JP2020074755A - Alkaline phosphatase composition, method for producing dephosphorylated nucleic acid and labeled nucleic acid - Google Patents

Abstract

To provide a high-quality composition containing alkaline phosphatase, and to provide a method for producing a dephosphorylated nucleic acid and a method for producing a labeled nucleic acid using the composition.SOLUTION: There is provided a composition containing alkaline phosphatase and a peptide fragment group (A) consisting of two or more peptide fragments comprising consecutive 5 to 50 amino acid residues selected from positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4. A ratio of the content of the peptide fragment group (A) to the content of the alkaline phosphatase is the following formula (A): (X/Y)×100≤5.0000. In the formula, Xis a peak area value of the peptide fragment group (A), which is calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS/MS analysis of the composition, Y is a peak area value of the alkaline phosphatase calculated by the automatic integration method from the chromatogram obtained by an LC-UV analysis of the composition.SELECTED DRAWING: None

Description

  TECHNICAL FIELD The present invention relates to a composition containing alkaline phosphatase, a method for producing a dephosphorylated nucleic acid and a method for producing a labeled nucleic acid using the composition.

  Alkaline phosphatase has a catalytic function of hydrolyzing phosphate monoester, and is widely used in methods for measuring the amount of biological substances such as proteins and nucleic acids (eg, immunostaining method, ELISA, nucleic acid microarray method, etc.). There is. For example, in the field of genetic engineering research, for the purpose of pretreatment of labeling of nucleic acids such as DNA and RNA, prevention of vector self-ligation, etc., 5'end and / or 3'end of nucleic acid using alkaline phosphatase is used. It is dephosphorylated.

  As an industrial production method of alkaline phosphatase, a production method mainly using bovine small intestine or large intestine as a raw material is widely adopted because of its high specific activity. The evaluation of the specific activity of alkaline phosphatase is generally performed by measuring the absorbance at 405 nm derived from p-nitrophenol produced when p-nitrophenyl phosphate is decomposed.

  The quality of alkaline phosphatase is evaluated based on the alkaline phosphatase specific activity. Then, in order to obtain alkaline phosphatase having a higher specific activity than alkaline phosphatase derived from bovine intestine, the alkaline phosphatase having a high specific activity is isolated in the purification process, or the recombinant Escherichia coli having a high specific activity is used by a genetic engineering method. They also produce alkaline phosphatase.

  Patent Document 1 discloses a method for producing alkaline phosphatase having a high specific activity using recombinant Escherichia coli introduced with a gene derived from Bacillus badius. Further, Patent Document 2 discloses a method for producing alkaline phosphatase having high specific activity and thermostability using recombinant Escherichia coli into which a gene derived from the genus Shewanella is introduced.

Japanese Patent Laid-Open No. 10-262674 International Publication No. 2012/115023

  A dephosphorylation reagent containing alkaline phosphatase (eg, a commercially available alkaline phosphatase product) is a composition containing alkaline phosphatase and other components. The quality of dephosphorylation reagents containing alkaline phosphatase is evaluated on the basis of alkaline phosphatase specific activity.

  However, even if the dephosphorylation reagent has almost the same alkaline phosphatase specific activity, a labeled nucleic acid prepared by using the dephosphorylation reagent (the 5 ′ end of the nucleic acid and / or 3 (A labeled nucleic acid obtained by dephosphorylating the end and binding a labeling substance to the 5'end and / or the 3'end of the dephosphorylated nucleic acid) has a large difference in detection sensitivity in a nucleic acid detection method. Was found to occur. That is, it was revealed that the quality of the dephosphorylation reagent containing alkaline phosphatase cannot be accurately evaluated by using the alkaline phosphatase specific activity as an index.

  Therefore, an object of the present invention is to provide a high-quality composition containing alkaline phosphatase, a method for producing a dephosphorylated nucleic acid and a method for producing a labeled nucleic acid using the composition.

As a result of intensive studies to solve the above problems, the present inventors have found that a dephosphorylation reagent containing alkaline phosphatase (for example, a commercially available alkaline phosphatase product) contains one or more of the following impurities. We found that they could be mixed.
-Consists of two or more kinds of peptide fragments consisting of 5 to 50 consecutive amino acid residues selected from positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4 (amino acid sequence of bovine alkaline phosphatase) Peptide fragment group (A)
-Two or more kinds consisting of consecutive 5 to 50 amino acid residues selected from positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4 and including positions 86 to 90 of the amino acid sequence of SEQ ID NO: 4. Peptide fragment group composed of peptide fragments (B)
-Two or more kinds consisting of 13 to 50 consecutive amino acid residues selected from positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4 and including positions 93 to 105 of the amino acid sequence set forth in SEQ ID NO: 4. Peptide fragment group composed of peptide fragments (C)
First peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 1 Second peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2 Third peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 3

  Then, the present inventors have found that the content of the peptide fragment group (A), the content of the peptide fragment group (B), the content of the peptide fragment group (C) or the content of the second peptide fragment (preferably, Prepared using a dephosphorylation reagent by reducing the content of the second peptide fragment and the content of one or two peptide fragments selected from the first and third peptide fragments) Labeled nucleic acid (the 5'end and / or 3'end of the nucleic acid is dephosphorylated by the dephosphorylation reagent, and a labeling substance is bound to the 5'end and / or 3'end of the dephosphorylated nucleic acid It was found that the labeled nucleic acid obtained by the above can improve the detection sensitivity in the nucleic acid detection method, and has completed the present invention. That is, the present invention includes the following inventions.

[1] A group of peptide fragments consisting of alkaline phosphatase and two or more kinds of peptide fragments consisting of consecutive 5 to 50 amino acid residues selected from positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4 ( A) and a composition containing
The ratio of the content of the peptide fragment group (A) to the content of the alkaline phosphatase is represented by the following formula (A):
(X _A /Y)×100≦5.0000 (A)
[Wherein, X _A represents the peak area value of the peptide fragment group (A) calculated by the automatic integration method from the extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y Represents the peak area value of the alkaline phosphatase calculated by the automatic integration method from the chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, which satisfies:
[2] In the peptide fragment group (A), a first peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1, a second peptide fragment consisting of the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 3 The composition according to [1], which comprises one, two or three peptide fragments selected from the group consisting of a third peptide fragment consisting of the described amino acid sequence.
[3] The peptide fragment group (A) contains the first peptide fragment,
The ratio of the content of the first peptide fragment to the content of the alkaline phosphatase is represented by the following formula (1):
(X ₁ /Y)×100≦1.0000 (1)
[Wherein X ₁ represents a peak area value of the first peptide fragment calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y is , Is synonymous with the above. ]
The composition according to [2], which satisfies:
[4] The peptide fragment group (A) contains the second peptide fragment,
The ratio of the content of the second peptide fragment to the content of the alkaline phosphatase is represented by the following formula (2):
(X ₂ /Y)×100≦0.8000 (2)
[In the formula, X ₂ represents the peak area value of the second peptide fragment calculated by the automatic integration method from the extracted ion chromatogram obtained by the LC-MS / MS analysis of the composition, and Y is , Is synonymous with the above. ]
The composition according to [2] or [3], which satisfies:
[5] The peptide fragment group (A) contains the third peptide fragment,
The ratio of the content of the third peptide fragment to the content of the alkaline phosphatase is represented by the following formula (3):
(X ₃ /Y)×100≦2.3000 (3)
[Wherein X ₃ represents a peak area value of the third peptide fragment calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y is , Is synonymous with the above. ]
The composition according to any one of [2] to [4], which satisfies:
[6] consisting of alkaline phosphatase and 5 to 50 consecutive amino acid residues selected from positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4, and positions 86 to 90 of the amino acid sequence of SEQ ID NO: 4 A peptide fragment group (B) composed of two or more peptide fragments containing
The ratio of the content of the peptide fragment group (B) to the content of the alkaline phosphatase is represented by the following formula (B):
(X _B /Y)×100≦2.4000 (B)
[Wherein, X _B represents a peak area value of the peptide fragment group (B) calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y Represents the peak area value of the alkaline phosphatase calculated by the automatic integration method from the chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, which satisfies:
[7] The peptide fragment group (B) is selected from the group consisting of a first peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1 and a second peptide fragment consisting of the amino acid sequence of SEQ ID NO: 2. The composition according to [6], which comprises the one or two peptide fragments thus prepared.
[8] The peptide fragment group (B) contains the first peptide fragment,
The ratio of the content of the first peptide fragment to the content of the alkaline phosphatase is represented by the following formula (1):
(X ₁ /Y)×100≦1.0000 (1)
[Wherein X ₁ represents a peak area value of the first peptide fragment calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y is , Is synonymous with the above. ]
The composition according to [7], which satisfies:
[9] The peptide fragment group (B) contains the second peptide fragment,
The ratio of the content of the second peptide fragment to the content of the alkaline phosphatase is represented by the following formula (2):
(X ₂ /Y)×100≦0.8000 (2)
[In the formula, X ₂ represents the peak area value of the second peptide fragment calculated by the automatic integration method from the extracted ion chromatogram obtained by the LC-MS / MS analysis of the composition, and Y is , Is synonymous with the above. ]
The composition according to [7] or [8], which satisfies:
[10] consisting of alkaline phosphatase and 13 to 50 consecutive amino acid residues selected from positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4, and positions 93 to 105 of the amino acid sequence of SEQ ID NO: 4. A peptide fragment group (C) composed of two or more peptide fragments containing
The ratio of the content of the peptide fragment group (C) to the content of the alkaline phosphatase is represented by the following formula (C):
(X _C /Y)×100≦4.5000 (C)
[Wherein, X _C represents a peak area value of the peptide fragment group (C) calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y C Represents the peak area value of the alkaline phosphatase calculated by the automatic integration method from the chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, which satisfies:
[11] The peptide fragment group (C) is selected from the group consisting of a first peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1 and a third peptide fragment consisting of the amino acid sequence of SEQ ID NO: 3. The composition according to [10], which comprises one or two peptide fragments prepared as described above.
[12] The peptide fragment group (C) contains the first peptide fragment,
The ratio of the content of the first peptide fragment to the content of the alkaline phosphatase is represented by the following formula (1):
(X ₁ /Y)×100≦1.0000 (1)
[Wherein X ₁ represents a peak area value of the first peptide fragment calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y is , Is synonymous with the above. ]
The composition according to [11], which satisfies:
[13] The peptide fragment group (C) includes the third peptide fragment,
The ratio of the content of the third peptide fragment to the content of the alkaline phosphatase is represented by the following formula (3):
(X ₃ /Y)×100≦2.3000 (3)
[Wherein X ₃ represents a peak area value of the third peptide fragment calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y is , Is synonymous with the above. ]
The composition according to [11] or [12], which satisfies:
[14] A composition comprising alkaline phosphatase and a second peptide fragment consisting of the amino acid sequence of SEQ ID NO: 2,
The ratio of the content of the second peptide fragment to the content of the alkaline phosphatase is represented by the following formula (2):
(X ₂ /Y)×100≦0.8000 (2)
[In the formula, X ₂ represents the peak area value of the second peptide fragment calculated by the automatic integration method from the extracted ion chromatogram obtained by the LC-MS / MS analysis of the composition, and Y is Shows the peak area value of the alkaline phosphatase calculated by the automatic integration method from the chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, which satisfies:
[15] The composition further contains a first peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1,
The ratio of the content of the first peptide fragment to the content of the alkaline phosphatase is represented by the following formula (1):
(X ₁ /Y)×100≦1.0000 (1)
[Wherein X ₁ represents a peak area value of the first peptide fragment calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y is , Is synonymous with the above. ]
The composition according to [14], which satisfies:
[16] The composition further contains a third peptide fragment consisting of the amino acid sequence of SEQ ID NO: 3,
The ratio of the content of the third peptide fragment to the content of the alkaline phosphatase is represented by the following formula (3):
(X ₃ /Y)×100≦2.3000 (3)
[Wherein X ₃ represents a peak area value of the third peptide fragment calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y is , Is synonymous with the above. ]
The composition according to [14] or [15], which satisfies:
[17] The composition according to any one of [1] to [16], wherein the composition has an alkaline phosphatase specific activity of 2000 U / mg or more.
[18] The alkaline phosphatase has the following (a) and (b):
(A) an alkaline phosphatase having a protein molecule consisting of the amino acid sequence set forth in SEQ ID NO: 4; and (b) having 70% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4, The composition according to any one of [1] to [17], which is selected from alkaline phosphatases having a protein molecule consisting of an amino acid sequence containing positions 71 to 130 of the amino acid sequence.
[19] The composition according to any one of [1] to [18], wherein the composition further contains a nucleic acid.
[20] The composition according to [19], wherein the composition is a composition for dephosphorylating the nucleic acid.
[21] The composition according to any one of [1] to [18], wherein the composition further contains a dephosphorylated nucleic acid.
[22] The composition according to [21], wherein the composition is a composition for preparing a labeled nucleic acid comprising the dephosphorylated nucleic acid and a labeling substance bound to the dephosphorylated nucleic acid. ..
[23] The composition according to any one of [1] to [18], further comprising a labeled nucleic acid comprising a dephosphorylated nucleic acid and a labeling substance bound to the dephosphorylated nucleic acid. Composition.
[24] The composition according to [23], which is a nucleic acid sample to be subjected to a nucleic acid detection method.
[25] The composition according to [24], wherein the nucleic acid detection method is a nucleic acid detection method using a nucleic acid microarray.
[26] The following steps:
A step of preparing the composition according to any one of [1] to [18];
A method for producing a dephosphorylated nucleic acid, comprising the steps of preparing a nucleic acid; and treating the nucleic acid with the composition to dephosphorylate the nucleic acid.
[27] The following steps:
A step of preparing the composition according to any one of [1] to [18];
Preparing a nucleic acid;
Preparing a labeling substance;
A method for producing a labeled nucleic acid, comprising the steps of treating the nucleic acid with the composition to dephosphorylate the nucleic acid; and binding the labeling substance to the dephosphorylated nucleic acid.

