JP6455920B2 - Method for predicting immunostimulatory activity of microorganisms or DNA - Google Patents

Description

  The present invention includes a method for predicting the immunostimulatory activity of a microorganism or DNA, a method for screening a microorganism or DNA predicted to have immunostimulatory activity using the method, and the microorganism or DNA as an active ingredient. It relates to an immunostimulator. More specifically, among the immunostimulatory activities of microorganisms or DNA, the immunostimulatory activity through TLR9 receptor stimulation, which is represented by the activity of inducing interferon α (IFN-α) production in pDC cells, is characterized by the characteristics of the DNA sequence. Basically, the present invention relates to a method for efficiently screening highly active microorganisms and DNA.

Viruses and bacteria that invade humans and animals are recognized and engulfed by cells of the immune system. The immune system can be divided into the innate immune system and the acquired immune system. B cells, T cells, etc. are immune cells of the acquired immune system and are involved in long-term antigen-specific reactions. In contrast, macrophages, natural killer (NK) cells, dendritic cells, and the like are considered to be cells of the innate immune system and are involved in short-term and antigen-nonspecific reactions. The innate immune system is primarily responsible for primary responses in bacterial and viral infections, among which dendritic cells are powerful and important constituent cells. There are numerous subtypes of dendritic cells. Myeloid dendritic cells (myeloid dendritic cells = mDC), CD8 ⁺ dendritic cells (CD8 ⁺ dendritic cells = CD8 ⁺ DC) and plasmacytoid dendritic cells It can be roughly classified into (plasmacytoid dendritic cell = pDC). Among them, pDC is a main production cell of IFN-α that exhibits growth inhibitory activity against viruses, and has a very important role in antiviral biodefense.

  pDC expresses TLR7 and TLR9, which are Toll-like receptors (TLRs) localized in endosomes, and these receptors are activated by being stimulated by their ligands. -Produces α. IFN-α induces the expression of viral replication inhibitors such as MxA, RNase that degrades the virus, and other major immune cells such as macrophages, NK cells, cytotoxic T cells, and B cells. It is known to activate cells and induce strong virus resistance (Non-patent Document 1). It is also known that a DNA sequence containing a virus-derived single-stranded RNA or imidazoquinoline as an antiviral agent functions as a TLR7 ligand, and an unmethylated CpG as a TLR9 ligand. That is, activation of pDC and production of IFN-α occur in the body during virus infection, and the above-described chain virus resistance reaction is induced. For this reason, in the treatment of viral diseases such as hepatitis B and hepatitis C, treatments for parenteral administration such as intramuscular or subcutaneous administration of IFN-α protein produced by a technique such as genetic recombination are performed. . On the other hand, many examples have been reported in which the resistance reaction of immune cells against viruses is induced orally by ingesting safe edible microorganisms such as lactic acid bacteria and fermented foods of those microorganisms (Non-patent Document 2). However, many of these reports activate macrophages, mDCs, and NK cells, and do not directly activate pDCs.

  Recently, it has been reported that only a few lactic acid bacteria such as Lactococcus lactis JCM 5805 activate pDC and induce IFN-α production, and that its IFN-α production-inducing activity is dependent on TLR9. (Patent Document 1 and Non-Patent Document 3). In these reports, the authors screen IFN-α production-inducing active strains from a wide variety of lactic acid bacteria selected at random. However, there are only a few high-activity strains, and it is said that IFN-α production-inducing active strains of 100 pg / mL or more were obtained only in 3 out of 125 strains (2.4%). In addition, in Patent Document 1 and Non-Patent Document 3, as a technique for screening lactic acid bacteria having high IFN-α production-inducing activity, co-cultured dendritic cells (DC) in mouse bone marrow cells and candidate lactic acid bacteria, A method for measuring IFN-α in a culture solution by ELISA is disclosed. Similar experiments can also be performed using mouse spleen. However, in any method, it is necessary to purely culture each lactic acid bacterium for several days, then collect, wash, and freeze-dry, and it is necessary to obtain and culture pDC cells from mice of the appropriate age, Since it is necessary to measure the amount of IFN-α by ELISA, there is a drawback that it requires enormous time and cost.

  By the way, in a method for screening DNA such as oligonucleotide having immunostimulatory activity, a screening method focusing on unmethylated CpG motif-containing nucleotides known as TLR9 ligands has been reported. For example, Non-Patent Document 4 includes an oligonucleotide containing an unmethylated CpG motif derived from lactic acid bacteria and an unmethylated CpG motif derived from lactic acid bacteria in animal cells (transfectants) in which porcine TLR9 is forcibly expressed. A method of screening an oligonucleotide having immunostimulatory activity by contacting a non-existing oligonucleotide or the like with the degree of stimulation of TLR9 in the transfectant as an index of the degree of activation of NF-κB is disclosed in Patent Document 2 ( JP 2005-027665 A) describes a sample derived from a test microorganism such as lactic acid bacteria (cell wall, DNA fragment having an unmethylated CpG motif, etc.) on animal cells (transfectants) in which porcine TLR9 is forcibly expressed. ) To indicate the degree of increase in TLR9 activity in the transfectant. As a method for evaluating whether or not the sample or the test microorganism activates the intestinal tract immune system, Patent Document 3 (Japanese Translation of PCT International Publication No. 2002-517156) discloses an oligonucleotide containing an unmethylated CpG motif, etc. A method is disclosed in which an oligonucleotide is co-cultured with macrophages and an oligonucleotide having human immunostimulatory activity is screened using the amount of Th1 cytokine production in the culture supernatant as an index.

International Publication No. 2012/091081 JP 2005-027665 A JP-T-2002-517156

Immunol Rev (2010) 234 (1), 142-162 Br J Nutr (2011) 106 (4), 549-556 PLoS One. 2012; 7 (4): e32588 Animal Science Journal (2004) 75 (4), 377-382

  As described in the background art above, the conventional method of screening for lactic acid bacteria having high IFN-α production-inducing activity requires enormous time and cost. If there is a simple means for efficiently screening for microorganisms that stimulate pDC, it is possible to further expand the screening range, and as a result, selection of bacteria with higher growth efficiency and bacteria with excellent flavor can be performed in a short time. it is conceivable that. Furthermore, it is considered possible to apply to a wider range of foods and to obtain higher IFN-α production-inducing active strains. From these facts, there has been a strong demand for the elucidation of the activity inducer of microorganisms having high IFN-α production-inducing activity and the development of an efficient screening method for highly active microorganisms using the characteristics. As described in the background art above, many of the conventional immunostimulators using edible microorganisms activate macrophages, mDCs, and NK cells, and do not directly activate pDCs. There wasn't. For this reason, there has been a demand for microorganisms that can directly stimulate pDC to induce strong virus resistance, and immunostimulators using such microorganisms.

  An object of the present invention is to provide a method for efficiently predicting the immunostimulatory activity (preferably IFN-α production-inducing activity) of a microorganism or DNA, and a microorganism or DNA having a high immunostimulatory activity (preferably IFN-α production-inducing activity). It is an object of the present invention to provide an efficient screening method and an immunostimulant (preferably an IFN-α production inducer) containing such a microorganism or DNA as an active ingredient. Another object of the present invention is to provide a method for efficiently screening microorganisms and DNA fragments that are highly active for immunostimulation reactions through TLR9 receptor stimulation among immunostimulation reactions other than IFN-α production-inducing activity. And providing an immunostimulant (preferably an IFN-α production inducer) containing, as an active ingredient, a microorganism or DNA that directly stimulates pDC.

  As described in Background Art, Patent Document 1 and Non-Patent Document 3 report that IFN-α production-inducing activity such as Lactococcus lactis JCM 5805 is dependent on TLR9. This has shown that DNA is essential for IFN- (alpha) production induction activity among the structural components of these lactic acid bacteria. The present inventors pay attention to this, and the lactic acid bacterium producing IFN-α has a special structure in its chromosomal DNA. If the structure is clarified, the genome information of the microorganism and the DNA sequence of the gene It is possible to search for DNA and microorganisms containing the structure using public databases such as information, etc., and consider that DNA and microorganisms with high IFN-α-inducing activity can be screened more efficiently. Started.

  As described above, DNA fragments containing unmethylated CpG motifs are known to have TLR9 stimulating activity. In Patent Document 1 and Non-Patent Document 3, it is also reported that an oligo DNA containing an unmethylated CpG motif activates pDC. From the above, the present inventor has shown that DNA with high IFN-α production-inducing activity contains a large number of unmethylated CpG motifs, and microorganisms with high IFN-α production-inducing activity are present in genomic DNA compared to microorganisms with low IFN-α production-inducing activity. Were expected to contain a higher number of unmethylated CpG motifs.

  Based on this hypothesis, the present inventors synthesized DNAs having the same nucleotide length with different numbers of unmethylated CpG motifs, and investigated the correlation between the number of unmethylated CpG motifs and IFN-α production-inducing activity. As a result, no activity was observed for DNA fragments having no unmethylated CpG motifs or fragments having a significantly low number of unmethylated CpG motifs per unit length. On the other hand, contrary to expectation, even if the number of unmethylated CpG motifs increased, IFN-α production-inducing activity did not necessarily increase, and no clear positive correlation was observed between the two.

  Furthermore, the present inventors searched the total number of unmethylated CpG motifs in the genome for microbial strains that were considered to have high IFN-α production-inducing activity in Patent Document 1 and Non-Patent Document 3 using genomic information. The number of total unmethylated CpG motifs in the genome of the strain with low IFN-α production-inducing activity was never higher. From these results, the present inventors inferred that some DNA structure different from the number of unmethylated CpG motifs is necessary in order to be a DNA fragment with high IFN-α production-inducing activity, and diligently elucidated it. Repeated research. As a result, a DNA fragment having a high IFN-α production-inducing activity has a large number of unmethylated CpG motifs, and at the same time, the GC content of the entire DNA fragment is low, at most 45% or less. Was found to have a negative correlation between the GC content and the IFN-α production-inducing activity between 45% and 35%.

  Furthermore, since the high proportion of the number of unmethylated CpG motifs and the low GC content are contradictory events, the present inventors have found that unmethylated CpG of a DNA fragment having a high IFN-α production-inducing activity. The number of motifs (or the ratio thereof) and the GC content were respectively defined, and the present invention was completed. Furthermore, the present inventor examined the genome structure of various microorganisms in accordance with this definition, and microorganisms having a high number of unmethylated CpG motifs in the low GC content region of genomic DNA or a high ratio thereof have IFN-α production-inducing activity. It proved that it was a high microorganism, and completed this invention.

