JPH06237775A - Slo expression vector, strain transformed with the same vector, production of the vector and slo - Google Patents

Abstract

PURPOSE:To obtain an SLO(streptolysin O) expression vector, having a larger production than that by the culture of Streptococcus pyogenes and capable of expressing the SLO, a transformed microorganism and a method for producing the SLO expression vector and SLO. CONSTITUTION:A DNA fragment, having the function of expression in a strain belonging to the genus Bacillus and containing a promoter of various genes derived from a strain belonging to the genus Bacillus or a DNA fragment containing the promoter and at least a pre-region is arranged on the downstream side of a mature slo structural gene to construct an SLO expression vector. A promoter derived from spoOA gene, alkaline protease gene and P-43 gene of Bacillus subtilis can be selected as the promoter. A pre-region derived from slo gene itself and alkaline protease can be selected as the pre-region. Thereby, SLO having the activity of about 3.5 to 32 times that produced by the original strain Streptococcus pyogenes is produced by investigating the combination and culture conditions. The figure illustrates the comparison of the hemolytic activity between the original strain Streptococcus pyogenes and a recombinant Bacillus subtilis.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a vector expressing streptolysin O, Bacillus subtilis transformed with the vector, and a method for producing streptolysin O.

[0002]

2. Description of the Related Art Streptolysin O (hereinafter simply referred to as SLO
Is abbreviated as S ) of type A streptococcus, which is a type of streptococcus.
It is one of the hemolytic toxins produced by Treptococcus pyogenes (hereinafter simply referred to as S. pyogene ), and is known to be a causative agent of bacterial infections such as throat inflammation.

SLO is found in the serum of patients infected with this bacterium.
There is an increase in the antibody titer against. Therefore, S. pyo
SLO is used as an antigen as a diagnostic agent for infectious diseases against so-called type A streptococcus including genes . SLO
The method of cloning the gene encoding is described in Kehoe et al., Infect. Immu
n. 804 43 (1984). The gene sequence encoding SLO is
(Kehoe) et al., Infect. Immun. 5
5 (1987) No. 12, P3228-3232.

[0004]

However, the SLO
In order to obtain SLO by culturing S. pyogenes , which is a producing bacterium, this SLO-producing bacterium has a problem in that the production efficiency is poor and the production cost is relatively high due to that. ing. Therefore, the present invention provides an SLO expression vector capable of expressing SLO having a higher production amount than the culture of S. pyogenes , and provides a microorganism transformed by introducing the SLO expression vector, It is an object to provide a method for producing an SLO expression vector, and a method for producing SLO by culturing a microorganism transformed with the SLO expression vector.

[0005]

The above object can be achieved by adopting the following constitution. That is, according to the present invention, a DNA sequence containing a promoter of a gene of a strain belonging to the genus Bacillus capable of functioning the expression of SLO in a strain belonging to the genus Bacillus is arranged upstream of a structural gene encoding SLO. The SLO expression vector is characterized by:

The present invention also relates to a promoter of a gene of a strain belonging to the genus Bacillus capable of functioning the expression of SLO in a strain belonging to the genus Bacillus, and a DNA sequence encoding at least a pre of a signal sequence of the strain belonging to the genus Bacillus. The SLO expression vector is characterized in that the DNA fragment containing and is arranged upstream of the mature structural gene encoding SLO.

The present invention is also characterized in that a DNA fragment containing a promoter of the spoOA gene of Bacillus subtilis is arranged upstream of the structural gene encoding SLO.
This is a LO expression vector. The present invention also provides a DNA fragment comprising a promoter of the B. subtilis spoOA gene and a DNA sequence encoding at least a pre of a signal sequence of Bacillus subtilis alkaline protease, which is arranged upstream of a mature structural gene encoding SLO. The SLO expression vector is characterized in that

The present invention also relates to a mature structural gene encoding SLO, wherein a DNA fragment containing a promoter of the P-43 gene of Bacillus subtilis and a DNA sequence encoding at least a pre of the signal sequence of the alkaline protease of Bacillus subtilis. The SLO expression vector is characterized by being arranged upstream. The present invention also provides strains belonging to the genus Bacillus transformed with each of the above vectors.

The present invention also provides a DNA fragment comprising a promoter, SD sequence, and / or a DNA sequence encoding a signal sequence containing at least a pre of a gene of a strain belonging to the genus Bacillus in a strain belonging to the genus Bacillus. The types of genes are selected and combined so that the expression of SLO can function, and the combined DNA
A method for producing an SLO expression vector, which comprises arranging a fragment upstream of a region containing a mature structural gene encoding SLO.

The present invention also provides a method for producing SLO, which comprises culturing a strain belonging to the genus Bacillus transformed with each of the above vectors and collecting SLO from the medium. As a host into which the SLO expression vector in the present invention is introduced, a strain belonging to the genus Bacillus, preferably Bacillus subtilis is used. The reason for this is that Bacillus subtilis is a bacterium whose safety has been confirmed, and because it is a host with a high extracellular substance production ability, it is industrially advantageous for SLO production.

In order to express SLO in Bacillus subtilis and secrete it outside the cells, it is necessary to introduce the following SLO expression vector that can function in Bacillus subtilis. The vector is RN
A DNA sequence encoding a promoter that directs the binding of A polymerase and promotes transcription, an SD sequence involved in the initiation complex formation reaction of protein biosynthesis, and a signal sequence for SLO secretion (hereinafter simply referred to as a signal DNA sequence) A DNA fragment composed of
It is essential that the mature structural gene (hereinafter, simply referred to as mature slo structural gene) that encodes is encoded.

FIG. 1 shows the constitution of a gene for expressing SLO (hereinafter simply referred to as slo gene). sl
The o gene is composed of a promoter, an SD sequence, a signal DNA sequence, and a mature slo structural gene. Signal DN among the components of the slo gene
The DNA sequence consisting of the A sequence and the mature slo structural gene constitutes the slo structural gene. The signal DN
The A sequence is composed of pre and pro.

The present inventors have found that various promoters, SD sequences, signal DNA sequences derived from Bacillus subtilis, and S. pyo
slo structural gene from Genes (or deletions processed s
The expression unit consisting of a combination of the (lo structural gene) was genetically engineered into the plasmid pUB110 capable of replication in Bacillus subtilis to prepare various vectors for SLO expression. Among them, five highly productive SLO expression vectors, namely pUBSLO, pAS, p
OAS, pOASΔA and pPA were obtained.

For the SLO expression vector pUBSLO, the promoter is the spoOA gene of Bacillus subtilis (Proc.
tl. Acad. Sci. USA 82 (1985) 2647-2651), and the SD sequence and the pre and pro of the signal DNA sequence are derived from the slo gene of S. pyogenes . S
For LO expression vector pAS, promoter, SD
A sequence and a signal DNA sequence precoding a Bacillus subtilis alkaline protease gene (hereinafter, simply referred to as an alkaline protease gene) (Proc. Natl. Acad.
Sci. USA 81 (1984) 1184) and the signal DNA sequence pro is derived from the Bacillus subtilis alkaline protease gene and the S. pyogenes slo gene.

Regarding the SLO expression vector pOAS,
The promoter is derived from the spoOA gene of Bacillus subtilis, the SD sequence and the pre of the signal DNA sequence are derived from the alkaline protease gene of Bacillus subtilis,
The signal DNA sequence pro is derived from the Bacillus subtilis alkaline protease gene and the S. pyogenes slo gene.

Regarding the SLO expression vector pOASΔA, the promoter is derived from the spoOA gene of Bacillus subtilis, the SD sequence and the pre of the signal DNA sequence are derived from the alkaline protease gene of Bacillus subtilis, and the preregion thereof is Was synthesized by a DNA synthesizer .
It is derived from the nes lo gene.

Regarding the SLO expression vector pPA, the promoter and SD sequence are derived from the P-43 gene of Bacillus subtilis, and the pre of the signal DNA sequence is derived from the alkaline protease gene of Bacillus subtilis. , SD sequence, and DNA sequence consisting of pre are DNA
The signal DN was synthesized by a synthesizer.
The A sequence pro is from the slo gene of S. pyogenes .

