JPH0576382A - Method for producing hepatitis B virus protein - Google Patents

Abstract

(57) [Summary] [Objective] A core or surface structural protein of hepatitis B virus is produced by utilizing a selective amplification technique of a specific DNA and a gene recombination technique. A first primer having an NcoI recognition sequence and having a nucleotide sequence capable of specifically binding to a start codon-containing region of DNA encoding a natural core structure protein, and GGATC
CTA and a combination with a second primer having a base sequence capable of specifically binding to the stop codon-containing region of the core structure protein-encoding DNA, or an NcoI recognition sequence, and a start codon-containing region of the natural surface structure-encoding DNA. The combination of the first primer having a base sequence capable of specifically binding to AGCTTTA and the second primer containing AAGCTTTA and having a base sequence capable of specifically binding to the stop codon-containing region of the surface structure protein-encoding DNA is used.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a core structure protein or surface structure protein of hepatitis B virus by utilizing a technique for selectively amplifying a specific DNA and a gene recombination technique. The present invention is particularly directed to two types of primer DNAs used when utilizing a technique of selectively amplifying DNAs encoding those structural proteins, which includes a start codon and is cleaved with a specific restriction enzyme. And an oligonucleotide that contains a stop codon and can be cleaved with another specific restriction enzyme.

[0002]

2. Description of the Related Art Hepatitis B virus (hepatitis)
B virus: hereinafter, abbreviated as HBV may be used) is an infectious agent that causes human liver disease.
It has been revealed that patients with chronic hepatitis infected with BV lead to patients with cirrhosis or liver cancer. Infection due to blood transfusion is also a problem. A screening test for whether or not hepatitis B is infected is necessary for prevention and treatment of infection, and various diagnostic tests for hepatitis B infection have been attempted.

However, purified H used for the above-mentioned diagnostic reagents
It was necessary to use a large amount of human plasma in order to obtain the Bs antigen, and since this purification step was extremely dangerous, improvement was desired. In addition, since the number of HBs antigen-positive patients is gradually decreasing, it is expected that HBs antigen will be produced using gene recombination technology.

Various methods for producing HBs antigens using gene recombination technology have been reported, but all of them require collection and processing of plasma containing a large amount of Dane particles, and radioactive isotopes (eg, radioisotopes). , ³² P), it requires special equipment and was not common.

As a conventional method utilizing the gene recombination technique, for example, Fujizawa, Y. et al. Et al, Nucleei
c Acids Res. 11 , 3581-3590
The following method is described in (1983). That is, H
A DNA fragment obtained by cutting a circular DNA of about 3.2 Kb isolated from BV with a restriction enzyme (EcoRI, BamHI, etc.) was incorporated into a plasmid vector, and the transformant with this recombinant vector was spread on an agar plate. The colonies are screened to obtain hepatitis B virus DNA clones. Then, after growing this plasmid in E. coli,
The plasmid is isolated, and a DNA fragment containing the HBs protein gene (or HBc protein gene) is cut out using an appropriate restriction enzyme. Furthermore, the DNA thus obtained
The fragment was incorporated into an expression vector, and a transformant with the expression vector was cultured to isolate the target HBs protein or HBc protein.

However, in the above method, the DNA fragment to be inserted into the expression vector is HBs protein or HBs protein.
Larger than the gene encoding the Bc protein,
It had extra DNA sequences upstream of the 5'end and downstream of the 3'end. This is because there is no restriction enzyme that can be cleaved immediately before the start codon ATG, and the stop codons TAA and TA.
This was because no restriction enzyme that could be cleaved immediately after G or TGA was found. Furthermore, the HBs antigen has four subtypes (adr, adw, ayr and a).
yw) exists and the base sequences that can be cleaved by the same restriction enzymes (EcoRI, PstI, BamHI, etc.) are not stored if the subtypes differ, so it was necessary to confirm whether each subtype could be cleaved. .. In addition, the cleavage site may be lost due to the mutation, and thus cloning could not be performed in some cases.

[0007]

Therefore, the object of the present invention is to eliminate the need to use a large amount of plasma, to obtain an insert DNA fragment consisting of just before the start codon to immediately after the stop codon, and to obtain HBs. It is an object of the present invention to provide a method adaptable to the difference between antigen subtypes and the loss of cleavage site due to mutation.

[0008]

[Means for Solving the Problems] The above-mentioned problems are solved by the first method of the present invention, namely, (1) (a) a recognition base sequence CCAT of a restriction enzyme NcoI.
The natural human hepatitis B virus, which contains GG and has a base sequence ATG present in the recognition base sequence CCATGG, corresponds to the initiation codon ATG in the base sequence of the DNA encoding the natural human hepatitis B virus core structural protein. A first DNA primer having a base sequence capable of specifically binding to a region containing a start codon ATG of a DNA encoding a core structure protein or a complementary base sequence thereof (hereinafter sometimes referred to as 1C primer)
And (b) the base sequence CTA containing the base sequence GGATCCTA, and the base sequence CTA present at the end of the base sequence is complementary to the stop codon TAG in the base sequence of the DNA encoding the natural human hepatitis B virus core structural protein. Corresponding to CTA, the stop codon TAG of the DNA encoding the natural human hepatitis B virus core structural protein
A second DNA primer having a base sequence capable of specifically binding to a region containing or a complementary base sequence thereof (hereinafter sometimes referred to as 2C primer),
(C) a DNA polymerase and (d) a hepatitis B virus DNA mixture is subjected to a DNA amplification step to amplify the DNA encoding the hepatitis B virus core structure protein, and (2) the hepatitis B virus core structure. The amplified DNA encoding the protein is treated with the restriction enzymes NcoI and B
NcoI / BamHI digested DNA after digestion with amHI
Producing a fragment, (3) ligating the NcoI / BamHI digested DNA fragment to a replicable expression vector to form a recombinant DNA, (4) transforming a microorganism or cell with the recombinant DNA, 5) The resulting transformant is cultured, and the transformant DN encoding the hepatitis B virus core structural protein
Hepatitis B characterized by including each step of expressing A to produce hepatitis B virus core structural protein, and (6) isolating the produced hepatitis B virus core structural protein from the transformant. It can be solved by a method for producing a viral core structural protein.

