JPH06105697A - Purification of antigen protein specific to non-a, non-b hepatitis - Google Patents

Abstract

PURPOSE:To readily and efficiently obtain the subject protein of high purity useful as a reagent for clinical analysis of non-A, non-B hepatitis only by one purification, by purifying a solution containing an antigen protein specific to non-A, non-B hepatitis using Phenyl-Sepharose(R) as a carrier. CONSTITUTION:A solution containing an antigen protein (preferably an antigen protein obtained by gene recombination) specific to non-A, non-B hepatitis is brought into contact with Phenyl-Sepharose(R) or Octyl-Sepharose(R) as carrier so as to adsorb other excessive proteins onto the carrier. Thereby, the objective protein can be obtained from a fraction passing through without being absorbed.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying a non-A non-B hepatitis virus-related antigen (also referred to as "non-A non-B hepatitis-specific antigen protein"). In particular, the present invention also relates to a method for purifying a non-A non-B hepatitis virus-related antigen obtained using a gene recombination technique.

[0002]

2. Description of the Related Art Among viral hepatitis, with regard to hepatitis A, which is an epidemic hepatitis that is orally transmitted, and hepatitis B virus, which is transmitted via blood, causative viruses have been isolated and their properties have been clarified. . As a result, diagnostic methods have been established for both of them, and infection prevention methods have been established. However, non-A non-B hepatitis that is neither type A nor type B is said to account for about 90% of post-transfusion hepatitis in Japan (Nippon Clinic, Vol. 35, p. 2724 (1977); Journal. According to an estimate compiled by the Ministry of Health and Welfare, which accounts for nearly 40% of deaths from liver disease, hepatitis C is considered to be the majority of non-A non-B hepatitis. ing. The main routes of infection are blood transfusion and blood products, which can prevent infection if a proper test method can be established, and therefore there are great expectations for the development of test agents.

Recently, Chiron Inc. of the US injected chimpanzee with serum from a non-A non-B hepatitis patient, and used RNA derived from the serum of chimpanzee causing non-A non-B hepatitis as a starting material for HCV (C Hepatitis C virus) cDNA was cloned (QL Ku et al., Science, 244).
Volume, 359 (1989); European Pat.
ent Application # 8831092
2.5). Various antigens have been investigated based on this sequence, but it is difficult to say that they are effective in preventing HCV infection so far.

However, in the retrospective study presented in the symposium "Advances in Non-A, Non-B Hepatitis Research-Basics and Clinical Practice" of the 1990 General Meeting of the Japanese Society of Hepatology, the test agents currently in use were 8 years ago. Even if it was developed, 10 out of 14 people could not prevent the infection, indicating that the infectious virus was not reflected in the test results even though it was in the blood. As a cause of this, it is considered that the antigen used in this test drug is used by producing a portion that is considered to correspond to the nuclear protein of the virus particle by genetic engineering means.
It can be said that it is necessary to develop more than one type of antigen in order to carry out the CV test reliably.

For this purpose, many researchers have used H
We are trying to clone the CV genome, but both are PCR based on the HCV nucleotide sequence announced by Chiron.
Method (Y. Kubo et al., Nucleic Acids Research, 17: 10367 (1989);
H. Okamoto et al., Japan Journal of Experimental Medicine, Volume 60, 167 (199
(0) etc.), the PCR method can only clone HCV sequences that have a high degree of commonality with HCV manufactured by Chiron. Therefore, there is a problem in cloning the HCV genome in which the nucleotide sequence is likely to change. In addition, since the amino acid sequence encoded by the sequence changes with the change in the nucleotide sequence, the antigenicity of the virus is also likely to change, which causes a problem in the antibody diagnostic agent using a single antigen. In order to solve this, the present inventors specifically react with patient serum rather than hepatitis C patient serum 2
We have already isolated a clone and developed a highly sensitive ELISA-based drug that combines this clone (PCT / J
P91 / 1662).