These promoters have different influences on the production of SLO, such as the expression timing of SLO and the strength of transcription activity. For example, the promoter derived from the spoOA gene has the property of strongly expressing SLO during the sporulation period, and the promoter derived from the P-43 gene has the property of strongly expressing SLO during the logarithmic growth period. In Table 1 below,
The preferred combinations of the types of pre and pro origins of the promoter, SD sequence and signal DNA sequence that constitute each of the SLO expression vectors of the present invention are shown, and further, each of the SLO expression vectors of the present invention is introduced into Bacillus subtilis. 2 shows the lytic activity of SLO-producing bacteria transformed with.

[0019]

[Table 1]

The ratio of hemolytic activity to original strain ratio shown in Table 1 is
Assuming that the hemolytic activity in the culture solution obtained by culturing S. pyogenes at 30 ° C. as the original strain was 1, SLO-producing bacteria transformed by introducing each SLO expression vector into Bacillus subtilis 37
The original strain ratio of hemolytic activity of the culture solution obtained by culturing at 0 ° C is shown. Bacillus subtilis transformed with the SLO expression vector pUBSLO has 3.5 times the hemolytic activity against S. pyogenes .

Bacillus subtilis transformed with the SLO expression vector pAS has 6-fold hemolytic activity against S. pyogenes . Bacillus subtilis transformed with the SLO expression vector pOAS has 7-fold hemolytic activity against S. pyogenes . This SLO expression vector pOAS is
In the construction of the LO expression vector pAS, the promoter was replaced with that derived from the spoOA gene of Bacillus subtilis, and this construction increases the hemolytic activity of SLO-producing bacteria.

Bacillus subtilis transformed with the SLO expression vector pOASΔA has 8-fold hemolytic activity against S. pyogenes . This SLO expression vector pOASΔA
In the vector construction of the SLO expression vector pOAS, the S. pyogene
s are those that instead of the one derived from slo structural gene,
It can be seen that this configuration increased the hemolytic activity of SLO-producing bacteria.

Bacillus subtilis transformed with the SLO expression vector pPA has 9-fold hemolytic activity against S. pyogenes . This SLO expression vector pPA is
In the construction of the O expression vector pOASΔA, the promoter and SD sequence are replaced with those derived from the P-43 gene of Bacillus subtilis, and this construction increases the hemolytic activity of SLO-producing bacteria.

In the present invention, the DNA fragment to be incorporated into the expression vector may be D
It includes both cases that are synthesized by the NA synthesizer.

[0025]

【Example】

[Example 1] Preparation of SLO expression vector pUBSLO and generation of SLO
Present (1) Preparation of slo gene Cloning of the slo gene is known by Kehoe, M
& K, TimmisInfect. Immun. 8
According to the method disclosed in 04 43 (1984), the slo gene was introduced into a known λgt10 (manufactured by Amersham) to newly prepare it.

30 μg of λgt10 in which the obtained slo gene was cloned was collected, and 10 mM Tris-HCl buffer (pH 7.5) and 7 mM MgCl ₂ were added,
It was dissolved in 50 μl of a solution containing 100 mM NaCl, 10 units of restriction enzyme EcoRI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. Then, using the DNA separation method by agarose gel electrophoresis, the sludge was removed from the above reaction solution.
About 3 μg of a DNA fragment of about 3.2 kb containing the gene was obtained.

(2) Preparation of slo gene from which promoter part has been removed When a DNA fragment is inserted and integrated into a vector, it is inserted to facilitate integration or to remove an extra region. It is necessary to prepare the gene of interest to an appropriate length. The gene to be inserted in the present invention was the slo gene from which the promoter portion was removed. Example 1
Then, a DNA fragment of about 3.2 kb containing the slo gene obtained in the above step (1) was digested with a restriction enzyme SfaNI to an appropriate length and then deleted with ExoIII to obtain an appropriate DNA. The length In this case, (1)
When about 140 bp is deleted from the restriction enzyme SfaNI cleavage site existing on the 5′-end side of the slo gene obtained in the step (1), the open reading frame of the slo gene is started and the slo structural gene is obtained. Therefore, the slo gene from which the promoter portion was removed was prepared by the method shown below.

That is, s obtained in the above step (1)
Approximately 3 μg of a 3.2 kb DNA fragment containing the lo gene
7 mM with 10 mM Tris-HCl buffer (pH 7.5)
It was dissolved in 50 μl of a solution containing mMMgCl ₂ and 100 mM NaCl, 10 units of restriction enzyme SfaNI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. The reaction solution was extracted with phenol to inactivate the enzyme, and then the DNA was purified by a precipitation method with ethanol to obtain a DNA fragment. The DNA fragment obtained by this restriction enzyme SfaNI was subjected to the operations described below.

The obtained DNA fragment was treated with 50 mM Tris-HCl buffer (pH 8.0) and 100 mM NaCl.
Dissolve in 100 μl of a solution containing 5 mM MgCl ₂ and 10 mM mercaptoethanol, and add Ex
180 units of oIII (Takara Shuzo Co., Ltd.) was added, and 37
Digested at C for 2 minutes. After that, the reaction solution was heated at 65 ° C. for 10 minutes to inactivate ExoIII to obtain a digestion reaction solution.

40 mM sodium acetate (pH 4.5) and 100 mM NaCl were added to this digestion reaction solution.
And 100 μl of a solution containing ₂ mM ZnCl ₂ and 10% glycerol was added, and another 50 units of Mu was added.
ng Bean nuclease (manufactured by Takara Shuzo Co., Ltd.) was added, and a digestion reaction was carried out at 37 ° C. for 1 hour. After the reaction was completed, this reaction solution was extracted with phenol to inactivate the enzyme, and then the DNA was purified by the ethanol precipitation method to obtain 1 μg of a DNA fragment with blunt ends. That is, 1 μ each of the blunt-ended DNA fragments expected to contain the slo gene from which the desired promoter was removed.
g was obtained.

(3) slo from which the promoter portion has been removed
Blunt-ended D expected to contain the gene
Addition of Linker to NA Fragment The following two types of polylinkers having recognition sites for the restriction enzyme XbaI and the restriction enzyme BamHI were prepared. 5′TCTAGAAC 3 ′ 3′AGATCTTTG 5 ′ 5′ACTGGATCC 3 ′ 3′TGACCTAGG 5 ′ That is, each single-stranded DN consisting of 9 nucleotides
A, that is, 5'TCT-AGAAAC 3 ', 5'AC
TGGATCC 3 ', 3'AGATCTTTG 5'and 3'TGACCTAGG 5'are DNA synthesizers (A
(Manufactured by BI Co., Ltd.), single-stranded DNA 50 pm
is added to 50 mM Tris-HCl buffer (pH 7.5),
10 mM MgCl ₂ , 10 mM DTT, 1 mM
It was dissolved in 50 μl of a solution containing ATP, 5 units of T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) was added, and phosphorylation reaction was carried out at 37 ° C. for 1 hour to carry out the above DN.
Two types of linkers with A sequences were obtained.

DNA fragment 1 obtained in the above step (2)
μg to 70 mM Tris-HCl buffer (pH 7.5)
And 10 mM MgCl ₂ and 10 mM DTT and 1
Dissolve in 50 μl of a solution containing mMATP, and further add 1 μl each of the above-mentioned phosphorylation reaction solution of the synthetic DNA linker.
Was added. Five units of T4 DNA ligase was added to this mixed solution, and the mixture was held at 16 ° C. for 18 hours to carry out a ligation reaction.

(4) End Treatment of DNA Fragment to which Linker is Added 10 units of the restriction enzyme XbaI and the restriction enzyme BamHI are added to the reaction solution obtained in the step (3), and further 37
The digestion reaction was carried out at ℃ for 2 hours. Then, unreacted linker DNA, restriction enzyme XbaI and restriction enzyme BamH
I and the slo gene from which the desired promoter portion has been removed from the reaction solution by a DNA separation method using agarose gel in order to remove the DNA generated by the ExoIII digestion in the process (2) above. 0.5 μg of a 1.5-2 kb DNA fragment expected to contain was obtained.

(5) Preparation of plasmid pUCSLO1 In order to examine the degree of deletion of the slo gene deleted in the step (2), 0.5 μg of this DNA fragment and the restriction enzyme BamHI and the restriction enzyme XbaI of plasmid pUC19 were examined. 0.1 μg of the decomposed product of 70 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl ₂ and 1
50μ solution containing 0 mM DTT and 1 mM ATP
It was dissolved in 1. Add 5 parts of T4 DNA ligase to this mixture.
A unit was added and the mixture was kept at 16 ° C. for 18 hours to carry out a ligation reaction. Escherichia coli (JM109) using the obtained reaction solution
Were transformed to obtain ampicillin-resistant colonies.