That is, the present invention
(1) A method for predicting the immunostimulatory activity of a microorganism, comprising the following steps (A) to (F):
(A) Step A of obtaining the base sequence data of the genomic DNA of the microorganism from a database:
(B) In the base sequence data of the genomic DNA of the microorganism, step B of obtaining base sequence data of an arbitrary group of DNA fragments within a range of 50 bp to 1000 bp that are continuous or partially overlap each other:
(C) Checking the GC content of each DNA fragment in the DNA fragment group C:
(D) Of the DNA fragment group, a low GC content DNA fragment having a GC content of 35% or less, 40% or less, or 45% or less is arranged in a corresponding portion on the base sequence of the genomic DNA, and the low A step D in which a region of genomic DNA of the microorganism covered by a GC content DNA fragment is a low GC content region in the microorganism genome:
(E) Step E of totaling the number of unmethylated CpG motifs contained in the low GC content region to calculate the total number of low GC content region unmethylated CpG motifs:
(F) A step of predicting a microorganism having immunostimulatory activity when the total number of low GC content region unmethylated CpG motifs calculated in the step (E) is 100 or more per 10,000 bp of genomic DNA of the microorganism. F:
(2) The immunostimulatory activity characterized by including the process H which selects the microorganism estimated as the microorganism which has immunostimulatory activity in the method as described in said (1) as a microbe which is estimated to have immunostimulatory activity. A method for screening microorganisms predicted to have
(3) The method according to (1) or (2) above, wherein the GC content in step D is 40% or less,
(4) The method according to any one of (1) to (3) above, wherein the microorganism is a lactic acid bacterium,
(5) The method according to (4) above, wherein the lactic acid bacterium is a cocci,
(6) The method according to any one of (1) to (5) above, wherein the immunostimulatory activity is IFN-α production-inducing activity via plasmacytoid dendritic cells.

The present invention also provides:
(7) A method for screening DNA predicted to have immunostimulatory activity, comprising the following steps (Q) to (S):
(Q) Step Q of obtaining base sequence data of an arbitrary group of DNA fragments within the range of 50 bp to 1000 bp:
(R) Checking the GC content of each DNA fragment in the DNA fragment group, and selecting a DNA fragment having a GC content of 35% or less, 40% or less, or 45% or less R:
(S) The number of unmethylated CpG motifs contained in the DNA fragment selected in the step R is calculated, and a DNA fragment having 0.017 or more unmethylated CpG motifs per nucleotide is immunostimulatory. Selecting as a predicted DNA having
(8) In the nucleotide sequence data of the genomic DNA of the microorganism that is predicted to have immunostimulatory activity by the method according to any one of (1) to (6) above, any Q in the range of 50 bp to 1000 bp The method according to (7) above, which is a step of obtaining base sequence data of the DNA fragment group.

Furthermore, the present invention provides
(9) DNA having a GC content of 35% or less, 40% or less, or 45% or less, a number of unmethylated CpG motifs per nucleotide of 0.017 or more, and a range of 50 bp to 1000 bp An immunostimulant containing as an active ingredient,
(10) The DNA is selected from the group consisting of SEQ ID NOs: 173 to 177, 179 to 181, 183 to 185, 187 to 190, 194, 213, 214, 216 to 220, 225, 229, 231 and 233 The immunostimulator according to (9) above, which is DNA comprising the nucleotide sequence of
(11) By the method according to any one of (1) to (6) above, an immunostimulant containing a microorganism predicted to be an immunostimulatory activity or a processed product thereof as an active ingredient,
(12) The microorganism is a microorganism having IFN-α production-inducing activity, and the IFN-α production-inducing activity is such that the dried product of the microorganism is 10 μg / mL in the culture medium of bone marrow-derived dendritic cells. IFN-α in which the concentration of IFN-α in the supernatant of the culture medium after culturing the bone marrow-derived dendritic cells for 2 days at 37 ° C. and 5% CO ₂ is 100 pg / mL or more. The immunostimulator according to (11) above, which is a production-inducing activity,
(13) The immunostimulant according to (11) or (12) above, which contains, as an active ingredient, a lactic acid bacterium belonging to the genus Leuconostoc or genus Pediococcus or a processed product thereof.

  According to the present invention, a method for efficiently predicting the immunostimulatory activity (preferably IFN-α production-inducing activity) of a microorganism or DNA, and the microorganism or DNA having a high immunostimulatory activity (preferably IFN-α production-inducing activity) are efficiently performed. Can be provided, and an immunostimulant (preferably an IFN-α production inducer) containing such a microorganism or DNA as an active ingredient can be provided. Immunostimulants, including microorganisms or DNA that can activate pDC, can directly stimulate pDC to induce strong viral resistance.

  In addition, the screening method for microorganisms and DNA provided by the present invention is considered to be effective for screening immunostimulatory reactions induced by TLR9 receptor stimulation among immunostimulatory reactions other than pDC IFN-α production-inducing activity. It is done. Typical examples of these include suppression of allergies such as asthma by IL-12 production induction and accompanying improvement of Th1 / 2 balance, adjuvant effect at the time of vaccine administration, activation of B cells, and the like. In these immunostimulation reactions, oligo DNA containing an unmethylated CpG motif and specific microorganisms are considered to be effective (Nat Rev Immunol (2004) 4 (4), 249-258, J Immunol ( 2013) 190 (4), 1591-1602) does not reveal how to screen highly active sequences or strains. Furthermore, DNA has a skeleton into which a non-natural PS (phosphorothioate) bond is introduced, so that problems remain in terms of synthesis cost and safety. The DNA or microorganism screened by the present invention is considered to be effective for solving the remaining problem and enhancing the above-described immunostimulatory activity other than the IFN-α production-inducing activity of pDC.

  As can be seen from Table 14 described later, the number of low GC content region unmethylated CpG motifs contained in the entire genome shows a tendency opposite to the total number of unmethylated CpG motifs. On the other hand, it has been reported that the appearance frequency of the number of unmethylated CpG motifs in the entire genome shows a very strong correlation with the GC content of genomic DNA (J. Med. Microbiol. 63 (2), pp. 293 -308). That is, the number of low GC content unmethylated CpG motifs shows a certain negative correlation with the GC content of genomic DNA. In many microorganisms, the GC content of genomic DNA exceeds 40%, and the ratio of the total number of unmethylated CpG motifs in the low GC content region to the total number of unmethylated CpG motifs is usually not so high. The percentage of microorganisms having a low GC content region unmethylated CpG motif (more than a certain percentage of the number of bases in the genome) is not so high. By doing so, it is possible to greatly narrow down the microorganisms to be analyzed by prediction based on the index that the total number of unmethylated CpG motifs with low GC content relative to the number of bases in the genome is a specific ratio or more. It can be said that the screening method is extremely useful as a screening method.

In experiment of this-application Example 1, it is a figure which shows the correlation with the number of unmethylated CpG motifs of each DNA fragment, and IFN- (alpha) production induction activity. It is the figure which divided and showed the correlation of the number of unmethylated CpG motifs, and IFN- (alpha) production induction activity depending on whether the GC content of a DNA fragment is 45% or 40% or less among the experimental results of FIG. FIG. 2A is a diagram showing the results of DNA fragments having a GC content of 45% or less. FIG. 2B shows the results for DNA fragments with a GC content greater than 45%. FIG. 2C is a diagram showing the results of DNA fragments having a GC content of 40% or less. FIG. 2D shows the results for DNA fragments with a GC content greater than 40%. The experimental results in FIG. 1 are classified into 6 groups (“˜30” (30% or less), “30-35” (above 30% and 35% or less), “35-40”) depending on the degree of GC content of the DNA fragment. 35 to 40%), “40 to 45” (40 to 45%), “45 to 50” (45 to 50%), “50 to” (50 to 50%)) FIG. 4 is a diagram showing an average value of “activity per unmethylated CpG motif” obtained for each group. In experiment of this-application Example 2, it is a figure which shows the correlation with the number of unmethylated CpG motifs of each DNA fragment, and IFN- (alpha) production induction activity. FIG. 5 is a diagram showing the correlation between the number of unmethylated CpG motifs and IFN-α production-inducing activity separately depending on whether the GC content of the DNA fragment is 40% or 35% or less in the experimental results of FIG. FIG. 5A is a diagram showing the results of DNA fragments having a GC content of 40% or less. FIG. 5B shows the results for DNA fragments with a GC content greater than 40%. FIG. 5C is a diagram showing the results of DNA fragments having a GC content of 35% or less. FIG. 5D shows the results for DNA fragments with a GC content greater than 35%. It is a figure which shows the average value of "the activity per unmethylated CpG motif" which divided | segmented the result of the experiment of this-application Example 3 into 3 groups according to the grade of the GC content of a DNA fragment, and was calculated | required for each group. . FIG. 6A is a diagram divided into three groups of “˜45” (45% or less), “45 to 55” (higher than 45% and 55% or less), and “55” (higher than 55%). FIG. 6B is a diagram divided into three groups of “40˜” (40% or less), “40-50” (above 40% and 50% or less), and “50˜” (above 50%).

  The present invention relates to a method for predicting the immunostimulatory activity of a microorganism (hereinafter also referred to as “method for predicting the microorganism of the present invention”) and a method for screening a microorganism predicted to have immunostimulatory activity (hereinafter referred to as “the present”). A method for predicting the immunostimulatory activity of DNA (hereinafter also referred to as “method for predicting the DNA of the present invention”), and a DNA predicted to have an immunostimulatory activity. A screening method (hereinafter also referred to as “the DNA screening method of the present invention”) and an immunostimulant containing such a microorganism or DNA as an active ingredient (hereinafter also referred to as “the immunostimulatory agent of the present invention”) About.

In the present specification, an unmethylated CpG motif means a 6 nucleotide sequence (hexamer) represented by NNCGNNN (where N represents any nucleotide of A, T, G, and C, respectively). In the present invention, unless otherwise specified, “DNA”, “genomic DNA”, and “DNA fragment” include a nucleotide sequence (base sequence) in addition to DNA having a substance as a compound, genomic DNA, or DNA fragment. ) Data, DNA, genomic DNA and DNA fragments, that is, DNA sequences, genomic DNA sequences and DNA fragment sequences are also included. “DNA” in the present invention may be single-stranded or double-stranded, and when the DNA is derived from a microorganism, it may be a sense strand or an antisense strand. May be. In the present specification, the “microorganism-derived DNA” includes (x) DNA collected from the microorganism, and (y) DNA that is not collected from the microorganism, but all of the genomic DNA of the microorganism and its transcript. DNA consisting of nucleotide sequences identical or complementary to some nucleotide sequences is included. When DNA such as genomic DNA is derived from microorganisms such as bacteria, viruses or fungi, unlike CDNA derived from mammals, most CpG motifs are not methylated. All motifs are considered unmethylated CpG motifs. In addition, the immunostimulatory activity in the present invention is not particularly limited as long as it is an immunostimulatory activity, but immunostimulatory activity through activation of TLR9 in plasmacytoid dendritic cells or B cells is preferable. Immunostimulatory activity through activation of TLR9 in dendritic cells is more preferable. Among them, activity to induce type I interferon (interferon α and / or interferon β) production through activation of TLR9 in plasmacytoid dendritic cells is preferred. More preferably, interferon α production-inducing activity through activation of TLR9 in plasmacytoid dendritic cells is particularly preferable.