Further, the above-mentioned problem is the second method of the present invention, namely, (1) (a) the recognition base sequence CCAT of the restriction enzyme NcoI.
The natural human hepatitis B virus, which contains GG and has a base sequence ATG present in the recognition base sequence CCATGG, corresponds to the initiation codon ATG in the base sequence of the DNA encoding the natural human hepatitis B virus surface structural protein. A first DNA primer having a base sequence capable of specifically binding to a region containing a start codon ATG of a DNA encoding a surface structural protein or a complementary base sequence thereof (hereinafter sometimes referred to as 1S primer)
And (b) the base sequence TTA containing the base sequence AAGCTTTTA, and the base sequence TTA present at the end of the base sequence is complementary to the stop codon TAA in the base sequence of the DNA encoding the natural human hepatitis B virus surface structural protein. The stop codon TAA of the DNA encoding the natural human hepatitis B virus surface structural protein corresponding to TTA
A second DNA primer having a base sequence capable of specifically binding to a region containing or a complementary base sequence thereof (hereinafter, may be referred to as 2S primer),
A mixed solution containing (c) DNA polymerase and (d) hepatitis B virus DNA is subjected to a DNA amplification step to amplify DNA encoding a hepatitis B virus surface structure protein, and (2) hepatitis B virus surface structure. The amplified DNA encoding the protein is treated with restriction enzymes NcoI and H
Digested with indIII, NcoI / HindIII digested D
Producing an NA fragment, (3) ligating the NcoI / HindIII digested DNA fragment to a replicable expression vector to form a recombinant DNA, (4) transforming a microorganism or cell with the recombinant DNA, (5) The resulting transformant is cultured, and the transformant DN encoding the hepatitis B virus surface structural protein
Hepatitis B characterized by including each step of expressing A to produce hepatitis B virus surface structural protein, and (6) isolating the produced hepatitis B virus surface structural protein from the transformant. It can be solved by a method for producing a virus surface structural protein. In the description of the present specification, A means an adenine residue, C means a cytosine residue, G means a guanine residue, and T means a thymine residue.

The DNA gene sequence of HBV has already been elucidated [Nucleic Acids Research].
ch, No. 6, 8, 13 (1983)], molecular weight 2.
It is known to consist of about 3200 base pairs at 1 × 10 ⁶ daltons. Of these, the core protein gene (HBc
The (gene) region exists between about 1900th base and about 2400th base. In the first step of the first method of the present invention, only this HBc gene region, that is, the DNA region encoding HBc is selectively amplified. This includes PCR (polymerase chainrea).
method) can be used. By using the PCR method, it is possible to automatically amplify only a target DNA region from a small amount of DNA up to about 1,000,000 times (S
Science, 239 , 487-491, 1988).
In the PCR method, a cycle of amplification is repeated using a primer for the + strand, a primer for the − strand, and a thermostable DNA polymerase with the DNA region to be amplified interposed therebetween.

In the first method of the present invention, the 1C primer (for the + strand) used in the PCR method in the first step contains the recognition base sequence CC ATG G of the restriction enzyme NcoI, and the start thereof is within the base sequence. It has a codon ATG (underlined portion of the base sequence). The 1C primer preferably includes at least 20 bases (preferably 20 to 40 bases, more preferably 28 to 32 bases) including the recognition base sequence CCATGG of the restriction enzyme NcoI. When it is less than 20 bases, the specificity as a primer is lowered, and it becomes extremely difficult to achieve the object of the method of the present invention. Also, the larger the number of bases,
Although the specificity as a primer is improved, the production of the primer is costly, and the double structure between the primer molecules or within the molecule is easily formed, which is not preferable.

A preferred 1C primer is the above-mentioned when the base sequence ATG present in the recognition base sequence CCATGG corresponds to the initiation codon ATG in the base sequence of the DNA encoding the natural human hepatitis B virus core structural protein. Upstream of the recognition base sequence CCATGG and / or
Or, downstream, the same as the base sequence of the region containing the start codon ATG of the DNA encoding the natural human hepatitis B virus core structural protein, or at most 3 bases substituted,
It has a deleted or inserted base sequence. For example, the 1C primer has a sequence GA of preferably 9 bases downstream from the initiation codon, as in the natural human HBc gene.
CATTGAC, more preferably a 19-base sequence GAC
It may have a structure in which ATTGACCCCGTATAAA is linked. In addition, the 1C primer was prepared by adding natural human HBc upstream of the recognition base sequence of the restriction enzyme NcoI.
It may also have a 6-base sequence TTTGGG similar to the gene. Furthermore, a sequence having a substitution, deletion, or insertion of 2-3 bases in the 19-base sequence downstream of the start codon, for example,
GACATT (or C) GAC (or T) A C T (or
C ) TATAAAG may be used (the underlined portion is replaced).