Of these, 12 transformants were selected and DN
The 5'and 3'side nucleotide sequences of the slo gene lacking the A sequence were analyzed by the dideoxy method (Sanger, F. et.a.
l. Proc. Natl. Acad. Sci. USA
5463 74 (1977)). Of the resulting deletions, the 5'-end side was 62 bp upstream of methionine at the start codon of the slo gene and the 3'-end side was SL.
Some encoded up to the last 571 lysine at the C-terminus of the O protein. The resulting plasmid having the deletion-treated slo gene was designated as pUCSLO1. Although no promoter is present in this plasmid pUCSLO1, it is derived from S. pyogenes slo gene
It has a D sequence, a signal DNA sequence, and a mature slo structural gene.

(6) Construction of SLO Expression Vector pUBSLO and Expression of SLO This step is carried out by using the plasmid p obtained in the step (5) above.
The slo gene produced by adding the promoter of the spoOA gene of Bacillus subtilis to the upstream of the DNA sequence consisting of the SD sequence, the signal DNA sequence, and the mature slo structural gene contained in UCSLO1 is replicable in Bacillus subtilis and This is a step of producing the SLO expression vector pUBSLO by introducing it into the plasmid pUB110 having a drug resistance marker. The outline of the production process is shown in FIG. 2, and the details are shown below.

Plasmid pSPTA1 (Chibazakura, carrying a promoter region of spoOA gene,
T. et. al. J. Bacteriol. 2625
173 (1991)) was digested with restriction enzymes BamHI and HpaI, and a DNA fragment of about 230 bp containing the promoter region of the spoOA gene was obtained by a DNA separation method using agarose gel.

On the other hand, downstream of this DNA fragment carrying the promoter region of the obtained spoOA gene, the above (5)
The following operation was performed to add the DNA sequence containing the SD sequence, the signal DNA sequence, and the mature slo structural gene contained in the plasmid pUCSLO1 obtained in the step of. First, Xba of the plasmid pUCSLO1
3 μg of the degradation product of I was extracted with phenol and then precipitated with ethanol to obtain a precipitate. This precipitate was mixed with 67 mM calcium phosphate (pH 7.4) and 6.7 mM MgCl 2.
₂ , 1 mM 2-mercaptoethanol, 50 μM
The solution was dissolved in 20 μl of a solution containing dNTPs and 10 units of Klenow fragment (Takara Shuzo Co., Ltd.), and reacted at 37 ° C. for 1 hour. Then, it was left at 70 ° C. for 5 minutes to deactivate the enzyme.

The reaction mixture obtained had a final concentration of 100 mM.
NaCl was added so as to become NaCl, 10 units of the restriction enzyme BamHI was added to this solution, and the mixture was heated at 37 ° C. for 2 hours. This enzyme reaction solution was used for DN using agarose gel.
S. pyogenes slo gene-derived SD sequence, signal DNA sequence, mature sl separated by A separation method
0.5μ of about 1.8 kb DNA fragment containing o structural gene
g was obtained.

Next, pUB110 (Mckenzi,
T. et. al. Plasmid93 15 (198
6)) 0.5 μg of the BamHI degradation product and spo above
Approximately 230 bp DN containing promoter region of OA gene
A mixture of 0.5 μg of the A fragment and the DNA fragment of about 1.8 kb containing the SD sequence, the signal DNA sequence and the mature slo structural gene obtained in the above step was added to 70 mM.
Tris-HCl buffer (pH 7.5) and 10 mM Mg
It was dissolved in 50 μl of a solution containing Cl ₂ , 10 mM DTT and 1 mM ATP. Add T4DN to this mixture.
5 units of A ligase was added and the ligation reaction was performed at 16 ° C. for 18 hours to obtain a reaction solution containing the recombinant plasmid.

The recombinant plasmid in this reaction solution was introduced into Bacillus subtilis as follows. That is, Bacillus subtilis DB40
3 strains (Wang, LF et al. J.
Gen. , Appl. Microbiol. 487 3
5 (1989)) to 5 ml of CI medium for transformation (An
agnostpoulos, C.I. & J. Spizin
J. Bact. Was inoculated into 741 81 (1961)), 5 hours at 37 ° C., and shake culture was centrifuged collection bacteria. This bacterium is suspended in 10 ml of a CII medium for transformation (see the above-mentioned document) and further shake-cultured at 37 ° C. for 30 minutes. The reaction solution containing the recombinant plasmid obtained in (6) above was added to 0.5 ml of this bacterial solution, and the mixture was shake-cultured at 37 ° C. for 1 hour.

0.1 ml of the obtained culture broth was added to a final concentration of 1 mM DDT, 0.4 ml of sheep defibrinated blood, a final concentration of 5 μg / ml of kanamycin, and a final concentration of 0.7. 4 ml Dulbecco's PBS with% agar
It was added to the buffer (-) (manufactured by Nissui Pharmaceutical Co., Ltd.) heated to 48 ° C. The resulting solution is kanamycin 5
The cells were overlaid on TBAB medium (manufactured by Difco) containing μg / ml and defibrinated sheep blood (manufactured by Toho Pharmaceutical Co., Ltd.) having a final concentration of 5%, and cultured overnight at 37 ° C. By this culture, SLO was produced extracellularly, and SLO hemolyzed the red blood cells in the medium to form a transparent zone around the colony on the red agar medium. A strain carrying the SLO expression vector pUBSLO was isolated from the portion where the transparent zone was formed, and this strain was designated as an SLO-producing bacterium.

SLO expression vector pUBS of this Example 1
The origin of the nucleotide sequence of the SLO expression unit contained in LO is shown in FIG. Since the nucleotide sequence of the spoOA gene promoter of Bacillus subtilis and the nucleotide sequence of the slo gene of S. pyogenes are already known, the intermediate display in each nucleotide sequence is omitted. [Example 2] Preparation of SLO expression vector pAS and expression of SLO SLO expression vector pUBSL obtained in Example 1 above
SLO produced by Bacillus subtilis transformed with O is S. pyoge
SLO with about 3.5 times more hemolytic activity than SLO produced by nes , but with even higher hemolytic activity
In order to develop Bacillus subtilis transformed with the expression vector and the SLO expression vector thereof, in this Example 2, S having a promoter and a signal DNA sequence different from those in Example 1 was used.
It was decided to prepare an LO expression vector. In this Example 2, an SLO expression vector was prepared as described below using the promoter and signal DNA sequence region of the gene encoding the Bacillus subtilis alkaline protease.

(1) Preparation of slo gene The slo gene was prepared in the same manner as in Example 1 (1) above to obtain about 3 μg of a 3.2 kb DNA fragment containing the slo gene. (2) Preparation of slo structural gene from which the pre-region has been removed The preparation method described below is carried out by deleting the DNA fragment containing the slo gene obtained in step (1) above with ExoIII, an exonuclease. It is a method of preparing each to an appropriate length. In addition, DN by this ExoIII
Prior to the deletion treatment of the A fragment, the length of the DNA fragment was previously digested with the restriction enzyme BstEII. In this case, the restriction enzyme BstEII recognition site of the slo gene obtained in the step (1) was cleaved with the restriction enzyme BstEII, and about 30 bp was deleted with ExoIII from the cleaved site, so that the preregion was removed. Become a gene.

An example of preparation of the slo gene from which the pre-region has been removed is shown below. That is, about 3 μg of a DNA fragment of about 3.2 kb containing the slo gene obtained in the above step (1)
With 10 mM Tris-HCl buffer (pH 7.5) and 7 m
It was dissolved in 50 μl of a solution containing M MgCl ₂ and 100 mM NaCl, 10 units of the restriction enzyme BstEII was added, and the digestion reaction was carried out at 60 ° C. for 2 hours. The reaction solution was extracted with phenol to inactivate the enzyme, and then the DNA was purified by a precipitation method with ethanol to obtain a DNA fragment.

The obtained DNA fragment was treated with 50 mM Tris-HCl buffer (pH 8.0) and 100 mM NaCl.
Dissolve in 100 μl of a solution containing 5 mM MgCl ₂ and 10 mM mercaptoethanol, and add Ex
180 units of oIII (Takara Shuzo Co., Ltd.) was added, and 37
Digested at C for 2 minutes. Then, the reaction solution was heated at 65 ° C. for 10 minutes to deactivate ExoIII, and a digestion reaction solution was obtained.