  The present invention provides a high-quality composition containing alkaline phosphatase, a method for producing a dephosphorylated nucleic acid and a method for producing a labeled nucleic acid using the composition.

FIG. 1 shows the extracted ion chromatogram for the first peptide fragment obtained by LC-MS / MS analysis of composition C2 in Comparative Example 2. FIG. 2 shows an extracted ion chromatogram for the second peptide fragment obtained by LC-MS / MS analysis of composition C2 in Comparative Example 2. FIG. 3 shows an extracted ion chromatogram for the third peptide fragment obtained by LC-MS / MS analysis of composition C2 in Comparative Example 2. FIG. 4 shows an extracted ion chromatogram for the first peptide fragment obtained by LC-MS / MS analysis of composition E1 (purified product of composition C2) in Example 1. FIG. 5 shows an extracted ion chromatogram for the second peptide fragment obtained by LC-MS / MS analysis of composition E1 (purified product of composition C2) in Example 1. FIG. 6 shows an extracted ion chromatogram for the third peptide fragment obtained by LC-MS / MS analysis of composition E1 (purified product of composition C2) in Example 1. FIG. 7 shows a chromatogram for alkaline phosphatase obtained by LC-UV analysis of composition E1 (purified product of composition C2) in Example 1.

  Hereinafter, the present invention will be described in detail. It is possible to combine two or more of the aspects described below and to combine two or more of the exemplary embodiments described below, and such combinations are also included in the present invention. The expression “numerical value M to numerical value N” used in the present specification means a range of numerical value M or more and numerical value N or less.

  The present invention provides, as the composition according to the first aspect, a composition containing alkaline phosphatase and a peptide fragment group (A).

  The present invention provides, as the composition according to the second aspect, a composition containing alkaline phosphatase and a peptide fragment group (B).

  The present invention provides, as the composition according to the third aspect, a composition containing alkaline phosphatase and a peptide fragment group (C).

  The present invention provides, as a composition according to the fourth aspect, a composition comprising alkaline phosphatase and a second peptide fragment comprising the amino acid sequence set forth in SEQ ID NO: 2.

  Hereinafter, the compositions according to the first to fourth aspects are collectively referred to as the “composition of the present invention”. Therefore, the description regarding the “composition of the present invention” applies to any of the compositions according to the first to fourth aspects, unless otherwise specified.

[Alkaline phosphatase]
The composition of the present invention contains alkaline phosphatase. The composition of the present invention may contain one type of alkaline phosphatase, or may contain two or more types of alkaline phosphatase.

  The alkaline phosphatase contained in the composition of the present invention is not particularly limited as long as it has alkaline phosphatase activity. The alkaline phosphatase activity is an activity of hydrolyzing a phosphate monoester bond in alkaline conditions (pH 8 to 11, for example, pH 8 to 10 or pH 9 to 11), and its reaction form is classified into EC 3.1.3.1. To be done.

  The structure of the alkaline phosphatase contained in the composition of the present invention (eg, primary structure, secondary structure, tertiary structure, quaternary structure, etc.) is not particularly limited. For example, alkaline phosphatase may or may not have a sugar chain. Further, the alkaline phosphatase may be any isozyme that can exist based on the difference in the structure of the protein molecule (for example, the amino acid sequence of the protein molecule), sugar chain modification, and the like. Further, the alkaline phosphatase may be a monomer (monomer) formed from one subunit, or an oligomer (eg dimer, tetramer, etc.) formed from two or more subunits. ). The oligomer may be a homo-oligomer or a hetero-oligomer.

  The animal from which the alkaline phosphatase contained in the composition of the present invention is derived is not particularly limited. Examples of animals from which alkaline phosphatase is derived include bovine, shrimp, and microorganisms into which a gene encoding alkaline phosphatase has been introduced. Since bovine alkaline phosphatase has high alkaline phosphatase activity, bovine is preferable as the animal from which alkaline phosphatase is derived. When the alkaline phosphatase is derived from bovine, the organ from which alkaline phosphatase is derived is preferably the small intestine or the large intestine.

  The alkaline phosphatase contained in the composition of the present invention may be a wild type or a mutant type. The mutant alkaline phosphatase has, for example, a protein molecule having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence of the protein molecule of the wild-type alkaline phosphatase. The amino acid sequence of the protein molecule possessed by the mutant alkaline phosphatase is preferably 70% or more, more preferably 75% or more, even more preferably 80% or more, with respect to the amino acid sequence of the protein molecule possessed by the wild-type alkaline phosphatase. More preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more sequence identity.

In a preferred embodiment, alkaline phosphatase has the following (a) and (b):
(A) alkaline phosphatase comprising a protein molecule consisting of the amino acid sequence of SEQ ID NO: 4; and (b) having 70% or more sequence identity with the amino acid sequence of SEQ ID NO: 4, It is selected from alkaline phosphatases having a protein molecule consisting of an amino acid sequence comprising positions 71 to 130 of the described amino acid sequence. In this embodiment, the composition of the present invention may contain one alkaline phosphatase selected from the above (a) and (b), or 2 selected from the above (a) and (b). It may contain more than one type of alkaline phosphatase.

  The amino acid sequence of the protein molecule of alkaline phosphatase (a) (that is, the amino acid sequence set forth in SEQ ID NO: 4) corresponds to the amino acid sequence of the protein molecule of bovine alkaline phosphatase. Therefore, the alkaline phosphatase of the above (a) includes alkaline phosphatase derived from bovine.

  The amino acid sequence of the protein molecule possessed by alkaline phosphatase of the above (b) is preferably 70% or more, more preferably 75% or more, even more preferably 80% or more, still more preferably the amino acid sequence of SEQ ID NO: 4. It has a sequence identity of 85% or more, even more preferably 90% or more, even more preferably 95% or more.

  The alkaline phosphatase of the above (b) includes both wild-type alkaline phosphatase (for example, alkaline phosphatase derived from animals other than bovine, alkaline phosphatase derived from bovine having polymorphism, etc.) and mutant alkaline phosphatase. The positions at which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 4 are positions other than positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4.

  The alkaline phosphatases of the above (a) and (b) can produce a peptide fragment group (A), a peptide fragment group (B), a peptide fragment group (C), a first to a third peptide fragment, etc. by decomposition thereof. ..

[Peptide fragment group (A)]
The composition according to the first aspect contains the peptide fragment group (A). The peptide fragment group (A) is composed of two or more kinds of peptide fragments, and each peptide fragment constituting the peptide fragment group (A) is a continuous sequence selected from positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4. It consists of 5 to 50 amino acid residues. The peptide fragment group (A) is a peptide fragment contained in the composition according to the first aspect, in which 5 to 50 consecutive amino acid residues selected from positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4 remain. It is composed of all peptide fragments corresponding to the peptide fragment consisting of the base. In a preferred embodiment, the peptide fragment group (A) is composed of 3 or more kinds of peptide fragments.

  The amino acid sequence of each peptide fragment constituting the peptide fragment group (A) is a part (position consisting of a plurality of consecutive amino acid residues) at positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. That is, the peptide fragment group (A) can be generated by the decomposition of positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. This does not mean that the alkaline phosphatase contained in the composition according to the first aspect needs to include positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. The alkaline phosphatase contained in the composition according to the first aspect may not include positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. However, the alkaline phosphatase contained in the composition according to the first aspect preferably contains positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4.

  The amino acid sequence of each peptide fragment constituting the peptide fragment group (A) is not particularly limited as long as it is a part of positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4, but preferably it is set forth in SEQ ID NO: 4. Part of the 75-130 position of the amino acid sequence, more preferably part of the 75-125 position of the amino acid sequence set forth in SEQ ID NO: 4, and even more preferably, positions 75-120 of the amino acid sequence set forth in SEQ ID NO: 4. Is part of.

  The number of amino acid residues constituting each peptide fragment constituting the peptide fragment group (A) is not particularly limited as long as it is 5 to 50, but preferably 5 to 45, more preferably 10 to 40, and further It is preferably 10 to 30 pieces.

  In a preferred embodiment, the peptide fragment group (A) comprises a first peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 1, a second peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2, and SEQ ID NO: 3 1 type, 2 types or 3 types of peptide fragments selected from the group consisting of the third peptide fragment consisting of the amino acid sequence described in 1. In this embodiment, the peptide fragment group (A) may or may not include peptide fragments other than the first to third peptide fragments.

  The amino acid sequence described in SEQ ID NO: 1 (EAEAEFLIPAEEENPAFWNRQAAQ) corresponds to positions 86 to 109 of the amino acid sequence described in SEQ ID NO: 4. The amino acid sequence (EGVSLEKREAEAE) set forth in SEQ ID NO: 2 corresponds to positions 78 to 90 of the amino acid sequence set forth in SEQ ID NO: 4. The amino acid sequence (IPAEEENPAFWNR) described in SEQ ID NO: 3 corresponds to positions 93 to 105 of the amino acid sequence described in SEQ ID NO: 4.

  Peptide fragment group (A) can be produced by degradation of alkaline phosphatase containing positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. The peptide fragment group (A) may be one generated by decomposition of alkaline phosphatase not contained in the composition according to the first aspect, but usually, it is an alkali contained in the composition according to the first aspect. It is caused by the decomposition of phosphatase. Therefore, in a preferred embodiment, the alkaline phosphatase contained in the composition according to the first aspect is an alkaline phosphatase capable of producing the peptide fragment group (A). In a preferred embodiment, the alkaline phosphatase capable of producing the peptide fragment group (A) is selected from the alkaline phosphatases of (a) and (b) above. In this embodiment, the composition according to the first aspect contains one or more alkaline phosphatases selected from the alkaline phosphatases of (a) and (b) above.

[Peptide fragment group (B)]
The composition according to the second aspect contains the peptide fragment group (B). The peptide fragment group (B) is composed of two or more kinds of peptide fragments, and each peptide fragment constituting the peptide fragment group (B) is a continuous sequence selected from positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4. It consists of 5 to 50 amino acid residues and includes positions 86 to 90 (EAEAE) of the amino acid sequence set forth in SEQ ID NO: 4. Peptide fragment group (B) is a peptide fragment contained in the composition according to the second aspect, which has 5 to 50 consecutive amino acid residues selected from positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. It is composed of all the peptide fragments corresponding to the peptide fragment containing the base group and containing the 86th to 90th positions of the amino acid sequence of SEQ ID NO: 4.

