JP4270466B2 - Method for producing a protein expressed from an operon containing NADase, SNI and SLO genes, protein obtained thereby and use thereof - Google Patents

Description

  The present invention relates to a method for producing each protein expressed from an operon containing NADase, SNI, and SLO genes, a protein obtained by the production method and an antibody thereof, a vector containing a polynucleotide encoding the protein, and a transformant thereof, and Provide their use etc.

  Hemolytic streptococci is a pathogenic bacterium that causes pharyngitis, skin infections, scarlet fever, glomerulonephritis, rheumatic fever, sepsis, etc., and forms hemolytic plaques on blood agar plates containing red blood cells However, it is divided into groups A to G based on the immunochemical specificity of the polysaccharide antigen localized in the cell wall. Hemolytic streptococcal infection is a disease that is frequently derived as a childhood infection. Also, since the 1980s, fulminant infections with a high mortality rate have occurred in advanced countries such as Japan, Europe and the United States due to hemolytic streptococcal necrosis of muscle tissue, multi-organ damage, shock symptoms, etc. Therefore, it is often reported as human-eating bacteria.

  Hemolytic streptococci produce more than 20 types of various physiologically active substances outside the cells. Among them, the hemolytic toxin, streptolysin O (SLO), has been an important virulence factor since ancient times. Detailed research has been undertaken. Recently, it has been confirmed that a pathogenic factor such as NADase is transferred into a host cell from a hole formed on the cell membrane of the host by SLO. This discovery has received great attention as an indication that the type III secretion mechanism that is highly conserved among gram-negative pathogenic bacteria such as enterohemorrhagic Escherichia coli also exists in hemolytic streptococci, which are gram-positive bacteria. Yes.

  The present inventors have clarified that the SLO gene has been polycistronically transcribed by readthrough from the NADase gene (nga) located upstream thereof (see Non-Patent Document 1). This nga-slo operon is highly conserved between group A and group C hemolytic streptococci and is thought to play an important role in hemolytic streptococcal infections, but between the nga and slo genes Was known to have the gene orf1. However, the function of this gene orf1 is completely unknown.

  NADase has (a) activity to hydrolyze β-nicotinamide-adenine-dinucleotide (NAD) to produce adenosine diphosphoribose (ADPR) and nicotinamide (NAD glycohydrolase activity), (b) ) Activity to synthesize cyclic ADP ribose (cADPR) from NAD (ADP ribosyl cyclase activity), and (c) Activity to hydrolyze cADPR to produce ADPR (cADPR hydrolase activity). Since NADase is considered to enter the host cell from the pore formed on the host cell membrane by SLO, exert its toxicity by these activities, and bear the pathological condition of streptococcal infection. Very important in understanding infections.

By the way, as a method for producing NADase, there have been several reports so far.
For example, Non-Patent Documents 2 and 3 describe that NADase was purified from the culture supernatant of hemolytic streptococci while maintaining its activity. However, expression of NADase in heterologous host cells such as E. coli has not been successful. This is because NADase is extremely toxic to heterologous host cells.

  In addition, it is known that the SLO gene is highly conserved in different hemolytic streptococci groups, and in particular, there is no substantial difference between the SLOs of group A (GAS) and group C (GCS). In both groups of SLOs, 31 amino acid residues (3.3 kDa) including the N-terminal signal peptide are removed and secreted, and the molecular weight is gradually reduced by proteases secreted by themselves. SLO has a strong antigenicity, and it is easy to produce neutralizing antibodies, and anti-streptridine O antibody (ASLO) in serum rises during infection. Therefore, measurement of antibody titer is widely used for diagnosis of infection by this bacterium ( Anti-SLO test). SLO is not only used as a test antigen in the anti-SLO test, but also forms a huge ring-shaped hole on the cell membrane, and can send protein-sized molecules into living cells. In recent years, it has been widely used as a tool for improving the transparency of the skin. SLO can also be used as an antigen for ASLO purification. Therefore, development of a method for manufacturing SLO in large quantities and at low cost is demanded.

  In addition, ASLO is useful as a reagent such as a clinical test reagent, but commercially available ASLO is prepared using SLO partially purified from the culture supernatant of hemolytic streptococci as an antigen. However, since preparations partially purified from the culture supernatant also contain NADase in addition to SLO, antibodies produced using such preparations cross-react with not only SLO but also NADase. It is known (Non-Patent Documents 1 and 4). Therefore, development of a method for producing high-purity SLO is also required from the viewpoint of producing an antibody with low cross-reactivity.

There have been several reports on the manufacturing method of SLO.
For example, Patent Document 1 describes a method for producing (purifying) full-length SLO from a culture supernatant of hemolytic streptococci. However, there is a problem that the amount of SLO produced by Streptococcus pyogenes is low and the purification yield of full-length SLO from the culture supernatant of Streptococcus pyogenes is low due to degradation by the protease produced by itself.

In addition, the following report has been made as a method for producing SLO using Escherichia coli as a host.
In Non-Patent Document 5, when the SLO gene was subcloned into a high copy number plasmid, the host E. coli was easily lysed and also slow in growth, and further, the SLO gene itself was unstable and dropped out, and It is described that when the SLO gene was transferred to a low copy number plasmid, the SLO gene was stabilized but the production amount of SLO was small.
Non-Patent Document 6 discloses a specific N-terminal deletion type SLO (SLO corresponding to 496 amino acid residues excluding the N-terminal 75 amino acid residues including the signal peptide sequence) as a maltose binding protein (MBP, 42 kDa). It is described that a large amount of SLO was successfully expressed in E. coli by using a fusion protein. Although this is not full length, this is the first successful mass expression of SLO in E. coli.
Non-Patent Document 7 describes the large-scale expression of a specific N-terminal deletion type SLO in Escherichia coli (SLO encoding a region corresponding to 468 amino acid residues excluding 103 amino acid residues on the N-terminal side including the signal peptide sequence). Has been described as successful.
However, mass expression of full-length SLO in E. coli has not yet been successful. This is because a region near 32-75 amino acid residues near the N-terminus of SLO is considered to exhibit some toxicity to E. coli (Non-patent Documents 5-7).

  Furthermore, there are several reports on the large-scale expression of full-length SLO using Bacillus subtilis as a host (for example, Non-Patent Document 8, Patent Documents 2 and 3). However, Bacillus subtilis has a high ability to produce protease, and despite the fact that it produces a plurality of proteases, the production of a complete protease-deficient strain has not yet been successful. Furthermore, since Bacillus subtilis secretes various proteins, the purification process of SLO is complicated, and it is difficult to obtain high-purity SLO.

JP-A-5-331198 JP-A-5-184372 JP-A-6-237775 Kimoto et al., Biochim Biophys Acta 1681: 134-149 (2005) Karasawa et al., FEMS Microbiology Letters 130: 201-204 (1995) Gerlach et al., FEMS Microbiology Letters 136: 71-78 (1996) Madden et al., Cell 104: 143-152 (2001) Kehoe et al., Infect Immun 43: 804-810 (1984) Weller et al., Eur J Biochem 236: 34-39 (1996) Yamamoto et al., Biosci Biotechnol Biochem 65: 2682-2689 (2001) Yamada et al., Biosci Biotechnol Biochem 59: 363-366 (1995)

  As described above, several methods for producing streptococcal NADase and SLO have been reported. As a method for producing a recombinant protein with high purity and low cost, a method using a heterologous host cell such as Escherichia coli is known. However, a method for producing full-length NADase and SLO with high purity in large quantities and at low cost has not yet been developed for the following reasons.

  Since streptococcal NADase is highly toxic to heterologous host cells such as Escherichia coli, a method for producing a large amount of full-length NADase or a NADase fragment containing the toxic region using heterologous host cells has been developed. Not. Since analysis of NADase is considered to be very important for understanding streptococcal infection and its application, development of a method for producing NADase in large quantities is required.

Streptococcus-derived SLO is considered to have some toxicity to E. coli in the region near 32-75 amino acid residues near its N-terminus, and a large amount of SLO fragment containing the full-length SLO or the above region using E. coli. A manufacturing method has not been developed at a low cost.
Cholesterol-dependent cytolysin, such as SLO, forms a large group with similar molecular structure and function, not only Streptococcus genus Streptococcus but also Bacillus, Clostridium, Listeria It is known to produce bacteria such as genera, and their amino acid sequences exhibit high homology. However, SLO has the highest molecular weight among these groups, and its N-terminal region does not exist in other groups and is characteristic of hemolytic streptococci. For this reason, full-length SLO and its N-terminal fragment are not only used for clinical testing for hemolytic streptococcal infections (as antigens for antibody production), but also for clarifying the pathogenic mechanism at the molecular level. It is considered useful as a research tool. Therefore, if a method for producing full-length SLO or an SLO fragment containing the above region can be developed, it will be possible to obtain a large amount of highly pure antigen and to produce ASLO having substantially no cross-reactivity. Therefore, development of a method for producing them in high purity and in large quantities at low cost has been demanded.

  As a result of intensive studies, the present inventors succeeded in the large-scale expression of full-length NADase by expressing the NADase gene (nga) together with the unknown gene orf1 existing downstream thereof in heterologous host cells. Based on this result, the orf1 gene product was further analyzed, and it was found that it can act as an inhibitor of NADase by forming a complex with NADase in the host cell. The present inventors propose to label this orf1 gene product as a streptococcal NADase inhibitor (SNI). SNI that co-express and complex with NADase is believed to be able to protect its reservoir from NAD depletion. Based on this finding, it is considered that a therapeutic drug for streptococcal infection can be developed by screening a substance that dissociates the SNI / NADase complex in streptococci. In addition, since SNI binds very strongly and specifically to NADase, it is considered that the same effect as a purification tag such as a His tag that is currently widely used can be expected. Furthermore, the SNI was very stable to heat. Moreover, SNI inhibited not only bacteria-derived NADase but also mammalian-derived NADase. Therefore, it is considered that SNI can also be applied to the prevention and treatment of diseases that may involve mammalian-derived NADase. As described above, according to the knowledge of the present inventors, various applications are possible using SNI.

  In addition, the present inventors use an inducible promoter capable of strictly controlling gene expression in Escherichia coli to effectively suppress basal level gene expression and enable stable culture, and to induce against the promoter. In combination with the agent, full-length SLO was successfully expressed in large quantities. The present inventor has also succeeded in easily and inexpensively producing a high-purity full-length SLO by adding an appropriate tag to the SLO. The inventors further succeeded in producing an anti-SLO antibody that does not show cross-reactivity with NADase by using the high-purity full-length SLO prepared as described above.

Based on the above, the present inventors have completed the present invention. That is, the present invention provides the following inventions and the like:
[1] A polypeptide having 95% or more homology with the amino acid sequence of SEQ ID NO: 2 and having NADase inhibitory activity, or deletion or substitution of one or two amino acids in the amino acid sequence of SEQ ID NO: 2, A polypeptide having one or more modifications selected from the group consisting of addition and insertion and having NADase inhibitory activity.
[2] The polypeptide according to 1 above, wherein the NADase is a hemolytic streptococcal NADase.
[3] NADase is a polypeptide having 95% or more homology with the amino acid sequence represented by SEQ ID NO: 4, or deletion or substitution of 1 or 2 amino acids in the amino acid sequence of SEQ ID NO: 4 The polypeptide according to [1] or [2] above, wherein the polypeptide is subjected to one or more modifications selected from the group consisting of: addition, and insertion.
[4] The polypeptide according to any one of [1] to [3], which is derived from hemolytic streptococci.
[5] 80% or more of activity after heat treatment at 60 ° C. for 15 minutes, or 70% or more of activity after heat treatment at 100 ° C. for 15 minutes, The polypeptide according to any one of [1] to [4].
[6] A polynucleotide or sequence that is a polynucleotide having a homology of 95% or more with the polynucleotide represented by SEQ ID NO: 1 and encodes the polypeptide according to any one of claims 1 to 5 A polynucleotide that is capable of hybridizing with the complementary sequence of the polynucleotide represented by No. 1 under high stringency conditions and encodes the polypeptide according to any one of [1] to [5] above.
[7] The polynucleotide according to [6], which is derived from hemolytic streptococci.
[8] An expression vector having the polynucleotide according to [6] or [7] above.
[9] A transformant comprising the expression vector according to [8] above.
[10] The transformant according to [9] above, wherein the transformant is Escherichia coli.
[ 11 ] A NADase inhibitor comprising any one of the following (a) or (b) .
(A) The polypeptide according to any one of claims 1 to 5.
(B) A pharmaceutical comprising the expression vector [ 12 ] according to claim 8 and the NADase inhibitor according to [11] above .
[ 13 ] A reagent comprising the NADase inhibitor according to [11 ] above .
[ 14 ] The medicament according to 12 above, which is a therapeutic drug for hemolytic streptococcal infection.
[15] An antibody against the polypeptide according to any one of [1] to [5] above.
[16] a polypeptide according to any one of [1] to [5], and a NADas e, complex.
[ 17 ] The complex according to [ 16] above, wherein the NADase is a hemolytic streptococcal NADase.
[18] NADase is a polypeptide having 95% or more homology with the amino acid sequence represented by SEQ ID NO: 4, or deletion or substitution of 1 or 2 amino acids in the amino acid sequence of SEQ ID NO: 4 The complex according to [16] or [17] above, wherein the complex is a polypeptide having one or more modifications selected from the group consisting of addition, and insertion.
The polypeptide of any one of [19] above [1] to [5], comprising contacting the NADas e, composite according to any one of [16] - [18] Body manufacturing method.
[20] A method for producing NADase including the following steps (a) to (d):
(A) A step of preparing a co-expression vector having a first unit and a second unit, wherein the polynucleotide according to [6] or [7] is a first unit and the polynucleotide encoding NADase is a second unit. ;
(B) creating a transformant introduced with the co-expression vector;
(C) A step of co-expressing the polypeptide of any one of [1] to [5] with NADase in the transformant to obtain the complex of [16];
(D) A step of dissociating the polypeptide according to any one of [1] to [5] from the complex obtained in the above step to obtain an intact NADase:
[21] The production method of the above-mentioned [20], wherein the NADase is a hemolytic streptococcal NADase.
[22] NADase is a polypeptide having 95% or more homology with the amino acid sequence represented by SEQ ID NO: 4, or deletion or substitution of one or two amino acids in the amino acid sequence of SEQ ID NO: 4 The production method according to [20] or [21] above, wherein the polypeptide is subjected to one or more modifications selected from the group consisting of addition, and insertion.
[23] The production method according to [20] above, wherein the transformant is Escherichia coli.
[24] The production method of the above-mentioned [23], wherein the Escherichia coli is TOP10 strain.
[25] A method for producing an anti-NADase antibody, comprising a step of producing NADase by the production method according to any one of claims 20 to 24.
[ 26 ] A screening method for a therapeutic agent for infectious diseases caused by hemolytic streptococci, comprising the following steps (a) and (b):
(A) step test substance, to assess whether capable of suppressing the polypeptide of any one of [1] to [5], the formation of the complex containing the N ADAS e;
(B) A step of selecting a test substance that can suppress the formation of the complex.
[ 27 ] A screening method for a therapeutic agent for infectious diseases caused by hemolytic streptococci, comprising the following steps (a) and (b):
(A) a step of evaluating whether or not the test substance can cancel inhibition of N ADase activity by the polypeptide according to any one of [1] to [5] above ;
(B) selecting a test substance capable of releasing the inhibition.
[28] The screening method according to [26] or [27] above, wherein the NADase is a hemolytic streptococcal NADase.
[29] NADase is a polypeptide having 95% or more homology with the amino acid sequence represented by SEQ ID NO: 4, or deletion or substitution of 1 or 2 amino acids in the amino acid sequence of SEQ ID NO: 4 The screening method according to any one of [26] to [28] above, wherein the polypeptide is subjected to one or more modifications selected from the group consisting of: addition, and insertion.

The polypeptide of the present invention can be used in various applications. Specifically, the SNI of the present invention and its partial peptides and their expression vectors are useful for treating bacterial infections such as streptococci and various diseases other than infections, as well as inhibition of NADase and leukocyte chemotaxis. Useful for sex inhibition. The non-natural NADase and partial peptide thereof and expression vectors thereof of the present invention are useful as, for example, research on streptococcal NADase and development of a drug capable of suppressing its toxicity using the NADase, and an antibacterial or anticellular agent .
The expression vector of the present invention is useful for, for example, large-scale expression of a drug or reagent or a target polypeptide. Specifically, the SNI and NADase expression vectors of the present invention can be used not only as pharmaceuticals and / or reagents as described above, but also in large-scale expression of SNI and NADase in host cells, and themselves can function as a purification tag. It is also useful for producing fusion proteins. The SLO expression vector of the present invention is useful for large-scale expression of a full-length SLO or a partial peptide containing a toxic region thereof. Therefore, a transformant containing such an expression vector and a method for producing a target polypeptide using the transformant are also useful.
The antibody of the present invention is useful, for example, as a drug or reagent by detecting / quantifying SNI, NADase or SLO, or inhibiting their functions. Specifically, the SLO antibody of the present invention is particularly useful as a reagent because it has high specificity for SLO and does not cross-react.
The complex of the present invention and the production method thereof are useful for, for example, production of NADase, purification of a protein having NADase or SNI or a partial peptide thereof, and the screening method of the present invention.
The screening method of the present invention is useful for, for example, development of a therapeutic agent for streptococcal infection.

