JP3305342B2 - Method for producing hepatitis B virus protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a core structural protein or a surface structural protein of hepatitis B virus using a technique for selectively amplifying specific DNA and a gene recombination technique. The present invention particularly includes the use of a technique for selectively amplifying DNAs encoding those structural proteins, the use of two types of primer DNAs, including an initiation codon and cleavage with a specific restriction enzyme. And an oligonucleotide containing a stop codon and cleavable by another specific restriction enzyme.

[0002]

2. Description of the Related Art Hepatitis B virus (hepatitis)
B virus: hereinafter abbreviated as HBV) is an infectious agent that causes human liver disease.
It has been revealed that BV-infected chronic hepatitis patients lead to cirrhosis or liver cancer patients. Transmission by blood transfusion is also a problem. Screening tests for infection with hepatitis B are necessary for prevention and treatment of infection, and various tests for diagnosis of hepatitis B infection have been attempted.

However, purified H used in the above-mentioned diagnostic reagents
In order to obtain the Bs antigen, it is necessary to use a large amount of human plasma, and furthermore, this purification step is very dangerous, and thus an improvement has been desired. In addition, since HBs antigen-positive patients are gradually decreasing, it is expected that HBs antigens will be produced using genetic recombination technology.

[0004] Various methods for producing HBs antigens using genetic recombination techniques have already been reported, but all require the collection and treatment of plasma containing a large amount of Dane particles, and also require the use of radioisotopes (eg, , because it is performed using ³² P), require special facilities, it was not uncommon.

[0005] As a conventional method using the gene recombination technique, for example, Fujisawa, Y .; Et al., Nuclei
c Acids Res. 11 , 3581-3590
(1983) describe the following method. That is, H
A DNA fragment obtained by cutting about 3.2 Kb of the circular DNA 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 a clone of hepatitis B virus DNA. Subsequently, 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 using the expression vector was cultured to isolate the desired HBs protein or HBc protein.

[0006] 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,
There were 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 just before the start codon ATG, and the stop codons TAA, TA
This was due to the fact that a restriction enzyme that could be cleaved immediately after G or TGA was not found. Furthermore, the HBs antigen has four subtypes (adr, adw, ayr and a
yw) is present, and if the subtype is different, the base sequence that can be cleaved by the same restriction enzyme (EcoRI, PstI, BamHI, etc.) is not conserved, so it was necessary to confirm whether or not each subtype could be cleaved. . In addition, since the cleavage site may be lost due to the mutation, cloning may not be performed in some cases.

[0007]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a DNA fragment for insertion consisting of immediately before a start codon to immediately after a stop codon without using a large amount of plasma, and It is an object of the present invention to provide a method that can be adapted to differences in antigen subtypes and loss of cleavage sites due to mutation.

[0008]

The object of the present invention is to provide a first method of the present invention, that is, (1) (a) the recognition base sequence CCAT of the restriction enzyme NcoI.
GG, the nucleotide sequence ATG present in the recognition nucleotide sequence CCATGG corresponds to the start codon ATG in the nucleotide sequence of the DNA encoding the natural human hepatitis B virus core structural protein, and the natural human hepatitis B virus A first DNA primer having a base sequence capable of specifically binding to a region containing the initiation codon ATG of the DNA encoding the core structural protein or a complementary base sequence thereof (hereinafter, may be referred to as a 1C primer)
And (b) a nucleotide sequence containing the nucleotide sequence GGATCCTA, wherein the nucleotide sequence CTA at the end of the nucleotide sequence is complementary to the stop codon TAG in the nucleotide sequence of the DNA encoding the natural human hepatitis B virus core structural protein. Stop codon TAG of DNA encoding the natural human hepatitis B virus core structural protein corresponding to CTA
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 a 2C primer),
(C) subjecting a mixture containing DNA polymerase and (d) hepatitis B virus DNA to a DNA amplification step to amplify DNA encoding hepatitis B virus core structural protein; (2) hepatitis B virus core structure The amplified DNA encoding the protein was digested with the restriction enzymes NcoI and B
amNl digested with NcoI / BamHI digested DNA
(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) culturing the resulting transformant and adding the transformant to a DNA encoding hepatitis B virus core structural protein;
Expressing hepatitis B virus core structural protein by expressing A; and (6) isolating the produced hepatitis B virus core structural protein from the transformant. The problem can be solved by a method for producing a viral core structural protein.

