JP2020048557A - Alkaline phosphatase compositions and methods for producing dephosphorylated nucleic acids and labelled nucleic acids - Google Patents

Abstract

To provide high quality alkaline phosphatase compositions, methods for producing dephosphorylated nucleic acids and labelled nucleic acids using the compositions.SOLUTION: Disclosed is a composition comprising an alkaline phosphatase and a group of peptide fragments having specific amino acid sequences of consecutive 5-20 amino acids, where the content ratio of the peptide fragment group to the alkaline phosphatase satisfies the following formula: (X/Y)×100≤0.1800 (A) where Xrepresents the peak area value of the peptide fragment group calculated by an automatic integral technique from an extracted chromatogram obtained by LC-MS/MS analysis of the composition, and Y represents the peak area value of the alkaline phosphatase calculated by an automatic integral technique from an extracted chromatogram obtained by LC-UV analysis of the composition.SELECTED DRAWING: None

Description

  The present invention relates to a composition containing alkaline phosphatase, and 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, ELISA, nucleic acid microarray, etc.). I have. For example, in the field of genetic engineering research, the 5′-end and / or 3′-end of nucleic acid using alkaline phosphatase for the purpose of pretreatment of labeling of nucleic acid such as DNA and RNA, prevention of self-ligation of vector, etc. Dephosphorylation is taking place.

  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 used because of its high specific activity. Evaluation of the specific activity of alkaline phosphatase is generally performed by measuring the absorbance at 405 nm derived from p-nitrophenol generated when p-nitrophenyl phosphate is decomposed.

  Alkaline phosphatase quality has been evaluated based on alkaline phosphatase specific activity. Then, in order to obtain alkaline phosphatase having a higher specific activity than that of bovine intestine-derived alkaline phosphatase, high specific activity alkaline phosphatase is isolated in the purification process, or high specific activity is obtained by recombinant Escherichia coli using genetic engineering techniques. It produces alkaline phosphatase.

  Patent Document 1 discloses a method for producing alkaline phosphatase having high specific activity using recombinant Escherichia coli into which a gene derived from Bacillus badius has been introduced. Patent Document 2 discloses a method for producing alkaline phosphatase having high specific activity and heat resistance using recombinant Escherichia coli into which a gene derived from the genus Shewanella has been introduced.

JP-A-10-262677 International Publication No. 2012/115023

  A dephosphorylation reagent containing alkaline phosphatase (for example, a commercially available alkaline phosphatase product) is a composition containing alkaline phosphatase and other components. The quality of the dephosphorylating reagent containing alkaline phosphatase is evaluated based on the specific activity of alkaline phosphatase.

  However, even with a dephosphorylation reagent having substantially the same specific activity of alkaline phosphatase, a labeled nucleic acid prepared using the dephosphorylation reagent (the 5 ′ end and / or 3 ′ (A labeled nucleic acid obtained by dephosphorylating the 'end and attaching a labeling substance to the 5' end and / or 3 'end of the dephosphorylated nucleic acid) in the detection sensitivity in the nucleic acid detection method. Was found to occur. That is, it was found that the quality of the dephosphorylating reagent containing alkaline phosphatase could not be accurately evaluated using 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 using the composition, and a method for producing a labeled nucleic acid.

The present inventors have conducted intensive studies to solve the above-mentioned problems. As a result, one or more of the following impurities were contained in a dephosphorylation reagent containing alkaline phosphatase (for example, a commercially available alkaline phosphatase product). It has been found that they can be mixed.
-Consists of one or more peptide fragments consisting of 5 to 20 consecutive amino acid residues selected from positions 451 to 490 of the amino acid sequence of SEQ ID NO: 2 (amino acid sequence of bovine alkaline phosphatase) Peptide fragment group (A)
One or more kinds of amino acid sequences consisting of consecutive 9 to 20 amino acid residues selected from positions 451 to 490 of the amino acid sequence of SEQ ID NO: 2 and including positions 469 to 477 of the amino acid sequence of SEQ ID NO: 2 Peptide fragment group (B) composed of peptide fragments
A first peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1

  Then, the present inventors use the dephosphorylation reagent by reducing the content of the peptide fragment group (A), the content of the peptide fragment group (B), or the content of the first peptide fragment. Prepared labeled nucleic acid (the 5 'end and / or 3' end of the nucleic acid is dephosphorylated with the dephosphorylation reagent, and a labeling substance is added to the 5 'end and / or 3' end of the dephosphorylated nucleic acid. It has been found that the detection sensitivity of the labeled nucleic acid obtained by binding) in the nucleic acid detection method can be improved, and the present invention has been completed. That is, the present invention includes the following inventions.

[1] A peptide fragment group consisting of alkaline phosphatase and one or more peptide fragments consisting of consecutive 5 to 20 amino acid residues selected from positions 451 to 490 of the amino acid sequence of SEQ ID NO: 2 ( A) a composition comprising:
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≦0.1800 (A)
Wherein, X _A is the calculated from the extracted ion chromatogram obtained by LC-MS / MS analysis of the composition by an automatic integration method, represents the peak area value of the peptide fragment group (A), Y Represents a peak area value of the alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, wherein
[2] The composition according to [1], wherein the peptide fragment group (A) includes a first peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1.
[3] 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≦0.1800 (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 represents Has the same meaning as described above. ]
The composition according to [2], which satisfies the following.
[4] Consisting of alkaline phosphatase and 9 to 20 consecutive amino acid residues selected from positions 451 to 490 of the amino acid sequence of SEQ ID NO: 2, and positions 469 to 477 of the amino acid sequence of SEQ ID NO: 2 A peptide fragment group (B) composed of at least one peptide fragment comprising:
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≦0.1800 ··· (B)
Wherein, X _B is the calculated by the automatic integration method from the extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, represents the peak area value of the peptide fragment group (B), Y Represents a peak area value of the alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, wherein
[5] The composition according to [4], wherein the peptide fragment group (B) includes a first peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1.
[6] 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≦0.1800 (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 represents Has the same meaning as described above. ]
The composition according to [5], which satisfies the following.
[7] A composition comprising alkaline phosphatase and a first peptide fragment having 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≦0.1800 (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 represents Represents the peak area value of the alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, wherein
[8] The composition according to any one of [1] to [7], wherein the composition has a specific activity of alkaline phosphatase of 2000 U / mg or more.
[9] The alkaline phosphatase has the following (a) and (b):
(A) alkaline phosphatase having a protein molecule consisting of the amino acid sequence of SEQ ID NO: 2; and (b) having 70% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, The composition according to any one of [1] to [8], wherein the composition is selected from alkaline phosphatase having a protein molecule consisting of an amino acid sequence containing positions 451 to 490 of the amino acid sequence.
[10] The composition according to any one of [1] to [9], wherein the composition further contains a nucleic acid.
[11] The composition according to [10], wherein the composition is a composition for dephosphorylating the nucleic acid.
[12] The composition according to any one of [1] to [9], wherein the composition further contains a dephosphorylated nucleic acid.
[13] The composition according to [12], 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. .
[14] The composition according to any one of [1] to [9], wherein the composition further comprises a labeled nucleic acid comprising a dephosphorylated nucleic acid and a labeling substance bound to the dephosphorylated nucleic acid. Composition.
[15] The composition according to [14], wherein the composition is a nucleic acid sample subjected to a nucleic acid detection method.
[16] The composition according to [15], wherein the nucleic acid detection method is a nucleic acid detection method using a nucleic acid microarray.
[17] The following steps:
Preparing the composition according to any one of [1] to [9];
A method for producing a dephosphorylated nucleic acid, comprising: preparing a nucleic acid; treating the nucleic acid with the composition, and dephosphorylating the nucleic acid.
[18] The following steps:
Preparing the composition according to any one of [1] to [9];
Providing a nucleic acid;
Preparing a labeling substance;
A method for producing a labeled nucleic acid, comprising: 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, and a method for producing a dephosphorylated nucleic acid and a method for producing a labeled nucleic acid using the composition.