1. Polypeptide and partial peptide thereof The present invention provides streptococcal NADase inhibitor (SNI) or a partial peptide thereof. The present inventors succeeded for the first time in the expression of SNI, and succeeded in demonstrating its function. The presence of bacterial inhibitors for NADase was known in Bacillus subtilis, Proteus vulgaris, Proteus rettgeri and Mycobacterium butyricum, but is the first in streptococci. Moreover, with respect to thermal stability, conventional NADase inhibitors are completely inactivated by heating at 60 ° C. for 15 minutes (Acta Path. Microbiol. Scandinav. 69: 277-286 (1967)), whereas SNI. Can retain activity even under such conditions. The SNI of the present invention is not particularly limited as long as it is a Streptococcus derived polypeptide having NADase inhibitory activity. For example, the SNI is a polypeptide comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. possible.

  The present invention also provides a non-natural NADase derived from streptococci or a partial peptide thereof. So far, it has been reported that NADase was purified from the culture supernatant of hemolytic streptococci while maintaining its activity (for example, FEMS Microbiology Letters 130: 201-204 (1995); FEMS Microbiology Letters 136: 71-78 (1996)). However, since streptococcal NADase is highly toxic to host cells such as E. coli, expression of NADase in the host cell, that is, preparation of a recombinant protein has not been successful. This time, the present inventors succeeded for the first time in the preparation of a streptococcal NADase recombinant protein. Based on the research results of the present inventors, not only preparation of a recombinant protein of Streptococcus-derived natural NADase using host cells but also preparation of a recombinant protein of non-natural NADase (mutant NADase or genetically modified NADase) is possible. This is possible for the first time. In addition, by using the recombination technique in this way, preparation of a partial peptide of NADase is facilitated. The non-natural NADase of the present invention is not particularly limited as long as it is a NADase other than the previously reported natural NADase and has a NADase activity. For example, the amino acid sequence represented by SEQ ID NO: 4 or the sequence An amino acid sequence in which the amino acid sequence encoding the secretory signal sequence is removed from the amino acid sequence represented by No. 4 (hereinafter referred to as “the amino acid sequence represented by SEQ ID No. 4 or its secretory signal-removed amino acid sequence”) It may be a polypeptide consisting of an amino acid sequence substantially identical to (omitted) (except for the amino acid sequence represented by SEQ ID NO: 4 or the same amino acid sequence as the secretory signal-removed amino acid sequence).

  As used herein, “NADase activity” refers to any activity possessed by NADase, and unless otherwise specified, (a) hydrolyzes nicotinamide-adenine-dinucleotide (NAD) and adenosine. Activity to produce diphosphoribose (ADPR) and nicotinamide (NAD glycohydrolase activity), (b) activity to synthesize cyclic ADP ribose (cADPR) from NAD (ADP ribosyl cyclase activity), and (c) hydrolyze cADPR It means any activity of cleaving and generating ADPR (cADPR hydrolase activity) (see FIG. 1). This is because streptococcal NADase is considered to have the activities (a) to (c) above.

  As used herein, “NADase inhibitory activity” refers to an activity that suppresses any activity possessed by NADase, and unless otherwise specified, (d) inhibitory activity of NAD glycohydrolase activity, (e) It means the inhibitory activity of any of the inhibitory activity of ADP ribosyl cyclase activity and (f) the inhibitory activity of cADPR hydrolase activity. This is because SNI inhibits the activity of NADase by forming a very stable and strong complex with streptococcal NADase, and is therefore considered to have the inhibitory activities of (d) to (f) above. It is.

NADase having NAD glycohydrolase activity is present in many organisms from bacteria to mammals. Many eukaryotic NADases further combine ADP ribosyl cyclase activity and cADPR hydrolase activity. cADPR is involved in the regulation of signal transduction, and in fact, when eukaryotic cell surface NADase (eg, CD38, CD157) synthesizes cADPR from NAD, intracellular stored Ca ²⁺ concentration is released into the cytoplasm, This increase in calcium ions is a signal that causes various biological responses. Most prokaryotic NADases do not have ADP ribosyl cyclase activity and cADPR hydrolase activity. However, Streptococcus NADase disclosed in the present specification is known to have both of these activities despite being prokaryotic NADse (FEMS Microbiol. Lett. 130: 201-204 (1995). )reference). In addition, this streptococcal NADase is a secreted protein and is different from normal NADase localized in the cytoplasm, cell membrane and the like. For details of streptococcal NADase, see, for example, FEMS Microbiol. Lett. 191: 235-241 (2000).

  In one embodiment, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, or the amino acid sequence represented by SEQ ID NO: 4 or the amino acid sequence substantially identical to the secretory signal elimination amino acid sequence thereof, The amino acid sequence represented by SEQ ID NO: 2, or the amino acid sequence represented by SEQ ID NO: 4 or the amino acid sequence having a predetermined amino acid sequence identity with the secretory signal-removed amino acid sequence may be used. The degree of amino acid sequence identity is, for example, about 70%, preferably about 80%, more preferably about 90%, even more preferably about 95%, most preferably about 97%, about 98% or about 99% or more. possible. Amino acid sequence identity can be determined by a method known per se. For example, amino acid sequence identity (%) can be determined using a program commonly used in the art (eg, BLAST, FASTA, etc.) by default. In another aspect, identity (%) is determined by any algorithm known in the art, such as Needleman et al. (1970) (J. Mol. Biol. 48: 444-453), Myers and Miller (CABIOS, 1988, 4: 11-17). Needleman et al.'S algorithm is incorporated into the GAP program in the GCG software package (available at www.gcg.com) and the percent identity is, for example, BLOSUM 62 matrix or PAM250 matrix, and gap weight: 16, It can be determined by using either 14, 12, 10, 8, 6 or 4 and length weight: 1, 2, 3, 4, 5 or 6. The Myers and Miller algorithms are also incorporated into the ALIGN program that is part of the GCG sequence alignment software package. When the ALIGN program is used to compare amino acid sequences, for example, PAM120 weight residue table, gap length penalty 12, and gap penalty 4 can be used. The amino acid sequence identity may be determined as long as it is determined by any of the above methods, but preferably, a method showing the lowest value among the above methods can be adopted in the calculation.

  In another embodiment, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, or the amino acid sequence represented by SEQ ID NO: 4 or the amino acid sequence substantially identical to the secretory signal-removed amino acid sequence is The amino acid sequence represented by SEQ ID NO: 2, or the amino acid sequence represented by SEQ ID NO: 4 or the amino acid sequence in which one or more amino acids are substituted, added, deleted and / or inserted in the amino acid sequence from which the secretion signal is removed obtain. The number of amino acids to be substituted, added, deleted and / or inserted is not particularly limited as long as it is 1 or more. For example, 1 to about 100, preferably 1 to about 50, more preferably 1 to about 30. Even more preferably, it may be 1 to about 20, most preferably 1 to about 10, 1 to about 5, or 1 or 2.

  The SNI of the present invention may have NADase inhibitory activity and NADase binding activity. When the SNI of the present invention is a polypeptide comprising an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, the degree of activity is determined from the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. The polypeptide may be quantitatively equivalent to the polypeptide, but may be different within an acceptable range (for example, about 0.1 to about 5 times, preferably about 0.5 to about 2 times).

  The SNI of the present invention is also characterized by high thermal stability. Conventional NADase inhibitors have been completely inactivated by heating at 60 ° C. for 15 minutes. However, the SNI of the present invention can retain activity even under such conditions. More specifically, the SNI of the present invention can retain 80% or more activity after heat treatment at 60 ° C. for 15 minutes and 70% or more after heat treatment at 100 ° C.

  The non-natural NADase of the present invention may have NADase activity (NAD glycohydrolase activity, ADP ribosyl cyclase activity, and cADPR hydrolase activity) and SNI binding activity. When the non-natural NADase of the present invention is a polypeptide consisting of an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 4, the degree of activity is the same as that of the amino acid sequence represented by SEQ ID NO: 4. Although it may be quantitatively equivalent to a polypeptide consisting of an amino acid sequence, it may differ within an acceptable range (eg, about 0.1 to about 5 times, preferably about 0.5 to about 2 times).

  The non-natural NADase of the present invention may have the same characteristics as the previously reported natural NADase derived from streptococcus as enzymatic characteristics.

  The partial peptide of the present invention can be a partial peptide of the SNI polypeptide of the present invention or a partial peptide of the NADase polypeptide of the present invention. The length of the partial peptide of the present invention is not particularly limited as long as it has a predetermined activity. For example, at least about 8, preferably at least 10, and more preferably at least 12 selected from the amino acid sequence encoding the polypeptide. Or even more preferably at least 15 and most preferably 20 or more consecutive amino acids. Specifically, examples of the partial peptide of the present invention include a partial peptide of SNI having an ability to bind to NADase and a partial peptide of NADase (including a natural type and a mutant type) having an ability to bind to SNI.

In addition, by producing a polypeptide to which a purification tag or the like is added or a partial peptide thereof in a transformant and using a substance having affinity for the tag (for example, Ni ²⁺ resin, an antibody specific for the tag), The polypeptide of the present invention or its partial peptide can be more easily isolated and purified. Therefore, the polypeptide of the present invention or a partial peptide thereof can have such a purification tag as necessary. The purification tag will be described later.

  The polypeptide of the present invention and its partial peptide can be prepared by a method known per se. Details thereof will be described later.

2. Polynucleotide and its Partial Nucleotide The present invention provides a polynucleotide encoding the SNI of the present invention or a partial nucleotide thereof. The SNI polynucleotide of the present invention may be a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 1. The SNI polynucleotide of the present invention or a partial nucleotide thereof can be useful, for example, for producing the SNI of the present invention or a partial peptide thereof.

  The present invention also includes a polynucleotide encoding a natural NADase, a polynucleotide encoding the non-natural NADase of the present invention (hereinafter, abbreviated as the NADase polynucleotide of the present invention, if necessary), and portions thereof. Nucleotides are provided. Until now, streptococcal NADase is highly toxic to heterologous host cells such as Escherichia coli, and thus has not yet been successful in expressing NADase in a heterologous host cell, that is, preparing a recombinant protein. This time, the present inventors succeeded for the first time in the preparation of a streptococcal NADase recombinant protein. The present inventors' research results enable for the first time a meaningful use of the polynucleotide of the present invention that can be used for the preparation of recombinant proteins of Streptococcus-derived natural NADase and non-natural NADase using heterologous host cells. became. The NADase polynucleotide of the present invention includes, for example, a nucleotide sequence represented by SEQ ID NO: 3 or a nucleotide sequence obtained by removing a nucleotide sequence encoding a secretory signal sequence from the nucleotide sequence represented by SEQ ID NO: 3 (hereinafter referred to as necessary). Accordingly, it may be a polynucleotide comprising the same or substantially the same nucleotide sequence as the “nucleotide sequence represented by SEQ ID NO: 3 or its secretion signal-removed nucleotide sequence”. The NADase polynucleotide of the present invention or a partial nucleotide thereof can be useful, for example, for producing the NADase of the present invention or a partial peptide thereof.

  In one embodiment, the nucleotide sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 1, or the nucleotide sequence represented by SEQ ID NO: 3 or the nucleotide sequence substantially identical to the secretion signal elimination nucleotide sequence thereof, It may be the nucleotide sequence represented by SEQ ID NO: 1, or the nucleotide sequence represented by SEQ ID NO: 3 or a nucleotide sequence having a predetermined sequence identity with the nucleotide sequence from which the secretory signal is removed. The degree of nucleotide sequence identity is, for example, about 70%, preferably about 80%, more preferably about 90%, even more preferably about 95%, most preferably about 97%, about 98% or about 99% or more. possible. Nucleotide sequence identity can be determined by methods known per se. For example, nucleotide sequence identity (%) can be determined in the same manner as amino acid sequence identity (%) described above.

  In another embodiment, the nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 1, or the nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 3 or its secretion signal elimination nucleotide sequence is: , A nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence represented by SEQ ID NO: 3 or a nucleotide sequence in which one or more nucleotides are substituted, added, deleted and / or inserted in the nucleotide sequence from which a secretory signal is removed obtain. The number of nucleotides to be substituted, added, deleted and / or inserted is not particularly limited as long as it is 1 or more. For example, 1 to about 300, preferably 1 to about 150, more preferably 1 to about 100. Even more preferably from 1 to about 50, most preferably from 1 to about 30, from 1 to about 20, from 1 to about 10 or from 1 to about 5.

  In yet another embodiment, the nucleotide sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 1, or the nucleotide sequence represented by SEQ ID NO: 3 or the nucleotide sequence substantially identical to the secretory signal elimination nucleotide sequence thereof Can be a polynucleotide capable of hybridizing under high stringency conditions to the nucleotide sequence represented by SEQ ID NO: 1, or the nucleotide sequence represented by SEQ ID NO: 3 or the complementary sequence of the secretory signal elimination nucleotide sequence thereof. (However, the nucleotide sequence represented by SEQ ID NO: 1, or the nucleotide sequence represented by SEQ ID NO: 3 or its secretory signal-removed nucleotide sequence is excluded). Hybridization conditions under high stringency conditions can be set with reference to previously reported conditions (Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999). For example, hybridization conditions under high stringency conditions include hybridization at 6 × SSC (sodium chloride / sodium citrate) / 45 ° C., then 0.2 × SSC / 0.1% SDS / 50 to 65 ° C. Or two or more washes.

  The NADase polynucleotide of the present invention can encode a non-natural NADase of the present invention or a polypeptide having the same activity as that of the natural NADase. When the NADase polynucleotide of the present invention is a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3 or a nucleotide sequence substantially the same as the secretory signal-removed nucleotide sequence, the activity of the polypeptide encoded by the polynucleotide Can be quantitatively equivalent to the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 or its secretory signal-removed amino acid sequence, but within an acceptable range (for example, about 0.1 to about 5 times, preferably May be about 0.5 to about 2 times).

  The partial nucleotide of the present invention can be a nucleotide encoding the SNI partial peptide of the present invention or the NADase partial peptide of the present invention. Specifically, the partial nucleotide of the present invention consists of the nucleotide sequence represented by SEQ ID NO: 1, or the nucleotide sequence represented by SEQ ID NO: 3, or the same or substantially the same nucleotide sequence as the secretion signal elimination nucleotide sequence thereof. It can be a partial nucleotide of a polynucleotide. As the partial nucleotide of the present invention, for example, a polynucleotide encoding a partial peptide of SNI having an ability to bind to NADase, and a partial peptide of NADase (including a natural type and a mutant) having an ability to bind to SNI are encoded. A polynucleotide is mentioned.

  The polynucleotide of the present invention can be prepared by a method known per se. For example, a polynucleotide comprising the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 1, or the nucleotide sequence represented by SEQ ID NO: 3 or the same nucleotide sequence as the secretory signal elimination nucleotide sequence is Can be extracted, and cDNA can be prepared from mRNA, followed by PCR using appropriate primers. In addition, a polynucleotide comprising a nucleotide sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence represented by SEQ ID NO: 3 or a nucleotide sequence substantially identical to the secretory signal elimination nucleotide sequence thereof, These can be prepared by introducing mutations into the cloned polynucleotide as described above. Examples of the mutagenesis method include, for example, a synthetic oligonucleotide-directed mutagenesis method, a gapped duplex method, a method of randomly introducing point mutations (for example, treatment with nitrous acid or sulfite), a cassette mutagenesis method, a linker scanning method, Examples include a mismatch primer method.

3. Expression Vector The present invention provides an expression vector useful for the preparation of a protein expressed from an operon containing NADase, SNI and SLO genes. The expression vector of the present invention can be prepared by a molecular biological technique known per se by referring to the following. For example, for the molecular biological ^{techniques, Molecular Cloning, 2 nd edition,} J. Sambrook et al., Cold Spring Harbor Lab. Press (1989) reference.

3.1. Expression vector of SNI or a partial peptide thereof The present invention provides an expression vector of SNI or a partial peptide thereof (hereinafter abbreviated as SNI expression vector if necessary).

