JP2016121081A - Antiviral drug and antimicrobial agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To primarily provide a novel antiviral drug, and to secondarily provide a novel antimicrobial agent.SOLUTION: By an agent containing, as an active ingredient, a peptide selected from the group consisting of the following (A) and (B), the above subject is solved; (A): a peptide consisting of an amino acid sequence represented by LNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYGSSFYYGKGLINV (sequence ID number 1), and (B) a peptide consisting of an amino acid sequence having deletion, substitution, or addition of one or several amino acids in the amino acid sequence (A), the peptide having antiviral activity.SELECTED DRAWING: Figure 3

Description

  The present invention relates to an antiviral agent and an antibacterial agent comprising a peptide having a predetermined amino acid sequence as an active ingredient.

  In recent years, against the epidemic of viral diseases such as influenza, it has been desired to develop effective preventive and therapeutic methods for viral diseases. Viruses called envelope types such as influenza viruses and herpes viruses have a lipid bilayer membrane called an envelope. Many enveloped viruses are thought to enter cells by fusing the envelope and the cell membrane of cells in contact with the virus.

  As another countermeasure against some viral diseases, pharmaceuticals (antiviral drugs) such as acyclovir, tamivir, or zanamivir are used. However, there are no effective treatments for many viral diseases, and generally preventive measures are taken by vaccination.

  In addition, for example, it has begun to be reported that a peptide having a predetermined sequence exhibits antiviral activity, such as a pharmaceutical composition for treating viral diseases described in Patent Document 1, and new prevention and prevention of viral diseases. Expected as a treatment.

  On the other hand, unlike viruses, microorganisms are generally treated with antibiotics medically. Furthermore, the existence of peptides having antibacterial activity (antibacterial peptides) is also known (for example, Patent Document 2), and some antibacterial peptides are inherently provided as a defense means, such as defensin.

Special table 2008-509969 Special table 2010-517986 gazette

  As for vaccine therapy, immunization may not be achieved by a single vaccination, and multiple vaccinations are an economical burden on the recipient and are highly invasive. Furthermore, immunotherapy only produces antibodies that function specifically against the specific antigen used in the vaccine. Therefore, for example, there is a problem that a sufficient preventive effect cannot be obtained against a virus of a type different from that used for the vaccine. In addition, for drugs such as acyclovir, problems such as the generation of resistant strains and the possibility of causing serious side effects have been pointed out. Accordingly, a first object of the present invention is to provide a novel antiviral agent that replaces a conventionally known pharmaceutical such as a vaccine or acyclovir.

  Regarding microorganisms, the generation of resistant bacteria due to the use of antibiotics has become a major problem, and the development of antibacterial agents such as antibacterial peptides that replace antibiotics is desired. Accordingly, a second object of the present invention is to provide a novel antibacterial agent that replaces conventional antibiotics.

  As a result of intensive studies to solve the above problems, the present inventors have found that a peptide having a predetermined sequence exhibits high antiviral activity against an antiviral activity, particularly against an enveloped virus. Further, the present inventors have found that a peptide having a predetermined sequence exhibits high antibacterial activity, particularly against Streptococcus pneumoniae.

  According to the present invention, it is an object to provide a novel antiviral agent, particularly an antiviral agent used against enveloped viruses. Moreover, according to this invention, the novel antibacterial agent, especially the antibacterial agent used with respect to Streptococcus pneumoniae (Streptococcus pneumoniae) can be provided.

It is the result of having confirmed each fraction which fractionated the peptide extracted from the soybean fermented product of Bacillus subtilis by hydrophobic chromatography by SDS / Tris-tricine system electrophoresis. It is the result of having confirmed the peptide which concerns on this invention extracted from the soybean fermented material of Bacillus subtilis by SDS / Tris-tricine system electrophoresis. The antiviral activity with respect to the herpesvirus of the peptide which concerns on this invention is shown.

  Embodiments of the present invention will be described below. In addition, this invention is not limited only to the following embodiment.

  Further, in this specification, “X to Y” indicating a range means “X or more and Y or less”, and unless otherwise specified, measurement of operation and physical properties is room temperature (20 to 25 ° C.) / Relative humidity 40 to 50. Measure under the condition of%.

  In addition, “(A) LNGTSMASPHVAGAAAALILSKHPNLLSASQVRNRLSSTATYGSSFYYGKGLINV (SEQ ID NO: 1) peptide included in the antiviral agent or antibacterial agent according to the present invention” and “(B) in the amino acid sequence (A)” A “peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added and having antiviral activity” is also referred to as “a peptide according to the present invention”. Further, “(A) LNGTSMASPHVGAAAALILSKHPNLLSASQVRNRLSSTATYGSSFYYGKGLINV (SEQ ID NO: 1)” is simply referred to as “peptide (A)” and “(B) one or several amino acids in the amino acid sequence (A)”. A “peptide consisting of a deleted, substituted or added amino acid sequence and having antiviral activity” is also referred to as “peptide (B)”.

<Antiviral agent>
1st embodiment of this invention is related with the antiviral agent which contains the peptide chosen from the group which consists of following (A) and (B) as an active ingredient.
(A) a peptide consisting of an amino acid sequence represented by LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1),
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antiviral activity.

  The presumed mechanism of action of the antiviral agent according to the present invention is shown below. The following mechanism is speculation and does not limit the technical scope of the present invention. Many of the conventionally known antiviral drugs do not destroy the virus itself, but express antiviral activity by suppressing the growth of the virus. That is, acyclovir and ribavirin inhibit viral growth by inhibiting viral nucleic acid synthesis, and Tamivir and zanamivir inhibit viral neuraminidase. Interferon is also a drug used for the treatment of viral diseases, but it exhibits antiviral activity by stimulating immune function and does not destroy viruses. On the other hand, as described below, the peptide according to the present invention is presumed to interact with a virus and express antiviral activity by destroying the capsid and releasing the nucleic acid.