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

  Hereinafter, the present invention will be described in detail. It is possible to combine two or more aspects of the aspects described below, and to combine two or more aspects of the 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 from numerical value M to numerical value N.

  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 a composition comprising alkaline phosphatase and a peptide fragment group (B) as the composition according to the second aspect.

  The present invention provides, as the composition according to the third aspect, a composition containing alkaline phosphatase and a first peptide fragment having the amino acid sequence of SEQ ID NO: 1.

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

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

  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 under alkaline conditions (pH 8 to 11, for example, pH 8 to 10 or pH 9 to 11), and its reaction format is classified into EC 3.1.3.1. Is done.

  The structure (eg, primary structure, secondary structure, tertiary structure, quaternary structure, etc.) of the alkaline phosphatase contained in the composition of the present invention is not particularly limited. For example, alkaline phosphatase may have a sugar chain or may not have a sugar chain. Alkaline phosphatase may be any isozyme that can exist based on differences in protein molecule structure (eg, amino acid sequence of protein molecule), sugar chain modification, and the like. The alkaline phosphatase may be a monomer formed from one subunit, or an oligomer formed from two or more subunits (eg, dimer, tetramer, etc.). ). 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 cows, shrimp, and microorganisms into which a gene encoding alkaline phosphatase has been introduced. Since bovine alkaline phosphatase has high alkaline phosphatase activity, bovine is preferred as the animal from which alkaline phosphatase is derived. When alkaline phosphatase is derived from cattle, the organ from which alkaline phosphatase is derived is preferably the small or 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 consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added in the amino acid sequence of a protein molecule possessed by wild-type alkaline phosphatase. The amino acid sequence of the protein molecule of the mutant alkaline phosphatase is preferably 70% or more, more preferably 75% or more, still more preferably 80% or more, based on the amino acid sequence of the protein molecule of the wild-type alkaline phosphatase. More preferably it has a sequence identity of 85% or more, even more preferably 90% or more, even more preferably 95% or more.

In a preferred embodiment, the alkaline phosphatase comprises the following (a) and (b):
(A) an alkaline phosphatase comprising a protein molecule consisting of the amino acid sequence of SEQ ID NO: 2; and (b) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2, It is selected from alkaline phosphatase having a protein molecule consisting of an amino acid sequence containing positions 451 to 490 of the described amino acid sequence. In this embodiment, the composition of the present invention may contain one kind of alkaline phosphatase selected from the above (a) and (b), or may contain 2 kinds of alkaline phosphatase selected from the above (a) and (b). It may contain more than one alkaline phosphatase.

  The amino acid sequence of the protein molecule of the alkaline phosphatase (a) described above (that is, the amino acid sequence of SEQ ID NO: 2) corresponds to the amino acid sequence of the protein molecule of the alkaline phosphatase derived from bovine. Therefore, the alkaline phosphatase (a) includes bovine alkaline phosphatase.

  The amino acid sequence of the protein molecule contained in the alkaline phosphatase (b) is preferably 70% or more, more preferably 75% or more, still more preferably 80% or more, and still more preferably the amino acid sequence of SEQ ID NO: 2. 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 (eg, alkaline phosphatase derived from animals other than bovine, alkaline phosphatase derived from bovine having polymorphism, etc.) and mutant alkaline phosphatase. The positions where one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 2 are positions other than positions 451 to 490 of the amino acid sequence of SEQ ID NO: 2.

  The alkaline phosphatase of the above (a) and (b) can generate a peptide fragment group (A), a peptide fragment group (B), a first peptide fragment and the like by decomposition.

[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 one or more peptide fragments, and each peptide fragment constituting the peptide fragment group (A) is a continuous peptide selected from positions 451 to 490 of the amino acid sequence described in SEQ ID NO: 2. Consisting of 5 to 20 amino acid residues. The peptide fragment group (A) may be composed of one kind of peptide fragment, or may be composed of two or more kinds of peptide fragments. Peptide fragment group (A) is a peptide fragment contained in the composition according to the first embodiment, which includes 5 to 20 consecutive amino acid residues selected from positions 451 to 490 of the amino acid sequence described in SEQ ID NO: 2. It is composed of all peptide fragments corresponding to the peptide fragment comprising the group.

  The amino acid sequence of each peptide fragment constituting the peptide fragment group (A) is a part of the amino acid sequence described in SEQ ID NO: 2 at positions 451 to 490 (a part consisting of a plurality of consecutive amino acid residues). That is, the peptide fragment group (A) can be generated by degradation of the amino acid sequence shown in SEQ ID NO: 2 at positions 451 to 490. This does not mean that the alkaline phosphatase contained in the composition according to the first aspect needs to include positions 451 to 490 of the amino acid sequence shown in SEQ ID NO: 2. Alkaline phosphatase contained in the composition according to the first aspect may not include positions 451 to 490 of the amino acid sequence described in SEQ ID NO: 2. However, the alkaline phosphatase contained in the composition according to the first aspect preferably contains the amino acid sequence of positions 451 to 490 of SEQ ID NO: 2.