Further, the above-mentioned problem is solved by the second method of the present invention, that is, (1) (a) the nucleotide sequence CCAT which recognizes the restriction enzyme NcoI.
GG, wherein the base sequence ATG present in the recognition base sequence CCATGG corresponds to the start codon ATG in the base sequence of the DNA encoding the natural human hepatitis B virus surface structural protein, and the natural human hepatitis B virus A first DNA primer having a base sequence capable of specifically binding to a region including the initiation codon ATG of DNA encoding a surface structural protein or a complementary base sequence thereof (hereinafter, may be referred to as a 1S primer)
And (b) a nucleotide sequence containing the nucleotide sequence AAGCTTTTA, wherein the nucleotide sequence TTA present at the end of the nucleotide sequence is complementary to the stop codon TAA in the nucleotide 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 a complementary base sequence thereof corresponding to A. A mixed solution containing a primer (hereinafter sometimes referred to as 2S primer), (c) DNA polymerase, and (d) hepatitis B virus DNA
Through an amplification step, a DNA encoding the hepatitis B virus surface structural protein is amplified, and (2) the amplified DNA encoding the hepatitis B virus surface structural protein is converted into a restriction enzyme NcoI and a restriction enzyme H.
After digestion with indIII, NcoI / HindIII digested D
Generating 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) culturing the obtained transformant, and transforming the transformant into a DNA encoding a hepatitis B virus surface structural protein;
Expressing hepatitis B virus surface structural protein by expressing A; and (6) each step of isolating the produced hepatitis B virus surface structural protein from a transformant. The problem can be solved by a method for producing a virus surface structural protein. In addition, the present invention relates to (1) a nucleotide sequence consisting of at least 20 nucleotides of the nucleotide sequence shown in SEQ ID NO: 1 of the sequence listing and including the nucleotide sequence CCATGG recognized by the restriction enzyme NcoI, or a complementary nucleotide sequence thereof, or 2) At least 3 bases are substituted, deleted or inserted at least 20 bases in the base sequence described in SEQ ID NO: 1 of the sequence listing, and the restriction enzyme Nc
a base sequence containing the oI recognition base sequence CCATGG, or a DNA primer having a base sequence complementary thereto;
Thus, the DNA probe for use in the first method of the present invention
It also concerns Reimer . Furthermore, the present invention relates to (1) a base sequence consisting of at least 20 bases represented by SEQ ID NO: 2 in the sequence listing, and further comprising a base sequence GGATCCTA
Or (2) at least 20 bases in which at least 3 bases are substituted, deleted or inserted in the base sequence of SEQ ID NO: 2 in the sequence listing, or And the base sequence GG
A DNA primer having a nucleotide sequence containing ATCCTA or a nucleotide sequence complementary thereto, which is a first primer of the present invention.
The present invention also relates to the aforementioned DNA primer for use in the method . Furthermore, the present invention relates to (1) a nucleotide sequence consisting of at least 20 nucleotides of the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing and further comprising the nucleotide sequence CCATGG recognized by the restriction enzyme NcoI, or a complementary nucleotide sequence thereof, or (2) At least three nucleotides in the nucleotide sequence of SEQ ID NO: 3 in the sequence listing consist of at least 20 nucleotides of a substituted, deleted or inserted nucleotide sequence, and the restriction enzyme Nc
a base sequence containing the oI recognition base sequence CCATGG, or a DNA primer having a base sequence complementary thereto;
Thus, the DNA probe for use in the second method of the present invention
It also concerns Reimer . Furthermore, the present invention relates to (1) a base sequence consisting of at least 20 bases of the base sequence represented by SEQ ID NO: 4 in the sequence listing, and further comprising the base sequence AAGCTTTTA
Or (2) at least 3 bases in which at least 3 bases are substituted, deleted or inserted in at least 20 bases in the base sequence described in SEQ ID NO: 4 in the sequence listing. And the base sequence AA
A DNA primer having a base sequence containing GCTTTA or a base sequence complementary thereto,
The present invention also relates to the aforementioned DNA primer for use in the method . In the description of the present specification, A is an adenine residue, C is a cytosine residue, G is a guanine residue, and T is a thymine residue.

