JPH03218397A - Polypeptide and its production - Google Patents

Abstract

NEW MATERIAL:Polypeptides containing an amino acid sequence represented by the formula in its molecule. USE:A preventive and treating agent for herpes simplex virus (HSV)-infectious diseases. PREPARATION:For example, following the infection of Vero cells (African green monkey nephrocyte) with herpes simplex virus type-1 (HSV-1 type) Miyama strain, culturing the infected cells at 37 deg.C, subsequently separating virus by conventional method, extracting the DNA thereof, treating the extracted DNA with a restriction enzyme, binding the treated DNA to a vector and transforming colibacilli using the recombinant DNA, transformed colibacilli are isolated and cultured. The cultured transformed colibacilli are subjected to a cloning using a base segment of gB gene such as of a known HSV-1 type KOS strain as a DNA probe and positive clones are selected in order to extract DNA therefrom. The extracted DNA is treated with a restriction enzyme and bound to a vector to transform its host. The resultant transformant is cultured, thus obtaining the objective polypeptide containing an amino acid sequence of the formula.

Description

[Detailed Description of the Invention] The present invention relates to herpes simplex virus type 1 (hereinafter referred to as HSV).
The present invention relates to recombinant DNA for producing surface protein gB of Miyama strain (abbreviated as -1) and its derivatives.

Due to changes in the social environment and lifestyle, herpes simplex virus (HSV) infections such as herpes encephalitis and genital herpes have become a problem.

Most HSV infections are subclinical infections. This infected virus remains latent in the trigeminal ganglion, and sometimes becomes active and causes symptoms. Today, there is a desire to develop a vaccine to prevent this onset and prevent incubation.

Due to the latent nature of HSV and its suspected carcinogenicity, live and inactivated vaccines are inappropriate, and component vaccines are considered preferable. Glycoproteins gB and gD partially purified from HSV particles and HSV-infected cells have epitopes common to HSV-1 and -2 types.
It is a promising immunogen for vaccines. In fact, HSV-1
A component vaccine using gB purified from infected Vero cells has been reported to protect against acute HSV infection in BALB/c mice and subsequent latent infection in the trigeminal ganglion [Yoichiro Jono: Clinical and Virology, 12, 441 (1984)).

Recently, gB and gD have been prepared by genetic engineering techniques, and their potential as vaccine immunogens has been investigated. That is, P. W. Berman et al. [Science, 227.1490 (19
[84] reported that gD produced in Chinese hamster ovary cells protected against intravaginal HSV-2 infection in guinea pigs. Also, C. Nozaki
[Virus Res., 4
, 107 (1985)) demonstrate that gB produced in yeast protects against intraperitoneal infection with HSV type 11 in mice. Furthermore, recently Y. Kino et al. (Vaccine, 7, 155 (1989)) reported that this gB protects against HSV type 1 corneal infection in mice and HSV type 2 intracavitary infection in guinea pigs.

However, with the methods known so far, gB and gD obtained are in trace amounts and cannot be said to be at optimal expression levels.

The present inventors have used genetic engineering techniques to produce larger amounts of H.
The present invention was completed as a result of intensive research aimed at obtaining the surface protein gB of the SV-1 type Miyama strain.

7 As mentioned above, it has been desired to develop a technology to produce large quantities of HSV surface proteins.

The present inventors used the already known HSV-1 type KOS strain (DJ Bzik et al., Virol.)
, 133, 301 (1984)) and F strain (P, E, Pe
llett et al., Journal of Virology (J. Vi
ro1), 53, 243, (1985)) was synthesized as a DNA probe, and used to isolate the HSV-1 type Miyama strain [Nii, S. , & Ka
mahora, J. : Biken Journal
J. ), 4.75 (1961)).

