JP2002101887A - Method for preparing recombinant hepatitis b virus surface antigen, and the resulting antigen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing a recombinant hepatitis B virus surface antigen that permits, in a large amount, producing a recombinant hepatitis B virus antigen having a high antigenic activity, and to provide a recombinant hepatitis B virus surface antigen. SOLUTION: This method for preparing a recombinant hepatitis B virus surface antigen is such a method that the antigen is prepared using a gene modified by deleting a region encoding 1 to 23 amino acid (s) from 78th amino acid from the N-terminal in S antigen of the hepatitis B virus surface antigen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for preparing a recombinant hepatitis B virus surface antigen capable of obtaining a hepatitis B virus surface antigen having high antigen activity, and a recombinant hepatitis B virus surface antigen.

[0002]

2. Description of the Related Art At present, there is a demand for a method capable of inexpensively supplying an immunologically highly active antigen protein of hepatitis B virus, particularly a surface antigen protein useful for diagnosis.

For example, a method has been developed for producing a hepatitis B virus surface antigen as a recombinant antigen using yeast, animal cells, etc. (JP-A-6-90781 and JP-A-2-150292). Kaihei 2-16308
No. 9).

[0004] However, in the above-mentioned method, the productivity of the recombinant was not so excellent, and its antigenicity was lower than that of the natural antigen obtained from the blood of infected patients. In addition, a system using animal cells is more expensive than using bacteria in terms of culturing, maintaining strains, and the like,
The operation is also complicated, and it takes a long time from the start of culture to purification.

The hepatitis B virus surface antigen is expressed in a recombinant system using Escherichia coli because a part of the transmembrane region (hydrophobic region) of the antigen protein is considered to be toxic to Escherichia coli. (Gene, 30, 1984, 201-210, Gen.
e, 160, 1995, 179-184). In some cases, a gene lacking a portion that is considered to be toxic to Escherichia coli is produced by various methods to express a protein in Escherichia coli.
Acids Research, 11, 1983, 35
81-3591, Gene, 30, 1984, 201-
210) or C-terminal (Gene, 160, 199).
5,179-184) in which some residues were deleted, and the expression level was not always large.

[0006]

SUMMARY OF THE INVENTION In view of the above, the present invention provides a method for preparing an antigen having a structure as close as possible to a natural antigen while deleting a portion which is considered to be toxic to Escherichia coli. An object of the present invention is to provide a method for preparing a recombinant hepatitis B virus surface antigen and a recombinant hepatitis B virus surface antigen that can produce a large amount of a recombinant hepatitis B virus surface antigen having antigen activity.

[0007]

The method for preparing the recombinant hepatitis B virus surface antigen according to the present invention comprises the steps of: 1 to 23 amino acids from the 78th amino acid from the N-terminal to the S antigen of the hepatitis B virus surface antigen; Is prepared using a gene in which the region encoding is deleted. (Hereinafter referred to as the present invention 1)

The method for preparing the recombinant hepatitis B virus surface antigen according to the present invention comprises the steps of:
This is prepared by using a gene in which amino acids are substituted with a region encoding an amino acid forming an α-helix. (Hereinafter referred to as the present invention 2)

The method for preparing a recombinant hepatitis B virus surface antigen according to the present invention comprises the steps of: providing at least 30 bases in a region coding from the 78th amino acid to the 100th amino acid from the N-terminal in the S antigen of the hepatitis B virus surface antigen; It is prepared by using a pair-deficient gene. (Hereinafter referred to as the present invention 3)

The method for preparing a recombinant hepatitis B virus surface antigen according to the present invention comprises the steps of: providing at least 30 bases in the region encoding the amino acid from the 78th to the 100th amino acid from the N-terminal in the S antigen of the hepatitis B virus surface antigen; The pair is prepared by using a gene in which a region encoding an amino acid forming an α-helix is substituted. (Hereinafter referred to as the present invention 4).

[0011] The recombinant hepatitis B virus surface antigen of the present invention is obtained by the method for preparing the recombinant hepatitis B virus surface antigen of the present invention 1-4. Hereinafter, the present invention will be described in detail.

