β-glucan measurement reagent, its manufacturing method and use

Abstract

The present invention addresses the problem of providing a high-performance reagent for measuring β-glucan without the need for horseshoe crab blood cell extract. By using factor D derived from horseshoe crab in a reagent for measuring β-glucan, the present invention can provide a high-performance reagent for measuring β-glucan without the need for horseshoe crab blood cell extract.

Description

[Technical Field]

[0001] The present invention relates to the provision and use of a novel reagent for measuring β-glucan. [Background technology]

[0002] Mycoses are diseases caused by fungal infections. There are two types of mycoses: superficial mycoses, which infect the surface of the body, such as the skin or mucous membranes, and deep mycoses, which infect deeper parts of the body, such as the lungs or blood. The polysaccharides that make up fungal cell walls are unique in that they contain β-glucan. For this reason, β-glucan detection is used as a testing and diagnostic method for mycoses (especially deep mycoses).

[0003] Amebocyte lysate from horseshoe crabs is known to coagulate in response to β-glucan and is used as a reagent for measuring β-glucan. When β-glucan comes into contact with the hemocyte extract, factor G present in the hemocyte extract is activated, generating activated factor G with serine protease activity. This activated factor G cleaves proclotting enzyme present in the hemocyte extract to generate activated proclotting enzyme (i.e., clotting enzyme) with serine protease activity. This activated proclotting enzyme cleaves coagulogen present in the hemocyte extract, and the resulting coagulin causes the hemocyte extract to clot (Figure 1). Taking advantage of this cascade reaction, a reagent for measuring β-glucan has been devised, containing a measurement substrate containing the cleavage sequence of activated proclotting enzyme.

[0004] Attempts have been made to artificially produce the above-mentioned Factor G and proclotting enzyme using recombinant technology, etc., to create reagents for measuring β-glucan that do not rely on horseshoe crab hemocyte extract. For example, International Publication No. 95 / 001432 (Patent Document 1) describes an invention relating to a polypeptide containing the (1→3)-β-D-glucan binding site of Factor G and the cDNA encoding it. International Publication No. 2008 / 004674 (Patent Document 2) describes an invention relating to a nucleic acid encoding a proclotting enzyme derived from horseshoe crab. Japanese Patent Application Laid-Open No. 2006-271384 (Patent Document 3) describes an invention relating to a virus carrying DNA encoding the α subunit of horseshoe crab-derived Factor G. International Publication No. 2021 / 117841 (Patent Document 4) describes an invention relating to a heterodimer combining a Factor G α subunit having a specific amino acid sequence and a Factor G β subunit having a specific amino acid sequence. WO 2021 / 117841 (Patent Document 4) also describes an invention relating to recombinant Factor G derived from Limulus polyphemus expressed in insect cells as a host. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 95 / 001432 [Patent Document 2] International Publication No. 2008 / 004674 [Patent Document 3] JP 2006-271384 [Patent Document 4] International Publication No. 2021 / 117841 Summary of the Invention

[0006] Conventional β-glucan measurement reagents that use artificially produced factor G or proclotting enzyme do not have sufficient performance. For example, Example 3 of Patent Document 2 discloses the results of confirming the expression of activity by β-glucan using recombinant Factor G and recombinant Proclotting Enzyme. However, the invention described in Patent Document 2 requires a high concentration of β-glucan, at least 1 ng / mL, to express activity.

[0007] In Patent Document 3, even when 0.25 ng of β-glucan was used in a total volume of 125.1 μl (i.e., 2 ng / mL of β-glucan), the reaction took as long as 24 hours, which was not satisfactory for diagnosing fungal infections.

[0008] Furthermore, Example 3 of Patent Document 4 describes that it was confirmed that lentinan (a type of β-glucan) can be significantly detected down to a lower limit of 0.35 pg / mL by using a supernatant containing a co-expression product of specific Factor G α subunit and β subunit. However, the invention described in Patent Document 4 also requires a long reaction time of 200 minutes after mixing the reagent with the sample.

[0009] An object of the present invention is to provide a high-performance reagent for measuring β-glucan that does not require an extract from horseshoe crab hemocytes, and raw materials useful for producing the reagent. [Means for solving the problem]

[0010] According to one aspect of the present invention, by allowing specific components to coexist during the reaction between β-glucan and the reagent, a high-performance reagent for measuring β-glucan can be provided without the need for an extract from horseshoe crab hemocytes. Another aspect of the present invention provides a method for efficiently producing recombinant Factor G using mammalian cells. That is, the present invention includes the following aspects.

[0011] [1-1] A method for assaying a soluble protein comprising: horseshoe crab-derived Factor D; horseshoe crab-derived Factor G; horseshoe crab-derived proclotting enzyme; and a measurement substrate; A reagent for measuring β-glucan that does not contain horseshoe crab hemocyte extract. [1-2] The reagent for measuring β-glucan according to [1-1], wherein the horseshoe crab-derived Factor D, the horseshoe crab-derived Factor G, and the horseshoe crab-derived proclotting enzyme are recombinant proteins. [1-3] A reagent for measuring β-glucan according to [1-1] or [1-2], in which the detection limit of pachyman is 20 pg / mL or less within a reaction time of 60 minutes. [1-4] A reagent for measuring β-glucan according to any one of [1-1] to [1-3], wherein the detection limit of pachyman is 16 pg / mL or less within a reaction time of 40 minutes or less. [1-5] The reagent for measuring β-glucan according to any one of [1-1] to [1-4], wherein the horseshoe crab-derived factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70; (2) It has an amino acid sequence that is about 85% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70, and has the function of Factor D. [1-6] The reagent for measuring β-glucan according to any one of [1-1] to [1-4], wherein the horseshoe crab-derived factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33; (2) It has an amino acid sequence that is about 85% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33, and has the function of Factor D. [1-7] The reagent for measuring β-glucan according to any one of [1-1] to [1-6], wherein the horseshoe crab-derived factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70; (2) It has an amino acid sequence that is about 90% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70, and has the function of Factor D. [1-8] The reagent for measuring β-glucan according to any one of [1-1] to [1-6], wherein the horseshoe crab-derived factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33; (2) It has an amino acid sequence that is about 90% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33, and has the function of Factor D. [1-9] The reagent for measuring β-glucan according to any one of [1-1] to [1-8], wherein the horseshoe crab-derived factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70; (2') It has an amino acid sequence that is about 85% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70, and has the function of Factor D. [1-10] The reagent for measuring β-glucan according to any one of [1-1] to [1-8], wherein the horseshoe crab-derived factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33; (2') It has an amino acid sequence that is about 85% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33, and has the function of Factor D. [1-11] The reagent for measuring β-glucan according to any one of [1-1] to [1-10], wherein the horseshoe crab-derived factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70; (2') It has an amino acid sequence that is approximately 90% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70, and has the function of Factor D. [1-12] The reagent for measuring β-glucan according to any one of [1-1] to [1-10], wherein the horseshoe crab-derived factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33; (2') It has an amino acid sequence that is about 90% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33, and has the function of Factor D. [1-13] The reagent for measuring β-glucan according to any one of [1-1] to [1-12], wherein the horseshoe crab-derived factor D is derived from Tachypleus tridentatus, Limulus polyphemus, Carcinoscorpius rotundicauda, ​​or Tachypleus gigas. [1-14] The reagent for measuring β-glucan according to any one of [1-1] to [1-12], wherein the horseshoe crab-derived factor D is derived from Tachypleus tridentatus or Limulus polyphemus. [1-15] The reagent for measuring β-glucan according to any one of [1-1] to [1-14], wherein the measurement substrate is represented by the general formula YXZ: In the above general formula, Y is a hydrogen atom or a protecting group, X is a peptide containing the substrate sequence of the horseshoe crab-derived proclotting enzyme, and Z is a labeling substance. [1-16] The reagent for measuring β-glucan according to [1-15], wherein the labeling substance is an optical labeling substance. [1-17] The reagent for measuring β-glucan according to any one of [1-1] to [1-16], wherein the horseshoe crab-derived factor G is derived from Tachypleus tridentatus or Limulus polyphemus. [1-18] The reagent for measuring β-glucan according to any one of [1-1] to [1-17], wherein the horseshoe crab-derived proclotting enzyme is derived from Tachypleus tridentatus or Limulus polyphemus.

[0012] [2-1] A method for producing a reagent for measuring β-glucan, comprising: Artificial production of horseshoe crab-derived Factor D, a production method comprising assembling the artificially produced horseshoe crab-derived Factor D into a kit together with at least horseshoe crab-derived Factor G, horseshoe crab-derived Proclotting Enzyme, and a measurement substrate; [2-2] The production method according to [2-1], wherein the horseshoe crab-derived Factor D is artificially produced by recombinant production using a host cell. [2-3] The manufacturing method according to [2-1] or [2-2], wherein the detection limit of pachyman using the β-glucan measurement reagent is 20 pg / mL or less within a reaction time of 60 minutes. [2-4] The manufacturing method according to any one of [2-1] to [2-3], wherein the detection limit of pachyman using the β-glucan measurement reagent is 16 pg / mL or less within a reaction time of 40 minutes or less. [2-5] The method according to any one of [2-1] to [2-4], wherein the horseshoe crab-derived Factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70; (2) It has an amino acid sequence that is about 85% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70, and has the function of Factor D. [2-6] The method according to any one of [2-1] to [2-4], wherein the horseshoe crab-derived Factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33; (2) It has an amino acid sequence that is about 85% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33, and has the function of Factor D. [2-7] The method according to any one of [2-1] to [2-6], wherein the horseshoe crab-derived Factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70; (2) It has an amino acid sequence that is about 90% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70, and has the function of Factor D. [2-8] The method according to any one of [2-1] to [2-6], wherein the horseshoe crab-derived Factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33; (2) It has an amino acid sequence that is about 90% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33, and has the function of Factor D. [2-9] The method according to any one of [2-1] to [2-8], wherein the horseshoe crab-derived Factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70; (2') It has an amino acid sequence that is about 85% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70, and has the function of Factor D. [2-10] The method according to any one of [2-1] to [2-8], wherein the horseshoe crab-derived Factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33; (2') It has an amino acid sequence that is about 85% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33, and has the function of Factor D. [2-11] The method according to any one of [2-1] to [2-10], wherein the horseshoe crab-derived Factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70; (2') It has an amino acid sequence that is approximately 90% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70, and has the function of Factor D. [2-12] The method according to any one of [2-1] to [2-10], wherein the horseshoe crab-derived Factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33; (2') It has an amino acid sequence that is about 90% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33, and has the function of Factor D. [2-13] The method according to any one of [2-1] to [2-12], wherein the horseshoe crab-derived Factor D is derived from Tachypleus tridentatus, Limulus polyphemus, Carcinoscorpius rotundicauda, ​​or Tachypleus gigas. [2-14] The method according to any one of [2-1] to [2-12], wherein the horseshoe crab-derived Factor D is derived from Tachypleus tridentatus or Limulus polyphemus. [2-15] The method according to any one of [2-1] to [2-14], wherein the measurement substrate is represented by the general formula YXZ: In the above general formula, Y is a hydrogen atom or a protecting group, X is a peptide containing the substrate sequence of the horseshoe crab-derived proclotting enzyme, and Z is a labeling substance. [2-16] The manufacturing method according to [2-15], wherein the labeling substance is an optical labeling substance. [2-17] The method according to any one of [2-1] to [2-16], wherein the horseshoe crab-derived Factor G is derived from Tachypleus tridentatus or Limulus polyphemus. [2-18] The method according to any one of [2-1] to [2-17], wherein the horseshoe crab-derived proclotting enzyme is derived from Tachypleus tridentatus or Limulus polyphemus.