  In the amino acid sequence of each peptide fragment constituting the peptide fragment group (B), the position including positions 86 to 90 of the amino acid sequence set forth in SEQ ID NO: 4 is not particularly limited. The position containing positions 86 to 90 of the amino acid sequence of SEQ ID NO: 4 is any of the N-terminal portion of the peptide fragment, the C-terminal portion of the peptide fragment, and the portion other than the N-terminal portion and the C-terminal portion of the peptide fragment. May be

  The amino acid sequence of each peptide fragment constituting the peptide fragment group (B) is a part (position consisting of a plurality of consecutive amino acid residues) at positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. That is, the peptide fragment group (B) can be generated by the decomposition of positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. This does not mean that the alkaline phosphatase contained in the composition according to the second aspect needs to include positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. The alkaline phosphatase contained in the composition according to the second aspect may not include positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. However, the alkaline phosphatase contained in the composition according to the second aspect preferably contains positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4.

  The amino acid sequence of each peptide fragment constituting the peptide fragment group (B) is a part of positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4, and positions 86 to 90 of the amino acid sequence set forth in SEQ ID NO: 4 It is not particularly limited as long as it includes, but is preferably a part of positions 71 to 125 of the amino acid sequence set forth in SEQ ID NO: 4, more preferably a part of positions 75 to 120 of the amino acid sequence set forth in SEQ ID NO: 4, and even more Preferably, it is a part of positions 75 to 115 of the amino acid sequence set forth in SEQ ID NO: 4.

  The number of amino acid residues constituting each peptide fragment constituting the peptide fragment group (B) is not particularly limited as long as it is 5 to 50, but preferably 5 to 40, more preferably 10 to 40, and further It is preferably 10 to 30 pieces.

  In a preferred embodiment, the peptide fragment group (B) is composed of a first peptide fragment consisting of the amino acid sequence shown in SEQ ID NO: 1 and a second peptide fragment consisting of the amino acid sequence shown in SEQ ID NO: 2. It contains one or two selected peptide fragments. In this embodiment, the peptide fragment group (B) may or may not include peptide fragments other than the first and second peptide fragments.

The amino acid sequence shown in SEQ ID NO: 1 ( EAEAE FLIPAEEENPAFWNRQAAQ) corresponds to positions 86 to 109 of the amino acid sequence shown in SEQ ID NO: 4 and includes positions 86 to 90 of the amino acid sequence shown in SEQ ID NO: 4 (the underlined portion is Corresponding to positions 86 to 90 of the amino acid sequence set forth in SEQ ID NO: 4). The amino acid sequence of SEQ ID NO: 2 (EGVSLEKR EAEAE ) corresponds to positions 78 to 90 of the amino acid sequence of SEQ ID NO: 4 and includes positions 86 to 90 of the amino acid sequence of SEQ ID NO: 4 (the underlined portion is Corresponding to positions 86 to 90 of the amino acid sequence set forth in SEQ ID NO: 4).

  Peptide fragment group (B) can be produced by degradation of alkaline phosphatase containing positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. The peptide fragment group (B) may be one generated by decomposition of alkaline phosphatase not contained in the composition according to the second aspect, but is usually an alkali contained in the composition according to the second aspect. It is caused by the decomposition of phosphatase. Therefore, in a preferred embodiment, the alkaline phosphatase contained in the composition according to the second aspect is an alkaline phosphatase capable of producing the peptide fragment group (B). In a preferred embodiment, the alkaline phosphatase capable of producing the peptide fragment group (B) is selected from the alkaline phosphatases of (a) and (b) above. In this embodiment, the composition according to the second aspect contains one or more alkaline phosphatases selected from the alkaline phosphatases of (a) and (b) above.

[Peptide fragment group (C)]
The composition according to the third aspect contains the peptide fragment group (C). The peptide fragment group (C) is composed of two or more kinds of peptide fragments, and each peptide fragment constituting the peptide fragment group (C) is a continuous sequence selected from positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4. Consisting of 13 to 50 amino acid residues, and includes positions 93 to 105 (IPAEEENPAFWNR) of the amino acid sequence set forth in SEQ ID NO: 4. The peptide fragment group (C) is a peptide fragment contained in the composition according to the third aspect, which has 13 to 50 consecutive amino acid residues selected from positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. It is composed of all the peptide fragments corresponding to the peptide fragment containing the base group and containing the 93rd to 105th positions of the amino acid sequence of SEQ ID NO: 4.

  In the amino acid sequence of each peptide fragment constituting the peptide fragment group (C), the position including positions 93 to 105 of the amino acid sequence set forth in SEQ ID NO: 4 is the N-terminal portion of the peptide fragment, the C-terminal portion of the peptide fragment, In addition, it may be any portion other than the N-terminal portion and the C-terminal portion of the peptide fragment.

  The amino acid sequence of each peptide fragment constituting the peptide fragment group (C) is a part (position consisting of a plurality of consecutive amino acid residues) at positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. That is, the peptide fragment group (C) can be generated by the decomposition of positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. This does not mean that the alkaline phosphatase contained in the composition according to the third aspect needs to include positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. The alkaline phosphatase contained in the composition according to the third aspect may not include positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. However, the alkaline phosphatase contained in the composition according to the third aspect preferably contains positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4.

  The amino acid sequence of each peptide fragment constituting the peptide fragment group (C) is a part of positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4, and positions 93 to 105 of the amino acid sequence set forth in SEQ ID NO: 4 It is not particularly limited as long as it includes, but is preferably a part of positions 75 to 130 of the amino acid sequence set forth in SEQ ID NO: 4, more preferably a part of positions 85 to 130 of the amino acid sequence set forth in SEQ ID NO: 4, and even more Preferably, it is a part of positions 85 to 120 of the amino acid sequence set forth in SEQ ID NO: 4.

  The number of amino acid residues constituting each peptide fragment constituting the peptide fragment group (C) is not particularly limited as long as it is 13 to 50, but preferably 13 to 45, more preferably 13 to 40, and even more It is preferably 13 to 30.

  In a preferred embodiment, the peptide fragment group (C) is composed of a first peptide fragment consisting of the amino acid sequence shown in SEQ ID NO: 1 and a third peptide fragment consisting of the amino acid sequence shown in SEQ ID NO: 3. It contains one or two selected peptide fragments. In this embodiment, the peptide fragment group (C) may or may not include peptide fragments other than the first and third peptide fragments.

The amino acid sequence of SEQ ID NO: 1 (EAEAEFL IPAEEENPAFWNR QAAQ) corresponds to positions 86 to 109 of the amino acid sequence of SEQ ID NO: 4 and includes positions 93 to 105 of the amino acid sequence of SEQ ID NO: 4 (underlined portion). Corresponds to positions 93 to 105 of the amino acid sequence set forth in SEQ ID NO: 4). The amino acid sequence (IPAEEENPAFWNR) described in SEQ ID NO: 3 corresponds to positions 93 to 105 of the amino acid sequence described in SEQ ID NO: 4.

  The peptide fragment group (C) can be produced by the decomposition of alkaline phosphatase containing positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. The peptide fragment group (C) may be one produced by decomposition of alkaline phosphatase not contained in the composition according to the third aspect, but usually, it is an alkali contained in the composition according to the third aspect. It is caused by the decomposition of phosphatase. Therefore, in a preferred embodiment, the alkaline phosphatase contained in the composition according to the third aspect is an alkaline phosphatase capable of producing the peptide fragment group (C). In a preferred embodiment, the alkaline phosphatase capable of producing the peptide fragment group (C) is selected from the alkaline phosphatases of (a) and (b) above. In this embodiment, the composition according to the third aspect contains one or more alkaline phosphatases selected from the alkaline phosphatases of (a) and (b) above.

[Second peptide fragment]
The composition according to the fourth aspect contains a second peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2. In a preferred embodiment, the composition according to the fourth aspect is a group consisting of a first peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a third peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 3. It further contains one or two peptide fragments selected from In this embodiment, the composition according to the fourth aspect may or may not contain peptide fragments other than the first to third peptide fragments.

  The second peptide fragment can be generated by degradation of alkaline phosphatase containing positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. The second peptide fragment may be generated by decomposition of alkaline phosphatase that is not contained in the composition according to the fourth aspect, but it is usually the alkaline phosphatase contained in the composition according to the fourth aspect. It was caused by the decomposition of. Therefore, in a preferred embodiment, the alkaline phosphatase contained in the composition according to the fourth aspect is an alkaline phosphatase capable of giving rise to the second peptide fragment. In a preferred embodiment, the alkaline phosphatase capable of producing the second peptide fragment is selected from the alkaline phosphatases of (a) and (b) above. In this embodiment, the composition according to the fourth aspect contains one or more alkaline phosphatases selected from the alkaline phosphatases of (a) and (b) above.

[Ratio of Content of Peptide Fragment Group (A) to Content of Alkaline Phosphatase]
In the composition according to the first aspect, the ratio of the content of the peptide fragment group (A) to the content of alkaline phosphatase is represented by the following formula (A):
(X _A /Y)×100≦5.0000 (A)
Meet

In the above formula (A), X _A is the peak of the peptide fragment group (A) calculated by the automatic integration method from the extracted ion chromatogram obtained by the LC-MS / MS analysis of the composition according to the first aspect. Represents an area value, and Y represents a peak area value of alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by LC-UV analysis of the composition according to the first aspect. The "peak area value of peptide fragment group (A)" means the total peak area value of all kinds of peptide fragments constituting the peptide fragment group (A). The "peak area value of alkaline phosphatase" means, when the composition according to the first aspect contains one type of alkaline phosphatase, the peak area value of the one type of alkaline phosphatase, and the composition according to the first aspect is When containing two or more types of alkaline phosphatase, it means the total peak area value of the two or more types of alkaline phosphatase (that is, the total peak area value of all types of alkaline phosphatase contained in the composition according to the first aspect). To do.

  LC-MS / MS analysis and LC-UV analysis were performed using a sample in which the ratio of the content of the peptide fragment group (A) to the content of alkaline phosphatase was the same as the composition according to the first aspect. It The LC-MS / MS analysis and the LC-UV analysis can be performed using, for example, an aqueous solution prepared from the composition according to the first aspect, and having an alkaline phosphatase concentration of 10% by weight. ..

  LC-MS / MS analysis is a type of hyphenated method. The hyphenated method is a method in which a chromatograph such as a gas chromatograph or a liquid chromatograph is connected to a mass spectrometer for analysis. LC-MS / MS analysis is a method of analyzing by connecting a liquid chromatograph (LC) and a tandem mass spectrometer (MS / MS). In LC-MS / MS analysis, the components to be analyzed separated by the liquid chromatograph are ionized, the generated ions are separated by a tandem mass spectrometer, and specific mass ions are fragmented and detected.

  The extracted ion chromatogram is a hyphenated method, in which a mass spectrum is measured at regular time intervals and stored in a computer, and then a relative intensity at a specific (not limited to one) m / z value is read out, It is a chromatogram expressed as a function of time. The ion m / z value of each peptide fragment constituting the peptide fragment group (A) used for detecting the peak of each peptide fragment constituting the peptide fragment group (A) is preferably 50 to 2200, and further It is preferably 200 to 1500, and even more preferably 300 to 1200. For the ion of the peptide fragment for which the m / z value is specified, an extracted ion chromatogram of the peptide fragment can be created based on the m / z value. The m / z value of the ion of the first peptide fragment was 920.7682, the m / z value of the ion of the second peptide fragment was 723.88572, and the m / z value of the ion of the third peptide fragment was 786.8857. Is. For peptide fragments whose m / z values are not specified, it is confirmed whether or not a certain peak corresponds to the peak of a predetermined peptide fragment by amino acid sequence analysis of the peptide fragment showing the peak, and then the m of the peak is determined. An extracted ion chromatogram of the peptide fragment can be created based on the / z value.

  LC-UV analysis is a method of analyzing by connecting a liquid chromatograph (LC) and an ultraviolet ray detector (UV detector). In the LC-UV analysis, alkaline phosphatase is detected as a component having an absorption at 214 nm.

  The conditions of LC-MS / MS analysis and LC-UV analysis are as follows.