  Some viruses (envelope viruses) have a structure in which a capsid is covered with a lipid bilayer membrane called an envelope. Since the envelope is a lipid bilayer membrane, it has a negative charge. The peptide according to the present invention contains basic amino acids such as arginine, lysine and histidine, but has no positive amino acid such as aspartic acid or glutamic acid, and therefore has a positive charge with an isoelectric point of about 10.2. This charge bias contributes to the fact that the peptide according to the present invention can interact electrically with the envelope. Accordingly, in one embodiment of the present invention, an antiviral agent is provided for use against enveloped viruses.

In addition, the ^{9th to 21st} amino acids (Pro ^{9 to} Lys ²¹ ) (helix region 1) and the ^{27th to 37th} amino acids (Ala ^{27 to} Thr ³⁷ ) (helix region 2) of the peptide according to the present invention are α -It is considered to have a helix structure. For this reason, after the peptide according to the present invention interacts with the virus, it is presumed that the above-mentioned helix region is perforated in the envelope or capsid, and the nucleic acid is released from the virus. In particular, it is considered that the helix region 1 close to the N-terminal side plays an important role in capsid perforation. Therefore, the antiviral agent according to the present invention is considered to have a function of destroying the virus itself, and it is presumed that the action mechanism is different from that of the conventional antiviral agent.

  It is known in the art that each amino acid can be substituted with an amino acid having similar properties based on differences in its side chains (conservative substitution). For example, non-polar neutral amino acids (Gly, Ala, Met, Val, Leu, Ile, Pro), hydrophilic neutral amino acids (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), Groups can be grouped into those with similar properties such as basic amino acids (Arg, Lys, His) and aromatic amino acids (Phe, Tyr, Trp), and the amino acids within these groups can be substituted for each other.

  It is known in the art that a peptide including a peptide having a physiological activity and having one or several amino acid sequences linked to one or both ends thereof can also maintain the physiological activity. Therefore, a peptide having the above-described antiviral activity as a partial sequence and having one or several amino acids linked to one or both ends thereof and having antiviral activity can also be used as an antiviral agent. is there.

  Therefore, a peptide (peptide (B)) comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antiviral activity is also an antiviral agent according to the present invention. It can be used as an active ingredient. The “substitution” in the above peptide (B) may be “conservative substitution”.

  In the present specification, “several” for amino acids is usually 2 to 5, preferably 2 to 3.

The peptide (B) may have a region homologous to the ^{9th to 21st} amino acids (Pro ^{9 to} Lys ²¹ ) in the amino acid sequence (A) from the viewpoint of maintaining the secondary structure (α-helix structure). Good. In one embodiment of the present invention, the peptide (B) consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A), and the 9th to 21st positions in the amino acid sequence (A) It has a region homologous to the amino acid (Pro ^{9 to} Lys ²¹ ) and has antiviral activity. Also, 9 to 21 amino acids in the amino acid sequence ^{(A) (Pro 9 ~Lys 21} ), and 27 to 37 amino acid ^(Ala 27 ^{~Thr 37)} and may have regions of homology. In one embodiment of the present invention, the peptide (B) consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A), and the 9th to 21st positions in the amino acid sequence (A) Have a region homologous to the amino acid (Pro ^{9 to} Lys ²¹ ) and / or the ^{27th to 37th} amino acids (Ala ^{27 to} Thr ³⁷ ), and have antiviral activity.

  As long as it has antiviral activity, the peptide according to the present invention includes peptides modified by a conventionally known method in consideration of cell membrane permeability, protease resistance, simplicity of peptide synthesis / purification process, and the like. Examples of the peptide modification include addition of polyethylene glycol (PEG, for example, molecular weight of about 100 to 50,000), addition of a membrane-permeable peptide (for example, Trans-Activator of Transcription Protein (Tat) peptide, oligoarginine, or penetratin). , Prenylation such as geranylation and farnesylation (eg prenyl group having 1 to 5 isoprene units), biotinylation, alkylation (eg alkyl group having 1 to 15 carbon atoms), acylation such as acetylation and palmitoylation (For example, an acyl group having 1 to 15 carbon atoms), benzyloxycarbonylation, 9-fluorenylmethyloxycarbonylation and the like.

  In addition, in this specification, "antiviral activity" is evaluated by the method as described in an Example, for example. Specifically, when the final concentration of the sample is 0.1 mg / ml, the PFU measured by the method of the embodiment may be 50% or less when the vehicle value is 100%.

  In one embodiment of the invention, peptide (B) has at least 70% (eg, 80%, 90%, 95%, 98% or 100%) homology with peptide (A) and is antiviral. Has activity. In the present specification, peptide homology is evaluated by the BLASTP program.

  In one embodiment of the present invention, (A) an amino acid sequence represented by (A) LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1) comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added, and has antiviral activity. Having a peptide. The “substitution” may be a “conservative substitution”. In another aspect of this embodiment, (A) has at least 70% (eg, 80%, 90%, 95%, 98%, or 100%) homology with the amino acid sequence represented by LNGTSMASPHVAGAALILSKHPNLLSASQVRNRLSSTATYGSSFYYGGKGLINV (SEQ ID NO: 1) And a peptide having antiviral activity.

