Polymer compound derived from insect belonging to bombycidae

Abstract

To provide a compound of insect origin exhibiting high immunostimulatory activity.SOLUTION: There is provided a polymer compound that is polyethylene glycol or a derivative thereof having an ability to bind to a Toll-like receptor 4.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a polymer compound derived from an insect belonging to the Bombycidae family, and an immunostimulant containing the polymer compound as an active ingredient. [Background technology]

[0002] Insects are considered important as an animal protein that is easy to artificially produce, and attempts have been made to use insect larvae or pupae as feed ingredients for livestock or aquaculture (WO 2011 / 007867 (Patent Document 1), JP 2003-210071 A (Patent Document 2)).

[0003] Furthermore, International Publication No. 2014 / 017451 (Patent Document 3) discloses insect-derived polysaccharides that have immunostimulatory activity. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2011 / 007867 [Patent Document 2] Japanese Patent Application Laid-Open No. 2003-210071 [Patent Document 3] International Publication No. 2014 / 017451 Summary of the Invention [Problem to be solved by the invention]

[0005] The polysaccharides described in Patent Document 3 have immunostimulating effects and are expected to be used as pharmaceuticals or health foods, or as feed additives for livestock or fisheries, but there is a demand for substances with even stronger immunostimulating effects.

[0006] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an insect-derived compound having a high immunopotentiating effect. [Means for solving the problem]

[0007] As a result of extensive research, the present inventors have discovered a polymer compound having a high immunopotentiating effect from an insect belonging to the Bombycidae family, and have completed the present invention.

[0008] [1] The polymer compound according to the present invention is polyethylene glycol or a derivative thereof that has the ability to bind to Toll-like receptor 4.

[0009] [2] In the above [1], the polymer compound is It has at least a group represented by -O-CH2-CH2-, a methyl group adjacent to a carbonyl group, and an aldehyde group, the respective molar ratios of the group represented by -O-CH2-CH2-, the methyl group adjacent to the carbonyl group, and the aldehyde group are 100:1.5-2.5:0.1-0.5, Average molecular weight is 1.0 x 10 5 Da or more 3.0 × 10 5 It may be less than Da.

[0010] [3] In the above [1] or [2], the polymer compound may be derived from an insect belonging to the Bombycidae family.

[0011] [4] In the above [3], the insect belonging to the Bombycidae family may be a silkworm moth.

[0012] [5] In any one of the above [1] to [4], the polymer compound may have an immunopotentiating effect.

[0013] [6] In the above [2], the molar ratio of the group represented by -O-CH2-CH2-, the methyl group adjacent to the carbonyl group, and the aldehyde group may be 100:2:0.3.

[0014] [7] In any of the above [2], the average molecular weight is 1.9 × 10 5 It may also be Da.

[0015] [8] In any of the above items [1] to [7], the polymer compound may be soluble in water.

[0016] [9] In any of the above items [1] to [8], the polymer compound may be insoluble in ethanol.

[0017]

[10] The immunostimulating agent according to the present invention comprises the polymer compound according to any one of [1] to [9] as an active ingredient.

[0018]

[11] A method for producing a composition containing a polymer compound according to the present invention includes the steps of: A method for producing a composition containing the polymer compound according to any one of [1] to [9], a suspending step of mixing one or more raw materials selected from the group consisting of eggs, larvae, pupae, and adults of insects belonging to the Bombycidae family with ethanol to obtain a suspension; a recovery step of recovering solid matter from the resulting suspension; Includes:

[0019]

[12] In the above

[11] , in the suspension step, the raw material may be powdered and then mixed with the ethanol.

[0020]

[13] In the above

[11] or

[12] , an extraction step may be further included in which the recovered solid matter is mixed with water to extract the polymer compound.