<< LC-MS / MS analysis conditions >>
<Device configuration>
Mass spectrometer: maXis impact (manufactured by Bruker Daltonics, Inc.)
<Mass spectrometry conditions>
Ionization method: ESI
Measurement ion: Positive ion Capillary voltage: 4500V
Nebulizer: 2.0 bar
Dry gas: 8.0L / min Detector voltage: 1823V
Measuring range (MS): m / z 50 to 2200
<MS / MS conditions>
Measuring range (MS): m / z 50 to 2200
Collision gas: Nitrogen

<< LC-UV analysis conditions >>
<Device configuration>
Liquid chromatograph: LC-30A system (manufactured by Shimadzu Corporation)
Detector: UV-Vis (190-900 nm, Shimadzu)
<Liquid chromatography conditions>
Column: Acquity BEH C18 1.7 μm (manufactured by Waters Corporation) Column size: 2.1 mm × 100 mm
Column temperature: 50 ° C
Mobile phase flow rate: 0.2 mL / min Mobile phase A: Water / formic acid mixture (1000: 1)
Mobile phase B: Acetonitrile / water / formic acid mixture (900: 100: 1)
Injection volume: 20 μL
Gradient program:

The value of (X _A / Y) × 100 is not particularly limited as long as it is 5.0000 or less, but the smaller the better. The value of (X _A / Y) × 100 is preferably 4.5000 or less, more preferably 4.0000 or less, and even more preferably 3.5000 or less. The lower limit value of (X _A / Y) × 100 is the detection limit value. However, from the viewpoint of obtaining an effect commensurate with the effort for reducing the value of (X _A / Y) × 100 (for example, separation and removal of the peptide fragment group (A) by purification), (X _A / Y) × 100 The value is preferably 0.0800 or more, more preferably 0.1000 or more, even more preferably 0.1500 or more.

  Calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis using an aqueous solution prepared from the composition according to the first aspect and having an alkaline phosphatase concentration of 10% by weight. In addition, the smaller the peak area value of the peptide fragment group (A), the more preferable. The peak area value of the peptide fragment group (A) is preferably 14,000 or less, more preferably 12,000 or less, still more preferably 10,000 or less. The lower limit of the peak area value of the peptide fragment group (A) is the detection limit value. However, from the viewpoint of obtaining an effect commensurate with the effort for reducing the peak area value of the peptide fragment group (A) (for example, separation and removal of the peptide fragment group (A) by purification), the peak area value of the peptide fragment group (A) is obtained. Is preferably 600 or more, more preferably 800 or more, and even more preferably 1000 or more.

  Alkaline phosphatase calculated from the chromatogram obtained by LC-UV analysis using an aqueous solution prepared from the composition according to the first aspect and having an alkaline phosphatase concentration of 10% by weight, by an automatic integration method. The peak area value of is preferably 200,000 or more, more preferably 220,000 or more, still more preferably 240000 or more. The upper limit of the peak area value of alkaline phosphatase is not particularly limited. The peak area value of alkaline phosphatase is preferably 500000 or less, more preferably 400000 or less, and even more preferably 350,000 or less.

[Ratio of content of peptide fragment group (B) to content of alkaline phosphatase]
In the composition according to the second aspect, the ratio of the content of the peptide fragment group (B) to the content of alkaline phosphatase is represented by the following formula (B):
(X _B /Y)×100≦2.4000 (B)
Meet

In the above formula (B), X _B is the peak of the peptide fragment group (B) calculated by the automatic integration method from the extracted ion chromatogram obtained by the LC-MS / MS analysis of the composition according to the second aspect. Represents an area value, and Y represents a peak area value of alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by LC-UV analysis of the composition according to the second embodiment. The “peak area value of peptide fragment group (B)” means the total peak area value of all kinds of peptide fragments constituting the peptide fragment group (B). The “peak area value of alkaline phosphatase” means, when the composition according to the second aspect contains one type of alkaline phosphatase, the peak area value of the one type of alkaline phosphatase, and the composition according to the second aspect is In the case of containing two or more types of alkaline phosphatase, it means the total peak area value of the two or more types of alkaline phosphatase (that is, the total peak area value of all types of alkaline phosphatase contained in the composition according to the second aspect). To do. The description regarding the above formula (A) (including the description regarding the LC-MS / MS analysis and the LC-UV analysis) also applies to the above formula (B) unless otherwise specified.

  The ion m / z value of each peptide fragment constituting the peptide fragment group (B) used for detecting the peak of each peptide fragment constituting the peptide fragment group (B) is preferably 50 to 2200, and further It is preferably 200 to 1500, and even more preferably 300 to 1200.

The value of (X _B / Y) × 100 is not particularly limited as long as it is 2.4000 or less, but the smaller the better. The value of (X _B / Y) × 100 is preferably 2.2000 or less, more preferably 2.0000 or less, and even more preferably 1.5000 or less. The lower limit of (X _B / Y) × 100 is the detection limit value. However, from the viewpoint of obtaining an effect commensurate with the effort for reducing the value of (X _B / Y) × 100 (for example, separation and removal of peptide fragment group (B) by purification), (X _B / Y) × 100 The value is preferably 0.0800 or more, more preferably 0.1000 or more, even more preferably 0.1500 or more.

  Calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis using an aqueous solution prepared from the composition according to the second aspect, the aqueous solution having an alkaline phosphatase concentration of 10% by weight. Moreover, the smaller the peak area value of the peptide fragment group (B), the more preferable. The peak area value of the peptide fragment group (B) is preferably 6000 or less, more preferably 5000 or less, and even more preferably 4000 or less. The lower limit of the peak area value of the peptide fragment group (B) is the detection limit value. However, from the viewpoint of obtaining an effect commensurate with the effort for reducing the peak area value of the peptide fragment group (B) (for example, separation and removal of the peptide fragment group (B) by purification), the peak area of the peptide fragment group (B) is The value is preferably 500 or more, more preferably 800 or more, even more preferably 1000 or more.

  Alkaline phosphatase calculated from the chromatogram obtained by LC-UV analysis using an aqueous solution prepared from the composition according to the second aspect and having an alkaline phosphatase concentration of 10% by weight, by an automatic integration method. The peak area value of is preferably 200,000 or more, more preferably 220,000 or more, still more preferably 240000 or more. The upper limit of the peak area value of alkaline phosphatase is not particularly limited. The peak area value of alkaline phosphatase is preferably 500000 or less, more preferably 400000 or less, and even more preferably 350,000 or less.

  The composition according to the second aspect comprises, in addition to the peptide fragment group (B), a peptide fragment consisting of 5 to 50 consecutive amino acid residues selected from positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. In the containing embodiment, the composition according to the second aspect may or may not satisfy the above formula (A).

[Ratio of Content of Peptide Fragment Group (C) to Content of Alkaline Phosphatase]
In the composition according to the third aspect, the ratio of the content of the peptide fragment group (C) to the content of alkaline phosphatase is represented by the following formula (C):
(X _C /Y)×100≦4.5000 (C)
Meet

In the above formula (C), X _C is the peak of the peptide fragment group (C) calculated by the automatic integration method from the extracted ion chromatogram obtained by the LC-MS / MS analysis of the composition according to the third aspect. Represents an area value, and Y represents a peak area value of alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by LC-UV analysis of the composition according to the third aspect. The "peak area value of peptide fragment group (C)" means the total peak area value of all kinds of peptide fragments constituting the peptide fragment group (C). The "peak area value of alkaline phosphatase" means, when the composition according to the third aspect contains one type of alkaline phosphatase, the peak area value of the one type of alkaline phosphatase, and the composition according to the third aspect is When it contains two or more kinds of alkaline phosphatase, it means the total peak area value of the two or more kinds of alkaline phosphatase (that is, the total peak area value of all kinds of alkaline phosphatase contained in the composition according to the third aspect). To do. The description regarding the above formula (A) (including the description regarding the LC-MS / MS analysis and the LC-UV analysis) also applies to the above formula (C), unless otherwise specified.

  The m / z value of the ion of each peptide fragment constituting the peptide fragment group (C) used for detecting the peak of each peptide fragment constituting the peptide fragment group (C) is preferably 50 to 2200, further It is preferably 200 to 1500, and even more preferably 300 to 1200.

The value of (X _C / Y) × 100 is not particularly limited as long as it is 4.5000 or less, but the smaller the better. The value of (X _C / Y) × 100 is preferably 4.0000 or less, more preferably 3.0000 or less, and even more preferably 2.5000 or less. The lower limit value of (X _C / Y) × 100 is the detection limit value. However, from the viewpoint of obtaining an effect commensurate with the effort to reduce the value of (X _C / Y) × 100 (for example, separation and removal of the peptide fragment group (C) by purification), (X _C / Y) × 100 The value is preferably 0.0800 or more, more preferably 0.1000 or more, even more preferably 0.1500 or more.

  Calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis using an aqueous solution prepared from the composition according to the third aspect and having an alkaline phosphatase concentration of 10% by weight. Moreover, the smaller the peak area value of the peptide fragment group (C), the more preferable. The peak area value of the peptide fragment group (C) is preferably 12,000 or less, more preferably 10,000 or less, and even more preferably 8,000 or less. The lower limit of the peak area value of the peptide fragment group (C) is the detection limit value. However, from the viewpoint of obtaining an effect commensurate with the effort for reducing the peak area value of the peptide fragment group (C) (for example, separation and removal of the peptide fragment group (C) by purification), the peak area of the peptide fragment group (C) is obtained. The value is preferably 500 or more, more preferably 800 or more, even more preferably 1000 or more.

  Alkaline phosphatase calculated from the chromatogram obtained by LC-UV analysis using an aqueous solution prepared from the composition according to the third aspect and having an alkaline phosphatase concentration of 10% by weight, by an automatic integration method. The peak area value of is preferably 200,000 or more, more preferably 220,000 or more, still more preferably 240000 or more. The upper limit of the peak area value of alkaline phosphatase is not particularly limited. The peak area value of alkaline phosphatase is preferably 500000 or less, more preferably 400000 or less, and even more preferably 350,000 or less.

  In addition to the peptide fragment group (C), the composition according to the third aspect provides a peptide fragment consisting of 5 to 50 consecutive amino acid residues selected from positions 71 to 130 of the amino acid sequence set forth in SEQ ID NO: 4. In the containing embodiment, the composition according to the third aspect may or may not satisfy the above formula (A).

[Ratio of the content of the first peptide fragment to the content of alkaline phosphatase]
In the embodiment in which the composition according to the first, second, third or fourth aspect contains the first peptide fragment, the ratio of the content of the first peptide fragment to the content of alkaline phosphatase is represented by the following formula ( 1):
(X ₁ /Y)×100≦1.0000 (1)
It is preferable to satisfy.

In the above formula (1), X ₁ was calculated by the automatic integration method from the extracted ion chromatogram obtained by the LC-MS / MS analysis of the composition according to the first, second, third or fourth aspect. Represents the peak area value of the first peptide fragment, and Y is calculated by an automatic integration method from a chromatogram obtained by LC-UV analysis of the composition according to the first, second, third, or fourth aspect. Moreover, the peak area value of alkaline phosphatase is represented. The description regarding the above formula (A) (including the description regarding the LC-MS / MS analysis and the LC-UV analysis) also applies to the above formula (1), unless otherwise specified.

The value of (X ₁ / Y) × 100 is not particularly limited as long as it is 1.0000 or less, but the smaller the better. The value of (X ₁ / Y) × 100 is preferably 0.9000 or less, more preferably 0.8000 or less, and even more preferably 0.7000 or less. The lower limit of (X ₁ / Y) × 100 is the detection limit value. However, from the viewpoint of obtaining an effect commensurate with the effort for reducing the value of (X ₁ / Y) × 100 (for example, separation and removal of the first peptide fragment by purification), the value of (X ₁ / Y) × 100 Is preferably 0.0800 or more, more preferably 0.1000 or more, and even more preferably 0.1500 or more.

  Extraction ions obtained by LC-MS / MS analysis using an aqueous solution prepared from the composition according to the first, second, third or fourth aspect, wherein the alkaline phosphatase concentration is 10% by weight. The smaller the peak area value of the first peptide fragment calculated from the chromatogram by the automatic integration method, the better. The peak area value of the first peptide fragment is preferably 3000 or less, more preferably 2500 or less, and even more preferably 2000 or less. The lower limit of the peak area value of the first peptide fragment is the detection limit value. However, from the viewpoint of obtaining an effect commensurate with the effort for reducing the peak area value of the first peptide fragment (for example, separation and removal of the first peptide fragment by purification), the peak area value of the first peptide fragment is It is preferably 500 or more, more preferably 800 or more, and even more preferably 1000 or more.