<1. Method for Predicting Immunostimulatory Activity of Microorganism>
The method for predicting the immunostimulatory activity of microorganisms presented by the present invention includes the following steps (A) to (F).
(A) Step A of obtaining the base sequence data of the genomic DNA of the microorganism from a database:
(B) In the base sequence data of the genomic DNA of the microorganism, step B of obtaining base sequence data of an arbitrary group of DNA fragments within a range of 50 bp to 1000 bp that are continuous or partially overlap each other:
(C) Checking the GC content of each DNA fragment in the DNA fragment group C:
(D) Of the DNA fragment group, a low GC content DNA fragment having a GC content of 35% or less, 40% or less, or 45% or less is arranged in a corresponding portion on the base sequence of the genomic DNA, and the low A step D in which a region of genomic DNA of the microorganism covered by a GC content DNA fragment is a low GC content region in the microorganism genome:
(E) Step E of totaling the number of unmethylated CpG motifs contained in the low GC content region to calculate the total number of unmethylated CpG motifs in the low GC content region:
(F) A step of predicting a microorganism having immunostimulatory activity when the total number of low GC content region unmethylated CpG motifs calculated in the step (E) is 100 or more per 10,000 bp of genomic DNA of the microorganism. F:

  The merit of the method for predicting microorganisms of the present invention, which is that the immunostimulatory activity of microorganisms can be efficiently predicted is that the immunostimulatory activity of various types of microorganisms (for example, 10 types or more, preferably 20 types or more) is particularly effective. You can enjoy more when you predict.

The microorganism in the present invention is preferably a microorganism selected from the group consisting of bacteria, viruses and fungi, more preferably a microorganism selected from the group consisting of bacteria and viruses, more preferably bacteria, and more preferably human bodies. From the viewpoint of obtaining immunostimulatory activity while ensuring safety, lactic acid bacteria and Bacillus subtilis are more preferable. As representative examples, the genus Lactococcus (Lactococcus), Pedioccoccus (Pediocccus), Leuconostoc (Leuconostoc), Streptococcus (Streptococcus), Tetragenococcus (Tetragenococcus), Lactobacillus (Lactobacillus), Examples include lactic acid bacteria such as Enterococcus. Among them, since the cells are small, they are more easily incorporated into pDC and have a high possibility of obtaining a better immunostimulatory activity (preferably IFN-α production-inducing activity). Therefore, Lactococcus, Pediococcus, Leuco More preferred are lactic acid cocci such as Nostock, Streptococcus, Tetragenococcus and Enterococcus. Representative of these are Lactococcus lactis, Leuconostoc mesenteroides, and Pediococcus pentosaceus.

  The step A is not particularly limited as long as it is a step of obtaining the base sequence (nucleotide sequence) data of the genomic DNA of the microorganism from the database, and the database used may be a public database such as NCBI. It may be a database for use. The base sequence data to be used is not particularly limited as long as it is the base sequence data of the genomic DNA of the microorganism of the same genus and the same species as the target microorganism for predicting immunostimulatory activity. However, the base sequence data of the genomic DNA of the same genus and the same strain as the target microorganism is preferable. In addition, the “microorganism genomic DNA base sequence data” when the microorganism prediction method of the present invention is used in the microorganism screening method of the present invention described later is the microorganism to be screened from the viewpoint of expanding the screening target. The nucleotide sequence data of the genomic DNA of the same genus and homologous strain is also preferably as high as the nucleotide sequence data of the genomic DNA of the same genus and same strain. In the method for predicting microorganisms and the method for screening microorganisms of the present invention, the base sequence data of the genomic DNA of the same gene species can be used. This is because the GC content and the number of unmethylated CpG motifs in the low GC content region are quite similar (for example, see Table 16 below). Similarly, the results of the method for predicting the microorganism of the present invention and the screening method for the microorganism of the present invention described later are preferably applied to the same strain and the same strain as the microorganism from which the genomic DNA used is derived. It can also be applied to other strains of the same genera.

  The “microorganism genomic DNA base sequence data” used in the method of predicting the microorganism of the present invention and the microbe screening method of the present invention described later is not necessarily the base sequence data of the total genomic DNA of the microorganism. However, from the viewpoint of obtaining higher accuracy in predicting the immunostimulatory activity, at least 50% or more, more preferably 70% or more, more preferably 80% or more, more preferably 80% or more of the base sequence data of the whole genome DNA of the microorganism. Is 90% or more, more preferably 95% or more of genomic DNA base sequence data, most preferably total genomic DNA base sequence data.

  The names of lactic acid bacteria whose base sequences of total genomic DNA are known and the accession numbers of the nucleotide sequences in sequence databases such as NCBI are exemplified in Tables 1 to 3 below.

  The step B is not particularly limited as long as it is a step of obtaining base sequence data of an arbitrary DNA fragment group within a range of 50 bp to 1000 bp that are continuous or partially overlapping each other in the base sequence data of the genomic DNA of the microorganism. The above-mentioned “obtaining base sequence data of an arbitrary group of DNA fragments within a range of 50 bp to 1000 bp mutually” means a base sequence of a group of DNA fragments such that DNA fragments within a range of 50 bp to 1000 bp are continuous with each other For example, a DNA fragment consisting of nucleotide numbers 1 to 200 of genomic DNA, nucleotide numbers 201 to 201, which means obtaining data and obtaining base sequence data of 200 bp DNA fragment groups that are continuous with each other. Examples include obtaining base sequence data of a group of DNA fragments continuous to each other, such as a DNA fragment consisting of 400 nucleotides and a DNA fragment consisting of nucleotides 401 to 600. In addition, “obtaining base sequence data of an arbitrary group of DNA fragments within a range of 50 bp to 1000 bp partially overlapping each other” means that DNA fragments within a range of 50 bp to 1000 bp partially overlap each other. This means that the base sequence data of the DNA fragment group is obtained, and the case where the base sequence data of the 200 bp DNA fragment group partially overlapping each other is obtained by sliding every 100 bp. For example, nucleotides of genomic DNA Nucleotide sequences of a group of DNA fragments that partially overlap each other, such as a DNA fragment composed of nucleotides of numbers 1 to 200, a DNA fragment composed of nucleotides of nucleotide numbers 101 to 300, a DNA fragment composed of nucleotides of nucleotide numbers 201 to 400 Get data.

  From the viewpoint of obtaining higher accuracy in the evaluation and prediction of immunostimulatory activity, it is possible to obtain base sequence data of arbitrary DNA fragment groups partially overlapping each other in Step B above, instead of arbitrary DNA fragment groups consecutive to each other. preferable.

  The length of the arbitrary DNA fragment group in the above step B is not particularly limited as long as it is in the range of 50 bp to 1000 bp, but is preferably in the range of 60 bp to 800 bp, more preferably in the range of 80 bp to 600 bp, and 100 bp to 500 bp. Is more preferably within the range of 150 bp to 400 bp (preferably within the range of 180 bp to 355 bp), more preferably within the range of 150 bp to 230 bp or within the range of 230 bp to 400 bp, and within the range of 150 bp to 230 bp. Is more preferable, and 200 bp is more preferable. Moreover, as long as the length of the arbitrary DNA fragment group in the process B is in the range of 50 bp to 1000 bp, all may be the same or different ones may be included, but they are all the same length. It is preferable.

  As a preferable embodiment of “obtaining base sequence data of an arbitrary group of DNA fragments within the range of 50 bp to 1000 bp partially overlapping each other”, the starting nucleotide of the DNA fragment of any length within the range of 50 bp to 1000 bp Can be obtained by sliding the DNA sequence every 1 to 20 bp (preferably every 1 bp) on the base sequence data of the genomic DNA of the microorganism to obtain the base sequence data of the DNA fragment group.

  The step C is not particularly limited as long as it is a step of confirming the GC content (%) of each DNA fragment of the DNA fragment group in the step B. As a method for confirming the GC content of a DNA fragment, the base sequence data of each DNA fragment is known from the base sequence data of the genomic DNA of the microorganism obtained in step A above. A method of calculating and confirming the GC content (%) based on the above is mentioned. A typical method is to count the number of guanine and cytosine bases in the base sequence using base sequence analysis software such as GENETYX or BioEdit, and divide the base number by the total number of base sequences. The GC content (%) can be calculated by obtaining the ratio (%).

  As the step D, a low GC content DNA fragment having a GC content of 35% or less, 40% or less or 45% or less in the DNA fragment group of the step C is used as a corresponding part on the base sequence of genomic DNA. There is no particular limitation as long as it is a step of arranging and making the region of the genomic DNA of the microorganism covered by the low GC content DNA fragment a low GC content region in the microorganism genome. The “corresponding portion on the base sequence of genomic DNA” in “Placing a low GC content DNA fragment on the corresponding portion on the base sequence of genomic DNA” refers to the low GC content DNA in the base sequence of genomic DNA. This means the part that matches the base sequence of the fragment. In addition, when the “low GC content DNA fragments are arranged in corresponding portions on the base sequence of genomic DNA”, when the base sequences of the low GC content DNA fragments are continuous on the genomic DNA, When low GC content DNA fragments are continuously arranged on the base sequence, and when the base sequences of the low GC content DNA fragments partially overlap, they partially overlap on the genomic DNA base sequence. Will be placed. Further, “placement” in the process D may be placed on the array data.

  In addition, although the GC content of the DNA fragment used at the said process D should just be 35% or less, 40% or less, or 45% or less, it evaluates or estimates that it is a microorganism which has a more superior immunostimulatory activity with a higher probability. From the viewpoint, the GC content is preferably 40% or less, and more preferably 35% or less. However, when the GC content of the DNA fragment used in Step D is higher than 35% and 40% or less, or when the GC content is higher than 40% and 45% or less, it is evaluated as having excellent immunostimulatory activity. Since the microorganisms that can be used are contained, the standard of the GC content used in the step D can be appropriately selected depending on the situation.

  As long as the step E is a step of totaling the number of unmethylated CpG motifs contained in the low GC content region specified in the step D in the microbial genomic DNA, and calculating the total number of low GC content region unmethylated CpG motifs There is no particular limitation. As a representative method, the number of unmethylated CpG motifs contained in the target base sequence can be counted using base sequence analysis software such as GENETYX or BioEdit.

  In the method for predicting microorganisms of the present invention, the number of unmethylated CpG motifs of each DNA fragment itself may be measured for the first time in step E after performing steps C and D, or in steps B and C. May be performed between Step C and Step D, and any aspect is included in the method for predicting a microorganism of the present invention including Steps A to F, but the number of unmethylated CpG motifs between DNA fragments It is preferable to identify the low GC content region in Step D, and then measure and sum the number of unmethylated CpG motifs contained in the region in that it is simpler in that it is not necessary to eliminate duplication of .

  In the step F, when the total number of low GC content region unmethylated CpG motifs calculated in the step E is 100 or more per 10,000 bp of genomic DNA of the microorganism, the microorganism has immunostimulatory activity. Although it is not particularly limited as long as it is a process for predicting a microorganism, the low GC content region non-methyl per 10,000 bp of the genomic DNA of the microorganism from the viewpoint of predicting a microorganism having a higher immunostimulatory activity with a higher probability. The total number of modified CpG motifs is preferably 125 or more, and more preferably 150 or more. The upper limit of the total number of unmethylated CpG motifs in the low GC content region per 10,000 bp of microbial genomic DNA is not particularly limited. However, if the total number of unmethylated CpG motifs increases too much, the GC content of genomic DNA increases, Since there is a relation that the GC content region unmethylated CpG motif is reduced, there is an upper limit naturally, and the total number of the low GC content region unmethylated CpG motifs per 10,000 bp of genomic DNA of microorganisms is usually 500. Or less.