  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 451 to 490 of the amino acid sequence described in SEQ ID NO: 2, but preferably is the amino acid sequence described in SEQ ID NO: 2. Part of the amino acid sequence at positions 455 to 490, more preferably part of the amino acid sequence of positions 461 to 490 of SEQ ID NO: 2, even more preferably position 465 to 490 of the amino acid sequence of SEQ ID NO: 2 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 20, but is preferably 6 to 20, more preferably 7 to 20, and still more. Preferably it is 8-20.

  In a preferred embodiment, the peptide fragment group (A) includes a first peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1. In this embodiment, the peptide fragment group (A) may or may not include a peptide fragment other than the first peptide fragment.

  The amino acid sequence of SEQ ID NO: 1 (APGKALDSK) corresponds to positions 469 to 477 of the amino acid sequence of SEQ ID NO: 2.

  The peptide fragment group (A) can be generated by the decomposition of alkaline phosphatase containing positions 451 to 490 of the amino acid sequence shown in SEQ ID NO: 2. The peptide fragment group (A) may be generated by the decomposition of alkaline phosphatase that is not contained in the composition according to the first embodiment, but is usually the alkali contained in the composition according to the first embodiment. It is generated 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 generating 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 described in (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 one or more peptide fragments, and each peptide fragment constituting the peptide fragment group (B) is a continuous fragment selected from positions 451 to 490 of the amino acid sequence described in SEQ ID NO: 2. And comprises positions 469 to 477 (APGKALDSK) of the amino acid sequence of SEQ ID NO: 2. The peptide fragment group (B) may be composed of one type of peptide fragment, or may be composed of two or more types of peptide fragments. The peptide fragment group (B) includes, among the peptide fragments contained in the composition according to the second embodiment, 9 to 20 consecutive amino acid residues selected from positions 451 to 490 of the amino acid sequence described in SEQ ID NO: 2. And all peptide fragments corresponding to the peptide fragments containing positions 469 to 477 of the amino acid sequence described in SEQ ID NO: 2.

  In the amino acid sequence of each peptide fragment constituting the peptide fragment group (B), the position including positions 469 to 477 of the amino acid sequence described in SEQ ID NO: 2 is not particularly limited. The positions including positions 469 to 477 of the amino acid sequence described in SEQ ID NO: 2 may be 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. It may be.

  The amino acid sequence of each peptide fragment constituting the peptide fragment group (B) is a part of the amino acid sequence described in SEQ ID NO: 2 at positions 451 to 490 (a part consisting of a plurality of consecutive amino acid residues). That is, the peptide fragment group (B) can be generated by degradation of the amino acid sequence of SEQ ID NO: 2 at positions 451 to 490. This does not mean that the alkaline phosphatase contained in the composition according to the second aspect needs to include positions 451 to 490 of the amino acid sequence described in SEQ ID NO: 2. Alkaline phosphatase contained in the composition according to the second aspect may not include positions 451 to 490 of the amino acid sequence described in SEQ ID NO: 2. However, it is preferable that the alkaline phosphatase contained in the composition according to the second embodiment includes positions 451 to 490 of the amino acid sequence described in SEQ ID NO: 2.

  The amino acid sequence of each peptide fragment constituting the peptide fragment group (B) is a part of the amino acid sequence of positions 451 to 490 of SEQ ID NO: 2, and the amino acid sequence of positions 469 to 477 of the amino acid sequence of SEQ ID NO: 2 Although it is not particularly limited as long as it includes, preferably, a part of positions 455 to 490 of the amino acid sequence of SEQ ID NO: 2, more preferably, a part of positions 461 to 490 of the amino acid sequence of SEQ ID NO: 2, further more Preferably, it is a part of the amino acid sequence described in SEQ ID NO: 2 at positions 465 to 490.

  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 9 to 20, but is preferably 9 to 19, more preferably 9 to 18, and still more. Preferably it is 9-17.

  In a preferred embodiment, the peptide fragment group (B) includes a first peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1. In this embodiment, the peptide fragment group (B) may or may not include a peptide fragment other than the first peptide fragment.

  The peptide fragment group (B) can be generated by the decomposition of alkaline phosphatase containing positions 451 to 490 of the amino acid sequence shown in SEQ ID NO: 2. The peptide fragment group (B) may be one produced by the decomposition of alkaline phosphatase not contained in the composition according to the second aspect, but usually the alkali fragment contained in the composition according to the second aspect. It is generated 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 generating the peptide fragment group (B). In a preferred embodiment, the alkaline phosphatase that can generate the peptide fragment group (B) is selected from the alkaline phosphatases described in (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.

[First peptide fragment]
The composition according to the third aspect contains a first peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1. The composition according to the third aspect may or may not contain a peptide fragment other than the first peptide fragment.

  The first peptide fragment can be generated by the decomposition of alkaline phosphatase containing amino acid positions 451 to 490 of the amino acid sequence set forth in SEQ ID NO: 2. The first peptide fragment may be one produced by the decomposition of alkaline phosphatase not contained in the composition according to the third aspect, but usually the alkaline phosphatase contained in the composition according to the third aspect. Is caused by the decomposition of Thus, in a preferred embodiment, the alkaline phosphatase contained in the composition according to the third aspect is an alkaline phosphatase capable of producing a first peptide fragment. In a preferred embodiment, the alkaline phosphatase capable of producing the first peptide fragment 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.

[Ratio of content of peptide fragment group (A) to content of alkaline phosphatase]
In the composition according to the first embodiment, 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≦0.1800 (A)
Meet.

In the above formula (A), X _A is calculated by the automatic integration method from the resulting extracted ion chromatogram by LC-MS / MS analysis of the composition according to the first embodiment, the peak of the peptide fragment group (A) Y 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 embodiment. The “peak area value of the peptide fragment group (A)”, when the peptide fragment group (A) is composed of one kind of peptide fragment, means the peak area value of the one kind of peptide fragment, and the peptide fragment group ( When A) is composed of two or more peptide fragments, the total peak area value of the two or more peptide fragments (that is, the total peak area value of all the peptide fragments of the peptide fragment group (A)) )). "Peak area value of alkaline phosphatase", when the composition according to the first aspect contains one kind of alkaline phosphatase, means the peak area value of the one kind of alkaline phosphatase, the composition according to the first aspect When two or more alkaline phosphatases are contained, the total peak area of the two or more alkaline phosphatases (that is, the total peak area of all kinds of alkaline phosphatases contained in the composition according to the first embodiment) is meant. I do.