In the first method of the present invention, the 2C primer (for the minus strand) used in the PCR method in the first step contains the nucleotide sequence GGATCCTA. Of this 8-base sequence, the first 6-base sequence GGATCC is the restriction enzyme Bam.
This is the recognition base sequence of HI and the sequence of the last 3 bases CTA
Is a sequence complementary to the stop codon TAG. This 2C primer also includes the above-mentioned base sequence GGATCCTA,
At least 20 bases (preferably 20 to 40 bases,
It is more preferably 28 to 32 bases). When it is less than 20 bases, the specificity as a primer is lowered, and it becomes extremely difficult to achieve the object of the method of the present invention. Further, as the number of bases increases, the specificity as a primer is improved, but the production of the primer is costly, and further, a double structure between the primer molecules or in the molecule is easily formed, which is not preferable.

A preferred 2C primer is the last nucleotide sequence CTA in the nucleotide sequence GGATCCTA.
To the complementary base sequence CTA of the stop codon TAG in the base sequence of the DNA encoding the natural human hepatitis B virus core structural protein, the natural human B is located upstream and / or downstream of the base sequence GGATCCTA. DN encoding the hepatitis C virus core structural protein
It has the same nucleotide sequence as the complementary nucleotide sequence of the region containing the stop codon TAG of A, or has at most 3 nucleotides replaced, deleted or inserted. For example, the 2C primer is preferably a 6-base sequence ACATTG, more preferably a 16-base sequence ACA downstream of the stop codon.
It has a structure in which TTGAGATTCCCCGA is linked.
These base sequences are base sequences complementary to the + chain of the natural human HBc gene. In addition, the base sequence GGATC
Upstream of CTA, preferably the 6-base sequence TCCAAG
Can also have This base sequence is also natural human HB
It is a nucleotide sequence complementary to the + strand of the c gene. Furthermore, a sequence having a substitution, deletion, or insertion of 2 to 3 bases in the 16 bases sequence upstream of the stop codon, for example, ACAT TG GAT
It may be TCCCGA (the underlined part is replaced).

The surface protein gene (HBs gene) region prepared by the second method of the present invention exists between the about 150th base and the about 840th base of the HBV DNA gene sequence. In the first step of the second method of the present invention, only this HBs gene region is selectively amplified. For this, the PCR method can be used as described above.

The 1S primer (for the + strand) contains the recognition base sequence CC ATG G of the restriction enzyme NcoI, and has the start codon ATG (underlined portion of the base sequence) in the base sequence. This 1S primer includes the recognition base sequence CCATGG of the restriction enzyme NcoI,
At least 20 bases (preferably 20 to 40 bases,
It is more preferably 28 to 32 bases). When it is less than 20 bases, the specificity as a primer is lowered, and it becomes extremely difficult to achieve the object of the method of the present invention. Further, as the number of bases increases, the specificity as a primer is improved, but the production of the primer is costly, and further, a double structure between the primer molecules or in the molecule is easily formed, which is not preferable.

A preferred 1S primer is the above-mentioned when the base sequence ATG present in the recognition base sequence CCATGG corresponds to the initiation codon ATG in the base sequence of the DNA encoding the natural human hepatitis B virus surface structural protein. Upstream of the recognition base sequence CCATGG and / or
Or downstream, the same as the base sequence of the region containing the initiation codon ATG of the DNA encoding the natural human hepatitis B virus surface structural protein, or at most 3 bases substituted,
It has a deleted or inserted base sequence. For example, the 1S primer has a sequence G of preferably 10 bases downstream of the initiation codon, as in the natural human HBs gene.
AGAACACAA, more preferably a sequence G of 20 bases
It may have a structure in which AGAACACAACACATCAGGATT is linked. Further, the 1S primer may have a 5-base sequence ACCGA similar to that of the natural human HBs gene, upstream of the recognition base sequence of the restriction enzyme NcoI. Furthermore, a sequence having a substitution, deletion or insertion of 2-3 bases in the 20 base sequence downstream of the start codon, for example, GAGAA (or G ) CA TCG CATCAGGA.
It may be T (or C ) T (underlined part is replaced). Further, GA (or T) A (or T) GA may be linked in place of the 5-base sequence ACCGA upstream of the recognition base sequence of the restriction enzyme NcoI.

The 2S primer (for the minus strand) contains the nucleotide sequence AAGCTTTTA. Of this 8 base sequence,
The first 6-base sequence AAGCTT is the restriction enzyme HindII.
It is a recognition base sequence of I, and the last three base sequence TTA is a sequence complementary to the stop codon TAA. The 2S primer is also preferably composed of at least 20 bases (preferably 20 to 40 bases, more preferably 28 to 32 bases) including the base sequence AAGCTTTA. When it is less than 20 bases, the specificity as a primer is lowered, and it becomes extremely difficult to achieve the object of the method of the present invention. Further, as the number of bases increases, the specificity as a primer is improved, but the production of the primer is costly, and further, a double structure between the primer molecules or in the molecule is easily formed, which is not preferable.