[0006]

However, even if the reactivity of the clone itself is excellent, it is important to prepare the antigen itself to be used in constructing an antibody test drug. By the way, in many cases, as an antibody diagnostic agent, the one obtained as a fusion protein with a heterologous protein prepared by a gene recombination method is directly used as an antigen. For example, Choo
Human-derived superoxide desmutase, Va
Llari et al. uses CMP-KDO synthetase derived from Escherichia coli, which has another big problem. For example, there is a possibility that antibodies against these heterologous proteins are present in human serum, and it is possible that an accurate antibody test may not be possible due to this. In addition, false positives due to nonspecific binding of serum antibodies to these foreign proteins and the presence of foreign proteins
The antigen-antibody reaction can be physically inhibited resulting in false negatives. Such problems must be eliminated in order to carry out a more reliable inspection. In order to avoid these situations, the method of expressing the antigen protein independently in the microorganism and the method of using the synthetic peptide have been adopted on the one hand, but the former method is concerned with the stability of the antigen polypeptide in the cells. In addition, the latter case may be impractical because it is difficult to synthesize a peptide having a long sequence. Under these circumstances, in order to construct a highly accurate test drug, conventionally, a carrier column (gel 3 permeation method, ion exchange chromatography, etc.) was combined to purify the antigen protein, but the yield and In many cases, problems may occur in the purity of the obtained antigenic protein. An object of the present invention is to purify a non-A non-B hepatitis specific antigen simply, in high yield and with high purity.

[0007]

In order to achieve the above-mentioned object of the present invention, the present inventors have extensively researched and developed an efficient and excellent purification method for a non-A non-B hepatitis-specific antigen protein. That is, the present invention is a method for purifying a non-A non-B hepatitis specific antigen protein, which comprises contacting a solution containing the non-A non-B hepatitis specific antigen protein with a phenyl sepharose carrier or an octyl sepharose carrier.

The antigenic protein used in the present invention is a natural type,
There is no particular limitation such as a gene recombinant type. An antigen protein obtained by gene recombination is preferably used in the present invention. For example, as a preparation method using gene recombination technology, E. coli using gene recombination technology,
Examples of the method include expression in yeast, insect cells and animal cells. As an expression method, cDNA encoding this antigen
Alternatively, a protein synthesis initiation signal or termination signal is added to a part thereof, and then another peptide gene (for example, mouse interferon β, lipocortin, interleukin 2
Alternatively, any protein can be used, such as β-galactosidase, baculovirus polyhedrin, and trpE, as long as it is a protein stably expressed in cells or cells.
That is, any part of each gene that can be stably expressed is used in the present invention. 5) or 3)
Examples include a method in which a gene encoding a non-A non-B hepatitis-specific antigen is linked to the terminal side and expressed in a microorganism, insect cell, animal cell or the like. As a method for expressing a gene in E. coli, yeast, insect cells, animal cells, etc., a promoter,
A ribosome binding sequence, a transcription termination factor, etc. must be linked to the target gene as necessary. The promoter is trp, tac, etc. in E. coli, and sv in animal cells.
For example, in insect cells such as 40, polyhedrin and the like can be mentioned.
In this way, a gene encoding a non-A non-B hepatitis-specific antigen is ligated to the 5'-terminal or 3'-terminal side of the expressed protein gene by ligase so that the frames thereof match.

At this time, a protease (for example, lysine protease, collagenase, enterokinase, Factor X which is a blood coagulation factor) is present between the fusion protein and the heterologous protein.
a)) and cyanogen bromide recognition sites are genetically designed, and during purification, they can be digested using an enzymatic or chemical method to purify and use only the antigen portion of the present invention. Is. A preferable recognition site that can be enzymatically digested is a recognition sequence by Factor Xa. Furthermore, the fusion protein side can be designed so that an amino acid and / or peptide sequence that facilitates the purification treatment of the antigen portion of the present invention after the digestion treatment is present. A preferable sequence useful for such purification treatment is a sequence containing a histidine residue that enables efficient application of metal chelate chromatography. Examples of the metal chelate carrier used in such metal chelate chromatography include transition metal chelating carriers such as nickel chelating carriers. As these, commercially available products can be used, and as the carrier used therefor, polyacrylamide, cellulose and the like which are widely known and used can be used without limitation.