  LC-MS / MS analysis and LC-UV analysis are performed using a sample in which the ratio of the content of the peptide fragment group (A) to the content of alkaline phosphatase is the same as that of the composition according to the first embodiment. You. The LC-MS / MS analysis and the LC-UV analysis can be performed, for example, using an aqueous solution prepared from the composition according to the first embodiment 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 technique of connecting a chromatograph such as a gas chromatograph or a liquid chromatograph to a mass spectrometer for analysis. LC-MS / MS analysis is a technique of connecting a liquid chromatograph (LC) and a tandem mass spectrometer (MS / MS) for analysis. In the LC-MS / MS analysis, a component to be analyzed separated by liquid chromatography is ionized, generated ions are separated by a tandem mass spectrometer, and specific mass ions are fragmented and detected.

  In the extracted ion chromatogram, in the hyphenated method, a mass spectrum is measured at regular time intervals, stored in a computer, and then the relative intensity at a specific (not limited to one type) m / z value is read out. 5 is a chromatogram expressed as a function of time. The m / z value of the ion 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 more preferably 50 to 2200. Preferably it is 200-1500, Still more preferably, it is 300-1200. With respect to the ions 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 is 443.7533. For a peptide fragment whose m / z value is not specified, it is checked 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 prepared based on the / z value.

  LC-UV analysis is a technique of connecting a liquid chromatograph (LC) and an ultraviolet detector (UV detector) for analysis. In 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.

{Conditions for LC-MS / MS analysis}
<Apparatus configuration>
Mass spectrometer: maXis impact (Bruker Daltonics, Inc.)
<Mass spectrometry conditions>
Ionization method: ESI
Measurement ion: positive ion Capillary voltage: 4500V
Nebulizer: 2.0 bar
Dry gas: 8.0 L / min Detector voltage: 1823 V
Measurement range (MS): m / z 50-2200
<MS / MS conditions>
Measurement range (MS): m / z 50-2200
Collision gas: nitrogen

<< Conditions for LC-UV analysis >>
<Apparatus configuration>
Liquid chromatograph: LC-30A system (manufactured by Shimadzu Corporation)
Detector: UV-Vis (190-900 nm, manufactured by Shimadzu Corporation)
<Liquid chromatography conditions>
Column: Acquity BEH C18 1.7 μm (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 0.1800 or less, but the smaller the value, the better. The value of (X _A / Y) × 100 is preferably 0.1500 or less, more preferably 0.1200 or less, and even more preferably 0.1000 or less. The lower limit of (X _A / Y) × 100 is the detection limit. However, from the viewpoint that 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) is obtained, (X _A / Y) × 100 The value is preferably at least 0.0500, more preferably at least 0.0600, even more preferably at least 0.0700.

  An aqueous solution prepared from the composition according to the first embodiment, which has an alkaline phosphatase concentration of 10% by weight, is calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis. 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 400 or less, more preferably 380 or less, and even more preferably 350 or less. The lower limit of the peak area value of the peptide fragment group (A) is the detection limit. However, in order to obtain 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 100 or more, more preferably 130 or more, and still more preferably 150 or more.

  An alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by LC-UV analysis using an aqueous solution prepared from the composition according to the first embodiment and having an alkaline phosphatase concentration of 10% by weight. Is preferably 200,000 or more, more preferably 220000 or more, and still more preferably 240,000 or more. The upper limit of the peak area of alkaline phosphatase is not particularly limited. The peak area value of alkaline phosphatase is preferably 500,000 or less, more preferably 400,000 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 embodiment, 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≦0.1800 ··· (B)
Meet.

In the above formula (B), X _B is calculated by the automatic integration method from the resulting extracted ion chromatogram by LC-MS / MS analysis of the composition according to the second embodiment, the peak of the peptide fragment group (B) Y 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 the peptide fragment group (B)”, when the peptide fragment group (B) is composed of one type of peptide fragment, means the peak area value of the one type of peptide fragment. When B) is composed of two or more peptide fragments, the total peak area value of the two or more peptide fragments (that is, the total peak area value of all the peptide fragments of the peptide fragment group (B)) ). "Peak area value of alkaline phosphatase", when the composition according to the second aspect contains one kind of alkaline phosphatase, means the peak area value of the one kind of alkaline phosphatase, the composition according to the second aspect When two or more alkaline phosphatases are contained, the total peak area of the two or more alkaline phosphatases (that is, the total peak area of all kinds of alkaline phosphatases contained in the composition according to the second embodiment) is meant. I do. The description regarding the above formula (A) (including the description regarding the LC-MS / MS analysis and the LC-UV analysis) is also applied to the above formula (B), unless otherwise specified.

  The m / z value of the ion 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 more preferably 50 to 2200. Preferably it is 200-1500, Still more preferably, it is 300-1200.

The value of (X _B / Y) × 100 is not particularly limited as long as it is 0.1800 or less, but the smaller the value, the better. The value of (X _B / Y) × 100 is preferably 0.1500 or less, more preferably 0.1200 or less, and even more preferably 0.1000 or less. The lower limit of _(X B / Y) × 100 is the detection limit. 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 the peptide fragment group (B) by purification), (X _B / Y) × 100 The value is preferably at least 0.0500, more preferably at least 0.0600, even more preferably at least 0.0700.

  An aqueous solution prepared from the composition according to the second embodiment, which has an alkaline phosphatase concentration of 10% by weight, is calculated by an automatic integration method from an extracted ion chromatogram obtained by LC-MS / MS analysis. In addition, 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 400 or less, more preferably 380 or less, and even more preferably 350 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 obtained. The value is preferably at least 100, more preferably at least 130, even more preferably at least 150.

  An aqueous solution prepared from the composition according to the second embodiment, wherein the alkaline phosphatase is calculated by an automatic integration method from a chromatogram obtained by LC-UV analysis using an aqueous solution having an alkaline phosphatase concentration of 10% by weight. Is preferably 200,000 or more, more preferably 220000 or more, and still more preferably 240,000 or more. The upper limit of the peak area of alkaline phosphatase is not particularly limited. The peak area value of alkaline phosphatase is preferably 500,000 or less, more preferably 400,000 or less, and even more preferably 350,000 or less.