A preferred 2S primer is the last nucleotide sequence TTA in the nucleotide sequence AAGCTTTTA.
To the complementary base sequence TAA of the stop codon TAA in the base sequence of the DNA encoding the natural human hepatitis B virus surface structural protein, the natural human B is located upstream and / or downstream of the base sequence AAGCTTTTA. DN encoding a hepatitis C virus surface structural protein
It has the same nucleotide sequence as the complementary nucleotide sequence of the region containing the stop codon TAA of A, or has at most 3 nucleotides replaced, deleted or inserted. For example, the 2S primer may have a structure in which a 6-base sequence TTTTATT is linked upstream of the base sequence AAGCTTTTA. This base sequence is a base sequence complementary to the + chain of natural human HBc gene. Further, a sequence A of 6 bases is preferably provided downstream of the base sequence AAGCTTTTA.
ATGTA, more preferably a 16 base sequence AATGT
It may also have ATACCCAAAGA. These base sequences are also base sequences complementary to the + chain of the natural human HBc gene. Further, it may be a sequence having 2-3 bases substituted, deleted or inserted in the 16-base sequence downstream of the stop codon, for example, AATGTAT G CCCA G AGA (substitution in the underlined portion). In addition, the base sequence AAGCTT
Instead of the 6-base sequence TTTTATT upstream of TA, TTT
G (or C) TG can also be linked.

For each of the above primers, the 1C and 1S primers for the + strand and the 2C for the − strand
Although the C primer and the 2S primer have been described,
Similar results can be obtained by using an oligonucleotide having a nucleotide sequence complementary to them instead of them. Each of the above-mentioned primers can be prepared by a known DNA synthesis method (for example, phosphoamidite method) using an ordinary automatic DNA synthesizer.

Next, each step of the first method of the present invention will be described. Step 1: Amplification of DNA [FIG. 1] Any virus can be used as the hepatitis B virus, for example, HBs subtypes adr and adw.
May be used. The HB virus DNA is heat-denatured at about 95 ° C. using a PCR device and further heated at about 60 ° C. to anneal to make the circular double-stranded DNA into single-stranded DNA. Subsequently, when the above-mentioned 1C primer (for + strand) and 2C primer (for − strand) are added, each primer specifically binds to the region of the DNA encoding the HBc antigen (1C).
The primer binds to the + strand and the 2C primer binds to the-strand). Next, DNA polymerase, especially thermostable D
NA polymerase (for example, commercially available Taq polymerase) is added, and the following PCR amplification cycle is performed. One cycle includes (i) a denaturation step of DNA [about 90 to 95 ° C., about 10 seconds to about 2 minutes], and (ii) an annealing step of single-stranded DNA with 1C primer and 2C primer [about 37 to 70 ° C. For about 30 seconds to about 3 minutes], and (iii) a DNA synthesis step with a DNA polymerase [about 65 seconds to about 80 seconds for about 30 seconds to about 5 minutes]. Amplified,
It is amplified 2 ⁿ times after n cycles. In the first method of the present invention, the cycle is repeated 10 to 60 times, preferably 20 to 40 times. In the last cycle, the heating time in step (iii) is preferably extended to about 5-10 minutes to ensure complete DNA synthesis.

Next, Klenow DNA Polymerase I was added, and both ends were blunt-ended to give a DNA of about 600 bp.
The presence or absence of the fragment is confirmed by performing electrophoresis on an agarose gel.

Step 2: Digestion with restriction enzyme [FIG. 2] Next, a buffer solution is added to the reaction solution obtained in the above step (1), and a restriction enzyme NcoI is further added to the reaction solution at an appropriate temperature (eg, temperature). The enzymatic reaction is carried out at 20 to 40 ° C.) for a time sufficient for digestion with the enzyme (for example, 30 minutes to 5 hours). The DNA in this treated solution is extracted with phenol / chloroform, precipitated with ethanol and collected. This precipitate was dissolved in a buffer solution and the restriction enzyme BamHI was added.
And digest as above.

Step 3: Preparation of recombinant plasmid [Fig. 2] As an expression vector, a plasmid, E. coli phage λ,
Yeast, virus and the like can be used, but a plasmid is preferably used. For example the plasmid pTV119N
[Takazake] is digested with restriction enzymes NcoI and BamHI, the larger fragment is isolated by electrophoresis, and the DNA fragment obtained in step (2) is ligated with ligase to prepare a recombinant plasmid. As a plasmid, pTrc9
9A (Pharmacia) can also be used. Also,
Animal cell expression vectors using viral genes include pMSG (Pharmacia) and pMAMneo (Cl
ontech) or the like can be used. In this case, after the DNA fragment digested in the step (2) is blunt-ended, an appropriate linker (for example, 5 ′ phosphorylated XhoI linker or 5 ′ phosphorylated SalI linker) is used.
Is attached to both ends of the DNA, this DNA fragment is further digested with a restriction enzyme XhoI or SalI, and then introduced into the above vector digested with the same restriction enzyme to give a recombinant DN.
A can also be created.