40 mM sodium acetate (pH 4.5) and 100 mM NaCl were added to this digestion reaction solution.
And 100 μl of a solution containing ₂ mM ZnCl ₂ and 10% glycerol was added, and another 50 units of Mu was added.
ng Bean nuclease (manufactured by Takara Shuzo Co., Ltd.) was added, and a digestion reaction was carried out at 37 ° C. for 1 hour. After the reaction was completed, the reaction solution was extracted with phenol to inactivate the enzyme, and then the DNA was purified by the ethanol precipitation method to obtain 1 μg of a DNA fragment with blunt ends. That is, 1 µg of a blunt-ended DNA fragment expected to contain the slo structural gene from which the target pre-region was removed was obtained.

(3) blunt-ended DNA expected to contain the slo structural gene from which the preregion has been removed
Addition of Linker to Fragment The following two types of linkers having the recognition sites for the restriction enzyme XbaI and the restriction enzyme BamHI were prepared. 5′TCTAGAAC 3 ′ 3′AGATCTTTG 5 ′ 5′ACTGGATCC 3 ′ 3′TGACCTAGG 5 ′ That is, each single-stranded DN consisting of 9 nucleotides
A, that is, 5'TCT-AGAAAC 3 ', 5'AC
TGGATCC 3 ', 3'AGATCTTTG 5'and 3'TGACCTAGG 5'are DNA synthesizers (A
(Manufactured by BI Co., Ltd.), each single-stranded DNA 50 pm
is added to 50 mM Tris-HCl buffer (pH 7.5),
10 mM MgCl ₂ , 10 mM DTT, 1 mM
It was dissolved in 50 μl of a solution containing ATP, 5 units of T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) was added, and phosphorylation reaction was carried out at 37 ° C. for 1 hour to carry out the above DN.
Two types of linkers with A sequences were obtained.

DNA fragment 1 obtained in step (2) above
μg to 70 mM Tris-HCl buffer (pH 7.5)
And 10 mM MgCl ₂ and 10 mM DTT and 1
It was dissolved in 50 μl of a solution containing mMATP, and 1 μl of the phosphorylation reaction solution of the above synthetic DNA linker was further added. Five units of T4 DNA ligase was added to this mixed solution, and the mixture was held at 16 ° C. for 18 hours to carry out a ligation reaction.

(4) End Treatment of DNA Fragment to which Linker is Added 10 units of restriction enzyme XbaI and restriction enzyme BamHI are added to the reaction solution obtained in the step (3), and 37
The digestion reaction was carried out at ℃ for 2 hours. Then, unreacted linker DNA, restriction enzyme XbaI and restriction enzyme BamH
I and the slo structure in which the target pre-region is removed from the reaction solution by a DNA separation method using agarose gel in order to remove the DNA generated by the ExoIII digestion in the process (2) above. 0.5 μg of a 1.5-2 kb DNA fragment expected to contain the gene
Got

(5) Preparation of pUCSLO2 Among the 12 kinds of DNA fragments obtained in the above step (4), there were those in which the preregion was deleted.
The A fragment was selected. This DNA fragment is treated with the restriction enzyme XbaI.
And a 1.6 kb DNA digested with the restriction enzyme BamHI
A so-called mature slo structural gene is present in the fragment without the signal DNA sequence region of the slo structural gene. In the DNA fragment containing the mature slo structural gene, the pre-region of the signal DNA sequence is cleaved and removed, and the pro region and the mature slo structural gene begin 3
From the fourth amino acid, asparagine, the last 57
Coding up to the first lysine. The resulting plasmid was named pUCSLO2. This plasmid pUC
SLO2 does not have a promoter, an SD sequence, and a signal region pre-region, but has a signal sequence pro region and a mature slo structural gene.

(6) Preparation of plasmid pASN containing promoter, SD sequence and signal DNA sequence of gene encoding alkaline protease (hereinafter referred to as "alkaline protease gene") Cloning of alkaline protease gene of Bacillus subtilis was performed by DS Goldfarb et al. Method (Mol. Gen. Genet., 319
191 (1983)) according to the restriction enzyme HindIII.
It was cloned into pGR71, which has a recognition region of about 1.3 kb.

Next, 30 μg of pGR71 in which the alkaline protease gene was cloned was added to 10 mM.
Tris-HCl buffer (pH 7.5) and 7 mM MgCl
50 μl of a solution containing ₂ and 100 mM NaCl
10 units of the restriction enzyme HindIII was added and digestion reaction was carried out at 37 ° C. for 2 hours. Using the DNA separation method by agarose gel electrophoresis, about 3 μg of a DNA fragment of about 1.3 kb containing the promoter, SD sequence and signal of the alkaline protease gene was obtained from the above reaction solution.

The 1.3 kb DNA fragment obtained in the above step comprises the promoter, SD sequence and signal DNA sequence of the desired alkaline protease gene, and
It includes extra areas other than these. Therefore, in order to obtain the DNA sequence of only the desired portion, the 1.3 kb DNA fragment obtained above was digested with the restriction enzyme DdeI, and the restriction within the alkaline protease gene existing on the 5'-terminal side was restricted. Cleavage at the enzyme DdeI recognition site, deletion of approximately 150 bp from this cleavage site to the promoter of the alkaline protease gene with ExoIII, and 3 '
-At the terminal side, from the restriction enzyme HindIII recognition site to the pro region of the alkaline protease gene and mature sl
o About 120 bp up to the boundary with the structural gene was deleted, the promoter of the alkaline protease gene,
It is necessary to obtain the SD sequence and the signal DNA sequence. Therefore, the operation was performed under the following conditions.

About 1.3 kb DNA obtained in the above step
About 3 μg of the fragment was added to 10 mM Tris-HCl buffer (pH
7.5), 7 mM MgCl ₂ , and 100 mM Na
It is dissolved in 50 μl of a solution containing Cl and the restriction enzyme Dde is added.
10 units of I was added and digestion reaction was carried out at 37 ° C. for 2 hours, the resulting reaction solution was extracted with phenol to inactivate the enzyme, and then the DNA fragment was purified by the ethanol precipitation method.

Next, this DNA fragment was treated with 50 mM Tris-HCl buffer (pH 8.0) and 100 mM NaCl.
Dissolved in 100 μl of a solution containing 5 mM MgCl ₂ and 10 mM mercaptoethanol, and 180 units of Exo III (Takara Shuzo Co., Ltd.) was added to the solution to give 3
Digested at 7 ° C for 2 minutes. Then, the reaction solution was heated at 65 ° C. for 10 minutes to deactivate ExoIII. In this reaction solution,
40mM sodium acetate (pH4.5), 100m
100 μl of a solution containing M NaCl, 2 mM ZnCl ₂ , and 10% glycerol was added, 50 units of Mung Bean nuclease (Takara Shuzo Co., Ltd.) was added, and digestion reaction was carried out at 37 ° C. for 1 hour. The reaction solution thus obtained was extracted with phenol to inactivate the enzyme, and then the DNA fragment was purified by the ethanol precipitation method to obtain a promoter of the alkaline protease gene, SD.
1 μg of a blunt-ended DNA fragment containing a sequence and a signal DNA sequence was obtained.

Next, the following linker having a restriction enzyme XbaI recognition site and a restriction enzyme BamHI recognition site was prepared. 5′GGATCCTAA 3 ′ 3′CCTAGGATT 5 ′ 5′CAATCTAGA 3 ′ 3′GTTAGATCT 5 ′ That is, each single-stranded DNA consisting of 9 deoxyribonucleotides was synthesized using a DNA synthesizer (ABI), and each 50 pmol of single-stranded DNA was added to 50 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl ₂ , and 1
Solution 5 containing 0 mM DTT and 1 mM ATP
It was dissolved in 0 μl, 5 units of T4 polynucleotide kinase (Takara Shuzo Co., Ltd.) was added, and phosphorylation reaction was carried out at 37 ° C. for 1 hour.