The present invention is based on this cloning, and the base sequence of the gB gene analyzed this time and the amino acid sequence deduced from it are new. The present invention relates to a polypeptide contained in the gB gene, a recombinant DNA containing a base sequence encoding the polypeptide, a transformant carrying the recombinant DNA, and the production of the polypeptide by culturing the transformant. Regarding the law. That is, the present invention provides (1) a polypeptide (I) containing an amino acid sequence represented by the following formula in its -8 molecule: Ala Pro Ser Ser Pro Gly Thr Pro G
Val Ala Ala Ala Thr Gin
Ala Ala AsnGly Gly Pro Al
a Thr Pro Ala Pro Pro Ala
Pro Gly Pro Ala Pro ThrG
ly Asp Thr Lys Pro Lys Ly
s Asn Lys Lys Pro Lys Asn
Pro Pro Pro Pro Arg Pro A
la Gly Asp Asn Ala Thr Va
l Ala Ala Gly His Ala Thr
Leu Arg Glu His Leu Arg A
sp Ile Lys Ala lys Asn Th
r Asp Ala AsnPhe Tyr Val
Cys Pro Pro Pro Thr Gly A
la Thr Val Val Gln Phe Gl
uGln Pro Arg Arg Cys Pro
Thr Arg Pro Glu Gly Gln A
sn Tyr Thr GluGly Ile Ala
Val Val Phe Lys Glu Asn
Ile Ala Pro Tyr Lys Phe L
ysAla Thr Met Tyr Tyr Lys
Asp Val Thr Val Ser Gin
Val Trp Phe GlyHis Arg Ty
r Ser Gln Phe Met Gly Ile
Phe Glu Asp Arg Ala Pro
ValPro Phe Glu Glu Val Il
e Asp Lys Ile Asn Ala Lys
Gly Val Cys ArgSer Thr A
la Lys Tyr Val Arg Asn As
n Leu Glu Thr Thr Ala Phe
HisArg Asp Asp His Glu T
hr Asp Met Glu Leu Lys Pr
o Ala Asn Ala Ala Thr Arg
Thr Ser Arg Gly Trp His T
hr Thr Asp Leu Lys Tyr As
n ProSer Arg Val Glu Ala
Phe }Iis Arg Tyr Gly Thr
Thr Val Asn Cys Il■ Val Glu Glu Val Asp Ala A
rg Ser Val Tyr Pro Tyr As
n Glu Phe ValLeu Ala Thr
Gly Asp Phe Val Tyr Met S
er Pro Phe Tyr Gly Tyr Ar
gGlu Gly Ser His Thr Glu
His Thr Ser Tyr Ala Ala A
sp Arg Phe LysGin Val Asp
, (2) Recombinant DNA containing the base sequence encoding polypeptide (1) (II): (3) Recombinant DNA
(u) A transformant carrying: and (4) recombinant D
The present invention provides a method for producing polypeptide (1), which comprises culturing a transformant retaining NA (II), producing and accumulating polypeptide (1) in the culture, and collecting the polypeptide (1). be.

An example of polypeptide (1) is a polypeptide containing the amino acid sequence shown in FIG.

D containing the base sequence encoding polypeptide (1)
An example of NA is shown in Figure 1.
No. 341 to No. The part up to 2962 corresponds to the essential part.

Among the recombinant DNAs containing the base sequence encoding polypeptide (I) in the method of the present invention, for example, the expression vector can be prepared by (a) isolating genomic DNA from HSV-1 type Miyama strain, DNA with restriction enzyme Bam
Digest with HI, integrate the digested product into a plasmid that can be replicated in E. coli, (c) transform host E. coli with the obtained recombinant plasmid, (ii) culture the obtained transformant, and then transform from the transformant. A plasmid containing the desired DNA is isolated by an appropriate method, such as colony hybridization using a DNA probe, (e) the desired cloned DNA is excised from the plasmid, and (f) the cloned DNA is extracted if necessary. After subjecting the cloned DNA to restriction enzyme treatment, nuclease treatment, and addition reaction of an appropriate linker, the cloned DNA or the cloned DNA
A DNA encoding all or part of a signal peptide that operates in yeast is attached to the 5' end of NA in place of the gB original signal sequence, and is read downstream of the promoter in a foreign gene expression vehicle. It can be produced by connecting the frames so that they match.

The vector (eg, plasmid) used in the present invention may be any vector as long as it can replicate in the host. When the host is E. coli, for example, p B R3
22 [Sutcliffe, J. G. , Cold Spring Harbor Symposium
Harbor Symposium), 43.77 (1
A vehicle for foreign gene expression can be obtained by inserting a promoter into 979)). When the host is yeast, for example, psH19 (Harasima, S. et al., Molecular Cellular Biology (Mol.
l. Biol. ), 4, 771 (1984)), p.
sH19-1 (European Patent Application Publication EP-A-12
35430), and by inserting a promoter into these, a vehicle for foreign gene expression can be obtained. When the host is an animal cell, for example, p B R3
By inserting a promoter derived from SV40, a retrovirus promoter, etc. into 22, a vehicle for foreign gene expression can be obtained.

The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. If the host is E. coli, λ
P promoter, λPRL-12-promoter, trp promoter, tac promoter. T7 promoter etc. are preferably used.

If the host is yeast, use the GLD (GAPH) promo
one-tar, PH05 promoter, PGK promoter,
ADH promoter, GAP promoter, PH081 promoter, etc. are preferably used. When the host is an animal cell, examples include promoters derived from SV40 and retrovirus promoters.

Promoters can be enzymatically prepared from the corresponding gene. It can also be chemically synthesized.

An expression plasmid for expressing the DNA encoding polypeptide (I) can be obtained by inserting the DNA encoding polypeptide (1) downstream of the promoter of the above expression vector.

As mentioned above, the DNA shown in FIG. 1 is an example of this DNA.