Step 4: Transformation [FIG. 2] Using the recombinant DNA obtained in the step (3), transformation is carried out by a known means. That is, when a plasmid vector is used, it is introduced into E. coli treated with Ca ²⁺ . When a λ phage vector is used, a λ phage particle incorporating the recombinant is prepared by an in vitro packaging reaction, and E. coli is infected with the λ phage particle. Furthermore, when an animal cell expression vector is used, it is introduced into, for example, COS-1 cells by the electroporation method, and each is transformed.

Step 5: Expression of protein Each of the transformed cells obtained in the above step (4) is cultured by a suitable means. That is, Escherichia coli having the HBc gene is inoculated into an L-broth medium containing ampicillin and cultured at 30 to 40 ° C, preferably 37 ° C overnight (or at least about 3 hours). Subsequently, an inducer (for example, IPTG that acts on the lac promoter) is added to express the HBc protein. After a lapse of at least about 3 hours, a protease inhibitor (for example, PMSF) is added and the culture is continued. When an animal cell expression vector is used, it is cultured in, for example, G418-containing MEM medium, and HBc is added using dexamethasone.
It is also possible to perform protein expression.

Step 6: Isolation of protein The culture solution obtained in the above step (5) is centrifuged to collect the cells, for example, a surfactant is added and sonication is performed, or Lysozyme Sucrose Tris H
By adding a Cl and then performing freeze-thaw treatment,
Obtain the cytoplasmic fraction of E. coli. The obtained fraction is treated with a column (for example, DEAE cellulose column) to remove contaminants, HBc protein is eluted, and then this eluate is treated with an anti-HBc antibody column to obtain HBc.
Purify the protein.

Next, each step of the second method of the present invention will be described with a focus on the points different from the first method. Step 1: Amplification of DNA [Fig. 3] There is no difference from the first method except that the 1S primer and 2S primer described above are used as primers instead of the 1C primer and 2C primer described above. The 1S primer and the 2S primer specifically bind to the region of DNA encoding the HBs antigen. Among these, the 1S primer binds to the + strand, and the 2S primer binds to the − strand. A PCR method amplification cycle was performed, Klenow DNA polymerase I was added, and both ends were blunt-ended to determine the presence or absence of a DNA fragment of about 700 bp.
Confirm by performing electrophoresis on a 2% agarose gel.

HBs antigen has four subtypes of ad
r, adw, ayr, and ayw exist, and their base sequences are slightly different. However, since all of these mutations exist between the 1S primer and the 2S primer, they can be produced by the second method of the present invention using the 1S primer and the 2S primer.

Step 2: Digestion with restriction enzyme [FIG. 4] Next, a buffer solution is added to the reaction solution obtained in the above step (1), and a restriction enzyme NcoI is further added to the reaction solution at an appropriate temperature (eg, The enzymatic reaction is carried out at 20 to 40 ° C.) for a time sufficient for digestion with the enzyme (for example, 30 minutes to 5 hours). The DNA in this treated solution is extracted with phenol / chloroform, precipitated with ethanol and separated. This precipitate was dissolved in a buffer solution and the restriction enzyme HindIII was added.
And digest as above.

Step 3: Preparation of recombinant plasmid [Fig. 4] As an expression vector, a plasmid, E. coli phage λ,
Yeast, virus and the like can be used, but a plasmid is preferably used. For example, the plasmid pTV118N
[Takazake] is digested with restriction enzymes NcoI and HindIII, the larger fragment is isolated by electrophoresis, and the DNA fragment obtained in step (2) is ligated with ligase to prepare a recombinant plasmid. As a plasmid, pKK2
33-2 (Pharmacia) can be used.

As animal cell expression vectors utilizing viral genes, pMSG (Pharmacia) and p
MAMneo (Clontech) or the like can be used. In this case, D digested in step (2) above
After blunt-ending the NA fragment, appropriate linkers (for example, 5′-phosphorylated SalI linker) were attached to both ends of the DNA, and this DNA fragment was further digested with the restriction enzyme SalI, and then the vector was digested with the same restriction enzyme. Recombinant DNA can also be prepared by introducing it.

Step 4: Transformation [FIG. 4] Transformation is carried out by a known means using the above recombinant DNA. That is, when a plasmid vector is used, it is introduced into E. coli treated with Ca ²⁺ . When a λ phage vector is used, a λ phage particle incorporating the recombinant is prepared by an in vitro packaging reaction, and E. coli is infected with the λ phage particle. Furthermore, when an animal cell expression vector is used, it is introduced into, for example, COS-1 cells by the electroporation method, and each is transformed.

Step 5: The transformed cells obtained in the protein expression step (4) are cultured by suitable means. That is, Escherichia coli having the HBs gene is inoculated into an L-broth medium containing ampicillin,
Culture is performed overnight (or at least about 3 hours) at 30 to 40 ° C, preferably 37 ° C. Then, an inducer (eg, IPTG that acts on the lac promoter) is added to express the HBs protein. And at least 3
After a lapse of time, a protease inhibitor (eg PMSF) is added and the culture is continued. When an animal cell expression vector is used, for example, G418-containing ME
It is also possible to culture in M medium and express HB protein using dexamethasone.