[0021]

[14] The method for producing an immunostimulant according to the present invention comprises: A method for producing an immunostimulant containing the polymer compound as an active ingredient, comprising the step of obtaining the composition by the method for producing a composition according to any one of

[11] to

[13] above. [Effects of the Invention]

[0022] According to the present invention, it is possible to provide insect-derived compounds that have a high immunopotentiating effect. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 is a flowchart showing a method for producing a polymer compound according to this embodiment. [Figure 2] 2 is a graph showing the immunostimulatory activity (NO Assay), absorbance at 280 nm (UV 280 nm), refractive index (RI), total sugar, and sodium chloride elution concentration for each fraction obtained by separating the water-extracted fraction (C) of the Example on an anion exchange column. The horizontal axis shows the fraction number, and the vertical axis shows the absorbance corresponding to each index. [Figure 3] 3 is a graph showing the immunostimulatory activity (NO Assay), absorbance at 280 nm (UV 280 nm), refractive index (RI), and total sugar content of each fraction obtained by gel filtration chromatography of the fraction eluted with 0.5 M sodium chloride in the Example. The horizontal axis shows the fraction number, and the vertical axis shows the absorbance corresponding to each index. [Figure 4] 4 shows the H NMR spectrum of the polymer compound obtained in the example. The horizontal axis shows the chemical shift when trimethylsilylpropanoic acid is used as the reference (0 ppm), and the vertical axis shows the relative signal intensity. [Figure 5] 5 shows the H NMR spectrum of the polymer compound obtained in the example. The horizontal axis shows the chemical shift when trimethylsilylpropanoic acid is used as the reference (0 ppm), and the vertical axis shows the relative signal intensity. [Figure 6] 6 shows the C NMR spectrum of the polymer compound obtained in the example. The horizontal axis shows the chemical shift when trimethylsilylpropanoic acid is used as the reference (0 ppm), and the vertical axis shows the relative signal intensity. [Figure 7] 7 shows the C NMR spectrum of the polymer compound obtained in the example. The horizontal axis shows the chemical shift when trimethylsilylpropanoic acid is used as the reference (0 ppm), and the vertical axis shows the relative signal intensity. [Figure 8]FIG. 8 is a photograph showing the results of the ELISA like-test in the example. [Figure 9] FIG. 9 is a graph showing the evaluation results of immunopotentiating effects in the examples. [Figure 10] FIG. 10 is a graph showing the evaluation results of immunopotentiating effects in the examples. [Figure 11] FIG. 11 is a schematic diagram showing the procedure of "Comprehensive gene expression analysis using a next-generation sequencer" in the examples. [Figure 12] FIG. 12 is a volcano plot showing the results of gene expression analysis in the examples. [Figure 13A] FIG. 13A is a graph showing the results of KEGG pathway analysis in an example. [Figure 13B] FIG. 13B is a graph showing the results of KEGG pathway analysis in the example. [Figure 13C] FIG. 13C is a graph showing the results of KEGG pathway analysis in the example. DETAILED DESCRIPTION OF THE INVENTION

[0024] An embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited to this. In this specification, the notation in the format "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0025] <High polymer compound derived from an insect belonging to the Bombyx mori family> The polymer compound according to this embodiment is polyethylene glycol or a derivative thereof that has the ability to bind to Toll-like receptor 4.

[0026] In one aspect of this embodiment, the polymer compound is It has at least a group represented by -O-CH2-CH2-, a methyl group adjacent to a carbonyl group, and an aldehyde group, the molar ratio of the group represented by -O-CH2-CH2-, the methyl group adjacent to the carbonyl group, and the aldehyde group is 100:1.5-2.5:0.1-0.5, Average molecular weight is 1.0 x 10 5 Da or more 3.0 × 10 5 It may be less than Da.

[0027] <Insects used as sources> In this embodiment, "insect" refers to an organism belonging to the phylum Arthropoda and the class Insecta in the animal kingdom. The insect from which the polymer compound according to this embodiment is derived may be an insect belonging to the Bombycidae family. That is, the polymer compound may be derived from an insect belonging to the Bombycidae family.

[0028] Examples of insects belonging to the Bombycidae family include Bombyx mori, Bombyx mandarina, Oberthuria falcigera, Pseudandraca gracilis, Prismosticta hyalinata, etc. In one aspect of this embodiment, the insect belonging to the Bombycidae family may be a silkworm moth.

[0029] <Chemical structure> The polymer compound according to this embodiment is polyethylene glycol or a derivative thereof. In one embodiment, the polymer compound has at least a group represented by -O-CH2-CH2-, a methyl group adjacent to a carbonyl group, and an aldehyde group. Here, the "methyl group adjacent to a carbonyl group" refers to a methyl group directly bonded to a carbonyl group (a group represented by -C(=O)-). In one aspect of this embodiment, "having a methyl group adjacent to a carbonyl group" can be understood to be the same as "having an acetyl group."

