JPH0552190B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to the production of a herpes simplex virus protein useful for the production of a vaccine for the prevention of herpes simplex virus infections by applying genetic engineering technology to enhance the desired protein. The present invention is intended to be produced with high purity, and relates to a recombinant pramid into which a herpes simplex virus gene has been integrated, and to a transformed yeast obtained by transformation using the recombinant pramid. More specifically, we use a so-called shuttle vector that can grow in both Escherichia coli and yeast, and downstream of the inhibitory acid phosphatase expression regulatory region (hereinafter referred to as acid phosphatase promoter or acid phosphatase gene) carried by the vector. Herpes simplex virus (hereinafter referred to as
A recombinant plasmid incorporating the HSV (abbreviated as HSV) gene, especially the HSV gB gene, was obtained, and this was given to yeast to cause transformation to produce transformed yeast.
The present invention relates to a method for mass-producing HSV proteins, particularly HSV membrane protein gB, with high purity by culturing this yeast under normal culture conditions or under conditions in which the acid phosphatase promoter is not suppressed. Technical background and conventional technology Recently, the number of people with antibodies against HSV has been decreasing in developed countries, and in such countries,
HSV infections such as genital herpes, neonatal herpes, and herpes encephalitis are becoming a major problem.
Vaccines are effective in protecting against such HSV infections, and attenuated vaccines made of attenuated HSV and inactivated vaccines containing HSV DNA have already been proposed. However, HSV is known to have the risk of latent infection and carcinogenicity, and conventional attenuated vaccines and inactivated vaccines pose a practical problem because of the risk of such side effects. On the other hand, when HSV infects cells, several types of glycoproteins (gB, gC, gD, gE, etc.)
The name gB was the 1983 International Herpes Virus.
Workshop (Oxford, UK) produced gB, and these glycoproteins
It has been revealed that these glycoproteins are important as protective antigens against HSV infection, and research is being conducted on so-called component vaccines containing these glycoproteins as components. For example, Capel et al. HSV
It has been reported that glycoproteins extracted from infected cells or virus particles are effective as infection-protective antigens [Cappel el al., Arch. Virol.
73, 61 (1982)]. However, such component vaccines consisting of glycoproteins extracted from HSV-infected cells or virus particles contain many host cell proteins, and therefore side effects caused by these unnecessary proteins are a problem. Therefore, in order to obtain a component vaccine without side effects, it is necessary to use highly purified glycoproteins. From this perspective, the present inventors focused on gB, a type of HSV glycoprotein, highly purified it, and confirmed its effectiveness in experiments on mice [Jono, Department of Cell Engineering , 3 , 120 (1984)]. By the way, in order to obtain such glycoprotein gB, a method is adopted in which a virus is inoculated into cultured cells and the virus is then cultured to produce it. However, in such a method, the manipulation is difficult because it deals with an infectious agent. It is difficult and the process is extremely complicated, and it is almost impossible to prove that the DNA carrying the oncogenic gene has been completely removed. Producing component vaccines is extremely difficult. Under the above circumstances, the present inventors
We thought that if we could isolate the gB gene and express it in yeast, we would be able to obtain a safe vaccine that does not contain oncogenes.As a result of extensive research, we isolated the desired gB gene and expressed it in yeast. A new recombinant DNA is prepared by integrating the HSV gene under the control of the phosphatase promoter into a specific plasmid vector containing the gene and the E. coli gene and carrying the yeast inhibitory acid phosphatase gene. , succeeded in producing the desired HSV protein (i.e., HSV gB) by transforming yeast and culturing such transformed yeast, and has already filed a patent application (see Japanese Patent Application No. 151766/1983). ). Purpose of the Invention As a result of repeated research on the above-mentioned HSV gB gene integration plasmid, the present inventors discovered that restriction enzyme
The tail region of the gB gene obtained by treatment with Sac I was deleted, and the gene corresponding to approximately 90% of the total gB gene was inserted into the Sac I cleavage site of the plasmid vector carrying the above-mentioned repressive acid phosphatase gene. Prepare a recombinant plasmid that has been integrated with
When yeast was transformed using this, the HSV gB protein was expressed as a chimeric protein by culturing the transformed yeast, and the chimeric protein was transformed into gB.