[0010] The DNA gene sequence of HBV has already been elucidated [Nucleic Acids Research.
ch, No. 6, 8, 13 (1983)], molecular weight 2.
It has been found that 1 × 10 ⁶ daltons consist of about 3200 base pairs. Of these, the core protein gene (HBc
The gene) region is located between the about 1900th base and about the 2400th base. In the first step of the first method of the present invention, only the HBc gene region, that is, only the DNA region encoding HBc is selectively amplified. This includes PCR (polymerase chainrea)
ction) method can be used. By using the PCR method, it is possible to automatically amplify only a target DNA region from a trace amount of DNA up to about 1,000,000 times (S
science, 239 , 487-491, 1988).
In the PCR method, a cycle of amplifying using a primer for the + strand, a primer for the -strand, and a heat-resistant DNA polymerase across the DNA region to be amplified is repeated.

[0011] In the first method of the present invention, the 1C primer (for the + strand) used in the PCR method of the first step contains a recognition base sequence CC ATG G of a restriction enzyme NcoI, and has a start sequence within the base sequence. It has a codon ATG (the underlined portion of the base sequence). The 1C primer is preferably composed of at least 20 bases (preferably 20 to 40 bases, more preferably 28 to 32 bases), including the above-mentioned restriction enzyme NcoI recognition base sequence CCATGG. If it is less than 20 bases, the specificity as a primer will be reduced, and it will be 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 a double structure between primer molecules or within a molecule is easily formed, which is not preferable.

[0012] A preferred 1C primer is such that, when the nucleotide sequence ATG present in the recognition nucleotide sequence CCATGG is made to correspond to the start codon ATG in the nucleotide 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 including the start codon ATG of the DNA encoding the natural human hepatitis B virus core structural protein, or substitution of at most 3 bases,
It has a deleted or inserted base sequence. For example, the 1C primer is preferably a 9-base sequence GA downstream of the initiation codon, similar to the native human HBc gene.
CATTGAC, more preferably a 19-base sequence GAC
It may have a structure in which ATTGACCCGTATAAAG is linked. In addition, the 1C primer contains a natural human HBc upstream of the recognition base sequence of the restriction enzyme NcoI.
It may also have the same 6-base sequence TTTGGGG as the gene. Furthermore, a sequence having a substitution, deletion or insertion of 2 to 3 bases in the 19 base sequence downstream of the initiation codon, for example,
GACATT (or C) GAC (or T) A C T (or
C ) It may be TATAAAAG (underlined parts are replaced).

[0013] In the first method of the present invention, the 2C primer (for the minus strand) used in the PCR method of the first step contains a base sequence GGATCCTA. Of the eight base sequence, the first six base sequence GGATCC is the restriction enzyme Bam.
HI recognition base sequence, last three bases sequence CTA
Is a sequence complementary to the stop codon TAG. This 2C primer also contains the base sequence GGATCCTA,
At least 20 bases (preferably 20 to 40 bases,
(More preferably 28 bases to 32 bases). If it is less than 20 bases, the specificity as a primer will be reduced, and it will be extremely difficult to achieve the object of the method of the present invention. Also, as the number of bases increases, the specificity of the primer improves, but the production of the primer is costly, and a double structure between primer molecules or within a molecule is easily formed, which is not preferable.

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

[0015] The surface protein gene (HBs gene) region prepared by the second method of the present invention exists between the approximately 150th base and the approximately 840th base of the HBV DNA gene sequence. In the first step of the second method of the present invention, only the HBs gene region is selectively amplified. For this, the PCR method can be used as described above.

The 1S primer (for the + chain) contains the recognition base sequence CC ATG G of the restriction enzyme NcoI, and has an initiation codon ATG (underlined portion of the base sequence) in the base sequence. This 1S primer also contains the recognition base sequence CCATGG of the restriction enzyme NcoI,
At least 20 bases (preferably 20 to 40 bases,
(More preferably 28 bases to 32 bases). If it is less than 20 bases, the specificity as a primer will be reduced, and it will be extremely difficult to achieve the object of the method of the present invention. Also, as the number of bases increases, the specificity of the primer improves, but the production of the primer is costly, and a double structure between primer molecules or within a molecule is easily formed, which is not preferable.