  In addition to the peptide fragment group (B), the composition according to the second aspect comprises a peptide fragment consisting of consecutive 5 to 20 amino acid residues selected from positions 451 to 490 of the amino acid sequence described in SEQ ID NO: 2. In the embodiment containing, the composition according to the second aspect may or may not satisfy the above formula (A).

[Ratio of content of first peptide fragment to content of alkaline phosphatase]
In the composition according to the third aspect, 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≦0.1800 (1)
Meet.

In the embodiment in which the composition according to the first or second aspect contains the first peptide fragment, the ratio of the content of the first peptide fragment to the content of alkaline phosphatase is determined by the following formula (1):
(X ₁ /Y)×100≦0.1800 (1)
It is preferable to satisfy the following.

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

The value of (X ₁ / Y) × 100 is not particularly limited as long as it is 0.1800 or less, but the smaller the value, the better. The value of (X ₁ / Y) × 100 is preferably 0.1500 or less, more preferably 0.1200 or less, and even more preferably 0.1000 or less. The lower limit of (X ₁ / Y) × 100 is the detection limit. 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 obtained. Is preferably at least 0.0500, more preferably at least 0.0600, even more preferably at least 0.0700.

  From an extracted ion chromatogram obtained by LC-MS / MS analysis using an aqueous solution prepared from the composition according to the first, second or third aspect, wherein the aqueous solution has an alkaline phosphatase concentration of 10% by weight. The smaller the peak area value of the first peptide fragment calculated by the automatic integration method, the better. The peak area value of the first peptide fragment is preferably 400 or less, more preferably 380 or less, and even more preferably 350 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 at least 100, more preferably at least 130, even more preferably at least 150.

  An aqueous solution prepared from the composition according to the first, second or third aspect, wherein the concentration of alkaline phosphatase is 10% by weight. The calculated peak area value of alkaline phosphatase is preferably 200,000 or more, more preferably 220000 or more, and still more preferably 240,000 or more. The upper limit of the peak area of alkaline phosphatase is not particularly limited. The peak area value of alkaline phosphatase is preferably 500,000 or less, more preferably 400,000 or less, and even more preferably 350,000 or less.

[Alkaline phosphatase specific activity]
The composition of the present invention preferably has a specific activity of alkaline phosphatase of 2000 U / mg or more. The specific activity of the alkaline phosphatase of the composition of the present invention is more preferably 2500 U / mg or more, and even more preferably 3000 U / mg or more. The specific activity of the alkaline phosphatase 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 generated by adding alkaline phosphatase to the aqueous solution of p-nitrophenyl phosphate.

[Other ingredients]
The composition of the present invention may contain one or more other components. Examples of the other components include an aqueous medium such as water, a metal salt such as a magnesium salt and a sodium salt, a surfactant, an organic acid, and glycerin.

  The composition of the present invention can be used for various uses requiring alkaline phosphatase activity, and can contain one or more other components selected according to the use.

  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 to label 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 obtained by esterifying a carboxyl group of alkaline phosphatase with succinimide with an amino group of the protein.

  In one embodiment, the compositions of the present invention contain a substrate for alkaline phosphatase. In this embodiment, the compositions of the present 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. Substrates for alkaline phosphatase include, for example, nucleic acids, phospholipids, pyrophosphate and the like. When an alkaline phosphatase substrate is treated with the composition of the present invention, the phosphate monoester bond of the alkaline phosphatase substrate is hydrolyzed by alkaline phosphatase, resulting in dephosphorylation of the alkaline phosphatase substrate.

In a preferred embodiment, the compositions of the invention contain a nucleic acid. In this embodiment, the compositions of the present 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 nucleic acids. The peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment mixed in the alkaline phosphatase may have an adverse effect when dephosphorylating nucleic acids with alkaline phosphatase. In this regard, in the composition of the present invention, the ratio of the content of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment to the content of alkaline phosphatase satisfies the above-mentioned predetermined expression. That is, in the composition of the present invention, the relative content of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment is reduced. Therefore, by using the composition of the present invention, the adverse effects of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment which can be generated when dephosphorylating nucleic acid with alkaline phosphatase are reduced. Thus, the efficiency of dephosphorylation of nucleic acids can be improved. By treating a nucleic acid with the composition of the present invention, the 5 ′ end and / or 3 ′ end of the nucleic acid can be dephosphorylated. By dephosphorylating the 5 'end and / or 3' end of the nucleic acid, the labeling efficiency when labeling the 5 'end and / or 3' end of the nucleic acid with a labeling substance can be improved. This effect is particularly remarkable when ³² P is used as a labeling substance. In addition, self-ligation of the vector can be prevented by dephosphorylating the 5 'end and / or the 3' end of the vector used for DNA cloning, thereby reducing the background of the transformed cells. be able to.

  The nucleic acid is, for example, a nucleic acid such as DNA, RNA, PNA (peptide nucleic acid), or LNA (Locked Nucleic Acid) or a nucleic acid derivative. Examples of the nucleic acid derivative include modified nucleotides (for example, reconstitution of nucleotides and bases containing groups such as alkyl such as halogen and methyl, alkoxy such as methoxy and the like, thio and carboxymethyl, saturation of double bond, deamination, oxygenation Nucleic acid derivative) which is substituted with a sulfur molecule in the molecule. The nucleic acid may be single-stranded or double-stranded. Examples of the DNA include chromosomal DNA, viral DNA, DNA such as bacteria and fungi, cDNA, and fragments thereof. Examples of the 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 such as pathogenic bacteria and viruses or a part thereof, and genes causing a genetic disease or a part thereof.

  The nucleic acid can be prepared by, for example, extracting from a biological material, a virus, a bacterium, a mold, a food or the like by a conventional method. Examples of the biomaterial include body fluids such as blood, serum, plasma, urine, stool, cerebrospinal fluid, saliva, wiping fluid, and tissue fluid, cells, and tissues. Biomaterials may be of animal or plant origin.

  The amount of the nucleic acid contained in the composition of the present invention can be appropriately adjusted according to the intended use of the composition of the present invention (for example, detection of a target nucleic acid) and the like. 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 performed using the nucleic acid contained in the composition of the present invention as a template. Thereby, 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 depending on the purpose of use of the composition of the present invention (for example, detection of a target nucleic acid) and the like. For example, when detecting nucleic acids, the length of the nucleic acids is usually 10 to 300 bases, preferably 10 to 100 bases, and more preferably 15 to 50 bases. The length of the nucleic acid can be appropriately adjusted by fragmentation. The length of the nucleic acid is, for example, a length capable of hybridizing with the probe. When the nucleic acid is long (for example, 1500 bases or more, especially 4000 bases or more), it is preferable to adjust the length of the nucleic acid to an appropriate length by subjecting the nucleic acid to fragmentation. When performing the fragmentation treatment, it is not necessary to select a specific nucleic acid fragment from the generated nucleic acid fragments, and the fragmentation-treated product can be used as it is.