[0030] The presence or absence of the above three functional groups in the polymer compound depends on the 1 H NMR and 13This can be confirmed by C NMR. Measurement conditions include, for example, those described in the Examples below. When a group represented by -O-CH-CH is present, 1 1H NMR spectrum shows a peak at 3.70 ppm and 13 In the C NMR spectrum, peaks are observed at 62.94 ppm and 63.28 ppm. If a methyl group adjacent to the carbonyl group is present, peaks are observed at 1.92 ppm and 2.04 ppm. If an aldehyde group is present, a peak is observed at 8.46 ppm.

[0031] The molar ratio of the group represented by -O-CH2-CH2-, the methyl group adjacent to the carbonyl group, and the aldehyde group may be 100:1.5-2.5:0.1-0.5, 100:1.8-2.2:0.27-0.33, or 100:2:0.3. The molar ratio of each of the three functional groups is determined by the above-mentioned H 1 It can be calculated from the ratio of the peak areas of the chemical shifts corresponding to each functional group in the NMR spectrum.

[0032] In one aspect of this embodiment, the polymer compound is a compound having a molecular weight of 1000 or more, and a molecular weight of 1000 or more, which is determined using trimethylsilylpropanoic acid as a reference substance. 1 In the H NMR spectrum, peaks at 1.92 ppm, 2.04 ppm, 3.70 ppm, and 8.46 ppm were observed. 13 It can also be understood that peaks at 62.94 ppm and 63.28 ppm are observed in the C NMR spectrum. 1 In the H NMR spectrum, the area ratio of the peaks at 2.04 ppm, 3.70 ppm, and 8.46 ppm may be 1.63:100:0.07.

[0033] The polymer compound has an average molecular weight of 1.0×10 5 Da or more 3.0 × 10 5 Da or less, 1.8 × 10 5 Da or more 2.0×10 5Da, 1.9 x 10 5 The average molecular weight may be determined by gel filtration high performance liquid chromatography as described in the Examples below.

[0034] <Properties of polymer compounds> In this embodiment, the polymer compound may be soluble in water.

[0035] In one aspect of this embodiment, the polymer compound may be insoluble in ethanol.

[0036] The polymer compound may have an immunopotentiating effect. "Immunopotentiating effect" refers to the effect of activating the immune function of a living organism. In this embodiment, the immunopotentiating effect is evaluated using the amount of nitric oxide produced when a substance to be evaluated (such as the polymer compound) is contacted with a mouse macrophage-like cell line (RAW264 cells) as an indicator. More specifically, the immunopotentiating effect is evaluated according to the procedures and conditions described in "1.4 Evaluation of immunopotentiating effect" in the Examples section described below.

[0037] The immunostimulatory effect of the polymer compound is mediated by Toll-like receptor 4. That is, the polymer compound has the ability to bind to Toll-like receptor 4, and the binding of the polymer compound to Toll-like receptor 4 activates immune function in vivo. In one embodiment, the polymer compound does not bind to Toll-like receptor 2 and / or Dectin-1.

[0038] <Immunostimulant containing a polymer compound as an active ingredient> The immunostimulant according to this embodiment contains the polymer compound as an active ingredient.

[0039] The immunostimulant may contain other ingredients within the scope of the effects of the present invention, such as excipients, binders, wetting agents, disintegrants, lubricants, diluents, flavoring agents, fragrances, solubilizers, suspending agents, emulsifiers, stabilizers, preservatives, and isotonicity agents.

[0040] <Method for producing a composition containing a polymer compound> The method for producing a composition containing a polymer compound according to this embodiment includes the steps of: A method for producing a composition containing the polymer compound, comprising: a suspending step of mixing one or more raw materials selected from the group consisting of eggs, larvae, pupae, and adults of insects belonging to the Bombycidae family with ethanol to obtain a suspension; a recovery step of recovering solid matter from the resulting suspension; Includes.