A preferred 1S primer is the one described above, wherein the nucleotide sequence ATG present in the recognition nucleotide sequence CCATGG corresponds to the initiation codon ATG in the nucleotide 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 substitution of at most 3 bases,
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, similar to the native human HBs gene.
AGAACACAA, more preferably a 20 base sequence G
It may have a structure in which AGAACACAACATCAGGATT is linked. Also, the 1S primer may have a 5-base sequence ACCGA similar to 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 to 3 bases in the 20 base sequence downstream of the initiation codon, for example, GAGAA (or G ) CA TCG CATCAGGA
T (or C 2 ) T (the underlined part is replaced). Further, GA (or T) A (or T) GA may be ligated 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 base sequence AAGCTTTTA. Of this 8-base sequence,
The first six bases sequence AAGCTT is a restriction enzyme HindII.
This is the recognition base sequence of I, and the last three base sequence TTA is a sequence complementary to the stop codon TAA. This 2S primer also preferably comprises at least 20 bases (preferably 20 to 40 bases, more preferably 28 to 32 bases), including the base sequence AAGCTTTA. If it is less than 20 bases, the specificity as a primer will be reduced, and it will be extremely difficult to achieve the object of the method of the present invention. Also, as the number of bases increases, the specificity of the primer improves, but the production of the primer is costly, and a double structure between primer molecules or within a molecule is easily formed, which is not preferable.

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

For each of the above primers, a 1C primer and a 1S primer for the + strand and a 2C primer for the − strand
As described for the C primer and the 2S primer,
Similar results can be obtained by using oligonucleotides having base sequences complementary to them instead of them. Each of the above primers can be prepared by a known DNA synthesis method (for example, a phosphoramidite 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] As the hepatitis B virus, any virus can be used. 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 to about 60 ° C. for annealing to convert the circular double-stranded DNA into a single-stranded DNA. Subsequently, when the above-mentioned 1C primer (for the + chain) and 2C primer (for the − chain) are added, each primer specifically binds to the HBc antigen-encoding DNA region (1C
The primer binds to the + strand and the 2C primer binds to the -strand). Next, a DNA polymerase, especially a heat-resistant D
NA polymerase (eg, commercially available Taq polymerase) is added and the following PCR amplification cycle is performed. One cycle includes (i) a denaturation step of DNA (at about 90 to 95 ° C. for 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. About 30 seconds to about 3 minutes], and (iii) a DNA synthesis step using a DNA polymerase (about 65 to 80 ° C. for about 30 seconds to about 5 minutes), and the amount of DNA is doubled every cycle. Amplified
After n cycles, it is amplified by 2 ⁿ times. In the first method of the present invention, the above 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 to 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 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 thereto. The enzymatic reaction is carried out at a temperature of (20 to 40 ° C.) for a time sufficient for digestion by the enzyme (for example, 30 minutes to 5 hours). The DNA in this treatment solution is extracted with phenol / chloroform, precipitated with ethanol and separated. This precipitate was dissolved in a buffer, and the restriction enzyme BamHI was removed.
And digestion as before.

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

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

Step 5: Expression of protein The transformed cells obtained in the above step (4) are cultured by suitable means. That is, Escherichia coli having the HBc gene is inoculated into an L-broth medium containing ampicillin, for example, and cultured at 30 to 40 ° C., preferably 37 ° C., overnight (or at least about 3 hours). Subsequently, an inducer (for example, IPTG acting 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, for example, the cells are cultured in a G418-containing MEM medium, and HBc is cultured using dexamethasone.
Protein expression can also be performed.

Step 6: Isolation of protein The culture obtained in the above step (5) is centrifuged to separate cells.
Collect and sonicate, for example, with the addition of a surfactant, or Lysozyme sucrose Tris H
By performing a freeze-thaw treatment after adding Cl,
Obtain the cytoplasmic fraction of E. coli. The obtained fraction is treated with a column (for example, a DEAE cellulose column) to remove impurities, and then the HBc protein is eluted. Then, the eluate is treated with an anti-HBc antibody column, whereby the HBc protein is eluted.
Purify the protein.