  As a method for cutting nucleic acid for fragmentation, for example, a method for cutting by irradiation with ultrasonic waves, a method for cutting with an enzyme, a method for cutting with a restriction enzyme, a method using a nebulizer, a method for cutting with an acid or alkali Method and the like. In the case of the method of cutting with an ultrasonic wave, it is possible to cut the nucleic acid into a desired length by controlling the output intensity and the irradiation time of the ultrasonic wave applied to the nucleic acid.

  In a preferred embodiment, the compositions of the present invention contain a dephosphorylated nucleic acid. The description regarding nucleic acids is the same as above. Dephosphorylated nucleic acids have a 5 'end and / or a 3' end 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 label 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) or the first peptide fragment mixed in alkaline phosphatase is used when dephosphorylating a nucleic acid with alkaline phosphatase and / or a labeling substance is added to the dephosphorylated nucleic acid. Can be adversely affected when binding. In this regard, in the composition of the present invention, the ratio of the content of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment to the content of alkaline phosphatase satisfies the above-mentioned predetermined expression. That is, in the composition of the present invention, the relative content of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment is reduced. Therefore, by using the composition of the present invention, a peptide fragment group (A) that can be generated when dephosphorylating a nucleic acid with alkaline phosphatase and / or binding a labeling substance to the dephosphorylated nucleic acid In addition, the adverse effect of the peptide fragment group (B) or the first peptide fragment can be reduced, and the efficiency of dephosphorylation of nucleic acid and / or the efficiency of labeling of dephosphorylated nucleic acid 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 a 5 'end and / or a 3' end dephosphorylated with alkaline phosphatase. The labeling substance binds to the 5 'end and / or 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, a metal fine particle, biotin, a radioisotope and the like can be used. The reaction conditions for labeling the target nucleic acid can be appropriately adjusted according to the type of the labeling substance. When the labeling substance is a fluorescent dye, the fluorescent dye can be detected using a fluorescent microscope, a fluorescent 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, Organic fluorescent dyes such as pyrene dyes, Texas Red dyes, and cyanine dyes are exemplified.

  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, A exa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581 / 5930IP 665 (all manufactured by Invitrogen), methoxy coumarin, eosin, NBD, pyrene, Cy5, Cy5.5, and the like Cy7.

  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. 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 subjected to a 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, a target nucleic acid contained in a nucleic acid sample can be detected using a labeling substance of 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 target nucleic acid can be detected using a probe capable of hybridizing with the target nucleic acid. In one embodiment of a nucleic acid detection method using a probe, the probe is contacted with a nucleic acid sample containing the target nucleic acid, the probe is hybridized with the target nucleic acid, the target nucleic acid hybridized with the probe, the target nucleic acid is The detected labeling substance can be detected as an index. When a nucleic acid other than the target nucleic acid is contained in the sample, it is preferable that the nucleic acid not hybridized with the probe is removed by washing or the like after the target nucleic acid is brought into contact with the probe.

  The reaction conditions for hybridization between the target nucleic acid and the probe can be appropriately adjusted according to the length of the target nucleic acid, the length of the probe, and the like. The reaction time is generally 30-1200 minutes, preferably 60-360 minutes. The reaction temperature is usually from 25 to 60 ° C, preferably from 30 to 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. It can be appropriately adjusted according to the length, the type of the labeling substance, and the like.

  As the probe, for example, a nucleic acid or a nucleic acid derivative such as DNA, RNA, PNA (peptide nucleic acid), and LNA (Locked Nucleic Acid) can be used. Examples of the nucleic acid derivative include modified nucleotides (for example, reconstitution of nucleotides and bases containing groups such as alkyl such as halogen and methyl, alkoxy such as methoxy and the like, thio and carboxymethyl, saturation of double bond, deamination, oxygenation Nucleic acid derivative) which is substituted with a sulfur molecule in the molecule.

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

  The length (base number) 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, and more preferably 20 to 70 bases. It 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, and more preferably 15 to 100 bases.

  The probe may be any of a commercially available product, a synthetic product, a preparation from a living body, and the like. A nucleic acid called an oligonucleic acid having a length of up to 200 bases can be easily and 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 to the target nucleic acid, and the target nucleic acid hybridized to the probe is labeled with the target nucleic acid. The substance can be detected as an index. When a nucleic acid other than the target nucleic acid is contained in the sample, it is preferable that the target nucleic acid is brought into contact with the nucleic acid microarray, and then the nucleic acid not hybridized with the probe on the nucleic acid microarray is removed by washing or the like. By using a nucleic acid microarray provided with a plurality of probes, two or more types of target nucleic acids can be simultaneously detected.

  The support is not particularly limited as long as the probe can be immobilized. Examples of the support include a slide glass, a membrane, and beads. Examples of the material of the support include inorganic materials such as glass, ceramic, and silicon, and polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate, and silicone rubber.

  The 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 an oligonucleic acid on the upper surface of the support, and a method of dropping and fixing an oligonucleic acid synthesized in advance to the upper surface of the support, and the like. The former method includes, for example, 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. 7,037,659), and the like. Is mentioned. In these methods, since an organic solvent is used during the DNA synthesis reaction, the support is preferably made of a material that is resistant to the organic solvent. For example, a glass support having a concavo-convex structure manufactured using the method described in Japanese Patent Application Laid-Open No. 10-503841 can be used. In particular, in the method of Francesco et al., The support is preferably made of a light-transmitting 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), a method using a glass capillary, and the like. As an example of the glass capillary, a commercially available product such as a self-made glass capillary or a micropipette (MP-005, manufactured by Micro Support Co., Ltd.) can be used.

  As a method for detecting a target nucleic acid, a sandwich hybridization method can be used. In the sandwich hybridization method, a first probe (capture probe) fixed to a support and a second probe (detection probe) not fixed to a 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 to 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. By detecting the labeling substance contained in the complex, the target nucleic acid can be detected.