The present invention was completed based on the knowledge that it has activity and can be used in herpes simplex virus vaccines and the like. That is, the present invention provides a method for deleting the tail region.
The present invention provides a novel recombinant plasmid incorporating the HSV gB gene and transformed yeast using the novel recombinant plasmid. The recombinant plasmid of the present invention uses a shuttle vector that contains genes from both E. coli and yeast and can be propagated in either of them. HSV that has deleted its tail region after removing part or all of it or even a certain part upstream of it
Obtained by integrating the gB gene. A desired transformed yeast can be obtained by allowing the thus obtained HSV gene expression plasmid to act on yeast to cause transformation by a conventional method. By culturing this transformed yeast preferably under conditions in which the acid phosphatase promoter is not suppressed, the desired HSV chimeric protein can be stably mass-produced. Below, the recombinant DNA of the present invention, the transformed yeast, and the production of HSV chimeric protein using the same will be explained in more detail. (1) Cloning and nucleotide sequence of HSV gB gene-containing fragment The location (0.348 to 0.366 map units) of the gB gene of HSV (KOS strain) on the viral DNA and its nucleotide sequence have been determined by Buchik et al. (David J. .Bjik et al., Virology. 133 , 301-314
(1984)). The HSV gB gene to be integrated into the shuttle vector used in the present invention is obtained by converting HSV DNA with restriction enzymes.
Approximately 8Kb (0.345~
0.399 map unit) fragment (BamHI
-G fragment). Preparation of HSV gB gene-containing fragment is
This is carried out by cloning the BamHI-G fragment obtained by cleaving HSV DNA with the restriction enzyme BamHI, as described below. Viral DNA is isolated from HSV grown in Vero cells (African green monkey kidney cells), this viral DNA is cut with the restriction enzyme BamHI, and the BamHI-G fragment is separated and extracted by agarose electrophoresis. This BamHI-G fragment is subjected to a ligation reaction with E. coli plasmid pBR322, which has been previously treated with BamHI, using _T4 ligase. E. coli χ1776 was transformed with this reaction solution, and among the transformants, ampicillin resistant (Apr) and tetracycline sensitive (Tcs)
By selecting and culturing the bacteria,
Plasmid pG containing the BamHI-G fragment is obtained. This plasmid pG has the structure shown in FIG. Next, using this plasmid pG as a material, the base sequence of the SmaI-SacI region (approximately 3.3 kb) containing the gB gene was determined using the dideoxy method.
Vol. 29, No. 4, 294-306 (1984)). In other words, as shown in Figure 2, SmaI
- Divide the SacI fragment into small fragments using various restriction enzymes, and separate each fragment (←→ mark) into M13 for base sequencing.
Insert into a vector (Pharmacia Japan KK) and perform sequencing. As a result, in the SmaI-SacI region,
As shown in E, there is a 2712 bp open reading frame (ATG to TGA), which encodes 903 amino acids. When this base sequence is compared with the base sequence of Buchiku et al.
Six bases differ, and two differ at the amino acid level. The nucleotide sequence results show that basic amino acids are localized in the tail region of the gB gene. Therefore, in order to express the gB gene in yeast, the region excluding this tail region is designed to be linked downstream of a promoter for expression in yeast (acid phosphatase promoter). That is, the above plasmid pG was digested with the restriction enzyme SacI.
A 2.7kb fragment (referred to as the SacI fragment) with the tail region removed is obtained. This gene contains approximately 90% of the gB gene and encodes 816 amino acids. In other words, ATG (Met) in line A4 of Figure 3
It includes the base sequence from CTC (Leu) at the bottom of Figure 3D, which encodes the 816th amino acid Leu (see Figure 6). This is used to construct the HSV gB gene expression plasmid described later. (2) Shuttle Vector The shuttle vector used in the present invention is a plasmid vector that contains a yeast gene and an E. coli gene, and carries the yeast inhibitory acid phosphatase gene. These yeast genes generally include DNA sequences necessary for plasmids to propagate independently of chromosomes in yeast, such as those necessary for autonomous reproduction in yeast.
