Methods and kits for detecting and quantifying beta-1,6 branched beta-1,3-glucan or beta-1,3-glucan

Abstract

In the first process, in the case where the test sample contains β-1,6-branched β-1,3-glucan, by binding the β-1,6-branched β-1,3-glucan to the first fusion protein and the second fusion protein, one and the other of the split reporter proteins form an active-type reporter protein, in the case where the test sample contains β-1,3-glucan, by binding the β-1,3-glucan to the first fusion protein and the third fusion protein, one and the other of the split reporter proteins form an active-type reporter protein, in the second process, the active-type reporter protein formed in the first process is detected / quantified.

Description

Technical Field

[0001] This invention relates to a method for detecting / quantifying β-1,6-branched β-1,3-glucan or β-1,3-glucan, as well as a detection / quantification kit. Background Technology

[0002] β-glucan is abundant in the cell walls and intracellular spaces of yeast, algae, mushrooms, molds, lactic acid bacteria, bacteria, lichens, and other microorganisms. It is also secreted as a soluble molecule outside the cells. β-glucan exhibits strong immune-enhancing effects, and its applications in food, cosmetics, and animal feed are developing. In particular, soluble β-glucan from basidiomycetes is used in pharmaceuticals in Japan.

[0003] In addition, among polysaccharides produced by basidiomycetes such as mushrooms or yeasts, β-1,6-branched β-1,3-glucan with β-1,3-glucosidic bonds in the main chain and β-1,6-glucosidic bonds in the side chain is known to exhibit particularly characteristic physiological activities.

[0004] Analytical methods such as NMR (nuclear magnetic resonance), HPLC (high-performance liquid chromatography), and GC (gas chromatography) are suitable for analyzing and evaluating the structure of these β-glucans, but they require highly purified methods involving complex procedures. Furthermore, these analytical methods require the purification of soluble β-glucans, making them unsuitable for the analysis of crudely purified samples.

[0005] Therefore, various methods for quantifying β-1,3- / β-1,6-glucan in crude purified samples have been developed. Most of these methods combine saccharide-degrading enzymes such as α-glucanase, β-1,3-glucanase, and β-1,6-glucanase with acid-hydrolysis methods such as the sulfuric acid method. By quantifying the amount of glucose produced, the amount of β-1,3- / β-1,6-glucan in the test sample can be estimated, regardless of whether the sample is insoluble or soluble (e.g., Patent Document 1).

[0006] In addition, quantitative methods for β-1,3-glucan and β-1,6-glucan were developed.

[0007] As a quantitative method for β-1,3-glucan, the horseshoe crab method or enzymatic method using horseshoe crab body fluid is known, or the quantitative method based on ELISA (enzyme-linked immunosorbent assay) is a combination of β-glucan-binding proteins from horseshoe crabs, antibodies, mammalian β-glucan receptor Dectin-1, insect β-glucan recognition protein (BGRP), glycosphingolipids, etc. (e.g., Patent Documents 2, 3, 4, Non-Patent Documents 1, 2, 3, 4).

[0008] As a quantitative method for β-1,6-glucan, known methods include enzymatic methods, or ELISA-based quantitative methods that combine antibodies, lectins, or β-1,6-glucanase variants with specific binding activity to β-1,6-glucan (e.g., Non-Patent Literature 5).

[0009] Furthermore, by combining these β-1,3-glucan-binding proteins and β-1,6-glucan-binding proteins and constructing a sandwich ELISA-based quantitative method, β-1,6-branched β-1,3-glucan can also be quantified.

[0010] Existing technical documents

[0011] Patent documents

[0012] Patent Document 1: Japanese Patent No. 5467251.

[0013] Patent document 2: International publication WO92 / 16651.

[0014] Patent document 3: US Patent US9885726B2.

[0015] Patent document 4: International publication WO2010 / 107068.

[0016] Patent document 5: International publication WO2018 / 212095.

[0017] Non-patent literature

[0018] Non-patent literature 1: Appl Environ Microbiol. 2001 Dec; 67(12): 5420-5424.

[0019] Non-patent literature 2: Int J Mol Sci. 2019 Jul 16; 20(14):3498.

[0020] Non-patent literature 3: J Immunol Methods. 2006 Jul 31; 314(1-2):164-9.

[0021] Non-patent literature 4: J Immunol Methods. 2011 Feb 28; 365(1-2):158-65.

[0022] Non-patent literature 5: J Biol Chem. 2020 Apr 17; 295(16):5362-5376. Summary of the Invention

[0023] The problem that the invention aims to solve

[0024] However, the method in Patent Document 1 uses an enzymatic quantification method and does not detect the side chains of β-glucan (β-1,3-glucan with side chains formed by β-1,6-glucosidic bonds within one molecule). Furthermore, while soluble β-glucan can be quantified using ELISA-based methods, insoluble β-glucan cannot be quantified due to the need for multiple washing steps.

[0025] The present invention was made in view of the above circumstances, and its object is to provide a method and kit for the efficient detection / quantification of β-1,6-branched β-1,3-glucan or β-1,3-glucan without the need for washing steps, regardless of the purification purity or soluble / insoluble properties of β-glucan.