[0013] [3-1] A method for assaying β-glucan, comprising measuring β-glucan in a sample using a reagent for measuring β-glucan described in any one of [1-1] to [1-18] or a reagent for measuring β-glucan produced by the production method described in any one of [2-1] to [2-18]. [3-2] A method for assaying β-glucan, comprising measuring β-glucan in a sample using a reagent for measuring β-glucan described in any one of [1-1] to [1-13] or a reagent for measuring β-glucan produced by the production method described in any one of [2-1] to [2-13]. [3-3] A method for testing for mycosis, comprising: [3-1], A method wherein the sample is a biological sample derived from a subject suspected of suffering from a fungal disease. [3-4] A method for testing for mycosis, comprising: [3-2], A method wherein the sample is a biological sample derived from a subject suspected of suffering from a fungal disease. [3-5] A data acquisition method for diagnosing a fungal disease, comprising: [3-1] performing the β-glucan assay method described above to obtain data for diagnosing whether or not a subject is suffering from a fungal disease; The method, wherein the sample is a biological sample derived from the subject. [3-6] A data acquisition method for diagnosing a fungal disease, comprising: [3-2] performing the β-glucan assay method described above to obtain data for diagnosing whether or not a subject is suffering from a fungal disease; The method, wherein the sample is a biological sample derived from the subject.

[0014] [4-1] A method for enhancing the activity of a reagent for measuring β-glucan, the reagent comprising horseshoe crab-derived factor G, horseshoe crab-derived proclotting enzyme, and a measurement substrate, the method comprising: A method comprising allowing horseshoe crab-derived Factor D to coexist with the reagent for measuring β-glucan. [4-2] The method according to [4-1], wherein the horseshoe crab-derived Factor D is a recombinant protein. [4-3] The method described in [4-1] or [4-2], wherein the detection limit of pachyman using the activity-enhanced β-glucan measurement reagent is 20 pg / mL or less within a reaction time of 60 minutes. [4-4] A method according to any one of [4-1] to [4-3], wherein the detection limit of pachyman using the activity-enhanced β-glucan measurement reagent is 16 pg / mL or less within a reaction time of 40 minutes or less. [4-5] The method according to any one of [4-1] to [4-4], wherein the horseshoe crab-derived Factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70; (2) It has an amino acid sequence that is about 85% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70, and has the function of Factor D. [4-6] The method according to any one of [4-1] to [4-4], wherein the horseshoe crab-derived Factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33; (2) It has an amino acid sequence that is about 85% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33, and has the function of Factor D. [4-7] The method according to any one of [4-1] to [4-6], wherein the horseshoe crab-derived Factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70; (2) It has an amino acid sequence that is about 90% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70, and has the function of Factor D. [4-8] The method according to any one of [4-1] to [4-6], wherein the horseshoe crab-derived Factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33; (2) It has an amino acid sequence that is about 90% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33, and has the function of Factor D. [4-9] The method according to any one of [4-1] to [4-8], wherein the horseshoe crab-derived Factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70; (2') It has an amino acid sequence that is about 85% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70, and has the function of Factor D. [4-10] The method according to any one of [4-1] to [4-8], wherein the horseshoe crab-derived Factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33; (2') It has an amino acid sequence that is about 85% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33, and has the function of Factor D. [4-11] The method according to any one of [4-1] to [4-10], wherein the horseshoe crab-derived Factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70; (2') It has an amino acid sequence that is approximately 90% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70, and has the function of Factor D. [4-12] The method according to any one of [4-1] to [4-10], wherein the horseshoe crab-derived Factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33; (2') It has an amino acid sequence that is about 90% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33, and has the function of Factor D. [4-13] The method according to any one of [4-1] to [4-12], wherein the horseshoe crab-derived factor D is derived from Tachypleus tridentatus, Limulus polyphemus, Carcinoscorpius rotundicauda, ​​or Tachypleus gigas. [4-14] The method according to any one of [4-1] to [4-12], wherein the horseshoe crab-derived Factor D is derived from Tachypleus tridentatus or Limulus polyphemus. [4-15] The method according to any one of [4-1] to [4-14], wherein the measurement substrate is represented by the general formula YXZ: In the above general formula, Y is a hydrogen atom or a protecting group, X is a peptide containing the substrate sequence of the horseshoe crab-derived proclotting enzyme, and Z is a labeling substance. [4-16] The method according to [4-15], wherein the labeling substance is an optical labeling substance. [4-17] The method according to any one of [4-1] to [4-16], wherein the horseshoe crab-derived factor G is derived from Tachypleus tridentatus or Limulus polyphemus. [4-18] The method according to any one of [4-1] to [4-17], wherein the horseshoe crab-derived proclotting enzyme is derived from Tachypleus tridentatus or Limulus polyphemus. [5-1] A method for producing a raw material for a reagent for measuring β-glucan, comprising culturing mammalian cells transformed with DNA encoding both the α subunit and the β subunit of Factor G derived from Tachypleus tridentatus, and recovering the culture supernatant after culturing. [5-2] The production method according to [5-1], further comprising allowing the culture supernatant to contain a proclotting enzyme derived from Limulus polyphemus. [5-3] The method according to [5-1] or [5-2], wherein the mammalian cells are CHO cells, preferably CHO DG44 cells. [6-1] Culture supernatant of mammalian cells transformed with both DNA encoding the α and β subunits of Factor G derived from Tachypleus tridentatus. [6-2] The culture supernatant according to [6-1], further containing a proclotting enzyme derived from Limulus polyphemus. [6-3] The culture supernatant according to [6-1] or [6-2], wherein the mammalian cells are CHO cells, preferably CHO DG44 cells. [6-4] A raw material for a reagent for measuring β-glucan, comprising the culture supernatant according to any one of [6-1] to [6-3]. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the cascade reaction of horseshoe crab hemocyte extracts triggered by β-glucan and endotoxin. [Figure 2]FIG. 2 shows the results of β-glucan measurements in the presence and absence of Factor D. [Figure 3] FIG. 3 shows the results of β-glucan measurements in the presence and absence of recombinant Factor D. [Figure 4] FIG. 4 shows the results of β-glucan measurements in the presence and absence of recombinant Factor D. [Figure 5] FIG. 5 shows the results of β-glucan measurements in the presence and absence of recombinant Factor D. DETAILED DESCRIPTION OF THE INVENTION

[0016] According to the present invention, it is possible to provide a high-performance reagent for measuring β-glucan and raw materials useful for producing the reagent, even without requiring a horseshoe crab hemocyte extract. The present inventors have discovered a specific component that, when present during the reaction between β-glucan and a reagent for measuring the same, can improve the performance of the reagent. More specifically, they found that the responsiveness of a β-glucan measurement reagent containing horseshoe crab-derived Factor G, horseshoe crab-derived Proclotting Enzyme, and a measurement substrate to β-glucan can be improved by further adding horseshoe crab-derived Factor D when reacting the reagent with β-glucan. Horseshoe crab-derived Factor D is a protein first discovered by Kawabata et al. (FEBS Letters 398 (1996) 146-150) and is known to have the function of inhibiting the growth of Gram-negative bacteria. However, the relationship between horseshoe crab-derived Factor D and fungi or β-glucan is unknown.

[0017] Herein, the three proteins, horseshoe crab-derived factor D, horseshoe crab-derived factor G, and horseshoe crab-derived proclotting enzyme, may be referred to individually or collectively as "glucan-associated factors."

[0018] Furthermore, in this specification, horseshoe crab-derived Factor D may be referred to simply as "Factor D," horseshoe crab-derived Factor G may be referred to simply as "Factor G," horseshoe crab-derived Pro-clotting enzyme may be referred to simply as "Pro-clotting enzyme," horseshoe crab-derived Factor C may be referred to simply as "Factor C," and horseshoe crab-derived Factor B may be referred to simply as "Factor B." It is clear from the concept of the present invention and the entire description of this specification that the meaning of the terms "horseshoe crab-derived" and "derived from horseshoe crab" used in reference to proteins in this specification is not intended to limit the raw material from which the protein is recovered to horseshoe crab.

[0019] Also, references herein to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention, and indicate that not all embodiments necessarily include the particular feature, structure, or characteristic. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0020] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the following embodiments. <β-glucan measurement reagent> One aspect of the present invention relates to a reagent for measuring β-glucan, which contains horseshoe crab-derived Factor D. The reagent for measuring β-glucan is preferably a recombinant reagent containing horseshoe crab-derived Factor D (i.e., both Factor G and Proclotting Enzyme contained in the reagent for measuring β-glucan are recombinant proteins).

[0021] In one embodiment, a reagent for measuring β-glucan is provided, which comprises horseshoe crab-derived Factor D, horseshoe crab-derived Factor G, horseshoe crab-derived proclotting enzyme, and a measurement substrate, and does not contain horseshoe crab hemocyte extract.

[0022] In one embodiment, a reagent for measuring β-glucan is provided, comprising a horseshoe crab-derived Factor D recombinant protein, a horseshoe crab-derived Factor G recombinant protein, a horseshoe crab-derived Proclotting Enzyme recombinant protein, and a measurement substrate. In one embodiment, a reagent for measuring β-glucan is provided, comprising a measurement substrate, and containing only a horseshoe crab-derived Factor D recombinant protein, a horseshoe crab-derived Factor G recombinant protein, and a horseshoe crab-derived Proclotting Enzyme recombinant protein as glucan-related factors.

[0023] As used herein, the species of horseshoe crab from which the glucan-related factor is derived is not particularly limited. Four species of horseshoe crab are known: Tachypleus tridentatus, Limulus polyphemus, Carcinoscorpius rotundicauda, ​​and Tachypleus gigas. Among these horseshoe crabs, each glucan-related factor is preferably independently selected from the group consisting of Tachypleus tridentatus, Carcinoscorpius rotundicauda, ​​and Limulus polyphemus, and more preferably independently selected from the group consisting of Tachypleus tridentatus and Limulus polyphemus.

[0024] As used herein, "Factor D" is not limited to a polypeptide having the function of Factor D, and also includes variants of Factor D found in natural horseshoe crabs. The "function of Factor D" refers to the ability of a β-glucan assay reagent containing Factor G, proclotting enzyme, and a measurement substrate to improve the responsiveness of the reagent to β-glucan when the reagent is reacted with the polypeptide, compared to when the reagent is not coexistent. If the responsiveness of a certain polypeptide to β-glucan is improved when the polypeptide is coexistent with the β-glucan assay reagent compared to when the polypeptide is not coexistent, the polypeptide can be determined to have the "function of Factor D." A method for confirming improved responsiveness can be appropriately selected by those skilled in the art based on the assay technique employed. Specifically, improved responsiveness can be confirmed by an increase in the amount of released labeled substance in an endpoint assay or by an increase in the release rate of labeled substance in a kinetic assay. More specifically, improved responsiveness can be confirmed by the methods described in the Examples.

[0025] In one embodiment, Factor D is derived from Tachypleus tridentatus. In one embodiment, the amino acid sequence of Factor D derived from Tachypleus tridentatus is the sequence shown in SEQ ID NO: 1 (GenBank: BAA13312.1) in the Sequence Listing.

[0026] In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 2 in the sequence listing, which is SEQ ID NO: 1 with the signal sequence cleaved.

[0027] In one embodiment, the amino acid sequence of Factor D derived from Tachypleus tridentatus is the sequence shown in SEQ ID NO: 3 in the Sequence Listing.

[0028] In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 4 in the sequence listing, which is SEQ ID NO: 3 with the signal sequence cleaved.

[0029] In one embodiment, the amino acid sequence of Factor D derived from Tachypleus tridentatus is the sequence shown in SEQ ID NO: 5 in the sequence listing (SEQ ID NO: 3 with the N-terminal secretion signal changed and a His tag added to the C-terminus).

[0030] In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 6 in the sequence listing, which is SEQ ID NO: 5 with the signal sequence cleaved.

[0031] In one embodiment, Factor D is derived from Limulus polyphemus. In one embodiment, the amino acid sequence of Factor D derived from Limulus polyphemus is the sequence shown in SEQ ID NO: 7 in the Sequence Listing.

[0032] In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 8 in the sequence listing, which is SEQ ID NO: 7 with the signal sequence cleaved.

[0033] In one embodiment, the amino acid sequence of Factor D derived from Limulus polyphemus is the sequence shown in SEQ ID NO: 9 in the Sequence Listing.

[0034] In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 10 in the sequence listing, which is SEQ ID NO: 9 with the signal sequence cleaved.