Next, each step of the second method of the present invention will be described, focusing on the differences from the first method. Step 1: Amplification of DNA [FIG. 3] There is no difference from the first method except that the above-mentioned 1S primer and 2S primer are used instead of the above-mentioned 1C primer and 2C primer as primers. The 1S and 2S primers specifically bind to a region of DNA encoding the HBs antigen. Among them, the 1S primer binds to the + strand and the 2S primer binds to the -strand. A PCR 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.
Perform electrophoresis on a 2% agarose gel and confirm.

The HBs antigen has four subtypes, ad
There are r, adw, ayr and ayw, each of which has a slightly different base sequence. However, since these mutations are all present 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 further a restriction enzyme NcoI is added thereto. The enzymatic reaction is carried out at a temperature of (20 to 40 ° C.) for a time sufficient for digestion by the enzyme (for example, 30 minutes to 5 hours). The DNA in this treatment solution is extracted with phenol / chloroform, precipitated with ethanol, and separated. This precipitate was dissolved in a buffer, and the restriction enzyme HindIII was used.
And digestion as before.

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

Animal cell expression vectors utilizing viral genes include pMSG (Pharmacia) and pMSG.
MAMneo (Clontech) or the like can be used. In this case, D digested in step (2)
After blunt-end of the NA fragment, an appropriate linker (for example, 5'-phosphorylated SalI linker) was attached to both ends of the DNA, and this DNA fragment was further digested with the restriction enzyme SalI, and then digested with the same restriction enzyme. Recombinant DNA can also be prepared by introduction.

Step 4: Transformation [FIG. 4] Transformation is performed by a known means using the above-mentioned recombinant DNA. That is, when a plasmid vector is used, it is introduced into Ca ^{2+ -treated} Escherichia coli. When a λ phage vector is used, a λ phage particle incorporating a recombinant is prepared by an in vitro packaging reaction, and this is used to infect Escherichia coli. Further, when animal cell expression vectors are used, they are introduced into, for example, COS-1 cells by electroporation, 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, for example,
Cultivation is carried out at 30 to 40 ° C, preferably 37 ° C, overnight (or at least about 3 hours). Then, an HBs protein is expressed by adding an inducer (eg, IPTG acting on the lac promoter). And at least 3
After about a lapse of time, a protease inhibitor (for example, PMSF) is added, and the culture is continued. When an animal cell expression vector is used, for example, a G418-containing ME
Were cultured in M medium, it is also possible to effect expression of HB s protein using dexamethasone.

Step 6: Isolation of the protein The culture solution obtained in the step (5) is centrifuged to collect the cells, and for example, a surfactant is added thereto, followed by sonication or lysozyme. After adding sucrose / tris-HCl and subjecting to freeze-thaw treatment, a cytoplasmic fraction of E. coli is obtained. The obtained fraction is applied to a column (for example, D
After removing contaminants by treating with an EAE cellulose column), 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 antigens for measuring anti-HBc antibody titers and anti-HBs antibody titers in patient sera.

[0037]

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

Example 1 Hepatitis B Virus Core Tamper
Preparation of quality (1) Preparation of human plasma sample Plasma of a hepatitis patient who is HBs antigen-positive (by the RPHA method) and HBe antigen-positive (by the Ouchterlony method) is selected, and the HBs antigen subtype adr and subtype ad are selected.
About 80 ml of each plasma of w was collected. Place about 30 ml of plasma on 30 ml of 20% sucrose and ultracentrifuge (Hitachi RP-
45, 37000 rpm × 16 hours) at 4 ° C. to collect pellets of Dane particles. This pellet was mixed with about 5 ml of 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, and the final concentration was 0.5% SD.
S, 10 mM @ EDTA and 10 mM Tris HCl (pH
After performing protease treatment at 68 ° C. for 20 hours in 7.4), phenol extraction was repeated three times, followed by precipitation with ethanol to isolate HBV DNA.