  Automatic from a chromatogram obtained by LC-UV analysis using an aqueous solution prepared from the composition according to the first, second, third or fourth aspect, wherein the aqueous solution has an alkaline phosphatase concentration of 10% by weight. The peak area value of alkaline phosphatase calculated by the integration method is preferably 200,000 or more, more preferably 220,000 or more, still more preferably 240000 or more. The upper limit of the peak area value of alkaline phosphatase is not particularly limited. The peak area value of alkaline phosphatase is preferably 500000 or less, more preferably 400000 or less, and even more preferably 350,000 or less.

[Ratio of content of second peptide fragment to content of alkaline phosphatase]
In the composition according to the fourth aspect, the ratio of the content of the second peptide fragment to the content of alkaline phosphatase is represented by the following formula (2):
(X ₂ /Y)×100≦0.8000 (2)
Meet

In an embodiment in which the composition according to the first, second or third aspect contains the second peptide fragment, the ratio of the content of the second peptide fragment to the content of alkaline phosphatase is represented by the following formula (2):
(X ₂ /Y)×100≦0.8000 (2)
It is preferable to satisfy.

In the above formula (2), X ₂ was calculated by the automatic integration method from the extracted ion chromatogram obtained by the LC-MS / MS analysis of the composition according to the first, second, third or fourth aspect. , Y represents the peak area value of the second peptide fragment, and Y was calculated by the automatic integration method from the chromatogram obtained by the LC-UV analysis of the composition according to the first, second, third, or fourth aspect. Moreover, the peak area value of alkaline phosphatase is represented. The description regarding the above formula (A) (including the description regarding the LC-MS / MS analysis and the LC-UV analysis) also applies to the above formula (2) unless otherwise specified.

The value of (X ₂ / Y) × 100 is not particularly limited as long as it is 0.8000 or less, but the smaller the better. The value of (X ₂ / Y) × 100 is preferably 0.7000 or less, more preferably 0.6000 or less, and even more preferably 0.5000 or less. The lower limit value of (X ₂ / Y) × 100 is the detection limit value. However, from the viewpoint of obtaining an effect commensurate with the effort for reducing the value of (X ₂ / Y) × 100 (for example, separation and removal of the second peptide fragment by purification), the value of (X ₂ / Y) × 100 Is preferably 0.0800 or more, more preferably 0.1000 or more, and even more preferably 0.1500 or more.

  Extraction ions obtained by LC-MS / MS analysis using an aqueous solution prepared from the composition according to the first, second, third or fourth aspect, wherein the alkaline phosphatase concentration is 10% by weight. The smaller the peak area value of the second peptide fragment calculated from the chromatogram by the automatic integration method, the more preferable. The peak area value of the second peptide fragment is preferably 2000 or less, more preferably 1800 or less, even more preferably 1500 or less. The lower limit of the peak area value of the second peptide fragment is the detection limit value. However, from the viewpoint of obtaining an effect commensurate with the effort for reducing the peak area value of the second peptide fragment (for example, separation and removal of the second peptide fragment by purification), the peak area value of the second peptide fragment is It is preferably 500 or more, more preferably 800 or more, and even more preferably 1000 or more.

  Automatic from a chromatogram obtained by LC-UV analysis using an aqueous solution prepared from the composition according to the first, second, third or fourth aspect, wherein the aqueous solution has an alkaline phosphatase concentration of 10% by weight. The peak area value of alkaline phosphatase calculated by the integration method is preferably 200,000 or more, more preferably 220,000 or more, still more preferably 240000 or more. The upper limit of the peak area value of alkaline phosphatase is not particularly limited. The peak area value of alkaline phosphatase is preferably 500000 or less, more preferably 400000 or less, and even more preferably 350,000 or less.

[Ratio of content of third peptide fragment to content of alkaline phosphatase]
In an embodiment in which the composition according to the first, second, third or fourth aspect contains a third peptide fragment, the ratio of the content of the third peptide fragment to the content of alkaline phosphatase is represented by the following formula ( 3):
(X ₃ /Y)×100≦2.3000 (3)
It is preferable to satisfy.

In the above formula (3), X ₃ was calculated by the automatic integration method from the extracted ion chromatogram obtained by the LC-MS / MS analysis of the composition according to the first, second, third or fourth aspect. , Y represents the peak area value of the third peptide fragment, and Y is calculated by an automatic integration method from the chromatogram obtained by LC-UV analysis of the composition according to the first, second, third, or fourth aspect. Moreover, the peak area value of alkaline phosphatase is represented. The description regarding the above formula (A) (including the description regarding the LC-MS / MS analysis and the LC-UV analysis) also applies to the above formula (3), unless otherwise specified.

The value of (X ₃ / Y) × 100 is not particularly limited as long as it is 2.3000 or less, but the smaller the better. The value of (X ₃ / Y) × 100 is preferably 2.0000 or less, more preferably 1.5000 or less, and even more preferably 1.0000 or less. The lower limit of (X ₃ / Y) × 100 is the detection limit value. However, from the viewpoint of obtaining an effect commensurate with the effort for reducing the value of (X ₃ / Y) × 100 (for example, separation and removal of the third peptide fragment by purification), the value of (X ₃ / Y) × 100 Is preferably 0.1500 or more, more preferably 0.1000 or more, still more preferably 0.0800 or more.

  Extraction ions obtained by LC-MS / MS analysis using an aqueous solution prepared from the composition according to the first, second, third or fourth aspect, wherein the alkaline phosphatase concentration is 10% by weight. The smaller the peak area value of the third peptide fragment calculated from the chromatogram by the automatic integration method, the better. The peak area value of the third peptide fragment is preferably 6000 or less, more preferably 3000 or less, and even more preferably 1000 or less. The lower limit of the peak area value of the third peptide fragment is the detection limit value. However, from the viewpoint of obtaining an effect commensurate with the effort for reducing the peak area value of the third peptide fragment (for example, separation and removal of the third peptide fragment by purification), the peak area value of the third peptide fragment is It is preferably 500 or more, more preferably 800 or more, and even more preferably 1000 or more.

  Automatic from a chromatogram obtained by LC-UV analysis using an aqueous solution prepared from the composition according to the first, second, third or fourth aspect, wherein the aqueous solution has an alkaline phosphatase concentration of 10% by weight. The peak area value of alkaline phosphatase calculated by the integration method is preferably 200,000 or more, more preferably 220,000 or more, still more preferably 240000 or more. The upper limit of the peak area value of alkaline phosphatase is not particularly limited. The peak area value of alkaline phosphatase is preferably 500000 or less, more preferably 400000 or less, and even more preferably 350,000 or less.

[Specific activity of alkaline phosphatase]
The composition of the present invention preferably has an alkaline phosphatase specific activity of 2000 U / mg or more. The alkaline phosphatase specific activity of the composition of the present invention is more preferably 2500 U / mg or more, still more preferably 3000 U / mg or more. The alkaline phosphatase specific activity of the composition of the present invention is measured as follows. The specific activity of alkaline phosphatase can be calculated by measuring the absorbance at 405 nm derived from p-nitrophenol produced by adding alkaline phosphatase to the p-nitrophenyl phosphate aqueous solution.

[Other ingredients]
The compositions of the present invention may contain one or more other ingredients. Examples of other components include aqueous media such as water, metal salts such as magnesium salts and sodium salts, surfactants, organic acids, and glycerin.

  The composition of the present invention can be used in various applications in which alkaline phosphatase activity is required, and may contain one or more other components selected according to the application.

  In one embodiment, the composition of the present invention contains a protein such as an antigen or an antibody. In this embodiment, the composition of the present invention can be used for labeling proteins such as antigens and antibodies with alkaline phosphatase. That is, in one embodiment, the composition of the present invention is a composition for labeling a protein with alkaline phosphatase. Labeling of a protein with alkaline phosphatase can be performed by reacting an alkaline phosphatase succinimide ester product obtained by esterifying a carboxyl group of alkaline phosphatase with succinimide and an amino group of the protein.

In one embodiment, the composition of the present invention contains a substrate for alkaline phosphatase.
In this embodiment, the compositions of the invention can be used to dephosphorylate a substrate for alkaline phosphatase. That is, in one embodiment, the composition of the present invention is a composition for dephosphorylating a substrate of alkaline phosphatase. The substrate of alkaline phosphatase is not particularly limited as long as it is a compound having a phosphate monoester bond.
Examples of the substrate for alkaline phosphatase include nucleic acid, phospholipid, pyrophosphate and the like. Treatment of a substrate of alkaline phosphatase with the composition of the present invention results in hydrolysis of the phosphate monoester bond of the substrate of alkaline phosphatase by alkaline phosphatase, resulting in dephosphorylation of the substrate of alkaline phosphatase.

In a preferred embodiment, the composition of the invention contains nucleic acid. In this embodiment, the compositions of the invention can be used to dephosphorylate nucleic acids. That is, in a preferred embodiment, the composition of the present invention is a composition for dephosphorylating a nucleic acid. The peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment mixed in alkaline phosphatase may adversely affect the dephosphorylation of nucleic acid by alkaline phosphatase. is there. In this respect, in the composition of the present invention, the ratio of the content of the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment to the content of alkaline phosphatase is as described above. Meets the given formula. That is, in the composition of the present invention, the relative content of the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment is reduced. Therefore, by using the composition of the present invention, a peptide fragment group (A), a peptide fragment group (B), a peptide fragment group (C), or a second peptide fragment group (A) that can be generated when dephosphorylating a nucleic acid with alkaline phosphatase. The adverse effect of the peptide fragment can be reduced, and the nucleic acid dephosphorylation efficiency can be improved. By treating the nucleic acid with the composition of the present invention, the 5 ′ end and / or the 3 ′ end of the nucleic acid can be dephosphorylated. By dephosphorylating the 5 ′ end and / or the 3 ′ end of the nucleic acid, the labeling efficiency when labeling the 5 ′ end and / or the 3 ′ end of the nucleic acid with a labeling substance can be improved. This effect is particularly remarkable when ³² P is used as the labeling substance. In addition, dephosphorylation of the 5'end and / or 3'end of the vector used for DNA cloning can prevent self-ligation of the vector, thereby reducing the background of transformed cells. be able to.

  The nucleic acid is, for example, DNA, RNA, PNA (peptide nucleic acid), LNA (Locked Nucleic Acid) or the like, or a nucleic acid derivative. Examples of the nucleic acid derivative include modified nucleotides (for example, alkyl such as halogen and methyl, alkoxy such as methoxy, nucleotide and base containing a group such as thio and carboxymethyl, saturation of double bond, deamination, oxygen, etc. Nucleic acid derivatives including nucleotides that have undergone substitution of sulfur molecules for molecules). The nucleic acid may be single-stranded or double-stranded. Examples of DNA include chromosomal DNA, viral DNA, DNA of bacteria and molds, cDNA, fragments thereof and the like. Examples of RNA include mRNA, rRNA, small RNA, and fragments thereof. The nucleic acid may be chemically synthesized DNA, RNA or the like. Specific examples of the nucleic acid include genes of pathogenic bacteria, viruses and the like or a part thereof, genes causing causative diseases or a part thereof, and the like.

  The nucleic acid can be prepared, for example, by extracting it from a biomaterial, virus, bacterium, mold, food or drink, etc. by a conventional method. Examples of biomaterials include body fluids such as blood, serum, plasma, urine, feces, spinal fluid, saliva, swab, tissue fluid, cells, tissues and the like. The biomaterial may be animal-derived or plant-derived.

  The amount of nucleic acid contained in the composition of the present invention can be appropriately adjusted depending on the purpose of use of the composition of the present invention (for example, detection of target nucleic acid). For example, when the purpose is to detect a certain nucleic acid (target nucleic acid) among the nucleic acids contained in the composition of the present invention, a nucleic acid amplification method such as PCR is carried out using the nucleic acid contained in the composition of the present invention as a template. Thus, the target nucleic acid can be amplified. Thereby, the detection sensitivity of the target nucleic acid can be improved.

  The length (the number of bases) of the nucleic acid can be appropriately adjusted according to the purpose of use of the composition of the present invention (for example, detection of target nucleic acid). For example, for the purpose of detecting a nucleic acid, the length of the nucleic acid is usually 10 to 300 bases, preferably 10 to 100 bases, more preferably 15 to 50 bases. The length of the nucleic acid can be appropriately adjusted by the fragmentation treatment. The length of the nucleic acid is, for example, a length that allows hybridization with the probe. When the nucleic acid is long (for example, 1500 bases or more, particularly 4000 bases or more), it is preferable to subject the nucleic acid to a fragmentation treatment to adjust the length of the nucleic acid to an appropriate length. When carrying out the fragmentation treatment, it is not necessary to select a specific nucleic acid fragment from the generated nucleic acid fragments, and the fragmented product can be used as it is.