[0035] In one embodiment, the amino acid sequence of Factor D derived from Limulus polyphemus is the sequence shown in SEQ ID NO: 11 in the sequence listing (SEQ ID NO: 9 with the N-terminal secretion signal changed and a His tag added to the C-terminus).

[0036] In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 12 in the sequence listing, which is SEQ ID NO: 11 with the signal sequence cleaved.

[0037] In one embodiment, the amino acid sequence of Factor D derived from Limulus polyphemus is the sequence shown in SEQ ID NO: 32 in the Sequence Listing (SEQ ID NO: 9 with the N-terminal secretion signal altered).

[0038] In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 33 in the sequence listing, which is SEQ ID NO: 32 with the signal sequence cleaved. In one embodiment, Factor D is derived from Carcinoscorpius rotundicauda. In one embodiment, the amino acid sequence of Factor D derived from Carcinoscorpius rotundicauda is the sequence shown in SEQ ID NO: 67 in the Sequence Listing (corresponding to the coding region of GenBank: GILQ01008045.1; SEQ ID NO: 71). In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 68 in the Sequence Listing, which is SEQ ID NO: 67 with the signal sequence cleaved. In one embodiment, Factor D is derived from Tachypleus gigas. In one embodiment, the amino acid sequence of Factor D derived from Tachypleus gigas is the sequence shown in SEQ ID NO: 69 in the Sequence Listing (corresponding to the coding region of GenBank: GILM01006809.1; SEQ ID NO: 72). In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 70 in the Sequence Listing, which is SEQ ID NO: 69 with the signal sequence cleaved.

[0039] As used herein, Factor D also includes proteins in which one or more amino acid residues have been substituted, deleted, inserted, and / or added in the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70. In one embodiment, Factor D is a protein in which one or more amino acid residues have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 33, 68, or 70. As used herein, the term "multiple" refers to the number (total number) of amino acid residues that can be substituted, deleted, inserted, and / or added without causing the protein to lose its Factor D function. The term "multiple" may refer, for example, to a number that is preferably about 15% or less, more preferably about 10% or less, even more preferably about 5% or less, and particularly preferably about 1% or less of the total number of amino acid residues constituting the protein. In the case of the amino acid sequences of the proteins shown in SEQ ID NOs: 1 to 12, "multiple" is preferably 2 to 55, more preferably 2 to 35, even more preferably 2 to 15, and particularly preferably 2 to 3. In this specification, the term "about" indicates a range of ±5% of the value that follows. For example, the expression "about 10%" in this specification is interpreted as "9.5% or more and 10.5% or less."

[0040] As used herein, "substitution, deletion, insertion, and / or addition" refers to, for example, conservative mutation. A typical example of a conservative mutation is conservative substitution. Conservative substitution refers to, for example, a mutual substitution between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid residue; between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid residue; between Gln and Asn when the substitution site is a polar amino acid; between Lys, Arg, and His when the substitution site is a basic amino acid residue; between Asp and Glu when the substitution site is an acidic amino acid residue; and between Ser and Thr when the substitution site is an amino acid residue having a hydroxyl group. Specific examples of substitutions that are considered to be conservative substitutions include substitutions of Ala with Ser or Thr, substitutions of Arg with Gln, His, or Lys, substitutions of Asn with Glu, Gln, Lys, His, or Asp, substitutions of Asp with Asn, Glu, or Gln, substitutions of Cys with Ser or Ala, substitutions of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitutions of Glu with Gly, Asn, Gln, Lys, or Asp, substitutions of Gly with Pro, substitutions of His with Asn, Lys, Gln, Arg, or Tyr, substitutions of Ile Examples include substitutions of Lys with Leu, Met, Val, or Phe, Leu with Ile, Met, Val, or Phe, Lys with Asn, Glu, Gln, His, or Arg, Met with Ile, Leu, Val, or Phe, Phe with Trp, Tyr, Met, Ile, or Leu, Ser with Thr or Ala, Thr with Ser or Ala, Trp with Phe or Tyr, Tyr with His, Phe, or Trp, and Val with Met, Ile, or Leu.

[0041] The results of evaluating the sequence identity between the amino acid sequences of Factor D shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68 and 70 are shown in Table 1 below.

[0042] [Table 1]

[0043] The amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12 and 33 all have an amino acid sequence similar to each other of about 85% or more (e.g., 84%). % or more ) identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 33, 68, and 70, and SEQ ID NO: 1, 3, 5, 7, 9, 11, 32, 67, and 69, which are sequences of these sequences with added signal sequences (i.e., at least one of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70), will function as Factor D.

[0044] In one embodiment, Factor D is a protein described in (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70; (2) It has an amino acid sequence that is preferably about 85% or more, more preferably about 90% or more, even more preferably about 95% or more, still more preferably about 98% or more, and particularly preferably about 99% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70, and has the function of Factor D. In one embodiment, Factor D is a protein described in (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33; (2) It has an amino acid sequence that is preferably about 85% or more, more preferably about 90% or more, even more preferably about 95% or more, still more preferably about 98% or more, and particularly preferably about 99% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, and 33, and has the function of Factor D.

[0045] In one embodiment, Factor D is a protein of the following (1') or (2'): (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70; (2') It has an amino acid sequence that is preferably about 85% or more, more preferably about 90% or more, even more preferably about 95% or more, still more preferably about 98% or more, and particularly preferably about 99% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70, and has the function of Factor D. In one embodiment, Factor D is a protein of the following (1') or (2'): (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33; (2') It has an amino acid sequence that is preferably about 85% or more, more preferably about 90% or more, even more preferably about 95% or more, still more preferably about 98% or more, and particularly preferably about 99% or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33, and has the function of Factor D.

[0046] In a preferred embodiment, Factor D is a protein selected from the group consisting of the following (1") to (10"): (1") comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70; (2) Having an amino acid sequence that is about 86% or more identical to the amino acid sequence shown in SEQ ID NO: 2 and has the function of Factor D; (3) Having an amino acid sequence that is about 86% or more identical to the amino acid sequence shown in SEQ ID NO: 4 and has the function of Factor D; (4) Having an amino acid sequence that is about 84% or more identical to the amino acid sequence shown in SEQ ID NO: 6 and has the function of Factor D; (5) Having an amino acid sequence that is about 84% or more identical to the amino acid sequence shown in SEQ ID NO: 8 and has the function of Factor D; (6) Having an amino acid sequence that is about 87% or more identical to the amino acid sequence set forth in SEQ ID NO: 10 and has the function of Factor D; (7) Having an amino acid sequence that is about 85% or more identical to the amino acid sequence shown in SEQ ID NO: 12 and has the function of Factor D; (8)"Has an amino acid sequence that is about 86% or more identical to the amino acid sequence shown in SEQ ID NO: 33 and has the function of Factor D. (9) Having an amino acid sequence that is about 84% or more identical to the amino acid sequence set forth in SEQ ID NO: 68 and has the function of Factor D; (10)" Has an amino acid sequence that is about 85% or more identical to the amino acid sequence shown in SEQ ID NO: 70, and has the function of Factor D. In a more preferred embodiment, Factor D is a protein selected from the group consisting of the following (1") to (8"): (1") comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33; (2) Having an amino acid sequence that is about 86% or more identical to the amino acid sequence shown in SEQ ID NO: 2 and has the function of Factor D; (3) Having an amino acid sequence that is about 86% or more identical to the amino acid sequence shown in SEQ ID NO: 4 and has the function of Factor D; (4) Having an amino acid sequence that is about 84% or more identical to the amino acid sequence shown in SEQ ID NO: 6 and has the function of Factor D; (5) Having an amino acid sequence that is about 84% or more identical to the amino acid sequence shown in SEQ ID NO: 8 and has the function of Factor D; (6) Having an amino acid sequence that is about 87% or more identical to the amino acid sequence set forth in SEQ ID NO: 10 and has the function of Factor D; (7) Having an amino acid sequence that is about 85% or more identical to the amino acid sequence shown in SEQ ID NO: 12 and has the function of Factor D; (8)"Has an amino acid sequence that is about 86% or more identical to the amino acid sequence shown in SEQ ID NO: 33 and has the function of Factor D.

[0047] Specific examples of the amino acid sequence of SEQ ID NO: 2 in which one amino acid residue has been substituted include the sequences shown in SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, or 42 in the Sequence Listing. In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 39, 40, 41, or 42 in the Sequence Listing.

[0048] Specific examples of the amino acid sequence of SEQ ID NO: 4 in which one amino acid residue has been substituted include the sequences shown in SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, or 50 in the Sequence Listing. In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 47, 48, 49, or 50 in the Sequence Listing.

[0049] Specific examples of the amino acid sequence of SEQ ID NO: 8 in which one amino acid residue has been substituted include the sequences shown in SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, or 58 in the Sequence Listing. In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 55, 56, 57, or 58 in the Sequence Listing.

[0050] Specific examples of the amino acid sequence of SEQ ID NO: 10 in which one amino acid residue has been substituted include the sequences shown in SEQ ID NO: 59, 60, 61, 62, 63, 64, 65, or 66 in the Sequence Listing. In one embodiment, the amino acid sequence of Factor D is the sequence shown in SEQ ID NO: 63, 64, 65, or 66 in the Sequence Listing.

[0051] In one embodiment, Factor D is a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, and 35 to 66.

[0052] In one embodiment, Factor D is a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 39 to 42, 47 to 50, 55 to 58, and 63 to 66.

[0053] In one embodiment, Factor D is a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, and 47-50.

[0054] In one embodiment, Factor D is a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 33.

[0055] Amino acid sequence identity can be calculated using well-known computer software, for example, by aligning sequences using the BLAST algorithm (Karlin, S., Altschul, S. F. (1993) Proc. Natl. Acad. Sci. USA 90, 5873-7). Specifically, amino acid sequence identity can be calculated using, for example, GENETYX (Genetyx).

[0056] In one embodiment, a reagent for measuring β-glucan is provided, wherein Factor D is derived from Tachypleus tridentatus, Limulus polyphemus, Carcinoscorpius rotundicauda, ​​or Tachypleus gigas. In a preferred embodiment, a reagent for measuring β-glucan is provided, wherein Factor D is derived from Tachypleus tridentatus or Limulus polyphemus.

[0057] Natural Factor D does not have the serine residue required for the active center of a serine protease, and therefore does not have serine protease activity like Factor G or proclotting enzyme. While not limiting the scope of the present invention, if Factor D is made to have serine protease activity by introducing a mutation into a serine residue, etc., it may result in the degradation of the protein's molecular weight due to autolysis or degradation of other contaminating proteins during the purification process. The relationship between the responsiveness of a reagent to β-glucan and the presence or absence of serine protease activity of Factor D is unknown. However, from the viewpoint of preventing degradation of the molecular weight of materials used in a β-glucan assay reagent, it is preferable to use a Factor D that does not have serine protease activity in the present invention. The absence of serine protease activity of Factor D can be confirmed by electrophoretically separating Factor D under reducing and non-reducing conditions and observing that the molecular weight of Factor D detected under reducing conditions is not reduced compared to the molecular weight of Factor D detected under non-reducing conditions.