  The antiviral agent according to the present invention is used against various viruses, but it is particularly preferable from the viewpoint of antiviral activity that it is used against enveloped viruses. Examples of enveloped viruses include, but are not limited to, herpes simplex virus, varicella-zoster virus, koi herpes virus, salmon herpes virus, bovine herpes virus, influenza virus, human immunodeficiency virus, hepatitis B Examples include viruses, hepatitis C virus, Ebola virus, SARS coronavirus, mumps virus, lassa virus, dengue virus, rubella virus, measles virus, yellow fever virus, and Japanese encephalitis virus. Among these, the antiviral agent according to the present invention is effectively used against herpes viruses such as herpes simplex virus, varicella-zoster virus, koi herpes virus, salmon herpes virus, and bovine herpes virus; and influenza virus. In particular, it can be used effectively against herpes viruses.

(Method for producing peptide)
The peptide according to the present invention can be produced by a conventionally known means using an amino acid synthesizer or the like. The present inventors have also found that the peptide according to the present invention is contained as a peptide having a mass of 5 to 6 kDa in a fermented product of Bacillus subtilis. In one Embodiment of this invention, the manufacturing method of the said antiviral agent including collect | recovering peptides with a mass of 5-6 kDa from the fermented material of Bacillus subtilis is provided.

  In addition, although the mass of said peptide can be measured by well-known methods, such as mass spectrometry, size exclusion chromatography, or electrophoresis, in this specification, sodium dodecyl sulfate (SDS) / Tris-tricine system on condition of the following. It is a value confirmed by electrophoresis.

  The peptide according to the present invention has a mass of 5 to 6 kDa, and has a relatively low molecular weight as compared to proteins in vivo. In order to improve the separation of the low molecular weight region in the electrophoresis method, it is necessary to use a gel having a high acrylamide concentration. In an SDS / Tris-glycine system that is a general electrophoresis method, a gel with a higher acrylamide concentration is more likely to be cracked or mixed with bubbles, making it difficult to produce the gel. Moreover, in an SDS / Tris-glycine gel having a high acrylamide concentration, it is difficult to control the heat generation during electrophoresis, and the separation of the low molecular weight region may not be good. Therefore, in the present invention, a gel can be prepared with an acrylamide concentration lower than that of the SDS / Tris-glycine system, and the mass of the peptide is confirmed by SDS / Tris-tricine electrophoresis suitable for separation of a low molecular weight region. .

(Electrophoresis method for confirmation of peptide mass)
A gel is prepared with the following composition.

  Specifically, the separation gel is polymerized using an electrophoresis plate (50% (v / v) 2-propanol aqueous solution is layered during polymerization). After the separation gel is polymerized, the electrophoresis plate is washed with water to polymerize the spacer gel (during polymerization, 50% (v / v) 2-propanol aqueous solution is overlaid). The electrophoresis plate is washed again with water, and the concentrated gel is polymerized with the comb inserted in the electrophoresis plate. The gel prepared by such a method is washed with water and used for electrophoresis.

Electrophoresis is performed under the following conditions.
Anode-side electrophoresis buffer: 0.2 M Tris-HCl buffer (pH 8.9)
Cathode-side electrophoresis buffer: 0.1 M Tris-0.1 M Tricine-0.1% (w / v) SDS
Electrophoresis: A constant voltage of 50 V for 30 minutes from the start, and a constant voltage of 150 V thereafter (electrophoresis for 90 minutes with a mini gel).

  The sequence of the peptide obtained by fermentation of Bacillus subtilis can be confirmed by a conventionally known amino acid sequence analysis method.

  Subtilisins, which are a kind of protease produced by the genus Bacillus, contain amino acid sequences corresponding to the peptides of the present invention. The results of comparing alignment with peptide (A) using the clustalW program are shown below, taking as an example subtilisins (nattokinase and keratinase) produced by Bacillus subtilis. In the alignment comparison results below, “peptideA” is the “peptide (A)”, “nattokinase [B. subtilis]” is the Bacillus subtilis nattokinase (GenBank: ACJ48969.1) sequence, “keratinase [B. licheniformis]”. "Indicates the Bacillus licheniformis keratinase (GenBank: AID16241.1) sequence. Peptide (A) shows 83% homology with Bacillus subtilis nattokinase (GenBank: ACJ48969.1) and 98% homology with Bacillus licheniformis keratinase.

  Although not limiting the technical scope of the present invention, it is presumed that the 5 to 6 kDa peptide contained in the fermentation product of Bacillus subtilis is derived from a protease of the subtilisins produced by Bacillus subtilis. .

  The fermentation product of Bacillus subtilis is preferably a fermented soybean product. The soy fermented product may be, for example, a culture obtained by cultivating Bacillus subtilis in a medium containing soybean powder, soybean meal, defatted soybean meal, soybean peptide, soybean hydrolyzate, soybean, or soybean peptone as a nitrogen source. Moreover, fermented foods, such as natto and miso, may be sufficient. Bacillus subtilis fermented soy foods such as natto have long been eaten and are particularly excellent in terms of safety. In one Embodiment of this invention, the manufacturing method of said antiviral agent including collect | recovering peptides with a mass of 5-6 kDa from natto is provided.

  The Bacillus subtilis used for the production of the peptide according to the present invention is not particularly limited as long as it produces the peptide according to the present invention, but those that produce subtilisins are preferable, for example, ATCC 15245 strain, NCIB 3610 strain , And ATCC 6051 strain, etc., and may be a natto bacterium (Bacillus subtilis Natto) which is a variant. Of these, Bacillus subtilis Natto, which has been used for the production of fermented foods since ancient times, is particularly preferable from the viewpoint of safety. These microorganisms can be obtained from ATCC, National Institute of Technology and Evaluation (NITE), etc.

