JP2717799B2 - Hybrid antigen particles comprising HBs antigen and foreign peptide and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to hybrid antigen particles obtained by applying a gene recombination technique, and a method for producing the same. More specifically, the present invention relates to a hybrid antigen particle composed of an HBs antigen and a foreign peptide expressed by a transformed yeast and a method for producing the same.

BACKGROUND OF THE INVENTION AND PRIOR ART With the advance of genetic recombination technology, the production of useful substances using genetic recombination has been rapidly progressing in recent years, and some of these are used in clinical reagents, vaccines and therapeutic drugs. It is spreading as a product industrially.

In general, it is considered that an antigen for immunization used as a vaccine is desirably an antigen aggregate or an antigen particle having a large molecular weight in view of problems such as immunogenicity. When such an antigen for immunization is prepared using gene recombination technology, it is most preferable that such spherical particles or antigen aggregates are directly expressed by the constructed transformant. However, there are not so many exogenous genes in which antigen molecules expressed by the constructed transformant naturally form particles. According to previous reports, hepatitis B virus surface antigen (hepatitis B virus-related antigen gene) has been reported. The HBs) gene and the viral core antigen (HBc) gene are known as the few genes having such properties.

Like the HBs antigen, the characteristic that the expression product is spheroidized when expressed by the transformant has the advantage that the target product is excellent as an immunogen, as well as the advantage of purifying the target product from the transformant. In addition, it is considered that there are advantages that the operation is facilitated and that the protease is less affected by host cell-derived proteases and the like in the transformant.

Utilizing the advantages of such a spheroidized HBs antigen gene, some studies have been conducted in which antigen proteins of other viruses are bound to the HBs antigen and expressed. For example, P. Valenzuela et al. Proposed that these fusion structural genes be constructed in order to express a fusion peptide of an HBs antigen and a herpes virus gD antigen, and that these be expressed as particles [Bio-Technology3, 323 (1985)]. On the other hand, F. Delpeyroux et al. Attempted to express a modified spherical particle having the desired antigenicity by replacing the amino acid sequence of the hydrophilic region of the HBs antigen with a sequence encoding an epitope of a poliovirus infection protective antigen. [Science Vol.233, p472-475 (1986)].

The method as described above proposes a technique for expressing a part of the foreign peptide as an antigen fused with the HBs antigen. Using such a conventional method, Imamura et al.
An attempt was made to express a large S peptide as a spherical particle in which a pre-S1 peptide consisting of 174 amino acids was bound to the end of the HBs polypeptide (226 amino acids). However, as a result, it has been shown that when the large S peptide is expressed in transformed yeast, the expression product does not form particles [Imamura et al., Journal of Virol-ogy Vol.
1, No 11, p3543- 3549 (1987)]. That is, in the prior art, there is a limit to a foreign peptide to be expressed by being fused to an HBs peptide, and it has been difficult to express a peptide such as a pre-S1 peptide as spherical particles in a transformed yeast.

Objectives of the Invention In order to solve such a problem, the present inventors have repeated research aiming at the development of a novel yeast expression system capable of efficiently expressing the desired foreign gene in the form of particles together with the HBs antigen. As a result, a transformant in which the HBs gene was efficiently incorporated into the yeast chromosome gene was produced, and such a transformant (integrated yeast) was autonomously propagated (non-integrated yeast) as used in a normal plasmid expression system. By introducing an expression plasmid for an exogenous peptide-HBs fusion polypeptide (integrated type), a polypeptide comprising a fusion of an exogenous peptide of interest and an HBs antigen, and H
The present invention succeeded in efficiently expressing a novel hybrid antigen spherical particle composed of a Bs polypeptide from the transformant, thereby completing the present invention.

Structure and Effect of the Invention The novel hybrid antigen particles provided by the present invention are a polypeptide in which an exogenous peptide and an HBs antigen are fused and a HBs antigen.
Hybrid antigen spherical particles composed of two types of Bs polypeptides. Such hybrid antigen particles are
It is expressed by a transformed yeast in which an exogenous peptide-HBs fusion peptide expression autonomously replicating plasmid has been introduced into an integration yeast in which the s antigen expression gene has been integrated into the yeast chromosome.

The foreign peptide fusion antigen using the HBs antigen provided by the conventional technique was an antigen composed of a single polypeptide in which the foreign peptide and the HBs peptide were fused, whereas the present invention The hybrid antigen particle comprises a fusion polypeptide of an exogenous peptide and an HBs peptide, and HBs
It is a novel hybrid antigen particle having two kinds of peptides as its constituent proteins. According to the present invention, it is possible to form hybrid antigen particles even for a relatively long peptide such as pre-S1-HBs antigen, which has been impossible to form particles in the expression product by the conventional techniques. Become.

The term “exogenous peptide” as used herein means a peptide other than the HBs peptide (major S peptide). Examples of such an exogenous peptide include various viral constituent proteins, particularly viral membranes that can serve as protective antigens for infection. And a peptide sequence encoding the epitope thereof. Therefore, by utilizing the technology of the present invention, it is possible to express various virus-constituting proteins produced by genetic recombination as hybrid antigen particles with HBs antigen, and to obtain a very excellent protein as an immunogen. Become.