Step 6: The culture solution obtained in the protein isolation step (5) is centrifuged to collect the bacterial cells, and, for example, a surfactant is added for ultrasonic treatment or lysozyme. A cytosolic fraction of E. coli is obtained by adding sucrose-Tris-HCl and then freeze-thawing. The obtained fractions are stored in a column (eg D
After treatment with an EAE cellulose column) to remove contaminants, the HBs protein is eluted, and then the eluate is treated with an anti-HBs antibody column to purify the HBs protein.

The HBc protein and HBs protein obtained by the method of the present invention can be used as an antigen for measuring the anti-HBc antibody titer and the anti-HBs antibody titer in patient serum.

[0037]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention.

Example 1: Hepatitis B virus core tamper
(1) Preparation of human plasma sample The plasma of HBs antigen-positive (by the RPHA method) and HBe antigen-positive (by the Ouchterlony method) hepatitis patient is selected, and HBs antigen subtype adr and subtype ad
About 80 ml of each plasma of w was collected. About 30 ml of plasma is placed on 30 ml of 20% sucrose and ultracentrifuged (Hitachi RP-
45, 37,000 rpm × 16 hours) at 4 ° C. to collect pellets of Dane particles. Approximately 5 ml of this pellet was added to 10 mM Tris HCl (pH 7.4) / 150 mM-NaCl.
And stored at -80 ° C. 200 μg of Proteinase K was added to 1 ml of this suspension to give a final concentration of 0.5% SD.
S, 10 mM-EDTA and 10 mM Tris HCl (pH
HBV DNA was isolated by performing protease treatment at 68 ° C. for 20 hours at 7.4 ° C., repeating phenol extraction three times, and then precipitating with ethanol.

(2) Preparation of HBc protein gene A base represented by the sequence shown in SEQ ID NO: 1 of the sequence listing containing an NcoI recognition site by a conventional method using a 381A type DNA automatic synthesizer (Applied Biosystems). Thirty
An oligonucleotide consisting of 30 nucleotides (hereinafter referred to as HBc-1) and an oligonucleotide consisting of 30 bases represented by the sequence shown in SEQ ID NO: 2 of the sequence listing including a BamHI recognition site (hereinafter referred to as CHBc-1) Was prepared. Using these HBc-1 and CHBc-1 as primers for the HBc gene, two types of HBV DNA (HBs subtypes adr and adw) isolated in the above (1)
Sample was heat-treated at 95 ° C for 10 minutes and then Ta
qDNA polymerase [Take Shuzo] was added, and each operation of enzymatic reaction (72 ° C .; 3 minutes), thermal denaturation (90 ° C .; 1 minute) and annealing (60 ° C .; 2 minutes) was performed with a PCR device [Take Shuzo]. Repeated times [Fig. 1].

The thus amplified DNA was cleaved at the 5'end with a restriction enzyme NcoI and at the 3'end with BamHI, and then electrophoresed on a 1.2% agarose gel.
A band of a DNA fragment of about 570 bp which could be detected by a UV lamp in the presence of ethidium bromide was cut out, and D
The DNA was purified using an NA recovery / purification kit (trade name: DNA PREP; Diayertron) (FIG. 2).

(3) Preparation of Plasmid Containing HBc Gene Commercially available expression vector pTV119N (Take Shuzo) was treated with restriction enzymes NcoI and BamHI to remove the larger fragment, and the larger fragment of about 570 bp obtained in the above (2) was taken. The DNA fragment was inserted using T4 DNA ligase to obtain a recombinant plasmid pTV119.HBc [Fig. 2].

(4) Transformation of Escherichia coli with the plasmid pTV119.HBc The pTV119.HBc obtained in the above (3) and the commercially available Escherichia coli MV1184 (Take Shuzo) were treated with 50 mM CaCl ₂ to prepare competent cells at 0 ° C. After incubating at 40 ° C for 40 minutes and heat-treating at 42 ° C for 3 minutes, the cells were cultured at 37 ° C for 1 hour in L-broth. This E. coli was treated with ampicillin (50 μg / ml), 0.5 mM IPTG and 0.00
It was spread on an L-broth agar plate medium containing 4% X-GAL and cultured overnight at 37 ° C. The resulting ampicillin-resistant and transparent colonies were respectively transferred to new ampicillin (10
It was cultured overnight at 37 ° C. in L-broth containing 0 μg / ml), and plasmid DNA was isolated by an alkaline lysis method (Molecular Cloning, A Labo).
library Manual 2nd Edition
n, J. Sambrook, E .; F. Fritsch,
T. Maniatis; Cold Spring Ha
rbor Laboratory, New Yor
k). This DNA was cleaved with restriction enzymes NcoI and BamHI, and then electrophoresed on 1.2% agarose gel, and the presence of HBcDNA of about 570 bp and the plasmid vector of 3100 bp was confirmed by a UV lamp in the presence of ethidium bromide. .. The Escherichia coli harboring the HBc gene of about 570 bp obtained here was transformed into pTV119.
-HBc-001 (3 clones as subtype adr-derived DNA) and pTV119-HBc-101 (3 clones as subtype-adw-derived DNA).