[0026] Technical solutions to the problem

[0027] To address the aforementioned issues, the present invention provides a method for detecting / quantifying β-1,6-branched β-1,3-glucan or β-1,3-glucan, characterized by comprising the following steps: a first step of contacting a test sample with a reagent; and a second step of detecting / quantifying the product from the first step. The reagent comprises: a first fusion protein having a cleavage reporter protein; and either a second fusion protein or a third fusion protein having a cleavage reporter protein. An active reporter protein can be formed by pairing the two separated split reporter proteins. The first fusion protein contains one of the β-1,3-glucan-binding proteins and the split reporter protein; the second fusion protein contains one of the β-1,6-glucan-binding proteins and the split reporter protein; and the third fusion protein contains one of the β-1,3-glucan-binding proteins and the split reporter protein. In the first step, if the test sample contains β-1,6-branched β-1,3-glucan, the active reporter protein is formed by binding β-1,6-branched β-1,3-glucan to the first and second fusion proteins, and by binding β-1,3-glucan to the first and third fusion proteins. In the second step, the active reporter protein formed in the first step is detected / quantified.

[0028] The β-1,6-branched β-1,3-glucan or β-1,3-glucan detection / quantification kit of the present invention is characterized by comprising: a first fusion protein containing a split reporter protein; and a reagent containing either a second fusion protein or a third fusion protein containing a split reporter protein. An active reporter protein can be formed by pairing the two separated split reporter proteins. The first fusion protein contains one of a β-1,3-glucan-binding protein and the split reporter protein; the second fusion protein contains the other of a β-1,6-glucan-binding protein and the split reporter protein; and the third fusion protein contains the other of a β-1,3-glucan-binding protein. The active reporter protein can be formed by binding β-1,6-branched β-1,3-glucan to the first fusion protein and the second fusion protein, and by binding β-1,3-glucan to the first fusion protein and the third fusion protein, the active reporter protein can be formed by binding one of the split reporter proteins to the other.

[0029] The effects of the invention

[0030] The detection / quantification method and kit for β-1,6-branched β-1,3-glucan or β-1,3-glucan according to the present invention do not require a washing process regardless of the purity or soluble / insoluble properties of β-glucan, and can efficiently detect / quantify β-1,6-branched β-1,3-glucan or β-1,3-glucan. Attached Figure Description

[0031] Figure 1 The results are obtained by polyacrylamide electrophoresis and CBB staining (Coomassie brilliant blue staining) of the cleavage reporter protein fusion supBGRP (first and third fusion proteins) and the cleavage reporter protein fusion β-1,6-glucanase variant (second fusion protein).

[0032] Figure 2 This diagram shows the results of observing the bioluminescence emitted by the activated cleavage reporter protein (enzyme) after mixing it with supBGRP (first and third fusion proteins) and a cleavage reporter protein fusion β-1,6-glucanase variant (second fusion protein) with particulate β-glucan and enzyme substrate.

[0033] Figure 3 This is a graph showing the results of studying the effects of different concentrations and incubation times on the activation levels of cleavage reporter proteins (enzymes).

[0034] Figure 4 This figure represents the results of quantifying the bioluminescence emitted by activated cleavage reporter proteins (enzymes) by mixing various β-glucans, cleavage reporter proteins fused with supBGRP (first and third fusion proteins), and cleavage reporter proteins fused with β-1,6-glucanase variants (second fusion protein) with enzyme substrates.

[0035] Figure 5 This figure shows the results of quantifying the bioluminescence emitted by the activated cleavage reporter protein (enzyme) by mixing live yeast-type Candida alibicans with different cell numbers, supBGRP (first and third fusion proteins), and a variant of β-1,6-glucanase (second fusion protein) with the enzyme substrate.

[0036] Figure 6 This is an image showing the microscopic morphology of cultured Candida albicans. Scale bar: 50 μm.

[0037] Figure 7This figure represents the results of quantifying the bioluminescence emitted by activated cleavage reporter proteins (enzymes) by mixing Candida albicans cultured for different times, a cleavage reporter protein fusion supBGRP (first and third fusion proteins), and a cleavage reporter protein fusion β-1,6-glucanase variant (second fusion protein) with enzyme substrates.

[0038] Figure 8 This figure represents the results of real-time monitoring of side chain structural changes in Zymosan A, a particulate β-glucan, under various β-glucan-degrading enzyme treatments using a protein-fragment complementation assay.

[0039] Figure 9 This graph shows the results of comparing the reactivity of CSBG (Candida albicans soluble β-glucan) as a soluble β-glucan and D-zymosan as an insoluble β-glucan in a assay using a cleavage reporter protein assay with that of a conventional ELISA assay. Detailed Implementation

[0040] The following describes one embodiment of the method for detecting / quantifying β-1,6-branched β-1,3-glucan or β-1,3-glucan of the present invention.

[0041] The method for detecting / quantifying β-1,6-branched β-1,3-glucan or β-1,3-glucan of the present invention (hereinafter, there are cases where it is only described as "detection / quantification method" or "protein fragment complementation determination") comprises the following steps: a first step of contacting the test sample with a reagent; and a second step of detecting / quantifying the product in the first step.