[0058] The positions of the serine residues that form the active center of the serine protease are: position 335 in the amino acid sequence of SEQ ID NO: 1, position 317 in the amino acid sequence of SEQ ID NO: 2, position 335 in the amino acid sequence of SEQ ID NO: 3, position 317 in the amino acid sequence of SEQ ID NO: 4, position 348 in the amino acid sequence of SEQ ID NO: 5, position 320 in the amino acid sequence of SEQ ID NO: 6, position 335 in the amino acid sequence of SEQ ID NO: 7, position 317 in the amino acid sequence of SEQ ID NO: 8, position 337 in the amino acid sequence of SEQ ID NO: 9, position 319 in the amino acid sequence of SEQ ID NO: 10, position 350 in the amino acid sequence of SEQ ID NO: 11, position 322 in the amino acid sequence of SEQ ID NO: 12, position 350 in the amino acid sequence of SEQ ID NO: 32, position 322 in the amino acid sequence of SEQ ID NO: 33, position 335 in the amino acid sequence of SEQ ID NO: 67, position 318 in the amino acid sequence of SEQ ID NO: 68, position 334 in the amino acid sequence of SEQ ID NO: 69, and position 316 in the amino acid sequence of SEQ ID NO: 70. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 1, the amino acid residue at position 335 in the amino acid sequence of SEQ ID NO: 1 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2, the amino acid residue at position 317 in the amino acid sequence of SEQ ID NO: 2 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 3, the amino acid residue at position 335 in the amino acid sequence of SEQ ID NO: 3 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 4, the amino acid residue at position 317 in the amino acid sequence of SEQ ID NO: 4 is preferably an amino acid residue other than serine.In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 5, the amino acid residue at position 348 in the amino acid sequence of SEQ ID NO: 5 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 6, the amino acid residue at position 320 in the amino acid sequence of SEQ ID NO: 6 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 7, the amino acid residue at position 335 in the amino acid sequence of SEQ ID NO: 7 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 8, the amino acid residue at position 317 in the amino acid sequence of SEQ ID NO: 8 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 9, the amino acid residue at position 337 in the amino acid sequence of SEQ ID NO: 9 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 10, the amino acid residue at position 319 in the amino acid sequence of SEQ ID NO: 10 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 11, the amino acid residue at position 350 in the amino acid sequence of SEQ ID NO: 11 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 12, the amino acid residue at position 322 in the amino acid sequence of SEQ ID NO: 12 is preferably an amino acid residue other than serine.In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 32, the amino acid residue at position 350 in the amino acid sequence of SEQ ID NO: 32 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 33, the amino acid residue at position 322 in the amino acid sequence of SEQ ID NO: 33 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 67, the amino acid residue at position 335 in the amino acid sequence of SEQ ID NO: 67 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 68, the amino acid residue at position 318 in the amino acid sequence of SEQ ID NO: 68 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 69, the amino acid residue at position 334 in the amino acid sequence of SEQ ID NO: 69 is preferably an amino acid residue other than serine. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 70, the amino acid residue at position 316 in the amino acid sequence of SEQ ID NO: 70 is preferably an amino acid residue other than serine.

[0059] In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 1, it is more preferred that the amino acid residue at position 335 in the amino acid sequence of SEQ ID NO: 1 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2, it is more preferred that the amino acid residue at position 317 in the amino acid sequence of SEQ ID NO: 2 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 3, it is more preferred that the amino acid residue at position 335 in the amino acid sequence of SEQ ID NO: 3 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 4, it is more preferred that the amino acid residue at position 317 in the amino acid sequence of SEQ ID NO: 4 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 5, it is more preferred that the amino acid residue at position 348 in the amino acid sequence of SEQ ID NO: 5 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 6, it is more preferred that the amino acid residue at position 320 in the amino acid sequence of SEQ ID NO: 6 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 7, it is more preferred that the amino acid residue at position 335 in the amino acid sequence of SEQ ID NO: 7 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 8, it is more preferred that the amino acid residue at position 317 in the amino acid sequence of SEQ ID NO: 8 is a glycine residue. It is more preferable that in a protein in which one or more amino acid residues in the amino acid sequence of SEQ ID NO: 9 have been substituted, deleted, inserted, and / or added, the 337th amino acid residue in the amino acid sequence of SEQ ID NO: 9 is a glycine residue.In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 10, it is more preferable that the amino acid residue at position 319 in the amino acid sequence of SEQ ID NO: 10 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 11, it is more preferable that the amino acid residue at position 350 in the amino acid sequence of SEQ ID NO: 11 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 12, it is more preferable that the amino acid residue at position 322 in the amino acid sequence of SEQ ID NO: 12 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 32, it is more preferable that the amino acid residue at position 350 in the amino acid sequence of SEQ ID NO: 32 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 33, it is more preferred that the amino acid residue at position 322 in the amino acid sequence of SEQ ID NO: 33 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 67, it is more preferred that the amino acid residue at position 335 in the amino acid sequence of SEQ ID NO: 67 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 68, it is more preferred that the amino acid residue at position 318 in the amino acid sequence of SEQ ID NO: 68 is a glycine residue. In a protein having one or more amino acid residues substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 69, it is more preferred that the amino acid residue at position 334 in the amino acid sequence of SEQ ID NO: 69 is a glycine residue. It is more preferable that in a protein in which one or more amino acid residues in the amino acid sequence of SEQ ID NO: 70 have been substituted, deleted, inserted, and / or added, the 316th amino acid residue in the amino acid sequence of SEQ ID NO: 70 is a glycine residue.

[0060] In the β-glucan measurement reagent, Factor G is activated in the presence of β-glucan to activate Proclotting enzyme. There are no particular limitations on the type of Factor G, as long as it has the function of Factor G. Generally, Factor G is a heterodimer of an α subunit capable of binding to β-glucan and a β subunit with serine protease activity. The function of Factor G is to cleave Proclotting enzyme in the presence of β-glucan. The function of Factor G for a given protein can be confirmed by combining the protein with functional Factor D, Proclotting enzyme, and a measurement substrate and detecting the progress of the cascade reaction in the presence of β-glucan.

[0061] The amino acid sequences of the α subunit (SEQ ID NO: 13, GenBank: BAA04044.1) and β subunit (SEQ ID NO: 14, GenBank: BAA04045.1) of Factor G derived from Tachypleus tridentatus, and the α subunit (SEQ ID NOs: 15 to 17) and β subunit (SEQ ID NOs: 18 to 23) of Factor G derived from Limulus polyphemus are shown in the Sequence Listing.

[0062] Preferably, Factor G is a protein selected from the group consisting of the following (3) to (5): (3) consisting of an α subunit containing the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence obtained by cleaving the signal sequence from the amino acid sequence of SEQ ID NO: 13, and a β subunit containing the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence obtained by cleaving the signal sequence from the amino acid sequence of SEQ ID NO: 14; (4) An α subunit having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 17, or any one amino acid sequence among the amino acid sequences of SEQ ID NOs: 15 to 17 from which the signal sequence has been cleaved, and a β subunit having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 23, or any one amino acid sequence among the amino acid sequences of SEQ ID NOs: 18 to 23 from which the signal sequence has been cleaved; (5) In (3) or (4), the amino acid sequence of at least one of the α subunit and the β subunit has one or more amino acid residues substituted, deleted, inserted, and / or added, and has the function of Factor G.

[0063] In the case of the amino acid sequence shown in SEQ ID NO: 13 or any of SEQ ID NOs: 15 to 17, "plurality" is preferably 2 to 95, more preferably 2 to 60, even more preferably 2 to 30, and particularly preferably 2 to 5. In the case of the amino acid sequence shown in SEQ ID NO: 14 or any of SEQ ID NOs: 18 to 23, "plurality" is preferably 2 to 40, more preferably 2 to 25, even more preferably 2 to 12, and particularly preferably 2.

[0064] In the β-glucan measurement reagent, the pro-clotting enzyme is activated by activated Factor G and cleaves the measurement substrate. The pro-clotting enzyme is not limited as long as it has the function of a pro-clotting enzyme. The function of the pro-clotting enzyme is to cleave the measurement substrate in the presence of activated Factor G. The function of the pro-clotting enzyme for a certain protein can be confirmed by combining the protein with functional Factor D, Factor G, and a measurement substrate and detecting the progress of the cascade reaction in the presence of β-glucan.

[0065] The amino acid sequences of the proclotting enzyme derived from Tachypleus tridentatus (SEQ ID NO: 24, GenBank: AAA30094.1) and the proclotting enzyme derived from Limulus polyphemus (SEQ ID NO: 25, NCBI Reference Sequence: XP_013783518.1) are shown in the sequence listing.

[0066] Preferably, the pro-clotting enzyme is a protein selected from the group consisting of the following (6) to (8): (6) Contains the amino acid sequence of SEQ ID NO: 24, or the amino acid sequence of SEQ ID NO: 24 from which the signal sequence has been cleaved; (7) The amino acid sequence of SEQ ID NO: 25 or the amino acid sequence of SEQ ID NO: 25 from which the signal sequence has been cleaved; (8) In (6) or (7), one or more amino acid residues are substituted, deleted, inserted, and / or added to the amino acid sequence, and the protein has the function of proclotting enzyme.

[0067] In the case of the amino acid sequence shown in SEQ ID NO: 24 or 25, "multiple" is preferably 2 to 50, more preferably 2 to 30, even more preferably 2 to 15, and particularly preferably 2 to 3.

[0068] The β-glucan measurement reagent may contain an artificially produced glucan-related factor. The method for artificially producing a glucan-related factor is not particularly limited, and examples include recombinant production, production using a peptide synthesizer, and production by isolation from a horseshoe crab hemocyte extract. In a preferred embodiment, each glucan-related factor is a recombinant protein. When an artificially produced glucan-related factor is used in the β-glucan measurement reagent, the amount used in the measurement system may be determined with reference to, for example, a known β-glucan measurement reagent using a horseshoe crab hemocyte extract.

[0069] The glucan-related factor may have an additional amino acid sequence, such as a signal sequence or a tag, at the terminal portion of the polypeptide, as per conventional methods. The signal sequence can be appropriately selected depending on the type of host cell used for recombinant production. Examples of tags include His tag, FLAG tag, c-myc tag, protein A tag, MBP tag, GST tag, V5 tag, SUMO tag, and PA tag.

[0070] The cascade that activates proclotting enzyme in hemocyte extracts involves not only a pathway triggered by β-glucan and involving Factor G, but also pathways triggered by endotoxin and involving Factor C and Factor B (Figure 1). Therefore, to prevent false positives due to the presence of endotoxin, it is preferable that the β-glucan assay reagent does not contain horseshoe crab hemocyte extract. The absence of a β-glucan assay reagent can be confirmed, for example, by the absence of a significant enhancement of the cascade reaction (enhancement of the cascade reaction relative to an endotoxin-free control sample) when endotoxin is applied to the β-glucan assay reagent. Alternatively, the absence of a β-glucan assay reagent can be confirmed, for example, by the absence of polynucleotides derived from the genomic DNA of horseshoe crab hemocytes in the β-glucan assay reagent. The presence of polynucleotides derived from genomic DNA can be analyzed by methods well known to those skilled in the art, such as amplifying polynucleotides contained in a β-glucan assay reagent by PCR and sequence-analyzing the resulting amplified fragments. Recombinant production of glucan-related factors using insect cells, mammalian cells, or other hosts, or production using a peptide synthesizer is preferred because it allows the preparation of glucan-related factors without using horseshoe crab hemocyte extract. Incidentally, the coexistence of an endotoxin assay reagent containing Factor C with Factor D does not result in any improvement in reagent performance against endotoxin.

[0071] Recombinant proteins of glucan-related factors can be obtained, for example, by transforming host cells with a gene encoding the amino acid sequence of each glucan-related factor and expressing the glucan-related factor. The nucleotide sequence of the gene encoding the glucan-related factor may be obtained from a known database such as NCBI (www.ncbi.nlm.nih.gov). The nucleotide sequence of the gene encoding the glucan-related factor may be a nucleic acid variant in which the codon combination is optimized for expression in the host cell.

[0072] In one embodiment, the nucleotide sequence of the gene encoding Factor D is selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 26 to 31, 34, 71, and 72 in the sequence listing. In one embodiment, the nucleotide sequence of the gene encoding Factor D is selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 26 to 31 and 34 in the sequence listing.

[0073] The genes encoding the amino acid sequences of the glucan-related factors are operably linked to a vector by known genetic engineering techniques. For example, any two restriction enzyme sites present in the multicloning site of the vector can be selected, and the vector and the nucleic acid of the present invention can be subjected to limited digestion with these restriction enzymes, followed by ligation, thereby operably linking the foreign gene to the vector. Here, "operably linked" with respect to a gene refers to a state in which the gene is bound to a promoter so as to be transcribed from the promoter.

