Bst DNA polymerase, reagent composition, and nucleic acid amplification method

Abstract

To provide a Bst DNA polymerase capable of reducing the effects of interfering substances and reactivity at temperatures lower than an optimal reaction temperature, to provide a reagent composition and a reagent kit containing the Bst DNA polymerase, and to provide a method for amplifying nucleic acid using the Bst DNA polymerase.SOLUTION: The above problems are solved by a Bst DNA polymerase, which contains a specific amino acid sequence or a consensus sequence Asn-X-(Ser / Thr) (X is any amino acid residue other than a proline residue), contains an amino acid sequence that has 80% or more identity with the specific amino acid sequence, has 5'→3' DNA polymerase activity and strand displacement activity, and contains an N-linked glycan in the consensus sequence, the N-linked glycan containing three or more mannose residues.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a Bst DNA polymerase having an N-linked sugar chain.The present invention relates to a reagent composition for use in a nucleic acid amplification method, comprising the Bst DNA polymerase.The present invention relates to a nucleic acid amplification method using a Bst DNA polymerase having an N-linked sugar chain. [Background technology]

[0002] Bst DNA polymerase is a gene encoding the enzyme Geobacillus stearothermophilus ) and has 5'→3' DNA polymerase activity and strand displacement activity. Since Bst DNA polymerase has strand displacement activity, it can carry out an isothermal nucleic acid amplification reaction. Isothermal nucleic acid amplification reactions are used, for example, in fields such as medicine, food hygiene, and environmental hygiene, for genetic testing for cancer, and genetic testing for detecting viruses or bacteria. Since the activity of Bst DNA polymerase is maximized at 60°C to 65°C, Bst DNA polymerase is also suitable for synthesizing a DNA strand with a high GC content. Patent Document 1 describes the production of a recombinant Bst DNA polymerase using an Escherichia coli expression system. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Pat. No. 5,814,506 Summary of the Invention [Problem to be solved by the invention]

[0004] In general, it is known that in a nucleic acid amplification reaction, the amplification efficiency of a target nucleic acid decreases when an interfering substance is mixed into the reaction solution. An interfering substance is a substance that inhibits a nucleic acid amplification reaction, reduces the reaction rate, etc. An interfering substance may be contained in a sample containing a target nucleic acid, such as a biological sample such as blood, cells, or fixed and embedded tissue. Reagents used to extract and purify a target nucleic acid from such a sample may also be interfering substances. In addition, it is known that in a nucleic acid amplification reaction, non-specific amplification that does not depend on the nucleotide sequence of the target nucleic acid is likely to occur at a temperature lower than the optimal reaction temperature of the DNA polymerase used. For example, when the reaction solution is heated to the optimal reaction temperature, non-specific amplification products may be generated before the optimal reaction temperature is reached. When an isothermal nucleic acid amplification reaction using Bst DNA polymerase is used to detect a target nucleic acid, the influence of interfering substances and reactivity at a temperature lower than the optimal reaction temperature may reduce the detection sensitivity.

[0005] The present invention aims to provide a Bst DNA polymerase capable of reducing the influence of interfering substances and reducing reactivity at temperatures lower than the optimal reaction temperature, as well as a reagent composition containing the Bst DNA polymerase and a method for amplifying nucleic acid using the Bst DNA polymerase. [Means for solving the problem]

[0006] The present inventors have found that, when isothermal nucleic acid amplification is performed using the Bst DNA polymerase prepared by the present inventors, the influence of interfering substances and the reactivity at a temperature lower than the optimal reaction temperature are reduced compared to the case where a conventional Bst DNA polymerase is used, and have completed the present invention. The present invention provides the following [1] to

[21] .

[0007] [1] A Bst DNA polymerase comprising (1) the amino acid sequence shown in SEQ ID NO:1, or (2) the consensus sequence Asn-X-(Ser / Thr) (X is any amino acid residue other than a proline residue) and having 80% or more identity to the amino acid sequence shown in SEQ ID NO:1, and having 5'→3' DNA polymerase activity and strand displacement activity, and comprising an N-linked glycan in the consensus sequence, the N-linked glycan containing three or more mannose residues.

[0008] [2] The Bst DNA polymerase according to [1], which comprises a consensus sequence in the finger domain.

[0009] [3] The Bst DNA polymerase according to [1] or [2], wherein the N-linked sugar chain contains 9 or less mannose residues.

[0010] [4] The Bst DNA polymerase according to any one of [1] to [3], wherein the N-linked sugar chain has a structure represented by the following formula (I):

[0011] [ka] (In the formula, Asn is an asparagine residue in the consensus sequence, Man is a mannose residue, GlcNAc is an N-acetyl-D-glucosamine residue, Fuc is a fucose residue, m and n are each independently 0 or 1, and S1, S2, S3 and S4 are each independently a sugar chain that is absent or contains 1 to 5 monosaccharide residues.)