The method for predicting a microorganism of the present invention may further include the following step G in addition to the above steps A to F. In particular, the microorganism from which the immunostimulatory activity is to be predicted and the microorganism from which the genomic DNA used is derived. It is preferable to have this process G, when it becomes the relationship of different strains of the same genus same kind. In addition, the microorganisms in this process G are restricted to the microorganisms which have an entity.
(G) Step G of measuring the immunostimulatory activity of the microorganism predicted to have the immunostimulatory activity in Step F:

  When the process G is further included, the immunostimulatory activity (preferably IFN-α production inducing activity) of the microorganism can be reliably evaluated. The microorganism used in Step G is a microorganism predicted to have immunostimulatory activity in Step F, and has an extremely high probability of immunostimulatory activity (preferably IFN-α production-inducing activity). Therefore, all candidate microorganisms are immunostimulated. Compared with the case where the activity (preferably IFN-α production inducing activity) is measured, the immunostimulatory activity (preferably IFN-α production inducing activity) of the microorganism can be measured efficiently.

  Microorganisms used in the present invention include, for example, RIKEN BioResource Center (1-3, Takanodai, Tsukuba City, Ibaraki, Japan), Biological Genetic Resources Department of the National Institute of Technology and Evaluation (http: // www. nbrc.nite.go.jp), American type culture collection (USA), Tokyo University of Agriculture, strain storage room (http://nodaiweb.university.jp/nric/; 1-1 1-1 Sakuragaoka, Setagaya-ku, Tokyo, Japan No.), Fermentation Research Institute (2-17-85 Jusohoncho, Yodogawa-ku, Osaka, Osaka, Japan), DANISCO, etc.

  The immunostimulatory activity to be measured in Step G is not particularly limited as long as it is an immunostimulatory activity. However, type I interferon (IFN-α and IFN-α and mediated by TLR9 activation in plasmacytoid dendritic cells (pDC)) // IFN-β) production-inducing activity is preferable, and among them, IFN-α production-inducing activity through activation of TLR9 in pDC is more preferable.

  The method for measuring the immunostimulatory activity in the above step G is not particularly limited and can be measured by a known method. For example, as a known method for measuring IFN-α production-inducing activity, the following method is used. Preferable examples can be given.

(Detailed measurement method of IFN-α production inducing activity of microorganisms using pDC)
Bone marrow cells are collected from the femur and the like of a mammal such as a mouse (preferably a mouse) and subjected to erythrocyte removal treatment to prepare bone marrow cells. The obtained bone marrow cells are suspended in RPMI medium (manufactured by SIGMA) containing 10% FCS, 2 μM β-mercaptoethanol so as to be 5 × 10 ⁵ cells / mL. To the obtained cell culture solution, a dried microorganism product (preferably a lyophilized product, more preferably a lyophilized bacterial cell) is added so as to be 10 μg / mL. After culturing in an incubator maintained at 37 ° C. and 5% CO ₂ for 2 days, the supernatant is collected, and the IFN-α concentration in the supernatant is measured. For the measurement of the IFN-α concentration, a kit (manufactured by PBL Assay Science) by ELISA method can be used. When viable cells are used as microorganisms, it is preferable that the cultured viable cells are once lyophilized and a liquid in which the cells are suspended in PBS is added to the cell culture solution of bone marrow-derived dendritic cells.

  Examples of mammals in the present invention include mice, rats, guinea pigs, humans, monkeys, dogs, cats, cows, horses, pigs, sheep, goats and the like.

  In the method for predicting a microorganism of the present invention, a preferred embodiment of the microorganism having IFN-α production-inducing activity is the IFN in the culture supernatant according to the above-mentioned “detailed method for measuring the IFN-α production-inducing activity of a microorganism”. Examples include microorganisms having an α concentration of 50 pg / mL or more, preferably 75 pg / mL or more, more preferably 100 pg / mL or more, further preferably 150 pg / mL or more, more preferably 200 pg / mL or more.

<2. Screening Method for Microorganisms of the Present Invention>
The method for screening a microorganism predicted to have immunostimulatory activity according to the present invention is a microorganism that is predicted to have immunostimulatory activity in the method for predicting a microorganism of the present invention as a microorganism predicted to have immunostimulatory activity. The process H to select is included, It is characterized by the above-mentioned. The microorganism that is predicted to have the immunostimulatory activity to be selected is not particularly limited as long as the total number of low GC content region unmethylated CpG motifs is 100 or more per 10,000 bp of genomic DNA of the microorganism, From the viewpoint of obtaining a microorganism having more excellent immunostimulatory activity with a higher probability, the number is preferably 125 or more, and more preferably 150 or more. The upper limit of the total number of low GC content region unmethylated CpG motifs per 10,000 bp of genomic DNA of microorganisms to be selected is not particularly limited, but is usually 500 or less.

The microorganism screening method of the present invention may further include the following steps I and J in addition to the above-mentioned step H. In particular, the microorganism to be selected and the microorganism from which the genomic DNA to be used is derived belong to the same genera It is preferable to have such a process I and a process J when it becomes the relationship of different strains. In addition, the microorganisms used for this process I and process J are restricted to the microorganisms which have an entity.
(I) Step I of measuring the immunostimulatory activity of a microorganism predicted to have the immunostimulatory activity selected in Step H:
(J) Step J for selecting the microorganisms whose immunostimulatory activity was confirmed in Step I as microorganisms having immunostimulatory activity:

  The immunostimulatory activity to be measured in Step I above is not particularly limited as long as it is an immunostimulatory activity, but it is a type I interferon (IFN-α and IFN-α and mediated by TLR9 activation in plasmacytoid dendritic cells (pDC)). // IFN-β) production-inducing activity is preferable, and among them, IFN-α production-inducing activity through activation of TLR9 in pDC is more preferable.

  When the process I and the process J are further provided, a microorganism having an immunostimulatory activity (preferably IFN-α production inducing activity) can be selected. The microorganism used in Step I is a microorganism selected as a microorganism predicted to have immunostimulatory activity (preferably IFN-α production-inducing activity) in Step H, and has an extremely high probability of immunostimulatory activity (preferably IFN-α). When selecting a microorganism having immunostimulatory activity (preferably IFN-α production-inducing activity) by measuring immunostimulatory activity (preferably IFN-α production-inducing activity) for all candidate microorganisms. In comparison with, microorganisms having immunostimulatory activity (preferably IFN-α production inducing activity) can be efficiently selected. Thus, the microorganism screening method of the present invention can be suitably used as a primary screening for microorganisms having immunostimulatory activity.

  The method of measuring the immunostimulatory activity (preferably IFN-α production-inducing activity) of the microorganism in the above step I is as described in the method of predicting the microorganism of the present invention.

  As described above, the above-mentioned step J is “step J in which the microorganism having immunostimulatory activity confirmed in step I is selected as a microorganism having immunostimulatory activity”. This is the step J ′ ”of selecting microorganisms having excellent immunostimulatory activity (preferably IFN-α production-inducing activity) among microorganisms whose activation activity has been confirmed. In the “selecting a microorganism excellent in immunostimulatory activity (preferably IFN-α production-inducing activity)” in the step J ′, the “IFN-α production-inducing activity of microorganisms in detail” described above is used. A microorganism having a concentration of -α of 50 pg / mL or more, preferably 75 pg / mL or more, more preferably 100 pg / mL or more, further preferably 150 pg / mL or more, more preferably 200 pg / mL or more, or The IFN-α concentration according to “Detailed method for measuring the IFN-α production-inducing activity of microorganisms” is 12.5% or more, preferably 18.75 with respect to the IFN-α concentration of Lactococcus lactis JCM5805 strain. % Or more, more preferably 25% or more, further preferably 37.5% or more, more preferably 50% or more, and further preferably 75 %, More preferably 100% or more of microorganisms are selected. In addition, it is known that Lactococcus lactis JCM5805 strain has extremely high IFN-α production-inducing activity (Patent Document 1).

  In the aforementioned method for predicting the microorganism of the present invention and the method for screening a microorganism of the present invention, the microorganism predicted to have immunostimulatory activity (preferably IFN-α production-inducing activity), and immunostimulatory activity (preferably IFN- Microorganisms selected as microorganisms that are predicted to have (α production-inducing activity), in particular, immunostimulatory activity (preferably IFN-α production-inducing activity) is actually measured, and immunostimulatory activity (preferably IFN-α production induction) A microorganism selected as a microorganism having an activity) or an immunostimulatory activity (preferably an IFN-α production-inducing activity) is used as an immunostimulator (preferably an IFN-α production-inducing agent) described later. Can be used.

<3. Method for predicting DNA of the present invention>
The method for predicting the immunostimulatory activity of the DNA of the present invention comprises the following steps K to N.
(K) Step K of obtaining DNA base sequence data:
(L) Step L for confirming the GC content of DNA:
(M) Step M for confirming the number of unmethylated CpG motifs contained in DNA:
(N) DNA having a GC content of 35% or less, 40% or less, or 45% or less, and having the number of unmethylated CpG motifs per nucleotide of 0.017 or more, DNA having immunostimulatory activity Predicting process N:

  The merit of the method for predicting DNA of the present invention is that it can efficiently predict the immune activity of DNA. Particularly, the immunostimulatory activity of various types (for example, 10 types or more, preferably 20 types or more) of DNA is evaluated. It can be enjoyed more in the case of equality.

  The DNA used in the method for predicting the DNA of the present invention and the screening method for the DNA of the present invention described later may be DNA derived from microorganisms such as bacteria, viruses and fungi, or is not derived from any microorganism. The DNA may be arbitrarily designed, and as a preferred embodiment, the method of predicting the microorganism of the present invention or the method of screening for a microorganism of the present invention is preferably used for the microorganism predicted to have immunostimulatory activity. An arbitrary DNA fragment within the range of 50 bp to 1000 bp in the base sequence data of genomic DNA can be mentioned.

  The length of the DNA used in the method for predicting the DNA of the present invention and the DNA screening method of the present invention described later is not particularly limited as long as it is within the range of 50 bp to 1000 bp, but preferably within the range of 60 bp to 800 bp. More preferably within the range of 80 bp to 600 bp, more preferably within the range of 100 bp to 500 bp, more preferably within the range of 150 bp to 400 bp (preferably within the range of 180 bp to 355 bp), within the range of 150 bp to 230 bp, or 230 bp to 400 bp Is more preferable, the range of 150 bp to 230 bp is more preferable, and the range of 180 bp to 214 bp is more preferable.

  The step K is not particularly limited as long as it is a step of obtaining DNA base sequence data. For example, the DNA base sequence data may be obtained from a public database such as NCBI or a private database. The DNA base sequence data may be microbial-derived DNA base sequence data, or may be microbial-derived DNA, instead of microbial-derived DNA.