(5) Expression of HBc protein of pTV119.HBc gene in E. coli The gene expression of plasmid pTV119.HBc was 0.
Using L-broth containing 5 mM IPTG, the cells were cultured at 37 ° C. for 7 hours or overnight, and the cells were treated with 20% sucrose / 30 mM Tris HCl (pH 8.0) containing lysozyme (1 mg / ml). After treatment at 0 ° C. for 20 minutes, the cells were further subjected to freeze-thaw treatment three times (the above-mentioned Molecular Cloning, AL).
Laboratory Manual 2nd Edit
ion), and the resulting supernatant was used as a crude fraction of HBc protein. The results of the HBc protein (3 clones derived from adr) by the RPHA method are shown in Table 1. In Table 1, when cultured overnight, clones 1 and 2 produced a good amount of HBc protein, and clone 3 was degraded by a proteolytic enzyme, resulting in a decrease. Also adw
3 clones from each 7 hours and cultured overnight together 2 ¹⁰
It was ~ 2 ¹¹ .

[0044]

[Table 1]

(6) Confirmation of HBc protein by Western blot The crude fraction of HBc protein obtained in the above (5) was used for 12.5.
% Electrophoresis on a polyacrylamide gel (in the presence of 0.4% SDS), followed by blotting buffer [25 mM Tris HCl (pH 8.3), 192 mM glycine and 20
% Methyl alcohol] was used to transfer to a nitrocellulose membrane (50 V, 70 minutes). Nitrocellulose membrane 1
10 mM Tris HCl (pH 7.4) containing 0% FCS,
After blocking with 150 mM NaCl, mouse anti-HBc monoclonal antibody (subclass Ig
M) (Chemicon International
Inc. ), And a phosphatase-labeled goat anti-mouse IgM (μ) antibody was used as the second antibody. BCI as a substrate
When activity staining was performed using P (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitroblue tetrazolium), HBc protein bands of 45 Kd (dimer) and 21 Kd (monomer) were strongly stained. It was

(7) Separation and Purification of HBc Protein The crude fraction of HBc protein obtained in the above (5) is DE52.
It was applied to a (Whatman Paper Ltd.) column (2.5 × 15 cm) and eluted with 0.5 M NaCl. After dialyzing this fraction with 0.15 M NaCl,
Apply 4M-Mg to anti-HBc antibody column (1 x 10 cm)
The HBc protein was purified by elution with Cl ₂ .

Example 2: Surface tampering of hepatitis B virus
Sample preparation (1) Preparation of plasma sample The same sample as the sample described in Example 1 (1) above was used.

(2) Preparation of HBs gene Using a 381A type DNA automatic synthesizer (Applied Biosystems) in a conventional manner, the base 30 represented by the sequence shown in SEQ ID NO: 3 of the sequence listing containing the NcoI recognition site.
An oligonucleotide consisting of 30 bases (hereinafter referred to as HBs-1) and an oligonucleotide consisting of 30 bases represented by the sequence described in SEQ ID NO: 4 of the sequence listing including a HindIII recognition site (hereinafter referred to as CHBs-1) Was prepared. Using these HBs-1 and CHBs-1 as primers for the HBs gene, the two HBVs of the above (1)
DNA (HBs subtypes adr and adw) was used as a sample and treated in the same manner as in the method described in Example 1 (2) to give P.
The CR method was performed.

The 5'end of the thus amplified DNA was cleaved with the restriction enzyme NcoI, and the 3'end was digested with the restriction enzyme Hind.
After cleaving with III, electrophoresis on 1.2% agarose gel was performed, and a band of a DNA fragment of about 670 bp that could be detected by a UV lamp in the presence of ethidium bromide was cut out to obtain a DNA recovery and purification kit (trade name). DNA PR
DNA was purified using EP (Diatron) (FIG. 3).

(3) Preparation of Plasmid Containing HBs Gene Commercially available gene expression vector pTV118N (Take Shuzo) was treated with the restriction enzymes NcoI and HindIII to remove the larger fragment, and about 670 bp obtained in the above (2) was taken. Of D
The NA fragment (HBs gene) was inserted using T4 DNA ligase to obtain a recombinant plasmid pTV118.HBs [Fig. 4].

(4) Plasmid pTV118N.HBs
Transformation of E. coli with pTV118N.HBs obtained in the previous section (3) and L-
Escherichia coli MV1184 cultured overnight at 37 ° C in broth
Competent cells prepared by treating (Taka Shuzo) with 50 mM CaC1 ₂ were incubated at 0 ° C. for 40 minutes,
After heat treatment at 42 ° C for 3 minutes, L-at 37 ° C for 1 hour
Cultured in broth. This E. coli was ampicillin (50
μg / ml), 0.5 mM IPTG and 0.004% X-G
The cells were plated on L-broth agar plate medium containing AL and cultured overnight at 37 ° C. The resulting ampicillin-resistant, transparent colonies were each treated with a new ampicillin (100 μg / ml)
It was cultured overnight at 37 ° C. in L-broth containing the enzyme and the plasmid DNA was isolated by the alkaline lysis method (Mole described above).
color Cloning, A Laborator
y Manual 2nd Edition). This D
After cleaving NA with the restriction enzymes NcoI and HindIII, electrophoresis was performed on 1.2% agarose gel, and it was detected by a UV lamp in the presence of ethidium bromide to about 67%.
The presence of 0 bp HBsDNA and 3100 bp plasmid vector was confirmed. About 670bp obtained here
Escherichia coli having the HBs gene of pTV118.HBs-
001 (16 clones as subtype adr-derived DNA) and pTV118.HBs-101 (subtype a
The dw-derived DNA was named 12 clones).