[0012] [5] The Bst DNA polymerase according to [4], wherein S1 contains at least one monosaccharide residue selected from the group consisting of mannose residues, N-acetyl-D-glucosamine residues and glucose residues.

[0013] [6] The Bst DNA polymerase according to [4] or [5], wherein S2 contains at least one monosaccharide residue selected from the group consisting of mannose residues, N-acetyl-D-glucosamine residues and glucose residues.

[0014] [7] The Bst DNA polymerase according to any one of [4] to [6], wherein S3 contains at least one monosaccharide residue selected from the group consisting of mannose residues, N-acetyl-D-glucosamine residues and glucose residues.

[0015] [8] The Bst DNA polymerase according to any one of [4] to [7], wherein S4 contains at least one monosaccharide residue selected from the group consisting of mannose residues, N-acetyl-D-glucosamine residues and glucose residues.

[0016] [9] The Bst DNA polymerase according to [4], which is free of S1, S2, S3 and S4.

[0017]

[10] The Bst DNA polymerase according to any one of [4] to [8], wherein at least one of the sugar chains S1, S2, S3 and S4 is an N-acetyl-D-glucosamine residue.

[0018]

[11] The Bst DNA polymerase according to any one of [4] to [8], wherein at least one of the sugar chains S1, S2, S3 and S4 has a structure represented by the following formula (II):

[0019] [ka] (In the formula, S5 and S6 are each independently a sugar chain that is absent or contains 1 to 3 mannose residues.)

[0020]

[12] The Bst DNA polymerase according to any one of [1] to

[11] , wherein at least one non-reducing end of the N-linked sugar chain is a mannose residue or an N-acetyl-D-glucosamine residue.

[0021]

[13] The Bst DNA polymerase according to any one of [1] to

[12] , wherein m is 1 and the n is 0, or m is 0 and the n is 1.

[0022]

[14] A reagent composition for use in a method for amplifying nucleic acid, comprising the Bst DNA polymerase according to any one of [1] to

[13] .

[0023]

[15] The reagent composition according to

[14] , wherein the nucleic acid amplification method is carried out under substantially isothermal conditions.

[0024]

[16] The reagent composition according to

[14] or

[15] , wherein the nucleic acid amplification method is the LAMP method, the RCA method, the NASBA method, the SDA method, the TRC method or the ICAN method.

[0025]

[17] A reagent kit for use in a method for amplifying nucleic acid, comprising the reagent composition according to any one of

[14] to

[16] and dNTPs.

[0026]

[18] The reagent kit according to

[17] , wherein the nucleic acid amplification method is carried out under substantially isothermal conditions.

[0027]

[19] The reagent kit according to

[17] or

[18] , wherein the nucleic acid amplification method is the LAMP method, the RCA method, the NASBA method, the SDA method, the TRC method or the ICAN method.

[0028]

[20] A method for amplifying a target nucleic acid, comprising amplifying the target nucleic acid using the Bst DNA polymerase according to any one of [1] to

[13] .

[0029]

[21] The method for amplifying nucleic acid according to

[20] , wherein the amplification reaction of the target nucleic acid is carried out under substantially isothermal conditions. Effect of the Invention

[0030] According to the present invention, there is provided a Bst DNA polymerase capable of reducing the influence of interfering substances and reducing reactivity at temperatures lower than the optimal reaction temperature. Furthermore, according to the present invention, there are provided a reagent composition containing the Bst DNA polymerase and a method for amplifying nucleic acid using the Bst DNA polymerase. [Brief description of the drawings]

[0031]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0032] The Bst DNA polymerase of this embodiment (hereinafter also referred to as "the polymerase of this embodiment") is characterized in that it has an N-linked glycan containing three or more mannose residues in a consensus sequence in its amino acid sequence. The consensus sequence is a sequence containing an asparagine residue (Asn) to which an N-linked glycan is bound, and is composed of three amino acid residues, Asn-X-(Ser / Thr). "X" is any amino acid residue other than a proline residue. "Ser / Thr" represents a serine residue or a threonine residue. The consensus sequence is expressed in one letter as NX(S / T). The glycan will be described later.