Hepatitis B virus (HBV) has a double structure consisting of core particles and an envelope (surface) surrounding them. Hepatitis B virus surface antigen (hereinafter referred to as HBs antigen) is an S antigen. , Pre-S1 antigen and pre-S2 antigen. The HBs antigen is encoded by the S antigen gene of HBV-DNA, the pre-S1 antigen gene and the pre-S2 antigen gene upstream of the S antigen gene. In the present specification, the HBs antigen refers to an S antigen only, an S antigen and a pre-S1 antigen linked thereto, and an S antigen, a pre-S1 antigen and a pre-S2 antigen linked thereto. .

The S antigen has an antigen-determining region containing four antigen sites and four transmembrane regions.

The above four antigen sites are sites that determine four types of HBs antigen subtypes, ie, adw type, adr type, ayw type and ayr type, and are antigen sites that determine d / y. 12 from the S antigen N-terminal of a certain HBs antigen
The second amino acid, the 160th amino acid from the N-terminus of the S antigen which is an antigen site determining r / w, and the 126th and 145th amino acids from the N-terminus of the S antigen which is a site determining the common a antigen Say. HBs antigen a
The dr-type S antigen gene was sequenced in SEQ ID NO: 1 (Lonc
arevic, I .; F. et al. , 1990, Nu
cleic Acids Res. 18, 4940), and the sequence replaced with a codon frequently used in Escherichia coli,
It is shown in SEQ ID NO: 2.

The S antigen referred to in the present invention preferably contains the above four antigen sites in order to have antigen activity. Moreover, as long as the antigen site is included, modifications such as deletion, substitution, addition, and insertion may be made.

The four transmembrane regions are defined as transmembrane region I (hereinafter referred to as T) of the 9th to 27th amino acids from the N-terminus of the S antigen.
MI), transmembrane domain II (hereinafter referred to as TMII) of the 77th to 100th amino acids from the N-terminus of S antigen, S
The transmembrane region III of the 170th to 187th amino acids from the N-terminus of the antigen (hereinafter referred to as TMIII), the transmembrane region IV of the 189th to 209th amino acids from the N-terminus of the S antigen
(Hereinafter referred to as TMIV). The transmembrane region is
It can be estimated by literature or calculation software, and may be around several residues. Since the transmembrane region of the protein disrupts the membrane structure of Escherichia coli, the retention of the expression vector in Escherichia coli is extremely reduced. Above all, TMII has the highest transmembrane property and is considered to have high toxicity to Escherichia coli.

On the other hand, since the transmembrane region has a structure partially buried in the lipid membrane, the structure of the antigen site is stabilized,
Since it is considered that a recombinant hepatitis B virus surface antigen having high antigen activity can be prepared, the expression level of the recombinant HBs antigen is reduced when deletion is performed without restriction. The transmembrane region can be estimated experimentally or computationally (see Stirk, HJ et al.,
1992, Intervirology, 33, 148-158; Berting, A. et
al., 1995, Intervirology, 38, 8-15).

The gene of the present invention, in which the region encoding 1 to 23 amino acids from the 78th amino acid from the N-terminus is deleted, refers to a gene which is 1 to 23 amino acids from the 78th amino acid from the N-terminal of the S antigen. Gene, preferably a gene in which a region encoding 1 to 10 amino acids has been deleted. This region corresponds to a part or the whole of the transmembrane region of TMII. If there are too many regions to be deleted, the expression level of the recombinant HBs antigen will decrease.

The gene of the present invention in which 1 to 23 amino acids from the 78th amino acid from the N-terminus are substituted by a region encoding an amino acid forming an α-helix refers to the 78th amino acid from the N-terminus of the S antigen. Refers to a gene in which a region encoding 1 to 23 amino acids, preferably 1 to 10 amino acids counted from the amino acid is replaced with a region encoding an amino acid capable of forming an α-helical structure. α
-Substitution with a region encoding an amino acid capable of forming a helical structure makes it possible to maintain the three-dimensional structure of the natural HBs antigen as it is, thereby reducing the possibility of toxicity to E. coli and the like.