  The step L is not particularly limited as long as it is a step for confirming the GC content of DNA. As a method for confirming the GC content of DNA, the base sequence of the DNA base sequence data obtained in the step K can be used. A method of calculating and confirming the GC content (%) based on the above is mentioned. A typical method is to count the number of guanine and cytosine bases in the base sequence using base sequence analysis software such as GENETYX or BioEdit, and divide the base number by the total number of base sequences. The GC content (%) can be calculated by obtaining the ratio (%).

  The step M is not particularly limited as long as it is a step for confirming the number of unmethylated CpG motifs contained in DNA. As a representative method, a target sequence analysis software such as GENETYX or BioEdit can be used. The number of unmethylated CpG motifs contained in the base sequence can be counted.

  Either of the steps L and M may be performed first. In addition, when the step L is performed first, only the DNA whose GC content is confirmed to be 35% or less, 40% or less, or 45% or less can be confirmed in the next step M by confirming the number of unmethylated CpG motifs. On the other hand, when Step M is performed first, only the DNA confirmed to have 0.017 or more unmethylated CpG motifs per nucleotide of DNA is checked for GC content in the next Step L. May be.

  As the above step N, a DNA having a GC content of 35% or less, 40% or less, or 45% or less and the number of unmethylated CpG motifs per nucleotide is 0.017 or more is used as an immunostimulatory activity. There is no particular limitation as long as it is a step for predicting a DNA having. The GC content of DNA that is predicted to have immunostimulatory activity is 35% or less, 40% or less, or 45% or less, from the viewpoint of evaluating or predicting that the DNA has superior immunostimulatory activity with a higher probability. 40% or less of DNA is preferably selected, and 35% or less of DNA is more preferably selected. However, DNA having a GC content higher than 35% and 40% or lower, and DNA having a GC content higher than 40% and 45% or lower include DNA having excellent immunostimulatory activity. The standard of the GC content of DNA can be appropriately selected according to the situation.

  The number of unmethylated CpG motifs per nucleotide in the above step N is not particularly limited as long as it is 0.017 or more, but it has more excellent immunostimulatory activity (preferably IFN-α production inducing activity). Is preferably 0.025 or more, more preferably 0.0375 or more, still more preferably 0.05 or more, and 0.0625 or more from the viewpoint of obtaining a higher probability. More preferably, it is more preferably 0.075 or more. The upper limit of the number of unmethylated CpG motifs per nucleotide in the DNA to be predicted is not particularly limited, but there is an upper limit naturally because the GC content is 45% or less, and usually 0.125 or less.

The method for predicting DNA of the present invention may further include the following step O in addition to the above steps K to N. In addition, DNA used for this process O is restricted to DNA which has the substance as a compound.
(O) Step O of measuring the immunostimulatory activity of the DNA predicted to have the immunostimulatory activity in Step N:

  When the process O is further included, the immunostimulatory activity (preferably IFN-α production inducing activity) of DNA can be reliably evaluated. The DNA used in step O is DNA that is predicted to have immunostimulatory activity in step N, and has an extremely high probability of immunostimulatory activity (preferably IFN-α production-inducing activity). Compared with the case where the activity (preferably IFN-α production inducing activity) is measured, the immunostimulatory activity (preferably IFN-α production inducing activity) of DNA can be measured efficiently.

  Examples of the method for obtaining the DNA used in Step O include a method for preparing DNA based on known base sequence information, and representative methods include amplification of a target DNA fragment by PCR, A recombinant DNA that expresses the DNA fragment is produced using the amplified DNA fragment, the recombinant DNA is introduced into a host microorganism, and the target DNA fragment is expressed in the host microorganism, or by a chemical method. Synthesis of the target oligonucleotide can be mentioned.

  The immunostimulatory activity to be measured in the above step O is not particularly limited as long as it is an immunostimulatory activity, but type I interferon (IFN-α and IFN-α and mediated by TLR9 activation in plasmacytoid dendritic cells (pDC)) // IFN-β) production-inducing activity is preferable, and among them, IFN-α production-inducing activity through activation of TLR9 in pDC is more preferable.

  The method for measuring the immunostimulatory activity in the above step O is not particularly limited, and can be measured by a known method. For example, as a known method for measuring IFN-α production-inducing activity, the following method is used. Preferable examples can be given.

(Detailed method for measuring IFN-α production-inducing activity of DNA using pDC)
Bone marrow-derived dendritic cells are prepared by the method described in the above detailed method for measuring the IFN-α production-inducing activity of microorganisms. The DNA for which IFN-α production-inducing activity is to be measured is added to the cell culture solution of the cells so as to be 2 μg / mL, and the culture supernatant is recovered after 48 hours. The concentration of IFN-α contained in the culture supernatant is measured. For the measurement of the IFN-α concentration, a kit (manufactured by PBL Assay Science) by ELISA method can be used.

  In the method for predicting DNA of the present invention, a preferred embodiment of DNA having IFN-α production-inducing activity is the IFN in the culture supernatant according to the above-mentioned “detailed method for measuring IFN-α production-inducing activity of DNA”. -The α concentration is 50 pg / mL or more, preferably 75 pg / mL or more, more preferably 100 pg / mL or more, further preferably 150 pg / mL or more, more preferably 200 pg / mL or more, more preferably 300 pg / mL or more, more A DNA that is preferably 400 pg / mL or more can be mentioned.

<4. DNA screening method of the present invention>
The method for screening DNA that is predicted to have immunostimulatory activity of the present invention includes the method of predicting the immunostimulatory activity of the DNA of the present invention, wherein the DNA that is predicted to have immunostimulatory activity has the immunostimulatory activity. It includes a step P of selecting as a predicted DNA. Among the DNA screening methods of the present invention, a preferred embodiment includes a screening method comprising the following steps (Q) to (S).
(Q) Step Q of obtaining an arbitrary DNA fragment group within the range of 50 bp to 1000 bp:
(R) Checking the GC content of each DNA fragment in the DNA fragment group, and selecting a DNA fragment having a GC content of 30%, 40% or 45% or less R:
(S) The number of unmethylated CpG motifs contained in each DNA fragment selected in the step R is calculated, and a DNA fragment having a number of unmethylated CpG motifs per nucleotide of 0.017 or more is determined as IFN- Step S of selecting as DNA predicted to have α production-inducing activity:

  As a preferred embodiment of step Q, in the genomic DNA of a microorganism predicted to be a microorganism having immunostimulatory activity (preferably IFN-α production-inducing activity) by the method of predicting a microorganism of the present invention or the method of screening a microorganism, There is a step of obtaining base sequence data of an arbitrary group of DNA fragments within a range of 1000 bp.

  In the DNA screening method of the present invention, as the DNA to be selected, the GC content is 35% or less, 40% or less, or 45% or less, and the number of unmethylated CpG motifs per nucleotide is 0. Although it is not particularly limited as long as it is DNA of 017 or more, from the viewpoint of obtaining a DNA having a better immunostimulatory activity with a higher probability, it is preferable to select DNA of 40% or less for the GC content, More preferably, 35% or less of DNA is selected. However, DNA having an excellent immunostimulatory activity is selected from DNAs having a GC content higher than 35% and not higher than 40% and DNAs having a GC content higher than 40% and not higher than 45%. The standard of the GC content of DNA can be appropriately selected according to the situation. In addition, the number of unmethylated CpG motifs per nucleotide in the DNA to be selected is not particularly limited as long as it is 0.017 or more, but from the viewpoint of obtaining a DNA having superior immunostimulatory activity with a higher probability. , 0.025 or more, more preferably 0.0375 or more, further preferably 0.05 or more, more preferably 0.0625 or more, and 0.0. More preferably, it is 075 or more. The upper limit of the number of unmethylated CpG motifs per nucleotide in the DNA to be selected is not particularly limited, but there is an upper limit naturally because the GC content is 45% or less, and usually 0.125 or less.

The DNA screening method of the present invention may further include the following steps T and U in addition to the step P (or the steps Q to S). It should be noted that the DNA used in the steps T and U is limited to DNA having an entity as a compound.
(T) Step T of measuring the immunostimulatory activity of DNA that is predicted to have the immunostimulatory activity selected in Step P (or Steps Q to S above):
(U) Step U of selecting the DNA having immunostimulatory activity confirmed in Step T as DNA having immunostimulatory activity:

  When the process T and the process U are further included, DNA having an immunostimulatory activity (preferably IFN-α production-inducing activity) can be selected. The DNA used in Step T is DNA selected as DNA expected to have immunostimulatory activity in Step P (or Steps Q to S above), and has an extremely high probability of immunostimulatory activity (preferably induction of IFN-α production) Compared with the case of selecting DNA having immunostimulatory activity (preferably IFN-α production inducing activity) by measuring immunostimulatory activity (preferably IFN-α production inducing activity) for all candidate DNAs. Thus, DNA having immunostimulatory activity (preferably IFN-α production inducing activity) can be efficiently selected. Thus, the DNA screening method of the present invention can be suitably used as a primary screening for DNA having immunostimulatory activity (preferably IFN-α production-inducing activity).

  The method of measuring the immunostimulatory activity (preferably IFN-α production inducing activity) of DNA in the above step T is as described in the above method for predicting the DNA of the present invention.

  As described above, the process U is “the process U for selecting the DNA whose immunostimulatory activity is confirmed in the process T as the DNA having the immunostimulatory activity”. This is the step U ′ ”of selecting DNA having excellent immunostimulatory activity (preferably IFN-α production-inducing activity) among DNAs whose activation activity has been confirmed. In the “selecting DNA excellent in immunostimulatory activity (preferably IFN-α production inducing activity)” in the step U ′, the above-mentioned “Detailed method for measuring IFN-α production inducing activity of DNA” is used. The concentration of -α is 50 pg / mL or more, preferably 75 pg / mL or more, more preferably 100 pg / mL or more, further preferably 150 pg / mL or more, more preferably 200 pg / mL or more, more preferably 300 pg / mL or more, More preferably, the DNA of 400 pg / mL or more is selected, or the concentration of IFN-α according to the above-mentioned “detailed method for measuring IFN-α production-inducing activity of DNA” is a CpG oligonucleotide ODN 1585 (murine TLR9 ligand). When 0.5 μM (final concentration in the medium) of InvivoGen (SEQ ID NO: 242) is added 50% or more, preferably 75% or more, more preferably 100% or more, still more preferably 125% or more, more preferably 150% or more, and further preferably 175% or more with respect to the (control) IFN-α concentration. This involves selecting the proportion of DNA. In addition, 2 g on the 5 'end side and 6 g on the 3' end side of the ODN 1585 are oligonucleotides linked by phosphorothioate.

  In the aforementioned method for predicting the DNA of the present invention and the method for screening DNA of the present invention, DNA predicted to have immunostimulatory activity (preferably IFN-α production-inducing activity) and immunostimulatory activity (preferably IFN) DNA selected as DNA having -α production-inducing activity) can be used as an immunostimulatory activity (preferably an IFN-α production-inducing agent) described later.

<5. Immunostimulatory Agent of the Present Invention>
The immunostimulant of this invention contains a specific microorganism or its processed material, or specific DNA as an active ingredient. The specific microorganism is a microorganism predicted to have immunostimulatory activity by the method for predicting the microorganism of the present invention or the screening method for the microorganism of the present invention, and the specific DNA has a GC content of 35% or less, The DNA is 40% or less or 45% or less, the number of unmethylated CpG motifs per nucleotide is 0.017 or more, and is within the range of 50 bp to 1000 bp.