As one example, by expressing a gene encoding a pre-S antigen that is a hepatitis B virus-related surface antigen by the expression system of the present invention, HBs-related antigen particles that are extremely excellent in preventing hepatitis B infection can be obtained. It can be prepared.

That is, HBs antigen and pre-S-similar to HBs antigen particles excellent as an immunogen purified from human HBe antigen positive serum
It becomes possible to obtain hybrid antigen particles composed of HBs antigen. Further, regarding the pre-S gene, it has become possible to efficiently express and express the pre-S1 antigen consisting of 174 amino acids, which has been extremely difficult to be granulated by the conventional techniques.

Hereinafter, the hybrid antigen particles of the present invention and a method for expressing the same will be described in detail.

(1) Production of HBs expression integration yeast The HBs expression gene fragment used herein is a gene sequence that does not have a replication origin sequence for autonomous propagation of a plasmid gene and is integrated into a yeast chromosome. Such a plasmid is called YIp (Yeast Integrated Plasmi
d) Type plasmid [Experimental Medicine Vol.5, No.11
P136-141 (1987)]. However, it is known that, when transformation is usually performed using such a gene, it is extremely difficult to produce a transformant (integrated yeast) that efficiently expresses the target gene. In order to solve such a problem, in the present invention, the HBs-expressing gene fragment for integration is linearized, and a gene sequence encoded on the yeast chromosome at both ends, for example, a gene encoding yeast ribosomal RNA, , A gene encoding transboson
Transformation is performed using the HBs expression gene fragment. According to the present invention, it is particularly preferable to use a gene encoding yeast ribosomal RNA. Such a ribosomal RN
The gene encoding A can be easily obtained from a yeast gene bank. That is, after extracting a chromosomal gene from yeast, this is cut with an appropriate restriction enzyme (for example, XmaI), and the ribosome RNA is encoded by a hybridization method using a probe consisting of the gene sequence of the ribosome RNA already analyzed. Gene can be obtained. The preparation of such a ribosomal RNA was previously reported in detail by Pablo Valenzuela et al. [The Journal of Biological Chemistry vol.25.
2 p-8118-8125 (1977)].

The HBs expression gene fragment referred to here is a gene fragment in which an HBs structural gene is incorporated downstream of a yeast-derived promoter gene.If necessary, a terminator gene sequence is incorporated downstream of the HBs structural gene, A gene sequence that can serve as a marker at the time of conversion, for example, a G418 gene fragment that is a neomycin resistance gene [Nature 2
87, p869-871 (1980)], or a gene fragment incorporating a gene or the like that produces an amino acid (eg, a gene for producing an amino acid such as histidine or tryptophan). As the yeast-derived promoter used here, a promoter that can act in the cells of the host yeast is used. As an example, yeast tryptophan (trp) promoter, acid phosphatase (PH05) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and the like are used as the most effective promoters. Since the PHO5 promoter can be controlled by controlling the concentration of phosphate in the culture medium, if the expression of the target product adversely affects the host, the yeast must be grown sufficiently before induction of the promoter. It is a very effective promoter for expressing the target product. In addition, the GAP promoter is one of the strongest promoters that function in yeast cells, and is very effective when the expression of the target product does not adversely affect the host.

Such a gene fragment can be easily integrated into the chromosome gene of the host yeast by using a conventional yeast transformation technique such as the lithium acetate method. As a host yeast, Saccharomyces cere
As an example of a particularly preferred host cell using visiae,
And those derived from Saccharomyces cerevisiae AH22 strain.

Further, according to the present invention, it is possible to screen for a transformant that further expresses the HBs antigen at a higher level by mutating the transformant (integrated yeast) obtained as described above. For example, as a method by a chemical treatment, there is a method of adding ethylene methanesulfonate, nitrosoguanidine, or the like, and as a physical treatment, a method by ultraviolet irradiation or the like is given.

The transformed yeast thus obtained is a conventional integration (YIp) type plasmid (non-integrated yeast) having no gene encoded on the yeast chromosome such as a gene encoding ribosomal RNA at both ends of a gene fragment for transformation. Expression of HBs antigen is much higher than that of transformed yeast obtained using an autonomously replicating plasmid, and the HBs antigen expression level is almost equivalent to that of a normal expression system using an autonomously replicating plasmid. Can be.

(2) Construction of Transformant Producing Hybrid Antigen Particles of the Present Invention The fusion antigen particles of the present invention are obtained by adding the above-mentioned transformed yeast (integrated yeast) to an autonomously growing foreign gene-HB
It can be obtained by constructing and culturing a transformed yeast into which the s fusion gene expression plasmid has been introduced. As used herein, the autonomously replicating plasmid refers to a gene encoding a replication origin derived from yeast (for example, 2 μori, ars1).
Etc.), yeast selectable marker genes (amino acid production genes,
A gene that expresses a foreign peptide-HBs fusion protein (a gene fragment in which a foreign peptide-HBs fusion structural gene is incorporated downstream of a suitable yeast promoter region gene) as a basic gene. is there. When an exogenous peptide is fused to the HBs gene, a gene fragment encoding the entire peptide consisting of 226 amino acids of the HBs gene or a region thereof within an appropriate range from the N-terminal to about the 80th amino acid of the HBs peptide is used. A gene encoding an exogenous peptide is connected to the 5 'end of the HBs gene from which the gene encoding is deleted. When both genes are fused, it is necessary to match codons when translated into amino acids. Representative examples of such foreign peptides include various viral antigens or partial peptides thereof. Specific examples include a protective antigen such as influenza virus and herpes virus, or a peptide containing an antigenic epitope thereof.
Further, examples of the exogenous peptide are not limited to the viral proteins as described above, and include peptides other than the viral constituent proteins. As the promoter gene for expressing the fusion structural gene, the same promoter gene as that used in the aforementioned gene fragment for integration yeast construction can be used. Such a plasmid can be introduced into the above-mentioned transformed yeast (integrated yeast) using a usual transformation technique. The thus constructed transformed yeast is cultured by a conventional culturing method, collected, and the HBs antigen is purified from the transformant by a conventionally known method (for example, Japanese Patent Publication No. 61-1986). 55950
The foreign peptide of interest according to the method described in
It is possible to purify HBs antigen hybrid antigen particles.