[0033] The polymerase of the present embodiment has 5'→3' DNA polymerase activity and strand displacement activity as enzyme activities. The 5'→3' DNA polymerase activity refers to the activity of the polymerase synthesizing a DNA strand complementary to a single-stranded DNA template in the 5' to 3' direction. The strand displacement activity refers to the activity of the polymerase synthesizing a complementary DNA strand while dissociating the hydrogen bonds of the double-stranded DNA in the extension direction in the process of synthesizing a DNA strand complementary to the template DNA. Since the polymerase of the present embodiment has strand displacement activity, a heat treatment at a high temperature (for example, 90°C to 99°C) for dissociating the double-stranded DNA template in a nucleic acid amplification reaction is not required. That is, the polymerase of the present embodiment can perform an amplification reaction using double-stranded DNA as a template under substantially isothermal conditions.

[0034] In the present specification, "substantially isothermal" refers to a reaction temperature that is approximately constant. Since the optimal reaction temperature of the polymerase of this embodiment is about 60°C to 65°C, the reaction temperature is preferably 55°C to 70°C, more preferably 58°C to 67°C, and even more preferably 60°C to 65°C. Amplification reaction under substantially isothermal conditions can be performed by incubation using a device such as an incubator or a thermal cycler set to keep the temperature constant. When incubation is performed using a device, the amplification reaction can be performed by raising the temperature to the above reaction temperature and then setting it to keep it constant within the above optimal reaction temperature range. The temperature during the amplification reaction does not need to be completely constant, and may vary within the above optimal reaction temperature range.

[0035] The polymerase of this embodiment may be a protein comprising the amino acid sequence shown in SEQ ID NO: 1. The protein comprising the amino acid sequence shown in SEQ ID NO: 1 may be, for example, a protein consisting of an amino acid sequence in which one or more amino acid residues have been added to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 1. When multiple amino acid residues have been added, the multiple amino acid residues may constitute, for example, a tag peptide as described below. Alternatively, the polymerase of this embodiment may be a protein consisting of the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 1 is as follows. In the sequence, the underlined portion indicates a consensus sequence.

[0036] AEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAA YLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSEL AEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRL SSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNL NIT RKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK (SEQ ID NO: 1)

[0037] The amino acid sequence shown in SEQ ID NO:1 is identical to that of Geobacillus stearothermophilus ( Geobacillus stearothermophilus ) corresponds to the portion from 290 to 876 of the amino acid sequence (SEQ ID NO: 2) of the full-length protein encoded by the Bst DNA polymerase gene. The amino acid sequence shown in SEQ ID NO: 1 is also called the large fragment of the full-length protein, and lacks the 5'→3' exonuclease domain of the full-length protein. Therefore, Bst DNA polymerase containing the amino acid sequence shown in SEQ ID NO: 1 does not have 5'→3' exonuclease activity. The amino acid sequence shown in SEQ ID NO: 2 is as follows. In the sequence, the underlined portion is the amino acid sequence shown in SEQ ID NO: 1.

[0038] MKKKLVLIDGNSVAYRAFFALPLLHNDKGIHTNAVYGFTMMLNKILAEEQPTHLLVAFDAGKTTFRHETFQEYKGGRQQTPPELSEQFPLLRELLKAYRIPAYELDHYEADDIIGTLAARAEQEGFEVKIISGDRDLTQLASRH VTVDITKKGITDIEPYTPETVREKYGLTPEQIVDLKGLMGDKSDNIPGVPGIGEKTAVKLLKQFGTVENVLASIDEVKGEKLKENLRQHRDLALLSKQLASICRDAPVELSLDDIVYEGQDREKVIALFKELGFQSFLEKMAAPA AEGEKPLEEMEFAIVDVITEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFFMRPETALADSQFLAWLADETKKKSMFDAKRAVVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDIAAVAKMKQYEAVRSDEAVYGKGVKRSLPDEQTLAEHLVRKAAAIWALEQPFMDDLRNNEQDQLLTKLEQPLAAILAEMEFTGVNVDTKRLEQMGSELAEQLRAIEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTGKVHTMFNQALTQTGRLSSAEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFQRDLDIHTKTAMDIFHVSEEEVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEEQLQARLLLQVHDELILEAPKEEIERLCELVPEVMEQAVTLRVPLKVDYHYGPTWYDAK (SEQ ID NO:2)

[0039] The polymerase of this embodiment may be a protein containing an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, so long as it contains the consensus sequence Asn-X-(Ser / Thr) and has the above-mentioned N-linked glycan and enzyme activity. Preferably, the polymerase of this embodiment may be a protein containing an amino acid sequence having 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 1. Alternatively, the polymerase of this embodiment may be a protein consisting of an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 1. The identity of the amino acid sequence can be determined by sequence alignment. Sequence alignment itself is known and refers to a process of aligning two amino acid sequences to be compared and detecting amino acid residues that match between these amino acid sequences. Sequence alignment indicates the number of identical amino acid residues between two amino acid sequences as a percentage. Sequence alignment can be performed using known multiple alignment programs such as ClustalW, TREBMAL, and BLAST, or commercially available protein analysis software such as Discovery Studio.