When the host is E. coli, the expression vector is, for example, pTRP801 (Fujisa+ya,
Y et al., Nucleic Acids Research (NuC)
．． ACjdSRes. ), 11. 3581 (1983
)), p TB281 (Taniyama, Y.
et al., Journal of the Takeda Research Laboratories (
J. Takeda Res. Labs. ), 45, 1
36 (1.986)), pKK233-2 (Phar
macia LKB, Sweden). Examples of expression vectors when the host is yeast include pPHO17, pGLD906, and pGLD906.
-1, l: Itoh, Y. et al., Biochemical and Biophysical Research Communication (Bioc)
hem. Biophys. Res. Commu
n. ), 138, 268, (1986)
) can be given. When the host is an animal cell, expression vectors include, for example, pcDx (Pharmac
ia LKB, Sweden) and p MAMneo (T
oyobo Co, Ltd. , Japan). In this case, the expression level of the DNA encoding polypeptide (1) can be increased by inserting a terminator downstream of the DNA. As this terminator, for example, in the case of yeast, the phosphoglycerate kinase gene (PG
K), 2μDNA FLP gene, invertase gene (SUS2), and other terminators.

Methods for constructing the expression plasmids of the present invention are known and can be found in the literature, for example [Molecular Cloning (1982)].
, Cold Spring Harbor Laboratory (C01d
Spring Harbor Laboratory
) (1982)].

Examples of hosts that can be transformed with recombinant DNA (II) include E. coli, vinegar mother, animal cells, and the like. As E. coli, C600, MM294, N4830-1,
Examples include HMS174(DE3)pLysS.

Examples of yeast include Satsurikuchi Myces cerevisiae (
Saccharomyces cerevisiae)
AH22R-, NA74-3A (ρ-) +NA8
7-11. A. , DKD-5D, etc., but especially S. A respiratory-defective strain (ρ-) of S. cerevisiae is suitable as a host.

The method of obtaining a respiration-defective strain (ρ1) from a yeast having respiration ability (parent strain ρ+) is known, for example, [[Laboratory Course Manual Form-15- Sods in Yeast Genetics
ry Course Manual for Meth
od in Yeast Genetj. cS)”, Cold Spring Harbor Laboratory (Col.d
Spring Harbor Laboratory
), (1986): 1. That is, a respiratory-defective strain can be easily obtained by culturing the parent strain in a medium containing ethidium bromide and then isolating a strain that can grow in a medium containing glucose as a carbon source but cannot grow in a medium containing glycerol. Although it is rare, a respiratory-deficient strain can be obtained from a single colony of the parent strain. When the parent strain referred to herein is a transformed strain (recombinant) having an expression plasmid, a recombinant with a high expression level of the desired gene can be directly obtained by obtaining the respiration-defective strain. In addition, if the parent strain does not retain the expression plasmid, the desired recombinant can be obtained by obtaining a respiration-deficient strain and then introducing the expression plasmid into it.

Examples of animal cells include monkey cells COS-7, Ve
ro, Chinese hamster cells CH-16-O, mouse L cells, mouse myeloma cells, and human FL cells.

The method of transforming E. coli using an expression plasmid (recombinant DNA) is a method known per se, for example, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA),
69, 2110 (1972), Gene, 1.7, 1
07 (1982) and others.

The method of transforming yeast using recombinant DNA (plasmid) is a method known per se, such as the lithium method (H.
Ito et al., “Journal of Bacteriology (J
．． Bacterio1. ), 153, 163 (1983
)), protoplast method (A. tIinnen et al., Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci, U.
SA), 75, 1927 (1978)). Animal cells can be transformed, for example, according to the method described in Virology 52, 456 (1973).

The transformant (recombinant) thus obtained is cultured by a method known per se.

When culturing a transformant, a liquid medium is suitably used for culturing, and contains carbon sources, nitrogen sources, inorganic substances, and other substances necessary for the growth of the transformant. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn steep, etc.
Inorganic or organic substances such as liquor peptone, casein, meat extract, soybean meal, and potato extract; examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride.

Further, yeast, vitamins, growth promoting factors, etc. may be added.

The pH of the medium is preferably about 6-8.

As a medium for E. coli, for example, LB medium [Molecular cloning
g) (1982), Cold Spring H
arbor Laboratory), M9 medium containing glucose and casamino acids (Miller, Journal
Journal of Experiments in Molecular Fortress Genetics
nts in Molecular Genetics
), 431-433, Cold Spring H
arbor Laboratory, New York
1972) is preferred. If necessary, in order to make the promoter work efficiently, for example, IAA
(3β-indolyl acrylic acid), IPTG (isoprobyl-1-thio β-D-galacside) can be added.

Cultivation is usually carried out at about 15 to 43°C for about 3 to 24 hours, and aeration and stirring can be added if necessary.

As a medium for yeast, for example, Perkholder (B
urkholder) minimal medium [American Journal of Botany (Amer. J. Bot.), 30,
206 (1943)) or its modified medium [A. To
he et al., Journal of Bacteriology (J. Bac.
triol. ), IIL727 (1973)). or low phosphate medium (A. Tohe et al., Journal of Bacteriol., 1)
13, 727 (1973)). Cultivation is usually carried out at about 15°C to 40°C, preferably 24°C to 37°C for 10
-168 hours, preferably 24-72 hours. Shaking culture or static culture may be used, but aeration and stirring may be added as necessary. When culturing a transformant whose host is an animal cell, the medium may be, for example, MEM medium containing about 5 to 20% fetal bovine serum [Science 12
2,501 (1952)), DMEM medium [Viro-1ogy, 8,396 (1959)
), RP MI 16 40 medium [Journal of the American Medical Association (The Journal of the Ameri
can Medical Association)
199, 519 (1967)), 199 Medium [Procedure of the Society for the Biological Medicine (Pro-ceeding of
the Society for the Biol
73. 1 (19
50)].

