with PBS-T buffer was added and reacted. After washing with a PBS-T buffer, a peroxidase-labeled human IgG antibody (manufactured by Biogenesis) was allowed to act. After the reaction is completed, add 4 parts of PBS-T buffer.
After washing twice, a substrate color developing solution (containing phenyldiamine and hydrogen peroxide) was added and reacted. After stopping the reaction by adding 4N sulfuric acid, the absorption at 490 nm was measured. Table 4 shows the results. As a control in this example, the same antigenicity evaluation was performed for the standard serum.

[0090]

[Table 4]

As shown in Tables 1-4, natural HBs
An antigen or an inactive recombinant HBs antigen, HBc antigen or HCVc antigen is acted on by a reducing agent such as dithiothreitol or a surfactant such as octylglucoside in the presence of a denaturing agent such as urea or guanidium hydrochloride. Then, it was found that it is possible to obtain an antigen activity stronger than the antigenicity of the natural HBs antigen by reacting with PPIase after the molecular weight reduction treatment.

[0092]

According to the present invention, since it has the above-mentioned constitution, a virus antigen having enhanced antigen activity can be obtained, and the use of the virus antigen is useful for production of immunoassay reagents and the like.
In addition, the method for preparing a virus antigen of the present invention can be applied to a virus antigen obtained by a genetic recombination method using a bacterium or yeast as a host.
A large amount of virus antigens can be provided by simple operations.

[0093]