  Examples of the method for cleaving nucleic acid for fragmentation include, for example, a method of irradiating with ultrasonic waves, a method of cleaving with an enzyme, a method of cleaving with a restriction enzyme, a method of using a nebulizer, and a method of cleaving with acid or alkali. Methods and the like. In the case of the method of cutting with ultrasonic waves, the nucleic acid can be cut into a desired length by controlling the output intensity and irradiation time of ultrasonic waves with which the nucleic acid is irradiated.

  In a preferred embodiment, the composition of the invention contains dephosphorylated nucleic acid. The description regarding nucleic acids is the same as above. Dephosphorylated nucleic acids have 5'and / or 3'termini dephosphorylated with alkaline phosphatase. In this embodiment, the composition of the present invention can be used to prepare a labeled nucleic acid comprising a dephosphorylated nucleic acid and a labeling substance bound to the dephosphorylated nucleic acid. That is, in a preferred embodiment, the composition of the present invention is a composition for preparing a labeled nucleic acid comprising a dephosphorylated nucleic acid and a labeling substance bound to the dephosphorylated nucleic acid. The peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment mixed in alkaline phosphatase is used when dephosphorylating a nucleic acid by alkaline phosphatase and / or There is a possibility of adversely affecting the binding of the labeling substance to the oxidized nucleic acid. In this respect, in the composition of the present invention, the ratio of the content of the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment to the content of alkaline phosphatase is as described above. Meets the given formula. That is, in the composition of the present invention, the relative content of the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment is reduced. Therefore, by using the composition of the present invention, a peptide fragment group (A) that can be produced when dephosphorylating a nucleic acid with alkaline phosphatase and / or when a labeling substance is bound to the dephosphorylated nucleic acid , The peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment can be reduced, and the dephosphorylation efficiency of the nucleic acid and / or the labeling efficiency of the dephosphorylated nucleic acid can be reduced. Can be improved.

  In a preferred embodiment, the composition of the present invention contains a labeled nucleic acid comprising a dephosphorylated nucleic acid and a labeling substance bound to the dephosphorylated nucleic acid. The description regarding nucleic acids is the same as above. Dephosphorylated nucleic acids have 5'and / or 3'termini dephosphorylated with alkaline phosphatase. The labeling substance binds to the 5'end and / or the 3'end of the dephosphorylated nucleic acid.

  As the labeling substance, for example, a fluorescent dye, a fluorescent protein, a chemiluminescent substance, a metal complex, fine metal particles, biotin, a radioisotope or the like can be used. The reaction conditions for labeling the target nucleic acid can be appropriately adjusted depending on the type of labeling substance. When the labeling substance is a fluorescent dye, the fluorescent dye can be detected using a fluorescence microscope, a fluorescence scanner or the like.

  Examples of fluorescent dyes include fluorescein dyes, rhodamine dyes, Alexa Fluor (Invitrogen) dyes, BODIPY (Invitrogen) dyes, cascade dyes, coumarin dyes, eosin dyes, NBD dyes, Examples include organic fluorescent dyes such as pyrene dyes, Texas Red dyes, cyanine dyes and the like.

  Specific examples of the organic fluorescent dye include 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ', 4,4', 5 ', 7,7'-. Hexachlorofluorescein, 6-carboxy-2 ', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2 ', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine , 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514. , Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 603, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635, Alexa Fluor 635. Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIP6 / 530B550, BODIPY 630/550, BODIPY 530/550. Invitrogen), methoxycoumarin, eosin, NBD, pyrene, Cy5, Cy5.5, Cy7 and the like.

  In an embodiment in which the composition of the present invention contains a labeled nucleic acid comprising a dephosphorylated nucleic acid and a labeling substance bound to the dephosphorylated nucleic acid, the composition of the present invention is subjected to a nucleic acid detection method. It can be used as a nucleic acid sample. That is, in a preferred embodiment, the composition of the present invention is a nucleic acid sample used in the nucleic acid detection method. The labeled nucleic acid can contain a target nucleic acid to be detected and a nucleic acid other than the target nucleic acid. In the nucleic acid detection method, the target nucleic acid contained in the nucleic acid sample can be detected using the labeling substance contained in the target nucleic acid as an index. The nucleic acid detection method is not particularly limited and can be appropriately selected from known nucleic acid detection methods. The target nucleic acid can be detected using, for example, a hybridization method. In one embodiment of the hybridization method, a probe capable of hybridizing with the target nucleic acid can be used to detect the target nucleic acid. In one embodiment of a nucleic acid detection method using a probe, the probe and a nucleic acid sample containing the target nucleic acid are contacted, the probe and the target nucleic acid are hybridized, and the target nucleic acid hybridized with the probe is the target nucleic acid. The labeling substance possessed can be detected as an index. When the sample contains a nucleic acid other than the target nucleic acid, it is preferable to remove the nucleic acid that has not hybridized with the probe by washing or the like after bringing the target nucleic acid and the probe into contact with each other.

  The reaction conditions for hybridizing the target nucleic acid with the probe can be appropriately adjusted depending on the chain length of the target nucleic acid, the chain length of the probe, and the like. The reaction time is generally 30 to 1200 minutes, preferably 60 to 360 minutes. The reaction temperature is generally 25-60 ° C, preferably 30-40 ° C. The reaction is usually performed in an aqueous medium such as water.

  The amount of the target nucleic acid and the probe used is not particularly limited as long as the target nucleic acid and the probe can be hybridized and the labeling substance bound to the target nucleic acid can be detected, and the target nucleic acid chain length, the probe chain It can be appropriately adjusted depending on the length, the type of labeling substance, and the like.

As the probe, for example, a nucleic acid such as DNA, RNA, PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), or a nucleic acid derivative can be used.
Examples of the nucleic acid derivative include modified nucleotides (for example, alkyl such as halogen and methyl, alkoxy such as methoxy, nucleotide and base containing a group such as thio and carboxymethyl, saturation of double bond, deamination, oxygen, etc. Nucleic acid derivatives including nucleotides that have undergone substitution of sulfur molecules for molecules).

  The probe has a base sequence complementary to at least a part of the base sequence of the target nucleic acid, and can hybridize with the target nucleic acid. When the target nucleic acid is double-stranded, the probe may hybridize to the sense strand or to the antisense strand. The base sequence of the probe may be complementary to any part of the base sequence of the target nucleic acid, but is preferably complementary to a part of the base sequence of the target nucleic acid having high specificity. That is, the base sequence of the probe is preferably complementary to the base sequence of the target nucleic acid, which is not contained in the other nucleic acids contained in the sample.

The length (the number of bases) of the portion complementary to the base sequence of the target nucleic acid in the base sequence of the probe is not particularly limited, but is usually 10 to 150 bases, preferably 20 to 100 bases, more preferably 20 to 70 bases. Is. The probe may be composed of a base sequence complementary to the base sequence of the target nucleic acid, or may include a base sequence that is not complementary to the base sequence of the target nucleic acid.
The total length (total number of bases) of the probe is usually 10 to 300 bases, preferably 20 to 200 bases, more preferably 15 to 100 bases.

  The probe may be a commercially available product, a synthetic product, a preparation from a living body, or the like. A nucleic acid having a length of up to 200 bases, which is called an oligo nucleic acid, can be easily artificially synthesized by a synthesizer.

  The probe is preferably immobilized on a support. That is, in a preferred embodiment, the nucleic acid detection method is a nucleic acid detection method using a nucleic acid microarray. The nucleic acid microarray has a support and a plurality of probes immobilized on the surface of the support. In a nucleic acid detection method using a nucleic acid microarray, a labeled target nucleic acid is brought into contact with a nucleic acid microarray having a probe capable of hybridizing with the target nucleic acid, and the target nucleic acid hybridized with the probe is labeled with the target nucleic acid. The substance can be detected as an index. When the sample contains a nucleic acid other than the target nucleic acid, it is preferable to remove the nucleic acid that has not hybridized with the probe on the nucleic acid microarray by washing after contacting the target nucleic acid with the nucleic acid microarray. By using a nucleic acid microarray provided with a plurality of probes, two or more kinds of target nucleic acids can be detected simultaneously.

  The support is not particularly limited as long as it can immobilize the probe. Examples of the support include slide glass, membranes, beads and the like. Examples of the material for the support include inorganic materials such as glass, ceramics and silicon, polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethylmethacrylate and silicone rubber.

  Immobilization of the probe on the support can be performed according to a conventional method. Known methods for immobilizing the probe on the support include a method of synthesizing oligonucleic acid on the upper surface of the support and a method of dropping and preliminarily synthesizing oligonucleic acid on the upper surface of the support to immobilize it. Examples of the former method include the method of Ronald et al. (US Pat. No. 5,705,610), the method of Michel et al. (US Pat. No. 6,142,266), the method of Francesco et al. (US Pat. No. 7037659), and the like. Is mentioned. Since an organic solvent is used in the DNA synthesis reaction in these methods, the support is preferably made of a material resistant to the organic solvent. For example, it is possible to use a glass support having a concavo-convex structure produced by the method described in Japanese Patent Publication No. 10-503841. In particular, in the method of Francesco et al., The support is preferably made of a translucent material in order to control DNA synthesis by irradiating light from the back surface of the support. Examples of the latter method include the method of Hirota et al. (Japanese Patent No. 3922454) and the method of using a glass capillary. As an example of the glass capillaries, commercially available products such as self-made glass capillaries and micropipettes (MP-005 manufactured by Micro Support Co., Ltd.) can be used.

  A sandwich hybridization method can be used as a method for detecting a target nucleic acid. In the sandwich hybridization method, a first probe (capture probe) fixed to a support and a second probe (detection probe) not fixed to the support are used. Each of the capture probe and the detection probe has a base sequence complementary to a different portion of the target nucleic acid and can hybridize with a different portion of the target nucleic acid. A complex is formed by hybridizing the target nucleic acid with the detection probe and the capture probe. The target nucleic acid can be detected by detecting the labeling substance contained in this complex.

  It is preferable that the base sequence of the detection probe and the base sequence of the capture probe have low sequence identity. Sequence identity is preferably 20% or less, more preferably 10% or less. Here, the identity of two base sequences is determined by aligning the two sequences so as to match as many bases as possible (gaps as needed), and determining the number of matched bases as the total number of bases (bases of two base sequences). When the numbers are different, it is a numerical value obtained by dividing by the larger number of bases), and can be easily calculated with commercially available software such as FASTA and BLAST (also provided on the Internet).

  The signal detected in the method for detecting a target nucleic acid (for example, the intensity of the detected labeling substance) is compared with ambient noise. Specifically, the signal value obtained from the position on the support where the probe is fixed is compared with the signal value (noise value) obtained from the position on the support where the probe is not fixed, and the former The ratio between the numerical value and the noise value is defined as the S / N ratio. The detection accuracy can be represented by the S / N ratio. That is, the larger the S / N ratio, the higher the detection accuracy, and the smaller the S / N ratio, the lower the detection accuracy.

  In an embodiment in which the composition of the present invention contains a labeled nucleic acid comprising a dephosphorylated nucleic acid and a labeling substance bound to the dephosphorylated nucleic acid, the composition of the present invention is used as a nucleic acid sample in a nucleic acid detection method. By using it, the detection sensitivity of the target nucleic acid can be improved. This effect is remarkable in a nucleic acid detection method in which a very small amount (preferably 5-1000 μL, more preferably 5-500 μL) of a nucleic acid sample is used. In a nucleic acid detection method in which a very small amount of nucleic acid sample is used, the peptide fragment group (A), peptide fragment group (B), peptide fragment group (C) or second peptide fragment contained in the nucleic acid sample adversely affects the detection sensitivity. May give. In this respect, in the composition of the present invention, the ratio of the content of the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment to the content of alkaline phosphatase is as described above. Meets the given formula. That is, in the composition of the present invention, the relative content of the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment is reduced. Therefore, in a nucleic acid detection method in which a trace amount of a nucleic acid sample is used, by using the composition of the present invention as a nucleic acid sample, a peptide fragment group (A), a peptide fragment group (B), a peptide fragment group (C) or The adverse effect of the second peptide fragment can be reduced, and the detection sensitivity of the target nucleic acid can be significantly improved.