  In one embodiment, the SNI expression vector may be an expression vector intended for expression of SNI or a partial peptide thereof in a host cell. The SNI expression vector is not particularly limited as long as it can express the gene encoding the SNI polypeptide of the present invention or a partial peptide thereof in various host cells of prokaryotic cells and / or eukaryotic cells and produce them. However, for example, plasmid vectors and virus vectors can be mentioned. When the expression vector is used as a pharmaceutical, vectors suitable for administration to mammals such as humans include adenovirus, retrovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai. Examples thereof include virus vectors such as viruses and Epstein-Barr viruses.

  When a prokaryotic cell such as a bacterium (for example, E. coli) is used as a host cell for SNI production, the expression vector is, for example, a promoter-operator region, a start codon, a polynucleotide encoding the SNI polypeptide of the present invention or a partial peptide thereof. May contain elements such as a stop codon, terminator region, origin of replication and the like. The promoter-operator region for expressing the polypeptide of the present invention in bacteria contains a promoter, an operator and a Shine-Dalgarno (SD) sequence. As these elements, those known per se can be used.

  Moreover, when using a SNI expression vector as a pharmaceutical or research reagent, the expression vector which can utilize eukaryotic cells, such as yeast, an animal cell, or an insect cell, as a host cell can be used. In this case, the promoter used is not particularly limited as long as it can function in eukaryotes such as mammals to be administered. For example, when used as a medicament, SV40-derived early promoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived LTR, adenovirus-derived early promoter, etc., and β-actin gene promoter, PGK gene promoter, transferrin gene Examples include mammalian component protein gene promoters such as promoters.

  The SNI expression vector further includes a selection marker gene (a gene that confers resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, a gene that complements an auxotrophic mutation, etc.). Also good.

  In another embodiment, the SNI expression vector can be a fusion protein expression vector. In this case, SNI or a partial peptide thereof can be used as a purification tag. Therefore, the partial peptide of SNI can be a partial peptide of SNI having the ability to bind to NADase. Since the SNI of the present invention can bind to NADase even in the presence of a high concentration salt of 3M NaCl, the SNI or the partial peptide capable of binding to NADase or SNI can be used as a binding pair. The fusion protein to which the partial peptide is added can be purified. Such an SNI expression vector may contain a multiple cloning site (MCS) upstream or downstream of a polynucleotide encoding SNI or a partial peptide thereof in order to facilitate cloning of the target protein to be fused.

  The SNI expression vector may be in the same form as the NADase expression vector described later or the SLO expression vector described later. That is, “NADase” in the section “3.2. Expression vector of NADase or its partial peptide” and “SLO” in section “3.3. Expression vector of SLO or its partial peptide”, which will be described in detail later, It can be read as “SNI”, and such an expression vector can also be “an expression vector of SNI or a partial peptide thereof”.

3.2. Expression vector of NADase or a partial peptide thereof The present invention provides an expression vector of NADase or a partial peptide thereof (hereinafter abbreviated as NADase expression vector if necessary).

  In one embodiment, the NADase expression vector can be a co-expression vector that allows expression of NADase or a partial peptide thereof in a host cell. Since NADase is generally toxic to host cells, it is difficult to express NADase in host cells. In particular, NADase derived from streptococci is strongly toxic to host cells such as Escherichia coli. The preparation has not yet been successful. However, in order to enable the production of NADase in a host cell, the present inventors may co-express NADase with SNI or a partial peptide that has a neutralizing action against toxic effects on the host cell. I found.

  Thus, the NADase expression vector can include a polynucleotide encoding the first and second expression units, and a promoter operably linked to the polynucleotide encoding the expression unit. The first and second expression units can be NADase or a partial peptide thereof having a toxic effect on host cells, and SNI or a partial peptide thereof having a neutralizing effect on the toxic effect. A functional linkage of a promoter means that the promoter is bound to a polynucleotide encoding the gene so as to allow expression of the gene under its control.

  The host cell into which the NADase expression vector can be introduced is not particularly limited as long as it is an arbitrary host cell to be described later. For example, prokaryotic cells such as Escherichia coli and Bacillus (eg, Bacillus subtilis) are preferable. Examples of the toxic effect on the host cell include NADase activity. Examples of the neutralizing action against the toxic action on the host cell include NADase inhibitory activity.

  More specifically, the NADase expression vector can be a polycistronic mRNA expression vector, particularly when the NADase is a streptococcal NADase. The polycistronic mRNA expression vector is a polynucleotide encoding the first and second expression units that enables the expression of the polycistronic mRNA of the first and second expression units, and the expression of the polycistronic mRNA. A promoter operably linked to the polynucleotide may be included. In the polycistronic mRNA expression vector, the polynucleotide encoding the NADase or a partial peptide thereof having a toxic effect on the host cell and the polynucleotide encoding the SNI or a partial peptide thereof having a neutralizing action against the toxic effect are either upstream or Although it may be located downstream, the former is preferably located upstream from the viewpoint of reflecting the natural state (that is, the nga-slo operon).

  The NADase expression vector can also be a non-polycistronic mRNA expression vector. Non-polycistronic mRNA expression vector includes a polynucleotide encoding a first expression unit, a first promoter operably linked to the polynucleotide, and a polynucleotide encoding a second expression unit and the polynucleotide A second promoter operably linked to can be included.

  The NADase expression vector can be, for example, the polycistronic mRNA expression vector or the non-polycistronic mRNA expression vector described above, and preferably a polycistronic mRNA expression vector.

  The NADase expression vector can be similar to the features of the SNI expression vector except for the features described above. That is, the co-expression vector of the present invention may be a plasmid vector or a viral vector, and may contain an element such as a promoter similar to the expression vector of SNI or a partial peptide thereof, and a selection marker gene.

  In another embodiment, the NADase expression vector can be a fusion protein expression vector. In this case, NADase or a partial peptide thereof can be used as a purification tag. Therefore, the partial peptide of NADase may be a partial peptide of NADase having the ability to bind to SNI. Since NADase can bind to SNI even in the presence of a high concentration salt of 3M NaCl, NADase or its partial peptide can be obtained by using its partial peptide capable of binding to SNI or NADase as a binding pair. It is possible to purify the fusion protein to which is added. Such an expression vector may contain a multiple cloning site (MCS) upstream or downstream of the polynucleotide encoding NADase or a partial peptide thereof in order to facilitate cloning of the target protein to be fused.

  The NADase expression vector may be in the same form as the SNI expression vector described above or the SLO expression vector described later. That is, “SLO” in the section “3.1. Expression vector of SNI or its partial peptide” described in detail above, and “SLO” in the section “3.3. Expression vector of SLO or its partial peptide” described in detail later. "Can be read as" NADase ", and such an expression vector can also be" an expression vector of NADase or a partial peptide thereof ".

3.3. Expression vector of SLO or a partial peptide thereof The present invention is a full-length SLO derived from hemolytic streptococci or an expression vector of a partial peptide containing a region that can be toxic to Escherichia coli (hereinafter abbreviated as SLO expression vector if necessary). Provide). The SLO expression vector comprises a polynucleotide encoding a full-length SLO derived from hemolytic streptococci or a partial peptide containing a region that can be toxic to E. coli, and an inducible promoter operably linked to the polynucleotide.

  Streptolysin O (SLO) is a species of five genera (streptococcus, Bacillus, Clostridium, Listeria, Arcanobacterium). Belong to a highly homologous single family of cytolysin found (eg, Pharmacol Ther 11: 661-717 (1980); Int J Med Microbiol 290: 351-356 (2000); FEMS Microbiol Lett 182: 197-205 ( 2000); Arch Microbiol 165: 73-79 (1996); Infect Immun 47, 52-60 (1985); Toxicon 39: 1681-1689 (2001)). The SLO that can be the subject of the present invention is not particularly limited as long as it is an SLO derived from the above, but the SLO gene is highly conserved between different groups of hemolytic streptococci, so that the SLO derived from hemolytic streptococci is preferred. Examples of hemolytic streptococci include group A to group C and group G streptococci, but as onset factors such as purulent sore throat, wound infection and streptococcal toxic shock-like syndrome, scarlet fever, and acute glomerulonephritis Group A streptococci (GAS) with reports (eg, Clin Microbiol Rev 13: 470-511 (2000); FEMS Immunol Med Microbiol 34: 159-167 (2002); J Clin Microbiol 42: 186-192 (2004)) ), Group C streptococci (GCS), and group G streptococci (GGS) are preferred. In GAS and GCS, it has been reported that all SLOs are secreted by removing 31 amino acid residues including the N-terminal signal peptide, and gradually decrease in molecular weight by proteases secreted by themselves. (See, for example, Infect Immun 61: 2727-2731 (1993); Infect Immun 63: 2776-2779 (1995)). For these reasons, GAS and GCS full-length SLOs can be particularly difficult to purify, but the methods developed by the inventors allow these full-length SLOs to be purified. Therefore, among SLOs derived from the above streptococci, SLOs derived from GAS and GCS are more preferable. By the way, several subspecies are known for GAS and GCS. Examples of the GAS subspecies from which SLO that can be the subject of the present invention is derived include the D58X strain (ATCC 12383). Examples of the subtype of GCS from which SLO that can be the subject of the present invention is derived include the H46A strain (ATCC12449).

  Full-length SLO means SLO after removal of the N-terminal secretory signal peptide.

  A partial peptide of full-length SLO containing a region that can be toxic to E. coli means any fragment containing a peptide portion that can be toxic to E. coli. For example, SLO derived from hemolytic streptococci such as GAS and GCS has such a region in the region near the N-terminal, particularly in the region of the polypeptide consisting of amino acid residues of about 32 to about 75. (See, for example, Biosci Biotechnol Biochem 65: 2682-2689 (2001)). Therefore, a partial peptide containing a region that can be toxic to E. coli can include a peptide portion that can be toxic to E. coli contained in such a polypeptide.

  A full-length SLO or a partial peptide thereof that can be expressed from an SLO expression vector preferably has a purification tag. Thus, an SLO expression vector may comprise an in-frame ligation of a polynucleotide encoding a purification tag and a polynucleotide encoding SLO that allows expression of the purification tag fusion SLO. The purification tag is not particularly limited as long as it facilitates the purification of SLO from the culture supernatant of the transformant introduced with the expression vector of the present invention. For example, a His tag, maltose binding protein (MBP) ), Glutathione-S-transferase (GST), calmodulin-binding peptide (CBP), Strep-tag II, FLAG, and hey chain of protein C (HPC), but the added amino acid sequence is short and is often removed. The His tag is preferred because of its advantages that it does not need to be performed, and a plurality of commercially available products and specific antibodies and purification carriers are available.

  Inducible promoter means a promoter that enhances the expression of a gene under its control in the presence of an inducing agent. In the SLO expression vector, the inducible promoter can be operably linked to a polynucleotide encoding the SLO gene.

  Inducible promoters included in SLO expression vectors can also repress basal levels of gene expression in E. coli. Basal level gene expression means gene expression under non-inducible conditions. Since SLO can contain a region that can be toxic to E. coli, if the expression level of the SLO gene at the basal level is large, E. coli is likely to be damaged, so that it is difficult to massly express SLO containing such a region. was there. However, the present inventors have succeeded in expressing a large amount of full-length SLO or its partial peptide containing a region that can be toxic to E. coli in E. coli by using an inducible promoter capable of strictly suppressing basal level gene expression. did. Examples of such inducible promoters that can suppress basal level gene expression in E. coli include, for example, arabinose operon-regulated promoters (eg, araBAD using L-arabinose as an inducer: for example, pBAD series vectors can be used), xylose Operon-regulated promoter (eg, xylA promoter using xylose as an inducer: for example, pWH1520 can be used), salicylate-controllable promoter (eg, Pm promoter using salicylate as an inducer: for example, pCAS series vectors Available), promoters that can be controlled by low-temperature treatment (eg, cspA promoter that can be induced by low-temperature treatment: for example, pCold series of vectors can be used), from the viewpoint of ease of use, expression level, etc. Rabbi North operon control promoter is preferable.

  SLO expression vectors can also include sites for transcription initiation and termination, and ribosome binding sites that may be required for translation in the transcribed region, origins of replication, selectable marker genes, and the like. The selection marker gene is not particularly limited as long as it can select transformed E. coli when E. coli is used as a host. For example, ampicillin, tetracycline, kanamycin, spectinomycin, erythromycin, chloramphenicol And resistance genes to antibiotics such as carbenicillin.

4). Transformant The present invention provides a transformant useful for preparing a protein expressed from an operon containing NADase, SNI, and SLO genes, or a transformant capable of analyzing such a protein.

  The transformant of the present invention can be prepared by introducing the above-described expression vector of the present invention or another expression vector into a host cell. The host cell used for the preparation of the transformant is not particularly limited as long as it is compatible with the above expression vector and can be transformed. Natural cells commonly used in the technical field of the present invention or artificially used. Various cells such as established cell lines can be used. Preferred host cells according to each transformant are described below. Each transformant can be cultured by a method known per se (for example, see the below-mentioned culture method).

4.1. Transformant capable of expressing SNI or a partial peptide thereof The present invention provides a transformant capable of expressing SNI or a partial peptide thereof (hereinafter abbreviated as SNI transformant if necessary).

  The SNI transformant can be prepared by transforming a host cell with the expression vector of the SNI of the present invention or a partial peptide thereof.

  Examples of host cells for SNI transformants include E. coli (eg, E. coli described in detail in “4.3. Transformant capable of expressing SLO or its partial peptide”), Bacillus (eg, Bacillus subtilis). ), Eukaryotic cells such as actinomycetes, and eukaryotic cells such as yeast, insect cells, avian cells, mammalian cells (eg, cells derived from primates such as humans and monkeys, rodents such as mice and rats). Biological cells can be mentioned. In addition, when aiming at manufacturing SNI or its partial peptide in large quantities, it is preferable to use a prokaryotic cell as a host cell.

4.2. Transformant capable of expressing NADase or a partial peptide thereof The present invention provides a transformant capable of expressing NADase or a partial peptide thereof (hereinafter abbreviated as NADase transformant if necessary). Since NADase is generally toxic to host cells, it is difficult to express NADase in host cells. In particular, NADase derived from streptococci is strongly toxic to host cells such as Escherichia coli. The preparation has not yet been successful. However, in order to allow the production of NADase in a host cell or a partial peptide thereof having a toxic effect on the host cell, the present inventors co-administered SNI or the partial peptide having a neutralizing effect on the toxic effect. It has been found that a transformant to be expressed may be prepared.

  NADase transformants can be prepared by transforming host cells with NADase expression vectors (eg, the polycistronic mRNA expression vectors and non-polycistronic mRNA expression vectors described above). NADase transformants can also be produced by transforming host cells with both a vector that expresses only NADase and does not express SNI (ie, a NADase single expression vector) and an SNI expression vector.

  Examples of the host cell for the NADase transformant include the same cells as the SNI transformant described above. In addition, when it aims at manufacturing NADase or its partial peptide in large quantities, it is preferable to use a prokaryotic cell as a host cell.

4.3. Transformant capable of expressing SLO or a partial peptide thereof The present invention is a transformant capable of expressing a large amount of a full-length SLO derived from hemolytic streptococci or a partial peptide containing a region that can be toxic to Escherichia coli (hereinafter referred to as a transformant). Abbreviated as SLO transformant if necessary).

  An SLO transformant can be prepared by transforming a host cell with an expression vector of the SLO of the present invention or a partial peptide thereof.

  As the host cell of the SLO transformant, Escherichia coli is preferable in view of its purpose.

  Escherichia coli that can be used as a host may be a protease-deficient strain in view of one object of the present invention of producing full-length SLO with a large amount and high purity (in other words, suppressing SLO cleavage). preferable. Examples of such protease to be deleted in E. coli include lon and ompT. Examples of protease-deficient strains that can be used as hosts in the present invention include the BL21 strain.

  In addition, E. coli that can be used as a host exhibits the ability of an inducible promoter to a sufficient extent, and in consideration of another object of the present invention to produce a large amount of SLO containing a region that can be toxic to E. coli, It is preferred that the inducer is deficient in metabolic capacity. The metabolic ability to be deficient varies depending on the type of inducible promoter used in the present invention, but may be, for example, the metabolic ability of the above-mentioned inducer of the inducible promoter.

5). Method for Producing Polypeptide or Partial Peptide Using Transformant The present invention provides a method for producing a polypeptide or partial peptide thereof using the transformant of the present invention. For example, the production method of the present invention includes culturing the transformant of the present invention to produce a target polypeptide or a partial peptide thereof, and then recovering it. When the transformant of the present invention includes an expression vector having an inducible promoter, the production method of the present invention can also include adding an inducing agent for the inducible promoter to the medium and culturing.