[0042] (First process)

[0043] In the first step, the test sample comes into contact with the reagent.

[0044] The test sample only needs to contain purified or crudely purified β-1,6-branched β-1,3-glucan or β-1,3-glucan, and its properties can be arbitrarily soluble or insoluble.

[0045] In addition, there are no particular limitations on the substances contained in the test specimens. For example, cell walls or extracts of basidiomycetes such as mushrooms, bacteria, yeast, seaweed, and plants, β-glucan secreted outside the fungal body, and β-glucan present in environments such as indoors, soil, rivers, seawater, atmosphere, and outer space can be cited.

[0046] The reagent contains: a first fusion protein having a splitting reporter protein; and either a second fusion protein having a splitting reporter protein or a third fusion protein having a splitting reporter protein.

[0047] That is, the reagent contains a combination of a first fusion protein and a second fusion protein and / or a combination of a first fusion protein and a third fusion protein.

[0048] In this specification, a split reporter protein refers to a reporter protein that splits into two, and the active reporter protein is formed by bringing the two separated proteins together (close to or binding them).

[0049] Specifically, when the split reporter proteins exist separately from each other, they do not exhibit the function of reporter proteins. However, when one of the split reporter proteins approaches or binds to another, it exhibits the function of a reporter protein.

[0050] There are no specific restrictions on the structure of the split reporter protein, as long as it can be recognized when one of the split reporter proteins is close to or bound to another.

[0051] For example, the composition of a split reporter protein can be such that, with one split reporter protein and another in close proximity, structural complementarity is promoted to form an active reporter protein.

[0052] Furthermore, the composition of a splitting reporter protein can involve bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurring when one or more splitting reporter proteins are close together. In this case, an active reporter protein refers to a substance in which one or more splitting reporter proteins are close together and undergo BRET or FRET. When a reporter protein undergoes BRET or FRET, it can be composed of one or more splitting reporter proteins bound to a fluorescent substance.

[0053] Furthermore, the formation of a splitting reporter protein can involve the formation of an active reporter protein through protein splicing, with one and the other of the splitting reporter proteins in close proximity. This can be achieved by fusing one and the other of the splitting reporter proteins with a protein splicing domain, respectively. Examples of protein splicing domains include, for instance, the combination of DnaEn and DnaEc, which are protein splicing domains from the DnaE gene of Synechocystis sp.

[0054] More specifically, reporter proteins include enzymes and fluorescent proteins. Examples of enzymes include luciferase, alkaline phosphatase, horseradish peroxidase, invertase, β-galactosidase, β-glucuronidase, and variants of these enzymes. Examples of fluorescent proteins include green fluorescent protein (GFP) and variants of GFP.

[0055] The first fusion protein contains one of a β-1,3-glucan-binding protein and a cleavage reporter protein.

[0056] The second fusion protein contains one of the β-1,6-glucan-binding proteins and the split reporter protein.

[0057] The third fusion protein contains one of the β-1,3-glucan-binding proteins and the split reporter protein.

[0058] β-1,3-glucan-binding proteins are well-known, including antibodies, Dectin-1, lectins, horseshoe crab factor G protein, insect β-glucan recognition protein (BGRP), supBGRP (described in the examples below) and its derivatives, etc., with soluble recombinant proteins being preferred as they are easy to express.

[0059] β-1,6-glucan-binding proteins are known, including antibodies, lectins, β-1,6-glucanase variants with β-1,6-glucan cleavage activity and specific binding activity to β-1,6-glucan, and their derivatives, with soluble recombinant proteins being preferred for easy expression.

[0060] In the detection / quantification method of the present invention, reagents containing the first and second fusion proteins, or reagents containing the first and third fusion proteins, can be directly supplied to a test tube containing the test sample, or the method can be implemented using cells expressing these fusion proteins. In this case, the expression of the first and second fusion proteins, or the first and third fusion proteins, can be transient or constant. That is, stable cell lines expressing the first to third fusion proteins can be used.

[0061] Specifically, examples of the first to third fusion proteins include fusion proteins produced in *E. coli*, yeast, plants, animal cells, and cell-free expression systems. Fusion proteins produced in large quantities in *E. coli* are particularly preferred due to their ease of use. While crudely purified solutions such as cell lysates expressing the first to third fusion proteins can be used, fusion proteins fused with common low-molecular-weight peptide tags and purified to high purity via column purification or similar methods are preferred. Examples of such tags include GST tags, protein A tags, antibody Fc regions, multihistidine tags, V5 tags, Myc tags, SBP tags, Halo tags, and Strep tags. Furthermore, examples of cells include animal cells, insect cells, and plant cells; established cell lines are preferred due to their ease of use.