[0074] The vector can be selected appropriately depending on the type of host cell into which it is to be introduced. As used herein, "host cell" does not include horseshoe crab cells. For example, when eukaryotic cells such as insect cells or mammalian cells are used as hosts, examples of vectors include plasmids such as pIZ-V5 (Life Technologies), pCA7 (Takeda, M., et al. (2005) J. Virol. 79, 14346-54), and pCI-neo (Promega).

[0075] As the host cells, insect cells (for example, Sf9 cells, etc.) and mammalian cells (for example, HEK293 cells, CHO cells, etc.) can be used. Preferably, mammalian cells are used as the host.

[0076] Transformation of host cells with an expression plasmid can be carried out by a conventional method, such as the calcium phosphate method, lipofection, DEAE-dextran method, electroporation, or microinjection. Host cells can be cultured under conditions commonly used for culturing such cells.

[0077] The expressed glucan-related factors can be recovered by known methods for protein extraction and purification. For example, when the glucan-related factors are produced in a soluble form secreted into the culture supernatant, the culture supernatant can be recovered and used as a reagent as is. Furthermore, when the glucan-related factors are produced in a soluble form secreted into the cytoplasm or in an insoluble form, the glucan-related factors can be extracted by, for example, homogenization, bead milling, sonication, osmotic shock, freeze-thawing, extraction using a surfactant, or a combination thereof. From the viewpoint of production efficiency, it is preferable to recover the cell culture supernatant containing the expressed glucan-related factors. The glucan-related factors can be purified by conventionally known means such as salting out, ammonium sulfate fractionation, centrifugation, dialysis, ultrafiltration, various chromatographies, and combinations thereof.

[0078] In the β-glucan assay reagent, the assay substrate is cleaved by activated proclotting enzyme in the presence of β-glucan and is used to detect the progress of the cascade reaction. The assay substrate is a compound containing a substrate for activated proclotting enzyme (i.e., clotting enzyme). The assay substrate is not particularly limited as long as it serves as a substrate for proclotting enzyme. The assay substrate may be, for example, a protein, peptide, or a derivative thereof. An example of a protein used as the assay substrate is coagulogen, a substrate for clotting enzyme. Coagulogen can be prepared, for example, by isolating it from a blood cell extract. Alternatively, recombinant coagulogen can be prepared, for example, by referring to the method described in Miyata et al., Protein, Nucleic Acid, Enzyme, Special Issue No. 29, pp. 30-43, 1986. The assay substrate may be, for example, a chemically synthesized substance (synthetic substrate). The synthetic substrate is not particularly limited as long as it is a suitable substrate for measuring the progress of the cascade reaction. Preferably, the synthetic substrate is a derivative of a peptide.

[0079] In one embodiment, a reagent for measuring β-glucan is provided, wherein the measurement substrate is represented by the general formula YXZ; in this formula, Y is a hydrogen atom or a protecting group; X is a peptide containing the substrate sequence of horseshoe crab-derived proclotting enzyme; and Z is a labeling substance. Such a synthetic substrate may have the property that the covalent bond between X and Z is cleaved by activated proclotting enzyme, liberating the labeling substance Z. In the general formula YXZ, Y is preferably a protecting group for the N-terminal amino group of the peptide. In the general formula YXZ, the bond between Y and X is preferably an amide bond formed between the carboxy group of the protecting group and the α-amino group at the N-terminus of the peptide. Furthermore, in the general formula YXZ, the bond between X and Z is preferably an amide bond formed between the carboxy group at the C-terminus of the peptide and the amino group of the labeling substance Z.

[0080] The protecting group used as Y is not particularly limited, and any known protecting group applicable to the protection of peptides can be used. Examples of the protecting group include tert-butoxycarbonyl group (Boc), benzyloxycarbonyl group (Cbz), benzyl group (Bzl), benzoyl group (Bz), and acetyl group (Ac).

[0081] Peptide (X) is not particularly limited as long as it has an amino acid sequence that serves as a substrate for activated proclotting enzyme. The peptide is preferably a substrate suitable for measuring serine proteases, more preferably a peptide having an Arg(R) residue or a Lys(K) residue at the C-terminus, and even more preferably a peptide having an Arg(R) residue at the C-terminus. Examples of peptides having an Arg(R) residue at the C-terminus include Leu-Gly-Arg (LGR), Glu-Gly-Arg (EGR), Ile-Glu-Ala-Arg (IEAR), and Ile-Glu-Gly-Arg (IEGR). Among these, peptides containing the amino acid sequence A1-Gly-Arg (where A1 is any amino acid residue) are more preferred. Examples of peptides having a Lys(K) residue at the C-terminus include Ile-Glu-Gly-Lys (IEGK).

[0082] The labeling substance (Z) is not particularly limited, and any known labeling substance applicable to measuring protease activity can be used. Examples of labeling substances that can be used include compounds (optical labels) that can be optically detected (e.g., detected by color development, fluorescence, luminescence, etc.) when released from the peptide. Examples of such labeling substances include paranitroaniline (pNA), 7-methoxycoumarin-4-acetic acid, 2,4-dinitroaniline (DNP), 7-amino-4-methylcoumarin (AMC), 7-amino-4-trifluoromethylcoumarin, and luciferin. Furthermore, examples of labeling substances that can be used include compounds that can be detected by electrochemical measurement (e.g., voltammetry, amperometry, etc.) when released from the peptide. Examples of such labeling substances include p-aminophenol (pAP), p-methoxyaniline (pMA), N-methyl-p-phenylenediamine (MPDD), and N,N'-dimethyl-p-phenylenediamine (DMPD). In one preferred embodiment, a reagent for measuring β-glucan is provided, in which the labeling substance is an optical labeling substance.

[0083] The β-glucan to be measured by the β-glucan measurement reagent has a (1→3)-β-D-polyglucoside structure, and may be a straight-chain (1→3)-β-D-glucan or a branched (1→3)-β-D-glucan having an inner chain of the molecule, such as (1→6)-β-D- or (1→4)-β-D-. In one preferred embodiment, a reagent for measuring β-glucan is provided, which is used as a standard for (1→3)-β-D-glucan, and has a detection limit of 20 pg / mL or less for a reaction time of 60 minutes or less. The detection limit of β-glucan within a reaction time of 60 minutes or less is more preferably 16 pg / mL or less, even more preferably 5 pg / mL or less, and particularly preferably 1 pg / mL or less. In another embodiment, a reagent for measuring β-glucan is provided, which has a detection limit of β-glucan within a reaction time of 40 minutes or less, preferably 20 pg / mL or less, more preferably 16 pg / mL or less, even more preferably 5 pg / mL or less, and particularly preferably 1 pg / mL or less. The "reaction time" refers to the time from contacting Factor G, which is the most upstream component of the cascade reaction, with the sample to obtaining the final measured value. The reaction is usually carried out at 37°C. Pachyman can be, for example, a commercially available reagent. Pachyman is preferably a β-glucan standard. Examples of such a substance include β-glucan standard (Pachyman) (Associates of Cape Cod, Nissui Pharmaceutical Co., Ltd., etc.).

[0084] The β-glucan measurement reagent may contain optional additives such as a buffer in addition to the glucan-related factor and the measurement substrate. The β-glucan measurement reagent may contain each glucan-related factor and the measurement substrate in separate containers, or any combination of them may be contained in the same container. The dosage form of the β-glucan measurement reagent is not particularly limited and may be, for example, a solution or a lyophilized form.

[0085] In one embodiment, a method for enhancing the activity of a β-glucan assay reagent is provided, the method comprising allowing horseshoe crab-derived Factor D to coexist with the β-glucan assay reagent. In this method, horseshoe crab-derived Factor D may be allowed to coexist with the β-glucan assay reagent before reacting the sample with the β-glucan assay reagent. Alternatively, horseshoe crab-derived Factor D may be added after initiating the reaction between the sample and the β-glucan assay reagent. In this method, the β-glucan assay reagent with which Factor D is allowed to coexist may or may not contain a horseshoe crab hemocyte extract. The method for measuring β-glucan and the amount of Factor D used are described below regarding the application of the β-glucan assay reagent.

[0086] <Method for manufacturing β-glucan measurement reagent> One embodiment of the present invention relates to a method for producing a reagent for measuring β-glucan, which comprises artificially producing horseshoe crab-derived Factor D.

[0087] In one embodiment, there is provided a method for producing a reagent for measuring β-glucan, the method comprising artificially producing horseshoe crab-derived Factor D and assembling the produced horseshoe crab-derived Factor D into a kit together with at least horseshoe crab-derived Factor G, horseshoe crab-derived Proclotting Enzyme, and a measurement substrate. A "kit" refers to a collection containing multiple components.

[0088] The method for artificially producing horseshoe crab-derived Factor D is not particularly limited, and examples thereof include the recombinant production described above, production using a peptide synthesizer, and production by isolation from a horseshoe crab hemocyte extract. In one preferred embodiment from the viewpoint of production efficiency, a method for producing a reagent for measuring β-glucan is provided, in which the artificial production of horseshoe crab-derived Factor D is recombinant production using host cells.

[0089] The same descriptions as for the β-glucan measurement reagents apply to the horseshoe crab-derived Factor D, horseshoe crab-derived Factor G, horseshoe crab-derived proclotting enzyme, and measurement substrate used in the kit.

[0090] Since Factor G is composed of two subunits, α and β, when producing Factor G by recombinant DNA technology, it is preferable, from the viewpoint of production efficiency, to use a single host cell transformed to express both subunits and to use the resulting culture supernatant as a raw material for a β-glucan assay reagent. The present inventors discovered a new problem: when mammalian cells are used as hosts, even when transformed with both genes encoding the α and β subunits of Factor G derived from Limulus polyphemus, the β subunit is not secreted into the culture supernatant. Surprisingly, when these genes are derived from Tachypleus tridentatus, both the α and β subunits are secreted into the culture supernatant. This culture supernatant can be used as a raw material for the aforementioned β-glucan assay reagent. Therefore, one aspect of the present invention relates to an efficient method for producing recombinant Factor G using mammalian cells. In one embodiment, the present invention relates to a method for producing a raw material for a reagent for measuring β-glucan, comprising (1) culturing mammalian cells transformed with DNAs encoding both the α subunit and the β subunit of Tachypleus tridentatus Factor G and recovering the culture supernatant after the culturing. For the production of Tachypleus tridentatus Factor G, CHO cells, particularly CHO DG44 cells, are preferred among mammalian cells. In one embodiment, the method comprises culturing mammalian cells transformed with a vector incorporating DNAs encoding both the α subunit and the β subunit of Tachypleus tridentatus Factor G. In a preferred embodiment, the method further comprises allowing Limulus polyphemus proclotting enzyme to coexist in the culture supernatant. In another embodiment, a culture supernatant of mammalian cells transformed with DNAs encoding both the α subunit and the β subunit of Tachypleus tridentatus Factor G is provided. In a preferred embodiment, a raw material for a reagent for measuring β-glucan is provided, comprising the culture supernatant. In a preferred embodiment, the raw material for the β-glucan measurement reagent further contains Limulus polyphemus-derived proclotting enzyme.The "culture supernatant" in these embodiments contains Factor G (both the α and β subunits). The β-glucan measurement reagent may be packaged as a kit together with a dissolving solution, distilled water, a β-glucan standard, a microplate, a package insert containing product information, and the like.

[0091] <Applications of β-glucan measurement reagents> One embodiment of the present invention relates to the use of a reagent for measuring β-glucan, which contains horseshoe crab-derived factor D.

[0092] In one embodiment, a β-glucan assay method is provided, comprising measuring β-glucan in a sample using the above-mentioned β-glucan assay reagent or a β-glucan assay reagent produced by the above-mentioned method for producing a β-glucan assay reagent. The assay method includes quantitative or qualitative measurement of the presence or amount of β-glucan in a sample. The β-glucan assay method can be performed based on a conventional β-glucan assay method, except for using the above-mentioned β-glucan assay reagent or a β-glucan assay reagent produced by the above-mentioned method for producing a β-glucan assay reagent. Assay conditions are not limited, but for example, the pH of the reaction solution can be preferably 6.5 to 8.5. The reaction temperature can be, for example, 30°C to 40°C, preferably 37°C. The reaction time is also not particularly limited, but can be, for example, 5 minutes to 2 hours, preferably 30 to 60 minutes. The amount of each glucan-related factor used in the β-glucan assay reagent can be adjusted appropriately depending on the level of activity.