Furthermore, in constructing the transformant used in the present invention, it is preferable to select a clone having as high an expression level of the target gene as possible by effective screening. An example of such an effective screening method is the colony blotting method previously reported by the present inventors (the 8th Annual Meeting of the Molecular Biology Society of Japan p184 4E-26). According to this method, it is possible to screen approximately 10 ⁴ clones screening in one plate.

The foreign peptide-HBs hybrid antigen particles of the present invention can be obtained by using an integration yeast in which an HBs expression gene has been integrated into a yeast chromosome. The use of a host in which the HBs antigen-expressing gene has been integrated into the yeast chromosome as described above has the following advantages. That is, the hybrid antigen particles of the present invention are characterized in that the HBs peptide is used as a constituent peptide thereof in addition to the foreign peptide-HBs fusion peptide, and the HBs peptide, which is an important constituent peptide thereof, is used in the integration yeast. By doing so, stable expression can be achieved. Furthermore, when expressing foreign particles with HBs antigen particles by fusing various foreign peptides,
A plasmid expressing the HBs antigen fusion peptide may be constructed and introduced into the integration yeast, and the HBs expression integration yeast once created may be
It can be used many times as a host yeast for introducing plasmids for various purposes. This has the advantage that it is possible to extremely easily produce a transformant co-expressing the objective HBs and foreign peptide-HBs.

(3) Hybrid antigen particles composed of pre-S-HBs and HBs Regarding the preparation of HBs antigen, a method of expressing the antigen in a suitable host cell by applying gene recombination technology, and a method of expressing the antigen from blood of a person infected with hepatitis B virus Is known. For hepatitis B virus surface antigen, 3
It is known that there are antigens of different species sizes. That is, the most well-known HBs antigen consisting of 226 amino acids, a pre-S2-antibody having 55 amino acids further bound to the N-terminal of the peptide consisting of 226 amino acids.
There is an HBs antigen, and also a pre-S1-HBs antigen with 174 amino acids attached to the N-terminus of the HBs peptide.

Hepatitis B virus surface antigen-related particles present in human blood include spherical particles composed of HBs (major S) peptide in blood, and two types of HBs (major S) antigen and pre-S-HBs antigen. It is known that spherical particles composed of species peptides exist. However, an HBs antigen called a pre-S-added HBs vaccine in the present genetic recombination technology is a particle composed of only a pre-S-HBs peptide, and has a form different from that of a spherical particle in which two kinds of peptides are mixed as described above. Is different. Moreover, such an existing pre-S-added HBs vaccine has hardly seen any superiority in terms of effect as compared with a vaccine to which HBs has not been added. Under such circumstances, serum-derived pre-S mixed
It has been desired to establish a technique for producing HBs antigen particles having the same form as HBs antigen particles by genetic recombination.

In previous studies by genetic recombination of the hepatitis B virus surface antigen, HBs antigen (without pre-S) or pre-S-HBs antigen has been successfully expressed as spherical particles by a transformant. , Pre-S1-HB
s-spheroidizing for efficient expression and pre-S
-Expression of a hybrid antigen particle composed of HBs antigen and HBs antigen by a single transformant has not yet been reported.

The present inventors constructed an integration yeast in which the HBs expression gene was integrated into the chromosome gene of the host yeast, and further cultured the transformed yeast in which a plasmid having the pre-2-HBs expression gene was introduced into this transformant. With the transformed yeast, HBs antigen and pre-S2-HBs
We succeeded in expressing hybrid antigen particles composed of the antigen.

Further, when the plasmid incorporating the pre-S1-HBs gene was introduced into the integration yeast into which the HBs gene had been integrated into the chromosome, HB
Hybrid antigen particles composed of s-antigen and pre-S1-HBs antigen were successfully expressed by the transformed yeast. A gene such as pre-S1-HBs that does not form a spherical particle when expressed alone is expressed together with the HBs gene in the same host cell using the expression method of the present invention, so that both expression products become one spherical particle. Was an unexpected result.

In other words, even for a relatively large exogenous peptide, which did not achieve favorable results with the conventional method of expressing spherical particles composed of a single fusion peptide, it can be mixed with the HBs peptide by applying the expression method of the present invention. This makes it possible to efficiently express the desired spherical particles.