1 μg of a blunt-ended DNA fragment containing the promoter, SD sequence and signal DNA sequence of the alkaline protease gene obtained in the above step was added to 70
mM Tris-HCl buffer (pH 7.5) and 10 mM M
It is dissolved in 50 μl of a solution containing gCl ₂ , 10 mM DTT, and 1 mM ATP, and 1 μl of the phosphorylation reaction solution of the synthetic DNA linker obtained in the above step.
Was added. To this mixed solution, 5 units of T4 DNA ligase was added, and the ligation reaction was carried out at 16 ° C. for 18 hours. NaCl was added to the solution after the reaction so that the final concentration was 75 mM, 10 units each of the restriction enzyme XbaI and the restriction enzyme BamHI were added, and the digestion reaction was further performed at 37 ° C. for 2 hours. Expected to have promoter, SD sequence and signal DNA sequence of alkaline protease gene by DNA separation method using agarose gel in order to remove unreacted linker DNA and unnecessary DNA fragments generated by restriction enzyme digestion 0.5 μg of about 500 bp DNA fragment
Obtained.

To examine whether the obtained DNA fragment is a desired gene region obtained by removing the mature structural gene from the alkaline protease gene, 0.5 μg of this DNA fragment and the restriction enzyme B of the plasmid pUC19.
0.1 μg of a decomposed product of amHI and restriction enzyme XbaI,
70mM Tris-HCl buffer (pH 7.5) and 10m
M MgCl ₂ , 10 mM DTT, 1 mM AT
It was dissolved in 50 μl of a solution containing P and.

To this mixed solution, 5 units of T4 DNA ligase was added, and a ligation reaction was carried out at 16 ° C. for 18 hours. Escherichia coli (JM109) was transformed with the obtained reaction solution to obtain ampicillin-resistant colonies. 10 of these
Individual transformants were selected and the dideoxy method (Sanger,
F. et. al. Proc. Natl. Acad. Sc
i. USA 5463 74 (1977)), the nucleotide sequences on the 5'-terminal side and the 3'-terminal side of the deleted alkaline protease gene were determined, respectively. Of these, the most suitable alkaline protease gene deletion product was selected. The promoter and SD sequence of the alkaline protease gene of Bacillus subtilis are present in the DNA fragment of 460 bp that has been cleaved by this XbaI-BamHI, and the amino acid encoded by this is the pre- and pro-up to the 106th tyrosine (Tyr). Signal DN
A sequence, 107th alanine (Ala), and Xba
Glutamine (Gln) and serine (Se) at the I linker site
r) and arginine (Arg). The resulting plasmid was designated as pASN.

(7) Construction of SLO expression vector pAS The plasmid pUB110 which is replicable in Bacillus subtilis and has a drug resistance marker is added to the plasmid pUCSLO2 ( S. pyogenes SLO structural gene signal obtained in the above step (5). A DNA fragment having a pro region and a mature slo structural gene in which the pre-region of the sequence is removed, and the plasmid pASN (promoter, SD sequence and signal DNA sequence of the alkaline protease gene) obtained in the step (6) above. SL in which DNA fragments) are ligated
The O expression vector pAS was constructed as follows.

Restriction enzyme Bam of plasmid pUB110
After digestion with HI, 0.5 μg of this degradation product was added with 0.5 μg of pUCSLO2 obtained in the step (5) and 0.5 μg of the plasmid pASN obtained in (6). The mixture of each of these DNA fragments was treated with 70 mM Tris-HCl buffer (pH 7.5) and 10 mM MgCl ₂
50 μl of a solution containing 10 mM DTT and 1 mM ATP. To this mixed solution, 5 units of T4 DNA ligase was added and ligated at 16 ° C. for 18 hours to obtain a reaction solution containing a recombinant plasmid.

Next, the recombinant plasmid was introduced into Bacillus subtilis as follows. That is, 5 ml of the same Bacillus subtilis DB403 strain used in Example 1 was used for the CI medium for transformation (Anagnostpoulos, C. &
J. Spizin J. Bact. 741 81 (196
1)) was inoculated, shake-cultured at 37 ° C. for 5 hours, and collected by centrifugation. The obtained bacterium was suspended in 10 ml of a CII medium for transformation (see the above-mentioned document) and further shake-cultured at 37 ° C. for 30 minutes. To 0.5 ml of this bacterial solution, the reaction solution obtained by the above ligation reaction was added, and the mixture was shake-cultured at 37 ° C. for 1 hour. On the other hand, with a final concentration of 1 mM DTT,
0.4 ml sheep's defibrinated blood and final concentration 5 μg / ml
4 ml of Dulbecco's PBS (-) buffer (manufactured by Nissui Pharmaceutical Co., Ltd.) containing kanamycin of 10.7% and agar having a final concentration of 10.7% was heated to 48 ° C. Of this, 0.1 ml was added, and a mixed solution containing this culture solution was added to T containing 5 μg / ml of kanamycin and 5% of defibrinated sheep blood (manufactured by Toho Pharmaceutical Co., Ltd.).
The cells were overlaid on BAB medium (manufactured by Difco) and cultured overnight at 37 ° C. Bacillus subtilis harboring the desired SLO expression vector was able to hemolyze erythrocytes, resulting in clear zones around the colonies on red agar. The SLO expression vector pA is formed from the part where the transparent zone is formed.
A strain retaining S was isolated, and this strain was designated as an SLO-producing bacterium.

The origin of the nucleotide sequence of the SLO expression unit contained in the SLO expression vector pAS of this Example 2 is shown in FIG. Since the nucleotide sequence of the Bacillus subtilis alkaline protease gene and the nucleotide sequence of the S. pyogenes slo structural gene are already known, the intermediate representation of each nucleotide sequence is omitted. [Example 3] Preparation of SLO expression vector pOAS and expression of SLO In this Example 3, SLO with high SLO production efficiency in Bacillus subtilis.
The SL prepared in Example 2 above to obtain an expression vector
The present invention relates to an SLO expression vector pOAS having a DNA sequence in which the promoter of the Bacillus subtilis alkaline protease gene in the O expression vector pAS is replaced with the promoter of the Bacillus subtilis spoOA gene. Therefore, the SD sequence, the signal DNA sequence, and the mature slo structural gene are the same as in Example 2 above. FIG. 5 shows an outline of the production process of the SLO expression vector pOAS.

An example of preparation of the SLO expression vector pOAS is shown below. The plasmid pSPTA1 carrying the promoter region of the B. subtilis spoOA gene (the same plasmid used in Example 1 above) was the restriction enzyme BamHI.
And digested with the restriction enzyme HpaI, and about 230 bp containing the promoter region of the spoOA gene was obtained by the DNA separation method using agarose gel.

On the other hand, the SD sequence, pre- and pro-regions of the Bacillus subtilis alkaline protease gene were obtained as follows. That is, 3 μg of the AccI degradation product of the plasmid pASN obtained in (6) of Example 2 above is extracted with phenol. Next, the ethanol precipitate obtained by precipitating this extract with ethanol was added to 67 mM calcium phosphate (pH 7.4), 6.7 mM MgCl ₂ , and 1 mM.
2-mercaptoethanol and 50 μM dNTP
And 20 units of a solution containing 10 units of Klenow fragment (Takara Shuzo Co., Ltd.) at 37 ° C. for 1 hour. Then, it was left at 70 ° C. for 5 minutes to inactivate the enzyme. To the obtained reaction solution, NaCl was added so that the final concentration was 50 mM, and then 10 units of the restriction enzyme XbaI was added.
Digested by heating at ℃ for 2 hours. The enzyme digestion product thus obtained was subjected to a DNA separation method using agarose gel to obtain a DNA fragment containing the SD sequence of the alkaline protease gene and the pre- and pro-regions.

Using the same plasmid pUB110 as used in (6) of Example 1 above, this plasmid pU
B110 was digested with a restriction enzyme BamHI to obtain a degradation product. 0.5 μg of this BamHI degradation product was added to the above-mentioned Example 2.
0.5 μg of slo structural gene obtained by removing the pre-region from the plasmid pUCSLO2 obtained in
0.5 μg of the promoter of the A gene, 0.5 μg of the SD sequence of the alkaline protease gene of Bacillus subtilis and 0.5 μg of the pre- and pro regions were added. 70m of these DNA mixtures
M Tris-HCl buffer (pH 7.5) and 10 mM M
It was dissolved in 50 μl of a solution containing gCl ₂ , 10 mM DTT, and 1 mM ATP. Add T4D to this mixture
5 units of NA ligase was added and a ligation reaction was performed at 16 ° C. for 18 hours to obtain a solution containing a recombinant plasmid.