[0062] In the first fusion protein, one of the reporter proteins can fuse to the N-terminal side of the β-1,3-glucan-binding protein, and another can fuse to the C-terminal side of the β-1,3-glucan-binding protein. In the second fusion protein, another reporter protein can fuse to the N-terminal side of the β-1,6-glucan-binding protein, and another can fuse to the C-terminal side of the β-1,6-glucan-binding protein. Furthermore, in the third fusion protein, another reporter protein can fuse to the N-terminal side of the β-1,3-glucan-binding protein, and another can fuse to the C-terminal side of the β-1,3-glucan-binding protein. In this case, when fusing the β-1,3-glucan-binding protein or the β-1,6-glucan-binding protein with the reporter protein in the first, second, and third fusion proteins, it is preferable to insert a linker peptide of appropriate length to avoid steric hindrance.

[0063] Here, examples of linker peptides include peptides consisting of approximately 1 to 20 residues, whose amino acid sequence is identical to that of a general linker used in the production of fusion proteins. Specifically, examples include the GS linker composed of a repeating sequence containing Gly-Ser, the DDAKK linker composed of a repeating sequence of Asp-Asp-Ala-Lys-Lys (Sequence No. 1), and the EAAAK linker composed of a repeating sequence of Glu-Ala-Ala-Ala-Lys (Sequence No. 2). Furthermore, in the first, second, and third fusion proteins, the fusion region of one or the other of the reporter proteins is not limited to either the N-terminal or C-terminal side; it is preferable to determine the fusion region based on the characteristics of various β-1,3-glucan-binding proteins and β-1,6-glucan-binding proteins. Alternatively, fusion proteins can be fused at both the N-terminal and C-terminal sides.

[0064] In addition, in the first step, when the test sample contains β-1,6-branched β-1,3-glucan, by binding β-1,6-branched β-1,3-glucan to the first fusion protein and the second fusion protein, one of the reporter proteins is split and approaches or binds to the other to form an active reporter protein.

[0065] In addition, when the test sample contains β-1,3-glucan (including those with side chains), by binding β-1,3-glucan to the first fusion protein and the third fusion protein, one of the reporter proteins is split and brought close to or bound to the other to form an active reporter protein.

[0066] (Second process)

[0067] In the second step, the active reporter protein formed in the first step is detected / quantified. When the activity of the reporter protein is higher than in the absence of β-1,6-branched β-1,3-glucan or β-1,3-glucan, it indicates the presence of β-1,6-branched β-1,3-glucan or β-1,3-glucan in the test sample. Therefore, it is possible to easily detect / quantify β-1,6-branched β-1,3-glucan or β-1,3-glucan contained in the test sample. There are no particular limitations on the detection / quantification method; any known method can be appropriately used depending on the morphology of the reporter protein, etc.

[0068] As described in the examples below, the activity (background) of the inactive reporter protein observed in the absence of β-glucan, which has the target structure, is shown to be approximately 100-fold more sensitive than the activity of the recombinant reporter protein in the presence of β-glucan.

[0069] Furthermore, the detection / quantification method of the present invention eliminates the need for complex cleaning procedures, enabling high-throughput screening of the structure of β-glucan contained in the test sample. Therefore, by applying the detection / quantification method of the present invention, various β-glucans from different species / extraction / purification methods, exhibiting soluble / insoluble (gel-like / particulate, etc.) forms, can be analyzed extensively under the same conditions, allowing for the screening of more beneficial β-1,6-branched β-1,3-glucan or β-1,3-glucan. Additionally, the detection / quantification method of the present invention eliminates the need for test sample cleaning procedures, enabling efficient detection / quantification of β-1,6-branched β-1,3-glucan or β-1,3-glucan.

[0070] Furthermore, for example, for the same test specimen, by comparing the activity of the active reporter protein formed by the binding of β-1,6-branched β-1,3-glucan to the first and second fusion proteins, and the activity of the active reporter protein formed by the binding of β-1,3-glucan to the first and third fusion proteins, it is also possible to quantify β-1,3-glucan without β-1,6-branched β-1,3-glucan.

[0071] Next, an embodiment of the β-1,6-branched β-1,3-glucan or β-1,3-glucan detection / quantification kit of the present invention will be described. In the detection / quantification kit of the present invention, descriptions of aspects common to the detection / quantification methods described above are omitted.

[0072] The detection / quantification kit of the present invention is a detection / quantification kit for β-1,6-branched β-1,3-glucan or β-1,3-glucan, comprising: a reagent containing a first fusion protein having a splitting reporter protein, and any one of a second fusion protein and a third fusion protein having a splitting reporter protein.

[0073] An active reporter protein can be formed by pairing two splitting reporter proteins (by bringing them close together or binding them together).

[0074] The first fusion protein contains one of a β-1,3-glucan-binding protein and a cleavage reporter protein.

[0075] The second fusion protein contains one of the β-1,6-glucan-binding proteins and the split reporter protein.

[0076] The third fusion protein contains one of the β-1,3-glucan-binding proteins and the split reporter protein.

[0077] It can form an active reporter protein by binding β-1,6-branched β-1,3-glucan to the first and second fusion proteins, through the splitting of one and the other reporter proteins.

[0078] It can form an active reporter protein by binding β-1,3-glucan to the first and third fusion proteins, which are split from one of the reporter proteins.