  The immunostimulant of the present invention activates immunity in vitro and in vivo. The immunostimulatory activity exhibited by the immunostimulator of the present invention is not particularly limited as long as it is an immunostimulatory activity, but immunostimulatory activity through activation of TLR9 in plasmacytoid dendritic cells or B cells is preferred, More preferably, immunostimulatory activity through activation of TLR9 in plasmacytoid dendritic cells, among which type I interferon (IFN-α and / or IFN-) through activation of TLR9 in plasmacytoid dendritic cells is preferred. β) Production-inducing activity is more preferred, among which IFN-α production-inducing activity through activation of TLR9 in plasmacytoid dendritic cells is particularly preferred. Therefore, the immunostimulant of the present invention is preferably a type I interferon production inducer, and more preferably an IFN-α production inducer.

  The microorganism used for the immunostimulant of the present invention and preferred embodiments thereof are as described in the above [0026], but the microbe confirmed to have immunostimulatory activity is used for the immunostimulator of the present invention. As a suitable microorganism for use in the immunostimulant of the present invention, the IFN-α concentration in the supernatant of the culture medium after culturing in the “detailed method for measuring the IFN-α production-inducing activity of microorganisms” described in [0048] above Is 70 pg / mL or more, preferably 100 pg / mL or more, more preferably 150 pg / mL or more, still more preferably 200 pg / mL or more, more preferably 300 pg / mL or more, still more preferably 350 pg / mL or more, The IFN-α concentration according to “Detailed method for measuring the IFN-α production-inducing activity of microorganisms” is 12.5% or more, preferably 18.75 with respect to the IFN-α concentration of Lactococcus lactis JCM5805 strain. % Or more, more preferably 25% or more, still more preferably 37.5% or more, more preferably 50% or more. Preferably 75% or more, more preferably 100% or more microorganisms may be preferably mentioned. As the microorganisms having the above-mentioned IFN-α concentration of 100 pg / mL or more, NBRC 3832 and NBRC 102481 strains of Leuconostoc mesenteroides, NBRC 101982 strain, JCM 20109 strain, NBRC 101986 strain, NBRC 3892 strain of Pediococcus pentosaceus Is particularly preferred. These 6 strains, as shown in Table 15 to be described later, were strains that showed extremely excellent IFN-α production-inducing activity, with the IFN-α concentration in the culture supernatant in the assay being 100 pg / mL or more. is there. In the present specification, the specific microbial strain includes a microbial strain equivalent thereto. Here, the equivalent microbial strain means a microbial strain derived from the specific microbial strain, a microbial strain from which the specific microbial strain is derived, or a progeny microbial strain of the microbial strain.

  In the immunostimulant of the present invention containing a microorganism or a processed product thereof as an active ingredient, when the microorganism is a bacterium or a fungus, the microorganism or the processed product includes a living cell, a dead cell, a living cell, or a dead cell. Includes crushed cells of cells, lyophilized products of live or dead cells, crushed products of lyophilized products, etc., and not limited to all processed products of live or dead cells The processed product is also included. In addition, when the microorganism is a virus, the microorganism or a processed product thereof includes a virus, a crushed product of a virus, a lyophilized product of the virus, a crushed product of the lyophilized product, etc. It is not limited to processed products, and some processed products are included. Here, the treated product includes those treated by enzyme treatment, heat treatment, etc., or those obtained by ethanol precipitation of the treated product. Moreover, the DNA derived from the microorganism is also included in the processed product. A fraction containing abundant microorganism-derived DNA is produced, for example, by enzyme treatment and heat treatment (Japanese Patent Laid-Open No. 2001-46020) or by collecting a precipitate from ethanol (Japanese Patent Laid-Open No. 2000-262247). Can do. The immunostimulant containing abundant microorganism-derived DNA in the present invention can be used as a more effective immunostimulant with a concentrated active substance.

  Viable cells and viruses of the above-mentioned microorganism can be obtained from the culture by culturing the microorganism by a known method according to the type of the microorganism. For example, when the microorganism is a lactic acid bacterium, MRS medium, GAM medium, LM17 medium can be used as the medium, and inorganic salts, vitamins, amino acids, antibiotics, serum, and the like can be appropriately added and used. The culture can be performed at 25 to 40 ° C. for several hours to several days. By centrifuging or filtering the cultured product after culturing, it is possible to obtain viable cells or viruses of the above microorganisms. Dead cells can be obtained by heat-treating living cells with an autoclave or the like.

  As described above, the immunostimulant of the present invention may contain a specific DNA as an active ingredient instead of a specific microorganism or a processed product thereof. Such DNA has a GC content of 35% or less, 40% or less, or 45% or less, and the number of unmethylated CpG motifs per nucleotide is 0.017 or more (preferably 0.025 or more, more preferably 0.0375 or more, more preferably 0.05 or more, more preferably 0.0625 or more, more preferably 0.075 or more), and within a range of 50 bp to 1000 bp (preferably 60 bp to 800 bp). Within a range of 80 bp to 600 bp, more preferably within a range of 100 bp to 500 bp, more preferably within a range of 150 bp to 400 bp (preferably within a range of 180 bp to 355 bp), and even more preferably 150 bp to 230 bp. Within the range of 230 bp to 400 bp, more preferred Within 150Bp～230bp, more preferably include a DNA in the range of 180bp~214bp). The DNA used for the immunostimulant of the present invention is not limited in its origin and the like, but DNA that has been confirmed to have immunostimulatory activity is used. As suitable DNA used for the immunostimulant of the present invention, IFN-α in the supernatant of the culture medium after culturing in the “detailed method for measuring IFN-α production-inducing activity of DNA” described in [0073] above. Concentration is 50 pg / mL or more, preferably 75 pg / mL or more, more preferably 100 pg / mL or more, further preferably 150 pg / mL or more, more preferably 200 pg / mL or more, still more preferably 300 pg / mL or more, more preferably 400 pg. The concentration of IFN-α according to the above-mentioned “Detailed method for measuring IFN-α production-inducing activity of DNA” is CpG oligonucleotide ODN 1585 (manufactured by InvivoGen), which is a murine TLR9 ligand. 242) at 0.5 μM (final concentration in the medium) (control) IFN DNA having a ratio of 50% or more, preferably 75% or more, more preferably 100% or more, more preferably 125% or more, more preferably 150% or more, and further preferably 175% or more with respect to the α concentration. be able to.

  Specific examples of DNA suitable for use in the immunostimulant of the present invention include SEQ ID NOs: 173 to 177, 179 to 181, 183 to 185, 187 to 190, 194, 213, 214, 216 to 220, 225, 229, 231 and DNA comprising any nucleotide sequence selected from the group consisting of 233 can be mentioned, and more preferably, SEQ ID NOs: 176, 177, 180, 181, 183 to 185, 187 to 190, 194, 216, 217, DNA comprising any nucleotide sequence selected from the group consisting of 220 and 229 can be mentioned, more preferably, from SEQ ID NOs: 176, 180, 181, 184, 185, 188, 189, 190, 194 and 229. DNA comprising any nucleotide sequence selected from the group consisting of It can gel.

  In the immunostimulant of the present invention, the specific microorganism and / or its processed product and / or specific DNA may be used in combination, or two or more specific microorganisms or processed products thereof may be used in combination. Two or more specific DNAs may be used in combination.

  The immunostimulant of the present invention can be used as a medicament or pharmaceutical composition that enhances the immune activity of a living body. This medicine is already known as an indication for type I interferon, such as renal cancer, multiple myeloma, chronic myelogenous leukemia, hairy cell leukemia, glioblastoma, medulloblastoma, astrocytoma, malignant melanoma, Cancer, including mycosis fungoides, adult T-cell leukemia, subacute sclerosing panencephalitis, HTLV-1 myelopathy, hepatitis B, hepatitis C, chlamydia (sexually transmitted disease), mycobacterium (Tuberculosis), Listeria (sepsis, etc.), Staphylococcus (food poisoning), Helicobacter (gastritis) and other bacterial infections, multiple sclerosis and other autoimmune diseases can be used as preventive or therapeutic agents. In particular, the medicament is useful as a virus infection protection and virus infection therapeutic agent. In addition, since the function of inhibiting differentiation from osteoblasts to osteoclasts is known as the activity of type I interferon, it can also be used as a preventive or therapeutic agent for osteoporosis.

  Furthermore, the immunostimulant of the present invention can be used as an adjuvant that enhances the immune response of the living body and enhances the effect of the vaccine. When using the immunostimulant of this invention as an adjuvant, you may use only the immunostimulant of this invention as an adjuvant, and may use together with another adjuvant. In addition, a microorganism in which an antigen corresponding to a specific disease is expressed by a genetic engineering technique in a microorganism (preferably a lactic acid bacterium, more preferably a lactic acid bacterium) that is an immunostimulator of the present invention may be used as a vaccine. it can. In particular, since the cell wall of lactic acid bacteria has a function of protecting the antigen from stomach acid, such a heterologous antigen-expressing strain is suitable as a host for an oral vaccine. Generally, there are live vaccines, whole-particle inactivated vaccines, and split vaccines. However, live vaccines are at risk of virus poisoning, and all-particle inactivated vaccines have side effects due to impurities and are the safest. A high split split vaccine has problems with effectiveness. In order to overcome such problems, a recombinant vaccine that expresses only the target antigen has been developed. However, in the present invention, a microorganism having immunostimulatory activity (preferably lactic acid bacteria, more preferably lactic acid cocci) is used. If an antigen is expressed, an adjuvant effect is also obtained, which is extremely useful.

The form of the immunostimulant of the present invention is not particularly limited, and examples thereof include powders, granules, tablets, syrups, injections, drops, powders, suppositories, suspensions, ointments and the like. The immunostimulant of the present invention may be administered orally, and is administered parenterally such as intravenous administration, intramuscular administration, subcutaneous administration, rectal administration, transdermal administration, intradermal administration, intraperitoneal administration and the like. Oral administration is preferred. The immunostimulant may contain excipients, disintegrants, binders, lubricants, colorants and the like. Excipients include glucose, lactose, corn starch, sorbit, etc. Disintegrants include starch, sodium alginate, gelatin powder, calcium carbonate, calcium citrate, dextrin, and binders include dimethylcellulose, polyvinyl alcohol, and polyvinyl ether. , Methylcellulose, ethylcellulose, gum arabic, gelatin, hydroxypropylcellulose, polyvinylpyrrolidone and the like, and examples of the lubricant include talc, magnesium stearate, polyethylene glycol, hydrogenated vegetable oil and the like. The dosage can be appropriately determined according to the age, weight, sex, disease difference, and symptom of the patient to be administered, and may be administered once or divided into several times per day. The number of bacteria per administration Alternatively, the microorganism can be administered in an amount corresponding to 1 × 10 ⁹ to 1 × 10 ¹² cells or individual in terms of the number of viruses. Or 1-1000 mg per administration can be administered in conversion of the dry weight of microorganisms.