Preferably the pH is about 6-8. Culture is usually about 30
It is carried out for about 15 to 60 hours at ℃ to 40℃, with aeration and stirring added as necessary.

According to the present invention, the peptide having HSV gB activity can be obtained using conventional protein extraction and purification methods, such as cell disruption using glass beads, centrifugation, salting out, isoelectric focusing, gel filtration,
It can be easily purified by appropriately combining purification steps such as ion exchange chromatography, high performance liquid chromatography, Affinity 201 chromatography, sucrose gradient ultracentrifugation, and cesium chloride gradient ultracentrifugation.

In the specification and drawings of this application, when bases, amino acids, etc. are indicated by abbreviations, IUPAC-IUB Comm
ision on Biochemical Nome
The abbreviations are based on nclature abbreviations or common abbreviations in the field, examples of which are listed below. Furthermore, when an amino acid may have optical isomers, unless otherwise specified, the L-isomer is assumed to be indicated.

DNA: Deoxyribonucleic acid cDNA: Complementary deoxyribonucleic acid RNA: Ribonucleic acid mRNA: Messenger RNA A: Adenine T: Thymine G: Guanine C: Cytosine dATP: Deoxyadenosine triphosphate dTTP: Deoxythymidine triphosphate dGTP: Deoxyguanosine triphosphate Acid dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate aty: Glycine (G) Ala: Alanine (A) Val: Valine (V) Leu: Leucine (L) I1e: Isoleucine (I) Sep: Serine (S) Thr: Threonine (T) Cys: Cysteine (C) 1/2C ys: Half cystine Met: Methionine (M) Glu: Glutamic acid (E) Asp: Aspartic acid (D) Lys : Lysine (K) Arg : Arginine (R) His: Histidine (H) Phe : Phenylalanine (F) Tyr : Tyrosine (Y) Trp : Tryptophan (W) Pro : Proline (P) Asn : Asparagine (N) Gin : Glutamine (Q) APr: Ampicillin resistance gene Tcr: Tetracycline resistance gene ARS l Autonomous replication sequence 1 (a
autonomous replica S6qu
ence 1 ) In the peptide of the present invention, a part of its amino acid sequence may be modified (addition, deletion, substitution with other amino acids, etc.).

The HSV gB obtained by the present invention has the same biological activity as HSV gB produced from HSV-infected cells, and can be used as a vaccine for the prevention of HSV viruses and as a material for diagnostic kits. -23- Can be used.

By the method of the present invention, HSV gB can be produced more cheaply and safely than ever before, and in large quantities.

The present invention will be explained in more detail with reference to the following Reference Examples and Examples, but the present invention is not limited thereto.

The transformant Saccharomyces cerevisiae obtained in Example 4 described below
isiae) NA74-3A (ρ1)/pHSB106
was given the accession number FERMBP- to the Microbial Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry on June 8, 1999.
2457, and the microorganism has been deposited with the Institute for Fermentation Research (IFO) since May 26, 1999 under the accession number I.
Deposited as FO 10470.

The transformant Saccharomyces cerevisiae obtained in Example 6 described below
isiae)NA74-3A(/)-)/pHsB10
6ΔTth was given accession number FER to the Microbial Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry on November 27, 1999.
The microorganism was deposited as 124-M BP-2667, and since November 17, 1999, the microorganism has been deposited at the Fermentation Research Institute (IF○).
It has been deposited with accession number IF○ 10491.

Saccharomyces cerevis carrying plasmid pGFE213 obtained in Reference Example 1 described below
iae NA 74-3A(ρ-)/pGFE213
was given the accession number FERM to the Research Institute of Microorganisms (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry on October 11, 1985.
The microorganism was deposited as BP-2095, and the microorganism was deposited with the Fermentation Research Institute (IFO) on September 19, 1988, under accession number IFO 10460.

Reference Example 1 Construction of Expression Vector Amino acid sequence of known egg white lysozyme signal peptide (Jung, A et al., Proceedings of the National Academy of Sciences (Proc. Natl.
．． Acad. Sci. USA. ,77, 575
9 (1980)) as shown in Figure 5-1.
A synthetic nucleotide sequence is used that has a site for binding to the cut end of XhoI and HinfI at the 3' end. The entire sequence consists of six oligonucleotide blocks (
#1, #2, #3, #4, #9, #10),
These are based on the phosphoamidide method (Caruthers
, M. H. Tetrahedron Letters (Te
trahedron Letters) 22.1859
(1981)). These oligonucleotides were ligated with T4 DNA ligase to encode an improved signal peptide and the N-terminal side of human EGF76.
A bp Xho1-Hinfl fragment was prepared. Plasmid pTB370 (Taniyama et al., “Journal of Takeda Research Laboratories (J. Takeda Res.
．． Lab. ) J, 45, 136 (1986), JP-A-61-88881), a 0.16 kb Hinf encoding human EGF with a partial deletion on the N-terminal side.
The I-Pstl fragment was isolated and the 76
bp Xho I-Hi n f I fragment to T4DN
After ligation with A ligase, treatment with Xhol and PstI resulted in a 0.24kb XhoI-Pstl.
The fragment was isolated. After ligating the Pstl-Smal adapter to this fragment using T4 DNA ligase, it was treated with Xhol and Sma I to create a 0.24 kb Xh o I-S encoding the improved signal peptide and human EGF.
The ma I fragment was isolated.