[0041] <Suspension process> In this step, one or more raw materials selected from the group consisting of eggs, larvae, pupae, and adults of insects belonging to the Bombycidae family are mixed with ethanol to obtain a suspension.

[0042] The state of the raw material is not particularly limited, and may be, for example, a powder or a water extract. The raw material may be any of eggs, larvae, pupae, or adults of an insect belonging to the Bombycidae family, as long as it contains the polymer compound, but is preferably a pupa of an insect belonging to the Bombycidae family.

[0043] The amount of ethanol added is not particularly limited, but may be 10 ml or more and 50 ml or less per 1 g of the raw material.

[0044] <Recovery process> In this step, the solid matter is collected from the obtained suspension. Specifically, the ethanol is removed from the suspension by centrifugal separation or suction filtration, thereby obtaining the solid matter.

[0045] <Extraction process> In one aspect of this embodiment, the method may further include an extraction step of mixing the recovered solid matter with water to extract the polymer compound.

[0046] <Other processes> The method for producing a composition containing a polymer compound may further include other steps in addition to the suspending step, the recovering step, and the extracting step, such as a drying step of removing water from an extract containing the polymer compound after the extracting step.

[0047] The composition can be used as an immunostimulating composition. That is, the composition can be provided as an immunostimulant containing the polymer compound as an active ingredient, such as a pharmaceutical, health food, functional food, veterinary drug, feed, feed additive, or feed ingredient. The composition can be used not only for humans, but also for livestock such as pigs, cattle, sheep, goats, and chickens, pets such as dogs and cats, and aquaculture of red sea bream, yellowtail, amberjack, grouper, pufferfish, tuna, eel, shrimp, crab, and other species.

[0048] <Method for producing an immunostimulant containing a polymer compound as an active ingredient> The method for producing an immunostimulant according to the present embodiment includes the step of obtaining the composition by the method for producing the composition. In one aspect of the present embodiment, the method for producing an immunostimulant may further include the step of adding to the composition at least one selected from the group consisting of an excipient, a binder, a wetting agent, a disintegrant, a lubricant, a diluent, a flavoring agent, a fragrance, a solubilizing agent, a suspending agent, an emulsifier, a stabilizer, a preservative, and an isotonicity agent. [Example]

[0049] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0050] 1. Identification of immunostimulatory substances derived from Lepidoptera insects 1.1 Identification of immunostimulatory substances derived from Bombycidae insects We identified compounds with immunostimulatory properties contained in insects belonging to the order Lepidoptera. The insects used were pupae of the silkworm moth (Bombyx mori), a member of the Bombycinae family. The silkworm pupae were crushed into a paste and centrifuged to obtain the supernatant fraction (A). The supernatant fraction was then concentrated under reduced pressure using a rotary evaporator. A four-fold volume of ethanol was added to the resulting concentrate and stirred to obtain a suspension (suspension step). The resulting suspension was centrifuged to obtain the ethanol-extracted fraction (B) and a precipitate (recovery step). The alcohol-insoluble solids obtained as the precipitate were extracted with water (extraction step), yielding the water-extracted fraction (C) (Figure 1).

[0051] The aqueous extract fraction (C) was then eluted using a Hi Prep QXL 16 / 10 anion exchange column (GE Health) with 0M, 0.2M, 0.4M, and 1M sodium chloride solutions (Figure 2, NaCl_percentage graph). Each eluted fraction was collected as a 5-ml fraction. The collected fractions were diluted 200-fold or 2000-fold, and the immunostimulatory activity of each fraction was assessed using the amount of nitric oxide produced by a mouse macrophage-like cell line (RAW264 cells) as an indicator (Figure 2, NO_Assay_Dil_200 and NO_Assay_Dil_2000 graphs). Activated macrophages are known to produce nitric oxide, which was used as an indicator of immunostimulation. The specific procedure is described in Section 1.4 below.

[0052] Furthermore, the absorbance at 280 nm (UV_280 nm graph in Figure 2), differential refractive index (RI graph in Figure 2), and total sugar content (Total_sugar_A490 graph in Figure 2) of each collected elution fraction were measured. The absorbance at 280 nm was measured using a Biorad absorption spectrophotometer (product name: NGC Multi-wave length detector module). The differential refractive index was measured using a Shodex differential refractive index detector (product name: RI-501). The total sugar content was measured using the phenol-sulfuric acid method. The results are shown in Figure 2.