  The transformant can be cultured in a nutrient medium such as a liquid medium by a method known per se. The medium preferably contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. Here, examples of the carbon source include glucose, dextrin, soluble starch, and sucrose, and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, large extract, and the like. Examples include inorganic or organic substances such as soybean cake and potato extract, and examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. Moreover, you may add a yeast extract, vitamins, etc. to a culture medium. Culture conditions such as temperature, medium pH and culture time are appropriately selected so that the polypeptide of the present invention is produced in large quantities. The culture temperature is, for example, 30 to 37 ° C.

  When the transformant of the present invention contains an expression vector having an inducible promoter, the transformant of the present invention can be cultured in the presence of an inducing agent for the inducible promoter. Culturing of the transformant in the presence of the inducing agent can be carried out under an appropriate condition depending on the kind of the inducing agent, using a transformant grown to an appropriate number for large-scale expression under non-inducing conditions. For example, in the method for producing full-length SLO or its partial peptide using Escherichia coli as a host cell for the transformant, when the inducible promoter is an arabinose operon-controlled promoter, the concentration of L-arabinose added to the medium is 0. When the ratio is 002% (W / V) or more, sufficient expression of full-length SLO or a partial peptide thereof has been confirmed. Therefore, although the concentration of L-arabinose added to the medium is not particularly limited as long as it enables efficient expression of full-length SLO or a partial peptide thereof, it is preferably about 0.002% (W / V) or more. Preferably, it may be about 0.02% (W / V) or more. In addition, although the upper limit density | concentration of L-arabinose is not specifically limited, For example, it may be 0.2% (W / V) or less. The time for which the Escherichia coli of the present invention is placed under induction conditions is not particularly limited as long as it enables efficient expression of full-length SLO or a partial peptide thereof, and examples thereof include about 1 to about 24 hours. . Note that the culture under the induction condition can be performed in the same manner as the culture under the non-induction condition except that the inducer is used under a predetermined condition.

  A polypeptide produced by culturing a transformant or a partial peptide thereof can be recovered, preferably isolated and purified. For example, SNI or a partial peptide thereof can be recovered from the lysate after collecting the cultured transformants and subjecting them to a lysis treatment (eg, treatment with lysozyme, sonication, etc.). Moreover, NADase, SLO, or those partial peptides can be collect | recovered from the culture supernatant.

  As an isolation and purification method, for example, a method using solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, a method using molecular weight difference such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Methods using charges such as ion exchange chromatography and hydroxylapatite chromatography, methods using specific affinity such as affinity chromatography, methods using hydrophobic differences such as reversed-phase high-performance liquid chromatography, isoelectricity Examples thereof include a method using a difference in isoelectric point such as point electrophoresis.

  According to the production method of the present invention, it is possible to produce SNI, NADase, SLO and their partial peptides in high purity and in large quantities at low cost. For example, in the method for producing full-length SLO derived from hemolytic streptococci or its partial peptide, for example, about 1 mg or more, preferably about 1 mg to about 30 mg per liter of LB medium, depending on the medium used and induction conditions. Preferably, yields of about 5 mg to about 30 mg, even more preferably about 5 mg to about 20 mg, and most preferably about 5 mg to about 10 mg are possible. In addition, SLO obtained by the production method of the present invention has a specific activity similar to that of a natural protein obtained from the culture supernatant of hemolytic streptococci, despite being a recombinant protein produced in a heterologous host. Can do.

6). antibody

  The present invention provides antibodies against SNI, NADase, SLO and their partial peptides.

The antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a well-known immunological technique. This antibody may be not only a complete antibody molecule but also any fragment as long as it has an antigen binding site (CDR) for the polypeptide of the present invention, such as Fab, F (ab ′) ₂ , ScFv, minibody, etc. Is mentioned.

For example, polyclonal antibodies, the antigen (if desired, bovine serum albumin, KLH (may be a K eyhole L impet H emocyanin) complexes crosslinked to a carrier protein, etc.), commercial adjuvants (e.g., (Complete or incomplete Freund's adjuvant) and administered subcutaneously or intraperitoneally 2 to 4 times every 2 to 3 weeks (the antibody titer of the partially collected serum is measured by a known antigen-antibody reaction) It can be obtained by collecting the whole blood about 3 to 10 days after the final immunization and purifying the antiserum. Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

  A monoclonal antibody can be prepared by a cell fusion method. For example, the above antigen is administered to mice subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and spleen or lymph nodes are collected 3 days after the final administration, and white blood cells are collected. The leukocytes and myeloma cells (for example, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the factor. Cell fusion may be PEG or voltage pulse. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a well-known EIA or RIA method. The hybridoma producing the monoclonal antibody can be cultured in vitro, or in vivo, such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively.

  Furthermore, the antibody of the present invention may be a chimeric antibody, a humanized antibody, or a human antibody. Chimeric antibodies include, for example, “Experimental Medicine (Special Issue), Vol. 6, No. 10, 1988”, Japanese Patent Publication No. 3-73280, and humanized antibodies include, for example, Japanese Patent Publication No. 4-506458, JP-A-62-296890 and the like, human antibodies include, for example, `` Nature Genetics, Vol.15, p.146-156, 1997 '', `` Nature Genetics, Vol.7, p.13-21, 1994 '', JP 4-504365 Publication, International Application Publication No. WO 94/25585 Publication, `` Nikkei Science, June, 40 to 50, 1995 '', `` Nature, Vol.368, p.856-859, 1994 ", Japanese Patent Publication No. 6-500233, and the like. For example, when the antibody of the present invention is used as a medicine, a humanized antibody and a human type antibody are preferable.

  The antibody of the present invention (in particular, the SLO antibody described later) is useful as a reagent such as a clinical test reagent, a research reagent, and a medicine. When the antibody of the present invention is used as a reagent, for example, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), immunochromatography, luminescence immunoassay, spin immunoassay Can be used for detection and quantification of antigens (especially SLO) by Western blotting. Examples of the EIA method include a direct competition ELISA, an indirect competition ELISA, and a sandwich ELISA. On the other hand, when the antibody of the present invention is used as a medicine, it can be used, for example, as a therapeutic agent for hemolytic streptococcal infection.

  When the antibody of the present invention is used as a medicine, the medicine of the present invention may be formulated with a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier include those described later. The antibody of the present invention can be preferably administered parenterally according to the method described below at the dose described below.

6.1. Antibody to SNI or its partial peptide The present invention provides an antibody against SNI or its partial peptide (hereinafter abbreviated as SNI antibody as necessary).

  As an antigen used for production of an SNI antibody, a polypeptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof can be used. The partial peptide is not particularly limited as long as it has immunogenicity. For example, the partial peptide is selected from the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence encoding the above-mentioned domain. It may be a peptide consisting of at least about 8, preferably at least 10, more preferably at least 12, even more preferably at least 15, most preferably 20 or more consecutive amino acids.

  In addition, in the case of aiming to obtain a neutralizing antibody, it is preferable to use a partial peptide having NADase inhibitory activity as an antigen, and in the case of aiming to obtain a dissociator for the complex of the present invention described later. It is preferable to use a partial peptide thereof capable of binding to NADase as an antigen.

6.2. Antibody to NADase or its partial peptide The present invention provides an antibody to NADase or its partial peptide (hereinafter abbreviated as NADase antibody as necessary). Based on the research results of the present inventors, not only preparation of recombinant proteins of Streptococcus derived natural NADase using host cells, but also non-natural NADase (mutant NADase or genetically modified NADase) using host cells It is possible for the first time to prepare a replacement protein. In addition, by using the recombination technique in this way, preparation of a partial peptide of NADase is facilitated. Therefore, the production of NADase antibody can be facilitated as compared with the prior art.

  As an antigen used for preparation of the NADase antibody, a polypeptide comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4 or a partial peptide thereof can be used. The partial peptide is not particularly limited as long as it has immunogenicity. For example, the partial peptide is selected from the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence encoding the above-mentioned domain. It may be a peptide consisting of at least about 8, preferably at least 10, more preferably at least 12, even more preferably at least 15, most preferably 20 or more consecutive amino acids.

  In addition, when it is intended to obtain a neutralizing antibody, it is preferable to use a partial peptide having NADase activity as an antigen. When the purpose is to obtain a dissociator for the complex of the present invention described later. Preferably uses a partial peptide thereof capable of binding to SNI as an antigen.

6.3. Antibody to SLO or its partial peptide The present invention provides an antibody against SLO or its partial peptide (hereinafter abbreviated as SLO antibody as necessary).

  Antigens used for the production of SLO antibodies include full-length SLO derived from hemolytic streptococci or partial peptides containing regions that can be toxic to E. coli (for example, polymorphs containing amino acid residues from about 32 to about 75). A partial peptide of the peptide) can be used. The partial peptide is not particularly limited as long as it has immunogenicity. For example, at least 8, preferably at least 10, more preferably at least 12, and even more preferably at least 15 selected from any part of the SLO. It may be a polypeptide consisting of two or more consecutive amino acids.

  An SLO antibody is, for example, an antibody that exhibits specific reactivity against full-length SLO or a partial peptide containing a region that can be toxic to E. coli and does not exhibit cross-reactivity to NADase, particularly NADase derived from hemolytic streptococci. possible. The antibody of the present invention can also be a neutralizing antibody of full-length SLO or the activity of a partial peptide containing a region that can be toxic to E. coli, particularly hemolytic activity.

7). Other Inventions Related to SNI The present invention provides nucleic acids complementary to SNI polynucleotides or partial nucleotides (antisense nucleic acids), RNAi-derived nucleic acids (siRNA), ribozymes, amplification primer pairs, and the like. These may be useful, for example, for analysis of the SNI of the present invention, production of mutant streptococci, and the like.

8). Pharmaceutical or Reagent The present invention also provides a pharmaceutical or reagent comprising a partial peptide having SNI or NADase inhibitory activity or an expression vector thereof. The present inventors have succeeded for the first time in elucidating a part of the function of the unknown gene SNI. More particularly, the inventors have found that SNI can inhibit NADase. Therefore, the present inventors have made it possible for the first time to use the partial peptides having SNI and NADase inhibitory activity or their expression vectors as pharmaceuticals or reagents.

  The medicament of the present invention is useful as a therapeutic agent for infectious diseases of bacteria such as streptococci. Streptococcus NADase, which is a secreted protein, is secreted into biological fluids such as blood, enters the host cell through pores formed on the host cell membrane by SLO, and exhibits its toxicity by NADase activity. It is thought that it can carry the pathology of infectious diseases. Therefore, if the partial peptide having SNI or NADase inhibitory activity is present in the living body, a stable complex between Streptococcus NADase and SNI or the partial peptide is formed, thereby reducing the NADase activity of Streptococcus NADase. It is considered that the pathological condition of streptococcal infection can be reduced by obtaining. The infectious disease to which the medicament of the present invention can be applied is not particularly limited as long as it is an infectious disease that can be caused by a bacterium that exerts a toxic effect by NADase, but for example, by group A, group C, and group G hemolytic streptococci Infectious diseases that are caused include.

  The medicament of the present invention is also useful for the treatment of non-infectious diseases. The present inventors have found that SNI can inhibit not only bacterial NADase but also mammalian NADase. Therefore, the partial peptide having SNI or NADase inhibitory activity is treated symptomatically by diseases that can be brought about by hyperfunction of mammal-derived NADase (eg, CD38, CD157), or by inhibiting function of mammal-derived NADase. It is considered effective against a disease obtained (for example, a disease whose symptoms can be ameliorated, alleviated or alleviated by inhibiting the function of mammalian NADase). Examples of such diseases include autoimmune diseases (eg, ulcerative colitis), inflammatory diseases (eg, adult respiratory distress syndrome (ARDS), arthritis), allergic diseases, multiple myeloma , Ischemia, asthma, diabetes, transplant rejection, neurodegenerative diseases, but are not limited to these (eg, WO2002 / 032288; Nature Medicine 7 (11): 1209-16 (2001); The Journal of Immunology 172: 1896-1906 (2004)).

The medicament or reagent of the present invention is further useful as a NADase inhibitor. The NADase inhibitor means one having the above-mentioned NADase inhibitory activity. Because NADase is widely recognized from bacteria to mammals, and SNI inhibited bacteria and mammalian NADase, it is believed that SNI can inhibit NADse from any species. NADse that can be inhibited by a partial peptide having SNI or NADase inhibitory activity includes any NADase from prokaryotic to eukaryotic, including, for example, bacteria (eg, streptococci), mammals (eg, Humans, monkeys, chimpanzees, rabbits, cattle), insects, birds, fishes, mollusks (eg, snails), plants, and mammals such as bacteria and humans are preferred. Specifically, examples of streptococcal NADase that can be inhibited by a partial peptide having SNI or NADase inhibitory activity include group A, group C, and group G streptococcal NADase. Examples of mammalian-derived NADases that can be inhibited by a partial peptide having SNI or NADase inhibitory activity include CD38, CD157, rabbit ADP ribosyl cyclase, and bovine NAD ⁺ glycohydrolase, with CD38 and CD157 being preferred. NADases can also be classified into secreted, cell membrane-bound, cytoplasmic localized, etc., depending on their localization. The partial peptide having SNI or NADase inhibitory activity can inhibit any of these types of NADase, but preferably can inhibit secreted or cell membrane-bound NADase. In addition, since the enzymatic activity of CD38 (and generation of cADPR) can be involved in the release of Ca ²⁺ from intracellular Ca ²⁺ stores, cell proliferation, apoptosis, etc. (see, for example, WO2002 / 032288) Drugs and reagents can inhibit these effects. The NADase inhibitor of the present invention is also a cell line (eg, use of the transformant of the present invention) or a cell-free system (eg, use of the co-expression vector of the present invention, or a combination of NADase expression vector and SNI expression vector). It can be used as a preparation agent of NADase in use or addition of SNI in the NADase synthesis system).

  The medicament or reagent of the present invention is also useful as an inhibitor of leukocyte chemotaxis. Leukocyte chemotaxis refers to the recruitment of leukocytes to the site of inflammation. CD38 and CD157 are known to play an important role in leukocyte chemotaxis (for example, Blood vol. 104 (13): 4269-4278 (2004), Nat Med. Vol. 7 (11)). : 1209-16 (2001)). Examples of leukocytes whose chemotaxis can be inhibited by a partial peptide having SNI or NADase inhibitory activity include neutrophils, eosinophils, monocytes, lymphocytes, macrophages, and dendritic cells (for example, WO2002 / 032288), neutrophils are preferred.

  The medicament of the present invention may be formulated with a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier include excipients such as sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate , Aerosil, Talc, Lubricant such as sodium lauryl sulfate, Citric acid, Menthol, Glycyllysine / Ammonium salt, Glycine, Orange powder and other fragrances, Sodium benzoate Preservatives such as sodium, sodium bisulfite, methylparaben, propylparaben, stabilizers such as citric acid, sodium citrate, acetic acid, suspensions such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, dispersants such as surfactants, Examples include, but are not limited to, water, physiological saline, diluents such as orange juice, base waxes such as cacao butter, polyethylene glycol, and white kerosene.

  Formulations suitable for oral administration include a solution in which an effective amount of a ligand is dissolved in a diluent such as water, physiological saline, orange juice, a capsule containing an effective amount of the ligand as a solid or granule, a sachet or Examples thereof include tablets, suspensions in which an effective amount of an active ingredient is suspended in a suitable dispersion medium, and emulsions in which a solution in which an effective amount of an active ingredient is dissolved is dispersed in an appropriate dispersion medium and emulsified.

  Formulations suitable for parenteral administration (eg, intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions. May contain antioxidants, buffers, antibacterial agents, tonicity agents and the like. Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. Alternatively, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.

  The dosage of the preparation of the present invention varies depending on the type and activity of the active ingredient, the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age, etc. The amount of active ingredient can be about 0.001 to about 2.0 g / kg.

9. Complex and method for producing the same The present invention also includes a complex comprising a partial peptide capable of binding to SNI or NADase and a partial peptide capable of binding to NADase (eg, streptococcus-derived NADase) or SNI. I will provide a. The present inventors have found that SNI binds to NADase to form a complex and suppresses NADase activity.