  In a preferred embodiment, the nucleic acid detection method is a nucleic acid detection method using a nucleic acid microarray. In a nucleic acid detection method using a nucleic acid microarray, a very small amount (preferably 5-1000 μL, more preferably 5-500 μL) of a nucleic acid sample is used. Therefore, in a nucleic acid detection method using a nucleic acid microarray, by using the composition of the present invention as a nucleic acid sample, the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second fragment group The adverse effect of the peptide fragment can be reduced, and the detection sensitivity of the target nucleic acid can be significantly improved.

[Production method]
The composition of the present invention is an alkaline phosphatase extract from an organ such as bovine or shrimp, an alkaline phosphatase extract from a microorganism into which a gene encoding alkaline phosphatase has been introduced, or a bacterial cell of a microorganism into which a gene encoding alkaline phosphatase has been introduced. It can be produced by separating the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment from a crushed product, a commercially available alkaline phosphatase product or the like. Examples of the method for separating the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment include dialysis, salting out, gel filtration, ultrafiltration, membrane separation, and ion separation. Exchange, column chromatography, electrophoresis and the like can be mentioned. As the method for separating peptide fragments, one type may be used alone, or two or more types may be used in combination. For example, by purifying a commercially available alkaline phosphatase product by column chromatography or the like, a peptide fragment group (A), a peptide fragment group (B), a peptide fragment group (C) or a second peptide fragment with respect to the content of alkaline phosphatase. The content of can be in a desired range. Column chromatography is, for example, liquid chromatography. The column and mobile phase used in liquid chromatography are not particularly limited as long as they can separate the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment. It is preferred to use a reverse phase column loaded with

[how to use]
The compositions of the present invention can be used in various methods where alkaline phosphatase activity is sought.

In one embodiment, the composition of the present invention comprises the following steps:
Providing a composition of the invention;
It is used in a method for producing a dephosphorylated nucleic acid, which comprises the steps of preparing a nucleic acid; and treating the above nucleic acid with the above composition to dephosphorylate the above nucleic acid. The peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment mixed in alkaline phosphatase may adversely affect the dephosphorylation of nucleic acid by alkaline phosphatase. is there. In this respect, in the composition of the present invention, the ratio of the content of the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment to the content of alkaline phosphatase is as described above. Meets the given formula. That is, in the composition of the present invention, the relative content of the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment is reduced. Therefore, the nucleic acid dephosphorylation efficiency can be improved by treating the nucleic acid with the composition of the present invention.

In one embodiment, the composition of the present invention comprises the following steps:
Providing a composition of the invention;
Preparing nucleic acid;
Preparing a labeling substance;
It is used in a method for producing a labeled nucleic acid, which comprises a step of treating the nucleic acid with the composition to dephosphorylate the nucleic acid; and a step of binding the labeling substance to the dephosphorylated nucleic acid. The peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment mixed in alkaline phosphatase is used when dephosphorylating a nucleic acid by alkaline phosphatase and / or There is a possibility of adversely affecting the binding of the labeling substance to the oxidized nucleic acid. In this respect, in the composition of the present invention, the ratio of the content of the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment to the content of alkaline phosphatase is as described above. Meets the given formula. That is, in the composition of the present invention, the relative content of the peptide fragment group (A), the peptide fragment group (B), the peptide fragment group (C) or the second peptide fragment is reduced.
Therefore, by treating the nucleic acid with the composition of the present invention, the dephosphorylation efficiency of the nucleic acid and / or the labeling efficiency of the dephosphorylated nucleic acid can be improved.

  In the step of dephosphorylating the nucleic acid by treating the nucleic acid with the composition of the present invention, the reaction conditions can be appropriately adjusted. The reaction time is generally 10 to 60 minutes, preferably 20 to 50 minutes. The reaction temperature is generally 20-60 ° C, preferably 25-45 ° C. The reaction is usually performed in an aqueous medium such as water. The nucleic acid is, for example, DNA or RNA. Treatment of nucleic acid with the composition of the present invention results in dephosphorylation of the 5'and / or 3'ends of the nucleic acid.

  In the step of binding the labeling substance to the dephosphorylated nucleic acid, as the labeling substance, for example, a fluorescent dye, a fluorescent protein, a chemiluminescent substance, a metal complex, fine metal particles, biotin, a radioisotope or the like can be used. The reaction conditions can be appropriately adjusted depending on the type of labeling substance. The dephosphorylated nucleic acid has a 5 ′ end and / or a 3 ′ end dephosphorylated with alkaline phosphatase, and a labeling substance can be bound to the dephosphorylated 5 ′ end and / or the 3 ′ end. it can.

  Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following Examples.

<< LC-MS / MS analysis conditions >>
The conditions of the LC-MS / MS analysis used in the examples and comparative examples are as follows.
<Device configuration>
Mass spectrometer: maXis impact (manufactured by Bruker Daltonics, Inc.)
<Mass spectrometry conditions>
Ionization method: ESI
Measurement ion: Positive ion Capillary voltage: 4500V
Nebulizer: 2.0 bar
Dry gas: 8.0L / min Detector voltage: 1823V
Measuring range (MS): m / z 50 to 2200
<MS / MS conditions>
Measuring range (MS): m / z 50 to 2200
Collision gas: Nitrogen

<< LC-UV analysis conditions >>
The conditions for LC-UV analysis used in Examples and Comparative Examples are as follows.
<Device configuration>
Liquid chromatograph: LC-30A system (manufactured by Shimadzu Corporation)
Detector: UV-Vis (190-900 nm, Shimadzu)
<Liquid chromatography conditions>
Column: Acquity BEH C18 1.7 μm (manufactured by Waters Corporation) Column size: 2.1 mm × 100 mm
Column temperature: 50 ° C
Mobile phase flow rate: 0.2 mL / min Mobile phase A: Water / formic acid mixture (1000: 1)
Mobile phase B: Acetonitrile / water / formic acid mixture (900: 100: 1)
Injection volume: 20 μL
Gradient program:

<< Nucleic acid detection method >>
The nucleic acid detection methods used in Examples and Comparative Examples are as follows.
The nucleic acid detection method was performed using a DNA chip (DNA microarray). Specifically, it was performed using "3D-Gene" human miRNA oligo chip (compatible with miRBase release 21) manufactured by Toray Industries, Inc.

[Comparative Examples 1 to 8]
Eight types of alkaline phosphatase products (hereinafter referred to as “composition C1” to “composition C8”) purchased from five companies were used as the alkaline phosphatase compositions of Comparative Examples 1 to 8. The alkaline phosphatase contained in each of the compositions C1 to C8 is a bovine intestinal-derived alkaline phosphatase. When the respective alkaline phosphatase specific activities of the compositions C1 to C8 were measured, the composition C1 was 2238 U / mg, the composition C2 was 2492 U / mg, the composition C3 was 2431 U / mg, the composition C4 was 2519 U / mg, and the composition The substance C5 was 2411 U / mg, the composition C6 was 2552 U / mg, the composition C7 was 2448 U / mg, and the composition 8 was 2490 U / mg. The alkaline phosphatase specific activity was measured by a method using p-nitrophenyl phosphate. Specifically, it is as follows.

The following solutions A and B were prepared.
Solution A: 1M diethanolamine buffer (pH 9.8)
Solution B: 0.67 M p-nitrophenol phosphoric acid aqueous solution 2.9 mL of solution A and 0.1 mL of solution B were prepared in a cuvette (optical path length = 1 cm), and heated at 37 ° C. for 5 minutes. Next, 0.1 mL of an alkaline phosphatase aqueous solution was added, and the change in absorbance at 405 nm (37 ° C.) was measured for 3 to 5 minutes with a spectrophotometer to obtain the change in absorbance per unit time (ΔOD). As a control, the change in absorbance was determined using a sample to which water was added instead of the alkaline phosphatase aqueous solution (ΔOD blank). The alkaline phosphatase activity (U / mL) was calculated by the following formula.
Alkaline phosphatase activity (U / mL) = (ΔOD−ΔOD blank) × 3.1 / (18.2 × 0.1 × 1.0)

  The concentration of alkaline phosphatase in the alkaline phosphatase aqueous solution was calculated by measuring the absorbance at 214 nm. The alkaline phosphatase aqueous solution was diluted with distilled water so that the absorbance at 214 nm was 0.1 to 1.0, approximated to 1 Abs = 1 mg / mL, and the value obtained by multiplying the dilution ratio was taken as the concentration of alkaline phosphatase. The specific activity in the present invention is the activity per 1 mg of alkaline phosphatase (U / mg), and was calculated by the above measurement method.

  A 10 wt% alkaline phosphatase aqueous solution was prepared from each of the compositions C1 to C8, and this aqueous solution was used for LC-UV analysis and LC-MS / MS analysis. Based on the extracted ion chromatogram obtained by LC-MS / MS analysis, consisting of the first peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1 (EAEAEFLIPAEEENPAFWNRQAAQ) and the amino acid sequence of SEQ ID NO: 2 (EGVSLEKREAEAE) The peak area value of each of the second peptide fragment and the third peptide fragment consisting of the amino acid sequence (IPAEEENPAFWNR) described in SEQ ID NO: 3 was calculated by the automatic integration method. Further, the peak area value of alkaline phosphatase was calculated by the automatic integration method based on the chromatogram obtained by the LC-UV analysis. In LC-UV analysis, alkaline phosphatase was detected as a component having an absorption at 214 nm.

FIG. 1 shows the extracted ion chromatogram for the first peptide fragment obtained by LC-MS / MS analysis of composition C2 in Comparative Example 2.
FIG. 2 shows an extracted ion chromatogram for the second peptide fragment obtained by LC-MS / MS analysis of composition C2 in Comparative Example 2.
FIG. 3 shows an extracted ion chromatogram for the third peptide fragment obtained by LC-MS / MS analysis of composition C2 in Comparative Example 2.

  Nucleic acids were dephosphorylated using the alkaline phosphatase compositions of Comparative Examples 1 to 8, and the resulting dephosphorylated nucleic acids were labeled with a cyanine organic fluorescent dye. Specifically, the dephosphorylation reaction and the labeling reaction were performed as follows.

  Whole blood collected from a healthy person was centrifuged to obtain 1 mL of serum. MicroRNA was extracted from serum using "3D-Gene" RNA extraction reagent from liquid sample kit (manufactured by Toray Industries, Inc.). The extracted microRNA thus obtained was used as a mother liquor and labeled with a "3D-Gene" miRNA labeling kit (manufactured by Toray Industries, Inc.). 5 μL of the obtained extracted microRNA was added to a mixed solution of 0.4 μL of AP buffer and 1.0 μL of Spike Control of the above kit, and 0.4 μL of the composition C1 was further added to prepare a solution. Then, after incubating at 37 ° C. for 40 minutes, it was allowed to stand on ice for 2 minutes. Then, LE Buffer 1.2 μL, 3D-Gene Fluorescent Label 3.0 μL, Nuclease free water 2.5 μL, and Labeling enzyme 1.0 μL were added, and the mixture was incubated at 16 ° C. for 1 hour, and then incubated at 65 ° C. for 15 minutes. A labeled nucleic acid was obtained. The dephosphorylation reaction and the labeling reaction using the compositions C2 to C8 were performed in the same manner as above using the same extracted microRNA mother liquor.

  Nucleic acid was detected using the obtained labeled nucleic acid. The labeled sample RNA was subjected to hybridization using a DNA chip (“3D-Gene” miRNA chip, manufactured by Toray Industries, Inc.) according to its standard protocol. The DNA chip after hybridization was subjected to a microarray scanner (manufactured by Toray Industries, Inc.) to measure the fluorescence intensity. The scanner was set so that the laser output was 100% and the voltage of the photomultiplier was set to AUTO. Nucleic acid was detected using a DNA chip (DNA microarray) as described above. The number of effective spots on the DNA chip was determined, and the detection rate (%) was calculated. Of all 2,588 spots on the DNA chip, the one with a value of 100 or more obtained by subtracting noise (the signal value at a spot without a spot) from the detected signal value is defined as an effective spot, and the number of effective spots is divided by the total number of spots. The value obtained by multiplying 100 by 100 was taken as the detection rate. The results are shown in Table 4-2.