[0079] In addition to the reagents described above, the detection / quantification kit of the present invention may also include various materials and devices for detecting and quantifying β-1,6-branched β-1,3-glucan or β-1,3-glucan.

[0080] The detection / quantification method and detection / quantification kit for β-1,6-branched β-1,3-glucan or β-1,3-glucan of the present invention are not limited to the embodiments described above.

[0081] Example

[0082] The present invention will be described below with reference to the embodiments, but the detection / quantification method and detection / quantification kit of β-1,6-branched β-1,3-glucan or β-1,3-glucan of the present invention are not limited to the following embodiments at all.

[0083] <Example 1> Preparation of fusion protein

[0084] Expression vectors for the first, second, and third fusion proteins were prepared using the pColdI vector (manufactured by Takara Bio). These vectors were transformed into *E. coli* (Shuffle, New England Biolabs) or BL21(DE3) and expressed as ×6 histidine-tagged (His-Tag) fusion proteins in LB medium supplemented with ampicillin. After purification using TALON Metal Affinity Resin (manufactured by Takara Bio), the presence of the purified proteins was confirmed by SDS-PAGE. Figure 1 ).

[0085] As a β-1,3-glucan-binding protein, supBGRP, based on insect-derived BGRP, is used. The amino acid sequence of supBGRP is based on the amino acid sequences of β-glucan-binding proteins (BGRP) from various known insects such as silkworms, and the inventors' previous knowledge. It is an artificially produced β-1,3-glucan-binding protein by introducing an amino acid mutation of approximately 36 residues (23 residues in silkworms).

[0086] The following shows the amino acid sequence and DNA sequence of supBGRP.

[0087] (Serial Number 3)

[0088]

[0089]

[0090] (Serial Number 4)

[0091]

[0092] As a β-1,6-glucan-binding protein, a variant of β-1,6-glucanase from Neurospora (Neg1-E321Q) is used (Patent Document 5).

[0093] Additionally, a luciferase (NanoLuc, manufactured by Prometheus) from deep-sea shrimp (Oplophorus gracilirostris) is used as the reporter protein. The cleavage reporter protein (NanoLuc) used here consists of two subunits: a large fragment called LgBiT and a small fragment called SmBiT. These two subunits form a complex to recombinantly activate the luciferase. Therefore, the combination of LgBiT and SmBiT constitutes one and the other of the cleavage reporter protein.

[0094] In this embodiment, a first fusion protein (supBGRP-LgBiT) was prepared by fusing a 4×DDAKK sequence as a linker peptide with LgBiT at the N-terminus of supBGRP. Additionally, a second fusion protein (Neg1-E321Q-SmBiT) was prepared by fusing a GGSGGGSGG sequence (serial number 5) as a linker peptide with SmBiT at the N-terminus of Neg1-E321Q. Furthermore, a third fusion protein (supBGRP-SmBiT) was prepared by fusing a 4×DDAKK sequence as a linker peptide with SmBiT at the N-terminus of supBGRP.

[0095] To confirm that the fusion protein functions and recombinantly activates the reporter protein in the presence of β-glucan, crudely purified particulate β-glucan from baker's yeast, namely yeast polysaccharide A, supBGRP-LgBiT, and Neg1-E321Q-SmBiT, were mixed and NanoLuc substrate (NanoGlo, Promega) was added, and the presence or absence of bioluminescence was observed.

[0096] The results are shown in Figure 2 .

[0097] Bioluminescence was only observed in the presence of supBGRP-LgBiT, Neg1-E321Q-SmBiT, yeast polysaccharide A, and NanoLuc substrate.

[0098] These results demonstrate that protein fragment complementation assays can be used for simple structural analysis of β-glucan.

[0099] <Example 2> Optimization Study of Protein Fragment Complementation Determination

[0100] To construct and optimize a protein fragment complementation assay that does not require a washing step, reactivity was studied by varying the concentration of the dextran probe and the reaction time.

[0101] A mixed solution (5 μL) of supBGRP-LgBiT and Neg1-E321Q-SmBiT at different concentrations (50, 100, 200, 400 nM) was incubated with 10 μL of yeast polysaccharide A (0–1000 ng / mL) in a 96-well plate for 30 min. Then, 15 μL of NanoLuc substrate was added, and luciferase activity was evaluated. The results are as follows: Figure 3 As shown in Figure A, it was confirmed that the luciferase activity increased proportionally to the concentration of each probe. Appropriate reaction curves were obtained in the presence of at least 200 nM of each probe.

[0102] Next, a 200 nM mixed solution (5 μL) of supBGRP-LgBiT and Neg1-E321Q-SmBiT was added to a 96-well plate and incubated with 0–1000 ng / mL yeast polysaccharide A (10 μL) for 10 min, 30 min, and 60 min. Then, 15 μL of NanoLuc substrate was added, and luciferase activity was measured. The results confirmed that while 10 min of incubation was sufficient to obtain the reaction curve, further extending the incubation time increased luciferase activity. Figure 3 B).