In the complex of the present invention, the partial peptide capable of binding to SNI or NADase and the partial peptide capable of binding to NADase or SNI may be as described above, but each may be labeled. . The label is not particularly limited as long as the formation of the complex is not hindered, and examples thereof include labeling with the above-described purification tag and labeling with a radioisotope (for example, a radioisotope such as ³⁵ S). The unlabeled / labeled patterns of the components in the complex are as follows (a) to (d):
(A) a combination of a partial peptide thereof capable of binding to SNI or NADase (unlabeled) and a partial peptide thereof capable of binding to NADase or SNI (unlabeled);
(B) a combination of a partial peptide (labeled) capable of binding to SNI or NADase and a partial peptide (unlabeled) capable of binding to NADase or SNI;
(C) a combination of a partial peptide (unlabeled) capable of binding to SNI or NADase and a partial peptide (labeled) capable of binding to NADase or SNI;
(D) A combination of a partial peptide (label) having binding ability to SNI or NADase and a partial peptide (labeling) having binding ability to NADase or SNI.
When both are labeled, the type of label may be the same or different, but may preferably be different. Therefore, in this case, labeling with different purification tags, labeling with different radioisotopes, labeling with purification tags and radioisotopes can be used.

  A protein or peptide labeled with a purification tag can be produced by a method known per se. For example, a DNA construct is prepared by appropriately ligating a polynucleotide encoding a target protein or peptide to a polynucleotide encoding a purification tag, and this DNA construct is expressed in a host cell. Proteins or peptides can be made. On the other hand, a protein or peptide labeled with a radioisotope can be prepared by culturing the expression cell in a medium containing a radioisotope.

  The complex of the present invention binds very firmly and specifically. For example, the complex of the present invention cannot be dissociated even in the presence of a high concentration of 3M NaCl. Therefore, the complex of the present invention can be very stable.

  The present invention also provides a method for producing a complex comprising SNI or a partial peptide thereof and NADase or a partial peptide thereof. Such a production method includes, for example, contacting the partial peptide having the binding ability with SNI or NADase and the partial peptide having the binding ability with NADase or SNI.

  More specifically, the contact for producing the complex of the present invention includes (a) contact of the isolated SNI or its partial peptide with the isolated NADase or its partial peptide, (b) SNI or its partial Introduction of expression vector of NADase or partial peptide thereof into cells expressing partial peptide, (c) Introduction of expression vector of SNI or partial peptide thereof into expression cells of NADase or partial peptide thereof, (d) SNI into host cell Or the partial peptide expression vector and NADase or its partial peptide expression vector, or (e) the SNI or its partial peptide and NADase or its partial peptide expression vector (ie, the above-mentioned present invention Vector) It can be formed.

  The complex of the present invention and the production method thereof are useful, for example, for the development of a therapeutic agent for streptococcal infection, or for the development of a dissociator between SNI or its partial peptide and NADase or its partial peptide in the complex of the present invention. is there. Therefore, the present invention also provides a screening method for substances capable of regulating the formation of a complex, which enables the development of such therapeutic agents and dissociating agents.

10. Screening Method The present invention provides a screening method capable of developing a therapeutic agent for streptococcal infection or a dissociation agent of the complex of the present invention, a substance obtained by the screening method, and an agent containing the substance.

In one embodiment, the screening method of the present invention may comprise the following steps (a) and (b):
(A) a step of evaluating whether or not the test substance can suppress the formation of the complex of the present invention;
(B) A step of selecting a test substance that can suppress the formation of the complex of the present invention.
Hereinafter, this screening method is abbreviated as screening method I as necessary.

  The test substance used in step (a) of the screening method I of the present invention can be any known compound and novel compound, such as a low molecular weight organic compound, a compound library prepared using combinatorial chemistry techniques, a nucleic acid (Eg, nucleosides, oligonucleotides, polynucleotides), carbohydrates (eg, monosaccharides, disaccharides, oligosaccharides, polysaccharides), lipids (eg, saturated or unsaturated linear, branched and / or ring-containing fatty acids) ), Amino acids, proteins (eg, oligopeptides, polypeptides), random peptide libraries prepared by solid phase synthesis or phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.

Step (a) can be performed in any manner as long as the inhibition of formation of the complex of the present invention by the test substance can be evaluated. For example, a reconstitution system (ie, a non-cellular system), a cell system (eg, Cell surface engineering techniques using hybrid systems, yeast, bacteria, etc.). In this case, step (a) may include, for example, the following steps (a1) and (a2):
(A1) contacting a test substance, a partial peptide thereof capable of binding to SNI or NADase, and a partial peptide thereof capable of binding to NADase or SNI;
(A2) A step of measuring the amount of the complex of the present invention formed by the above (a1) and comparing the amount of the complex with the amount of the complex of the present invention formed in the absence of the test substance.

  In the step (a1), the contact can be performed by a method known per se that enables formation of the complex of the present invention. Moreover, the test substance, SNI or its partial peptide, and NADase or its partial peptide may be contacted simultaneously or sequentially. For example, first, SNI or a partial peptide thereof and NADase or a partial peptide thereof are contacted to form a complex of the present invention, and then a test substance may be contacted with the complex of the present invention. Such an embodiment can also be preferably performed in this step.

In the step (a2), first, the amount of the complex of the present invention formed in the presence of the test substance is measured. The amount of complex can be measured by a method known per se, for example, immunological techniques (eg, immunoprecipitation, ELISA), interaction analysis methods using surface plasmon resonance (eg, use of Biacore ^™ ) ), Cell surface engineering techniques using two-hybrid systems, yeast, bacteria, and the like.

  The amount of complex formed in the presence of the test substance is then compared with the amount of complex formed in the absence of the test substance. The comparison of the amount of complex is preferably performed based on the presence or absence of a significant difference. The amount of complex formed in the absence of the test substance is the amount of complex measured at the same time, even if the amount of complex was measured in advance, compared to the amount of complex formed in the presence of the test substance. Although it may be, it is preferable that it is the amount of the complex measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

  In step (b) of screening method I of the present invention, a test substance that reduces the amount of the complex of the present invention (suppresses the formation of the complex) can be selected. The test substance thus selected is useful, for example, for the treatment of infection caused by streptococci having both NADase and SNI genes, or for dissociating the complex of the present invention.

  When the screening method I of the present invention is carried out with particular intention to develop a therapeutic agent for streptococcal infection (for example, the above-mentioned streptococcal infection), the streptococcus that can be the target has both the NADase gene and the SNI gene. It can be streptococci. Streptococcus is thought to protect itself from NAD depletion by co-expressing NADase with SNI. Based on this finding, it is considered that a therapeutic drug for streptococcal infection can be developed by screening a substance that dissociates the SNI / NADase complex in streptococci. Examples of streptococci having both NADase gene and SNI gene include group A, group C and group G streptococci.

  In addition, when the screening method I of the present invention is carried out with the intention of developing a therapeutic agent for streptococcal infection, a partial peptide of SNI having the ability to bind to NADase from the viewpoint of more reflecting physiological conditions, It is preferable to retain NADase inhibitory activity. From the same viewpoint, it is preferable that the partial peptide of NADase having the ability to bind to SNI further retains NADase activity. More preferably, the screening method I of the present invention can be performed using NADase and SNI derived from streptococci having both the NADase gene and the SNI gene.

In another embodiment, the screening method of the present invention may also include the following steps (a) and (b):
(A) a step of evaluating whether or not the test substance can release the suppression of NADase activity of the partial peptide having NADase or NADase activity by the partial peptide having SNI or NADase inhibitory activity;
(B) A step of selecting a test substance that can release the suppression.
Hereinafter, this screening method is abbreviated as screening method II as necessary.

  The test substance used in step (a) of screening method II of the present invention can be the same as that used in screening method I described above.

The step (a) can be performed in any manner as long as it is possible to evaluate the cancellation of the suppression by the test substance with respect to the above-mentioned NADase activity. Cell surface engineering techniques using yeast, bacteria, etc.). In this case, step (a) may include, for example, the following steps (a1) and (a2):
(A1) contacting a test substance, a substrate, a partial peptide thereof having SNI or NADase inhibitory activity, and a partial peptide thereof having NADase or NADase activity;
(A2) NADase activity of the partial peptide having NADase or NADase activity in the presence of the test substance and the partial peptide having SNI or NADase inhibitory activity is measured, and the activity is measured in the absence of the test substance and SNI or NADase inhibitory activity Comparing with the NADase activity in the presence of its partial peptide.

  In the step (a1), the contact can be performed by a method known per se that enables detection of NADase activity. Further, the contact of the test substance, substrate, SNI or its partial peptide, and NADase or its partial peptide may be performed simultaneously or sequentially as long as it is appropriate for the measurement of activity. For example, NAD can be used as the substrate.

  In the step (a2), first, NADase activity in the presence of the test substance and its partial peptide having SNI or NADase inhibitory activity is measured. NADase activity can be measured by a method known per se. For example, NADase activity can be assessed based on a decrease in substrate or an increase in detectable product. Detectable products that can be converted from the substrate include, for example, ADPR, nicotinamide, cADPR. For measuring NADase activity, see, for example, FEMS Microbiol Lett 136, 71-78 (1996); J. Exp. Med. 104: 577-587 (1956); Appl. Microbiol. 14: 391-393 (1966) That.

  The NADase activity measured in the presence of the test substance and its partial peptide having SNI or NADase inhibitory activity is then measured in the absence of the test substance and its partial peptide having SNI or NADase inhibitory activity. Compared with activity. The comparison of NADase activity is preferably performed based on the presence or absence of a significant difference. The NADase activity measured in the absence of the test substance may be the NADase activity measured in advance or the NADase activity measured simultaneously with respect to the measurement of the NADase activity measured in the presence of the test substance. However, it is preferable that the NADase activity is measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

  In the step (b) of the screening method II of the present invention, a test substance capable of releasing the suppression of the NADase activity of the partial peptide having NADase or NADase activity by the partial peptide having SNI or NADase inhibitory activity can be selected. The test substance thus selected is useful, for example, for the treatment of infectious diseases caused by streptococci having both NADase and SNI genes, or for dissociation of the complex of the present invention.

  When the screening method II of the present invention is carried out specifically for the development of a therapeutic agent for streptococcal infection (for example, the above-mentioned streptococcal infection), the streptococcus that can be the target has both the NADase gene and the SNI gene. It may be the aforementioned streptococci. Streptococcus is thought to protect itself from NAD depletion by co-expressing NADase with SNI. Based on this finding, a therapeutic drug for streptococcal infection can be obtained by screening a substance capable of releasing the suppression of NADase activity of NADase or its partial peptide by its partial peptide having SNI or NADase inhibitory activity. Can be developed. Preferably, the screening method II of the present invention can be performed using NADase and SNI derived from streptococci having both the NADase gene and the SNI gene.

  The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. Note that “Materials and Methods” to Example 7 are examples relating to SNI and NADase related inventions, and Examples 8 to 9 are examples of SLO related inventions.

Materials and Methods (1) Materials Mutanolysin and lysozyme were purchased from Sigma Chemical Co. (St. Louis, MO. USA). Ampicillin sodium and L (+)-arabinose were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). DNA modification and restriction enzymes were obtained from Takara Shuzo Co., Ltd. (Kyoto, Japan), Toyobo Co., Ltd. (Osaka, Japan), or Nippon gene (Toyama, Japan). Protein molecular preparations were purchased from Bio-Rad (California, USA) and Amersham Biosciences Corp. (NJ, USA). Yeast extract and tryptone were purchased from Difco Laboratories (Detroit, Mich. USA). Horseradish peroxidase conjugate Ni-NTA was obtained from Qiagen (Hilden, Germany). All other chemicals were reagent grade or molecular biology grade.

(2) Strains and culture conditions
Streptococcus dysgalactiae subsp.equimilis H46A (Christensen, LR (1945) J Gen Physiol 28, 363-383) is a Todd Hewitt broth supplemented with 1% yeast extract (Becton Dickinson, Cockeysville, Md. USA) ( (THY medium) at 37 ° C. Escherichia coli TOP10 (Invitrogen, Frederick, USA) and E. coli BL21 (Novagen, Madison, Wisconsin) were used as cloning plasmid cloning hosts. These strains were cultured at 37 ° C. in Luria-Bertani Broth (LB). Escherichia coli TOP10 has the ability to transport L-arabinose but not its metabolic ability. Electroporation was used for introduction of plasmid DNA into E. coli TOP10 and BL21 (J Biochem (Tokyo) 122: 237-242 (1997)). Transformants were selected on LB plates containing 100 μg / ml ampicillin.

(3) Polymerase chain reaction (PCR)
Expand High Fidelity PLUS PCR System is a product from Roche Diagnostics (Penzberg, Germany). Enzymes and buffers were used according to manufacturer's recommendations. The total volume of the PCR mixture was approximately 50 μl. 40 cycles under normal denaturation (94 ° C for 90 seconds), strand denaturation (94 ° C for 30 seconds), annealing (55 ° C for 30 seconds), and extension (72 ° C for 60 seconds per kb) Amplification was performed.

(4) DNA manipulation
Plasmid DNA of E. coli TOP10 was extracted using QIA prep Spin Miniprep Kit (Qiagen, Hilden, Germany). Streptococci genomic DNA for PCR was prepared according to a previous report (Biochim Biophys Acta 1681: 134-149 (2005)).

(5) Plasmid and synthetic oligonucleotide primers Plasmid pBAD / His series was purchased from Invitrogen (Groningen, The Netherlands). The nga-orf1 region of the nga-orf1-slo operon was amplified from the chromosomal DNA of S. dysgalactiae subsp. equisimilis H46A by PCR using the following oligonucleotide primers.
5'-CG GGATCC GTTAGTGGCAAAGAAGGTAAAAAAAGCG-3 '(underlined part is BamHI site: SEQ ID NO: 7)
5'-GG GGTACC CTAAAATGTTTCTATTGTTCTTTCGAC-3 '(underlined portion is KpnI site: SEQ ID NO: 8)
This PCR product was then digested with BamHI and KpnI and ligated to pBAD / HisB pretreated with BglII and KpnI to create plasmid pBAD / HisI (FIG. 2A).
Similarly, the nga region of the nga-orf1-slo operon was amplified by PCR using the following oligonucleotide primers.
5'-CG GGATCC GTTAGTGGCAAAGAAGGTAAAAAAAGCG-3 '(underlined part is BamHI site: SEQ ID NO: 9)
5'-GG GGTACC TTACTTCCTATCTTGCATTTTCTTAATTTTG-3 '(underlined portion is KpnI site: SEQ ID NO: 10)
Next, this PCR product was ligated to pBAD / HisB to prepare plasmid pBAD / HisII (FIG. 2A).
Plasmid pQE30 was purchased from Qiagen (Hilden, Germany). The orf1 region of the nga-orf1-slo operon was amplified from the chromosomal DNA of S. dysgalactiae subsp. equisimilis H46A by PCR using the following oligonucleotide primers.
5'-CG GGATCC TATAAGGTGCCAAAGGGTTTAGAAC-3 '(underlined part is BamHI site: SEQ ID NO: 11)
5'-GG GGTACC CTAAAATGTTTCTATTGTTCTTTCGAC-3 '(underlined portion is KpnI site: SEQ ID NO: 12)
This PCR product was then digested with BamHI and KpnI and ligated to pQE30 pretreated with BamHI and KpnI to create Orf1 fused to 6 consecutive histidine tags.

(6) DNA sequencing and sequence data analysis The nucleotide sequence was determined by the dideoxy method using appropriate primers and the ABI PARISM ^™ 3100 genetic analyzer (PE Applied Biosystems, California, USA). Nucleotide sequence data was analyzed using GENETYX computer software (Software development Co. Ltd., Tokyo, Japan). The chromosomal DNA sequence database was searched using the BLASTN search algorithm (http://www.ncbi.nlm.hih.gov/BLAST/).
(7) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
The SDS-PAGE of the enzyme was performed under reducing conditions by the method of Laemmli (Nature 227, 680-685 (1970)). The protein band was stained with Coomassie Brilliant Blue (CBB) R-250. To prepare an E. coli whole cell lysate, centrifuge 0.5 ml of the culture (16,000 xg, 3 min, 4 ° C) and transfer the resulting bacterial pellet to 0.1 ml of 1 x It was dissolved in sample buffer (composition: 1% SDS, 1% 2-mercaptoethanol, 20% glycerol, 50 mM Tris-HCl, pH 6.8), and this lysate was incubated at 100 ° C. for 3 minutes. Residual bacterial particles and non-lysed cells were removed by centrifugation (16,000 × g, 5 minutes, room temperature). Similarly, 5 ml of S. dysgalactiae subsp. Equisimilis H46A culture is centrifuged (4,000 × g, 15 minutes, 4 ° C.), and the resulting bacterial pellet is lysed with lysis buffer (composition: 100 U / ml Mutanolysin, 10 Incubation was carried out at 37 ° C. for 30 minutes in mg / ml lysozyme, 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl, pH 8.0). After incubation, the cells were harvested by centrifugation (4,000 × g, 5 minutes, 4 ° C.) and the pellet was suspended in 0.1 ml ultrapure. Then 0.1 ml of 2 × sample buffer was added and the suspension was incubated at 100 ° C. for 3 minutes. Residual bacterial particles and non-lysed cells were removed by centrifugation (16,000 × g, 5 minutes, room temperature).