Alanine, glutamic acid, leucine, methionine and the like are known as amino acids capable of forming the α-helical structure. In addition, other amino acids may be added to the extent that the formation of the α-helix structure is not inhibited, and the ranges of the regions before and after substitution do not always need to match. In order to form an α-helical structure, it is preferable to substitute a region encoding at least 4 amino acids. In addition, the transmembrane property within the S antigen is low and α-
It is preferably a region encoding an amino acid capable of forming a helical structure.

The gene of the present invention 3 in which at least 30 base pairs are deleted from the region coding from the 78th amino acid to the 100th amino acid from the N-terminus is defined as the N antigen of the S antigen.
It refers to a gene in which at least 30 base pairs (10 amino acids) at an arbitrary position in the region from the 78th amino acid to the 100th amino acid from the terminal, that is, at any position in the transmembrane region of TMII, is deleted. If the region to be deleted is smaller than 30 base pairs, the expression level of the HBs antigen may decrease, and preferably 30 to 36 base pairs.

The gene in which at least 30 base pairs of the region encoding the 78th amino acid to the 100th amino acid from the N-terminus according to the present invention 4 is replaced by a region encoding an amino acid forming an α-helix is S N of antigen
A region from the 78th amino acid to the 100th amino acid from the terminal, that is, a region encoding at least 30 base pairs (10 amino acids) at an arbitrary position in the transmembrane region of TMII, which forms an α-helix-forming amino acid; Means the gene replaced by Substitution with a region encoding an amino acid capable of forming an α-helix structure allows the natural HBs antigen to maintain its three-dimensional structure as it is, thereby reducing the possibility of toxicity to E. coli and the like. Amino acids forming an α-helix include
The same ones as described above can be used.

The gene referred to in the present invention is preferably a gene in which a region encoding 1 to 38 amino acids from the 188th amino acid from the N-terminal is further deleted. N above
The region encoding 1 to 38 amino acids from the 188th amino acid from the terminal corresponds to a part or all of the transmembrane region of the TMIV, and if too many regions are deleted, the expression level of the recombinant HBs antigen May decrease. Since the transmembrane region of TMIV is considered not to be directly involved in the antigenicity of HBs antigen, the transmembrane region is deleted by deleting the transmembrane region harmful to Escherichia coli.
The expression level of the Bs antigen can be increased.

As the gene for the S antigen of the hepatitis B virus surface antigen used in the present invention, a gene cloned from the genomic DNA of the hepatitis B virus isolated from an infected patient can be used. It is also possible to use synthetic genes prepared by a combination of chemical synthesis and genetic engineering techniques.

In the method for preparing a recombinant HBs antigen of the present invention, the above-mentioned gene (hereinafter, referred to as a recombinant gene)
The method for producing is not particularly limited, and for example, ordinary genetic engineering techniques such as a method using a restriction enzyme and a method using PCR can be used. Above all, from the viewpoint of simplicity, a method in which appropriate primers having a restriction enzyme site are designed and prepared by PCR is preferable. Further, when designing a gene primer, a gene primer having a histidine site or a histidine site and a protease cleavage site such as thrombin is designed, and a PC using the primer is designed.
The recombinant gene used in the present invention may be prepared by R.

In the method for preparing a recombinant HBs antigen of the present invention, the method for expressing the above-mentioned recombinant gene is not particularly limited, and a method for co-expressing the above-mentioned recombinant gene together with a gene encoding a protein folding factor, and the like are preferred. Used. Since the protein folding factor improves the efficiency of normal three-dimensional structure formation of the protein, co-expression in a host can increase the expression level of the recombinant HBs antigen. Examples of the protein folding factor include disulfide bond isomerase, peptidyl prolyl cis trans isomerase (hereinafter, referred to as PPIase), chaperone and the like. In this specification,
Co-expression means expressing two or more foreign genes in a host.

The above disulfide bond isomerase promotes recombination of disulfide bonds in a protein in the presence of a redox agent. The above-mentioned disulfide bond isomerase is present in almost all organisms, and many of its genes have been cloned and can be used. For example, in eukaryotes, there is protein disulfide isomerase (PDI), and in bacteria, there is the Dsb family of thioredoxins having an active motif of protein disulfide isomerase.

As a method for preparing the above-mentioned disulfide bond isomerase gene, the methods exemplified as the above-mentioned methods for preparing the recombinant gene and the like can be mentioned. For example, genome D
Using NA as a template, a partial base sequence is synthesized so as to contain an appropriate restriction enzyme site to prepare a PCR primer, and the primer can be prepared by a PCR method using the primer.