(2) Preparation of HBc Protein Gene A base represented by the sequence shown in SEQ ID NO: 1 containing an NcoI recognition site by a conventional method using an automatic type 381A DNA synthesizer (Applied Biosystems). 30
(Hereinafter referred to as HBc-1) and an oligonucleotide comprising 30 bases represented by SEQ ID NO: 2 in 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, the two HBV DNAs (HBs subtypes adr and adw) isolated in (1) above were used.
As a sample, heat-treated at 95 ° C. for 10 minutes.
qDNA polymerase [Takara Shuzo] was added, and each of the enzymatic reaction (72 ° C; 3 minutes), heat denaturation (90 ° C; 1 minute) and annealing (60 ° C; 2 minutes) was performed using a PCR device (Takara Shuzo) for 30 minutes. This was repeated several times (FIG. 1).

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

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

(4) Transformation of Escherichia coli with plasmid pTV119.HBc pTV119.HBc obtained in the above section (3) and commercially available Escherichia coli MV1184 (Takara Shuzo) were treated with 50 mM CaCl ₂ , and the prepared competent cells were treated at 0 ° C. After heating at 42 ° C for 3 minutes, the cells were cultured in L-broth at 37 ° C for 1 hour. The E. coli was ampicillin (50 μg / ml), 0.5 mM IPTG and 0.00
The cells were plated on an L-broth agar plate medium containing 4% X-GAL, and cultured at 37 ° C. overnight. The resulting ampicillin-resistant and clear colonies were each picked with new ampicillin (10
(0 μg / ml) at 37 ° C. overnight, and plasmid DNA was isolated by alkaline lysis (Molecular Cloning, A Labo).
rattro Manual 2nd Edition
n, J .; Sambrook, E .; F. Fritsch,
T. Maniatis; Cold Spring Ha
rbor Laboratory, New York
k). After cutting this DNA with restriction enzymes NcoI and BamHI, electrophoresis was performed on a 1.2% agarose gel, and the presence of about 570 bp of HB cDNA and a 3100 bp plasmid vector was confirmed by a UV lamp in the presence of ethidium bromide. . Escherichia coli having the HBc gene of about 570 bp obtained here was transformed into pTV119.
HBc-001 (3 clones as DNA derived from subtype adr) and pTV119.HBc-101 (3 clones as DNA derived from subtype adw).

(5) Expression of HBc Protein in E. coli of pTV119.HBc Gene The gene expression of plasmid pTV119.
After culturing at 37 ° C. for 7 hours or overnight using L-broth containing 5 mM IPTG, the cells were cultured 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 (Molecular Cloning, AL described above).
laboratory Manual 2nd Edit
ion), and the obtained supernatant was used as a crude fraction of HBc protein. Table 1 shows the results of the RPHA method for the HBc protein (three clones derived from adr). In Table 1, when cultured overnight, clones 1 and 2 have good HBc protein production, and clone 3 has been reduced by degradation by proteolytic enzymes. Also, adw
The three clones were 2 ¹⁰ cells each for 7 hours and overnight culture.
It was to 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 12.5%.
After electrophoresis on a% polyacrylamide gel (in the presence of 0.4% SDS), a blotting buffer [25 mM Tris-HCl (pH 8.3), 192 mM glycine and 20%
% Methyl alcohol] (50 V, 70 minutes). 1 nitrocellulose membrane
10 mM Tris HCl (pH 7.4) containing 0% FCS,
After blocking with 150 mM NaCl, a mouse anti-HBc monoclonal antibody (subclass Ig) was used as the first antibody.
M) (Chemicon International)
Inc. ), And a phosphatase-labeled goat anti-mouse IgM (μ) antibody was used as the second antibody. BCI for substrate
When the activity staining was performed using P (5-bromo {4-chloro} 3-indolyl phosphate) and NBT (nitroblue tetrazolium), the bands of the HBc proteins of 45 Kd (dimer) and 21 Kd (monomer) were strongly stained. Was.

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

Example 2 Hepatitis B Virus Surface Tamper
Preparation of Protein (1) Preparation of Plasma Sample The same sample as the sample described in Example 1 (1) was used.