Human lysozyme gene expression plasmid pGEL 12
5 (Yoshimura, K. et al., Biochemical and Biophysical Research Communication (B
iochem. Biophys. Res. Com
mun. , 145, 712 (1987) After cleaving the XhoI site at the 3' end of the gene by partial digestion with XhoI, plasmid pERI8602 was ligated with a synthetic linker (5'TCGACCCGGG3') having a Small recognition site. This plasmid was cut with XhoI and Smal to create a DNA encoding the improved signal peptide and human lysozyme.
Remove the A fragment and replace it with the 0.24kb XhoI fragment above.
-Smal fragment was inserted to obtain plasmid PGFEIOI (Fig. 5-1).

PGK from plasmid pGLDP31-RcT (European Application Publication EP-A-0235430)
0.28kb including terminator Ah am-Xh
o I fragment was isolated and transformed into plasmid pSP
64 (Riboprobe 27, USA) into the Small-Sail site to obtain plasmid pSP 64-T (PGK). EcoRI-Pst containing the PGK terminator was extracted from the plasmid.
The PstI-Smal adapter and the Smal-EcoRI adapter were added to the PstI-Smal adapter and inserted into the Sma1 site of the above plasmid PGFE1 to obtain the human EGF secretion plasmid pGFE213 (Figure 5-2). .

Example 1 Virus DN of herpes simplex virus Miyama strain
Preparation of A V 0.0 to ero cells (African green monkey kidney cells)
5 to 0.5 PFU/ce 11 of herpes simplex virus type 1 (HSV-1) Miyama strain was infected, and 37
Cultured at ℃. When cell degeneration had sufficiently occurred, the suspended culture medium of the infected cells was collected, and the culture medium was centrifuged at low speed (3,000 rpm, 10 minutes) to separate the supernatant and the cells. The cells were frozen and thawed (-80°C and 37°C).
After disrupting the cells by repeating freezing and thawing three times at 0.degree. C., the supernatant was added in an amount equal to 10 times the number of cells. This mixture was centrifuged at low speed (3.000 rpm, 10 minutes) for 28 minutes.
After removing cell debris, the supernatant was added to the previously obtained supernatant. The supernatant was washed for 20 minutes. The cells were centrifuged at 000 rpm for 4 hours, the supernatant was removed, and a pellet containing virus particles was obtained. Add phosphate buffer to physiological saline to this pellet.
．． 8%NaCQ, 0.02%KCQ, 0.115%N
a 2H P O4, 0.02% KH 2 P O
4 (pH 7.2)] to prepare a virus suspension. First add 10p of DNasel to this solution.
g/mQ and RNAse A 0.3mg/m
Q was added and reacted for 1 hour at 37°C, then 175 volumes of 5XSTEP (0.5% SDS, 50mM Tri
s-HCQ, pH7.5, 0.4M EDTA,
0.1% proteinase K) was added and reacted at 50°C for 1 hour. Furthermore, this reaction solution was mixed with an equal amount of phenol-chloroform (1:1). The mixture was then treated with chloroform to obtain an aqueous layer containing DNA.

This aqueous layer was mixed with TE buffer (10 mM Tris
-HCQ (pH 8.0), 1mM EDTA) overnight.

Add cold ethanol to the dialysis solution and centrifuge (12.00
Orpm for 20 minutes) to remove the supernatant, air-dry the resulting precipitate (DNA), and add TE solution (10 mM T
r i s -HC Q , pH 8.0. 1mM
EDTA).

Example 2 Preparation of plasmid containing HSV-1 type Miyama strain DNA fragment 30 units of restriction enzyme BamHl (manufactured by Takara Shuzo Co., Ltd.) was added to about 2 μg of the obtained HSV-1 type DNA at 30 tons.
tQ reaction solution (lo+nM Tris-HCII (pH
8.0), 7mM MgCQ2, 100mM Na(i
ll, 2mM 2-mercaptoethanol] at 37°C.
After reacting for 1 hour, the protein was removed with phenol and precipitated with cold ethanol.

Approximately 100 ng of plasmid pBR 322, which was cleaved with the restriction enzyme BamHI in the same manner as above, and the above B
Approximately 200 ng of HSV-I DNA cut with amHI
20μQ of reaction solution (66mMTris
-HCQ (pH 7.6), 66mM MgCQ2,10
mM dithiothreitol, 1mM ATP, 300
Unit T4 DNA ligase [manufactured by Takara Shuzo Co., Ltd.],
The DNA was allowed to bind overnight at 16°C.