  Furthermore, the immunostimulant of the present invention can also be used by being included in foods and drinks, and when included in foods and drinks, the foods and drinks are immunostimulated foods and drinks, immune-enhancing foods and drinks, foods and drinks for virus infection protection Etc. can be used. The type of food or drink to be targeted is not particularly limited as long as the immunostimulatory activity of the immunostimulant of the present invention is not inhibited. For example, milk and dairy products; beverages; seasonings; alcoholic beverages; processed agricultural and forestry foods; confectionery and bread products; flour and noodles; processed marine products; processed livestock products; processed fats and oils; processed frozen products; Instant foods; food materials. Among these, it can use suitably for fermented milk products, such as yogurt and cheese, and a lactic acid bacteria drink. When used as a fermented food or drink, the required amount of lactic acid bacteria having immunostimulatory activity as dead bacteria can be added to the fermented food or drink, and the fermented food or drink can also be produced as a lactic acid bacteria starter.

  The foods and drinks in the present invention include health foods and drinks, foods and drinks for specified health use, nutritional function foods and drinks, health supplement foods and drinks, and the like. Here, the food and drink for specified health use refers to food and drink that is ingested for the purpose of specific health in the eating habits and displays that the purpose of the health can be expected by the intake. These foods and drinks, for example, enhance the body's immune function, activate the body's immune function, make it difficult to catch a cold, make it less susceptible to infection with viruses such as influenza virus, norovirus or rotavirus, and prevent carcinogenesis. An indication such as having an effect may be attached.

  EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[Evaluation of IFN-α production inducing activity of a 200 bp DNA fragment derived from Lactococcus lactis JCM 5805]
In order to evaluate whether the IFN-α production-inducing activity of Lc. Lactis JCM 5805 is simply dependent on the total number of unmethylated CpG motifs on the genome of the strain, DNA with different numbers of unmethylated CpG motifs Fragments were prepared by PCR. Specifically, based on the genome information of Lc. Lactis JCM 5805, 34 200 bp regions that contain 5 to 15 unmethylated CpG motifs are selected and required to amplify each of those regions. 34 types of oligonucleotide primer sets were prepared. The correspondence between each primer in these primer sets and its SEQ ID NO is shown in Tables 4 to 6 below.

  PCR was performed using the genomic DNA purified from the cells of Lc. Lactis JCM 5805 as a template and each of the primer sets described above. The amplified DNA fragment was purified from each reaction product obtained by each PCR, and the obtained pure DNA fragment was used as a DNA library. For the above PCR, TaKaRa Ex Taq (registered trademark) manufactured by Takara Bio Inc. was used, and 35 cycles of reaction were performed with an annealing temperature of 55 ° C. and an extension time of 20 seconds. The amplified DNA fragment was purified by removing contaminants such as primers from the PCR reaction product using QIAquick PCR Purification Kit (manufactured by QIAGEN).

The IFN-α production-inducing activity of each DNA fragment was measured by the following method using plasma cytoid dendritic cells (pDC). C57BL / 6 mouse bone marrow cells were collected from the femur according to a conventional method and subjected to red blood cell removal treatment. The obtained bone marrow cells were suspended in RPMI medium (manufactured by SIGMA) containing 10% FCS, 2 μM β-mercaptoethanol so as to be 5 × 10 ⁵ cells / mL. Flt-3L (manufactured by R & D Systems) as a pDC-inducing cytokine was added to the obtained cell suspension at a final concentration of 100 ng / mL. The bone marrow-derived dendritic cell (BM-DC) was obtained by culturing for 7 days at 37 ° C. and 5% CO ₂ in a CO ₂ incubator. Each DNA fragment in the DNA library obtained by the above PCR was added to 2 μg / mL, and the culture supernatant was collected after 48 hours. For measurement of the amount of IFN-α contained in the culture supernatant, a kit by ELISA (PBL Assay Science) was used.

  SEQ ID NO: showing the nucleotide sequence of the DNA fragment amplified by each primer set, GC content of the DNA fragment, and IFN-α concentration (pg / mL) contained in the culture supernatant in the above assay using the DNA fragment Table 7 below.

FIG. 1 shows the correlation between the number of unmethylated CpG motifs of each DNA fragment in Table 7 and the IFN-α production-inducing activity (IFN-α concentration (pg / mL)). As can be seen from FIG. 1, there was no clear correlation between the number of unmethylated CpG motifs of each DNA fragment and the IFN-α production inducing activity (decision coefficient R ² = 0.2716). From these results, it was suggested that factors other than the number of unmethylated CpG motifs affect the IFN-α production-inducing activity of Lc. Lactis JCM 5805. Accordingly, the present inventors have continued to examine each DNA fragment in the above-mentioned DNA library from various angles, and as a result of repeating trial and error, the difference in the GC content in each DNA fragment has an effect on the IFN-α production-inducing activity. Finally found that.

Among the experimental results shown in FIG. 1, the experimental result of a DNA fragment having a GC content of 45% or less is shown in FIG. 2A, and the experimental result of a DNA fragment having a GC content exceeding 45% is shown in FIG. 2B. As shown in FIG. 2A, when the correlation between the number of unmethylated CpG motifs and IFN-α production-inducing activity is shown after limiting the DNA fragment to a GC content of 45% or less, there is a strong relationship between the two. A correlation (decision coefficient R ² = 0.508) was observed. On the other hand, in the DNA fragment having a GC content exceeding 45%, as shown in FIG. 2B, a clear correlation was not recognized between the number of unmethylated CpG motifs and the IFN-α production inducing activity. In FIG. 2B, although the number of unmethylated CpG motifs is relatively large, IFN-α production-inducing activity is low, or the number of unmethylated CpG motifs is relatively small, but IFN-α production is low. Relatively many cases with low induction activity were observed.

Among the experimental results shown in FIG. 1, the experimental result of a DNA fragment having a GC content of 40% or less is shown in FIG. 2C, and the experimental result of a DNA fragment having a GC content exceeding 40% is shown in FIG. 2D. As shown in FIG. 2C, when the correlation between the number of unmethylated CpG motifs and IFN-α production-inducing activity is shown after limiting to DNA fragments with a GC content of 40% or less, there is a strong relationship between the two. A correlation (decision coefficient R ² = 0.4811) was observed. On the other hand, in a DNA fragment having a GC content exceeding 40%, as shown in FIG. 2D, a very clear correlation was not observed between the number of unmethylated CpG motifs and IFN-α production-inducing activity. In FIG. 2D, although the number of unmethylated CpG motifs is relatively large, the IFN-α production-inducing activity is low, or the number of unmethylated CpG motifs is relatively small, but IFN-α production is low. Relatively many cases with low induction activity were observed.

  Further, in the experimental results of FIG. 1, the value obtained by dividing the value of the IFN-α production inducing activity of each DNA fragment by the value of the number of unmethylated CpG motifs contained in each DNA fragment is the unmethylated in each DNA fragment. The activity per CpG motif was defined as the activity. This calculated activity was determined according to the degree of GC content of each DNA fragment, according to the 6 groups (“˜30” (30% or less), “30-35” (above 30% and 35% or less), “35-40” ( 35 to 40%), “40 to 45” (40 to 45%), “45 to 50” (45 to 50%), “50 to” (50 to 50%)) The average value of “activity per unmethylated CpG motif” was determined for each group. The result is shown in FIG. The results in FIG. 3 clearly showed that the lower the GC content of the DNA fragment, the higher the IFN-α production-inducing activity per unmethylated CpG motif. In particular, DNA fragments having a GC content of 45% or less show significantly higher IFN-α production-inducing activity per unmethylated CpG motif than DNA fragments having a GC content of more than 45%. It was done.

    From the results of Table 7 above, DNA fragment groups having an IFN-α concentration of 200 pg / mL or more and a GC content of 45% or less were used as SEQ ID NOs: 173 to 177, 179 to 181, 183 to 185, 187. DNA consisting of any nucleotide sequence selected from the group consisting of ˜190 and 194 is mentioned, DNA fragments having an IFN-α concentration of 300 pg / mL or more and a GC content of 45% or less, DNA consisting of any nucleotide sequence selected from the group consisting of Nos. 176, 177, 180, 181, 183-185, 187-190 and 194, the IFN-α concentration is 400 pg / mL or more, and As a group of DNA fragments having a GC content of 45% or less, SEQ ID NOs: 176, 180, 181, 184, 185, 188 DNA comprising any nucleotide sequence selected from the group consisting of 189, 190 and 194 are listed. Moreover, about DNA which consists of a nucleotide sequence of sequence number 195 or 196, although GC content exceeds 45%, IFN- (alpha) density | concentration was mentioned as a DNA fragment more than 200pg / mL. Since these DNA fragments are excellent in IFN-α production inducing activity, they can be suitably used as the immunostimulatory agent (preferably IFN-α production inducing agent) of the present invention.

[Evaluation of IFN-α production inducing activity of 300 bp DNA fragment derived from Lactococcus lactis JCM 5805]
In order to confirm that the trends and results shown in Example 1 are not specific to a DNA fragment having a length of 200 bp, 35 kinds of DNA of 300 bp instead of 200 bp were obtained by the same method as in Example 1. A library consisting of fragments was prepared. Tables 8 to 10 below show the correspondence between each primer in the 35 types of primer sets used in preparing this library and their SEQ ID NOs.

  With respect to 35 types of DNA fragments of 300 bp in the above library, IFN-α production-inducing activity was measured in the same manner as in Example 1. The following Table 11 and Table 11 show the SEQ ID No. indicating the nucleotide sequence of the DNA fragment amplified by each primer set, the GC content of the DNA fragment, and the IFN-α concentration contained in the culture supernatant in the assay using the DNA fragment. No.12.

  This measurement test was performed in two steps. The first time was performed on DNA fragments by primer sets B1, B5, B7, B8, B11 to B15, B18, B19, B23, B24, B26 to B29, B32, B33, B35, and the second time was performed on other DNA fragments. . The relative activity (%) in Tables 11 and 12 is shown as a relative value (%) relative to IFN-α (pg / mL) in the control (161.2 pg / mL for the first time and 232.1 pg / mL for the second time). It was. As such a control, a sample to which 0.5 μM (final concentration in the medium) of CpG oligonucleotide (ODN 1585: manufactured by InvivoGen) (sequence number 242), which is a murine TLR9 ligand, was used was used. FIG. 4 shows the correlation between the number of unmethylated CpG motifs contained in each DNA fragment and the IFN-α production inducing activity.

Among the experimental results shown in FIG. 4, the experimental result of a DNA fragment having a GC content of 40% or less is shown in FIG. 5A, and the experimental result of a DNA fragment having a GC content exceeding 40% is shown in FIG. 5B. When the GC content exceeded 40% (FIG. 5B), no correlation was observed between the number of unmethylated CpG motifs and IFN-α production-inducing activity (decision coefficient R ² = FIG. 4). 0.0575; coefficient of determination R ² = 0.149 in FIG. 5B), when the GC content is 40% or less (FIG. 5A), there is a strong positive between the number of unmethylated CpG motifs and the IFN-α production-inducing activity. A correlation (decision coefficient R ² = 0.5463) was observed.