Hereinafter, an example using an expression experiment of hepatitis B virus surface antigen (HBs antigen) and pre-S-HBs antigen will be described.
The present invention will be described in more detail.

Example 1: Expression of HBs antigen gene (1) Construction of a gene fragment for integration yeast construction (plasmid YIp7GR) Plasmid YRp7 [Gene, 10p-157 to 16 in which yeast-derived Trp1 gene was incorporated into the EcoRI site of plasmid pBR322]
6, (1980)], digested with the restriction enzyme PstI, treated with phenol and ethanol, dried, and then TE buffer (10 mM
Tris-HCl (pH 8.0, 1 mM EDTA) was suspended in 50 μl.

On the other hand, plasmid pAH301 having a structural gene for HBs antigen
[Pro. NAS USA Vol. 80 p1-5 (1983)] 1 μg was treated with restriction enzyme XhoI, treated with phenol and precipitated with ethanol, and then suspended in 20 μl of TE buffer. Furthermore, plasmid pYG100 consisting of yeast-derived GAP promoter, GAP terminator, 2 μori and leucine-producing gene (1eu2) and Escherichia coli-derived plasmid pBR322 [Experimental Medicine Vol.
o.11 136-141 (1987)] After treating 1 μl with the restriction enzyme SalI, phenol treatment and ethanol precipitation were carried out.
Resuspended in μl TE buffer. To 1 μl of this solution, add the above
5 µl of the solution DNA prepared from pAH301 was added, and 1 µl of T4 DNA ligase, 1 µl of 0.1 M ATP, and 0.1 M dithiothreitol 1
μl, 2μ of 10x concentrated buffer for nick translation
was added to make a total volume of 20 μl with sterilized water, and the mixture was reacted at 16 ° C. overnight. 1 μl of this reaction solution was transformed using Escherichia coli HB101, and the plasmid pYG100
Was screened for a plasmid pYG100s in which the XhoI fragment containing the HBs structural gene derived from pAH301 was incorporated into the SalI site of the plasmid pYG100s.

1 μg of this pYG100s was treated with the restriction enzyme HindIII, and finally suspended in 20 μl of TE buffer. To 10 μl of this reaction solution, 1 μl of DNA polymerase I, 1 μm of 2.5 mM dNTP
l, add 2 μl of 10 concentrated buffer for reaction, and make up the whole volume with sterile water.
After reaction at room temperature for 30 minutes in 20 μl, the reaction was stopped by heating at 70 ° C. for 5 minutes. After cooling, 1 μl of PstI linker, 1 μl of T4 DNA ligase, 0.1 M
Add 1 μl of ATP and 1 μl of 0.1 M dithiothreitol, and add
The reaction was carried out at 16 ° C. overnight with 30 μl. After the reaction, the mixture was heated at 70 ° C. for 5 minutes, cooled, and cooled with 3 μl of PstI buffer and PstI restriction enzyme.
Add 1 μl, react at 37 ° C for 1 hour,
After ethanol treatment, the suspension was dried under vacuum and suspended in 20 μl of TE buffer.

5 μl of this reaction solution was mixed with 1 μl of the PstI digestion reaction solution of the plasmid YRp7, and a ligation reaction was carried out in the same manner as described above. Using this reaction solution, Escherichia coli HB101 was transformed. From the Escherichia coli clones which became tetracycline resistant and ampicillin sensitive by this transformation, YRp7 PstI treated large fragment was converted to YRp7 PstI treated large fragment by Yst Select clones with integrated fragments. The plasmid thus obtained was designated as YIp7G.

On the other hand, plasmid pSF 2124 carrying the gene encoding yeast ribosomal RNA [The Journal of Biological
Chemistry vol.252 p8118-8125 (1977)]
Restriction enzyme HindIII (XmaI, XbaI or
BglI + II can be used) 1 μl, and 10 μl of a 10-fold concentration of HindIII buffer, add 100 μl with sterile water
and reacted at 37 ° C. for 1 hour. After the reaction, the mixture was treated with phenol and ethanol, and suspended in 20 μl of TE buffer. To this suspension, 3 μl of a 10-fold concentration buffer for nick translation (0.5 M Tris-HC1 pH 7.5, 0.1 M magnesium chloride, 10 mM dithiothreitol), 1 μl of DNA polymerase I (Klenow Fragment, manufactured by Takara), 2.5mM dN
After adding 1 μl of TP, making the volume 30 μl with sterilized water, the reaction was carried out at room temperature for 30 minutes. After the reaction, this solution was heated at 70 ° C. for 5 minutes to terminate the enzymatic reaction, immediately quenched, and then cooled to the aforementioned YIp7G
1 μl of a reaction solution obtained by cleaving the plasmid with the restriction enzyme PvuII was added, and 1 μl of T4 DNA ligase, 1 μl of 0.1 M ATP,
Add 1 μl of 0.1 M dithiothreitol, and add sterile water
The reaction was carried out at 16 ° C. overnight with 30 μl. After the reaction,
After performing phenol treatment and ethanol treatment and drying
It was suspended in 20 μl of TE buffer.

Using 1 μl of this reaction solution, E. coli HB101
The strain was transformed. In the same manner as above, a plasmid was prepared from the obtained colonies, and by judging from the results of agarose gel electrophoresis, the plasmid YIp7G
A plasmid in which a gene fragment encoding the ribosomal RNA gene derived from plasmid pSF2124 has been integrated into the uvII site.
YIp7GR (FIG. 1) was obtained.