E. coli DH-1 was transformed using this reaction solution, and plasmid DNA was extracted from the ampicillin-resistant transformants (HSV-ID DNA library) by alkaline extraction [
T. Maniatis et al., Molecular Cloning (
Ao1ecular C]. oning) Spring
g Harbor Laboratory 366 pages (
(1982)). The plasmid DN
After cleaving A with the restriction enzyme BamHI, the buffer solution (
0.04M Tris-acetate, 0.002
M E D T A. (pH 8.0)), IO
OV. Electrophoresis was performed for about 1 hour.

DNA fragments from approximately 7.0 kb to approximately 9.0 kb were filtered through a nitrocellulose filter (S&S, BA85).
Transferred to the top [Southern mouthing method, T. Mania
tis et al., Molecular Cloning
larCloning) Spring Harbor
Laboratory, page 382 (1982)].

On the other hand, P. E. Pellett et al. [Journal of Biology (J. Virol., 53, 24
3 (1985)).
Based on the salt 31-base sequence of type 1 F strain gB protein, a base strand complementary to the base sequence corresponding to the N-terminus of gB protein (5' GGCGCCCTG
GCGCATGGC 3') and a base strand complementary to the base sequence corresponding to the central part (5'GCACCCAGT
CCCAGGCCACGGTGAACTT 3') was chemically synthesized. These two types of oligonucleotides were synthesized using T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.).
0μΩ reaction solution [oligonucleotide 0.1μg, 5
0 mM Tris-HCQ, pH 8.0, lomM
MgCQ2.10mM 2-mercaptoethanol, 50μCiγ-32pATP (75000C
The oligonucleotide was reacted for 1 hour at 37° C. in 3 units of T4 polynucleotide kinase (3 units T4 polynucleotide kinase), and the 5' end of the oligonucleotide was labeled with 32P.

Two types of oligonucleotide probes labeled by the above method were separately associated with the DNA-immobilized filter. The association reaction was performed using 6X SSC containing 10 μCi of probe.
(150mM NaCQ, 15mM sodium
citrate (pH 7.0)), 5XDen
The reaction was carried out in 3 mQ of a solution containing hardt solution, 0.5% 32-SDS, 10 mM EDTA, 100 μg/rnQ denatured salmon sperm DNA at 37°C for 16 hours, and after the reaction, the filter was filtered in 5X SSC, 0.1% SD.
Washed with S solution twice at room temperature and then twice at 40°C for 30 minutes each (T. Maniatis et al., Molecular Cloning S
pring l{arbor Laboratory
309 pages (1982)).

From the radioautography of the washed filter, approximately 8
．． It was found that a 1 kb DNA fragment reacted with two types of probes. This approximately 8,1 kb DNA fragment was digested with the restriction enzyme XhoI (manufactured by Takara Shuzo Co., Ltd.) to prepare an approximately 3.6 kb DNA fragment, and the base sequence of this fragment was determined using the dideoxynucleotide synthesis chain termination method. (JJBssing et al., Nuleic Acids Research,
9, 309 (1981)) (Fig. 1).

From the results, k. H
It was found that the entire amino acid sequence of the gB protein of the SV11 type Miyama strain was encoded. The amino acid sequence deduced from the above base sequence is shown in FIG. Although the nucleotide sequence is very similar to the gB protein nucleotide sequence reported for the F strain, there are 11 base substitutions (5 amino acid residue differences) and 3 base additions. [D. J
．． Bik et al., Virol., 133,
301 (1984)), addition of 3 bases and substitution of 28 bases (differences in 27 amino acid residues) were confirmed. From these results, it was found that the 3.6 kb DNA fragment was isolated from HSV-1.
It was found that it encodes the gB protein of type Miyama strain.

pBR322 into which the above 3.6kb DNA fragment was inserted
was named pHSB 200.

Example 3 The plasmid vector pHSB 200 containing the HSV-1 type Miyama strain gB gene prepared in Example 2 was digested with restriction enzymes Notl and BamHI to give approximately 3.1
A DNA fragment of kb was obtained. 45 with an XhoI site on the 5′ side and a NotI site on the 3′ side as shown in Figure 3.
A bp DNA fragment was chemically synthesized, and this and the Not
Approximately 3.1k by reacting l-BamHI fragment
The XhoI-BamHI fragment of b was obtained and reacted with the XhoI-BamHI digest of plasmid vector pHsG397 to subclon plasmid pHsG39.
7-g B 106 was produced.

This plasmid was digested with restriction enzymes Xhol and Nhel to prepare a DNA fragment of approximately 2.4 kb.

On the other hand, the plasmid vector pHSE200 containing the HSV-1 type Miyama strain gB gene prepared in Example 2 was digested with restriction enzymes Nhel and Sphl to obtain a 239 bp DNA fragment, and the Nhel-Sp of plasmid pB R322
The subcloning plasmid p.
BR322-gB239 was obtained.