DNA sequence (ars1) and 2μm Required for DNA replication
There is a DNA sequence (2 μori) and, if desired, a gene that serves as a selection marker for transformed yeast. This selection marker includes a leucine-producing gene, a histidine-producing gene, a tryptophan-producing gene, a uracil-producing gene, an adenine-producing gene, and the like. One or more of these may be used. The genes on the E. coli side include the DNA sequence necessary for the plasmid to proliferate within the E. coli body;
For example, the replication origin DNA of ColEI-based plasmids
It preferably further contains a gene that serves as a selection marker for transformed E. coli. The selection marker genes include ampicillin resistance gene,
Examples include a kanamycin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, and one or more of these genes may be used. As such E. coli DNA, pBR322, which has an ampicillin resistance gene and a tetracycline resistance gene, is generally used. The shuttle vector used in the present invention is characterized by carrying a yeast repressible acid phosphatase promoter, and this acid phosphatase promoter normally contains 60,000 molecules that constitute phosphatase.
It is the promoter of Dalton's polypeptide (p60). A typical example of such a shuttle vector is
It is disclosed in Japanese Patent Application Laid-Open No. 59-31799. A shuttle vector that combines yeast DNA with ars1, 2μori, and leucine resistance gene (Leu2) as yeast genes and E. coli plasmid pBR322.
From pAT77, part or all of the acid phosphatase structural gene and an appropriate site upstream up to -100bp, usually +1 to -100bp,
Preferably, it is a plasmid with +1 to -50 bp deleted, such as shuttle vector pAM82 with -33 bp deleted. This pAM82 has an XhoI site downstream of the acid phosphatase promoter, and this site contains
HSV gB gene of the present invention (SacI fragment:
2.7kb), this pAM82 was cut with the restriction enzyme XhoI, converted to a blunt end using a DNA polymerase reaction, and SacI
A linker is attached and recircularization is performed to obtain plasmid pAM82 (Sac) having the structure shown in FIG. This plasmid pAM82 (Sac) is a shuttle vector that can express foreign genes in pure form under the control of the acid phosphatase promoter, and its integration site can be easily cleaved by treatment with the restriction enzyme SacI. , a DNA fragment with a SacI site can be inserted at that location. (3) Construction of HSV gB gene expression plasmid The recombinant plasmid of the present invention, that is, the plasmid incorporating the HSV gene except for the tail region, is prepared by first using the shuttle vector pAM82 (Sac).
is treated and cleaved with the restriction enzyme SacI, and the above-mentioned HSV gB gene (SacI fragment 2.7 kb) is allowed to act on this to ligate. This was amplified in E. coli and a recombinant plasmid with the structure shown in Figure 5 was created.
Obtain pAMGB1. In this plasmid, the gB gene is linked to the downstream of the acid phosphatase promoter, and the gB gene encodes 816 amino acids, followed by 4 more amino acids in the same frame using the base sequence on the vector side. is coded, then
The frame stops at TGA, resulting in a total of 820 amino acids being encoded (see Figure 6). (4) Transformation of yeast The yeast to be transformed is a mutant strain that has a mutation that is complemented by the selection marker gene of the transformed yeast carried on a plasmid, such as a leucine auxotrophic mutant strain, Satucharomyces.・Saccharomyces cerevisiae AH22 or AH22pho80 (a leu 2 his4 Can1, Ci ⁺ )
Use. After propagating the above recombinant plasmid in Escherichia coli, it is allowed to act on the yeast mutant strain in a conventional manner to, for example, become sphaeroplus, and then the calcium-treated bacterial cells and plasmid DNA are mixed to cause transformation. Transformed yeast are selected and isolated using the expression of a gene complementary to the mutation in the host yeast carried on the vector, such as a leucine-producing gene, as an indicator. In addition to leucine auxotrophic mutants, examples of yeast include histidine auxotrophic mutants, tryptophan auxotrophic mutants, lauyl auxotrophic mutants, adenine auxotrophic mutants, and the like. (5) Cultivation of transformed yeast and production of HSV gB The transformed yeast obtained by the above method was precultured under normal culture conditions in a medium containing phosphoric acid, and the cells in logarithmic growth were Transfer to a medium that does not contain , and culture under conditions that do not inhibit the acid phosphatase promoter. After culturing, the bacteria are collected by a conventional method and subjected to bacteriolytic treatment to obtain a lysate containing a large amount of the desired HSV chimeric protein. Depending on the type of yeast used, for example, pho80
When a mutant strain is used, there is no need to use conditions that do not suppress the acid phosphatase promoter, and the transformed yeast can be directly cultured to obtain the desired result.