Next, a recombinant plasmid was prepared as follows. That is, the same Bacillus subtilis DB403 strain as used in Example 1 was inoculated into 5 ml of the CI medium for transformation (the same medium as in Example 1), and shake-cultured at 37 ° C. for 5 hours, The cells were collected by centrifugation. This bacterium was suspended in 10 ml of a CII medium for transformation (the same medium as in Example 1), and further cultured by shaking at 37 ° C for 30 minutes. To 0.5 ml of this bacterial solution, the reaction solution obtained by the above ligation reaction was added, and the mixture was shake-cultured at 37 ° C. for 1 hour.

0.1 ml of the obtained culture broth was added to a final concentration of 1 mM DTT, 0.4 ml of sheep defibrinated blood, a final concentration of 5 μg / ml of kanamycin, and a final concentration of 0.7. % Of agar containing Double Becko PBS (-) buffer (manufactured by Nissui Pharmaceutical Co., Ltd.) (4 ml) was added to that heated to 48 ° C, and kanamycin (5 μg / ml) was further added.
And T containing defibrinated sheep blood (Toho Pharmaceutical) with a final concentration of 5%
Layers were overlaid on BAB medium (Difco) and cultured overnight at 37 ° C.

Bacillus subtilis harboring the desired SLO expression vector was able to hemolyze erythrocytes, so that a transparent zone was formed around the colony on the red agar medium. A strain carrying the SLO expression vector pOAS was isolated from the portion where the transparent zone was formed, and this strain was designated as an SLO producing bacterium. The origin of the nucleotide sequence of the SLO expression unit contained in the SLO expression vector pOAS is shown in FIG.

Example 4 Preparation of SLO Expression Vector pOASΔA and Generation of SLO
Pro region of SLO signals produced by SLO producing bacteria of the current Example 3, and those of the alkaline protease gene of B. subtilis, and a derived from slo structural gene of S. pyogenes. Therefore, the pro region of the SLO expression vector pOAS is presumed to be excessive because it is composed of two origins.

Therefore, in the present Example 4, S.
In order to obtain an SLO expression vector with high LO production efficiency, in the SLO expression vector pOAS prepared in the above Example 3, only the pro region derived from the alkaline protease gene of Bacillus subtilis constituting a part of the pro region of the signal DNA sequence. The SLO expression vector pOASΔA having the SLO expression unit in which is deleted. The SLO expression vector pOASΔA obtained in this Example 4 is the same as that of the SLO expression vector pOAS in Example 3 described above with respect to the promoter, SD sequence, pre region, part of the pro region, and mature slo structural gene. .

The outline of the procedure for constructing the SLO expression vector pOASΔA is as follows. The vector pASN2 was prepared by deleting the alkaline protease pro region from the plasmid pASN, and the SD sequence and the pre region of the alkaline protease gene were obtained from this, and the spoOA promoter was obtained. And the slo gene excluding the pre-region are fused to prepare an SLO expression vector pOASΔA having the spoOA promoter, the SD sequence and the pre-region of the alkaline protease gene, the pro-region of the slo gene and the mature slo structural gene. FIG. 7 shows the SLO expression vector pOASΔA.
An outline of the manufacturing process of is shown. Below, SLO expression vector p
An example of preparation of OASΔA is shown.

(1) Preparation of SD and pre-region DNA fragments of the alkaline protease gene from plasmid pASN Preparation of synthetic linker In order to provide a restriction enzyme XbaI site on the 3'side of the pre-region of the alkaline protease gene, In order to delete the region, a DNA sequence from the restriction enzyme HpaI site present in the pre-region of the alkaline protease gene to the end of the pre-region and a little into the pro-region was synthesized by DNA synthesis to obtain the sequence shown below. Made as a linker.

That is, each single-stranded DNA was prepared using a DNA synthesizer (manufactured by ABI), and each single-stranded DNA 50
pmol was added to 50 mM Tris-HCl buffer (pH 7.
5), 10 mM MgCl ₂ , and 10 mM DTT
And 50 μl of a solution containing 1 mM ATP, 5 units of T4 polynucleotide kinase (Takara Shuzo Co., Ltd.) was added, and phosphorylation reaction was carried out at 37 ° C. for 1 hour,
A synthetic linker having the above DNA sequence was obtained.

This synthetic linker has a restriction enzyme XbaI recognition site added to the 3'side, and has the base sequence shown in SEQ ID NO: 1 in [Sequence Listing]. The reverse chain of this synthetic linker has the base sequence shown in SEQ ID NO: 2 in [Sequence Listing]. Construction of vector pASN2 Meanwhile, promoter of alkaline protease gene, SD
A plasmid p containing a sequence and a signal DNA sequence
ASN was prepared by the method of Example 2 (6) above. This plasmid pASN is used as a restriction enzyme HpaI and a restriction enzyme Xb.
After digestion with aI, 0.5 µg of the obtained degradation product was added with 0.5 µg of a fragment obtained by phosphorylating the synthetic linker containing the pre-region of the alkaline protease gene.

The obtained mixture was mixed with 70 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl ₂ and 10 mM.
It was dissolved in 50 μl of a solution containing mM DTT and 1 mM ATP. To this mixed solution, 5 units of T4 DNA ligase was added, and the ligation reaction was carried out at 16 ° C. for 18 hours. Escherichia coli (JM109) was transformed with the obtained reaction solution to obtain ampicillin-resistant colonies. Of these, 10 transformants were selected to obtain plasmid pASN2 in which the pro region of the alkaline protease gene was removed from plasmid pASN.

Preparation of DNA Fragment Containing SD Sequence and Pre-Region of Alkaline Protease Gene The plasmid pASN2 obtained above was used as a restriction enzyme Ac.
After decomposing with cI and extracting 3 μg of the decomposed product with phenol, the extract is precipitated with ethanol to obtain an ethanol precipitate. This ethanol precipitate was mixed with 67 mM calcium phosphate (pH 7.4) and 6.7 mM MgC.
l ₂ and 1 mM 2-mercaptoethanol, 50 μ
MdNTP and Klenow fragment (Takara Shuzo Co., Ltd.)
The solution was added to 20 μl of a solution containing 10 units and reacted at 37 ° C. for 1 hour. Then, it was left at 70 ° C. for 5 minutes to deactivate the enzyme. To the resulting reaction solution, NaCl was added so that the final concentration was 50 mM, and the restriction enzyme XbaI was added to 10 mM.
A unit was added and the mixture was heated at 37 ° C for 2 hours. The reaction solution was separated by a DNA separation method using an agarose gel, and contained the SD sequence of the alkaline protease gene and the pre-region.
A bp DNA fragment was obtained.

(2) Preparation of DNA fragment containing promoter of spoOA gene The plasmid pSPTA1 shown in the above Example 1 (6) carrying the promoter of spoOA gene is inserted into restriction enzyme B.
After digestion with amHI and the restriction enzyme HpaI, a DNA fragment of about 230 bp containing the promoter region of the spoOA gene was obtained by the DNA separation method using agarose gel.

(3) Preparation of Vector pOASΔA by Ligation Reaction 0.5 μg of a degradation product obtained by digesting the plasmid pUB110 with the restriction enzyme BamHI was added to the plasmid pUCSLO2 obtained in Example 2 (5) by the restriction enzyme Xbal.
And 0.5 μg of 1.6 kb slo structural gene obtained by digestion with restriction enzyme BamHI from which the pre-region was removed, 0.5 μg of a DNA fragment containing the promoter of the spoOA gene obtained in (2) above, DN containing the SD sequence and pre-region of the alkaline protease gene obtained in (1)
0.5 μg of A fragment was added.

These mixtures were mixed with 70 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl ₂ and 10 mM.
Solution 50 containing mM DTT and 1 mM ATP
It was dissolved in μl. To this mixed solution, 5 units of T4 DNA ligase was added, and ligation reaction was performed at 16 ° C. for 18 hours to obtain a reaction solution containing a recombinant plasmid. The recombinant plasmid was introduced into Bacillus subtilis as follows.