The above-mentioned PPIase refers to the N-terminal of the proline residue.
It is an enzyme that has the effect of promoting the rotation of terminal peptide bonds. The PPIase is present in almost all organisms, and many of its genes have been cloned, so that it is possible to use them. The above PP
Examples of the method for preparing the Iase gene include the methods exemplified as the above-described methods for preparing the recombinant gene.

The chaperone is a kind of heat shock protein, is produced when cells are exposed to various environmental stresses such as temperature change, and is known to be involved in protein folding and stabilization in the presence of ATP. Have been. Examples of the chaperone include a bacterial Group I type,
In addition, eukaryotic and archaeal Group II type chaperones are known, and both are suitably used. As a method for preparing the chaperone gene, the HB
Examples of the method for preparing the s antigen gene include the methods exemplified above.

In the method for preparing a recombinant HBs antigen of the present invention, the host is not particularly limited.
Examples include bacteria such as Escherichia coli, yeast, insects, animal cells, and the like. Above all, the host is preferably a bacterium or a yeast from the viewpoint that the culture days are short and the culturing operation is simple.

In addition, Escherichia coli is particularly preferred in that it can express a large amount of foreign genes. Among Escherichia coli, Escherichia coli deficient in at least one enzyme that reduces a protein disulfide bond (hereinafter referred to as a disulfide bond reductase-deficient strain) .).
Examples of the enzyme that reduces the disulfide bond of the protein include thioredoxin reductase and glutathione reductase, which are enzymes involved in two types of disulfide bond reduction systems in Escherichia coli cells, the thioredoxin system and the glutathione system. Those in which at least one, and more preferably two of these enzymes are deleted are mentioned. When these disulfide bond reductase-deficient strains are used as a host, disulfide bonds are not reduced in Escherichia coli, and the S--S bond of the protein remains formed, and the protein is produced with the original structure of the protein maintained. Is done. As a result, the solubility of the protein is improved, and the production amount can be increased. In particular, the HBs antigen has a very high content of cysteine, which is an amino acid forming a disulfide bond, of 20 to 25%, so that the effect of using a disulfide bond reductase-deficient strain is very large.

In the method for preparing a recombinant HBs antigen of the present invention, the above-mentioned recombinant gene and the gene for a protein folding factor such as a disulfide bond isomerase gene, a PPIase gene and a chaperone gene can be separately expressed. And a method of inserting into the same vector. As a method for insertion into the above-mentioned separate vectors, first, the above-mentioned recombinant gene and the above-mentioned gene of the protein folding factor are inserted into separate vectors which can coexist, respectively, to produce two kinds of plasmids. Next, the host is transformed with the two plasmids. As the above-mentioned vector, a vector into which arbitrary drug resistance genes different from each other are introduced in advance is used.
By culturing the transformant in which the species plasmid is integrated in the presence of the drug, a transformant in which the two drug resistance genes are both expressed can be selected.

When Escherichia coli is used as the host, for example, the above-mentioned cloned recombinant gene is inserted into an expression vector having a pBR T7 promoter or the like.
A Bs antigen expression plasmid is prepared, and a gene for a protein folding factor such as a disulfide bond isomerase gene, a PPIase gene, a chaperone gene, together with a T7 promoter, a terminator, etc., is added to a pACYC vector or the like which can coexist with a pBR-based vector. Introduce. By introducing any different drug resistance genes into the HBs gene expression plasmid and the protein folding factor gene expression plasmid, it becomes possible to maintain the two types of expression plasmids in E. coli.

As a method for insertion into the same vector, there is a method in which the above-mentioned recombinant gene and the above-mentioned gene for the protein folding factor are inserted in the same vector in tandem or in a reverse direction.

When Escherichia coli is used as the host, for example, an expression vector having a pBR T7 promoter is used to express a protein folding factor such as a disulfide bond isomerase gene, a PPIase gene, and a chaperone gene under the control of the T7 promoter. The unit and the HBs antigen expression unit also under the control of the T7 promoter are placed in tandem. Alternatively, the protein folding factor expression unit and the HBs antigen expression unit may be placed in opposite directions. The transformant incorporating the expression vector is cultured in the presence of a drug having resistance to the vector, and the expression is induced, whereby the protein folding factor gene and the recombinant gene can be co-expressed in the host. it can.