HSV chimeric protein can be produced in large quantities. The chimeric protein obtained by the above culture is purified by a conventional protein purification method. For example, anti-gB
The gB-containing extract is passed through a column packed with antibody-bound gel and eluted with 3MKSCN to isolate the chimeric protein. The HSV chimeric protein obtained by the above method is
It is immunologically identical to natural gB obtained from HSV-infected cells and can be used as an HSV vaccine or diagnostic reagent. Next, the production of the recombinant plasmid, transformed yeast, and HSV chimeric protein of the present invention will be explained in more detail with reference to Examples. Example (1) Cloning and base sequence of HSV gB gene-containing DNA (i) Preparation of HSV-1 DNA Vero cells (African green monkey kidney cells) (approx.
Herpes ^simplex virus type 1 (HSV
-1) (KOS strain) was infected at an amount of 0.5 to 1 PFU/cel1, cultured at 37℃ for 20 to 24 hours, and when cell degeneration had sufficiently occurred, centrifuged at 28,000 rpm for 1 hour to separate the infected cells. The supernatant is separated to obtain a pellet (2-3 ml) of infected cells. Add this to 6.0ml of phosphate buffer-physiological saline (PH7.2) (hereinafter abbreviated as PBS).
This suspension was sonicated (9KHz,
200W, 5 minutes) or freeze-thaw (-50℃
Cells are disrupted by (acetone-dry ice) and repeated freezing and thawing three times at 37°C), and cell debris is removed by low-speed centrifugation (3000 rpm, 20 minutes). The resulting solution is layered on a glycerol cushion (50%, 40%) and centrifuged at 35,000 rpm for 1 hour. PBS1 0.5-1ml of the obtained pellets
Suspend in ~2 ml and add DNase 10 μg/ml and
RNase 0.3mg/ml was allowed to react at 37°C for 1 hour, then 1/5 volume of 5×STEP (0.5% SDS, 50mM Tris
-HCl, PH7.5, 0.4MEDTA, 0.1% proteinase K) and let it act at 50°C for 30 minutes. This treated solution is sequentially extracted with equal amounts of phenol, phenol-chloroform (1:1), and chloroform to obtain an aqueous layer containing DNA. This aqueous layer was dissolved in TE buffer (20mM Tris-HCl,
After dialysis against 1mM EDTA, PH7.5), add cold ethanol to precipitate the DNA. This DNA was taken, dried under vacuum, dissolved in 5 ml of cesium chloride aqueous solution (Rf1.3885, added with 0.04% ethidium bromide, and 0.4% lauroyl sarcosinate), centrifuged at 4000 rpm for 72 hours, and HSV-
Form a band of 1DNA. This band is collected, washed with isopropyl alcohol to remove ethidium bromide, and then dialyzed against TE buffer.
Add cold ethanol to this to precipitate HSV-
Obtain 1 DNA. (ii) Cloning of BamHI-cleaved G fragment of HSV-1 DNA Approximately 100 μg of HSV-1 DNA prepared by the above method
, 73mM Tris-HCl (PH8.0), 7mM _MgCl2 ,
In 0.75ml of a mixture of 100mM NaCl and 2mM 2-mercaptoethanol at 37°C with the restriction enzyme BamHI.
After treatment for 6 hours, each fragment is separated by 0.7% agarose electrophoresis, the portion of the gel corresponding to the G fragment (0.345 to 0.399 map units) is cut out, and the G fragment is recovered electrophoretically. 1/10 molar amount of pBR322 plasmid cleaved with the restriction enzyme BamHI in the same manner as above and the above G
Approximately 2 μg of the fragment was mixed with 50 mM Tris-HCl,
React at 16° C. for about 16 hours using T ₄ DNA ligase in a mixture of pH 7.9, 10 mM MgCl ₂ , 20 mM dithiothreitol, and 1 mM ATP. Prepare a culture solution of Escherichia coli χ1776 by the method described in "Gene Manipulation Experimental Methods", edited by Yasutaka Takagi, page 161, add the above reaction solution to 0.1 ml of the culture solution, and mix well.