[Sequence List] <110> SEKISUI CHEMICAL CO., LTD. Marine Biotechnology Institute Co., Ltd. <120> Preparation of virus antigen <130> 99P00844 <150> JP P1998-377103 <151> 1998-12-28 <160> 16 <210> 1 <211> 608 <212> DNA <213> Methanococcus thermolithotrophicus <220> <221> CDS <222> 82..543 <400> 1 tgtaataaat ccattaatcg tggtagcaat aataaaatat atataccaat acagtttgag 60 ttatgatatc gacataagca t gtg att ttt ttg gta gat aaa gga gtt aaa 111 Val Ile Phe Leu Val Asp Lys Gly Val Lys 1 5 10 ata aaa gta gac tac ata ggt aaa ctt gaa agt gga gat gtc ttt gac 159 Ile Lys Val Asp Tyr Ile Gly Lys Leu Glu Ser Gly Asp Val Phe Asp 15 20 25 act tcc ata gaa gag gta gca aaa gaa gct gga ata tac gcg cct gat 207 Thr Ser Ile Glu Glu Val Ala Lys Glu Ala Gly Ile Tyr Ala Pro Asp 30 35 40 aga gaa tat gaa cct cta gag ttc gtt gta gga gaa ggt cag ttg att 255 Arg Glu Tyr Glu Pro Leu Glu Phe Val Val Gly Glu Gly Gln Leu Ile 45 50 55 caa ggt t tt gaa gaa gct gtt tta gac atg gaa gtc ggg gac gaa aaa 303 Gln Gly Phe Glu Glu Ala Val Leu Asp Met Glu Val Gly Asp Glu Lys 60 65 70 acc gta aaa atc cct gca gag aaa gca tac ggt aac aga aat gaa 351 Thr Val Lys Ile Pro Ala Glu Lys Ala Tyr Gly Asn Arg Asn Glu Met 75 80 85 90 tta ata cag aaa ata cca aga gat gct ttt aaa gaa gct gat ttt gaa 399 Leu Ile Gln Lys Ile Pro Arg Asp Ala Phe Lys Glu Ala Asp Phe Glu 95 100 105 cct gaa gaa gga atg gta atc ctt gca gaa gga att cct gct aca ata 447 Pro Glu Glu GIy Met Val Ile Leu Ala Glu Gry Ile Pro Ala Thr Ile 110 115 120 acc gaa gtt aca gat aat gag gta act tta gac ttt aac cat gaa ctt 495 Thr Glu Val Thr Asp Asn Glu Val Thr Leu Asp Phe Asn His Glu Leu 125 130 135 gca gga aaa gat tta gta ttt aca att aaa att att gaa gtt gtc gaa 543 Ala Gly Lys Asp Leu Val Phe Thr Ile Lys Ile Ile Glu Val Val Glu 140 145 150 taattatcat tataacattc cgccgagatt aaataaaatc acttgtgatt ttatatcttt 603 acccg 608 <210> 2 <211> 154 <212> PRT <213> Methanococcus thermolithotrophiphicus <400> 2 Val Ile Phe Leu Val Asp Lys Gly Val Lys Ile Lys Val Asp Tyr Ile 1 5 10 15 Gly Lys Leu Glu Ser Gly Asp Val Phe Asp Thr Ser Ser Ile Glu Glu Val 20 25 30 Ala Lys Glu Ala Gly Ile Tyr Ala Pro Asp Arg Glu Tyr Glu Pro Leu 35 40 45 Glu Phe Val Val Gly Glu Gly Gln Leu Ile Gln Gly Phe Glu Glu Ala 50 55 60 Val Leu Asp Met Glu Val Gly Asp Glu Lys Thr Val Lys Ile Pro Ala 65 70 75 80 Glu Lys Ala Tyr Gly Asn Arg Asn Glu Met Leu Ile Gln Lys Ile Pro 85 90 95 Arg Asp Ala Phe Lys Glu Ala Asp Phe Glu Pro Glu Glu GIy Met Val 100 105 110 Ile Leu Ala Glu Gry Ile Pro Ala Thr Ile Thr Glu Val Thr Asp Asn 115 120 125 Glu Val Thr Leu Asp Phe Asn His Glu Leu Ala Gly Lys Asp Leu Val 130 135 140 Phe Thr Ile Lys Ile Ile Glu Val Val Glu 145 150 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Sequence Listing Free Text Forward primer <400> 3 ggccatggta gataaaggag ttaaaataa 29 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Column table free text river Primer <400> 4 ccggatcctt attcgacaac ttcaataatt 30 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <221> variation <222> 5 <221> variation <222> 16 <221> variation <222> 25 <223> Sequence Listing Free Text Primer <400> 5 gsccnwtggb gataangcha tccgnccgc 29 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <221> variation <222> 4 <221 > variation <222> 25 <223> Sequence listing free text primer <400> 6 atgncstcca tccgyatgcg atgcnggtcw 30 <210> 7 <211> 678 <212> DNA <213> Type B hepatitis virus <220> <221> CDS <222 > 1..678 <400> 7 atg gag aac atc aca tca gga ttc cta gga ccc ctg ctc gtg tta cag 48 Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 gcg ggg ttt ttc ttg ttg aca aga atc ctc aca ata ccg cag agt cta 96 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 gac tcg tgg tgg act tct ctc aat ttt cta ggg gga tca ccc gtg tgt 144 Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ser Pro Va l Cys 35 40 45 ctt ggc caa aat tcg cag tcc cca acc tcc aat cac tca cca acc tcc 192 Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser 50 55 60 tgt cct cca att tgt cct ggt tat cgc tgg atg tgt ctg cgg cgt ttt 240 Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 atc ata ttc ctc ttc atc ctg ctg cta tgc ctc atc ttc ttc tt ttg gtt 288 Ile Ple Phe Ple Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val 85 90 95 ctt ctg gat tat caa ggt atg ttg ccc gtt tgt cct cta att cca gga 336 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly 100 105 110 tca aca aca acc agt acg gga cca tgc aaa acc tgc acg act cct gct 384 Ser Thr Thr Thr Thr Thr Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 115 120 125 caa ggc aac tct aag ttt ccc tca tgt tgc tgt aca aaa cct acg gat 432 Gln Gly Asn Ser Lys Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp 130 135 140 gga aat tgc acc tgt att ccc atc cca tcg tcc tgg gct ttc gca aaa 480 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys 145 150 155 160 tac cta tgg gag tgg gcc tca gtc cgt ttc tct tgg ctc agt tta cta 528 Tyr Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu 165 170 175 gtg cca ttt gtt cag tgg ttc gta ctt tcc ccc act gtt tgg ctt 576 Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 tca gct ata tgg atg atg tgg tat tgg ggg cca agt ctg tac agc atc 624 Ser Ala Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile 195 200 205 gtg agt ccc ttt ata ccg ctg tta cca att ttc ttt tgt ctc tgg gta 672 Val Ser Pro Phe Ile Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 220 tac att 678 Tyr Ile 225 <210> 8 <211> 226 <212> PRT <213> Type B hepatitis virus <400> 8 Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ser Pro Val