After digesting this plasmid with restriction enzyme SphI, T4
It was treated with polymerase (Takara Shuzo Co., Ltd.) and further digested with restriction enzyme HinDm to obtain a 439 bp DNA fragment. A phosphorylated SacI linker d (P
CGAGCTCG), approximately 0.44 kb Hi
nd■-Sacl fragment was obtained and the plasmid vector pUc
18 HjndIII-Sac35-I digest, and subcloned plasmid pU
c18-gB0.44 was obtained. By digesting this plasmid with restriction enzymes Nhel and Sacl, approximately 0.2
A 4kb NheI and SacI fragment was obtained.

This DNA fragment is approximately 2.4 kb of xho1 prepared previously.
- The NheI fragment was transferred to the plasmid pGFE described in Reference Example 1.
gB gene expression plasmid pHSE106 was obtained by reacting with XhoI-Sacl digest of 213 (
Figure 4).

Example 4 The yeast Saccharomyces cerevisiae (Saccharomyces cerevisiae) was grown using the plasmid pHSB106 constructed in Example 3.
yces cerevisiae) A H 2 2
R1 and NA74-3Aρ1 were transformed, and the transformant Satsuriromyces cerevisiae AH22R/pHsB10
6 and N A 7 4. −3 Aρ−/ p H S
B 1 0 6 were obtained, respectively.

The obtained transformants were each cultured in the medium described in Example 15 of European Patent No. 36-Publication EP-A-0235430, and then the bacterial cells were collected. More specifically, the yeast transformant was grown in a 5-resistant culture medium [per IQ, K. HP 0.3g, glucose 30g, asparagine 4g, L-histidine 100nIg, KI O,lm
g, MgSO4.7H,0 500mg, CaCl.・
2H20330H, CuSO4・5H20 0.4+n
z, FeS0.7H. 0 2.5mg, MnSo4
・4H20 0.4mH, (NH4). P.O.・12
MoO,・3H20 0.2mg, Zn SO4・7H
20 3.1■, inositol 10■, thiamine 0.2
■, pyridoxine 0.2■, Ca-pantothenic acid 0.2
■, Niacin 0.2■, Biotin 0.002■) containing 2+
++1 in 18 ml fresh medium [per IQ, KH2F0
．． 300mg, L/sucrose 50g, asparagine 4
g + L-histidine 100■, KCI 1.5g
, K.I.O. 1mg, MgSO4・7H20 500
+ng, CaCl2・2H, 0 330■, glucose tog, tris-maleic acid (pH 6.5) 25m
M, Cu SO4・5 H,0 0.4ru,F
e SO4・7H20 2.5mg, Mn So4・4
H20 0.4m5, (NH.)3PO4・12Mo
O.・3 H20 0.2■, Z n SO
, ・7 H20 3.1mg, inositol 10■,
Thiamine 0.2■, pyridoxine 0.2■, Ca-pantothenic acid 0.2■, niacin 0.2■, biotin 0.
002■], cultured with shaking at 30°C for 2 days, and the bacterial cells were collected by centrifugation.

Approximately 200 mg of bacterial cells were added to sodium phosphate buffer [10
0mM sodium phosphate (pH 8.1), 7.4
Murea, 1% Triton X-100. 0.
Suspend in 500μ of 1mM (p-amidinophenyl)methanesulfonyl fluoride hydrochloride,
1 g of glass beads was added and mixed vigorously on a Vortex for about 20 minutes. A supernatant was obtained by centrifugation at 10,000 rpm for 5 minutes. Add to this extract an equal amount of twice the concentration of L.
aemmli buffer was added and heated at 100°C for 10 minutes. After cooling, it was centrifuged at 10,000 rpm for 5 minutes to obtain a supernatant. 40 μQ of the extract was added to SDS.
- Electrophoresed on polyacrylamide gels and electroblotted onto nitrocellulose filters. In this filter, DAKOPATTS anti-He
rpes virus type 1 (Maclrl
tyre) antibody (rabbit), then ho
React with rseradish peroxidase-labeled anti-rabbit antibody and develop development reag.
When color was developed using ENT (Bio rad), a specific band was detected.

Example 5 Construction of expression plasmid for truncated gB gene of HSV-1 type Miyama strain Subcloning plasmid pHS obtained in Example 3
G397-gB106 was digested with restriction enzymes XhoI and Baml to prepare a DNA fragment of about 3.1 kb, and this fragment was further digested with restriction enzyme Tthllll to obtain a DNA fragment of about 2.0 kb.
The Xh o I-Tt hllll fragment was obtained. A 14 bp DNA fragment having the stop codon shown in Figure 6 was chemically synthesized, reacted with the above Xhol-Tthllll fragment, and digested with restriction enzymes Sacl and Xhol to obtain an approximately 2.0 kb Xhol-SacI fragment. . This fragment was transferred to the plasmid vector pHsG397 XhoI-S
React with acl digest and subcloning plasmid pHSG397-gB106
ΔTth was prepared. About 2.0 kb of XhoISacI was obtained by digesting this plasmid with restriction enzymes SacI and xho1.
DNA fragment and plasmid pGFE21 described in Reference Example 1
Expression plasmid pHsB106ΔTth was obtained by reacting the Xhol-SacI digested product of 3 (IF○10460, derived from FERM BP-2095) (FIG. 6). Furthermore, the base sequence and deduced amino acid sequence of the obtained truncated g B gene are shown in FIG.