After standing at 0℃ for 45 minutes, ampicillin 100μ
Spread on agar plates containing 1.5 g/ml and incubate overnight at 37°C. The colonies that appear are plated on agar plates containing 100 μg/ml of ampicillin and 100 μg/ml of tetracycline, and cultured in the same manner to select colonies that grew only on the agar plates containing ampicillin. pBR322 has an ampicillin resistance gene and a tetracycline resistance gene, but the BamHI site in the tetracycline resistance gene is
Since the tetracycline resistance is abolished by insertion of the 1 DNA fragment, the selected colonies will retain the recombinant DNA of the BamHIG fragment of pBR322-HSV DNA. Plasmids are prepared from the colonies obtained by the above method according to the method described in "Preparation of Plasmid DNA" in "Metabolism" Vol. 17, No. 4, pp. 81-89 (1980). The obtained plasmid was digested with various restriction enzymes (BamHI, BstEII, KpnI,
By analyzing the cleavage pattern with SalI, SstI, XhoI), the BamHIG fragment of HSV-1 DNA was integrated into pBR322.
Obtain recombinant DNA of BamHI-G fragment (plasmid pG). (iii) Base sequence of HSV gB gene The base sequence of plasmid pG containing the above HSV gB gene is determined as follows. Dissolve 10 μg of plasmid pG in 100 μg of restriction enzyme reaction mixture of Sma I and Sac I, and add restriction enzyme to this.
The gB was treated with 10 units of SmaI and 10 units of SacI.
A fragment of the SmaI-SacI region containing the gene is obtained. This SmaI-SacI fragment was mixed with various restriction enzymes, SacI, SacII,
Using SmaI, PstI, SalI, PvuII, Sau3A and NarI, it is digested into small fragments as shown in Figure 2 (fragments marked with ←→).
(Sold by Japan Co., Ltd.) into the SalI site, and the dideoxy method (Proteins/Nucleic Acids/Enzymes, Vol. 29, No. 4, 294)
306 (1984) "Method for DNA Sequencing Using the Dideoxy Method"), the SmaI-SacI region was found to have a nucleotide sequence as shown in Figure 3 and encoded by it.
It was found to have the amino acid sequence of gB protein. This shows that the gB gene consists of 903 amino acids. (iv) Preparation of SacI fragment excluding the tail region 10 μg of plasmid pG obtained in () above,
6mM Tris-HCl (PH7.5), 6mM _MgCl2 ,
6mM2-meltocaptoethanol, 150mM
After reacting for 2 hours at 37°C in 100μ of a mixture of 10 units of NaCl and SacI, a 2.7kb DNA fragment (SacI fragment) containing about 90% of the gB gene was separated by 1% agarose electrophoresis in the same manner as above. Separate and extract. (2) Preparation of shuttle vector pAM82 (Sac) Using plasmid pAM82 prepared in the same manner as described in JP-A-59-31799, it was treated as follows to convert its XhoI site to SacI site. do. 2 μg of a fragment obtained by treating plasmid pAM82 with XhoI was added to 200 μM of dATP, dCTP,
67mM Tris-HCl (PH
8.6), 6.7mM _MgCl2 , 10mM2-mercaptoethanol, 6.7μM EDTA and 16.7mM ( _NH4 ) ₂
T ₄ DNA polymerase 0.1 in 50 µl of SO ₄
React with the unit at 37℃ for 30 minutes. After subjecting this reaction solution to phenol extraction and ethanol precipitation,
The resulting DNA and SacI linker are combined at a molar ratio of 1:10 using _T4 ligase at 16°C for 8 hours. Using this reaction solution, Escherichia coli plasmid χ1776 was transformed by the method described in "Gene Manipulation Experimental Methods" edited by Yasutaka Takagi, pages 161-162, and ampicillin-resistant bacteria were cultured, in the same manner as in the preparation of plasmid pGBX. , from the bacterial cells in the same manner as above.