In addition, among the experimental results of FIG. 4, the experimental result of a DNA fragment having a GC content of 35% or less is shown in FIG. 5C, and the experimental result of a DNA fragment having a GC content exceeding 35% is shown in FIG. 5D. As in Example 1 using a 200 bp DNA fragment, the number of unmethylated CpG motifs was obtained when all fragments were targeted (FIG. 4) or when the GC content exceeded 35% (FIG. 5D). is; (determined in FIG. 5D coefficient ^R 2 = 0.0122 determination coefficient ^R 2 = 0.0575 in FIG. 4), GC content whereas correlation was observed between the IFN-alpha production inducing activity In the case of 35% or less (FIG. 5C), a strong positive correlation (decision coefficient R ² = 0.642) was observed between the number of unmethylated CpG motifs and the IFN-α production inducing activity.

  From the results of Tables 11 and 12 above, DNA fragment groups having an IFN-α concentration of 200 pg / mL or more and a GC content of 45% or less were used as SEQ ID NOs: 213, 214, 216 to 220, 225, 229. DNA comprising any nucleotide sequence selected from the group consisting of 231 and 233, DNA fragment groups having an IFN-α concentration of 300 pg / mL or more and a GC content of 45% or less, DNA fragments comprising any nucleotide sequence selected from the group consisting of Nos. 216, 217, 220 and 229, DNA fragments having an IFN-α concentration of 400 pg / mL or more and a GC content of 45% or less As a group, DNA consisting of the nucleotide sequence of SEQ ID NO: 229 was mentioned. In addition, a DNA comprising any nucleotide sequence selected from the group consisting of SEQ ID NOs: 234, 238 and 240 is cited as a DNA fragment having a GC content of more than 45% but an IFN-α concentration of 200 pg / mL or more. It was. Since these DNA fragments are excellent in IFN-α production inducing activity, they can be suitably used as the immunostimulatory agent (preferably IFN-α production inducing agent) of the present invention.

[Evaluation of Lactobacillus rhamnosus ATCC 53103-derived 300 bp DNA fragment]
In order to confirm that the trends and results shown in Example 1 and Example 2 are also established with DNA fragments derived from lactic acid bacteria other than Lactococcus lactis JCM 5805, Lactobacillus rhamnosus ATCC 53103 (a typical immunostimulatory lactic acid bacterium) A library consisting of 17 types of DNA fragments of 300 bp derived from LGG) was prepared in the same manner as in Example 1. Table 13 below shows the correspondence between each primer in the 17 kinds of primer sets used in preparing this library and their sequence numbers. The primer was designed based on the genome information of the LGG strain.

With respect to 17 kinds of DNA fragments of 300 bp in the above library, IFN-α production-inducing activity was measured by the same method as in Example 1, and the activity per unmethylated CpG motif in each DNA fragment was calculated. . This calculated activity was determined according to the degree of GC content of each DNA fragment, according to 3 groups (“˜45” (45% or less), “45 to 55” (above 45% and 55% or less), “55” (55 The average value of “activity per unmethylated CpG motif” was determined for each group. The result is shown in FIG. 6A. From the results of FIG. 6A, it was clearly shown that the lower the GC content of the DNA fragment, the higher the IFN-α production-inducing activity per unmethylated CpG motif. In particular, a DNA fragment having a GC content of 45% or less per per unmethylated CpG motif compared to a DNA fragment having a GC content higher than 45% and 55% or less, or a DNA fragment having a GC content higher than 55%. It was shown that the IFN-α production-inducing activity of the. From this, it is not specific to the strain of Lc. Lactis JCM 5805 that the unmethylated CpG motif localized in the low GC region of genomic DNA has a remarkable IFN-α production-inducing activity. It was shown that.

  Also, the activity per 17 unmethylated CpG motifs of 17 types of DNA fragments of 300 bp was changed to “˜45” (45% or less), “45 to 55” (45% or less) depending on the degree of GC content of each DNA fragment. It was divided into 3 groups, higher than 45% and lower than 55%) and “55-” (higher than 55%). 50% or less) and “50 to” (higher than 50%), and the average value of “activity per unmethylated CpG motif” was determined for each group. The result is shown in FIG. 6B. The result of FIG. 6B clearly showed that the lower the GC content of the DNA fragment, the higher the IFN-α production-inducing activity per unmethylated CpG motif. In particular, a DNA fragment having a GC content of 40% or less per per methylated CpG motif compared to a DNA fragment having a GC content higher than 40% and 50% or lower, or a DNA fragment having a GC content higher than 50%. It was shown that the IFN-α production-inducing activity was significantly and significantly increased (p <0.05). Therefore, it is not specific to the strain of Lc. Lactis JCM 5805 that the unmethylated CpG motif localized in the low GC region of genomic DNA has a remarkable IFN-α production-inducing activity. It was shown that.

[Screening based on the number of unmethylated CpG motifs localized in the low GC region]
From the results of Examples 1 to 3, it was shown that an unmethylated CpG motif present in a region having a low GC content is important for IFN-α production-inducing activity. By utilizing this fact, it was considered that microorganisms with high IFN-α production-inducing activity can be efficiently screened based on genome information. In order to verify this, the number of unmethylated CpG motifs localized in the low GC region of various lactic acid bacteria was calculated. Specifically, it was calculated by the following method.

  A 200 bp DNA fragment group partially overlapping each other was obtained for the entire genome sequence of each strain, targeting strains whose genome sequences were disclosed (see Table 14 described later). When a 200 bp DNA fragment group partially overlapping each other was obtained, the starting nucleotide of the 200 bp fragment was slid on the genomic DNA sequence of the strain every 1 bp to obtain each DNA fragment. That is, a DNA fragment consisting of nucleotides 1 to 200 of the genomic DNA of the strain, a DNA fragment consisting of nucleotides 2 to 201, a DNA fragment consisting of nucleotides 3 to 202, and so on. A partially overlapping DNA fragment group was obtained. The GC content of each DNA fragment in these DNA fragment groups was confirmed. In Example 4, a DNA fragment having a GC content of 35% or less in the DNA fragment group was defined as a low GC content DNA fragment. Such a low GC content DNA fragment is arranged in a corresponding part on the base sequence of the genomic DNA of the strain, and the region of the genomic DNA of the strain covered by the low GC content DNA fragment is defined as a low GC in the strain genome. The content area. The unmethylated CpG motif contained in the low GC content region was defined as a low GC content region unmethylated CpG motif, and the number of low GC content region unmethylated CpG motifs on the genomic DNA sequence of the strain was measured. These series of analyzes were performed using a system in which the present inventors independently created source code using the Perl language. Similar systems can be easily created by those skilled in the art of computer programming.

  Table 14 shows the results of analyzing the number of low GC content region unmethylated CpG motifs based on public information of various lactic acid bacteria.

  As can be seen from Table 14, it became clear that the number of low GC content region unmethylated CpG motifs satisfying such conditions greatly varies depending on the bacterial species. In addition, there is no positive correlation between the total number of unmethylated CpG motifs contained in the entire genome and the number of low GC content region unmethylated CpG motifs distributed in the low GC region, rather it is contained in the entire genome. A tendency that the number of low GC content region unmethylated CpG motifs tended to be smaller as the bacterial species with a larger total number of unmethylated CpG motifs was observed. This is a sequence having a GC content of at least 33.3% in which the unmethylated CpG motif is NNCGNNN (where N is any nucleotide in A, T, G, C), and the unmethylated CpG motif Considering that there is a tendency that if the number is large, the region is not a low GC region, it is considered reasonable.

  From the results shown in Table 14, Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus pentosaceus as species containing a large amount of low GC content region unmethylated CpG motif on the genome, and conversely Lactobacillus plantarum, Lb. casei, Lb. rhamnosus, Lb as less species. Selected fermentum. By evaluating the IFN-α production-inducing activity of various strains belonging to each of these species, the usefulness of the screening method of the present invention using the number of low GC content region unmethylated CpG motifs as an index was examined. The IFN-α production-inducing activity of the strain was evaluated by the following method.

Each conical tube (15 mL volume) into which 10 mL of MRS medium (manufactured by OXOID) was dispensed was inoculated with each strain, and then sealed in each tube and incubated at 30 ° C. to 37 ° C. for 2 days. . The cells obtained by centrifuging the culture solution after the culture at high speed were washed twice with PBS to remove the supernatant, and frozen at −80 ° C. for 2 hours. Water was removed from the frozen cells by evaporation to obtain freeze-dried cells. Each freeze-dried microbial cell obtained was suspended in PBS to prepare a 10 mg / mL suspension, and then 10 μg / mL each of bone marrow-derived dendritic cells (BM-DC) prepared by the method described in Example 1. It added so that it might become mL. After culturing in an incubator maintained at 37 ° C. and 5% CO ₂ for 2 days, the supernatant was collected, and the IFN-α concentration in the supernatant was measured using an ELISA kit. The results are shown in Table 15 below.

  As shown in Table 15, 2 out of 3 strains (67%) of Lc. Lactis showed strong IFN-α production induction activity of 100 pg / mL or more, and 2 out of 10 strains of Leuconostoc mesenteroides (20 %), Pediococcus pentosaceus was found in 4 out of 18 strains (22% ratio). According to past reports of screening lactic acid bacteria at random (Non-patent Document 3), IFN-α production-inducing active strains of 100 pg / mL or more can be obtained only in 3 out of 125 strains (2.4% ratio). In view of this, the screening method of the present invention using the number of low GC content region unmethylated CpG motifs as an index is extremely useful as a method for efficiently screening for microorganisms exhibiting strong IFN-α production-inducing activity. It has been shown.

  In addition, even if it is a homologous homologous species, even if it is a different strain, how similar the GC content of genomic DNA, the total number of unmethylated CpG motifs on genomic DNA, and the number of low GC content region unmethylated CpG motifs are similar. Three strains of each of the same genus and homologous lactic acid bacteria in Table 15 were confirmed. The results are shown in Table 16 below.

  From the results of Table 16, it was shown that the GC content of genomic DNA, the total number of unmethylated CpG motifs on genomic DNA, and the number of low methyl content region unmethylated CpG motifs were quite similar among homologous species. . This is a natural result because the similarity of genomic DNA is quite high among the same species. Therefore, different strains of the same genus and homogenous strain may be used as the “genomic DNA” in the method for evaluating the microorganism of the present invention and the screening method for the microorganism of the present invention described later. The results of the method for evaluating the microorganism of the present invention and the screening method for the microorganism of the present invention described later can also be applied to other strains of the same genus and the same species as the microorganism from which the genomic DNA used is derived.

  According to the present invention, a method for efficiently predicting the immunostimulatory activity of a microorganism or DNA, a method for efficiently screening a microorganism or DNA having a high immunostimulatory activity, and an immunostimulator comprising such a microorganism or DNA as an active ingredient An agent can be provided.

Claims (2)

An immunostimulant comprising, as an active ingredient , DNA comprising any nucleotide sequence selected from the group consisting of SEQ ID NOs: 181, 184, 189 and 194 . A composition for immunostimulation comprising, as an active ingredient , DNA comprising any nucleotide sequence selected from the group consisting of SEQ ID NOs: 181, 184, 189 and 194 .