Example 6 Plasmid pHSB106Δ constructed in Example 5
Yeast Saccharomyces cerevisiae (Sacch)
aromyce Scerevisiae) N A 7
4-3Aρ- was transformed into a transformant Satsuromyces cerevisiae NA74-3Aρ-/pHS
B 106ΔTth was obtained.

The obtained transformant was published as a European patent application.
- After culturing in the medium described in Example 15 of A-0235430 for -40- days, the bacterial cells were collected. More specifically, yeast transformants were mixed with K2HPO4 per 4 ml of culture solution []Q.
3g, glucose 30g, asparagine 4gtL-histidine 100■, KI0.1■, MgS○4・7H20
500mg, C a C 1 2・2 H20 33
0mg, CuSO4・5H200.4■, FeS04・
7H202.5mg, MnS 04・4 H20
0.4111g + (NH4)3 PO.・12M
oO, ・3H20 0.2mg, Zn So4・7H
20 3.1■, inositol 10■, thiamine 0.2
■, pyridoxine 0.2■, Ca-pantothenic acid 0.2
■, Niacin 0.2■, Biotin 0.002■)) after shaking culture at 30℃ for 3 days, 0.5n
+I2 was transferred to 4.5 mQ of the same medium and cultured with shaking at 30°C for 1 day. Next, change that 2mQ to 18mΩ
of fresh medium (per IQ, K H2P 04300 mg
, sucrose 80g, asparagine 4g, L-histidine 1
00■, KCQ 2. Og, KI O. 1mg, M
gSO4・7H20500■, CaC12・2H203
30■, glucose 10g. Tris-maleic acid (p
H6.5) 25mM, CuSO.

5T{20 0.4+ng, F e So.・7H,
0 2.5■, MnSO4・4H20 0.4mg,
(NH4). P.O. -12Mo03・3H20 0
, 2■, ZnSO4・7H20 3.1■, Inositol 10■, Thiamine 0.2■, Pyridoxine 0.2
■, Ca-pantothenic acid 0.2■, Niacin 0.2■
, containing 0.002μ of biotin] and further incubated at 30°C.
The cells were cultured with shaking for 3 days, and the bacterial cells were collected by centrifugation.

Approximately 150 microbial cells were added to sodium phosphate buffer [100
mM sodium phosphate (pH 7.4), 7.0M urea, 1% Triton X-100. 0.1mM (p-
aminodiphenyl) methanesulfonyl fluoride hydrochloride, 10mM EDT A (p
H8.0)) Suspend in 500 μB and add 1 glass bead.
g and stirred vigorously on a Vortex for about 30 minutes.

A supernatant was obtained by centrifugation at 10.00 rpm for 5 minutes. Add 1/3 of the 4-fold concentration of Laem to this extract.
mli buffer was added and heated at 100°C for 10 minutes. After cooling, the mixture was centrifuged at 10,000 rpm for 5 minutes to obtain a supernatant. 80 μQ of the extract was subjected to SDS-polyacrylamide gel electrophoresis and then electrically blotted onto a nitrocellulose filter.

This filter has anti-Herp from DAKOPATTS.
es virus type 1 (Maclintyr
e) React with antibody (rabbit), then horse
A radish peroxidase-labeled anti-rabbit antibody was reacted, and development reagent (
A specific band was detected when the color was developed using Bio rad (Bio rad).

[Brief explanation of drawings]

FIG. 1 shows the base sequence of DNA containing the HSV-1 Miyama strain gB gene, and FIG. 2 shows the amino acid sequence of the HSV-1 Miyama strain gB gene. Figure 3 shows Example 3
Figure 4 shows the base sequence of the synthetic DNA fragment used in H.
It is a construction diagram of the SV-1 type Miyama strain gB gene expression plasmid. FIG. 5 is a diagram of the construction of pramid PGFE213. Figure 6 shows HSV-1 type Miyama strain truncated g
It is a construction diagram of a B gene expression plasmid. Figure 7 is H
1 shows the base sequence of the SV11 type Miyama strain truncated g B gene and the amino acid sequence deduced from it.

Claims (5)

[Claims] (1) A polypeptide containing an amino acid sequence represented by the following formula in its molecule. [There is a gene sequence] [There is a gene sequence] (2) The polypeptide according to claim 1, which contains an amino acid sequence represented by the following formula. [There is a gene sequence] [There is a gene sequence] [There is a gene sequence] [There is a gene sequence] (3) A recombinant DNA containing a base sequence encoding the polypeptide according to claim 1. (4) A transformant carrying the recombinant DNA according to claim 3. (5) A method for producing the polypeptide according to claim 1, which comprises culturing the transformant according to claim 4, producing and accumulating the polypeptide according to claim 1 in the culture, and collecting the same. .