Plasmid pAM82 (Sac) in which the XhoI site of pAM82 has been converted to a SacI site is isolated. (3) Preparation of HSV gB gene expression plasmid Plasmid pAM82 (Sac) obtained in (2) above,
1μg in 6mM Tris-HCl (PH7.5), 6mM
MgCl ₂ , 6mM 2-mercaptoethanol,
37 in 100μ of a mixture of 150mM NaCl, 10 units of SacI
℃ for 2 hours, and the reaction solution was subjected to phenol extraction and ethanol precipitation. 10ng of the fragment obtained above and the above (1)
100ng of the 2.7kb SacI fragment obtained in ()
and 66mM Tris-HCl (PH7.6), 6.6mM
_MgCl2 , 10mM dithiothreitol and 66μM
16 at 0.1 unit of _T4 ligase in 10μ of ATP mixture
℃ for 8 hours. Using this reaction solution, Escherichia coli plasmid χ1776 was transformed by the method described in "Gene Manipulation Experimental Methods" edited by Yasutaka Takagi, pages 161-162, ampicillin-resistant bacteria were selectively cultured, and ampicillin-resistant bacteria were selectively cultured. The HSV gB gene (approximately 90% of the total gB gene, 816 amino acids) with the tail region deleted downstream of the acid phosphatase promoter and the vector-derived gene (4 amino acids) were ligated downstream from the bacterial cell. Isolate the recombinant plasmid pAMGB1. (4) Preparation of transformed yeast Saccharomyces cerevisiae AH22 as yeast
[a leu2 his4 Can1 (Cir ⁺ )] (Feikoken Joyori No. 312
100% YPD medium (2% polypeptone, 1% yeast extract, 2% glucose).
ml, cultured overnight at 30°C, and centrifuged to collect bacteria. The bacterial cells are washed with 20 ml of sterilized water, then suspended in 5 ml of a solution of 1.2 M sorbitol and 100 μg/ml Zymolyase 60000 (manufactured by Seikagaku Corporation), and kept at 30° C. for about 30 minutes to form spheroplasts. The spheroplasts were then washed three times with 1.2M sorbitol solution, followed by 2M sorbitol,
10mM _CaCl2 and 10mM Tris-HCl (PH7.5)
Suspend in 0.6ml of solution and dispense 60μ aliquots into small test tubes. Add 10 μg of the recombinant plasmid pAMGB1 prepared in (3) above to this, mix thoroughly,
Add additional 0.1M CaCl ₂ (3μ) to final concentration of 10mM
Add _CaCl2 and leave at room temperature for 5-10 minutes. Next, add 20% polyethylene glycol 4000,
10mM _CaCl2 and 10mM Tris-HCl (PH7.5)
Add 1 ml of the solution, mix, and leave at room temperature for about 20 minutes. Add 0.2 ml of this mixture to a regeneration medium (22% sorbitol, 2% glucose, 0.7% yeast nitrogen-based amino acids,
2% YPD, 20 μg/ml histidine, 3% agar) 10
ml, mix gently and pre-prepared 1.2M
Sorbitol-containing minimal medium (0.7% yeast nitrogen-based amino acids, 2% glucose, 20μ
After layering on a plate (g/ml histidine, 2% agar) and solidifying, culture at 30°C to obtain colonies of leucine non-auxotrophic yeast. this colony
Bulk holder minimal edium containing 20 μg/ml histidine (Tohe, A. et al; J. Bachterol.,
113, 727-738 (1973)] to obtain the transformed yeast Saccharomyces cerevisiae. (5) Method for producing HSV gB using transformed yeast The transformed yeast colonies obtained in (4) above were further spread on a bulk holder minimal medium agar plate containing 20 μg/ml histidine,
Cultivate at 30°C to form colonies (to reconfirm transformants that have become non-leucine auxotrophic).
Next, bacterial cells were isolated from this colony and 20μ
The cells were inoculated into 10 ml of bulk holder minimal medium containing g/ml histidine, and cultured at 30°C. Approximately 24 hours later, the cells in the logarithmic growth phase are collected by centrifugation, and then transferred to phosphate-free minimal medium (contained in the bulk holder Minimal Medium).