(2) Preparation of host yeast From the yeast Saccharomyces cervisiae AH22 (Leu2His4), a Trp-requiring strain (Saccharomyces cer
evisiae AH22T).

Cut 5 μg of plasmid YRp7 with restriction enzyme HindIII,
After phenol treatment and ethanol precipitation, the cells were suspended in 16 μl of TE buffer. 2.5 mM NTP (dATP, dGTP, dCTP, dTTP) 1
μl, 2 μl of ligation buffer, and 1 μl of Escherichia coli DNA polymerase I (Klenow Fragment Takara), reacted at room temperature for 30 minutes, and heated at 70 ° C. for 5 minutes.
The polymerase was disqualified. Add 5 pM to 20 μl of this reaction solution.
Was added and a ligation reaction was performed. This was digested again with the restriction enzyme XhoI, and then treated with phenol and ethanol, followed by TE buffer.
The resultant was suspended in 20 μl to prepare a vector DNA solution.

On the other hand, plasmid YEp13 [Experimental Medicine Vol. 5, No. 11 p136
-141 (1987)], digested with restriction enzymes SalI and XhoI, and electrophoresed on 0.8% agarose to obtain Leu.
An XhoI-SalI gene fragment (small fragment) containing the two genes was separated, cut out from the gel, collected, precipitated with ethanol, and then suspended in 20 μl of a TE buffer.

XhoI-SalI gene fragment solution containing this Leu2 gene 10 μm
and 1 μl of the above vector DNA solution, and add T4D
After adding 1 μl of NA ligase, and making the total volume 20 μl with a ligation buffer, the reaction was carried out at 16 ° C. for 1 hour.
A transformant which became ampicillin-resistant was obtained by adding 100 μl of the 101 competent cell solution and using an ampicillin-containing medium. Plasmid DNA obtained from this transformant was
Rp7-Leu. Further, this DNA (YRp7-Leu) was digested with restriction enzymes BglII and SalI to obtain 20 μg of a gene fragment of about 3 Kbp in which the Leu gene was inserted between the Trp genes.

Yeast Saccharomyces cervisiae AH22R ^- strain and AH22R ⁺
Using this as a host, this DNA fragment was introduced and transformed using the lithium acetate method. The target transformant was selected using an agar selection plate in which tryptophan and histidine were added as amino acids to an MMP ⁺ medium (bulk holder minimum medium). The strain obtained in this manner is transformed into yeast Sacc
haromyces cerevisiae AH22T strain.

(3) Preparation of integrated yeast (introduction of plasmid YIp7GR into yeast AH22T strain) 20 μg of plasmid YIp7GR constructed in (1) above was digested with restriction enzyme KpnI to linearize DNA, and yeast AH22T produced in (2) above. Using the strain as a host, transformation was performed by the lithium acetate method. As the selection medium, the above-mentioned MMP ⁺ medium supplemented with histidine was used. On the other hand, the plasmid YIp7GR was not treated with the restriction enzyme KpnI, and the plasmid DNA as it was was also transformed.

(4) Screening of the integration yeast The transformed colonies on the selective medium plate obtained in the above (3) were transformed with 10 ml of YPD medium (1% yeast extract, 2% polypeptone, 2% D-glucose,
(pH 6.0) was inoculated into an L-shaped test tube and cultured at 37 ° C. with shaking for two days and nights. 5 ml of the culture solution thus obtained is collected by centrifugation, and glass beads (0.45 mm)
1 ml, lysis solution [50 mM potassium phosphate buffer (KPB)
(PH 7.5), 0.1% Triton X-100, 1 mM PMSF (phenylmethanesulfonyl fluoride)] and 1 ml of vibrating vigorously for 5 minutes to obtain a yeast solution. The supernatant obtained by centrifuging the lysate was subjected to a hepatitis B virus surface antigen (HBs) activity measurement reagent (Serocrit HBs kit: manufactured by Chemistry and Serum Therapy Laboratory) by rPHA (reverse passive hemagglutination). Was used to measure HBs antigen activity. This measurement is performed on serially diluted samples using a 96-well microtiter plate.
The limiting dilution factor indicating the agglutination reaction was determined.

As a result, the transformation method using linear gene fragments
A transformant of 55 clones was obtained.
2 ⁴ or more HBs antigen activity Transformants were obtained 7 clones that showed by RPHA. On the other hand, in the transformation method using the circular gene fragment, although 35 transformants were obtained,
it was not possible to obtain a strain which showed 2 ⁴ or more HBs antigen activity by RPHA.

From these results, it was confirmed that it is preferable to use a linear gene fragment when producing a transformed yeast (integrated yeast) in the present invention.

About 7 transformants thus obtained, YP
Passage was performed for 3 passages using D medium, and the stability was examined. As a result, three of these strains produced ²⁶ or more HBs antigens by the activity of r-PHA.

The chromosome of the thus obtained transformant (integrated yeast) was used to determine whether or not the introduced gene fragment was actually incorporated into the chromosome by pulse field electrophoresis (Pulsedfield gradient gel electrophoresis, [Cell 3
7, p67 (1984)]). As a result, it was confirmed that the gene fragment used for the transformation was integrated into chromosome 12 in which the ribosomal RNA gene forms a cluster, and further analysis by Southern blot revealed that there were 4 copies of these per genome. Was confirmed.