(2) Preparation of HBs Gene Using an automatic type 381A DNA synthesizer (Applied Biosystems), base 30 represented by the sequence shown in SEQ ID NO: 3 containing the NcoI recognition site by a conventional method.
(Hereinafter referred to as HBs-1) and an oligonucleotide comprising 30 bases represented by SEQ ID NO: 4 in 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 types of HBV
DNA (HBs subtypes adr and adw) was used as a sample, and treated in the same manner as described in Example 1 (2).
The CR method was performed.

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

(3) Preparation of Plasmid Containing HBs Gene A commercially available gene expression vector pTV118N (Takara Shuzo) was treated with restriction enzymes NcoI and HindIII to obtain a larger fragment, and the about 670 bp obtained in the above item (2) was obtained. 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 Escherichia coli with pTV118N · HBs obtained in (3) above and L-
E. coli MV1184 cultured overnight at 37 ° C. in a broth
(Takara Shuzo) was treated with 50 mM CaCl ₂ and the competent cells produced were incubated at 0 ° C for 40 minutes.
After heat treatment at 42 ° C for 3 minutes, L-
The cells were cultured in a broth. This E. coli was transformed into ampicillin (50
μg / ml), 0.5 mM IPTG and 0.004% X-G
The cells were plated on an L-broth agar plate medium containing AL, and cultured at 37 ° C. overnight. The resulting ampicillin-resistant and transparent colonies were each renewed with new ampicillin (100 μg / ml).
Overnight at 37 ° C. in an L-broth containing the plasmid, and plasmid DNA was isolated by the alkaline lysis method (Mole described above).
cultural Cloning, A Laboratory
y Manual 2nd Edition). This D
After digestion of NA with restriction enzymes NcoI and HindIII, electrophoresis was performed on a 1.2% agarose gel, and about 67% by a UV lamp in the presence of ethidium bromide.
The presence of 0 bp HBs DNA and 3100 bp plasmid vector was confirmed. About 670 bp obtained here
E. coli having the HBs gene of pTV118 · HBs-
001 (16 clones as subtype adr-derived DNA) and pTV118 HBs-101 (subtype a
(12 clones as dw-derived DNA).

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

[0053]

When the core structural protein or the surface structural 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 the DNA fragment for insertion comprising from immediately before the start codon to immediately after the stop codon. Can be obtained, and can be applied to the difference between HBs antigen subtypes and the disappearance of cleavage sites due to mutation.

[0054]

[Sequence list]

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

SEQ ID NO: 2 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Sequence: TCCAAGGGAT CCTAACATTG AGATTCCCGA

SEQ ID NO: 3 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Sequence: ACCGACCATG GAGAACACAA CATCAGGATT

SEQ ID NO: 4 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Sequence: TTTATTAAGC TTTAAATGTA TACCCAAAGA

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is an explanatory diagram showing a step of preparing an HBc gene.

FIG. 2 is an explanatory diagram showing a step of preparing a plasmid pTV119.HBc.

FIG. 3 is an explanatory diagram showing a step of preparing an HBs gene.

FIG. 4 is an explanatory diagram showing a step of preparing plasmid pTV118 · HBs.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI C12R 1:92) C12R 1:92) (56) References JP-A-3-27400 (JP, A) JP-A-2-57184 (JP, A) JP-A-3-108494 (JP, A) JP-A-64-67191 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/68 C12N 15 / 00-15/90 REGISTRY (STN) CA (STN) BIOSIS / WPI (DIALOG)

Claims (6)