[0053] As shown in Figure 2, the fraction eluted with 0.4M sodium chloride solution exhibited immunostimulating effects. The fraction eluted with 0.4M sodium chloride solution was confirmed to produce nitric oxide even when diluted 2000 times, demonstrating its strong immunostimulating effects. Since it was adsorbed to an anion exchange column and eluted with 0.4M sodium chloride solution, it was found that the substance with immunostimulating effects is acidic.

[0054] The fraction eluted with 0.4 M sodium chloride was further purified by gel filtration chromatography (Figure 3). An Enrich SEC 650 column (fractionation range: 0 CV to 1.2 CV) (Biorad) was used. Each elution fraction was collected in 5 ml fractions. The collected fractions were diluted 20-, 200-, and 2000-fold, and the immunostimulatory activity of each fraction was examined using nitric oxide production by a mouse macrophage-like cell line (graphs NO_Assay_Dil_20, NO_Assay_Dil_200, and NO_Assay_Dil_2000 in Figure 3). Furthermore, the absorbance at 280 nm (UV_280nm graph in Figure 3), differential refractive index (RI graph in Figure 3), and total sugar content (Total_sugar_A490 graph in Figure 3) of each elution fraction were examined. The results are shown in Figure 3. As shown in Figure 3, there were "high molecular weight fractions" (fractions 5-8) that exhibited strong immunostimulatory activity, and "low molecular weight fractions" (fractions 11-15) that exhibited weaker immunostimulatory activity than the "high molecular weight fractions." The "high molecular weight fractions" did not exhibit absorbance at 280 nm, differential refractive index, or total sugar content, suggesting that they were neither proteins nor polysaccharides. Further analysis of these "high molecular weight fractions" was performed below.

[0055] The "large molecular weight fraction" was subjected to gel filtration high performance liquid chromatography to measure the molecular weight. The column used was Showdex (registered trademark) SB-807 HQ (manufactured by Showa Denko K.K.), which has a fractionation range of 500 million to 10,000. As a result, the substance with immunopotentiating activity contained in the "large molecular weight fraction" had a molecular weight of approximately 190,000 (approximately 1.9 × 10 5 From 100 g of silkworm pupae, 70.76 mg of a polymer compound with immunostimulatory activity was purified.

[0056] 1.2 Structural analysis by NMR In order to understand the structure of the polymer compound isolated from the silkworm pupae, the following analytical conditions were used: 1 H NMR and 13 C NMR was performed. (common) Equipment :Bruker AVANCE NEO 700 Probe: 5mmφ Cryoprobe (CP DCH) Concentration: 10mg / 550μL of heavy water Temperature: 25℃ ( 1 H NMR) Observed nucleus: 1H (700.2MHz) Accumulation count: 32 times Wait time: 30 seconds Standard: TSP (trimethylsilylpropanoic acid) (0 ppm) ( 13 C NMR) Observation kernel: 13 C(176.1MHz) Accumulation count: 256 times Wait time: 30 seconds Standard:TSP(0ppm) Measurement mode: Inverse gated decoupling (quantitative method)

[0057] 1 The results of H NMR measurement are shown in Figures 4 and 5. 13 The results of C NMR measurements are shown in Figs. 6 and 7. 1 1H NMR spectrum shows a peak at 3.70 ppm and13 In the C NMR spectrum, peaks were observed at 62.94 ppm and 63.28 ppm. From these results, it was estimated that the main component of the polymer compound was a polymer whose repeating unit was a group represented by -O-CH2-CH2-, and that the polymer contained polyethylene glycol (PEG) in its structure. 1 From the H NMR spectrum, trace components were detected at around 2 ppm (1.92 ppm, 2.04 ppm) as a methyl group adjacent to the carbonyl group (RC(=O)-CH3) and at 8.46 ppm as a peak presumed to be an aldehyde group (HC(=O)-R'). 1 In the H NMR spectrum, the area ratio of the peaks at 2.04 ppm, 3.70 ppm, and 8.46 ppm was 1.63:100:0.07. From this area ratio, when the molar ratio of the group (repeating unit) represented by -O-CH2-CH2- was taken as 100, the molar ratios of the methyl group adjacent to the carbonyl group and the aldehyde group were estimated to be approximately 2 and approximately 0.3, respectively. From these results, it was considered that this polymer compound is a polymer compound whose main structure is PEG, contains a trace amount of aldehyde groups, and may also contain acetyl groups, etc.