(5) Mutation of integration yeast (mutation) (5-A) EMS mutation Mutation of the above integration yeast in 30 ml of 10 ml YPD medium.
The cells are cultured at 20 ° C. for 20 hours, and when the OD650 of the culture reaches 1.0, the cells are collected by centrifugation. The bacterial mass obtained in this way corresponds to approximately 1 × 10 ⁸ cells. 1
After washing with 0 ml of sterile water, the cells were centrifuged again to collect the cells, and 1 ml of distilled water, 7.8 m of 0.2 M sodium phosphate (pH 8.0) solution
l, 1.0 ml of a 20% glucose solution and 0.2 ml of a 99% ethyl methanesulfonate (Albrich Chemical) solution were added and stirred vigorously. Shake this at 30 ° C for 60 minutes,
Centrifugation was performed at 3000 rpm for 5 minutes. Discard the supernatant and discard the resulting precipitate.
The suspension was sufficiently suspended in 10 ml of sterilized water. This centrifugal washing operation is performed 3 times.
By repeating 4 times, the remaining EMS was completely removed. 10 ml of YPD medium was added to the precipitate, and cultured at 30 ° C. for 6 hours.

The culture solution was diluted 100-fold, and 10 μl was spread on a YPD medium plate, and a high HBs expression strain was produced by the following method.

Transformant Screening Method As the screening method, the colony blotting method presented by Shioji et al. At the 8th Annual Meeting of the Molecular Biology Society of Japan was used with some modifications as described below. Hereinafter, this method will be briefly described.

When the colonies on the plate become about 0.5 mm,
Using this plate as a parent plate, a replica (replica plate) is made using a nitrocellulose membrane. This nitrocellulose replica plate was placed in a 0.1 M Tris-HCl buffer (pH 8.
0), 1 M sorbitol, 10 mM EDTA (pH 8.0), 24 mM 2-
Mercaptoethanol, 100μg / ml Simolyase 100
It is placed on 3MM paper containing a sufficient solution of T, and the reaction is allowed to proceed at 30 ° C. for 2 hours.

The solution was replaced with 3MM paper sufficiently containing a solution composed of 50 mM potassium phosphate buffer (pH 7.5) and 0.1% Triton X-100, and reacted at room temperature for 1 hour.

After the reaction, a TNB buffer (10 mM Tris-HCl buffer (pH 7.
5) Wash 5 times with 0.5 M NaCl, 0.1% Brigi-58) for 5 minutes, wash off the cells on the filter, and further
Wash with a solution of 1% BSA (bovine serum albumin) in buffer.

The HBs antigen particles derived from human serum was obtained by immunizing a rabbit anti-H there are HBs antigen activity of 2 ^{13-2 15} hemagglutination
Bs rabbit serum was diluted to 1 / 1,000 and reacted at 0 ° C. overnight. After washing this with a TNB buffer for 5 minutes and 5 times, using a Bexstein ABC kit peroxidase rabbit IgG (Funakoshi Pharmaceutical Co., Ltd.), the color is developed by the method described in the ABC kit, and colonies with strong color development are selected. I do.

The cells corresponding to the strongly colored colonies in the above method are scraped off from the parent plate, about 10 ³ cells of the cells are suspended in the medium, and the above colony screening is performed again using the cells. Thus, a transformant that produces high HBs antigen is selected through two screenings.

As a result of this mutation manipulation and screening, the HBs expression level was increased about 4-fold as compared to before the mutation treatment.

(5-B) NTG Mutation The transformant whose expression level was increased by the EMS mutation was further treated with NTG (nitrosoguanidine).

The mutant strain obtained by the EMS treatment in YPD medium 10 ml,
After culturing at 30 ° C. for 20 hours, when the OD650 of the culture solution reached 1.0, the cells were collected by centrifugation. This amount of cells corresponds to approximately 1 × 10 ⁸ cells. This was washed with a 0.2 M sodium acetate buffer (pH 5.0), and then sufficiently suspended in 10 ml of a 0.2 M sodium acetate buffer (pH 5.0) containing 2 mg / ml NTG. This was cultured with shaking at 30 ° C. for 30 minutes, and the remaining NTG was removed by washing three times with a YPD medium. 100 μl of a solution obtained by suspending the cells in 10 ml of YPD medium was applied to a YPD medium plate.

Screening of the thus obtained transformant was carried out in the same manner by the colony blotting method described above.

By this operation, a transformant having an HBs expression level four times higher was produced.

(5-C) Mutation of ultraviolet irradiation Irradiation of the transformant obtained as described above in 10 ml of YPD medium
And incubate at 30 ° C for 20 hours. OD650 of the culture solution is 1.0
Then, apply this to the YPD medium plate,
Next, irradiate with an ultraviolet lamp at a distance of about 30 cm for 60 seconds,
The cells were cultured in the dark at 30 ° C. for 2 days. The thus obtained transformant was screened by the aforementioned colony blot method.

(6) Production of HBs antigen By such three-step mutagenesis treatment, the HBs expression level of the obtained transformant increased as shown in Table 2 below.