[0093] In the assay method, the glucan-related factors, measurement substrates, samples, and, if used, other substances (such as buffers) can be mixed in any order. The sample is not particularly limited, and examples include biological samples such as blood, body fluids, and tissues; consumer goods such as water for injection, pharmaceuticals, infusions, medical devices, quasi-drugs, cosmetics, foods, and reagents; environmental samples such as water and soil; and compounds such as proteins and nucleic acids. The sample is preferably a biological sample, and more preferably blood.

[0094] β-glucan in a sample can be measured by turbidimetry, for example, when coagulogen is used as the measurement substrate.

[0095] When a synthetic substrate containing a labeled substance is used as the measurement substrate, β-glucan in a sample can be measured using the labeled substance released from the measurement substrate as an indicator. During measurement using a synthetic substrate, an amount (molar number) of labeled substance corresponding to the protease activity (total activity) of proclotting enzyme activated by β-glucan is released from the measurement substrate. Depending on the type of labeled substance, the labeled substance released from the measurement substrate can be measured using, for example, optical analytical instruments such as spectrophotometers and fluorometers, or electrochemical measuring instruments such as voltammetry meters and amperometry meters. For example, β-glucan in a sample can be measured by comparing the measured value obtained by measuring the sample with a blank value (measurement value of a β-glucan-free control sample). In the assay method, the cascade reaction is preferably carried out in an aqueous solvent.

[0096] The assay method may further include any other optional steps. The assay method may include, for example, a step of converting the obtained measurement value into another value. An example of a step of converting the measurement value into another value is a step of calculating the amount of β-glucan based on the measurement value. Specifically, such a step is, for example, a step of converting the measurement value obtained when measuring a sample into the amount of β-glucan based on the relationship between the measurement value obtained when measuring a sample and a standard substance of known concentration (standard curve) and the concentration of the standard substance.

[0097] In one embodiment, a method for testing for a fungal disease is provided, which comprises performing the above-described β-glucan assay method using a biological sample derived from a subject suspected of having a fungal disease.

[0098] In one embodiment, a data acquisition method for diagnosing a fungal disease is provided, which includes performing the above-mentioned β-glucan assay method using a biological sample derived from a subject, thereby obtaining data for diagnosing whether the subject is suffering from a fungal disease.

[0099] As used herein, the subject includes humans and non-human animals (eg, dogs, cats, rabbits, rats, mice, etc.), but is preferably a human.

[0100] If the above-described β-glucan assay method detects the presence of β-glucan in a sample or the amount of β-glucan contained in the sample exceeds a predetermined cutoff value, the subject can be diagnosed as having or suspected of having a fungal infection. If the presence of β-glucan is not detected in the sample or the amount of β-glucan contained in the sample is equal to or less than the predetermined cutoff value, the subject can be diagnosed as not having a fungal infection or as being unlikely to have a fungal infection (e.g., follow-up observation is recommended). [Example]

[0101] Hereinafter, preferred embodiments of the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to the following examples.

[0102] (Test Example 1) Following the method of Muta et al. (The Journal of Biological Chemistry 270 (1995) 892-897), a fraction containing factor G (G fraction, not containing factor D) was collected from the hemocyte extract of Tachypleus tridentatus.

[0103] A fraction containing Factor D (SEQ ID NO: 2) (Fraction D) was prepared from the hemocyte extract of Tachypleus tridentatus, following the method described by Kawabata et al. (FEBS Letters 398 (1996) 146-150). The D fraction was then subjected to SDS-PAGE and stained with CBB. A band corresponding to Factor D (45 kDa) was detected, whereas bands corresponding to the α and β subunits of Factor G were not. To be sure, the N-terminal amino acid sequence of this 45 kDa protein (Factor D) was confirmed using a protein sequencer. The N-terminal pyroglutamic acid of the 45 kDa protein was deblocked by digestion with Pfu Pyroglutamate Aminopeptidase (Takara Bio Inc.). A BLAST search of the determined amino acid sequence confirmed that it matched the amino acid sequence of Factor D from Tachypleus tridentatus (SEQ ID NO: 1; GenBank: BAA13312.1). Furthermore, the N-terminus of Factor D from which the signal sequence had been removed was glutamic acid, which was consistent with the fact that the N-terminus of the 45 kDa protein was blocked. Furthermore, the amino acid sequence determined by protease digestion of the internal sequence of the 45 kDa protein also matched the reported sequence of Factor D.

[0104] β-glucan assay reagents were prepared using the G fraction, D fraction, and a mixture of the G and D fractions (G+D fraction). Specifically, each fraction was used together with Tachypleus tridentatus proclotting enzyme (a recombinant protein produced in insect cells according to the example in WO 2012 / 118226) and a synthetic substrate (Boc-LGR-pNA). This reagent was mixed with a sample containing β-glucan (Pachyman; Associates of Cape Cod) (final β-glucan concentration: 90 pg / mL). The reaction was carried out according to the method described in the example in Patent Document 3 (except that the reaction was carried out at 37°C for 60 minutes), and the absorbance (405 nm) was measured.

[0105] The measured absorbance (mAbs) was divided by the time (minutes) to calculate the activity of the proclotting enzyme. The results are shown in Figure 2. In the β-glucan measurement reagent using the G fraction, the activity of the proclotting enzyme was significantly lower. In addition, in the β-glucan measurement reagent using the D fraction, no progress of the cascade reaction was observed. On the other hand, in the β-glucan measurement reagent using the G+D fraction, significant activation of the proclotting enzyme was confirmed.

[0106] From the above, it was confirmed that the addition of horseshoe crab-derived Factor D to a β-glucan measurement reagent containing horseshoe crab-derived Factor G, horseshoe crab-derived proclotting enzyme, and a measurement substrate significantly promoted the β-glucan-triggered cascade reaction.

[0107] (Test Example 2) Referring to the Examples in WO 2014 / 092079, an expression plasmid was constructed by inserting a gene encoding the Limulus polyphemus proclotting enzyme (SEQ ID NO: 25) into a vector in place of the gene encoding Tachypleus tridentatus Factor C. CHO DG44 cells transformed with this expression plasmid were cultured in the presence of methotrexate, following the Examples in WO 2014 / 092079, and then monocloned. The resulting monoclonal cell line was cultured in serum-free medium. The culture supernatant containing the Limulus polyphemus proclotting enzyme recombinant protein was used as the raw material for producing the β-glucan measurement reagent described below.

[0108] With reference to the description in the Examples of WO 2014 / 092079, an expression plasmid was constructed by replacing the gene encoding Tachypleus tridentatus Factor C with genes encoding the α and β subunits of Limulus polyphemus Factor G (SEQ ID NOs: 15 and 23) into a vector. CHO DG44 cells were transformed with this expression plasmid. Western blotting showed that the β subunit was not detected in the culture supernatant. An expression plasmid was constructed by inserting genes encoding the α and β subunits of Tachypleus tridentatus Factor G (SEQ ID NOs: 13 and 14) into a vector in place of the gene encoding Limulus polyphemus Factor G. CHO DG44 cells were transformed with this expression plasmid. Western blotting detected the α and β subunits in the culture supernatant. The transformed cells were then cultured in the presence of methotrexate and monocloned, following the procedures described in the Examples of WO 2014 / 092079. The resulting monoclonal cell line was cultured in serum-free medium. This culture supernatant containing Tachypleus tridentatus Factor G recombinant protein was used as the raw material for producing a β-glucan assay reagent, as described below.

[0109] A reagent was prepared using the resulting Proclotting Enzyme recombinant protein and Factor G recombinant protein and a measurement substrate (synthetic substrate: Boc-LGR-pNA). This reagent was mixed with a sample containing β-glucan (curdlan; manufactured by Wako Pure Chemical Industries, Ltd.) (final β-glucan concentration: 200 pg / mL), and the reaction was carried out according to the method described in the Examples of Patent Document 3 (except that the reaction was carried out at 37°C for 40 minutes), and the absorbance (405 nm) was measured. A similar measurement was performed using a sample (blank) that did not contain β-glucan.

[0110] The proclotting enzyme activity of the blank was set at 1, and the relative activity when β-glucan was used as the sample was calculated to be 1.1. In other words, almost no activation of the proclotting enzyme was observed.

[0111] (Test Example 3) The Factor D gene was cloned by PCR using mRNA derived from hemocytes of Tachypleus tridentatus as a template. The amino acid sequence of the obtained Factor D (SEQ ID NO: 3) was confirmed to contain one mutation (serine at position 373 in SEQ ID NO: 1 was replaced with alanine) compared to the reported amino acid sequence (SEQ ID NO: 1).

[0112] We prepared recombinant Factor D protein from Tachypleus tridentatus. Specifically, we used pCA7 as a vector to construct an expression plasmid containing a gene (SEQ ID NO: 28) encoding the amino acid sequence of SEQ ID NO: 5 (TFD). The amino acid sequence of SEQ ID NO: 5 was prepared by replacing the signal sequence of the amino acid sequence of SEQ ID NO: 3 cloned above with a signal sequence derived from tyrosine phosphatase, which is commonly used in mammalian cell expression, and further adding a His tag to the C-terminus.

[0113] We constructed recombinant Factor D protein from Limulus polyphemus. Specifically, we used pCA7 as a vector to construct an expression plasmid containing a gene (SEQ ID NO: 31) encoding the amino acid sequence of SEQ ID NO: 11 (LFD). LFD is a recombinant protein derived from XP_013794271.1 (SEQ ID NO: 9), registered with NCBI as Limulus polyphemus Factor D. The signal sequence of this amino acid sequence has been replaced with a signal sequence derived from a tyrosine phosphatase, which is commonly used in mammalian cell expression. Furthermore, a His tag has been added to the C-terminus.

[0114] The expression plasmids prepared as described above were cultured using the Expi CHO Expression System (Thermo Fisher Scientific). The culture supernatant was collected as a sample, and extracellular secretion of recombinant Factor D proteins (TFD and LFD) was confirmed by Western blotting using an anti-His tag antibody.

[0115] The Factor D recombinant proteins (TFD, LFD) recovered as described above were used to prepare β-glucan measurement reagents together with Factor G recombinant protein, Proclotting enzyme recombinant protein, and measurement substrates in the same manner as in Test Example 2. Assays were performed using these reagents in the same manner as in Test Example 2, and the relative activity compared to the blank when β-glucan was used as the sample was calculated in the same manner as in Test Example 2.

[0116] The results confirmed that the presence of β-glucan significantly activated the proclotting enzyme in both reagents (relative activity of 21.0 for the β-glucan assay reagent containing TFD, and 21.5 for the β-glucan assay reagent containing LFD). In other words, TFD and LFD significantly enhanced the β-glucan-triggered cascade reaction in the β-glucan assay reagent.

[0117] (Test Example 4) Similar to Test Example 3, a β-glucan assay reagent (genetically recombinant reagent) containing LFD was prepared and its practicality evaluated. The amount of culture supernatant containing LFD added was 2% (v / v) or 4% (v / v) of the total reagent. A calibration curve was then created using the standard β-glucan (Pachyman) included with Glucatel (registered trademark, Associates of Cape Cod), a β-glucan assay reagent using horseshoe crab hemocyte extract. The reaction conditions (37°C, 40 minutes) and reaction volume (100 μL of reagent + 25 μL of sample) were as described in the Glucatel (registered trademark) package insert.

[0118] As a result, a calibration curve could be prepared for the recombinant reagent in the range of 0.6 pg / mL to 5.0 pg / mL, demonstrating that the reagent has the same sensitivity as Glucatel (registered trademark) (Figure 3). However, with the reagent of Test Example 2, which does not contain Factor D, activation of proclotting enzyme by β-glucan could not be confirmed, and β-glucan could not be quantified (Figure 3).