[0058] 1.3 Evaluation of antibody specificity Rabbit antibodies against the above polymer compounds were produced and their cross-reactivity with PEG4000, PEG6000, and PEG500000 (all manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was evaluated. The method for producing the rabbit antibodies and the method for evaluating the cross-reactivity are described below.

[0059] 1.3.1 Rabbit antibody production A rabbit (SPF grade, 1 rabbit) was immunized with the polymer compound according to the following immunization schedule. 56 days after the first immunization, the rabbit was euthanized and exsanguinated. Serum was collected from the collected blood by a known method, and the target antibody (sometimes referred to as "anti-novel functional substance IgG antibody") was purified from the serum by a known method. The purified antibody was used for the cross-reactivity evaluation described below. <Immunization Plan> Day 0: Preliminary blood collection, antigen administration (0.25 mg) Day 14 antigen administration (0.25 mg) Day 28 antigen administration (0.25 mg) Day 42 antigen administration (0.25 mg) Day56 Whole blood collection

[0060] 1.3.2 Assessment of cross-reactivity ELISA like-test was performed in the following order, using the above polymer compounds, PEG4000, PEG6000, and PEG500000 as antigens.

[0061] First, silkworm organs (samples) adjusted to 20 mg / ml were added to a 96-well plate (96-well CARBO-BIND, Costar) at 50 μl / well. The plate was then incubated at 38°C for 1 hour to coat the bottom of the plate. Each well was washed three times with 20 mM Tris-HCl buffer (pH 8.0). After washing, 100 μl / well of Blocking Reagent (Roche) dissolved in 0.1 g / 10 ml DIG I buffer was added to each well and incubated at 38°C for 1 hour. After washing each well again, 50 μl / well of anti-novel functional substance IgG antibody diluted 1,000-fold with Can Get Signal (TOYOBO)-reagent 1 was added to each well and incubated at 38°C for 1 hour. Each well was washed again. 50 μl / well of goat alkaline phosphatase antibody diluted 1:1,000 in Can Get Signal-reagent 2 was added to each washed well and incubated at 38°C for 1 hour. Each well was washed again, and 100 μl / well of PNPP tablets (Thermo Fiser Scientific) dissolved in 1× Diethanolamine Substrate (Thermo Fiser Scientific) at 1 mg / ml was added. The plate was incubated at 38°C for 30 minutes to allow color development, after which the reaction was stopped with 50 μl / well of 2 N NaOH. The absorbance at 405 nm was measured using a microplate reader, and the concentration of the polymer compound in the sample was calculated based on a calibration curve of the novel functional substance concentration (the polymer compound described above). Photographs of the color development on the plate were also saved; the results are shown in Figure 8.

[0062] The results in Figure 8 show that the rabbit antibodies obtained by immunization with the polymer compound bind to the polymer compound but do not bind to PEG 4000, PEG 6000, or PEG 500000. This suggests that the polymer compound has a chemical structure that is clearly different from that of general polyethylene glycol.

[0063] 1.4 Evaluation of immunostimulatory activity 1.4.1 Specific evaluation procedures The immunostimulatory effect of the polymer compounds was evaluated by the following procedure. First, a mouse macrophage-like cell line (RAW264 cells) was cultured at 1.0 × 10 6 The cells were seeded onto a 96-well plate at a density of 1000 cells / ml. MEM (Gibco) was used as the medium. The above polymer compounds, lipopolysaccharide (LPS), or PEG500000 (PEG 500Kd) were then added to each well at the specified concentrations, and the cells were cultured for 24 hours (37°C, 5% CO2). Lipopolysaccharide, a substance known to have immunostimulatory effects, was used as a positive control. PEG500000 was used as a negative control.

[0064] After the incubation, the concentration of nitric oxide was measured using the Griess Reagent method. The results are shown in Figure 9. When the concentration of nitric oxide was 2 µM or higher, it was determined to have immunostimulating activity. The results in Figure 9 demonstrate that the polymer compound exhibits immunostimulating activity in a concentration-dependent manner.