  When Bacillus subtilis is cultured to obtain a fermented product, the culture may be performed by a conventionally known method, for example, 20 to 50 ° C, preferably 30 to 40 ° C. The pH of the medium is usually 6-8.

  For cultivation of Bacillus subtilis, a synthetic medium or natural medium containing a carbon source, a nitrogen source, and an inorganic salt is usually used.

  For example, as the carbon source, corn starch, corn meal, starch, dextrin, malt, glucose, glycerin, sucrose, molasses and the like are used alone or as a mixture.

  Nitrogen sources include soybean meal, soybean meal, defatted soybean meal, soybean peptide, soybean hydrolysate, soyton, soy peptone, ammonium sulfate, sodium nitrate, corn steep liquor, gluten meal, meat extract, fat meat bone meal, yeast extract, dried Inorganic or organic nitrogen sources such as yeast, cottonseed meal, peptone, polypeptone, wheat germ, fish meal, meat meal, defatted rice bran, defatted meat and bone meal, malt extract, corn gluten meal and the like can be used alone or as a mixture. From the viewpoint of peptide productivity, preferably, the nitrogen source is derived from soybean, specifically from soybean flour, soybean meal, defatted soybean meal, soybean peptide, soybean hydrolysate, soyton, and soy peptone. Preferably the nitrogen source is selected from the group consisting of

  As the inorganic salt, various inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, sodium bromide, sodium borate or monopotassium phosphate, zinc sulfate, magnesium sulfate can be used alone or as a mixture.

  Moreover, if necessary, a trace amount of heavy metals such as iron, manganese, zinc, cobalt, and molybdic acid can be added. Furthermore, in order to suppress foaming during heat sterilization and culture, vegetable oils such as soybean oil and linseed oil, higher alcohols such as octadecanol, and various antifoaming agents such as silicon may be added.

  When the fermented product is a fermented product of soy food, for example, taking a natto production method as an example, the washed soybean is immersed in water as necessary (for example, at 10 to 20 ° C. for 15 to 30 hours). Cook. Then, the spore of Bacillus natto is sprinkled on soybeans and fermented (35-50 ° C., relative humidity 85-95%, 15-30 hours) to obtain a fermented product. After fermentation, it may be further ripened for about 24 to 72 hours at a low temperature (eg, 4 to 10 ° C.).

  A peptide having a mass of 5 to 6 kDa can be performed by appropriately combining conventionally known methods such as ammonium sulfate (ammonium sulfate) precipitation, column purification, ultrafiltration, dialysis, and salting out.

  As a method for purifying a peptide according to the present invention from a fermented product, for example, a fermented product of Bacillus subtilis such as natto as described above is homogenized in physiological saline or a buffer solution, and then the water-soluble fraction is separated by centrifugation or the like. Recover. As the buffer solution, besides the phosphate buffer solution and the Tris buffer solution, Good buffer solutions such as PIPES, TES, and HEPES can be used. The pH of the buffer used for extraction is preferably 6.5 to 8.5.

  When the water-soluble fraction is collected by centrifugation, the conditions are not particularly limited. For example, the supernatant is collected at 10,000 to 22,000 rpm for about 5 to 60 minutes. However, the operation here is for roughly separating the active ingredient from other ingredients, and is not particularly limited as long as the effects of the present invention can be finally obtained.

  Subsequently, a peptide having a mass of 5 to 6 kDa is recovered from the water-soluble fraction of the fermented product by appropriately combining conventionally known methods such as the above ammonium sulfate fractionation, column chromatography, ultrafiltration, dialysis, and salting out. . The peptide according to the present invention is preferably purified by an ammonium sulfate fraction because of the ease of purification operation.

  In one embodiment of the present invention, recovery of a peptide with a mass of 5-6 kDa yields a fraction (1) soluble in 30% saturated ammonium sulfate from the fermented product and is insoluble in 50% saturated ammonium sulfate from fraction (1). There is provided a method for producing the above antiviral agent, comprising obtaining fraction (2). In the present invention, the fermented material (for example, the above water-soluble fraction) was added with 30% of ammonium sulfate at a saturated concentration and centrifuged (for example, 15,500 rpm, 15 minutes at 4 ° C.). To precipitate (fraction (1)), 50% of saturated ammonium sulfate was added and centrifuged (for example, 15,500 rpm, 4 ° C. for 15 minutes). Also referred to as ~ 50% fraction. In the “fraction (2)” in the present specification, the precipitate obtained by adding 50% saturated ammonium sulfate and centrifuging as described above is further desalted by dialysis or the like, and / or Or dried ones are also included.

  The 30 to 50% fraction obtained by such a technique includes a peptide having a mass of 5 to 6 kDa, which is a kind of peptide according to the present invention. Accordingly, 30-50% fraction is desalted by dialysis or the like as necessary, and the crude product containing the peptide according to the present invention recovered from the fermented Bacillus subtilis is used as the antiviral agent according to the present invention. It may be used. That is, the antiviral agent according to the present invention does not prevent the inclusion of a component derived from a fermented product of Bacillus subtilis having a mass of 5 to 6 kDa as long as an antiviral effect is obtained.