The above Bacillus subtilis DB403 strain was treated with 5 ml of a CI medium for transformation (Anagnostpoulos, C. & J. Spiz).
in, J.K. Bact. It was inoculated into 741 81 (1961)), and 5 hours with shaking cultured at 37 ° C., and centrifuged collection bacteria. This bacterium was suspended in 10 ml of a CII medium for transformation (see the above-mentioned document) and further shake-cultured at 37 ° C. for 30 minutes. To 0.5 ml of the obtained bacterial solution, the reaction solution obtained by the above ligation reaction was added, and the mixture was cultured at 37 ° C. for 1 hour with shaking. 0.1 ml of the obtained culture solution was added to the final concentration of 1
Dulbecco's PBS (-) buffer containing mM DTT, 0.4 ml sheep defibrinated blood, final concentration 5 μg / ml kanamycin, and finally 0.7% agar (Nissui Pharmaceutical Co., Ltd.). 4 ml) was added to the solution heated to 48 ° C. The resulting mixture was added with 5 μg of kanamycin
/ Ml and finally 5% of defibrinated sheep blood (manufactured by Toho Pharmaceutical Co., Ltd.) in TBAB medium (manufactured by Difco), and the mixture was cultured overnight at 37 ° C. By this culture, a transparent zone was formed around the colony on the red agar medium. From this portion, the desired SLO expression vector pOA was obtained.
A strain carrying SΔA was isolated, and this strain was designated as an SLO-producing bacterium.

The origin of the nucleotide sequence of the SLO expression unit contained in the SLO expression vector pOASΔA is shown in FIG. [Example 5] Preparation of SLO expression vector pPA This Example 5 relates to an SLO expression vector capable of further increasing the production amount than the SLO production amount by the SLO-producing bacteria described in Examples 1 to 4 above. . The SLO expression vector of this Example 5 is the same as that of Bacillus subtilis in the SLO expression vector pOASΔA prepared in the above Example 4.
The promoter derived from the poOA gene and the SD sequence derived from the alkaline protease gene of Bacillus subtilis were added to P-4 of Bacillus subtilis.
The promoter and SD sequences derived from 3 genes were replaced, and the signal DNA sequence consisting of the pre and pro regions was the same as in Example 4 above.

The promoter derived from the P-43 gene of Example 5 has the property of strongly expressing SLO in the logarithmic growth phase. The outline of the construction process of the SLO expression vector pPA is
-43 gene-derived promoter and SD sequence and DNA corresponding to a pre-region derived from alkaline protease gene
The fragments were synthesized by a DNA synthesizer, and the synthesized DNA
The fragment is spo present in the SLO expression vector pOAS
It consists of replacing the promoter derived from the OA gene, the pre- and pro-regions of the SD sequence of the alkaline protease gene. FIG. 9 shows an outline of the production process of the SLO expression vector pPA.

An example of preparation of the SLO expression vector pPA will be shown below. (1) Preparation of Synthetic Linker DNA corresponding to promoter and SD sequence of P-43 gene and pre-region of alkaline protease gene 5 '
A synthetic linker having a restriction enzyme BamHI site on the side and a restriction enzyme XbaI site on the 3 ′ side was synthesized as follows.

That is, each single-stranded DNA was prepared using a DNA synthesizer (manufactured by ABI). 50 pmol of each obtained single-stranded DNA was added to 50 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl ₂ , and 10 mM.
50 μl of a solution containing DTT and 1 mM ATP
5 units of T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) was added, and phosphorylation reaction was carried out at 37 ° C. for 1 hour to obtain a synthetic linker. The nucleotide sequence of the obtained synthetic linker is shown in SEQ ID NO: 3 in [Sequence Listing]. The above DN
The reverse strand corresponding to A was made similarly. The base sequence is shown in SEQ ID NO: 4 in [Sequence Listing].

(2) Preparation of SLO Expression Vector pPA by Ligation Reaction 0.5 μg of the degradation product obtained by digesting the plasmid pUB110 with the restriction enzyme BamHI, the plasmid pUCSLO2 of Example 2 (5) was treated with the restriction enzyme Xbal and the restriction enzyme. The pre-region obtained by digestion with the enzyme BamHI was removed.
0.5 μg of a 6 kb slo structural gene and 0.5 μg of a synthetic linker corresponding to the promoter derived from the P-43 gene, the SD sequence and the pre-region derived from the alkaline protease gene obtained in (1) above. Was added. These mixtures were mixed with 70 mM Tris-HCl buffer (p
H7.5), 10 mM MgCl ₂ , and 10 mM D
It was dissolved in 50 μl of a solution containing TT and 1 mM ATP. To this mixed solution, 5 units of T4 DNA ligase was added, and ligation reaction was performed at 16 ° C. for 18 hours to obtain a reaction solution containing a recombinant plasmid.

The recombinant plasmid was introduced into Bacillus subtilis as follows. 5 of the above-mentioned Bacillus subtilis DB403 strain
ml of CI medium for transformation (Anagnostpoulos, C. & J. S
pizin, J. Bact. 741 81 (1961)), the mixture was shake-cultured at 37 ° C. for 5 hours, and the cells were collected by centrifugation. This bacterium was suspended in 10 ml of a CII medium for transformation (see the above-mentioned document) and further shake-cultured at 37 ° C. for 30 minutes. To 0.5 ml of the obtained bacterial solution, the reaction solution obtained by the above ligation reaction was added, and the mixture was cultured at 37 ° C. for 1 hour with shaking. 0.1 ml of the obtained culture solution was added to a final concentration of 1 m.
Dulbecco's PBS (-) buffer containing MTT of M, 0.4 ml of defibrinated sheep blood, 5 μg / ml final concentration of kanamycin, and 0.7% final concentration of agar (manufactured by Nissui Pharmaceutical Co., Ltd.). 4 ml) was added to the solution heated to 48 ° C. The resulting mixed solution was added with kanamycin 5 μg /
ml and 5% final concentration of defibrinated sheep blood (Toho Pharmaceutical Co., Ltd.)
Was added to TBAB medium (manufactured by Difco) and cultured overnight at 37 ° C. By this culture, a transparent zone was formed around the colony on the red agar medium. Therefore, a strain carrying the target SLO expression vector pPA was isolated from this region, and this strain was designated as an SLO producing bacterium.

S contained in the SLO expression vector pPA
The origin of the base sequence of the LO expression unit is shown in FIG. [Test Example] (1) Confirmation of hemolytic activity Kanamycin was added to each of the SLO-producing bacteria obtained in Examples 1 to 5 to a final concentration of 5 µg / ml.
× L medium (4% tryptone, 2% yeast extract (all manufactured by Difco), 0.5% NaCl)
100 ml was inoculated and cultured with shaking at 37 ° C. for 8 hours.
The obtained culture solution was centrifuged and the hemolytic activity was measured with the culture supernatant. Bacillus subtilis DB403 as control pUB11
A bacterium transformed by introducing 0 was used, and this bacterium was treated in the same manner as above. The original strain, S. pyogenes, was added to Todd-Hewitt medium in 1% tryptone and 1%.
Inoculated with yeast extract (all manufactured by Difco), shake-cultured at 30 ° C. for 15 hours,
The obtained culture solution was centrifuged to obtain a culture supernatant of each bacterial cell.

The following hemolytic activity was measured using these culture supernatants. For sheep's defibrinated blood, 20 times its physiological saline (125 mM NaCl, 10 mM Na ₂ HP
O ₄ , 20 mM KH ₂ PO ₄ ) was added, and 2000 rp
After centrifuging for 3 minutes, physiological saline was added again to the obtained precipitate and the mixture was centrifuged at 2000 rpm for 3 minutes. This operation was repeated three times or more, and finally a 2.5% blood solution was prepared with physiological saline. The culture supernatant of the bacterial cells was 40 times, 80 times, 160 times, 320 times, 6 times with physiological saline containing 0.1% BSA.
40x, 1280x, 2560x, 5120x, 102
Diluted 40 times and 20480 times to prepare each diluted solution.

On the other hand, SLO is oxidized by contact with air and loses its activity. Therefore, cysteine hydrochloride as a reducing agent is dissolved in physiological saline so that the final concentration is 125 mM, and the pH of the solution is adjusted to 6.5. Was adjusted with NaOH to prepare a cysteine hydrochloride solution. For measuring the hemolytic activity, 200 μl of each diluted solution sample of the above-mentioned culture supernatant was added to
200 μl of cysteine hydrochloride solution was added and left at room temperature for 15 minutes. Next, 200 μl of the blood solution was added to each of the solutions and heated at 37 ° C. for 45 minutes. The reaction solution obtained was added to 15
After centrifugation at 000 rpm for 3 minutes, the supernatant was
The absorbance was measured by m, and this was used as the hemolytic activity of SLO.
The higher the activity is, the more red the color is, and the higher the absorbance value is.