The HBs protein produced by the integration yeast mutant thus obtained was expressed using a conventional autonomously replicating plasmid (JP-A-59-31799).
In the same manner as in (1), particles were formed, and the molecular weight was exactly the same as a result of analysis by Western blotting.

The position of the messenger RNA band was confirmed by Northern blotting, but was completely the same as that of the strain before the mutation treatment, confirming that the amount of transcription changed in proportion to the expression amount.

In addition, the transformant screened through such a mutation operation was used for the yeast Saccharomyces cerevisiac.
AH22T INT-S: Deposited as No. 9944 of Microbial Laboratory Bacteria.

Example 2: Co-expression by introduction of plasmid (1) Construction of expression plasmid Plasmids pYG100-M and pYG100-L [Imamura et al., Jo
urnal of Virology Vol.61 No.11 p3543-3549 (198
7)] with the restriction enzyme HIndIII to obtain a pre-S2-HBs (middle S) gene, which is a gene encoding the pre-S of the hepatitis B virus surface antigen [55 B at the 5 'end in addition to the HBs gene] A gene encoding an amino acid] and a pre-S1-HBs (large S) gene [a gene encoding 174 amino acids at the 5 'end in addition to the HBs gene] were prepared. The DNA polymerase I (Klenow large fr
agmemnt) to make blunt ends, and plasmid pAJ50 [Na
ture287, p869-871 (1980)] and blunt-ended BamHI sites, and ligated using T4 DNA ligase. Thus, the autonomously replicating plasmid pAJ50M-S (FIG. 2) and plasmid pAJ50L- for expression of the middle S gene and the large S gene were obtained.
S (FIG. 3) was prepared.

Similarly, the plasmid pYG1 used in Example 1- (1) was used.
By treating 00-S with the restriction enzyme HindIII, an HBs-expressing gene fragment encoding an HBs antigen consisting of 226 amino acids was prepared. This was similarly blunt-ended using DNA polymerase I, and HBs was inserted into the BamHI site of plasmid pAJ50.
An autonomously replicating plasmid pAJ50S (for a control experiment) for the purpose of expressing the HBs gene into which the expressed gene was integrated was prepared.

(2) Introduction of Expression Plasmid Using the integration yeast mutant (EMS-NTG-UV mutant) obtained after the three-step mutation treatment in Example 1 as a host cell, each of the above plasmids is introduced by the lithium acetate method. Was transformed. Transformants were selected using 1 mg / ml G418 neomycin derivative (GIBCO
This was performed by using a YPD medium containing GENETICIN) and selecting a yeast transformed with neomycin resistance.

As a control test, Saccharomyces cerevisiac A
Using the H22 PHO80 strain (Microtechnical Research Laboratories No. 509) (10 ⁷ cells), each plasmid was similarly introduced and transformed.

(3) Analysis of Hybrid Antigen Particles of the Present Invention Plasmids pAJ50-S, pAJ50M-S and pAJ50L-S were introduced into the integration yeast mutant strain of the present invention, and a transformant obtained by selecting with neomycin (G418) was used.
Each was cultured in one YPD (containing 1 mg / ml G418) medium, and about 30 g of each bacterial cell was obtained by centrifugation. The cells were treated with a solution consisting of Zymolyase 100T (Zymolyase 100T, manufactured by Seikagaku Corporation) (100 μg / ml), 14 mM 2-mercaptoethanol, and 50 mM phosphate buffer (pH 7.2) to give sphero. Plastify, then 0.1% Tri
ton X-100, 50 mM phosphoric acid (pH 7.2) and 1 mM PMSF (phe
The expressed antigen particles were eluted with a lysate consisting of nylmethylsulfonyl fluorido). The precipitate was removed by centrifuging the yeast extract thus obtained. In order to purify the target antigen particles, the extract was subjected to 5% to 20% sucrose density gradient centrifugation and cesium chloride density gradient centrifugation to purify the antigen particles. Each of the three yeast extracts thus purified is an antigen particle exhibiting 1.2 g / cm ³ by cesium chloride density gradient centrifugation. When this fraction was observed under an electron microscope by a negative staining method, it was observed that it was the same spherical particle as the Australian antigen in the serum.

In order to prove that the expression product of the present invention is a hybrid antigen particle, the expression product of each of the expression products was analyzed by combining Western blotting with adsorption using human polymerized albumin and using a rabbit antibody against pre-S antigen. The constituent peptides were analyzed.

First, each yeast extract obtained above was directly analyzed by Western blot using a 15% SDS-polyacrylamide gel [A]. As an antibody for detecting a peptide having HBs antigen activity, an antibody (β-813F) obtained by immunizing rabbits with HBs antigen particles purified from human serum was used. The results are shown in FIG. Lanes and samples represent antigens prepared from transformants obtained by introducing plasmids pAJ50M-S, pAJ50-S and pAJ50L-S into the HBs antigen-producing integration yeast produced in this Example, respectively. is there. Lanes are samples of an HBs production integration yeast extract that does not incorporate an autonomously replicating plasmid. The antigen obtained from was used as a sample.