(8) NH _2- terminal amino acid sequence
Intact Orf1 (mature, without His tag) overexpressed in E. coli BL21 and intact orf1 highly purified from S. dysgalactiae subsp. Equisimilis H46A were subjected to 12.5% and 15% SDS-PAGE, PVDF membrane Transcribed to. PVDF membrane was stained with CBB R-250 and a 19 kDa protein band was excised from the membrane. Two bands of protein sequencing were performed by automated Edman degradation using the PE Applied Biosystems model 491 Procise protein sequencing system (PE Applied Biosystems, California, USA).

(9) Overexpression of NADase and Orf1 One of E. coli TOP10 carrying plasmid pBAD / HisB (vector only), pBAD / HisI (His-tagged NADase and intact Orf1) or pBAD / HisII (His-tagged NADase alone) The evening culture was diluted 1:20 in fresh medium and grown until OD ₆₆₀ reached 0.5. The culture was then induced with 0.002% or 0.02% L-arabinose for 4 hours. Cells are harvested by centrifugation (5,000 xg, 10 min, 4 ° C), washed with phosphate buffered saline, and resuspended in BugBuster ^TM protein extraction reagent (Novagen, Madison, Wisconsin) at room temperature did. BugBuster reagent was used according to manufacturer's recommendations. After protein extraction, insoluble cell debris was removed by centrifugation (12,000 × g, 30 minutes), and the supernatant was used as a protein source. Similarly, an overnight culture of E. coli BL21 carrying the recombinant plasmid pQE30 (His tag fusion Orf1) was diluted 1:20 in fresh medium and grown until OD ₆₆₀ reached 0.5. The culture was then induced with 1 mM IPTG for 3 hours, and His-tagged Orf1 was extracted using BugBuster reagent.

(10) TSKgel BioAssist Ni ²⁺ chelate column
To a TSKgel BioAssist chelate column (Tosoh Co., Tokyo, Japan) previously washed with 50 mM sodium phosphate buffer (pH 7.4) (buffer A) containing 0.01% sodium azide, 4 ml of 0.1 M Ni ₂ SO ₄ The solution was loaded and then washed with 10 ml of buffer A containing 500 mM imidazole, using a Tosoh HPLC system (Tosoh Co., Tokyo, Japan) equipped with a PEEK type PPCM pump at a flow rate of 1 ml / min. And equilibrated at room temperature with buffer A containing 20 mM imidazole.

(11) Purification of His-tagged NADase / Orf1 complex E.coli TOP10 BugBuster extract carrying recombinant plasmid pBAD / HisI (encoding His-tagged NADase and intact Orf1) Dialyzed overnight at ° C. After removal of the denatured product by centrifugation (10,000 × g, 20 minutes), 3.5 ml of the extract was repeatedly loaded onto a TSKgel BioAssist Ni ²⁺ chelate column equilibrated with buffer A containing 20 mM imidazole. To wash the non-specific adsorbate on the column, 4 ml of 0.1 M sodium phosphate buffer (pH 7.8) containing 3 M NaCl and 0.01% sodium azide was passed through. Elution was performed at 20 ° C. with a flow rate of 1.0 ml / min. The protein elution profile was obtained by monitoring the absorbance at 280 nm using UV8000 (Tosoh Co., Tokyo, Japan). His-tagged NADase / Orf1 complex was recovered with a linear gradient of 20-250 mM imidazole in buffer A over 30 minutes. The complex fractions were pooled and dialyzed against buffer A.

(12) Purification of intact Orf1 (mature form, without His tag) and His tag fusion NADase E.coli TOP10 BugBuster extract carrying recombinant plasmid pBAD / HisI (coding His tag fusion NADase and intact Orf1) Dialysis was performed overnight at 4 ° C. against 2 L of buffer A. After removal of the denatured product by centrifugation (10,000 × g, 20 minutes), the dialyzed extract was applied to a TSKgel BioAssist Ni ²⁺ chelate column equilibrated with buffer A containing 20 mM imidazole. To wash the non-specific adsorbate on the column, 4 ml of 0.1 M sodium phosphate buffer (pH 7.8) containing 3 M NaCl and 0.01% sodium azide was passed through. Orf1 was recovered by elution with 3 ml of 0.1 M sodium phosphate buffer (pH 7.8) containing 3 M sodium thiocyanate (NaSCN) and 0.01% sodium azide. Purified Orf1 was diluted with an equal volume of water and dialyzed against buffer A. The His-tagged NADase adsorbed on the column was then recovered with a linear gradient of 20-250 mM imidazole in buffer A. The purified enzyme was pooled and dialyzed against buffer A.

(13) Purification of His-tagged Orf1
The BugBuster extract of E. coli cells was dialyzed overnight at 4 ° C. against 2 L of buffer A. After removal of the denatured product by centrifugation (10,000 × g, 20 minutes), the dialyzed extract was applied to a TSKgel BioAssist Ni ²⁺ chelate column equilibrated with buffer A containing 20 mM imidazole. To wash the non-specific adsorbate on the column, 4 ml of 0.1 M sodium phosphate buffer (pH 7.8) containing 3 M NaSCN and 0.01% sodium azide was passed through. His-tagged Orf1 was recovered by a linear gradient of 20-250 mM imidazole in buffer A over 30 minutes. Purified His-tagged Orf1 was pooled and dialyzed against buffer A.

(14) Purification of intact NADase (mature, no His tag) from S. dysgalactiae subsp. Equisimilis H46A culture To the culture supernatant of strain H46A, ammonium sulfate was added to 80% saturation, and the resulting precipitate was added. It was recovered by centrifugation (10,000 × g, 20 minutes, 4 ° C.). The pellet was dissolved in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.01% sodium azide and dialyzed overnight at 4 ° C. against the same buffer. Hydrolyzed apatite Bio-gel HT gel (Bio-Rad Laboratories) packed in a ToyoScreen 5 ml column (Tosoh Co., Tokyo, Japan) was obtained by centrifuging (10,000 × g, 20 minutes). , CA) at a flow rate of 1.0 ml / min at room temperature. NADase was recovered by a linear gradient of 0.1 M to 0.5 M sodium phosphate buffer (pH 7.4) containing 0.01% sodium azide over 30 minutes. The active fractions were pooled and dialyzed against buffer A. Dialyzed and partially purified NADase was applied to a TSKgel BioAssist Ni ²⁺ chelate-His-tagged Orf1 affinity column. This column was preloaded with 1 mg of purified His-tagged Orf1 and equilibrated with buffer A containing 20 mM imidazole. To wash the non-specific adsorbate on the column, 4 ml of 0.1 M sodium phosphate buffer (pH 7.8) containing 3 M NaCl and 0.01% sodium azide was passed through. Intact NADase was recovered by loading 3 ml of 0.1 M sodium phosphate buffer (pH 7.8) containing 3 M NaSCN and 0.01% sodium azide at a flow rate of 0.5 ml / min. Each fraction (0.5 ml) was collected in a tube containing 0.5 ml water to prevent protein denaturation with high concentrations of NaSCN. NADase active fractions were pooled and dialyzed against buffer A as much as possible.

(15) Gel permeation chromatography of intact NADase (mature, no His tag), Orf1 and NADase / Orf1 complex
Gel permeation chromatography of intact NADase (mature, no His tag) Orf1 and NADase / Orf1 complex was performed using a TSKgel BioAssist G3SWXL column (Tosoh Co., Tokyo, Japan). The column was developed at room temperature with 0.15 M sodium phosphate buffer (pH 6.6) at a flow rate of 0.5 ml / min. The protein elution profile was obtained by monitoring the absorbance at 280 nm.

(16) Analysis of intracellular Orf1 of S. dysgalactiae subsp. Equisimilis H46A in lysis solution (composition: 100 U / ml mutanolysin, 10 mg / ml lysozyme, 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl, pH 8.0) The cell extract obtained by digestion and sonication was applied to an anti-Orf1 antibody-conjugated HiTrap NHS activated HP column (Amersham Biosciences Corp., NJ, USA). To wash the non-specific adsorbate on the column, 4 ml of 0.1 M sodium phosphate buffer (pH 7.8) containing 3 M NaCl and 0.01% sodium azide was applied, then Orf1 was added with 3 M NaSCN and Recovered by loading 4 ml of 0.1 M sodium phosphate buffer (pH 7.8) containing 0.01% sodium azide. Orf1 fractions were pooled and dialyzed against water. The dialysate was concentrated using a Speedvac Concentrator (Sarvant Instrument Inc., NY, USA) and subjected to SDS-PAGE. Proteins in the gel were transferred to a PVDF membrane and stained with CBB R-250. Protein bands were excised and analyzed by PE Applied Biosystems model 491 Procise protein sequencing systems (PE Applied Biosystems, California, USA).

(17) Antibody
His-tagged Orf1 was used to elicit rabbit anti-Orf1 antibodies. Briefly, rabbits were injected subcutaneously with approximately 2 mg of His-tagged Orf1 mixed with complete Freund's adjuvant. Booster injections consisting of 1 mg protein and incomplete Freund's adjuvant were given 3 times at 2 week intervals. The immunoglobulin G fraction was purified by affinity chromatography using protein A-Sepharose (Amersham Biosciences Corp., NJ, USA).
Rabbit polyclonal anti-SLO IgG (containing anti-NADase antibody) was purchased from American Research Product Inc. (Belmont, MA. USA).

(18) Immunoblot analysis (Western blot analysis)
After SDS-PAGE, proteins were transferred onto Sequi-Blot PVDF membrane (Bio-Rad, California, USA) by electroblotting. Membranes were blocked with 5% skim milk and 1% bovine serum albumin (BSA) in phosphate buffered saline (PBST) containing 0.05% Tween 20 for 1 hour. The membrane was washed with PBST, then primary antibody (diluted 1: 3,000 in PBST) was added and the blot was incubated for 1 hour. Next, goat affini-pure IgG peroxidase conjugate / anti-rabbit IgG (Wako Pure Chemical Industries, Ltd) diluted 1: 3,000 in PBST and incubated for 1 hour to obtain antibody-antigen complex (J Immunol Methods 95, 71-77 (1986)) and visualized with the POD immunostaining set (Wako Pure Chemical Industries, Ltd). Ni-NTA HRP conjugate (Qiagen, Hilden, Germany) (nickel-nitrilotriacetic acid (Ni-NTA) coupled with horseradish peroxidase) was used for direct detection of recombinant proteins with His tags. Used according to manufacturer's recommendations.

(19) Far Western analysis of the interaction between Orf1 and extracellular NADase
Culture supernatant and medium alone of S. dysgalactiae subsp. Equisimilis H46A were run on SDS-PAGE (10% acrylamide), transferred to PVDF membrane, and in the presence of purified His-tagged Orf1 (10 μg / ml in PBST) Or it incubated at room temperature for 1 hour in absence. Binding of purified His-tagged Orf1 to NADase was assayed by Western blot analysis using an anti-Orf1 antibody as described in “Immunoblot analysis”. Briefly, the membrane was washed with PBST, then rabbit anti-Orf1 antiserum was added and the blot was incubated for 1 hour. Subsequently, goat affii-pureIgG peroxidase conjugate / anti-rabbit IgG was added and incubated for 1 hour, and the antibody-antigen complex was visualized by POD immunostaining. NADase and culture medium alone (control) in the culture supernatant were assayed by Western blot analysis with anti-SLO polyclonal antibody containing anti-NADase antibody.

(20) NADase assay
NADase assay was performed by the method of Gerlach et al. (FEMS Microbiol Lett 136, 71-78 (1996)). One unit of enzyme cleaves 0.010 μM NAD in 7.5 minutes at 37 ° C.
-Preparation of whole cell extract:
Cells from a medium-phase culture (10 ml) in logarithmic growth phase were dissolved in a lysis solution (composition: 100 U / ml mutanolysin, 5 mg / ml lysozyme, 0.01% sodium azide, 150 mM sodium phosphate, pH 6.6). Incubated for 30 minutes at 37 ° C. After incubation, the cells were sonicated, the lysate was centrifuged at low temperature, and the supernatant was used for NADase assay.
・ Temperature stability:
The temperature stability of intact Orf1 (mature, no His tag) was determined at a protein concentration of 38 μg / ml (2.0 μM) in 0.15 M sodium phosphate buffer (pH 7.4). Orf1 solution was sealed in an Eppendorf tube and incubated at 20-100 ° C. for 15 minutes. Incubation was stopped by cooling the solution in ice water and 0.5 μl of each solution was added to 10 μl 5 μg / ml containing 70 μg / ml (1 μM) BSA in 50 mM potassium phosphate buffer (pH 7.5). (0.1 μM) Intact NADase (mature, no His tag) was added to the solution. The mixture was then incubated at room temperature for 10 minutes (the final molar ratio of Orf1 and NADase was 1: 2), and the remaining hydrolase activity was measured by NAD. As a control, 50 mM potassium phosphate buffer (pH 7.5) containing BSA alone was used instead of the incubated Orf1 solution.

Example 1: Expression of Streptococcus NADase in E. coli Previous work on the regulation of Streptococcal NAD glycohydrolase (NADase) -Streptocricin O (SLO) operon has been carried out by using pHY300PLK as a vector. Subcloning of gene clusters in Overexpression of active SLO was achieved in E. coli (Biosci Bisotechnol Biochem 65, 2682-2689 (2001)), but no colonies with NADase gene inserts were obtained (Biochim Biophys Acta 1681: 134-149 ( 2005)). Considering the potential host toxicity of this enzyme, the NADase gene nga was subcloned into an expression vector pBAD having an araBAD promoter (pBAD) that strictly controls gene expression in E. coli (FIG. 2A). However, E. coli TOP10 (pBAD / HisII) having only the NADase gene insert did not produce this enzyme protein upon induction by L-arabinose treatment (FIG. 2B). Growth of this recombinant strain was slow even in the absence of inducer and some of the cells aggregated (data not shown). On the other hand, the E. coli recombinant strain carrying pBAD / HisI with a longer insert spanning the 3 ′ end of nga to orf1 produced significant amounts of NADase (FIG. 2B). A database search revealed that Orf1 was similarly conserved in group A Streptococcus (GAS) strains but was deleted in other bacterial strains (data not shown). When the full-length NADase-SLO operon was placed under pBAD control and expressed in E. coli, active SLO was produced in addition to NADase and Orf1 (data not shown).
These results suggest that intracellular streptococcal NADase is probably toxic not only to E. coli but also to streptococci itself, and that co-expressed Orf1 may protect the reservoir from NAD depletion . In order to verify this possibility, the genetic and biochemical properties of the unknown gene product Orf1 of the NADase-SLO operon were examined in subsequent experiments.

Example 2: Expression of Orf1 and sequencing of its N-terminal region
When co-expressed in E. coli under P _BAD control, the amount of Orf1 was higher than that of NADase as judged by SDS-PAGE profile (FIG. 3A). The Orf1 band was excised from the gel and directly subjected to N-terminal amino acid sequencing. The obtained sequence MYKVPKGLEHYQKM (SEQ ID NO: 15) was identical to that estimated from the nucleotide alignment of the orf1 gene in the NADase-SLO operon (FIG. 3B). This gene consisted of 483 base pairs and encoded a predicted 161 amino acids. From the nucleotide sequence, the molecular weight of this Orf1 protein was estimated to be 18,800.
To demonstrate the expression of orf1 in S. dysgalactiae subsp. Equisimilis H46A, anti-Orf1 antibodies were prepared by immunizing rabbits with His-tagged Orf1 produced in recombinant E. coli cells (FIG. 4A). ). Western blot analysis using this antibody showed that Orf1 protein was produced and localized exclusively within H46A cocci (FIG. 4B). The molecular weight of Orf1 coincided with the molecular weight of the protein co-expressed with NADase in E. coli (FIG. 4B). Unlike SLO and NADase, Orf1 was not detected in the streptococcal culture supernatant (FIG. 4B).