[0065] 1.4.2 Exploring the mechanism of immunostimulatory effects To explore the mechanism underlying the immunopotentiating effect induced by the polymer compounds, we evaluated their immunopotentiating effect in the presence of anti-TLR2, anti-TLR4, or anti-Dectin-1 antibodies. Specifically, the same method as described in 1.4.1 above was used, except that the polymer compounds were added to each well at a concentration of 25 μg / mL, and anti-TLR2 (25 μg / mL), anti-TLR4 (25 μg / mL), anti-TLR2 and anti-TLR4 (25 μg / mL each), or anti-Dectin-1 (25 μg / mL) antibodies were added together with the polymer compounds. Control experiments were also performed without anti-TLR2, anti-TLR4, or anti-Dectin-1 antibodies. The results are shown in Figure 10. In Figure 10, the horizontal axis indicates the type of antibody added, and the vertical axis indicates the relative nitric oxide concentration compared to the nitric oxide concentration without antibody (no blocking). The notation "TLR2 blocked" on the horizontal axis indicates a sample to which anti-TLR2 antibody was added. The same applies to other notations.

[0066] The results in Figure 10 show that the concentration of nitric oxide was significantly reduced only when treated with anti-TLR4 antibodies ("TLR4 blocked" and "TLR2 / 4 blocked"), indicating that the above polymer compounds activate cellular immunity at least by binding to TLR4.

[0067] 1.5 Comprehensive gene expression analysis using next-generation sequencers 1.5.1 Cell culture and isolation of total RNA from cultured cells RAW264.7 cells were cultured in a 12-well plate (1 × 10 6 Cells were seeded at 2 mL / well (25 μg / mL; 2 mL / well) and pre-cultured at 37°C, 5% CO2, and humidified conditions for 2.5 hours. Then, a novel substance purified from silkworm pupae (25 μg / mL, 1 mL) or medium (control, 1 mL) was added to the wells, and the cells were cultured at 37°C, 5% CO2, and humidified conditions for 6 hours (Figure 11). After culture, total RNA was extracted from the RAW264.7 cells using the method described below and used for the following analysis. This experiment was performed three times in total.

[0068] Total RNA was isolated from the cultured RAW264.7 cells using ISOGEN II (Nippon Gene Co., Ltd., Toyama, Japan) according to the manufacturer's protocol. The resulting sample was adjusted to a total RNA amount of 90 ng or more and a volume of 25 μl or more. Then, the sample was purified using TURBO DNA-free chromatograph. TM The genomic DNA contained in the samples was completely digested using a kit (Invitrogen, Massachusetts, USA). The quantity and quality of the resulting samples (total RNA) were confirmed using an Implen NanoPhotometer P330 (Implen, Munchen, Germany), and the quality was evaluated by electrophoresis on a 1% agarose bleach gel.

[0069] 1.5.2 Library preparation and sequencing RNA library preparation and transcriptome sequencing were outsourced to Novogene (Beijing, China). The procedure is as follows: mRNA was purified from total RNA using magnetic beads conjugated with poly-T oligonucleotides. The purified mRNA was fragmented, and the first strand of cDNA was synthesized using random hexamer primers. The second strand of cDNA was then synthesized using dUTP instead of dTTP. The directional library was completed through end repair, poly(A) tailing, adapter addition, size selection, USER enzyme digestion, amplification, and purification. The library concentration was preliminarily tested using a Qubit 2.0 fluorometer (Thermo Fisher Scientific, Massachusetts, USA). The library was then quantified by real-time PCR, and its size distribution was detected using an Agilent 2100 Bioanalyzer (Agilent, California, USA). The resulting libraries were pooled to achieve the expected library concentration and data volume, and then sequenced using the MGI DNBSEQ-T7 sequencing platform (MGI Tech, Shenzhen, China). Adapter-containing reads, poly-N-containing reads, and low-quality reads were then removed from the raw data to obtain clean data.