  In addition, as a peptide having a mass of 5 to 6 kDa recovered from a fermentation product of Bacillus subtilis, the fraction (2) obtained above is dissolved again in the above-mentioned physiological saline, buffer solution, etc. What was collect | recovered through the process may be used. Examples of more sophisticated purification steps include column chromatography and ultrafiltration. When purifying by column chromatography, conventionally known methods such as ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption chromatography (hydroxyapatite carrier), reverse phase chromatography, and / or affinity chromatography May be combined as appropriate. Hereinafter, as an example, purification conditions by hydrophobic chromatography are exemplified, but the present invention is not limited to the following method as long as the peptide according to the present invention can be recovered.

(Purification conditions by hydrophobic chromatography)
Fraction (2) is added to 10 mM Tris-HCl buffer (pH 7.4) to a final concentration of 1% (w / v). Further, ammonium sulfate is added to the buffer so as to obtain a 25% saturation concentration, followed by centrifugation at 15,000 rpm for 15 minutes at 4 ° C. After centrifugation, the supernatant is collected and filtered through a filter having a pore size of 0.45 μm, and the filtrate is used as a sample for purification by the following hydrophobic chromatography. A butyl sepharose column (eg, Butyl Sepharose High Performance (GE Healthcare)) is equilibrated with an equilibration buffer (10 mM Tris-HCl buffer (pH 7.4) containing 25% saturated ammonium sulfate). . The above sample is applied to the column after equilibration, and the column is washed with a washing buffer (10 mM Tris-HCl buffer (pH 7.4) containing 25% saturated ammonium sulfate). Of fractions sequentially eluted with elution buffer (10 mM Tris-HCl buffer (pH 7.4) containing 20%, 15%, and 10% saturated ammonium sulfate), containing 10% saturated ammonium sulfate A fraction (fraction (3)) eluted with 10 mM Tris-HCl buffer (pH 7.4)) is obtained.

  Preferably, the method for producing an antiviral agent according to the present invention further includes separating the fraction (2) by hydrophobic chromatography to obtain a 10% saturated ammonium sulfate elution fraction (3).

  The fraction (3) containing a peptide having a mass of 5 to 6 kDa can be used as an antiviral agent. The “fraction (3)” in the present specification includes a 10% saturated ammonium sulfate elution fraction as described above, which is further desalted by dialysis and / or dried.

  Optionally, the peptide according to the present invention, which is a peptide having a mass of 5 to 6 kDa, is further purified by the above method from the fraction (3), and the peptide having a mass of 5 to 6 kDa is isolated and used as an antiviral agent. Good.

More preferably, in the method for producing an antiviral agent according to the present invention, the fraction (3) is separated by reverse phase chromatography, and a fraction having a retention time of 30 minutes (acetonitrile concentration of 30% (v / v)) is used. Further comprising obtaining a fraction, fraction (4)). In addition, the “fraction (4)” in the present specification includes those obtained by further drying the fraction having the acetonitrile concentration of 30% (v / v) as described above. Although the method of reverse phase chromatography is described below, it is not limited to the following method as long as the peptide according to the present invention can be recovered (purification conditions by reverse phase chromatography).
Column: Octadecyl (C18) silica gel column (for example, product name: Wakosil (registered trademark) -5C18HG, manufactured by Wako Pure Chemical Industries, Ltd.)
Mobile phase: Liquid A (H ₂ O for HPLC): Liquid B (acetonitrile for HPLC) (linear gradient from acetonitrile concentration 0% (v / v) to 60% (v / v))
Column temperature: 30 ° C
Flow rate: 1 ml / min.

  In one embodiment of the invention, the peptide according to the invention is isolated. As the isolated peptide, the fraction (4) obtained by the above method may be used.

  The amount of the peptide having a mass of 5 to 6 kDa contained in the recovered product from the fermented Bacillus subtilis is preferably 10 to 100% by mass in terms of the solid content of the recovered product, for example, from the viewpoint of antiviral activity. .

  After purification, the recovered material may be dried by freeze drying or the like and used for the antiviral agent according to the present invention.

<Antimicrobial agent>
In 2nd embodiment of this invention, the antibacterial agent which contains the peptide chosen from the group which consists of following (A) and (B) as an active ingredient is provided.
(A) a peptide consisting of an amino acid sequence represented by LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1),
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antibacterial activity.

  About the peptide used in this embodiment, the description mentioned above about an antiviral agent is modified suitably, and is applied.

  The antibacterial agent according to the present invention is preferably used against bacteria belonging to the genus Streptococcus, and more specifically, alpha-Streptococcus, Streptococcus sp. (Streptococcus sp.), Streptococcus agalactiae (S. agalactiae), Streptococcus anginosus (S. constellatus), Streptococcus gordonii (S. Examples include S. intermedius, S. oralis, S. pneumoniae, S. pyogenes, and S. sanguinis. .

  Among these, the antibacterial agent according to the present invention is particularly effectively used against Streptococcus pneumoniae. That is, in one embodiment of the present invention, the antibacterial agent according to the present invention is used against Streptococcus pneumoniae. That is, in one Embodiment of this invention, the said antibacterial agent is an anti-pneumonitis streptococcus agent.

<Application of antiviral and antibacterial agents>
The antiviral agent and / or antibacterial agent according to the present invention can be used, for example, as a mixture in various foods and beverages, or as a component of pharmaceuticals or various daily necessities.

  The dosage when the antiviral agent and / or antibacterial agent according to the present invention is used as a pharmaceutical varies depending on the symptom of the disease, the age of the patient, and the like. It is 10,000 mg / 60 kg body weight, preferably 10 to 10,000 mg / 60 kg body weight, more preferably 100 to 5000 mg / 60 kg body weight.