The fact that the recombinant gene and the gene for the protein folding factor are co-expressed in the host can be confirmed by SDS-polyacrylamide gel electrophoresis (hereinafter, referred to as SDS-polyacrylamide gel electrophoresis).
SDS-PAGE) and measuring the molecular weight of these gene products.

The method for purifying the expressed recombinant HBs antigen is not particularly limited. For example, conventional methods such as ammonium sulfate fractionation, gel filtration column chromatography, ion exchange column chromatography, hydrophobic column chromatography, affinity chromatography, etc. It can be performed by the method.

The method for measuring the activity of the obtained recombinant HBs antigen is not particularly limited. For example, a polyclonal antibody or a monoclonal antibody against a natural HBs antigen is prepared, and enzyme immunoassay (EIA) and R
It can be performed by IA or the like. For the method of measuring the activity of the HBs antigen, a commercially available analysis kit or the like can be used.

Since the method for preparing a recombinant HBs antigen of the present invention has the above-mentioned constitution, a large amount of recombinant HBs antigen having high antigen activity can be prepared easily. A recombinant HBs antigen obtained by the method for preparing a recombinant HBs antigen of the present invention is also one of the present invention.

[0041]

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

Example 1 (Preparation of plasmid) adr-type H shown in SEQ ID NO: 2
In the S antigen gene of the Bs antigen, a region encoding 23 amino acids from the 78th amino acid at the N-terminal (the T
MII) was deleted by PCR.
This was cut with NdeI-BamHI, and then introduced into an expression vector having a T7 promoter that had been treated with the same restriction enzyme to prepare pTHBM1.

(Evaluation of Protein Expression and Antigenicity by Escherichia coli) The Escherichia coli having the expression vector pTHBM1 was purified using 2XYT containing carbenicillin (100 μg / mL).
Medium (20 g of tryptone, 8 g of yeast extract, NaCl1
OD600nm at 0.6-1.0 at 5 g / L)
After culturing at 7 ° C., the culture was incubated at 20 ° C. for 30 minutes, expression was induced by adding 1 mM IPTG, and further cultivated at 20 ° C. overnight.
The cells were collected. About 5mg protein / m by sonication
1 ml of cell extract was obtained. A “Quick Chaser HBsAg” (manufactured by Mizuho Medi Co.) obtained by diluting the cell extract 100-fold with an E. coli control extract
Diluted by 25,000-fold by immunochromatography was evaluated using an EIA kit for measurement of HBs antigen "Enzygnost-HBsAg monoclonal" (Boehringer Mannheim). Table 1 shows the results of the antigenicity evaluation results. As a result of confirming the expression of the protein by SDS-PAGE, a band corresponding to a protein of about 22.4 kDa was detected.

Example 2 In the S antigen gene of the adr-type HBs antigen shown in SEQ ID NO: 2, a region encoding the 23 amino acids from the 78th amino acid at the N-terminus (TMII) and the 189th amino acid at the N-terminus A gene in which the region coding for 38 amino acids was deleted from the amino acids of No. 1 was prepared by PCR. HB was prepared in the same manner as in Example 1 except that this gene was used.
s antigen was obtained. Table 1 shows the results of the antigenicity evaluation.

Example 3 In the S antigen gene of the adr-type HBs antigen shown in SEQ ID NO: 2, a region (part of TMII) encoding 10 amino acids from the 78th amino acid at the N-terminal was deleted. The gene was created by PCR. An HBs antigen was obtained in the same manner as in Example 1 except that this gene was used. Table 1 shows the results of the antigenicity evaluation.

Example 4 In the S antigen gene of the adr-type HBs antigen shown in SEQ ID NO: 2, a region encoding 10 amino acids from the 78th amino acid at the N-terminus (part of the TMII) and N
A gene in which a region encoding 38 amino acids from the terminal 189th amino acid was deleted was prepared by PCR. An HBs antigen was obtained in the same manner as in Example 1 except that this gene was used. Table 1 shows the results of the antigenicity evaluation.