[0070] 1.5.3 RNA-seq and enrichment analysis All downstream analyses were based on high-quality, clean sequence data. The reference genome (GRCm39 / GCA_000001635.9) and gene model annotation files were downloaded directly from the ensemblegenom website. A reference genome index was constructed using STAR v2.7.11.b, and paired-end cleanliness was aligned to the reference genome using STAR v2.7.11b. The mapping output data was then converted using Samtools v1.19.2, and the abundance of each transcript was quantified using featureCounts v3.18. After normalization using the DEGES normalization method in the TCC package v1.38, differential gene expression analysis (DEGs analysis) was performed using the TMM-edgeR-TMM pipeline. Genes with an adjusted P value <0.05 detected by TCC were considered differentially expressed between groups. Multidimensional scaling analysis, heat map analysis, and volcano plot analysis (Fig. 12) were generated using the metaseqR2 v1.10 and ggplot2 v3.4.3 packages, respectively.

[0071] Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment in clustered differentially expressed genes were tested using the hypergeometric test in the clusterProfiler package v4.8.3. GO terms and KEGG pathways were tested for significance at a corrected P value < 0.05. Visualization of significant GO and KEGG pathways was performed using ggplot2 v3.4.3 [7] and the pathview v1.40 package (Figures 13A, 13B, and 13C). The results are shown in Figures 12–13C.

[0072] The heat map analysis revealed that the gene expression patterns were significantly different between the cell group cultured with the addition of the polymer compound (novel substance-added group) and the cell group cultured without the addition of the polymer compound (control group). These results demonstrate that the polymer compound significantly changes the gene expression pattern in mouse macrophage-like cell lines.

[0073] The results in Figure 12 show that the addition of the polymer compound significantly decreased the expression of 3,030 genes and significantly increased the expression of 2,116 genes. This indicates that changes in the expression of a relatively large number of genes were induced, particularly in immune system cells.

[0074] The results in Figures 13A, 13B and 13C show that the genes whose expression is induced by the polymer compounds are mainly genes involved in the immune system.

[0075] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0076] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims rather than the above-described embodiments and examples, and it is intended to include any modifications within the scope of the claims that are equivalent to the claims.

Claims

1. A polymeric compound which is polyethylene glycol or a derivative thereof and has the ability to bind to Toll-like receptor 4.

2. -O-CH ２ -CH ２ -, a methyl group adjacent to a carbonyl group, and an aldehyde group, The —O—CH ２ -CH ２ -, the methyl group adjacent to the carbonyl group, and the aldehyde group are in a molar ratio of 100:1.5 to 2.5:0.1 to 0.5, Average molecular weight is 1.0 x 10 ５ Da or more 3.0 x 10 ５ The polymer compound according to claim 1, having a molecular weight of 1 Da or less.

3. 3. The polymer compound according to claim 1, which is derived from an insect belonging to the Bombycidae family.

4. The polymer compound according to claim 3 , wherein the insect belonging to the Bombycidae family is a silkworm moth.

5. The polymer compound according to claim 1 or 2, which has an immunopotentiating effect.

6. The —O—CH ２ -CH ２ 3. The polymer compound according to claim 2, wherein the molar ratio of the group represented by -, the methyl group adjacent to the carbonyl group, and the aldehyde group is 100:2:0.

3.

7. The average molecular weight is 1.9×10 ５ The polymer compound according to claim 2, wherein the molecular weight is Da.

8. The polymer compound according to claim 1 or 2, which is soluble in water.

9. The polymer compound according to claim 1 or 2, which is insoluble in ethanol.

10. An immunostimulant comprising the polymer compound according to claim 1 or 2 as an active ingredient.

11. A method for producing a composition containing the polymer compound according to claim 1 or 2, comprising: a suspending step of mixing one or more raw materials selected from the group consisting of eggs, larvae, pupae, and adults of insects belonging to the Bombycidae family with ethanol to obtain a suspension; a recovery step of recovering solid matter from the resulting suspension; A method for producing a composition comprising:

12. The method for producing a composition according to claim 11, wherein in the suspending step, the raw material is powdered and then mixed with the ethanol.

13. The method for producing a composition according to claim 11, further comprising an extraction step of mixing the recovered solid matter with water to extract the polymer compound.

14. A method for producing an immunostimulant containing the polymer compound as an active ingredient, the method comprising the step of obtaining the composition by the method for producing a composition according to claim 11.

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