The results are shown in Table 1 above as a ratio of hemolytic activity to original strain. This value is a comparison of the activity of SLO produced by each SLO when the hemolytic activity of SLO produced by the original strain S. pyogenes is 1. Table 1 above
According to the above, SLO produced by the SLO-producing strain of the present invention against the original strain S. pyogenes is the SLO expression vector pU.
3.5 for BSLO, 6 for SLO expression vector pAS, 7 for SLO expression vector pOAS, 8 for SLO expression vector pOASΔA, SLO expression vector pPA.
Has a very high hemolytic activity of 9.

Hemolytic activity was not confirmed in the vector pUB110 used as a control. (2) Confirmation of SLO by Western blot The culture supernatant of each cell used in (1) above was added to 7.5% SDS.
-Acrylamide gel (Laemmli Nature
680 277 (1970)) and electrophoresed at 50 mA for 2 hours, followed by nitro filter (Amers).
It was transferred to Hybond ^™ -C) manufactured by ham. afterwards,
The filter was treated with human anti-SLO antibody and goat anti-human IgG antibody conjugated with alkaline phosphatase. FIG. 11 shows an immunoblot for the anti-SLO antibody of the culture supernatant of Bacillus subtilis carrying the SLO expression vector of the present invention. According to FIG. 11, it was confirmed that each SLO-producing bacterium of the present invention secretes SLO to the outside of Bacillus subtilis.

[Example 6] Examination of culture temperature condition for SLO expression In the test example (1), pPA was used among SLO expression vectors.
Since Bacillus subtilis transformed with had the highest hemolytic activity, using this strain, the culture temperature was examined,
We tried to obtain higher hemolytic activity. Bacillus subtilis harboring the SLO expression vector pPA was cultured in 4 L medium at 28 ° C. for 30
The cells were cultured at 33 ° C and 33 ° C for 12 to 18 hours, and the hemolytic activity of the culture supernatant was measured by the method of Test Example (1) above.

Bacillus subtilis harboring the SLO expression vector pPA was cultured in 4 L medium supplemented with kanamycin to a final concentration of 5 μg / ml at 30 ° C. for 15 hours to obtain a culture supernatant. Similarly, a culture supernatant was obtained from the one cultured at 37 ° C. On the other hand, S. pyogenes is Todd-Hewit
Culture was performed at 30 ° C. for 15 hours in a medium containing 1% tryptone and 1% yeast extract (all manufactured by Difco) in t medium to obtain a culture supernatant.

The hemolytic activity of each culture supernatant is shown in FIG. 12 as a curve of turbidity-dilution ratio. The plot of the dilution rate in the hemolysis curve is shown every two times at equal intervals. Therefore,
According to FIG. 12, in the culture at 30 ° C., the SLO activity produced by Bacillus subtilis harboring the SLO expression vector pPA was
About 32 more than that produced by the original strain S. pyogenes
It turns out that it is more than double. The hemolytic activity of SLO can be regarded as the amount of SLO produced. Therefore, according to the production method of Example 6, the amount of SLO produced was S. pyogen.
It can be seen that it is increased about 32 times or more with respect to es .

[0097]

INDUSTRIAL APPLICABILITY According to the present invention, the SLO activity, that is, SLO, is higher than that of SLO obtained by culturing S. pyogenes.
S capable of expressing 3.5 to 32 times as much as S
It is possible to provide a method for producing LO, a strain belonging to the genus Bacillus transformed with an SLO expression vector, and an expression vector.

Bacillus subtilis as a host is easily available,
Highly safe, easy to culture, and produced S
Since LO is secreted extracellularly, the present invention is suitable for commercial production of SLO.

[0099]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 56 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AACGTTAATC TTTACGATGG CATTCAGCAA CATGTCTGCG CAGGCTGCCG GAAAAT 56

SEQ ID NO: 2 Sequence length: 60 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CTAGATTTTC CGGCAGCCTG CGCAGACATG TTGCTGAATG CCATCGTAAA GATTAACGTT 60

SEQ ID NO: 3 Sequence length: 188 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbols: promotor, sig peptide Method of characterizing: P-sequence GATCCTTAGA AATGGGCGTG AAAAAAAGCG CGCGATTATG TAAAATATAA AGTGATAGCG 60 GTACCATTAT AGGTAAGAGA GGAATGTACA CGTGAGAAGC AAAAAATTGT GGATCAGCTT 120 GTTGTTTGCG TCAACGTTAA TCTTTACGAT GGCATTCAGC A

SEQ ID NO: 4 Sequence length: 188 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Characteristic symbols: promotor, sig peptide Method of characterizing: P-sequence CTAGATTTTC CGGCAGCCTG CGCAGACATG TTGCTGAATG CCATCGTAAA GATTAACGTT 60 AACGCAAACA ACAAGCTGAT CCACAATTTT TTGCTTCTCA CGTGTACATT CCTCTCTTAC 120 CTATAATGGT ACCGCTATCA CTTTATATTC TCACTCATTCG CGCAGATTCC CGCAGATTTG

[Brief description of drawings]

FIG. 1 shows the constitution of genes for expressing SLO.

FIG. 2 shows an outline of the production process of the SLO expression vector pUBSLO.

FIG. 3 shows the origin of the nucleotide sequence of the SLO expression unit contained in the SLO expression vector pUBSLO.

FIG. 4 SLO contained in the SLO expression vector pAS
The origin of the nucleotide sequence of the expression unit is shown.

FIG. 5 shows an outline of the production process of the SLO expression vector pOAS.

FIG. 6: SL contained in the SLO expression vector pOAS
The origin of the nucleotide sequence of the O expression unit is shown.

FIG. 7 shows an outline of the production process of the SLO expression vector pOASΔA.

FIG. 8 shows the origin of the nucleotide sequence of the SLO expression unit contained in the SLO expression vector pOASΔA.

FIG. 9 shows an outline of the production process of the SLO expression vector pPA.

FIG. 10: SL contained in SLO expression vector pPA
The origin of the nucleotide sequence of the O expression unit is shown.

FIG. 11 shows an immunoblot against the anti-SLO antibody of the culture supernatant of Bacillus subtilis carrying the SLO expression vector.

FIG. 12 shows the hemolytic activity of the culture supernatant as a turbidity-dilution ratio curve.

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Claims (8)

[Claims] 1. A DNA fragment containing a promoter of a gene of a strain belonging to the genus Bacillus, which is capable of functioning to express streptolysin O in a strain belonging to the genus Bacillus, is arranged upstream of a structural gene encoding streptolysin O. Streptolysin O characterized by
Expression vector. 2. A promoter of a gene of a strain belonging to the genus Bacillus capable of functioning the expression of streptolysin O in a strain belonging to the genus Bacillus, and a DNA sequence encoding at least a pre of a signal sequence of the strain belonging to the genus Bacillus. A streptolysin O expression vector in which a DNA fragment containing the above is arranged upstream of a mature structural gene encoding streptolysin O. 3. A streptolysin O expression vector, wherein a DNA fragment containing a promoter of Bacillus subtilis spoOA gene is arranged upstream of a structural gene encoding streptolysin O. 4. A DNA fragment containing a promoter of Bacillus subtilis spoOA gene and a DNA sequence encoding at least a pre of a signal sequence of Bacillus subtilis alkaline protease is arranged upstream of a mature structural gene encoding streptolysin O. And a streptolysin O expression vector. 5. A DNA fragment containing a promoter of the P-43 gene of Bacillus subtilis and a DNA sequence encoding at least a pre of the signal sequence of alkaline protease of Bacillus subtilis is upstream of a mature structural gene encoding streptolysin O. And a streptolysin O expression vector. 6. The streptolysin O according to claim 1 or 2.
A strain belonging to the genus Bacillus transformed with the expression vector. 7. A D comprising a DNA sequence encoding a promoter, an SD sequence, and / or a signal sequence containing at least a pre, of a gene of a strain belonging to the genus Bacillus.
NA fragments are selected and combined so that the expression of streptolysin O can be made to function in a strain belonging to the genus Bacillus, and the combined DNA
A method for producing a streptolysin O expression vector, which comprises arranging the fragment upstream of a region containing a mature structural gene encoding streptolysin O. 8. A method for producing streptolysin O, which comprises culturing the strain according to claim 6 and collecting streptolysin O from the medium.