(5) Expression of HBs protein of pTV118.HBs gene in E. coli Expression of plasmid pTV118.HBs was carried out under the same conditions as in the method described in Example 1 (5). The HBs protein was confirmed by the RPHA method using the HBs antigen kit (Welcome Diagnostics Japan). However, both pTV118.HBs-001 and pTV118.HBs-101 clones were HB.
production of s antigen protein amount was less 2 ^{0-2 2.}

[0053]

When the core structure protein or surface structure protein of hepatitis B virus is produced by the method of the present invention, it is not necessary to use a large amount of plasma, and a DNA fragment for insertion consisting of just before the start codon to immediately after the stop codon. It is also possible to adapt to the loss of the cleavage site due to the difference or mutation depending on the subtype of HBs antigen.

[0054]

[Sequence list]

SEQ ID NO: 1 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Sequence: TTTGGGCCAT GGACATTGAC CCGTATAAAG

SEQ ID NO: 2 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Sequence: TCCAAGGGAT CCTAACATTG AGATTCCCGA

SEQ ID NO: 3 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Sequence: ACCGACCATG GAGAACACAA CATCAGGATT

SEQ ID NO: 4 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Sequence: TTTATTAAGC TTTAAATGTA TACCCAAAGA

[Brief description of drawings]

FIG. 1 is an explanatory view showing a process of creating an HBc gene.

FIG. 2 is an explanatory view showing the steps for preparing plasmid pTV119.HBc.

FIG. 3 is an explanatory diagram showing a process of creating an HBs gene.

FIG. 4 is an explanatory view showing the steps for preparing plasmid pTV118.HBs.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location (C12P 21/02 C12R 1:19)

Claims (2)

[Claims] 1. (1) (a) The recognition base sequence CCATGG containing a recognition base sequence CCATGG of a restriction enzyme NcoI,
The nucleotide sequence ATG existing in GG corresponds to the initiation codon ATG in the nucleotide sequence of the DNA encoding the natural human hepatitis B virus core structural protein, and D encoding the natural human hepatitis B virus core structural protein.
A first DNA primer having a base sequence capable of specifically binding to a region containing the initiation codon ATG of NA or a complementary base sequence thereof, and (b) containing the base sequence GGATCCTA and present at the end of the base sequence The nucleotide sequence CTA is complementary to the stop codon TAG in the nucleotide sequence of the DNA encoding the natural human hepatitis B virus core structural protein.
Corresponding to A, a second DNA having a base sequence capable of specifically binding to a region containing the stop codon TAG of the DNA encoding the natural human hepatitis B virus core structural protein or a complementary base sequence thereof. A mixture containing a primer, (c) DNA polymerase, and (d) hepatitis B virus DNA is subjected to a DNA amplification step to amplify the DNA encoding the hepatitis B virus core structural protein, and (2) hepatitis B The amplified DNA encoding the viral core structural protein is treated with restriction enzyme NcoI and restriction enzyme B.
NcoI / BamHI digested DNA after digestion with amHI
Producing a fragment, (3) ligating the NcoI / BamHI digested DNA fragment to a replicable expression vector to form a recombinant DNA, (4) transforming a microorganism or cell with the recombinant DNA, 5) The resulting transformant is cultured, and the transformant DN encoding the hepatitis B virus core structural protein
Hepatitis B characterized by including each step of expressing A to produce hepatitis B virus core structural protein, and (6) isolating the produced hepatitis B virus core structural protein from the transformant. Method for producing virus core structural protein. 2. (1) (a) containing a recognition base sequence CCATGG of a restriction enzyme NcoI, wherein the recognition base sequence CCAT
The nucleotide sequence ATG existing in GG corresponds to the initiation codon ATG in the nucleotide sequence of the DNA encoding the natural human hepatitis B virus surface structural protein, and D encoding the natural human hepatitis B virus surface structural protein
A first DNA primer having a base sequence capable of specifically binding to a region containing the start codon ATG of NA or a complementary base sequence thereof, and (b) containing a base sequence AAGCTTTTA and present at the end of the base sequence. The base sequence TT that is complementary to the stop codon TAA in the base sequence of the DNA encoding the natural human hepatitis B virus surface structural protein TT
A second DNA having a base sequence capable of specifically binding to the region containing the stop codon TAA of the DNA encoding the natural human hepatitis B virus surface structural protein or its complementary base sequence corresponding to A. A mixture containing a primer, (c) DNA polymerase, and (d) hepatitis B virus DNA is subjected to a DNA amplification step to amplify the DNA encoding the hepatitis B virus surface structural protein, and (2) hepatitis B The amplified DNA encoding the viral surface structural protein is treated with restriction enzymes NcoI and H
Digested with indIII, NcoI / HindIII digested D
Producing an NA fragment, (3) ligating the NcoI / HindIII digested DNA fragment to a replicable expression vector to form a recombinant DNA, (4) transforming a microorganism or cell with the recombinant DNA, (5) The resulting transformant is cultured, and the transformant DN encoding the hepatitis B virus surface structural protein
Hepatitis B characterized by including each step of expressing A to produce hepatitis B virus surface structural protein, and (6) isolating the produced hepatitis B virus surface structural protein from the transformant. Method for producing virus surface structural protein.