Example 5 In the S antigen gene of the adr-type HBs antigen shown in SEQ ID NO: 2, a region encoding 10 amino acids from the N-terminal 78th amino acid (part of the TMII) was replaced with N
A gene in which the 13th amino acid at the end was replaced with a region encoding 10 amino acids (region forming an α-helix) was prepared by PCR. An HBs antigen was obtained in the same manner as in Example 1 except that this gene was used. Table 1 shows the results of the antigenicity evaluation.

Example 6 In the S antigen gene of the adr-type HBs antigen shown in SEQ ID NO: 2, the region encoding 10 amino acids from the N-terminal 78th amino acid (part of the TMII) was replaced with N
A gene obtained by substituting a region encoding 10 amino acids from the 13th amino acid at the terminal (a region forming an α-helix) and deleting a region encoding 38 amino acids from the 189th amino acid at the N-terminal Was prepared by PCR. An HBs antigen was obtained in the same manner as in Example 1 except that this gene was used. Table showing results of antigenicity evaluation
Shown in 1.

Comparative Example 1 Human serum-derived HBs antigen (2 mg / ml) diluted 100-fold with an E. coli control extract was 100,000-fold subjected to immunochromatography using "Quick Chaser HBsAg" (manufactured by Mizuho Mediy KK). An EIA kit for measuring HBs antigen "Enzygnost-
HBsAg monoclonal "(Boehringer Mannheim). Table 1 shows the results of the antigenicity evaluation.

Comparative Example 2 An HBs antigen was obtained in the same manner as in Example 1 except that the full length gene of the HBs antigen shown in SEQ ID NO: 2 was used.
Table 1 shows the results of the antigenicity evaluation.

[Table 1]

[0051]

Since the method for preparing a recombinant HBs antigen of the present invention has the above-mentioned constitution, a recombinant HBs antigen having a high antigen activity can be obtained at a low cost. Useful for etc.

[0052]

[Sequence List] <110> SEKISUI CHEMICAL CO., LTD. <120> Preparation method and antigen <130> 00P02015 <160> 2 <210> 1 <211> 681 <212> DNA <213> Type B hepatitis virus <220> <221> CDS <222> 1..678 <400> 1 atg gag aac aca aca tca gga ttc cta gga ccc ctg ctc gtg tta cag 48 Met Glu Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 gcg ggg ttt ttc ttg ttg aca aga atc cta aca ata cca cag agt cta 96 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 gac tcg tgg tgg tgg act tct ctc aat ttt cta ggg gga gca ccc acg tgt 144 Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr Cys 35 40 45 cct ggc caa aat tcg cag tcc cca acc tcc aat cag tca cca acc tct 192 Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn Gln Ser Pro Thr Ser 50 55 60 tgt cct cca att tgt cct ggc tat cgc tgg atg tgt ctg cgg cgt ttt 240 Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 atc ata ttc ctc ttc atc ctg ctg cta tgc ctc atc ttc ttg ttg gtt 2 88 Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val 85 90 95 ctt ctg gac tac caa ggt atg ttg ccc gtt tgt cct cta ctt cca gga 336 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly 100 105 110 aca tca act acc agc acg gga cca tgc aag acc tgc acg att cct gct 384 Thr Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile Pro Ala 115 120 125 caa gga acc tct atg ttt ccc tct tgt tgc tgt aca aaa cct tcg gac 432 Gln Gly Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp 130 135 140 gga aac tgc act tgt att ccc atc cca tca tcc tgg gct ttc gca aga 480 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg 145 150 155 160 ttc cta tgg gag ggg gcc tca gtc cgt ttc tcc tgg ctc agt tca cta 528 Phe Leu Trp Glu Gly Ala Ser Val Arg Phe Ser Trp Leu Ser Ser Leu 165 170 175 gtg cca ttt gtt cag tgg ttc gta ggg ctt tcc ccc act gtt tgg ctt 576 Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 tca gtt ata tgg atg atg tgg tat tgg gga cca agt ctg tac aac atc 624 Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn Ile 195 200 205 ttg agt ccc ttt tta cct cta tta cca att ttc ttt tgt ctt tgg gta 672 Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 220 tac att taa 681 Tyr Ile 225 <210> 3 <211> 678 <212> DNA <213> Type B hepatitis virus <220> <221> CDS <222> 1..678 <400 > 3 atg gaa aac act act tct ggt ttc ctg ggt ccg ctg ctg gta ctg cag 48 Met Glu Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 gca ggt ttc ttc ctg ctg aca cgt atc ctca att cca cag tct ctg 96 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 gac tct tgg tgg act tct ctc aat ttt ctg ggt ggt gca ccg act tgc 144 Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr Cys 35 40 45 cct ggc caa aat tct cag tcc cca acc tcc aat cac tct cca acc tct 192 Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser 50 55 60 tgc cct cca att tgc cct ggc tat cgc tgg atg tgc ctg cgt cgt ttt 240 Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 atc att ttc ctc ttc atc ctg ctg ctg tgc ctc atc ttc ctc ctg ctg gtt 288 Ile Ile Ile Phe Leu Phe Ile Leu Leu Leu 85 Hes 90 95 ctt ctg gac tac caa ggt atg ctg cca gtt tgc cct ctg ctt cca ggt 336 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly 100 105 110 aca tct acc acc agc act ggt cca tgc aag acc tgc act att cct gct 384 Thr Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile Pro Ala 115 120 125 caa ggt acc tct atg ttt ccg tct tgc tgc tgc aca aaa cct tct gac 432 Gln Gly Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp 130 135 140 ggt aac tgc act tgc att ccg atc cca tct tcc tgg gct ttc gca cgt 480 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg 145 150 155 160 ttc ctg tgg gag tgg gcc tct gtc cgt ttc tcc tgg ctc tct ctg ctg 528 Phe Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu 165 170 175 gtg cca ttt gtt cag tgg ttc gta ggt ctg tct ccg act gtt Val Pr o Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 tct gtt att tgg atg atg tgg tat tgg ggt cca tct ctg tac aac atc 624 Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn Ile 195 200 205 ctg tct ccg ttt ctg cct ctg ctg cca att ttc ttc tgc ctt tgg gta 672 Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 220 tac att 678 Tyr Ile 225