By using phenyl-sepharose or octyl sepharose carrier in the purification of the above-mentioned antigenic protein, highly purified non-A non-B hepatitis-specific antigen can be obtained in a single purification. That is, a solution containing a non-A non-B hepatitis-specific antigen protein is brought into contact with a phenyl-sepharose or octyl sepharose carrier to non-A non-B non-B
A highly purified non-A non-B hepatitis-specific antigen protein can be obtained by adsorbing impurities other than the hepatitis-specific antigen protein onto a carrier. It can also be used in combination with other columns, for example, a solution containing a non-A non-B hepatitis-specific antigen protein is passed through nickel chelate affinity chromatography to adsorb the antigen protein, and the adsorbate is eluted with an eluent, By contacting the eluate with a phenyl-sepharose or octyl sepharose carrier, high purity non-A non-B
A hepatitis-specific antigen protein can be obtained. Furthermore, a hydrocarbon-bonded sepharose carrier such as one selected from the group consisting of cyclohexyl, diphenyl, octadecyl or cyanopropyl could be used without departing from its purpose. In one purification method, the fused non-A non-B hepatitis-specific antigen protein produced by gene recombination technology is first purified with a metal chelating carrier. This purification treatment can be repeated once or plural times as necessary. Next, the fused non-A non-B hepatitis-specific antigen protein thus purified is treated with, for example, an enzyme to produce the desired non-A non-B hepatitis-specific antigen protein, and then obtained. The obtained protein is contacted with a phenyl sepharose carrier or an octyl sepharose carrier. This contact treatment can be repeated once or plural times as necessary.

The present invention will be described in more detail with reference to the following examples.

【Example】

Example 1 Expression of S4 Antigen in Escherichia coli The S4 gene has a histidine 6 residue at the EcoRI site of pEFS having a trp promoter and a sequence encoding the recognition site of trpE and the coagulation factor FactorXa. It was inserted to create pFHMS4 (FIGS. 1 and 2). First of all, pEFS trpE
The EcoRI site present on the C-terminal side of was blunt-ended with Klenow fragment (Takara Shuzo). Then, the S4 fragment containing the histidine cluster amplified by the PCR method was inserted (pFHMS4). This prepared pFHM
After transforming S4 into E. coli HB101, the resulting strain was
ml LB medium (tryptone 1%, yeast extract 0.5%, NaCl, 0.5% ampicillin 10
(0 μg / ml) and precultured overnight at 37 ° C. After overnight culturing, 200 μl of this cultivated bacterium was planted in 10 ml of M9 minimal medium containing ampicillin, and cultivated with shaking at 30 ° C. for 6 hours. After 6 hours, 0.2 ml of 1 mg / ml indole acrylic acid was added, and the mixture was further cultured overnight. The expression of the protein was confirmed by SDS-PAGE after centrifuging 1 ml of the culture solution, collecting the cells, adding 1% SDS and 2-mercaptoethanol to lyse the cells. The fusion protein of S4 and trpE has a molecular weight of 60000.
It was observed in the vicinity, and the amount of protein reached 20 to 30% of the total protein. Since the obtained fusion protein had formed insoluble granules, it was washed with a phosphate buffer or a guanidine solution,
Soluble protein was removed. After solubilization with 8M urea F
actorXa buffer (20 mM Tris-HCl
It was dialyzed against PH8, 100 mM NaCl). S4
As for the antigenicity of the fusion protein of TrpE and trpE, after electrophoresis, the protein was transferred to a nitrocellulose filter, and C
By examining the reactivity with hepatitis C patient serum, it was confirmed that the expressed protein did not react with the healthy person serum but specifically with the hepatitis C patient serum. 1/20 weight of this fusion protein
Factor Xa of 0 was added, and the mixture was reacted at room temperature for about 20 hours to cleave the S4 protein from the protected peptide. This S4 protein was purified by the following method and used as an antigen to be immobilized on a microplate.

First, the protein solution buffer was replaced with Facto.
rXa buffer (20 mM Tric-HCl PH
(8,100 mM NaCl) was exchanged with 0.1 M phosphate buffer of pH 8 containing 6 M guanidine hydrochloride, and S4 protein was adsorbed on the nickel chelating carrier. Subsequently, this column was washed with 0.1 M phosphate buffer of pH 6 containing 6 M guanidine hydrochloride to remove excess protein.
Elution of S4 protein was performed using 0.1 M phosphate buffer of pH 5 containing 6 M guanidine hydrochloride, and the recovery rate was about 8
At 0%, S4 protein with a purity of about 90% was obtained. Further, this fraction was again added to the Factor Xa buffer (20 mM Tri
It was dialyzed against s-HCl PH8, 100 mM NaCl) and purified by hydrophobic chromatography (phenyl sepharose). Other extra proteins are FactorXa
S4 protein was adsorbed on the phenyl sepharose carrier in the buffer, but S4 protein was not adsorbed, whereby S4 protein having a purity of about 95% or more could be obtained.