Cys 35 40 45 Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser 50 55 60 Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val 85 90 95 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly 100 105 110 Ser Thr Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 115 120 125 Gln Gly Asn Ser Lys Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp 130 135 140 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys 145 150 155 160 Tyr Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu 165 170 175 Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 Ser Ala Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile 195 200 205 Val Ser Pro Phe Ile Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 220 Tyr Ile 225 <210> 9 <211> 681 <212 > DNA <213> Type B hepatitis virus <220> <221> CDS <222> 1.678 <400> 9 atg gag aac aca aca tca gga ttc cta gga ccc ctg ctc gtg tta cag 48 Met Glu Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 gcg ggg ttt ttc ttg ttg aca aga atc cta aca ata cca cag agt cta 96 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 gac tcg tgg tgg act tct ctc aat ttt cta ggg gga gca ccc acg tgt 144 Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr Cys 35 40 45 cct ggc caa aat tcg cag tcc cca acc tcc aat cag tca cca acc tct 192 Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn Gln Ser Pro Thr Ser 50 55 60 tgt cct cca att tgt cct ggc tat cgc tgg atg tgt ctg cgg cgt ttt 240 Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 atc ata ttc ctc ttc atc ctg ctg cta tgc ctc atc ttc ttg ttg gtt 288 Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val 85 90 95 ctt ctg gac tac caa ggt atg ttg ccc gtt tgt cct cta ctt cca gga 336 Leyrn Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly 100 105 110 aca tca act acc agc acg gga cca tgc aag acc tgc acg att cct gct 384 Thr Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile Pro Ala 115 120 125 caa gga acc tct atg ttt ccc tct tgt tgc tgt aca aaa cct tcg gac 432 Gln Gly Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp 130 135 140 gga aac tgc act tgt att ccc atc cca tca tcc tgg gct ttc gca aga 480 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg 145 150 155 160 ttc cta tgg gag ggg gcc tca gtc cgt ttc tcc tgg ctc agt tca cta 528 Phe Leu Trp Glu Gly Ala Ser Val Arg Phe Ser Trp Leu Ser Ser Leu 165 170 175 gtg cca ttt gtt cag tgg ttc gta ggg ctt tcc ccc act gtt tgg ctt 576 Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 tca gtt ata tgg atg atg tgg tat tgg gga cca agt ctg tac aac atc 624 Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn Ile 195 200 205 ttg agt ccc ttt tta cct cta tta cca att ttc ttt tgt ctt tgg gta 672 Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 220 tac att taa 681 Tyr Ile 225 <210> 10 <211> 226 <212> PRT <213> Type B hepatitis virus <400> 10 Met Glu Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu V al Leu Gln 1 5 10 15 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr Cys 35 40 45 Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn Gln Ser Pro Thr Ser 50 55 60 Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val 85 90 95 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly 100 105 110 Thr Ser Thr Thr Ser Ser Gly Pro Cys Lys Thr Cys Thr Ile Pro Ala 115 120 125 Gln Gly Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp 130 135 140 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg 145 150 155 160 Phe Leu Trp Glu Gly Ala Ser Val Arg Phe Ser Trp Leu Ser Ser Leu 165 170 175 Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn Ile 195 200 205 Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 22 0 Tyr Ile 225 <210> 11 <211> 697 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (6) .. (689) <220> <223> Sequence Listing Free Text adr Double-chain synthetic gene encoding type HBs antigen <400> 11 ggcat atg gaa aac act act tct ggt ttc ctg ggt ccg ctg ctg gta ctg 50 Met Glu Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu 1 5 10 15 cag gca ggt ttc ttc ctg ctg aca cgt atc ctc aca att cca cag tct 98 Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser 20 25 30 ctg gac tct tgg tgg act tct ctc aat ttt ctg ggt gca ccg act 146 Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr 35 40 45 tgc cct ggc caa aat tct cag tcc cca acc tcc aat cac tct cca acc 194 Cys Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr 50 55 60 tct tgc cct cca att tgc cct ggc tat cgc tgg atg tgc ctg cgt cgt 242 Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg 65 70 75 ttt atc att ttc ctc ttc atc ctg ctg ctg tgc ctc atc ttc ctg ctg 290 Phe I le Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu 80 85 90 95 gtt ctt ctg gac tac caa ggt atg ctg cca gtt tgc cct ctg ctt cca 338 Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro 100 105 110 ggt aca tct acc acc agc act ggt cca tgc aag acc tgc act att cct 386 Gly Thr Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile Pro 115 120 125 gct caa ggt acc tct atg ttt ccg tct tgc tgc tgc aca aaa cct tct 434 Ala Gln Gly Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser 130 135 140 gac ggt aac tgc act tgc att ccg atc cca tct tcc tgg gct ttc gca 482 Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala 145 150 155 cgt ttc ctg tgg gag tgg gcc tct gtc cgt ttc tcc tgg ctc tct ctg 530 Arg Phe Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu 160 165 170 175 ctg gtg cca ttt gtt cag tgg ttc gta ggt ctg tct ccg act gtt tgg 578 Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp 180 185 190 ctg