  When used as a pharmaceutical, it is administered by oral administration or parenteral administration such as intramuscular, intradermal, subcutaneous, intravenous, lower body cavity and skin. Further, the antiviral agent and / or antibacterial agent according to the present invention may be composed of only the peptide according to the present invention, or a pharmaceutically acceptable carrier and / or diluent suitable for each dosage form. It can also be formulated. Formulation methods and various carriers are well known in the field of pharmaceutical formulation. The pharmaceutically acceptable carrier or diluent may be, for example, a buffer such as a physiological buffer or an excipient (sugar, lactose, corn starch, calcium phosphate, sorbitol, glycine, etc.), and a binder ( Syrup, gelatin, gum arabic, sorbitol, polyvinyl chloride, tragacanth, etc.), lubricant (magnesium stearate, polyethylene glycol, talc, silica, etc.) and the like may be appropriately mixed. As the dosage form, dosage forms such as tablets, granules, powders, capsules, syrups, eye drops, troches, injections, suppositories, ointments and the like can be adopted. These preparations can be produced by generally known production methods.

  Moreover, the standard of the quantity which uses the antiviral agent and / or antibacterial agent which concern on this invention for food is the same grade as the said pharmaceutical. When it is used as a health food, it is added in a ratio of 0.0005 to 10% by mass, preferably 0.1 to 5.0% by mass, based on the total amount of the health food material in terms of dry matter. The By setting it as 0.0005 mass% or more, the target effect is acquired effectively and it is preferable to set it as 10 mass% from a viewpoint of taste. The food and drink are prepared by using the antiviral agent and / or antibacterial agent according to the present invention alone or as a raw material generally used in foods, for example, excipients such as sugars, starches and lipids, and if necessary, binders, lubricants. A flavoring agent such as a flavoring agent, a coloring agent, and a fragrance can be used in combination, and it is produced by a conventional method. Examples of foods and drinks include candy, drops, tablet confectionery, chewing gum, capsules, soft drinks, milk drinks, fruit juice drinks, vegetable drinks, soy milk drinks, coffee drinks, tea drinks, powder drinks, concentrated drinks, nutrition drinks, alcoholic drinks, etc. Is included.

  From the viewpoint of convenience, the antiviral agent and / or antibacterial agent of the present invention is preferably in the form of a spray. The propellant is used, for example, in the form of a spray, aerosol or dry spray. The propellant is processed into a dosage form suitable for spraying and / or discharging with a compressed gas or a pump. For example, the peptide according to the present invention is dissolved in water, a buffer solution having a pH of about 6 to 8, or physiological saline, or is formulated in a suspension in a volatile solvent. The spray can be administered directly to a living body, such as an oral spray or a nasal spray, or can be applied to a textile product such as a face mask or clothing, or sprayed indoors with a humidifier or the like.

  In one embodiment according to the present invention, a textile product obtained by applying the antiviral agent and / or antibacterial agent is provided. Textile products include synthetic fibers, recycled fibers or natural fibers, or garments, hygiene products, bedding products, films, interior goods, and the like processed from these blends. Although not particularly limited, examples of textile products include shirts, cloaks, trousers, skirts, nightwear, underwear, socks, gloves, hats, white robes, nursing clothes, nursing clothes, work clothes, face masks, Examples include sheets, pillow covers, carpets, futons, cushions, wallpaper, various filters, curtains, carpets, and the like. What is necessary is just to apply | coat the antiviral agent and / or antibacterial agent which concern on this invention to the textiles or fiber before product processing by the method of spraying, immersion, impregnation, or coating etc., for example.

<Treatment methods>
In the third embodiment of the present invention, there is provided use of a peptide selected from the group consisting of the following (A) and (B) for treatment or prevention of viral infection or bacterial infection.
(A) a peptide consisting of an amino acid sequence represented by LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1),
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antibacterial activity.

  The embodiment is a method for treating or preventing a viral infection, comprising administering to a patient an effective amount of a peptide selected from the group consisting of (A) and (B).

  In the present specification, the “effective amount” is an amount of an active ingredient that is required at least for exerting a desired effect such as treatment or prevention of health damage caused by a viral infection.

  The patient refers to animals including mammals including, for example, humans, monkeys, mice, rats, hamsters, rabbits, guinea pigs, cows, pigs, dogs, horses, cats, goats and sheep, and preferably humans.

  In this embodiment, the peptide selected from the group consisting of the above (A) and (B) is orally administered or non-muscular such as intramuscular, intradermal, subcutaneous, intravenous, lower body cavity, and skin as described above. It is administered by oral administration. In addition, a peptide formulated with a pharmaceutically acceptable carrier and / or diluent as described above suitable for each dosage form may be used.

In the fourth embodiment of the present invention, there is provided use of a peptide selected from the group consisting of the following (A) and (B) in the manufacture of a medicament for the treatment or prevention of viral infection or bacterial infection. The
(A) a peptide consisting of an amino acid sequence represented by LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1),
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antibacterial activity.

  In the third embodiment and the fourth embodiment of the present invention, the description in the antiviral agent and the antibacterial agent is appropriately changed and applied.

  The virus infection in the third embodiment and the fourth embodiment of the present invention is a disease caused by the infection of the virus described in the description of the antiviral agent. The present invention is particularly useful for enveloped viral infections, particularly herpes virus infections.

  The bacterial infection in the third embodiment and the fourth embodiment of the present invention is a disease caused by infection of the above-mentioned Streptococcus spp. Described in the description of the antibacterial agent. The present invention is particularly effectively used for Streptococcus pneumoniae infections.

  The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples.