  It is preferable that the sequence identity between the base sequence of the detection probe and the base sequence of the capture probe is low. Sequence identity is preferably at most 20%, more preferably at most 10%. Here, the identity of the two base sequences is determined by aligning the two sequences so that the bases match as much as possible (with a gap if necessary), and reducing the number of matched bases to the total number of bases (bases of the two base sequences). This is a numerical value obtained by dividing by a larger number of bases when the numbers are different, and can be easily calculated by commercially available software such as FASTA or 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 label) is compared with ambient noise. Specifically, a signal value obtained from a position on the support where the probe is fixed is compared with a signal value (noise value) obtained from a position on the support where the probe is not fixed. Let the ratio between the numerical value of the above and the noise value be the S / N ratio. The detection accuracy can be represented by the S / N ratio. That is, the higher the S / N ratio, the higher the detection accuracy, and the lower 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 nucleic acid detection method uses the composition of the present invention as a nucleic acid sample. By using this, the detection sensitivity of the target nucleic acid can be improved. This effect is remarkable in a nucleic acid detection method using a small amount (preferably 5 to 1000 μL, more preferably 5 to 500 μL) of a nucleic acid sample. In a nucleic acid detection method using a small amount of a nucleic acid sample, the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment contained in the nucleic acid sample may adversely affect the detection sensitivity. In this regard, in the composition of the present invention, the ratio of the content of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment to the content of alkaline phosphatase satisfies the above-mentioned predetermined expression. That is, in the composition of the present invention, the relative content of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment is reduced. Therefore, by using the composition of the present invention as a nucleic acid sample in a nucleic acid detection method using a small amount of a nucleic acid sample, adverse effects of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment can be obtained. 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 small amount (preferably 5 to 1000 μL, more preferably 5 to 500 μL) of a nucleic acid sample is used. Therefore, in the nucleic acid detection method using the nucleic acid microarray, the adverse effect of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment is reduced by using the composition of the present invention as a nucleic acid sample. As a result, the detection sensitivity of the target nucleic acid can be significantly improved.

[Production method]
Compositions of the present invention include alkaline phosphatase extract from bovine and shrimp organs, alkaline phosphatase extract from a microorganism into which a gene encoding alkaline phosphatase has been introduced, and cells of a microorganism into which a gene encoding alkaline phosphatase has been introduced. It can be produced by separating a peptide fragment group (A), a peptide fragment group (B) or a first 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) or the first peptide fragment include dialysis, salting out, gel filtration, ultrafiltration, membrane separation, ion exchange, column chromatography, and electricity. Electrophoresis and the like. 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, the content of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment relative to the alkaline phosphatase content can be adjusted to a desired range. It can be. Column chromatography is, for example, liquid chromatography. The column and mobile phase used in the liquid chromatography are not particularly limited as long as the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment can be separated. It is preferred to use

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

In one embodiment, the composition of the present invention comprises the following steps:
Providing a composition of the present invention;
A step of preparing a nucleic acid; and a step of treating the nucleic acid with the composition and dephosphorylating the nucleic acid. The peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment mixed in the alkaline phosphatase may have an adverse effect on dephosphorylation of nucleic acid by alkaline phosphatase. In this regard, in the composition of the present invention, the ratio of the content of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment to the content of alkaline phosphatase satisfies the above-mentioned predetermined expression. That is, in the composition of the present invention, the relative content of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment is reduced. Therefore, by treating the nucleic acid with the composition of the present invention, the efficiency of dephosphorylation of the nucleic acid can be improved.

In one embodiment, the composition of the present invention comprises the following steps:
Providing a composition of the present invention;
Providing a nucleic acid;
Preparing a labeling substance;
It is used in a method for producing a labeled nucleic acid, comprising a step of treating the nucleic acid with the composition and dephosphorylating 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) or the first peptide fragment mixed in alkaline phosphatase is used when dephosphorylating a nucleic acid with alkaline phosphatase and / or a labeling substance is added to the dephosphorylated nucleic acid. Can be adversely affected when binding. In this regard, in the composition of the present invention, the ratio of the content of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment to the content of alkaline phosphatase satisfies the above-mentioned predetermined expression. That is, in the composition of the present invention, the relative content of the peptide fragment group (A), the peptide fragment group (B) or the first peptide fragment is reduced. Therefore, by treating the nucleic acid with the composition of the present invention, the efficiency of dephosphorylation of the nucleic acid and / or the efficiency of labeling of the dephosphorylated nucleic acid can be improved.

  In the step of treating a nucleic acid with the composition of the present invention and dephosphorylating the nucleic acid, reaction conditions can be appropriately adjusted. The reaction time is generally 10-60 minutes, preferably 20-50 minutes. The reaction temperature is usually 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, RNA, or the like. When a nucleic acid is treated with a composition of the present invention, the 5 'and / or 3' end of the nucleic acid is dephosphorylated.

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

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

<< Conditions for LC-UV analysis >>
The conditions of the LC-UV analysis used in Examples and Comparative Examples are as follows.
<Apparatus configuration>
Liquid chromatograph: LC-30A system (manufactured by Shimadzu Corporation)
Detector: UV-Vis (190-900 nm, manufactured by Shimadzu Corporation)
<Liquid chromatography conditions>
Column: Acquity BEH C18 1.7 μm (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 is as follows. The measurement was performed using “3D-Gene” human miRNA oligo chip (corresponding to miRBase release 21) manufactured by Toray Industries, Inc.

[Comparative Examples 1 to 8]
Eight kinds 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 an alkaline phosphatase derived from bovine intestine. When the specific activity of alkaline phosphatase of each of the compositions C1 to C8 was 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 was 2519 U / mg. The product C5 was 2411 U / mg, the composition C6 was 2552 U / mg, the composition C7 was 2448 U / mg, and the composition C8 was 2490 U / mg. The specific activity of alkaline phosphatase was measured by a method using p-nitrophenyl phosphate. Specifically, it is as follows.

The following solutions A and B were prepared.
Solution A: 1 M 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, and the change in absorbance per unit time was determined (ΔOD). As a control, the absorbance change was determined for 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 equation.
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 aqueous alkaline phosphatase solution was diluted with distilled water so that the absorbance at 214 nm was 0.1 to 1.0, and the value obtained by multiplying the dilution ratio by approximating 1 Abs = 1 mg / mL was defined as the alkaline phosphatase concentration. The specific activity in the present invention is the activity (U / mg) per 1 mg of alkaline phosphatase, and was calculated by the above-mentioned measurement method.