According to the results shown in FIG. 5, the antigen (lane and) obtained from the transformant using AH22T INT-S as the host contained an HBs peptide having a molecular weight of about 23 kDalton. 33k depending on the plasmid
Of the medium S peptide (lane) and the large S peptide (lane). On the other hand, only the band of the middle S peptide (lane) or the large S peptide (lane) was detected from the antigen obtained from the transformant using AH22 Pho80 as the host, depending on the introduced plasmid.

Next, a microtiter plate for ELISA coated with human polymerized albumin was prepared, and the same six kinds of samples as described above were added in 50 μl portions. After incubation for 1 hour, the plate was washed with a Tris buffer containing Tween 20 (0.05%). (TBS: Tris-buffer-saline), and the antigen complex was recovered with a denaturing buffer for electrophoresis containing SDS-2ME (2-mercaptoethanol). After performing electrophoresis by gel, Western blot was performed using an antibody against HBs, and the result [B] is shown in FIG. Each lane corresponds to the same antigen sample as the Western blot sample of [A].

Furthermore, peptides 120-153 of the pre-S region (pre-S
ELISA coated with rabbit antibody against S2 region)
Similarly, six kinds of samples were subjected to an adsorption reaction using a microtiter plate, collected, and subjected to the same Western blotting. The result [C] is shown in FIG. Each lane corresponds to the same antigen sample described above.

Such a method for purifying antigen particles and a method for Western blotting using immunoadsorption are described in detail in a report by Imamura et al. [Journal of Vir.
ology, Vol.61, No.11, p3643-3549 (1987)].

It is said that the binding activity to polymerized albumin is localized to the pre-S moiety. In the above reaction system, antigen particles consisting of only HBs (major S) antigen cannot form an antigen complex with polymerized albumin and pre-S antibody,
No peptide having HBs antigen activity was detected (B, C lanes). However, in the case where major S and middle S were co-expressed (lane) and the case where major S and large S were co-expressed (lane), an antigen complex was formed only with the activity against pre-S and the antibody and recovered. Nevertheless, in addition to the band of pre-S-HBs peptide (33K or 43K dalton),
A major S peptide band (23 K daltons) containing no was detected at almost the same level.

From these results, it is considered that the antigen particles of the present invention coexist with middle S (33K) and major S (23K) or large S (43K) and major S (23K) via the lipid membrane of the particles. Can be Lanes in Western blots B and C are positive controls indicating that the system for detecting the pre-S region used in the examples is reliable, and lanes are negative controls for confirming the specificity for pre-S.

From the results of Western blotting and electron microscopy described above, it was confirmed that the product expressed according to the present invention formed a spherical particle together with the major S antigen in each case.

[Brief description of the drawings]

FIG. 1 shows the structure of a plasmid YIp7GR used for producing an integration yeast. FIG. 2 shows the structure of the preS2-HBs expression plasmid pAJ50M-S. FIG. 3 shows the structure of the preS1-HBs expression plasmid pAJ50L-S. FIG. 4 is a schematic diagram showing the relationship between a major S (HBs) peptide and a pre-S peptide. FIG. 5 shows the results of Western blot analysis of each antigen particle expressed in Example 2. FIG. 6 shows the results of Western blot analysis of the adsorbed sample after performing immunoadsorption treatment on each antigen particle expressed in Example 2 using polymerized albumin. FIG. 7 shows that each antigen particle expressed in Example 2 was subjected to immunoadsorption treatment using an anti-pre-S2 antibody,
The result of having analyzed the adsorbed sample by Western blot is shown.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication (C12P 21/00 C12R 1: 865)

Claims (10)

(57) [Claims] 1. A hybrid antigen comprising a HBs polypeptide and a fusion polypeptide in which a foreign peptide is bound to the N-terminus of an HBs polypeptide or a partial polypeptide thereof, which is expressed from a transformed yeast. particle. 2. The hybrid antigen particle according to the above (1), wherein the foreign peptide is a protein constituting a virus or a partial peptide thereof. 3. The hybrid antigen particle according to claim 2, wherein said exogenous peptide is hepatitis B virus pre-S1 peptide. 4. The hybrid antigen particle according to (2), wherein said exogenous peptide is hepatitis B virus pre-S2 peptide. 5. An integrated yeast in which an HBs antigen-expressing gene has been integrated into a yeast chromosome gene,
N of the HBs polypeptide or a part thereof
A hybrid consisting of an HBs antigen and a foreign peptide, which comprises culturing a transformant obtained by introducing an autonomously replicating expression plasmid into the yeast, which expresses a fusion polypeptide having a foreign peptide bound to its end. Production of antigen particles. 6. The method for producing hybrid antigen particles according to the above (5), wherein the exogenous peptide is a protein constituting a virus or a partial peptide thereof. 7. The method for producing hybrid antigen particles according to (6), wherein the exogenous peptide is an HB pre-S1 peptide. 8. The method for producing hybrid antigen particles according to (6), wherein the exogenous peptide is an HB pre-S2 peptide. 9. The HBs antigen-expressing gene to be integrated into a chromosome,
(5) The method for producing hybrid antigen particles according to the above (5), which is a gene fragment having a gene encoded on a yeast chromosome at both ends and having an HBs antigen expression gene therebetween. 10. The method for producing hybrid antigen particles according to (9), wherein the gene encoded on the yeast chromosome is a gene encoding yeast ribosomal RNA.