(57) [Claims] (1) (1) (a) comprising the recognition base sequence CCATGG of the restriction enzyme NcoI, wherein the recognition base sequence CCAT
The nucleotide sequence ATG present in the GG corresponds to the start codon ATG in the nucleotide sequence of the DNA encoding the natural human hepatitis B virus core structural protein, and corresponds to the D sequence 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 start codon ATG of NA or a complementary base sequence thereof; and (b) a base sequence including GGATCCTA, which is present at the end of the base sequence. Base sequence CT which is complementary to the stop codon TAG in the base sequence of DNA encoding the natural human hepatitis B virus core structural protein
A second DNA having a base sequence capable of specifically binding to a region containing a stop codon TAG of DNA encoding the natural human hepatitis B virus core structural protein or a complementary base sequence thereof corresponding to A A mixture comprising a primer, (c) DNA polymerase, and (d) hepatitis B virus DNA
Amplifying the DNA encoding the hepatitis B virus core structural protein by subjecting the amplified DNA encoding the hepatitis B virus core structural protein to restriction enzymes NcoI and B;
amNl digested with NcoI / BamHI digested DNA
(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) culturing the resulting transformant and adding the transformant to a DNA encoding hepatitis B virus core structural protein;
Expressing hepatitis B virus core structural protein by expressing A; and (6) each step of isolating the produced hepatitis B virus core structural protein from a transformant. A method for producing a viral core structural protein. (1) (1) (a) comprising the recognition base sequence CCATGG of the restriction enzyme NcoI,
The nucleotide sequence ATG present in the GG corresponds to the start codon ATG in the nucleotide sequence of the DNA encoding the natural human hepatitis B virus surface structural protein, and corresponds to the D sequence 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 including the start codon ATG of NA or a complementary base sequence thereof; and (b) a base sequence comprising AAGCTTTTA, which is present at the end of the base sequence. Base sequence TT which is complementary to the stop codon TAA in the base sequence of the DNA encoding the natural human hepatitis B virus surface structural protein
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 a complementary base sequence thereof corresponding to A. A mixture comprising a primer, (c) DNA polymerase, and (d) hepatitis B virus DNA
Through an amplification step, a DNA encoding the hepatitis B virus surface structural protein is amplified, and (2) the amplified DNA encoding the hepatitis B virus surface structural protein is converted into a restriction enzyme NcoI and a restriction enzyme H.
After digestion with indIII, NcoI / HindIII digested D
Generating 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) culturing the obtained transformant, and transforming the transformant into a DNA encoding a hepatitis B virus surface structural protein;
Expressing hepatitis B virus surface structural protein by expressing A; and (6) each step of isolating the produced hepatitis B virus surface structural protein from a transformant. A method for producing a virus surface structural protein. 3. (1) Consisting of at least 20 bases of the nucleotide sequence of SEQ ID NO: 1 in the sequence listing, and further comprising a restriction enzyme N
a base sequence containing the coI recognition base sequence CCATGG, or a complementary base sequence thereof, or (2) a base having at most 3 bases replaced, deleted or inserted in the base sequence of SEQ ID NO: 1 in the sequence listing Consisting of at least 20 bases of the sequence and having the restriction enzyme Nc
a base sequence containing the oI recognition base sequence CCATGG, or a DNA primer having a base sequence complementary thereto;
Thus, the DNA for use in the method of claim 1
Primer . 4. A base sequence G comprising at least 20 bases of the base sequence shown in SEQ ID NO: 2 in the sequence listing.
(2) at least 20 bases in which at most 3 bases are substituted, deleted or inserted in the base sequence containing GATCCTA, or its complementary base sequence, or (2) the base sequence described in SEQ ID NO: 2 in the sequence listing And the base sequence GG
A DNA primer having a nucleotide sequence containing ATCCTA or a nucleotide sequence complementary thereto , which is described in claim 1.
The DNA primer for use in the method described above . 5. (1) Consisting of at least 20 bases of the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing, and further comprising a restriction enzyme N
a base sequence containing the coI recognition base sequence CCATGG, or a complementary base sequence thereof, or (2) a base having at most 3 bases replaced, deleted or inserted in the base sequence of SEQ ID NO: 3 in the sequence listing Consisting of at least 20 bases of the sequence and having the restriction enzyme Nc
a base sequence containing the oI recognition base sequence CCATGG, or a DNA primer having a base sequence complementary thereto;
Thus, the DNA for use in the method of claim 2
Primer . 6. A nucleotide sequence A comprising at least 20 nucleotides of the nucleotide sequence of SEQ ID NO: 4 in the sequence listing.
A nucleotide sequence containing AGCTTTA, or its complementary nucleotide sequence, or (2) at least 20 nucleotides of a nucleotide sequence in which at most 3 nucleotides have been substituted, deleted or inserted in the nucleotide sequence of SEQ ID NO: 4 in the sequence listing Consisting of the base sequence AA
A DNA primer having a base sequence containing GCTTTA, or a base sequence complementary thereto, as described in claim 2.
The DNA primer for use in the method described above .