  A 10% by weight aqueous solution of alkaline phosphatase was prepared from each of the compositions C1 to C8, and LC-UV analysis and LC-MS / MS analysis were performed using this aqueous solution. Based on the extracted ion chromatogram obtained by the LC-MS / MS analysis, the peak area value of the first peptide fragment consisting of the amino acid sequence represented by SEQ ID NO: 1 (APGKALDSK) was calculated by an automatic integration method. Further, based on the chromatogram obtained by LC-UV analysis, the peak area value of alkaline phosphatase was calculated by an automatic integration method. In the LC-UV analysis, alkaline phosphatase was detected as a component having an absorption at 214 nm.

  FIG. 1 shows an extracted ion chromatogram of the first 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-based organic fluorescent dye. Specifically, a dephosphorylation reaction and a 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 the serum using “3D-Gene” RNA extraction reagent from liquid sample kit (manufactured by Toray Industries, Inc.). The obtained extracted microRNA was used as a mother liquor, and labeled using a “3D-Gene” miRNA labeling kit (manufactured by Toray Industries, Inc.). 5 μL of the obtained extracted microRNA was added to a mixture of 0.4 μL of AP buffer and 1.0 μL of Spike Control of the above kit, and a solution was further prepared by adding 0.4 μL of composition C1. Next, after incubating at 37 ° C. for 40 minutes, the plate was allowed to stand on ice for 2 minutes. Next, 1.2 μL of LE Buffer, 3.0 μL of 3D-Gene Fluorescent Label, 2.5 μL of Nuclease free water, and 1.0 μL of Labeling enzyme were added, followed by incubation at 16 ° C. for 1 hour, followed by incubation at 65 ° C. for 15 minutes. Thus, 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 described 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 the 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 settings were 100% laser output, and the photomultiplier voltage setting was 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, those having a value obtained by subtracting noise (signal value of no spot) from the detected signal value are 100 or more are regarded as effective spots, and the number of effective spots is divided by the total number of spots. And multiplied by 100 to obtain a detection rate. The results are shown in Table 4-2.

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

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

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

(Hydrophobic column process)
The alkaline phosphatase fraction was recovered from the recovered solution after the 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, 3 M ammonium sulfate (50/50)
Mobile phase B: 20 mM disodium hydrogen phosphate gradient program:

Detector: UV 214nm

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

(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% by weight glycerin) using an ultrafiltration column (cutoff molecular weight 10 K). , To obtain a composition E1.

  Compositions E2, E3, and E4 were similarly obtained from compositions C3, C4, and C8, respectively, using methods similar to those described above.

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

  From each of the compositions E1 to E4, a 10% by weight aqueous solution of alkaline phosphatase was prepared, 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 peak area value of the first peptide consisting of the amino acid sequence (APGKALDSK) described in SEQ ID NO: 1 was calculated by an automatic integration method. Further, based on the chromatogram obtained by LC-UV analysis, the peak area value of alkaline phosphatase was calculated by an automatic integration method. In the LC-UV analysis, alkaline phosphatase was detected as a component having an absorption at 214 nm.

FIG. 2 shows an extracted ion chromatogram of the first peptide fragment obtained in Example 1 by LC-MS / MS analysis of the composition E1 (a purified product of the composition C2).
FIG. 3 shows a chromatogram of alkaline phosphatase obtained in Example 1 by LC-UV analysis of composition E1 (purified product of composition C2). In addition, the chromatogram regarding alkaline phosphatase obtained by LC-UV analysis of each composition in Examples 2 to 4 and Comparative Examples 1 to 8 was similar to FIG.

  The nucleic acid was dephosphorylated using the alkaline phosphatase compositions of Examples 1 to 4, and the obtained dephosphorylated nucleic acid was labeled with a cyanine-based organic fluorescent dye. The dephosphorylation reaction and the labeling reaction were performed in the same manner as described 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 Table 4-1 and Table 4-2, when the nucleic acid sample prepared using the alkaline phosphatase compositions of Comparative Examples 1 to 8 was 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. In addition, although the detection rates in Comparative Examples 1 to 8 were different although the alkaline phosphatase specific activities of the alkaline phosphatase compositions were almost the same, the detection rates in Examples 1 to 4 were Approximately the same, and higher than the detection rates of Comparative Examples 1 to 8.

As shown in Tables 4-1 and 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 the value of Comparative Examples 1 to 4. The alkaline phosphatase compositions of Examples 1 to 4 exceed 0.1800 (the minimum value is 0.1810 in the alkaline phosphatase composition of Comparative Example 4), whereas the alkaline phosphatase compositions of Examples 1 to 4 are 0.1800 or less. is there. This is considered to be the main cause of the difference in the effects between Examples 1 to 4 and Comparative Examples 1 to 8.

Claims (5)

Alkaline phosphatase,
A composition comprising a peptide fragment group (A) composed of one or more peptide fragments consisting of 5 to 20 consecutive amino acid residues selected from positions 451 to 490 of the amino acid sequence of SEQ ID NO: 2; 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≦0.1800 (A)
Wherein, X _A is the calculated from the extracted ion chromatogram obtained by LC-MS / MS analysis of the composition by an automatic integration method, represents the peak area value of the peptide fragment group (A), Y Represents a peak area value of the alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, wherein Alkaline phosphatase,
One or more peptides consisting of consecutive 9 to 20 amino acid residues selected from positions 451 to 490 of the amino acid sequence of SEQ ID NO: 2 and including positions 469 to 477 of the amino acid sequence of SEQ ID NO: 2 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≦0.1800 ··· (B)
Wherein, X _B is the calculated by the automatic integration method from the extracted ion chromatogram obtained by LC-MS / MS analysis of the composition, represents the peak area value of the peptide fragment group (B), Y Represents a peak area value of the alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, wherein Alkaline phosphatase,
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≦0.1800 (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 represents Represents the peak area value of the alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by LC-UV analysis of the composition. ]
The above composition, wherein The following steps:
Providing a composition according to any one of claims 1 to 3;
A method for producing a dephosphorylated nucleic acid, comprising: preparing a nucleic acid; treating the nucleic acid with the composition, and dephosphorylating the nucleic acid. The following steps:
Providing a composition according to any one of claims 1 to 3;
Providing a nucleic acid;
Preparing a labeling substance;
A method for producing a labeled nucleic acid, comprising: treating the nucleic acid with the composition to dephosphorylate the nucleic acid; and binding the labeling substance to the dephosphorylated nucleic acid.