[0103] <Example 3> Determination of various β-glucans using fusion proteins

[0104] Mix 5 μL of a mixture of the first fusion protein (supBGRP-LgBiT) and the second fusion protein (Neg1-E321Q-SmBiT) (200 nM each) or a mixture of the first fusion protein (supBGRP-LgBiT) and the third fusion protein (supBGRP-SmBiT) (200 nM each) with 10 μL of various polysaccharide samples (0–50 μg / mL) in a 96-well plate (white and flat-bottomed) and vibrate on a plate shaker.

[0105] The polysaccharide samples used are shown in Table 1.

[0106] [Table 1]

[0107]

[0108] After 30 minutes, 15 μL of NanoLuc substrate was added, and the bioluminescence level was measured using a photometer (Promega).

[0109] The results are shown in Figure 4 .

[0110] In the combination of the first fusion protein (supBGRP-LgBiT) and the second fusion protein (Neg1-E321Q-SmBiT) (supBGRP-LgBiT / Neg1-E321Q-SmBiT), luminescence was observed in samples containing β-1,6-branched β-1,3-glucan. In the combination of the first fusion protein (supBGRP-LgBiT) and the third fusion protein (supBGRP-SmBiT) (supBGRP-LgBiT / supBGRP-SmBiT), luminescence was observed in samples containing β-1,3-glucan.

[0111] As for the samples containing β-1,6-branched β-1,3-glucan, specifically examples include yeast glycan A, lecithin, sclerosing glucan, and heat-sterilized Candida albicans (HKCA), reactivity with these samples was observed in a mixture of the first and second fusion proteins (supBGRP-LgBiT / Neg1-E321Q-SmBiT). This demonstrates that not only purified, high-purity β-glucan, but also unpurified HKCA can be detected using the same procedure.

[0112] On the other hand, as a sample containing β-1,3-glucan, specifically examples include yeast polysaccharide A, glucan gum, lecithin, sclerotium glucan, Euglena starch, pachyman, laminarin, HKCA, etc., which showed reactivity with the mixture of the first and third fusion proteins (supBGRP-LgBiT / supBGRP-SmBiT).

[0113] In contrast, none of the mixtures showed reactivity with chitin, dextran, xylan, or mannan, which do not possess a β-glucan structure.

[0114] These results confirm that the method according to the present invention can efficiently perform structural analysis of β-glucan in multiple samples.

[0115] <Example 3> Structural Analysis of Natural Cell Walls in Fungi

[0116] In Example 2, it was demonstrated that β-1,3-glucan or long-chain β-1,6-branched β-1,3-glucan could be determined to be present on the surface of HKCA. Figure 4 H). However, since heat treatment often removes the mannan layer covering the β-glucan surface, HKCA does not necessarily mimic the native cell wall composition of Candida albicans. Therefore, the possibility of applying protein fragment complementation assays to the analysis of the native cell wall structure of live fungi was investigated.

[0117] First, different amounts of yeast-type Candida albicans (0, 10) were placed... 410 5 10 6 Cells (per well) were washed in a 96-well V plate and resuspended in 10 μL of PBS. This was then mixed with 5 μL of a mixture of the first and third fusion proteins (supBGRP-LgBiT / supBGRP-SmBiT) or a mixture of the first and second fusion proteins (supBGRP-LgBiT / Neg1-E321Q-SmBiT). After 30 minutes, 15 μL of NanoLuc substrate was added, and the activation level of the recombinant reporter protein on the cell surface was measured. The results showed that the activation levels of the recombinant reporter protein were higher in the combinations of supBGRP-LgBiT / supBGRP-SmBiT and supBGRP-LgBiT / Neg1-E321Q-SmBiT, corresponding to 10 cells / well. 5 Bioluminescence of yeast-type Candida albicans with a count of 1 or more per well is significantly increased. Figure 5 A, Figure 5 B).

[0118] Next, using this method, the possibility of directly analyzing the cell wall glucan structure of filamentous Candida albicans, which is generally difficult to analyze, was investigated. To induce hyphae formation, yeast-type Candida albicans (10g) was added to RPMI 1640 medium containing 10% inactivated fetal bovine serum in 96-well V-plates. 5 Candida albicans were cultured in 96-well plates at 37°C for different times (0, 2, 4, and 7 hours) to confirm hyphal formation. Microscopic morphology of Candida albicans at each culture time is shown in the figures. Figure 6 .

[0119] The washed hyphae were suspended in 10 μL of PBS and mixed with 5 μL of a mixture of the first and third fusion proteins (supBGRP-LgBiT / supBGRP-SmBiT) or a mixture of the first and second fusion proteins (supBGRP-LgBiT / Neg1-E321Q-SmBiT). After 30 minutes, 15 μL of NanoLuc substrate was added, and the bioluminescence level was measured. Luciferase activity, representing the amount of β-1,3-glucan or long-chain β-1,6-branched β-1,3-glucan on the surface, increased significantly in a hyphal growth-dependent manner. Figure 7 (A, B). These results demonstrate the usefulness of this assay in analyzing the dextran structure of untreated live fungal cell walls.