Example 2 In the same manner as in Example 1, S4 protein was cleaved from the protected peptide and purified by the following method. FactorXa
The S4 protein was cleaved from the protected peptide by digesting with, and DTT was added so that the final concentration was 2 mM. Thirty
After leaving at room temperature for 1 minute to 1 hour, it was purified with phenyl-Sepharose (manufactured by Pharmacia) equilibrated with a solution of FactorXa buffer to which DTT was added so that the final concentration was 2 mM. S4 and its degradation products were not adsorbed on the phenyl-sepharose carrier, but other mixed proteins were adsorbed on the carrier, and only S4 protein was specifically present in the flow-through fraction. The purification with phenyl-sepharose was repeated once more to obtain an S4 protein having a purity of 95% or more.
Similar effects were observed with octyl-Sepharose in addition to phenyl-Sepharose, but there was no effect with phenyl-Toyopearl (manufactured by Tosoh Corporation), which is a hydrophobic carrier. Thus, a highly pure S4 antigen could be obtained very simply by using the phenyl-sepharose carrier.

Example 3 Microplate E combining S29 and S4 antigens
Preparation of IA A microplate EIA was prepared by combining the purified recombinant antigen S4 described in Example 1 and the synthetic peptide S29-1 described in Sequence Listing 1. After immobilizing the synthesized peptide S29-1 and purified recombinant antigen S4 on a 96-well microplate, 100 μl of buffer solution and 10 μl of sample serum
The mixture was further reacted at 37 ° C. for 60 minutes. After washing the plate, it was reacted with a mouse anti-human IgG monoclonal antibody labeled with peroxidase (manufactured by Kappel) at 37 ° C. for 15 minutes. After washing the plate, a substrate solution was added and reacted at 37 ° C. for 15 minutes to stop the reaction by adding 1N sulfuric acid, and then the absorbance at 450 nm was measured. The result is shown in FIG. Twenty-nine of 30 serum samples could be determined to be positive and did not react with 7 healthy subjects. Similar results can be obtained by using the recombinant antigen S4 obtained by purification by the method of Example 2. According to the present invention, it is possible to prepare a large amount of purified non-A non-B hepatitis-specific antigen that does not contain an extra sequence using gene recombination technology and is applied to an actual non-A non-B hepatitis antibody test agent. It was shown that it was possible.

[0015]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to purify a large amount of non-A non-B hepatitis-specific antigen containing no extra sequence using gene recombination technology, and the antigen prepared by this method is non-A non-A. It was shown to be applicable to the development of a hepatitis B antibody test drug. Antibodies using this antigen do not contain sequences other than non-A non-B hepatitis-specific antigens, so it is expected that false positives and false negatives are unlikely to occur, and serum from patients can be determined with high sensitivity and blood screening. Is considered to be useful in doing

[Sequence list]

[Sequence list]

[Brief description of drawings]

FIG. 1 shows a schematic for making a ΔEcoRI fragment from plasmid pFHMS4.

FIG. 2 shows a schematic for making the plasmid pFHMS4.

FIG. 3 shows the results of ELISA using S4 and T064 as antigens. Sample numbers 1 to 30 in the figure are hepatitis C chronic patient sera and 31 to 37 are healthy human sera.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location G01N 33/53 D 8310-2J 33/531 A 8310-2J 33/576 Z 9015-2J // C12N 15/51 (C12P 21/02 C12R 1:19)

Claims (2)

[Claims] 1. A method for purifying a non-A non-B hepatitis specific antigen protein, which comprises contacting a solution containing a non-A non-B hepatitis specific antigen protein with a phenyl sepharose carrier or an octyl sepharose carrier. 2. The purification method according to claim 1, wherein the non-A non-B hepatitis-specific antigen protein is produced from a fusion protein obtained by recombination.