[Examples 1 to 4]
The alkaline phosphatase compositions of Comparative Examples 2 to 4 and 8 (compositions C2 to C4 and C8) were purified by the following method, and the alkaline phosphatase compositions of Examples 1 to 4 (hereinafter "composition E1" to "composition") were purified. Item E4 "). The purification method is as follows.

(Dialysis process)
Composition C2 (30 μL) was dialyzed 3 times with dialysis buffer (1 mL, 50 mM Tris-HCl, 2 mM MgCl ₂ , 0.2 mM ZnCl ₂ ) using a dialysis cup (cutoff molecular weight 3.5 K), The concentrated liquid was collected.

(Gel filtration step)
The concentrated solution after the dialysis treatment was collected by filtration with a buffer (2.5 mL, 10 mM Tris-HCl, 1 mM MgCl ₂ , 0.1 mM ZnCl ₂ , 50 mM KCl, 55 wt% glycerin) using a gel filtration column.

(Hydrophobic column process)
The alkaline phosphatase fraction was recovered from the recovered solution after gel filtration using a hydrophobic column under the following conditions.

Mobile phase flow rate: 1.0 mL / min Mobile phase A: 20 mM disodium hydrogen phosphate, 3M ammonium sulfate (50/50)
Mobile phase B: 20 mM disodium hydrogen phosphate gradient program:

Detector: UV214nm

(Dialysis process)
The recovered alkaline phosphatase fraction was dialyzed three times under the same conditions as in the above dialysis to collect a concentrated solution.

(Ultrafiltration process)
The collected concentrate was buffered (2.5 mL, 10 mM Tris-HCl, 1 mM MgCl ₂ , 0.1 mM ZnCl ₂ , 50 mM KCl, 55 wt% glycerin) using an ultrafiltration column (cutoff molecular weight 10 K). It was collected by filtration to obtain a composition E1.

  Compositions E2-E4 were similarly obtained from composition C3, composition C4 and composition C8, respectively, using a method similar to that described above.

  When the alkaline phosphatase specific activities of the alkaline phosphatase compositions of Examples 1 to 4 were measured, the composition E1 was 2490 U / mg, the composition E2 was 2420 U / mg, the composition E3 was 2522 U / mg, and the composition E4 was 2470 U / mg. It was mg. The alkaline phosphatase specific activity was measured in the same manner as above.

  A 10 wt% alkaline phosphatase aqueous solution was prepared from each of the compositions E1 to E4, and LC-UV analysis and LC-MS / MS analysis were performed using this aqueous solution. Based on the extracted ion chromatogram obtained by LC-MS / MS analysis, the first peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (EAEAEFLIPAEEENPAFWNRQAAQ) and the first peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (EGVSLEKREAEAE) The peak area value of each of the peptide of 2 and the third peptide consisting of the amino acid sequence (IPAEEENPAFWNR) described in SEQ ID NO: 3 was calculated by the automatic integration method. Further, the peak area value of alkaline phosphatase was calculated by the automatic integration method based on the chromatogram obtained by the LC-UV analysis. In LC-UV analysis, alkaline phosphatase was detected as a component having an absorption at 214 nm.

FIG. 4 shows an extracted ion chromatogram for the first peptide fragment obtained by LC-MS / MS analysis of composition E1 (purified product of composition C2) in Example 1.
FIG. 5 shows an extracted ion chromatogram for the second peptide fragment obtained by LC-MS / MS analysis of composition E1 (purified product of composition C2) in Example 1.
FIG. 6 shows an extracted ion chromatogram for the third peptide fragment obtained by LC-MS / MS analysis of composition E1 (purified product of composition C2) in Example 1.
FIG. 7 shows a chromatogram for alkaline phosphatase obtained by LC-UV analysis of composition E1 (purified product of composition C2) in Example 1. In addition, the chromatogram regarding alkaline phosphatase obtained by LC-UV analysis of each composition in Examples 2-4 and Comparative Examples 1-8 was the same as that of FIG. 7.

  Nucleic acids were dephosphorylated using the alkaline phosphatase compositions of Examples 1 to 4, and the resulting dephosphorylated nucleic acids were labeled with a cyanine-based organic fluorescent dye. The dephosphorylation reaction and the labeling reaction were performed in the same manner as above.

  Nucleic acid was detected using the obtained labeled nucleic acid. Nucleic acid was detected using a DNA chip (DNA microarray) as described above. The number of effective spots on the DNA chip was determined, and the detection rate (%) was calculated. The results are shown in Table 4-1.

  As shown in Tables 4-1 and 4-2, when the nucleic acid samples prepared using the alkaline phosphatase compositions of Comparative Examples 1 to 8 were used in the nucleic acid detection method, the number of effective spots was less than 1500. On the other hand, when the nucleic acid samples prepared using the alkaline phosphatase compositions of Examples 1 to 4 were used in the nucleic acid detection method, the number of effective spots was 1500 or more. Further, the detection rates in Comparative Examples 1 to 8 are different from each other in spite of the fact that the alkaline phosphatase specific activities of the alkaline phosphatase composition are almost the same, whereas the detection rates in Examples 1 to 4 are It was almost the same, and was higher than the detection rates of Comparative Examples 1-8.

As shown in Table 4-1 and Table 4-2, it is an index of the ratio of the total content of the first to third peptide fragments to the content of alkaline phosphatase ((X ₁ + X ₂ + X ₃ ) / Y). The value of × 100 exceeds 5.0000 in the alkaline phosphatase compositions of Comparative Examples 1 to 8 (minimum value is 5.4989 in the alkaline phosphatase composition of Comparative Example 1), while that of Examples 1 to 4 It is 5.0000 or less in the alkaline phosphatase composition. This is considered to be one of the main causes of the difference in the above effects between Examples 1 to 4 and Comparative Examples 1 to 8.

As shown in Table 4-1 and Table 4-2, it is an index of the ratio of the total content of the first and second peptide fragments to the content of alkaline phosphatase ((X ₁ + X ₂ ) / Y) × 100. The value of is more than 2.4000 in the alkaline phosphatase compositions of Comparative Examples 1 to 8 (minimum value is 2.4619 in the alkaline phosphatase composition of Comparative Example 3), while the alkaline phosphatase of Examples 1 to 3 is It is 2.4000 or less in the composition. This is considered to be one of the main causes of the difference in the above effects between Examples 1 to 4 and Comparative Examples 1 to 8.

As shown in Table 4-1 and Table 4-2, it is an index of the ratio of the total content of the first and third peptide fragments to the content of alkaline phosphatase ((X ₁ + X ₃ ) / Y) × 100. Of the alkaline phosphatase composition of Comparative Examples 1 to 8 exceeds 4.50 (the minimum value is 4.6492 in the alkaline phosphatase composition of Comparative Example 1), while the alkaline phosphatase of Examples 1 to 4 is It is 4.5000 or less in the composition. This is considered to be one of the main causes of the difference in the above effects between Examples 1 to 4 and Comparative Examples 1 to 8.

As shown in Table 4-1 and Table 4-2, the value of (X ₂ / Y) × 100, which is an index of the ratio of the content of the second peptide fragment to the content of the alkaline phosphatase, is Comparative Example 1 to The alkaline phosphatase composition of Example 8 had a value exceeding 0.8000 (the minimum value was 0.8497 in the alkaline phosphatase composition of Comparative Example 1), while the value of 0.8000 or less was obtained in the alkaline phosphatase compositions of Examples 1 to 4. is there. This is considered to be one of the main causes of the difference in the above effects between Examples 1 to 4 and Comparative Examples 1 to 8.

As shown in Table 4-1 and Table 4-2, the value of (X ₁ / Y) × 100, which is an index of the ratio of the content of the first peptide fragment to the content of alkaline phosphatase, is Comparative Example 1, In the alkaline phosphatase compositions of 2 and 4 to 8, it exceeds 1.0000 (however, in Comparative Example 3, it is 1.0000 or less), whereas in the alkaline phosphatase compositions of Examples 1 to 4, 1.0000 or less. Is. This is also considered to be one of the sub-factors that caused the above-mentioned difference in effect between Examples 1 to 4 and Comparative Examples 1, 2 and 4 to 8.

As shown in Table 4-1 and Table 4-2, the value of (X ₃ / Y) × 100, which is an index of the ratio of the content of the third peptide fragment to the content of alkaline phosphatase, is Comparative Example 2 to 2. The alkaline phosphatase composition of Examples 4, 5 and 6 exceeds 2.3000 (however, in Comparative Examples 1, 5 and 6, it is 2.3000 or less), while the alkaline phosphatase composition of Examples 1 to 4 has It is 2.3000 or less. This is also considered to be one of the sub-factors that caused the difference in effect between Examples 1 to 4 and Comparative Examples 2 to 4, 7 and 8.

Claims (8)

Alkaline phosphatase,
And a peptide fragment group (A) composed of two or more kinds of peptide fragments consisting of consecutive 5 to 50 amino acid residues selected from positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4. A thing,
The ratio of the content of the peptide fragment group (A) to the content of the alkaline phosphatase is represented by the following formula (A):
(X _A /Y)×100≦5.0000 (A)
[Wherein, X _A represents the peak area value of the peptide fragment group (A) calculated by the automatic integration method from the extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y Represents the peak area value of the alkaline phosphatase calculated by the automatic integration method from the chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, which satisfies: Alkaline phosphatase,
Two or more peptides consisting of 5 to 50 consecutive amino acid residues selected from positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4 and containing positions 86 to 90 of the amino acid sequence of SEQ ID NO: 4. A composition comprising a peptide fragment group (B) composed of fragments,
The ratio of the content of the peptide fragment group (B) to the content of the alkaline phosphatase is represented by the following formula (B):
(X _B /Y)×100≦2.4000 (B)
[Wherein, X _B represents a peak area value of the peptide fragment group (B) calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y Represents the peak area value of the alkaline phosphatase calculated by the automatic integration method from the chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, which satisfies: Alkaline phosphatase,
Two or more kinds of peptides consisting of 13 to 50 consecutive amino acid residues selected from positions 71 to 130 of the amino acid sequence of SEQ ID NO: 4 and containing positions 93 to 105 of the amino acid sequence of SEQ ID NO: 4. A composition comprising a peptide fragment group (C) composed of fragments,
The ratio of the content of the peptide fragment group (C) to the content of the alkaline phosphatase is represented by the following formula (C):
(X _C /Y)×100≦4.5000 (C)
[Wherein, X _C represents a peak area value of the peptide fragment group (C) calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y C Represents the peak area value of the alkaline phosphatase calculated by the automatic integration method from the chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, which satisfies: Alkaline phosphatase,
A composition comprising a second peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2,
The ratio of the content of the second peptide fragment to the content of the alkaline phosphatase is represented by the following formula (2):
(X ₂ /Y)×100≦0.8000 (2)
[In the formula, X ₂ represents the peak area value of the second peptide fragment calculated by the automatic integration method from the extracted ion chromatogram obtained by the LC-MS / MS analysis of the composition, and Y is Shows the peak area value of the alkaline phosphatase calculated by the automatic integration method from the chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, which satisfies: The composition further comprises a first peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 1,
The ratio of the content of the first peptide fragment to the content of the alkaline phosphatase is represented by the following formula (1):
(X ₁ /Y)×100≦1.0000 (1)
[Wherein X ₁ represents a peak area value of the first peptide fragment calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y is , Is synonymous with the above. ]
The composition of claim 4, which satisfies: The composition further comprises a third peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 3,
The ratio of the content of the third peptide fragment to the content of the alkaline phosphatase is represented by the following formula (3):
(X ₃ /Y)×100≦2.3000 (3)
[Wherein X ₃ represents a peak area value of the third peptide fragment calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, and Y is , Is synonymous with the above. ]
The composition according to claim 4 or 5, which satisfies the above condition. The following steps:
Providing a composition according to any one of claims 1 to 6;
A method for producing a dephosphorylated nucleic acid, comprising the steps of preparing a nucleic acid; and treating the nucleic acid with the composition to dephosphorylate the nucleic acid. The following steps:
Providing a composition according to any one of claims 1 to 6;
Preparing nucleic acid;
Preparing a labeling substance;
A method for producing a labeled nucleic acid, comprising the steps of treating the nucleic acid with the composition to dephosphorylate the nucleic acid; and binding the labeling substance to the dephosphorylated nucleic acid.