tct gtt att tgg atg atg tgg tat tgg ggt cca tct ctg tac a ac 626 Leu Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn 195 200 205 atc ctg tct ccg ttt ctg cct ctg ctg cca att ttc ttc tgc ctt tgg 674 Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp 210 215 220 gta tac att taa tag ggatccgg 697 Val Tyr Ile 225 <210> 12 <211> 226 <212> PRT <213> Artificial Sequence <400> 12 Met Glu Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr Cys 35 40 45 Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser 50 55 60 Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val 85 90 95 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly 100 105 110 Thr Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile Pro Ala 115 120 125 Gln Gly Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp 1 30 135 140 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg 145 150 155 160 Phe Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu 165 170 175 Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn Ile 195 200 205 Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 220 Tyr Ile 225 <210> 13 <211> 568 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (6) .. (560) <220> <223> Sequence Listing Free Text HBc antigen Double-stranded synthetic gene to encode <400> 13 ggcat atg gac atc gac ccg tac aaa gaa ttc ggt gct tct gtt gaa ctg 50 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu 1 5 10 15 ctg tct ttt ctg cct tct gac ttc ttt cct tct att cgt gat ctc ctc 98 Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu 20 25 30 gac acc gcc tct gct ctg tat cgt gag gcc ctc gag tct ccg gaa cat 146 Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His 35 40 45 tgc tct cct cac cat aca gca ctc cgt caa gct atc ctg tgc tgg ggt 194 Cys Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly 50 55 60 gag ctg atg aat ctg gcc acc tgg gtg ggt agc aat ctg gaa gac cca 242 Glu Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro 65 70 75 gca tcc agc gaa ctt gta gtc agc tat gtc aat gtt aat atg ggc Serg Gla Ala Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu 80 85 90 95 aaa atc cgt caa ctg ctg tgg ttt cac att tcc tgc ctt act ttt ggc 338 Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly 100 105 110 cgt gaa act gtt ctt gag tat ctg gtg tct ttt ggt gtg tgg att cgc 386 Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg 115 120 125 act cct ccg gct tac cgt cca cca aat gcc cct atc ctg tct aca ctt 434 Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu 130 135 140 ccg gaa act act gtt gtt cgt cgt cgt ggc cgt tcc cct cgt cgc cgt 482 Pro Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg 145 150 155 act ccg tct cct cgc cgt cgt cgt tct caa tct ccg cgt cgc cgt cgc 530 Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg 160 165 170 175 tct caa tct cgt gaa tct caa tgc taa tag ggatccgg 568 Ser Gln Ser Arg Glu Ser Gln Cys 180 185 <210> 14 <211> 183 <212> PRT <213> Artificial Sequence <400> 14 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Ser Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 1 40 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu Ser Gln Cys 180 <210 > 15 <211> 592 <212> DNA <213> Artificial Sequence <220> <223> Sequence Listing Free Text Double-stranded synthetic gene encoding HCVc antigen <220> <221> CDS <222>. (590) <400> 15 ggcat atg tct act aac ccg aaa ccg cag cgt aaa act aaa cgt aac act 50 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr 1 5 10 15 aac cgt cgc cca cag gac gtc aag ttc cct ggt ggt ggt cag atc gtt 98 Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gly Gln Ile Val 20 25 30 ggt ggc gtt tac ctg ctt cca cgc cgt ggc cca cgt ctg ggt gtg cgt 146 Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg 35 40 45 gcg act cgt aag act tcc gag cgc tct caa cct cgt ggt cgt cgt caa 194 Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln 50 55 60 cct atc ccg aag gct cgt cgt cca gag ggt cgt gc c tgg gct cag cca 242 Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro 65 70 75 ggt tac cct tgg cca ctc tat ggc aat gag ggc atg ggt tgg gca ggt 290 Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly 80 85 90 95 tgg ctc ctg tct cca cgc ggt tcc cgt cct agc tgg ggt ccg act gac 338 Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp 100 105 110 cca cgt cgt cgc tct cgt aac ctg ggt aag gtc atc gat acc ctc aca 386 Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr 115 120 125 tgc ggc ttc gcc gac ctc atg ggt tac att ccg gtc ggt 434 Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro 130 135 140 ctg ggt ggt gct gcc cgt gcc ctg gcg cat ggc gtc cgt gtt ctg gaa 482 Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Val Leu Glu 145 150 155 gac ggc gtg aac tat gca aca ggt aat ctg cca ggt tgc tct ttc tct 530 Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser 160 165 170 175 atc ttc ctc ctg gct ct tcc tgc c tg acc atc cca gcc tcc gct 578 Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala 180 185 190 taa tag gga tcc gg 592 Gly Ser 195 <210> 16 <211> 191 <212> PRT <213 > Artificial Sequence <400> 16 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro Gly 65 70 75 80 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp 85 90 95 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125 Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140 Gly Gly Ala Ala Arg Ala Lela Ala His Gly Val Arg Val Leu Glu Asp 145 150 155 160 Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175 Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala 180 185 190