[0119] (Test Example 5) The α and β subunits of Factor G (SEQ ID NOs: 13 and 14) were expressed using the Expi CHO Expression System (Thermo Fisher Scientific) with the expression plasmid for Tachypleus tridentatus Factor G prepared in Test Example 2. The culture supernatant containing the Tachypleus tridentatus Factor G recombinant protein was used as a raw material for producing a reagent for measuring β-glucan, as described below.

[0120] A recombinant Limulus polyphemus Factor D protein was produced. Specifically, a gene (SEQ ID NO: 34) encoding the amino acid sequence of SEQ ID NO: 32 (LFD2) was inserted into pCI-neo as a vector to create an expression plasmid. The amino acid sequence of SEQ ID NO: 32 was obtained by replacing the signal sequence of the amino acid sequence of XP_013794271.1 (SEQ ID NO: 9) registered with NCBI as Limulus polyphemus Factor D with a signal sequence derived from a tyrosine phosphatase commonly used for expression in mammalian cells.

[0121] The recombinant Factor D protein (LFD2) was expressed using the expression plasmid prepared as described above with the Expi CHO Expression System (Thermo Fisher Scientific). The culture supernatant was collected as a sample, and extracellular secretion of the recombinant Factor D protein (LFD2) was confirmed by Western blotting.

[0122] A β-glucan assay reagent was prepared using the recombinant Factor D protein (LFD2) recovered as described above, along with the Tachypleus tridentatus-derived recombinant Factor G protein, the Tachypleus tridentatus-derived proclotting enzyme (a recombinant protein produced in insect cells according to the example in International Publication WO 2012 / 118226), and a synthetic substrate (Boc-LGR-pNA). This reagent was mixed with a sample containing β-glucan (curdlan; Wako Pure Chemical Industries, Ltd.) (final β-glucan concentration: 100 pg / mL). The reaction was carried out according to the method described in the example in Patent Document 3 (except that the reaction was carried out at 37°C for 60 minutes), and the absorbance (405 nm) was measured. A similar assay was also performed using a sample (blank) containing no β-glucan.

[0123] The relative activity of the blank proclotting enzyme was calculated using β-glucan as the sample, with the activity set at 1. The relative activity of the β-glucan assay reagent without Factor D was 1.1. This means that there was almost no activation of the proclotting enzyme. On the other hand, the β-glucan assay reagent containing LFD2 showed significant activation of the proclotting enzyme (relative activity of 13.0). This confirms that LFD2 significantly enhances the cascade reaction triggered by β-glucan in the β-glucan assay reagent.

[0124] (Test Example 6) In the same manner as in Test Example 2, a culture supernatant containing a Limulus polyphemus-derived proclotting enzyme recombinant protein and a culture supernatant containing a Tachypleus tridentatus-derived Factor G recombinant protein were prepared.

[0125] Referring to the Examples in WO 2014 / 092079, CHO DG44 cells transformed with the expression plasmid prepared in Test Example 5 were monocloned and cultured in serum-free medium. The culture supernatant was collected as a sample, and Western blotting confirmed that recombinant Factor D protein (LFD2) was secreted extracellularly.

[0126] The Factor D recombinant protein (LFD2) recovered as described above was used to prepare a β-glucan measurement reagent together with Factor G recombinant protein, Proclotting enzyme recombinant protein, and a measurement substrate, as in Test Example 2. Then, as in Test Example 4, a calibration curve was prepared using β-glucan (Pachyman; Associates of Cape Cod; final β-glucan concentration: 0 to 20 pg / mL).

[0127] As a result, a calibration curve could be created within the above β-glucan concentration range (Figure 4). With the reagent that did not use LFD2, activation of proclotting enzyme by β-glucan could not be confirmed, and β-glucan could not be quantified (Figure 4).

[0128] (Test Example 7) Fractions containing Limulus polyphemus Factor G were isolated by column chromatography from hemocyte extracts. Western blot analysis confirmed that the isolated fractions contained neither Factor D nor proclotting enzyme.

[0129] A β-glucan assay reagent containing LFD was prepared using the Limulus polyphemus-derived Factor G fraction obtained above instead of the Tachypleus tridentatus-derived Factor G recombinant protein used in Test Example 3. This reagent was mixed with a sample containing β-glucan (Pachyman; Nissui Pharmaceutical Co., Ltd.) (final β-glucan concentration: 20 pg / mL) and incubated at 37°C for 30 minutes, after which the absorbance (405 nm) was measured. The measured absorbance (mAbs) was divided by the time (minutes) to calculate the proclotting enzyme activity. A similar assay was also performed using a sample (blank) containing no β-glucan.

[0130] The relative activity of the blank proclotting enzyme was calculated using β-glucan as the sample, with the activity set at 1. The relative activity of the β-glucan assay reagent without Factor D was 1.1. This means that there was almost no activation of the proclotting enzyme. On the other hand, the β-glucan assay reagent containing LFD showed significant activation of the proclotting enzyme (relative activity of 7.0). This confirms that LFD significantly enhances the cascade reaction triggered by β-glucan in the β-glucan assay reagent.

[0131] (Test Example 8) Referring to the description in the Examples (paragraphs

[0113] to

[0115] ) of WO 2014 / 092079, HEK293 GnTI cells transformed with the expression plasmid prepared in Test Example 5 were cultured. The culture supernatant was collected as a sample, and Western blotting was used to confirm that recombinant Factor D protein (LFD2) was secreted outside the cells.

[0132] The Factor D recombinant protein (LFD2) recovered as described above was used to prepare a β-glucan measurement reagent together with Factor G recombinant protein, Proclotting Enzyme recombinant protein, and a measurement substrate, as in Test Example 2. This reagent was mixed with a sample containing β-glucan (Pachyman; Nissui Pharmaceutical Co., Ltd.) (final concentration of β-glucan: 20 pg / mL) and incubated at 37°C for 40 minutes. anti After the reaction, the absorbance (405 nm) was measured. The measured absorbance (mAbs) was divided by the time (minutes) to calculate the activity of the proclotting enzyme. A similar measurement was also performed using a sample (blank) that did not contain β-glucan.

[0133] The relative activity of the blank proclotting enzyme was calculated using β-glucan as the sample, with the activity set at 1. The relative activity of the β-glucan assay reagent without Factor D was 1.1. This means that there was almost no activation of the proclotting enzyme. On the other hand, the β-glucan assay reagent containing LFD2 showed significant activation of the proclotting enzyme (relative activity 6.8). This confirms that LFD2 significantly enhances the cascade reaction triggered by β-glucan in the β-glucan assay reagent.

[0134] (Test Example 9) A β-glucan measurement reagent containing LFD was prepared in the same manner as in Test Example 3, except that the measurement substrate was changed to Ac-IEAR-pNA (Biorbyt). This reagent was mixed with a sample containing β-glucan (Pachyman; Associates of Cape Cod) (final concentration of β-glucan: 20 pg / mL) and incubated at 37°C for 40 minutes. anti After the reaction, the absorbance (405 nm) was measured. The measured absorbance (mAbs) was divided by the time (minutes) to calculate the activity of the proclotting enzyme. A similar measurement was also performed using a sample (blank) that did not contain β-glucan.

[0135] The relative activity of the blank proclotting enzyme was calculated using β-glucan as the sample, with the activity set at 1. The relative activity of the β-glucan assay reagent without Factor D was 1.1. This means that there was almost no activation of the proclotting enzyme. On the other hand, the β-glucan assay reagent containing LFD showed significant activation of the proclotting enzyme (relative activity of 5.9). This confirms that LFD significantly enhances the cascade reaction triggered by β-glucan in the β-glucan assay reagent.

[0136] (Test Example 10) A β-glucan measurement reagent (genetically recombinant reagent) containing LFD was prepared in the same manner as in Test Example 3. This reagent (160 μL) was mixed with a sample (40 μL) containing β-glucan (lentinan; manufactured by Ajinomoto Co., Inc.) (final concentration of β-glucan: 0 to 5 pg / mL), and incubated at 37°C for 60 minutes. anti After the reaction, the absorbance (405 nm) was measured. The measured absorbance (mAbs) was divided by the time (minutes) to calculate the activity of the proclotting enzyme. A similar measurement was also performed using a sample (blank) that did not contain β-glucan.

[0137] As a result, a calibration curve could be created within the above β-glucan concentration range (Figure 5). With reagents that did not use LFD, activation of proclotting enzyme by β-glucan could not be confirmed, and β-glucan could not be quantified (Figure 5).

[0138] (Test Example 11) We constructed four recombinant Factor D proteins, two from Tachypleus tridentatus and two from Limulus polyphemus. Specifically, four expression plasmids were constructed by incorporating the gene encoding the amino acid sequence of SEQ ID NO: 1 (TFD2), the gene encoding the amino acid sequence of SEQ ID NO: 3 (TFD3), the gene encoding the amino acid sequence of SEQ ID NO: 7 (LFD3), or the gene encoding the amino acid sequence of SEQ ID NO: 9 (LFD4) into the vector (pCI-neo).

[0139] Using the expression plasmids prepared as described above, four types of recombinant Factor D proteins were expressed using the Expi CHO Expression System (Thermo Fisher Scientific). Culture supernatants were collected as samples, and extracellular secretion of the recombinant Factor D proteins was confirmed by Western blotting.

[0140] TFD2 is a Tachypleus tridentatus Factor D gene registered with NCBI as BAA13312.1. TFD3 is encoded by a cDNA cloned from Tachypleus tridentatus (SEQ ID NO: 27). LFD3 is a Limulus polyphemus Factor D gene registered with NCBI as XP_013794266.1. LFD4 is a Limulus polyphemus Factor D gene registered with NCBI as XP_013794271.1.

[0141] Using each of the four recombinant Factor D proteins (TFD2, TFD3, LFD3, and LFD4), a Proclotting enzyme recombinant protein, a recombinant Factor G protein, and a measurement substrate (synthetic substrate: Boc-LGR-pNA) were used to prepare β-glucan measurement reagents in the same manner as in Test Example 2. This reagent was mixed with a sample containing β-glucan (Pachyman; Nissui Pharmaceutical Co., Ltd.) (final β-glucan concentration: 20 pg / mL), and the reaction was carried out according to the method described in the Examples of Patent Document 3 (except that the reaction was carried out at 37°C for 40 minutes), and the absorbance (405 nm) was measured. A similar measurement was also performed using a sample (blank) that did not contain β-glucan.

[0142] The relative activity of the proclotting enzyme in the blank was defined as 1, and the relative activity was calculated for β-glucan as a test sample. The relative activity of the β-glucan assay reagent without Factor D was 1.1. This means that there was almost no activation of the proclotting enzyme. In contrast, the β-glucan assay reagent containing recombinant Factor D protein exhibited significant activation of the proclotting enzyme (relative activities: TFD2 17.5, TFD3 7.2, LFD3 6.4, and LFD4 11.6). This confirms that TFD2, TFD3, LFD3, and LFD4 significantly enhance the β-glucan-triggered cascade reaction in the β-glucan assay reagent.

[0143] (Test Example 12) A β-glucan measurement reagent containing LFD was prepared in the same manner as in Test Example 3, except that the measurement substrate was changed to Ac-IEGK-pNA. This reagent was mixed with a sample containing β-glucan (Pachyman; Associates of Cape Cod) (final concentration of β-glucan: 20 pg / mL) and incubated at 37°C for 40 minutes. anti After the reaction, the absorbance (405 nm) was measured. The measured absorbance (mAbs) was divided by the time (minutes) to calculate the activity of the proclotting enzyme. A similar measurement was also performed using a sample (blank) that did not contain β-glucan.