<Production Example 1>
170 g of natto (manufactured by Yamada Foods) was weighed, 510 ml (3 times the amount of natto) of 10 mM Tris-HCl (pH 7.4) was added, and homogenized with a homogenizer (Polytron PT4000, Central Science Trade). The homogenized natto was centrifuged for 15 minutes (15,500 rpm, 4 ° C.) and separated into a water-soluble fraction and an insoluble fraction. The water-soluble fraction of the supernatant was collected, ammonium sulfate was added so that the saturation concentration was 30%, and the mixture was stirred at 4 ° C. for 30 minutes. After stirring, the solution was centrifuged for 15 minutes (15,500 rpm, 4 ° C.). To the supernatant collected after centrifugation, ammonium sulfate was added so that the saturation concentration was 50%, and the mixture was stirred at 4 ° C. for 30 minutes. After stirring, the solution was centrifuged for 15 minutes (15,500 rpm, 4 ° C.) to obtain a precipitate (30-50% fraction).

  The obtained precipitate was dissolved in 10 ml of 10 mM Tris-HCl buffer (pH 7.4). The solution of the 30-50% fraction was dialyzed against a 10 mM Tris-HCl buffer (pH 7.4) for 24 hours using a dialysis membrane (fraction (2)).

  The sample after dialysis was added to 10 mM Tris-HCl buffer (pH 7.4) so that the final concentration was 1% (w / v). Further, ammonium sulfate was added to the buffer so as to obtain a 25% saturation concentration, and centrifugation was performed at 15,000 rpm for 15 minutes at 4 ° C. After centrifugation, the supernatant was collected and filtered through a filter having a pore size of 0.45 μm, and the filtrate was used as a sample for purification by the following hydrophobic chromatography.

  A butyl sepharose column (Butyl Sepharose High Performance (manufactured by GE Healthcare)) was equilibrated with an equilibration buffer (10 mM Tris-HCl buffer (pH 7.4) containing 25% saturated ammonium sulfate). The sample was applied to the column after equilibration, and the column was washed with a washing buffer (10 mM Tris-HCl buffer (pH 7.4) containing 25% saturated ammonium sulfate). 10% saturation of fractions eluted sequentially with elution buffer (10 mM Tris-HCl buffer (pH 7.4) containing 20%, 15%, 10%, 5%, and 2% saturated ammonium sulfate concentration) A fraction (fraction (3)) eluted with 10 mM Tris-HCl buffer (pH 7.4) containing ammonium sulfate at a concentration was obtained.

  The result of developing the purified product by hydrophobic chromatography by SDS / Tris-Tricine electrophoresis is shown in FIG. In FIG. 1, “10% saturation elution”, “5% saturation elution”, and “2% saturation elution” mean the ammonium sulfate saturation concentration (%) in the elution buffer, respectively. Moreover, the arrow in FIG. 1 is a 5-6 kDa peptide.

  The peptide fraction (fraction (3)) eluted with 10% saturated ammonium sulfate was subjected to reverse phase chromatography under the following conditions, and the fraction with a retention time of 30 minutes (acetonitrile concentration 30% (v / The fraction of v) and the fraction (4)) were isolated as peptides according to the present invention.

(Purification conditions by reverse phase chromatography)
Column: Octadecyl (C18) silica gel column (Product name: Wakosil (registered trademark) -5C18HG, manufactured by Wako Pure Chemical Industries, Ltd.)
Equipment used: Gulliver series (manufactured by JASCO)
Mobile phase: Liquid A (H ₂ O for HPLC) / Liquid B (acetonitrile for HPLC) (linear gradient from acetonitrile concentration 0% (v / v) to 60% (v / v))
Column temperature: 30 ° C
Flow rate: 1 ml / min
Injection volume: 1.0ml
Detector: LKB 2158 Uvicord SD (206 nm).

  The molecular weight of the peptide was confirmed by sodium dodecyl sulfate (SDS) / Tris-Tricine electrophoresis under the above conditions. The results are shown in FIG. In FIG. 2, the arrows indicate the peptides according to the present invention.

  When the amino acid sequence of the peptide of mass 5 kDa confirmed by electrophoresis was confirmed by a protein sequencer (PPSQ-31B / 33B, manufactured by Shimadzu Corporation), the amino acid sequence represented by SEQ ID NO: 1 (mass: 5480 Da) was obtained. there were.

  The peptide collected by the above method was frozen and then lyophilized to obtain a sample (sample 1) in the following Examples. Sample 1 contains 90% by mass or more of the peptide according to the present invention.

<Example 1>
Using the sample 1 obtained under the above conditions, antiviral activity against herpes virus was evaluated by the following method.

  (1) A virus solution of HSV1 (Herpes simplex virus type 1) (VR3 strain, purchased from ATCC) was prepared by the following method.

(Method for preparing virus solution)
Cells: Vero cells (African green monkey kidney cells, IFN gene deficient)
Medium: RPMI1640 containing 10% fetal bovine serum (FBS), 10 μg / ml penicillin-streptomycin (hereinafter also referred to as “RPMI1640 medium” or simply “medium”) (manufactured by Wako Pure Chemical Industries, Ltd.)
(1) -1: Vero cells cultured in T75 flasks were inoculated with a virus containing HSV1.
(1) -2: The cells were cultured for 1 hour in a CO ₂ incubator, and the cells were contacted and infected with the virus.
(1) -3: The culture supernatant was removed, and the cells were washed once with PBS (−) (phosphate buffered saline).
(1) -4: 10 ml of the medium was placed in the flask and cultured in a CO ₂ incubator until a cytopathic effect (CPE) was observed. (About 2-3 days)
(1) -5: When CPE was observed in about 90% of the cultured cells, the cells were freeze-thawed. Thereafter, the supernatant was collected and centrifuged (2000 rpm, 5 minutes, 25 ° C.).
(1) -6: After centrifugation, the supernatant was recovered.
(1) -7: The recovered supernatant was used as a virus solution, dispensed in 500 μl aliquots into tubes, and stored at −80 ° C. until use.