[Brief description of the drawings]

FIG. 1 is a chart of gel filtration column chromatography of a natural HBs antigen (untreated) in Example 1. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

FIG. 2 shows a natural type H treated with a low molecular weight in Example 1.
It is a chart of the gel filtration column chromatography of a Bs antigen. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

FIG. 3 is a chart of gel filtration column chromatography of a natural HBs antigen that has been subjected to a PPIase reaction after a low molecular weight treatment in Example 1. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

FIG. 4 is a chart of gel filtration column chromatography of a natural HBs antigen that has been subjected to a PPIase reaction after a molecular weight reduction treatment in Example 2. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

FIG. 5 is a chart of gel filtration column chromatography of a recombinant adw-type HBs antigen subjected to a molecular weight reduction treatment in Example 3. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

FIG. 6 is a chart of gel filtration column chromatography of a recombinant adw-type HBs antigen that has been subjected to a PPIase reaction after depolymerization treatment in Example 3. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

FIG. 7 is a chart of gel filtration column chromatography of a recombinant adw-type HBs antigen that has been subjected to a PPIase reaction after depolymerization treatment in Example 4. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

FIG. 8 is a chart of gel filtration column chromatography of a recombinant adr-type HBs antigen subjected to a molecular weight reduction treatment in Example 5. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

FIG. 9 is a chart of gel filtration column chromatography of a recombinant adr-type HBs antigen that has been subjected to a PPIase reaction after depolymerization treatment in Example 5. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

FIG. 10 shows PPI after depolymerization treatment in Example 6.
It is a chart of the gel filtration column chromatography of the recombinant adr-type HBs antigen which carried out the ase reaction. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

FIG. 11 shows the PPI after the depolymerization treatment in Example 7.
4 is a chart of gel filtration column chromatography of a recombinant adr-type HBs antigen that has been subjected to an ase reaction. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

FIG. 12 shows a PPI after a low molecular weight treatment in Example 8.
It is a chart of the gel filtration column chromatography of the recombinant HBc antigen which underwent an ase reaction. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

FIG. 13 shows a PPI after a low molecular weight treatment in Example 9.
It is a chart of the gel filtration column chromatography of the recombinant HCVc antigen which underwent an ase reaction. The vertical axis is the intensity of the absorbance, and the horizontal axis is the unit indicating the retention time of the column.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Masahiro Furuya 2-1 Hyakuyama, Shimamoto-cho, Mishima-gun, Osaka Prefecture Sekisui Chemical Co., Ltd. (72) Inventor Akira Ideno 75th Hirata, Hirata, Kamaishi City, Iwate Prefecture 1 Marine Biotechnology Research Institute Kamaishi Laboratory

Claims (8)

[Claims] 1. A method for preparing a virus antigen, comprising the step of reacting a virus antigen with peptidyl prolyl cis trans isomerase. 2. A step of treating a virus antigen with at least one selected from the group consisting of a surfactant, a reducing agent and a protein denaturant, and reacting the treated virus antigen with peptidyl prolyl cis trans isomerase. A method for preparing a virus antigen, comprising the steps of: 3. The method for preparing a virus antigen according to claim 1, wherein the virus antigen is expressed by a recombinant method using bacteria and yeast as hosts. 4. The method for preparing a virus antigen according to claim 1, wherein the peptidyl prolyl cis trans isomerase is bound to an insoluble carrier. 5. The method for preparing a virus antigen according to claim 1, wherein the virus antigen is an HBs antigen, an HBc antigen or an HCVc antigen. 6. The peptidyl prolyl cis trans isomerase is prepared from archaebacteria or expressed from DNA prepared from archaebacteria.
The method for preparing a viral antigen according to 2, 3, 4 or 5. 7. The method according to claim 6, wherein the archaea is a bacterium belonging to the genus Methanococcus. 8. The bacterium belonging to the genus Methanococcus is Methanococcus thermolystrophycus (Methanococcus).
occus thermolithotropicu
s) or Methanococcus voltae (Methanoc)
8. The method for preparing a viral antigen according to claim 7, which is O. occus voltae.