Example 3: Purification and amino-terminal sequencing of Orf1 from S. dysgalactiae subsp. Equisimilis H46A
Orf1 protein was extracted from S. dysgalactiae subsp. Equisimilis H46A and purified by affinity chromatography using an anti-Orf1 antibody-immobilized column (FIG. 5). This eluted fraction was subjected to SDS-PAGE and blotted on a PVDF membrane. CBB R-250 staining detected five bands possibly derived from the intracellular Orf1 complex morphology (FIG. 5). Amino-terminal sequencing showed that the 19 kDa band was Orf1: its sequence MYKVPKGLEHYQKM (SEQ ID NO: 15) and the molecular size deduced from the nucleotide sequence, and Orf1 expressed in recombinant E. coli cells It was the same as that of protein. Two bands of calculated 50 kDa may have been precursors of NADase, but their amino acid sequencing was unsuccessful, probably due to amino-terminal blocking. After translation, a region of about 2 kDa has recently been found to be proteolytically removed from the C-terminus of NADase (Biochim Biophys Acta 1681: 134-149 (2005)). If the band of about 50 kDa is NADase, C-terminal removal must have occurred in cocci cells. The dark band of calculated 34 kDa was NADase protein (ΔN187) from which N-terminal 187 amino acids were deleted. It is unclear whether this deletion occurs due to abnormal transcription or proteolysis during preparation of the cocci extract. However, the fact that this truncated molecule was adsorbed to the anti-Orf1 antibody-immobilized column indicates that the NADase N-terminal region is not essential for interaction with Orf1. The 16 kDa band was egg white lysozyme used to lyse streptococcal cells. Although non-physiological, this lytic enzyme appeared to interact with either Orf1, NADase or the Orf1-NADase complex in vitro. As inferred from the SDS-PAGE profile (FIG. 5), the ratio of Orf1 to NADase precursor was not so high in Streptococcus compared to the ratio in E. coli (FIG. 3A).
The NADase-SLO operon member Orf1 was translated from the same start codon in recombinant E. coli and Streptococcus strain H46A. The fact that production of streptococcal NADase in E. coli requires coexpression of Orf1 suggests a self-protective role for this protein in the suppression of potentially toxic NADase activity. Therefore, to analyze the in vitro physical and functional interactions of NADase and Orf1, the following experiment was performed.

Example 4: Analysis of interaction between Orf1 and NADase
For analysis of interaction with NADase, Orf1 produced in E. coli as a His-tagged protein was purified by nickel-chelate column chromatography (FIG. 4A). The THY medium before and after the culture of S. dysgalactiae subsp. Equisimilis H46A was subjected to SDS-PAGE, blotted on a PVDF membrane, and divided into two parts. One of the membranes was directly subjected to Western blotting using anti-SLO polyclonal IgG, the other membrane was treated with purified Orf1 and then subjected to Western blotting using anti-Orf1 antiserum (FIG. 6). As shown in FIG. 4B, the spent media from Streptococcus H46A cultures did not contain Orf1 and its cross-reactive material. For detection of NADase, a commercially available anti-SLO polyclonal IgG that cross-reacts with this enzyme protein was used (Cell 104: 143-152 (2001); Biochim Biophys Acta 1681: 134-149 (2005)).
By Far Western blotting, Orf1 was detected at the exact position corresponding to the NADase band in the Orf1-treated PBDF membrane. Since this result indicates a physical interaction between NADase and Orf1, the effect of the latter protein on NADase activity was subsequently investigated.

Example 5: Inhibition of NADase by Orf1
Orf1 is an intracellular protein, and its cell content is not so high (FIG. 5). In order to overproduce this protein, co-expression with NADase was attempted in E. coli carrying pBAD / HisI. As shown in FIG. 4B and FIG. 5, the electrophoretic profile and N-terminal sequence of Orf1 from E. coli were the same as those from H46A. At the same time, His-tagged NADase coexpressed with Orf1 in E. coli was purified by nickel-chelate column chromatography. The co-expressed Orf1 molecule was present as expected as a complex with NADase, and separation from each other could not be achieved even with 3M NaCl treatment (FIG. 7A). The complex was dissociated using 3M NaSCN (FIG. 7B) and Orf1 was further purified by gel permeation chromatography. NADase in the recombinant E. coli extract was present as an enzymatically inactive complex with Orf1. N-terminal sequencing and molecular size determination indicate that this NADase protein is in a precursor form. When dissociated from Orf1, the NADase precursor showed almost the same specific activity as that of the secreted mature form of the enzyme (data not shown). Since the NADase precursor was small and the secreted mature enzyme also formed an inactive complex with Orf1, the extracellular form was used in the following experiments. Therefore, NADase was purified from the culture supernatant of S. dysgalactiae subsp. Equisimilis H46A by affinity column chromatography using a column on which Orf1 overproduced by E. coli as a His-tagged protein was immobilized (FIG. 7C). ). When purified NADase was mixed with Orf1 and incubated at room temperature for 10 minutes, this enzyme activity was almost completely blocked at a reasonable molar ratio (FIG. 7D). This inhibition reaction was very rapid and occurred even at 0 ° C. (data not shown).
To verify the possibility that the NADase precursor in Streptococcus exists as an inactive complex bound to Orf1, H46A lysate was mixed with an equal volume of 6M NaSCN. After the dissociation treatment, Orf1 was neutralized with anti-Orf1 antiserum. As shown in Table 1, no active NADase could be detected in streptococcal lysates, but an activity of about 10 ⁴ units per 1 g (wet weight) sample treated with NaSCN and then anti-Orf1 antiserum was observed. It was. When the activated extract was diluted with water, NADase activity was masked again (data not shown). This suggests that NADase has re-associated with Orf1. These results indicate that the initial NADase forms an inactive complex with Orf1 in streptococci, as in recombinant E. coli.

^a Activity was expressed as mean ± SD from three experiments.
^b Whole cell extract was prepared as described in “NADase Assay” in Materials and Methods, and 10 μl of extract was mixed with 10 μl of 0.1 M Na ₂ HPO ₄ (pH 7.4) and 80 μl of ultrapure.
^c 10 μl of whole cell extract was mixed with 10 μl of 6 M NaSCN, 0.1 M Na ₂ HPO ₄ (pH 7.4) at room temperature, and after 5 seconds vortexing, 80 μl ultrapure was added immediately.
^d 10 μl whole cell extract was mixed with 10 μl 0.1 M Na ₂ HPO ₄ (pH 7.4), 1 μl anti-Orf1 antiserum and 79 μl ultrapure.
^e 1 μl of anti-Orf1 antiserum was mixed with 10 μl 0.1 M Na ₂ HPO ₄ (pH 7.4) and 89 μl ultrapure.
^f 10 μl whole cell extract was mixed thoroughly with 10 μl 6 M NaSCN, 0.1 M Na ₂ HPO ₄ (pH 7.4) at room temperature, vortexed for 5 seconds, then 1 μl anti-Orf1 antiserum and 79 μl extra Pure was added immediately.

Example 6: Determination of molecular size of NADase / Orf1 complex
In order to determine the molecular size of the NADase / Orf1 complex, gel permeation chromatography was performed using a TSKgel BioAssist G3SWXL column (Tosho Co., Tokyo, Japan). As shown in FIG. 8A, each of NADase, Orf1, and NADase · Orf1 complex was detected as a distinct single peak. The molecular weight estimated from each retention time was 24 kDa (Orf1), 54 kDa (NADase) and 69 kDa (NADase / Orf1 complex). These values (FIG. 8B) were somewhat higher but approximated the values estimated from each nucleotide sequence. These results indicate that NADase and Orf1 exist as monomeric proteins and form a complex as a heterodimer when mixed.

Example 7: Temperature stability and protease sensitivity The bacterial NADaes described so far are intracellular enzymes and their inhibitory proteins are heat labile (Methods Enzymol 66; 137-144 (1980); Science 123 : 50-53 (1956)). Interestingly, Orf1 was heat stable and the relative inhibitory activity remaining after heat treatment for 15 minutes was 82% calculated at 60 ° C. and 72% calculated at 100 ° C. (FIG. 9). Purified Orf1 was sensitive to trypsin and was inactivated by lysyl endopeptidase (data not shown).

Considerations on Examples 1-7
The NADase-streptricin O operon is highly conserved in group A and C streptococci (Cell 104: 143-152 (2001); Biochim Biophys Acta 1681: 134-149 (2005); Infect Immun 70: 2730 -2733 (2002)). In addition to Nga and slo, this operon contained the unknown gene orf1. The above data indicate that this gene encodes a 18.8 kDa protein that functions as a streptococcal NADase inhibitor (SNI). Therefore, it is considered appropriate to mark this gene as sni. In contrast to failure of single nga subcloning in E. coli (Biochim Biophys Acta 1681: 134-149 (2005)), co-expression of this gene with orf1 (sni) has an nga-orf1 insert It was stably generated in a heterologous host carrying the recombinant plasmid. Production of active NADase in the microbial cells can cause toxic effects due to degradation of NAD essential for redox. The presence of bacterial inhibitors for this potentially harmful enzyme is known in Bacillus subtilis, Proteus vulgaris, Proteus rettgeri and Mycobacterium butyricum. These bacterial NADases are intracellular thermostable proteins complexed with thermolabile inhibitors (Methods Enzymol 66: 137-144 (1980); Science 123: 50-53 (1956)). Genetic and functional studies of these inhibitors have not been conducted so far. On the other hand, hemolytic streptococci secrete thermolabile NADase into the culture medium (J Exp Med 104: 577-587 (1956); J Exp Med 106: 15-26 (1957); FEMS Microbiol Lett 136: 71 -78 (1996)). Streptococcus cells that produce extracellular NADase contain an inhibitor protein for this enzyme (Acta Path. Microbiol. Scandinav. 69: 277-286 (1967)). Intracellular localization, molecular weight and trypsin sensitivity are similar to those of SNI. However, with respect to thermal stability, the inhibitors reported by Holm and Kaijser (Acta Path. Microbiol. Scandinav. 69: 277-286 (1967)) are different from SNI. Their preparations were completely inactivated by heating at 60 ° C. for 15 minutes, whereas SNI was rather heat stable (FIG. 9).

  The gene (sni) encoding SNI is cotranscribed with slo as a polycistronic mRNA from the nga (spn) promoter (Biochim Biophys Acta 1681: 134-149 (2005)). During translation of the nga-slo operon, the NADase precursor autotoxicity appears to be rapidly blocked by complex formation with SNI at a molar ratio of 1: 1 (FIG. 10). As demonstrated by Western blot analysis, the localization of SNI is completely intracellular (Figure 4B). Consistent with this result, the signal peptide region is defined by SOSUI (Bioinformatics 14: 378-379 (1998); Bioinformatics 18: 608-616 (2002)) and SignalP (Int J Neural Dsyst 8: 581-599 (1997)). In silico analysis using a transmembrane estimation algorithm was not found in SNI. The amount of NADase precursor and SLO in the cocci was small (data not shown). This suggests that the synthesized protein is secreted rapidly into the medium. However, since the NADase precursor forms a stable intracellular complex, this complex should dissociate during excretion through the cocci envelope. Whether this complex dissociation is caused by interaction with several membrane components, by proteolytic exclusion of SNI, or by other mechanisms is an issue to be elucidated.

Example 8 : Mass expression of full-length SLO in Escherichia coli According to the findings from the promoter analysis of the SLO gene by the present inventors (Biochim Biophys Acta 1681: 134-149 (2005)), the SLO gene does not have its own promoter, It has been found to be part of the operon that is basically transcribed by readthrough from the promoter of the NADase gene (nga) located upstream. In view of the fact that the SLO gene does not have its own promoter and the cytotoxicity of SLO, the present inventors expressed using the araBAD promoter (P _BAD ) that can strictly control gene expression in E. coli. A large-scale expression of full-length SLO fused with His tag was attempted using a vector. The amino acid sequence added by the His tag method is short and in many cases does not need to be removed. In addition, specific antibodies and purification carriers are advantageous in that a plurality of commercially available products are available and the cost is low. Furthermore, it can be purified in the presence of protein denaturing agents such as urea and guanidine hydrochloride, and has the great advantage that it can be refolded while bound to the column (Protein Expr Purif 41: 98-105 ( 2005)).

First, the present inventors are Streptococcus dysgalactiae subsp. Equisimilis H46A (J Gen Physiol 28: 363-383 (1945)), which is well known as an industrial production strain of streptokinase and streptodornase already used as pharmaceuticals. The SLO gene was amplified from the chromosomal DNA by PCR and cloned into His-tagged protein expression vector pBAD / HisB (Invitrogen) in order to execute the gene expression in Escherichia coli very strictly (FIG. 11 ). A region of the full-length SLO gene corresponding to 540 amino acid residues (60.4 kDa) excluding 31 amino acid residues (3.3 kDa) on the N-terminal side including the signal peptide sequence is inserted into the BamHI and KpnI sites of pBAD / His B. Inserted with frame. This recombinant plasmid was introduced into Escherichia coli TOP10 strain (L-arabinose metabolic capacity deficient strain), and His-tag fusion full-length SLO expression experiment using L-arabinose, which is a gene expression inducer, was performed. As a result, sufficient expression of SLO was confirmed when the concentration of L-arabinose in the medium was 0.002% (w / v) (FIG. 12 ). As a result of immunoblot analysis, the molecular weight of SLO expressed in Escherichia coli was estimated to be about 70 kDa, which was slightly larger than the expected 64.5 kDa combined with the 4.1 kDa region including the His tag on the vector side. It has also been reported by Gerlach et al. (Infect Immun 61: 2727-2731 (1993)) that the molecular weight determined from the electrophoretic analysis of SLO is larger than expected.
Next, the inventors changed E. coli from the TOP10 strain to the BL21 strain, which is a protease-deficient strain of both lon and ompT, and expressed SLO in large quantities. For purification, commercially available TSKgel BioAssist Ni ²⁺ -chelate column (Tosoh Co., Tokyo, Japan) was used, and high-purity SLO was purified in one step by this affinity column chromatography (FIG. 13 ). The expression level of His-tagged full-length SLO is about 6 mg of protein per liter of LB medium (about 3 mg of active protein). Its specific activity is 748,000 HU / mg protein and Gerlach et al. Streptococcus It almost coincided with 750,000 HU / mg of SLO purified from the culture supernatant of dysgalactiae subsp. equisimilis H46A (Infect Immun 61: 2727-2731 (1993)). The amount of protein obtained in this experiment was as described above, but at least due to appropriate changes in the culture conditions (eg, improvement of bacterial density by optimization of the medium and extension of induction time by inducer). It is considered possible to obtain a protein amount of about 10-30 mg.

Example 9: To full length SLO mass expression currently commercially available anti-streptavidin Li Thin O (ASLO) antibody in E. coli is used SLO purified culture supernatant portion of hemolytic streptococci as an antigen, as well It is known to cross-react with NAD glycohydrolase (NADase) secreted into the culture supernatant (Biochim Biophys Acta 1681: 134-149 (2005); Cell 104: 143-152 (2001)). Accordingly, the present inventors, the SLO purified to prepare a ASLO by immune rabbit, a culture supernatant of GAS Sa strains and GCS H46A strain was evaluated the antigen specificity of ASLO by immunoblot analysis (FIG. 14 ).
As a result, SLO in the culture supernatant of GCS H46A strain was detected as a clear band with high sensitivity by ASLO prepared by the present inventors. The two weak bands that appear below the major band appear to be low molecular weight SLO molecules. On the other hand, SLO could not be detected in GAS Sa strain. This is probably because the amount of SLO produced by GAS is generally small, and the molecular weight is quickly reduced by the protease produced by itself. In fact, the present inventors have reported that detection and purification of SLO in GAS Sa strains is difficult (Kimoto et al., Biosci Biotechnol Biochem 67: 2203-2209 (2003); Kimoto et al., Biochim Biophys Acta 1681: 134-149 (2005)). This ASLO neutralized the hemolytic activity of His-tag fusion full-length SLO purified from E. coli and SLO produced by Streptococcus pyogenes (data not shown).
Next, in order to examine the cross-reaction of ASLO with NADase, immunoblot analysis with anti-NADase antiserum was performed using the culture supernatant of GAS Sa strain and GCS H46A strain.
As a result, NADase was confirmed in the culture supernatants of both the GAS Sa strain and the GCS H46A strain. The reason why the molecular weight of NADase is slightly different between the two is that NADase produced by GCS H46A strain is cleaved after translation (Kimoto et al., Biochim Biophys Acta 1681: 134-149 (2005)) . In any case, SLO and NADase detected bands with the expected molecular weight, and the size between them was clearly different. In addition, no band was detected in the non-immunized control serum.
From the above, it was shown that the ASLO produced this time can detect SLO specifically and with high sensitivity, unlike conventional commercial products.