(KH ₂ PO ₄ replaced with KCl and 20 μg/ml histidine added) Approximately 4 x 10 ⁶ bacteria per 10 ml
Suspend at a concentration of cells/ml, continue culturing at 30°C for about 24 hours, and collect the bacterial cells by centrifugation at 4000 rpm for 10 minutes. The cells were suspended in 3 ml of a solution containing 1.2 M sorbitol, 50 mM phosphate buffer (PH7.2), 14 mM 2-mercaptoethanol, and 100 μg/ml Zymolyase 60000, and gently shaken at 30°C for 30 minutes to form a spheroid. It turns into a plastic and collects it by centrifugation. The spheroplasts are suspended in 1 ml of 50 mM phosphate buffer (PH7.2) containing 1% Triton X-100, and glass beads are added and stirred to disrupt the bacterial cells. This crushing liquid
After centrifugation at 5000 rpm for 10 minutes, the gB antigen activity of the supernatant was measured by enzyme antibody method. The results are shown in Table 1. Furthermore, when five guinea pigs were subcutaneously inoculated with 1 ml of the above-mentioned disrupted solution four times every week, it was observed that neutralizing antibodies appeared in all the guinea pigs. 【table】

[Brief explanation of drawings]

Figure 1 shows the HSV DNA used in the present invention.
Structure of plasmid pG containing the BamHI-G fragment, Figure 2 is a restriction enzyme map of the region containing the gB gene, Figure 3 A to E is the base sequence of the HSV gB gene, Figure 4 is the structure of plasmid pAM82 (Sac). ,
FIG. 5 shows the structure of plasmid pAMGB1, and FIG. 6 shows the base sequence of the gB gene-vector binding portion of plasmid pAMGB1.

Claims (1)

[Scope of Claims] 1. A restriction enzyme is added to a plasmid vector containing a yeast-derived gene and an Escherichia coli-derived gene and carrying a yeast inhibitory acid phosphatase expression control region under the control of the phosphatase promoter.
A recombinant plasmid characterized in that it has integrated a herpes simplex virus gB gene fragment obtained by treatment with Sac I and deleted in the tail region. 2 Herpes simplex virus with deleted tail region
The recombinant plasmid according to item 1 above, in which the gB gene fragment has the following base sequence encoding 816 amino acids. [Table] [Table] [Table] 3 The inhibitory acid phosphatase expression regulatory region is the gene for the 60,000 Dalton prepeptide (P60) that constitutes phosphatase, and part or all of the structural gene or its upstream The recombinant plasmid of item 1 above, in which various sites have been removed. 4. The recombinant plasmid of item 1 above, wherein the yeast-derived genes are ars1 and 2μori. 5. The recombinant plasmid of item 1 above, which has ars1 and 2μori as yeast-derived genes and a gene that serves as a selection marker for transformed yeast. 6. The recombinant plasmid according to item 5 above, wherein the gene serving as the selection marker is one or more selected from a leucine-producing gene, a histidine-producing gene, a tryptophan-producing gene, a uracil-producing gene, and an adenine-producing gene. 7. The recombinant plasmid of item 1 above, which is a DNA sequence necessary for the E. coli-derived gene to proliferate within the E. coli body. 8. The recombinant plasmid of item 1 above, which is a DNA sequence necessary for the E. coli-derived gene to proliferate in E. coli and a gene that serves as a selection marker for transformed E. coli. 9. The recombinant plasmid according to item 8 above, wherein the selectable marker gene is one or more selected from ampicillin resistance gene, kanamycin resistance gene, tetracycline resistance gene, and chloramphenicol resistance gene. 10 A plasmid vector containing a yeast-derived gene and an Escherichia coli-derived gene and carrying a yeast inhibitory acid phosphatase expression regulatory region is treated with the restriction enzyme Sac I under the control of the phosphatase promoter. 1. A transformed yeast, which is characterized in that the yeast is transformed by allowing a recombinant plasmid into which a herpes simplex virus gB gene fragment is deleted from the tail region of the yeast to transform the yeast. 11 The yeast is a leucine auxotrophic mutant, a histidine auxotrophic mutant, a tryptophan auxotrophic mutant,
The transformed yeast according to item 10 above, which is one selected from uracil auxotrophic mutant strains and adenine auxotrophic mutant strains. 12 The yeast is a leucine auxotrophic mutant Satucharomyces cerevisiae AH22a 1eu2 his4 Can1
(Cir ⁺ ), the transformed yeast according to item 10 above.