[0144] The relative activity of the blank proclotting enzyme was calculated using β-glucan as the sample, with the activity set at 1. The relative activity of the β-glucan assay reagent without Factor D was 1.2. This means that there was almost no activation of the proclotting enzyme. On the other hand, the β-glucan assay reagent containing LFD showed significant activation of the proclotting enzyme (relative activity of 5.4). This confirms that LFD significantly enhances the cascade reaction triggered by β-glucan in the β-glucan assay reagent. [Industrial Applicability]

[0145] The present invention provides a use of Factor D as a major factor that constitutes the reaction system, together with other glucan-related factors, in the Limulus reaction triggered by β-glucan. That is, the present invention can provide a high-performance reagent for measuring recombinant β-glucan. Furthermore, the present invention can enhance the activity of the reagent for measuring β-glucan. The present invention is expected to be permanently and widely used in the detection and quantification of β-glucan to assess the safety of injectable water, pharmaceuticals, medical devices, etc., in the serodiagnosis of fungal infections, etc., as a tool for detecting microbial contamination in the fields of environmental and food hygiene, and as a research reagent, etc. While the present invention has been described with reference to specific examples and various embodiments, it will be readily apparent to those skilled in the art that many modifications and adaptations of the embodiments described herein are possible without departing from the spirit and scope of the invention. This application claims priority based on Patent Application No. 2021-128613 filed with the Japan Patent Office on August 4, 2021, Patent Application No. 2021-175670 filed with the Japan Patent Office on October 27, 2021, and Patent Application No. 2022-10643 filed with the Japan Patent Office on January 27, 2022, the contents of which are incorporated by reference in their entireties into this application.

[0146] <Description of Sequence Listing> SEQ ID NO: 1: Amino acid sequence of Factor D from Tachypleus tridentatus 1 (TFD2) SEQ ID NO: 2: Amino acid sequence of Factor D from Tachypleus tridentatus (TFD2 (mature)) SEQ ID NO: 3: Amino acid sequence of Factor D 2 from Tachypleus tridentatus (TFD3) SEQ ID NO: 4: Amino acid sequence of Factor D 2 from Tachypleus tridentatus (TFD3 (mature)) SEQ ID NO: 5: Amino acid sequence of Factor D from Tachypleus tridentatus 3 (TFD) SEQ ID NO: 6: Amino acid sequence of Factor D from Tachypleus tridentatus 3 (TFD (mature)) SEQ ID NO: 7: Amino acid sequence of Limulus polyphemus-derived Factor D 1 (LFD3) SEQ ID NO: 8: Amino acid sequence of Limulus polyphemus-derived Factor D 1 (LFD3 (mature)) SEQ ID NO: 9: Amino acid sequence of Limulus polyphemus-derived Factor D 2 (LFD4) SEQ ID NO: 10: Amino acid sequence of Limulus polyphemus-derived Factor D 2 (LFD4 (mature)) SEQ ID NO: 11: Amino acid sequence of Limulus polyphemus-derived Factor D 3 (LFD) SEQ ID NO: 12: Amino acid sequence of Limulus polyphemus-derived Factor D (LFD (mature)) SEQ ID NO: 13: Amino acid sequence of the α subunit of Factor G from Tachypleus tridentatus SEQ ID NO: 14: Amino acid sequence of the β subunit of Factor G from Tachypleus tridentatus SEQ ID NO: 15: Amino acid sequence of the α subunit of Factor G from Limulus polyphemus 1 SEQ ID NO: 16: Amino acid sequence 2 of the α subunit of Factor G from Limulus polyphemus SEQ ID NO: 17: Amino acid sequence of the α subunit of Factor G from Limulus polyphemus 3 SEQ ID NO: 18: Amino acid sequence of the β subunit of Factor G from Limulus polyphemus 1 SEQ ID NO: 19: Amino acid sequence 2 of the β subunit of Factor G from Limulus polyphemus SEQ ID NO: 20: Amino acid sequence of the β subunit of Factor G from Limulus polyphemus 3 SEQ ID NO: 21: Amino acid sequence of the β subunit of Factor G from Limulus polyphemus 4 SEQ ID NO: 22: Amino acid sequence of the β subunit of Factor G from Limulus polyphemus 5 SEQ ID NO: 23: Amino acid sequence of the β subunit of Factor G from Limulus polyphemus 6 SEQ ID NO: 24: Amino acid sequence of proclotting enzyme from Tachypleus tridentatus SEQ ID NO: 25: Amino acid sequence of proclotting enzyme from Limulus polyphemus SEQ ID NO: 26: Nucleotide sequence 1 of Factor D derived from Tachypleus tridentatus SEQ ID NO: 27: Nucleotide sequence 2 of Factor D derived from Tachypleus tridentatus SEQ ID NO: 28: Nucleotide sequence 3 of Factor D derived from Tachypleus tridentatus SEQ ID NO: 29: Nucleotide sequence of Factor D derived from Limulus polyphemus 1 SEQ ID NO: 30: Nucleotide sequence 2 of Factor D derived from Limulus polyphemus SEQ ID NO: 31: Nucleotide sequence of Factor D derived from Limulus polyphemus 3 SEQ ID NO: 32: Amino acid sequence of Limulus polyphemus-derived Factor D 4 (LFD2) SEQ ID NO: 33: Amino acid sequence of Limulus polyphemus-derived Factor D 4 (LFD2 (mature)) SEQ ID NO: 34: Nucleotide sequence of Factor D derived from Limulus polyphemus 4 SEQ ID NO: 35: Amino acid sequence 4 of Factor D from Tachypleus tridentatus SEQ ID NO: 36: Amino acid sequence 5 of Factor D from Tachypleus tridentatus SEQ ID NO: 37: Amino acid sequence 6 of Factor D from Tachypleus tridentatus SEQ ID NO: 38: Amino acid sequence 7 of Factor D from Tachypleus tridentatus SEQ ID NO: 39: Amino acid sequence 8 of Factor D from Tachypleus tridentatus SEQ ID NO: 40: Amino acid sequence 9 of Factor D from Tachypleus tridentatus SEQ ID NO: 41: Amino acid sequence of Factor D from Tachypleus tridentatus SEQ ID NO: 42: Amino acid sequence of Factor D from Tachypleus tridentatus SEQ ID NO: 43: Amino acid sequence 12 of Factor D from Tachypleus tridentatus SEQ ID NO: 44: Amino acid sequence 13 of Factor D from Tachypleus tridentatus SEQ ID NO: 45: Amino acid sequence 14 of Factor D from Tachypleus tridentatus SEQ ID NO: 46: Amino acid sequence 15 of Factor D from Tachypleus tridentatus SEQ ID NO: 47: Amino acid sequence 16 of Factor D from Tachypleus tridentatus SEQ ID NO: 48: Amino acid sequence 17 of Factor D from Tachypleus tridentatus SEQ ID NO: 49: Amino acid sequence 18 of Factor D from Tachypleus tridentatus SEQ ID NO: 50: Amino acid sequence 19 of Factor D from Tachypleus tridentatus SEQ ID NO: 51: Amino acid sequence of Factor D from Limulus polyphemus SEQ ID NO: 52: Amino acid sequence of Factor D from Limulus polyphemus 6 SEQ ID NO: 53: Amino acid sequence 7 of Factor D from Limulus polyphemus SEQ ID NO: 54: Amino acid sequence of Factor D from Limulus polyphemus 8 SEQ ID NO: 55: Amino acid sequence of Factor D from Limulus polyphemus 9 SEQ ID NO: 56: Amino acid sequence of Factor D from Limulus polyphemus SEQ ID NO: 57: Amino acid sequence of Factor D from Limulus polyphemus 11 SEQ ID NO: 58: Amino acid sequence 12 of Factor D from Limulus polyphemus SEQ ID NO: 59: Amino acid sequence 13 of Factor D from Limulus polyphemus SEQ ID NO: 60: Amino acid sequence 14 of Factor D from Limulus polyphemus SEQ ID NO: 61: Amino acid sequence 15 of Factor D from Limulus polyphemus SEQ ID NO: 62: Amino acid sequence 16 of Factor D from Limulus polyphemus SEQ ID NO: 63: Amino acid sequence 17 of Factor D from Limulus polyphemus SEQ ID NO: 64: Amino acid sequence 18 of Factor D from Limulus polyphemus SEQ ID NO: 65: Amino acid sequence 19 of Factor D from Limulus polyphemus SEQ ID NO: 66: Amino acid sequence of Factor D from Limulus polyphemus SEQ ID NO: 67: Amino acid sequence 1 of Factor D from Carcinoscorpius rotundicauda SEQ ID NO: 68: Amino acid sequence 1 (mature) of Factor D from Carcinoscorpius rotundicauda SEQ ID NO: 69: Amino acid sequence 1 of Factor D from Tachypleus gigas SEQ ID NO: 70: Amino acid sequence 1 (mature) of Factor D from Tachypleus gigas SEQ ID NO: 71: Nucleotide sequence of Factor D from Carcinoscorpius rotundicauda 1 SEQ ID NO: 72: Nucleotide sequence 1 of Factor D derived from Tachypleus gigas

Claims

1. The assay comprises horseshoe crab-derived Factor D, horseshoe crab-derived Factor G, horseshoe crab-derived proclotting enzyme, and a measurement substrate; A reagent for measuring β-glucan that does not contain horseshoe crab hemocyte extract.

2. 2. The reagent for measuring β-glucan according to claim 1, wherein the horseshoe crab-derived factor D, the horseshoe crab-derived factor G, and the horseshoe crab-derived proclotting enzyme are recombinant proteins.

3. The reagent for measuring β-glucan according to claim 1, wherein the detection limit of pachyman is 20 pg / mL or less within a reaction time of 60 minutes.

4. The reagent for measuring β-glucan according to claim 1, wherein the horseshoe crab-derived factor D is a protein (1) or (2) below: (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70; (2) It has an amino acid sequence that is 84% ​​or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70, and has the function of Factor D.

5. The reagent for measuring β-glucan according to claim 1, wherein the horseshoe crab-derived factor D is a protein (1') or (2') below: (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70; (2') It has an amino acid sequence that is 84% ​​or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68 and 70, and has the function of Factor D.

6. 2. The reagent for measuring β-glucan according to claim 1, wherein the horseshoe crab-derived factor D is derived from Tachypleus tridentatus, Limulus polyphemus, Carcinoscorpius rotundicauda, ​​or Tachypleus gigas.

7. A method for producing a reagent for measuring β-glucan, comprising: Artificially producing horseshoe crab-derived Factor D, a production method comprising assembling the artificially produced horseshoe crab-derived Factor D into a kit together with at least horseshoe crab-derived Factor G, horseshoe crab-derived Proclotting Enzyme, and a measurement substrate;

8. The production method according to claim 7, wherein the horseshoe crab-derived Factor D is artificially produced by recombinant production using a host cell.

9. The detection limit of pachyman using the β-glucan measurement reagent is within 60 minutes of reaction time. The method of claim 7, wherein the concentration is 20 pg / mL or less.

10. 8. The method according to claim 7, wherein the horseshoe crab-derived Factor D is a protein of the following (1) or (2): (1) comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70; (2) It has an amino acid sequence that is 84% ​​or more identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 12, 32, 33, and 67 to 70, and has the function of Factor D.

11. The production method according to claim 7, wherein the horseshoe crab-derived Factor D is a protein represented by the following (1') or (2'): (1') comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68, and 70; (2') It has an amino acid sequence that is 84% ​​or more identical to at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 33, 68 and 70, and has the function of Factor D.

12. The production method according to claim 7, wherein the horseshoe crab-derived Factor D is derived from Tachypleus tridentatus, Limulus polyphemus, Carcinoscorpius rotundicauda, ​​or Tachypleus gigas.

13. The reagent for measuring β-glucan according to any one of claims 1 to 6, or claims 7 to 12. A test is carried out using a β-glucan measurement reagent produced by the production method described in any one of the above. A method for assaying β-glucan, comprising measuring β-glucan in a sample.

14. A method for testing for fungal disease, comprising: performing the β-glucan assay method of claim 13, A method wherein the sample is a biological sample derived from a subject suspected of suffering from a fungal disease.

15. 1. A data acquisition method for diagnosing a fungal disease, comprising: The β-glucan assay method according to claim 13 is carried out to determine whether a subject is suffering from a fungal disease. and obtaining data to diagnose whether or not The method, wherein the sample is a biological sample derived from the subject.

16. A method for enhancing the activity of a reagent for measuring β-glucan, comprising: horseshoe crab-derived factor G; horseshoe crab-derived proclotting enzyme; and a measurement substrate, and allowing horseshoe crab-derived Factor D to coexist with the β-glucan measurement reagent. method.

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