[0120] <Example 4> Application of protein fragment complementation assay in real-time monitoring of structural changes in β-glucan

[0121] For example, the structure of cell wall glucans dynamically changes during fungal growth due to the action of various enzymes. Therefore, this study investigated whether yeast glucan A, a particulate β-glucan, could be treated with endo-β-1,6-glucanase (Neg1) or endo-β-1,3-glucanase (Zymolyase) using this assay, and evaluated the time-dependent changes in the structure of β-1,6-bound β-glucan side chains or β-1,3-glucan chains.

[0122] Add 5 μL of a mixture of the first and third fusion proteins (supBGRP-LgBiT / supBGRP-SmBiT) or a mixture of the first and second fusion proteins (supBGRP-LgBiT / Neg1-E321Q-SmBiT) to 10 μL of yeast polysaccharide A (10 μg / mL) and incubate for 60 min. Add 15 μL of NanoLuc substrate containing PBS, endopeptidase (final concentration 0.5 μg / mL), or endopeptidase (final concentration 0.5 μg / mL), and measure the recombinant NanoLuc activity every 2 min for a total of 15 measurements (30 min).

[0123] like Figure 8 As shown in Figure A, in the combination of the first and third fusion proteins (supBGRP-LgBiT / supBGRP-SmBiT), the luminescence intensity in the untreated control sample gradually decreased over time (natural decay). On the other hand, in yeast glycan A treated with endo-β-1,3-glucanase, the luminescence intensity decreased rapidly, with the bioluminescence level (RLU measurement) after 30 minutes being approximately one-third of that in the untreated group. Even the degradation of the β-1,6-binding side chain of yeast glycan A did not affect the glucan-binding energy of supBGRP.

[0124] In contrast, the activity of NanoLuc recombined from the combination of the first and second fusion proteins (supBGRP-LgBiT / Neg1-E321Q-SmBiT) was significantly reduced upon treatment with endo-β-1,6-glucanase or endo-β-1,3-glucanase. Figure 8 B). After 30 minutes, the bioluminescence level (RLU measurement) decreased to approximately 1 / 6 in the endo-β-1,3-glucanase-treated group and approximately 1 / 14 in the endo-β-1,6-glucanase-treated group compared to the untreated group. These results confirm that protein fragment complementation assays are not only suitable for the structural analysis of β-glucans but also for real-time monitoring of structural changes in long-chain β-1,6-branched β-1,3-glucans.

[0125] <Example 5> Comparison of reactivity between the fusion protein assay and conventional ELISA

[0126] Soluble and insoluble β-glucans were determined using conventional ELISA and a method for determining fusion proteins, and their reactivity was compared. For soluble β-glucan, an aqueous solution of CSBG purified from Candida cell walls was used; for insoluble β-glucan, a suspension of depleted-Zymosan (D-zymosan, InvivoGen) washed with a hot alkaline solution was used (Table 1).

[0127] A mixture of 5 μL of the first fusion protein (supBGRP-LgBiT) and the second fusion protein (Neg1-E321Q-SmBiT) (200 nM each) was mixed with 10 μL of each soluble and insoluble sample (0–1000 ng / mL) in a 96-well plate (white with a flat bottom) and shaken on a plate shaker. After 30 minutes, 15 μL of NanoLuc substrate was added, and the bioluminescence level was measured using a photometer (Promega).

[0128] like Figure 9 As shown in Figure A, it was confirmed that regardless of whether it is soluble or insoluble, the reactivity increases depending on the concentration of β-glucan added.

[0129] On the other hand, the first fusion protein (supBGRP-LgBiT) was immobilized on a 96-well plate (white with a flat bottom) (2 μg / mL) and blocked with phosphate-buffered saline (PBS) containing bovine serum albumin. After washing the plate, soluble and insoluble samples (0–1000 ng / mL) were added. After washing for 60 minutes, biotin-labeled β-1,6-glucan-binding protein (Neg1-E321Q-Biotin, 2 μg / mL) was added and incubated for 60 minutes. After washing, streptavidin-labeled horseradish peroxidase was added and incubated for 20 minutes. After thorough washing, a commercially available luminescent substrate for peroxidase was added, and the luminescence level was measured using a spectrophotometer (Promega).

[0130] like Figure 9 As shown in B, in the conventional ELISA method, CSBG, as a soluble β-glucan, showed an increase in reactivity depending on the concentration added, but no reactivity was shown in D-yeast glucan, as an insoluble β-glucan.