It is a figure which shows the mode of activity of NADase. 1 is a schematic diagram of the NADase streptolysin O operon of S. dysgalactiae subsp. Equisimilis H46A. Promoters and terminators are indicated by “P” and “T”, respectively. The arrow labeled “orf1 (sni)” was not similar to any gene in the database. NADase expression plasmids pBAD / HisI and II each contain only the chromosomal region from NADase to orf1 and the NADase region. It is a figure which shows the western blotting analysis of Streptococcus NADase in E.coli TOP10 whole cell lysate. E. coli TOP10 cells were transformed with an expression plasmid encoding the indicated region modified to contain 6 consecutive histidine residues (His tag), induced with L-arabinose, NADase synthesis was then assayed. A lysate of E. coli TOP10 carrying pBAD / His B (vector only) was used as a control. To determine the relative expression level of NADase, whole cell lysates were subjected to Western blot analysis. It is a figure which shows the detection of the Orf1 protein of H46A. Proteins in whole cell lysates of recombinant E. coli TOP10 carrying pBAD / His I were analyzed by SDS-PAGE. NH ₂ terminal amino acid acid sequence is shown in parentheses. It is a figure which shows the nucleotide sequence and deduced amino acid sequence of orf1 of H46A. Underlined amino acids were determined by NH ₂ terminal amino acid sequencing of Orf1. It is a figure which shows the refinement | purification of His tag fusion Orf1. An extract from E. coli BL21 cells carrying the recombinant plasmid pQE30 (His-tagged fusion Orf1) was applied to a TSKgel BioAssist Ni ²⁺ chelate column (Tosoh Co., Tokyo, Japan). His-tagged Orf1 was recovered with a 20-250 mM linear gradient of imidazole and each elution fraction was examined by SDS-PAGE. It is a figure which shows the western blot analysis of the orf1 product in a recombinant E. coli cell and S. dysgalactiae subsp. Equisimilis H46A culture supernatant. In order to confirm the expression of Orf1, whole cell lysates and culture supernatants were subjected to Western blot analysis using rabbit anti-Orf1 antiserum obtained by immunization with Orf1 purified as described above. FIG. 4 shows purification and amino-terminal sequencing of Orf1 derived from H46A. Whole cell extract from H46A was applied to anti-Orf1 antibody / conjugate / HiTrap NHS activated HP column (Amercham Biosciences Corp., NJ, USA), and Orf1 was added with 3 M sodium thiocyanate (NaSCN) and 0.01% Recovered by loading 4 ml of 0.1 M sodium phosphate buffer (pH 7.8) containing sodium azide. Orf 1 fractions were pooled, dialyzed against water, then concentrated and subjected to SDS-PAGE. Proteins in the gel were transferred to a PVDF membrane and stained with CBB R-250. Protein bands were excised and analyzed with PE Applied Biosystems model 491 Procise protein sequencer (Foster City, CA, USA). Far Western analysis of the interaction between Orf1 and extracellular NADase of H46A. The culture supernatant of H46A and medium alone were run on SDS-PAGE, transferred to a PVDF membrane, and incubated in the presence or absence of purified His-tagged fusion Orf1. NADase in the culture supernatant was assayed by Western blot analysis with anti-SLO polyclonal IgG containing anti-NADase antibody (left panel). Binding of purified His-tagged Orf1 to NADase was assayed by Far Western analysis using anti-Orf1 antiserum (right panel). It is a figure which shows co-purification of His tag fusion NADase and intact Orf1 (mature type, No His tag) using TSKgel BioAssist Ni2 ⁺ chelate column. A cell extract derived from E. coli TOP10 carrying recombinant plasmid pBAD / His I (encoding His-tagged NADase and intact Orf1) was applied to a TSKgel BioAssist Ni ²⁺ chelate column and combined with His-tagged NADase / ORF1 The body was collected with a 20-250 mM linear gradient of imidazole. The purified NADase / Orf1 complex fractions were pooled, dialyzed and subjected to SDS-PAGE. It is a figure which shows the refinement | purification of intact Orf1 by TSKgel BioAssist G3SWXL gel permeation chromatography. A cell extract derived from E. coli TOP10 carrying recombinant plasmid pBAD / His I (encoding His-tagged NADase and intact Orf1) was applied to a TSKgel BioAssist Ni ²⁺ chelate column. Orf1 was recovered by elution with 3 ml of 0.1 M sodium phosphate buffer (pH 7.4) containing 3 M sodium thiocyanate (NaSCN) and 0.01% sodium azide. The His-tagged NADase adsorbed on the column was then recovered with a 20-250 mM linear gradient of imidazole in 50 mM sodium phosphate buffer (pH 7.4). The purified enzyme was pooled, dialyzed against 50 mM sodium phosphate buffer (pH 7.4) containing 0.01% sodium azide, and subjected to SDS-PAGE. It is a figure which shows the refinement | purification of intact NADase (a mature type, no His tag) from the culture supernatant of S. dysgalactiae subsp. Equisimilis H46A by affinity chromatography using the column to which His tag fusion Orf1 was fixed. After ammonium sulfate precipitation and dialysis, the protein from the supernatant was applied to a TSKgel BioAssist Ni ²⁺ chelate-His tag fusion Orf1 affinity column. NADase was recovered by loading with 0.1 M sodium phosphate buffer (pH 7.8) containing 3 M NaSCN and 0.01% sodium azide and dialyzed. It is a figure which shows inhibition of NADase by Orf1. Purified intact NADase was mixed with intact Orf1, incubated at room temperature for 10 minutes, and the remaining NADase activity was measured. It is a figure which shows the analysis by TSKgel BioAssist G3SWXL gel permeation chromatography of NADase, Orf1, and NADase-Orf1 complex. Bovine serum albumin (BSA) dimer (134 kDa), monomer (67 kDa), ovalbumin (OV, 43 kDa), carbonic anhydrase (CA, 29 kDa) and lysozyme (14.4 kDa) were used as molecular weight markers. Vo and Vc indicate the empty capacity and the column capacity, respectively. Intact NADase and Orf1 (mature, without His-tag) purified by affinity chromatography using His-tagged Orf1 immobilized column and anti-Orf1 antibody immobilized column in TSKgel BioAssist in 0.15 M sodium phosphate buffer (pH 6.6) G3SWXL gel permeation chromatography was used. It is a figure which shows the analysis by TSKgel BioAssist G3SWXL gel permeation chromatography of NADase, Orf1, and NADase-Orf1 complex. Bovine serum albumin (BSA) dimer (134 kDa), monomer (67 kDa), ovalbumin (OV, 43 kDa), carbonic anhydrase (CA, 29 kDa) and lysozyme (14.4 kDa) were used as molecular weight markers. Vo and Vc indicate the empty capacity and the column capacity, respectively. Purified NADase and Orf1 were mixed, incubated at room temperature for 10 minutes, applied to a TSKgel BioAssist G3SWXL column, and developed with 0.15 M sodium phosphate buffer (pH 6.6). It is a figure which shows the temperature stability of intact Orf1 (without His tag). The Orf1 solution was incubated at 20-100 ° C. for 15 minutes and immersed in ice water. Each solution was added to purified NADase without purified His tag. The mixture was then incubated for 10 minutes at room temperature (the final molar ratio of Orf1 and NADase was 1: 2) and the remaining hydrolase activity was measured by NAD. As a control, 50 mM sodium phosphate buffer (pH 7.4) containing BSA was used instead of the Orf1 solution. It is a model figure of the interaction between the products of nga-ni-slo operon. The gene (sni) encoding SNI is transcribed as polycistronic mRNA together with nga (NADase) and slo from the nga promoter. Upon translation, NADase autotoxicity can be quickly blocked by the formation of a stable intracellular complex with SNI at a molar ratio of 1: 1. The NADase-SNI complex dissociates during streptococcal envelope excretion, possibly by interaction with several membrane components, by proteolytic removal of SNI, or by other mechanisms. FIG. 2 is a schematic diagram showing the construction of a streptolysin O gene (SLO) and an SLO expression vector. Sense primer (5'-cg GGATCC gactcgaacaaacaaaacactgcc-3 ': SEQ ID NO: 13) and antisense primer (5'-cgg GGTACC cttttatgttatgctttgcttg-3': SEQ ID NO: 14) containing the BamHI and KpnI sites were added to the NH ₂ terminal signal peptide. It was designed to allow cloning of the missing His-tagged SLO sequence into the expression vector pBAD / HisB (Invitrogen, Frederick, USA). It is a figure which shows the immunoblot analysis of streptococcus SLO in E.coli TOP10 whole cell extract. The position of the pre-stained protein molecular preparation and the calculated molecular weight are shown on the left of the panel. The size is given in kilodaltons (kDa). The arrow indicates the position of SLO. E. coli TOP10 cells were transformed with an expression plasmid encoding an SLO region modified to contain 6 consecutive histidine residues (His tag) and then assayed for SLO synthesis. In the presence of L-arabinose, expression from pBAD is turned on, but in the absence of L-arabinose, the transcription level from pBAD is very low. Cultures were induced with 0.00002%-0.02% L-arabinose for 4 hours at 37 ° C. The lysate of E. coli TOP10 carrying pBAD / HisB (vector only) was used as a control (induced with 0.02% L-arabinose). To determine the relative expression level of SLO, whole cell lysates were subjected to Western blot analysis using Ni-NTA HRP conjugate (Qiagen, Hilden, Germany). This conjugate [nickel-nitrilotriacetic acid (Ni-NTA) coupled to horseradish peroxidase] was used for direct detection of recombinant proteins with His tags. Lane 1: 0.00002% L-arabinose; Lane 2: 0.0002% L-arabinose; Lane 3: 0.002% L-arabinose; Lane 4: 0.02% L-arabinose; Lane 5: Control (vector only, 0.02% L-arabinose Induction) It is a figure which shows the refinement | purification of His tag fusion SLO. A culture of E. Coli BL21 containing pBAD / His B-slo was induced with 0.2% L-arabinose at 37 ° C. for 4 hours. The cell extract was applied to a TSKgel BioAssist Ni ²⁺ -chelate column (Tosoh Co., Tokyo, Japan) equilibrated with 50 mM sodium phosphate buffer (pH 7.4) containing 20 mM imidazole (buffer A). To wash away nonspecific adsorbate from the column, 0.1 M sodium phosphate buffer (pH 7.8) containing 3 M NaCl was passed through. This His-tagged SLO was recovered with a linear gradient of 20-250 mM imidazole in buffer A. Purified His-tagged SLOs were pooled and dialyzed against buffer A. The dialyzate was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (CBB) R-250. The position of the protein molecular preparation and each calculated molecular weight are shown on the left of the panel. The size is given in kilodaltons (kDa). The arrow indicates the position of SLO. It is a figure which shows the immunoblot analysis of the culture supernatant derived from GAS strain Sa and GCS strain H46A. Rabbit anti-SLO antibody was raised using purified His-tag fused SLO. Approximately 2 mg of His-tagged SLO (mixed with complete Freund's adjuvant) was injected subcutaneously into rabbits. Booster injections consisting of 1 mg protein and incomplete Freund's adjuvant were administered 3 times at 2-week intervals. The immunoglobulin G fraction was purified by affinity chromatography on protein A-Sepharose (Amersham Biosciences Corp., NJ, USA). Rabbit antiserum produced by immunization with purified H46A NADase (Gerlach et al., 1996) was provided by Dr. D. Gerlach of Friedrich-Schiller University (Germany). The culture supernatant of Streptococcus was subjected to SDS-PAGE (10% total acrylamide), and the protein was transferred onto a Sequi-Blot PVDF membrane (Bio-Rad, CA, USA) by electroblotting. The membrane was blocked with 5% skim milk and 1% bovine serum albumin (BSA) for 1 hour in phosphate buffered saline (PBST) containing 0.05% Tween 20. The membrane was washed with PBST, then each primary antibody was added (diluted 1: 3,000 with PBST) and the blots were incubated for 1 hour. Next, goat affini pure IgG peroxidase conjugated to anti-rabbit IgG (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added at 1: 3,000 diluted with PBST, incubated for 1 hour, and antibody / antigen complex The body was visualized using a POD immunostaining set (Wako Pure Chemical Industries, Ltd., Osaka, Japan) as described by the manufacturer. The position of the pre-stained protein molecular preparation and each calculated molecular weight are shown on the left of the panel. The size is given in kilodaltons (kDa).

Claims (29)

A polypeptide having 95% or more homology with the amino acid sequence of SEQ ID NO: 2 and having NADase inhibitory activity, or deletion, substitution, addition of one or two amino acids in the amino acid sequence of SEQ ID NO: 2, and A polypeptide having one or more modifications selected from the group consisting of insertion and having NADase inhibitory activity. The polypeptide according to claim 1, wherein the NADase is a hemolytic streptococcal NADase. NADase is a polypeptide having 95% or more homology with the amino acid sequence represented by SEQ ID NO: 4, or deletion, substitution, addition of one or two amino acids to the amino acid sequence of SEQ ID NO: 4, And the polypeptide according to claim 1, which is a polypeptide subjected to one or more modifications selected from the group consisting of an insertion. The polypeptide according to any one of claims 1 to 3, which is derived from hemolytic streptococci. It is possible to retain 80% or more of activity even after 15 minutes of heat treatment at 60 ° C, or 70% or more of activity after 15 minutes of heat treatment at 100 ° C. The polypeptide according to any one of the above. A polynucleotide having a homology of 95% or more with the polynucleotide represented by SEQ ID NO: 1 and encoding the polypeptide according to any one of claims 1 to 5, or SEQ ID NO: 1 A polynucleotide that is capable of hybridizing under high stringency conditions with a complementary sequence of the polynucleotide that is represented and that encodes the polypeptide of any one of claims 1-5. The polynucleotide according to claim 6, which is derived from hemolytic streptococci. An expression vector comprising the polynucleotide according to claim 6 or 7. A transformant comprising the expression vector according to claim 8. The transformant according to claim 9, wherein the transformant is Escherichia coli. A NADase inhibitor comprising any one of the following (a) or (b):
(A) The polypeptide according to any one of claims 1 to 5.
(B) the expression vector according to claim 8 A medicament comprising the NADase inhibitor according to claim 11 . A reagent comprising the NADase inhibitor according to claim 11 . The medicament according to claim 12, which is a therapeutic drug for hemolytic streptococcal infection. An antibody against the polypeptide according to any one of claims 1 to 5. A polypeptide according to any one of claims 1-5, and a NADas e, complex. The complex according to claim 16, wherein the NADase is a hemolytic streptococcal NADase. NADase is a polypeptide having 95% or more homology with the amino acid sequence represented by SEQ ID NO: 4, or deletion, substitution, addition of one or two amino acids to the amino acid sequence of SEQ ID NO: 4, The complex according to claim 16 or 17, which is a polypeptide having one or more modifications selected from the group consisting of and insertion. A polypeptide according to any one of claims 1 to 5, comprising contacting the NADas e, method for manufacturing a composite body according to any one of claims 16 to 18. A method for producing NADase including the following steps (a) to (d):
(A) creating a co-expression vector having a first unit and a second unit, wherein the polynucleotide according to claim 6 or 7 is the first unit, and the polynucleotide encoding NADase is the second unit;
(B) creating a transformant introduced with the co-expression vector;
(C) causing the transformant to co-express the polypeptide of any one of claims 1 to 5 with NADase to obtain the complex of claim 16;
(D) Dissociating the polypeptide according to any one of claims 1 to 5 from the complex obtained in the above step to obtain intact NADase: The production method according to claim 20, wherein the NADase is a hemolytic streptococcal NADase. NADase is a polypeptide having 95% or more homology with the amino acid sequence represented by SEQ ID NO: 4, or deletion, substitution, addition of one or two amino acids to the amino acid sequence of SEQ ID NO: 4, The production method according to claim 20 or 21, which is a polypeptide having one or more modifications selected from the group consisting of: and insertion. The production method according to claim 20, wherein the transformant is Escherichia coli. The production method according to claim 23, wherein the Escherichia coli is a TOP10 strain. The manufacturing method of an anti- NADase antibody including the process of manufacturing NADase with the manufacturing method of any one of Claims 20-24. A screening method for a therapeutic agent for infectious diseases caused by hemolytic streptococci, comprising the following steps (a) and (b):
(A) step test substance, to evaluate the polypeptide according to any one of claims 1 to 5, whether or not capable of suppressing the formation of the complex containing the N ADAS e;
(B) A step of selecting a test substance that can suppress the formation of the complex. A screening method for a therapeutic agent for infectious diseases caused by hemolytic streptococci, comprising the following steps (a) and (b):
(A) a step of evaluating whether or not the test substance can cancel inhibition of N ADase activity by the polypeptide according to any one of claims 1 to 5 ;
(B) selecting a test substance capable of releasing the inhibition. 28. The screening method according to claim 26 or 27, wherein the NADase is a hemolytic streptococcal NADase. NADase is a polypeptide having 95% or more homology with the amino acid sequence represented by SEQ ID NO: 4, or deletion, substitution, addition of one or two amino acids to the amino acid sequence of SEQ ID NO: 4, The screening method according to any one of claims 26 to 28, which is a polypeptide having one or more modifications selected from the group consisting of: and insertion.