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA01 AA11 BA33 CA02 DA06 EA04 FA02 GA11 GA25 HA03 4B064 AG33 CA02 CA19 CC01 CC24 CE02 CE12 DA01 DA15 4H045 AA11 BA10 CA01 DA86 EA31 EA53 FA74

Claims (7)

[Claims] 1. A recombinant prepared by using a gene in which a region encoding 1 to 23 amino acids from the 78th amino acid from the N-terminus of the S antigen of the hepatitis B virus surface antigen is deleted. A method for preparing hepatitis B virus surface antigen. 2. An S antigen of hepatitis B virus surface antigen, which is prepared by using a gene in which a region encoding 1 to 10 amino acids from the 78th amino acid from the N-terminus has been deleted. Item 1. Recombinant type B according to item 1.
A method for preparing hepatitis B virus surface antigen. 3. In the S antigen of the hepatitis B virus surface antigen, the region encoding 1 to 23 amino acids from the 78th amino acid from the N-terminus is replaced by a region encoding an α-helix forming amino acid. A method for preparing a recombinant hepatitis B virus surface antigen, which is prepared using a gene. 4. An S antigen of hepatitis B virus surface antigen, which is prepared by using a gene in which at least 30 base pairs in the region encoding the amino acid from the 78th amino acid to the 100th amino acid from the N-terminus are deleted. A method for preparing a recombinant hepatitis B virus surface antigen. 5. The S antigen of the hepatitis B virus surface antigen, wherein at least 30 base pairs of a region coding from the 78th amino acid to the 100th amino acid from the N-terminus are:
A method for preparing a recombinant hepatitis B virus surface antigen, which is prepared using a gene substituted with a region encoding an amino acid that forms an α-helix. 6. The above-mentioned gene further comprises 188 from the N-terminal.
The method for preparing a recombinant hepatitis B virus surface antigen according to claim 1, wherein the gene is a gene in which a region encoding 1 to 38 amino acids from the second amino acid has been deleted. 7. A hepatitis B virus surface antigen obtained by the method for preparing a recombinant hepatitis B virus surface antigen according to claim 1.