[0131] These results confirm that the method according to the invention can accurately determine the amount of soluble and insoluble β-glucan under the same conditions. Sequence Listing <110> TOEI SHINYAKU CO.,LTD <120> A method for detection and quantification of β-1,6-branched β-1,3-glucan and β-1,3-glucan. <130> 21F007PCT <150> JP2020-149797 <151> 2020 / 09 / 07 <160> 5 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence <400> 1 Asp Asp Ala Lys Lys 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence <400> 2 Glu Ala Ala Ala Lys 1 5 <210> 3 <211> 149 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence <400> 3 Met Asn His Lys Val His His His His His His Ile Glu Gly Arg His 1 5 10 15 Met Glu Leu Gly Thr Tyr Glu Val Pro Asp Ala Lys Leu Glu Ala Ile 20 25 30 Tyr Pro Lys Gly Leu Arg Val Ser Ile Pro Asp Asp Gly Phe Ser Leu 35 40 45 Phe Ala Phe His Gly Lys Leu Asn Glu Glu Met Glu Gly Leu Glu Ala 50 55 60 Gly Thr Trp Ser Arg Asp Ile Thr Lys Ala Lys Asn Gly Arg Trp Thr 65 70 75 80 Phe Arg Asp Arg Asn Ala Glu Leu Lys Ile Gly Asp Lys Ile Tyr Phe 85 90 95 Trp Thr Tyr Val Ile Lys Asp Gly Leu Gly Tyr Arg Gln Asp Asn Gly 100 105 110 Glu Trp Thr Val Thr Gly Tyr Val Asp Glu Asp Gly Asn Pro Val Asp 115 120 125 Thr Asp Gly Pro Thr Thr Thr Pro Thr Gly Ser Glu Phe Lys Leu Val 130 135 140 Asp Leu Gln Ser Arg 145 <210> 4 <211> 450 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence <400> 4 atg aat cac aaa gtg cat cat cat cat cat cat atc gaa ggt agg cat 48 atg gag ctc ggt acc tat gaa gtg cct gat gcg aaa ctc gaa gcc att 96 tac ccc aaa ggg tta cgc gtt agc att ccg gat gat ggc ttt tcg ctg 144 ttt gcc ttc cat ggg aaa ctg aac gag gag atg gaa ggt ctg gaa gct 192 gga act tgg agt cgg gac atc acg aaa gcg aag aac ggt cgt tgg acc 240 ttt cgt gac cgc aat gca gag ctg aaa att ggc gac aag atc tac ttc 288 tgg acc tac gtc atc aaa gat ggc ttg ggt tat cgc cag gat aac gga 336 gaa tgg acc gta acg ggc tat gtg gac gaa gat ggc aat ccg gtt gat 384 acc gat ggt ccg act acg aca cca acc gga tcc gaa ttc aag ctt gtc 432 gac ctg cag tct aga tag 450 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence <400> 5 Gly Gly Ser Gly Gly Gly Ser Gly Gly 1 5

Claims

1. A method for the detection and / or quantification of β-1,6-branched β-1,3-glucan or β-1,3-glucan, characterized in that, The method includes the following steps: The first step involves bringing the test sample into contact with the reagents; and The second step involves detecting and / or quantifying the products from the first step. The reagent comprises: (A) a first fusion protein having a cleavage reporter protein and a second fusion protein having a cleavage reporter protein; And (B) the first fusion protein having a cleavage reporter protein and the third fusion protein having a cleavage reporter protein, By pairing the two separated reporter proteins, an active reporter protein can be formed. The first fusion protein contains one of a β-1,3-glucan-binding protein and the aforementioned cleavage reporter protein. The second fusion protein contains one of the β-1,6-glucan-binding proteins and the cleavage reporter protein. The third fusion protein contains another of the β-1,3-glucan-binding protein and the cleavage reporter protein. In the first step, if the test sample contains β-1,6-branched β-1,3-glucan, the active reporter protein is formed by binding β-1,6-branched β-1,3-glucan to the first fusion protein and the second fusion protein, through the splitting of one and the other of the reporter proteins. When the test sample contains β-1,3-glucan, the active reporter protein is formed by binding β-1,3-glucan to the first fusion protein and the third fusion protein, through the splitting of one and the other of the reporter proteins. In the second step, the active reporter protein formed in the first step is detected and / or quantified.

2. The method as described in claim 1, characterized in that, The first fusion protein is a fusion protein of the cleavage reporter protein fused to the N-terminal or C-terminal side of a β-1,3-glucan-binding protein. The second fusion protein is another fusion protein of the aforementioned cleavage reporter protein, fused to the N-terminal or C-terminal side of a β-1,6-glucan-binding protein. The third fusion protein is another fusion protein of the split reporter protein that is fused to the N-terminal or C-terminal side of the β-1,3-glucan-binding protein.

3. The method as described in claim 1 or 2, characterized in that, The test specimen is insoluble.

4. A kit for the detection and / or quantification of β-1,6-branched β-1,3-glucan or β-1,3-glucan, characterized in that, The kit contains reagents. The reagent contains: (A) a first fusion protein having a cleavage reporter protein and a second fusion protein having a cleavage reporter protein; And (B) the first fusion protein having a cleavage reporter protein and the third fusion protein having a cleavage reporter protein, By pairing the two separated reporter proteins, an active reporter protein can be formed. The first fusion protein contains one of a β-1,3-glucan-binding protein and the aforementioned cleavage reporter protein. The second fusion protein contains one of the β-1,6-glucan-binding proteins and the cleavage reporter protein. The third fusion protein contains another of the β-1,3-glucan-binding protein and the cleavage reporter protein. The active reporter protein can be formed by binding β-1,6-branched β-1,3-glucan to the first and second fusion proteins, through the splitting of one and the other of the reporter proteins. The active reporter protein can be formed by binding β-1,3-glucan to the first fusion protein and the third fusion protein, through the splitting of one and the other reporter proteins.

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