  (2) The virus solution prepared in (1) and sample 1 (final concentration in the following (4): 0.1 to 1 mg / ml) were mixed in an RPMI1640 medium to prepare a reaction solution.

  (3) The reaction solution was allowed to stand at room temperature (25 ° C.) for 30 minutes.

(4) The reaction solution was diluted with RPMI1640 medium to prepare a virus diluted solution (10 ² to 10 ⁹ (PFU (Plaque Forming Unit) / ml)).

  In the present specification, PFU is defined as follows. That is, 60 μl of virus solution and 540 μl of RPMI 1640 medium were mixed to prepare a 10-fold dilution series. Vero cells cultured in a monolayer on a 24-well plate were inoculated with the virus solution prepared above and washed with PBS. Thereafter, RPMI1640 medium in which carboxymethylcellulose (CMC) (final concentration 1% (w / v)) was dissolved was added and cultured at 37 ° C. for 48 hours. Thereafter, the cells were stained with crystal violet, and the number of transparent circles (plaques) not stained was visually measured. The amount of virus forming one plaque was defined as “1PFU”.

(5) Vero cells (cell number: cells) prepared by monolayer culture (48 hours in a 5% CO ₂ incubator at 37 ° C.) in advance on a 24-well plate (manufactured by Greiner) with the virus diluted solution (250 μl) prepared in (4). 1 × 10 ⁵ / well).

(6) Vero cells inoculated with the virus were allowed to stand for 1 hour in a 5% CO ₂ incubator at 37 ° C.

  (7) After standing for 1 hour, the supernatant was discarded, and the cells were washed with 500 μl of PBS (−).

(8) Cells were cultured in 500 μl / well of RPMI 1640 medium (containing 1% (w / v) CMC) in a 5% CO ₂ incubator at 37 ° C.

  (9) While changing the medium at a rate of 2 days / time, the culture was continued until day 5 when plaque appeared.

  (10) PFU (place forming unit) was calculated on the fifth day from the start of the culture.

The calculation result of PFU is shown in FIG. In FIG. 3, when the virus solution was allowed to stand in a reaction solution not containing sample 1, the PFU was 1.2 × 10 ⁹ / ml. In contrast, when the virus solution was allowed to stand in a reaction solution containing Sample 1 at 1 mg / ml or 0.1 mg / ml, the PFU was 8.0 × 10 ⁶ / ml and 2.8 × 10 ⁸ / ml, respectively. Met.
As shown in FIG. 3, antiviral activity was observed in Sample 1.

<Example 2>
Using sample 1 obtained under the same conditions as in Example 1, antibacterial activity against various bacteria was evaluated by the following method.

  Specifically, a 2-fold dilution series having a sample concentration from 1280 μg / ml to 1.25 μg / ml was prepared using the medium shown in Table 2 below, and contained in a 96-well plate (150 μl / well) together with the cells. The cells were cultured overnight in a 37 ° C. incubator, and the presence or absence of bacterial cell growth was confirmed with a microscope, and the minimum inhibitory concentration (MIC, μg / ml) of Sample 1 was determined. The results are shown in Table 2.

  Amoxicillin, a penicillin antibiotic used as a positive control, was purchased from Wako Pure Chemical Industries.

  From the above, it was shown that the antibacterial agent according to the present invention has a particularly strong antibacterial activity against S. pneumoniae.

Claims (10)

An antiviral agent comprising a peptide selected from the group consisting of the following (A) and (B) as an active ingredient;
(A) a peptide consisting of an amino acid sequence represented by LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1),
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antiviral activity.   The antiviral agent according to claim 1, which is used against an enveloped virus.   The enveloped virus is herpes simplex virus, varicella-zoster virus, koi herpes virus, salmon herpes virus, bovine herpes virus, influenza virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, Ebola virus, SARS The antiviral agent according to claim 2, selected from the group consisting of coronavirus, mumps virus, lassa virus, dengue virus, rubella virus, measles virus, yellow fever virus, and Japanese encephalitis virus.   The antiviral agent according to any one of claims 1 to 3, which is in the form of a spray.   The manufacturing method of the antiviral agent of any one of Claims 1-4 including collect | recovering peptides with a mass of 5-6 kDa from the fermented material of Bacillus subtilis (Bacillus subtilis).   The production method according to claim 5, wherein the fermented product is a fermented soybean product. Recovery of the 5-6 kDa peptide yielded a fraction (1) soluble in 30% saturated ammonium sulfate from the fermented product,
The production method according to claim 5 or 6, comprising obtaining a fraction (2) insoluble in 50% saturated ammonium sulfate from the fraction (1).   The textile product formed by apply | coating the antiviral agent of any one of Claims 1-4, or the antiviral agent manufactured by the manufacturing method of any one of Claims 5-7. An antibacterial agent comprising a peptide selected from the group consisting of the following (A) and (B) as an active ingredient;
(A) a peptide consisting of an amino acid sequence represented by LNGTSMASPHVVAGAALILSKHPNLLSASQVRNRLSSTATYGSFSYYKGLINV (SEQ ID NO: 1),
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (A) and having antibacterial activity.   The antibacterial agent of Claim 9 used with respect to Streptococcus pneumoniae (Streptococcus pneumoniae).