[0040] An amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 may be an amino acid sequence containing one or more amino acid residue modifications with respect to the amino acid sequence shown in SEQ ID NO: 1. The modification of an amino acid residue refers to the substitution, addition, deletion, and combinations thereof of an amino acid residue. The modification of an amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 may be, for example, a conservative substitution of an amino acid residue. Conservative substitution refers to the substitution of a certain amino acid residue in a sequence with another amino acid residue having similar properties such as structure, charge, polarity, or hydrophobicity. Examples of such a conservative substitution include substitution between a phenylalanine residue, a tryptophan residue, and a tyrosine residue, substitution between a serine residue and a threonine residue, substitution between a lysine residue, an arginine residue, and a histidine residue, and substitution between an aspartic acid residue and a glutamic acid residue. Alternatively, the modification of an amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 may be a modification corresponding to a mutation in a known mutant of Bst DNA polymerase. Bst DNA polymerase mutants are described, for example, in Ma Y. et al., BioMed Research International, (2016) vol.2016:2906484 and Paik I. et al., Biochemistry, (2023) vol.62, pp.410-418.

[0041] For example, the polymerase of this embodiment may have at least one modification selected from the group consisting of a deletion of the 1st alanine residue (A1), a substitution of the 311th glycine residue (G311), a substitution of the 366th serine residue (S366), a substitution of the 413th arginine residue (R413), a substitution of the 417th lysine residue (K417), a substitution of the 438th isoleucine residue (I438), a substitution of the 439th threonine residue (T439), a substitution of the 488th threonine residue (T488), a substitution of the 541st aspartic acid residue (D541), and a substitution of the 547th alanine residue (A547) in the amino acid sequence shown in SEQ ID NO:1.

[0042] In the amino acid sequence shown in SEQ ID NO: 1, the substitution of G311 may be, for example, an alanine residue (G311A) or a leucine residue (G311L). The substitution of S366 may be, for example, an aspartic acid residue (S366D). The substitution of R413 may be, for example, an alanine residue (R413A) or a glutamic acid residue (R413E). The substitution of K417 may be, for example, an alanine residue (K417A) or an aspartic acid residue (K417D). The substitutions of I438 and T439 are substitutions of consensus sequences. That is, the substitution of I438 is a substitution of any amino acid residue other than a proline residue, and the substitution of T439 is a substitution of a serine residue (T439S). The substitution of T488 may be, for example, an asparagine residue (T488N). Substitution at D541 may be, for example, with an alanine residue (D541A) or with a glutamic acid residue (D541E).Substitution at A547 may be, for example, with a glycine residue (A547G).

[0043] In general, DNA polymerases are known to have a highly conserved structure. Specifically, the three-dimensional structure of DNA polymerase resembles the human right hand, and the DNA polymerase includes, from the N-terminus, a thumb domain corresponding to the thumb, a palm domain corresponding to the palm, and a fingers domain corresponding to the fingers. The DNA polymerase binds to the template DNA by grasping it with these three domains. The polymerase of this embodiment preferably includes a thumb domain, a palm domain, and a fingers domain. In the amino acid sequence shown in SEQ ID NO: 1, the consensus sequence Asn-Ile-Thr is known to be included in the fingers domain. The polymerase of this embodiment preferably includes a consensus sequence Asn-X-(Ser / Thr) in the fingers domain. The fingers domain of the polymerase of this embodiment can be confirmed by examining the three-dimensional structure of the polymerase of this embodiment using, for example, commercially available protein analysis software such as Discovery Studio.

[0044] As described above, the polymerase of this embodiment has an N-linked glycan at the consensus sequence in its amino acid sequence. More specifically, the N-linked glycan is covalently bound to a nitrogen atom in the side chain of an asparagine residue in the consensus sequence. The number of N-linked glycans in the polymerase of this embodiment may be one or more. Bst DNA polymerase consisting of the amino acid sequence of SEQ ID NO: 1 has one consensus sequence and therefore has one N-linked glycan. When the polymerase of this embodiment has multiple consensus sequences, it may have one or more N-linked glycans.

[0045] The N-linked glycan contains three or more mannose residues. More specifically, the N-linked glycan contains at least a core structure represented by the following formula (III). Hereinafter, "Asn" in the formula indicates an asparagine residue in the consensus sequence. As represented in formula (III), the core structure is composed of three mannose residues (Man) and two N-acetyl-D-glucosamine residues (GlcNAc).

[0046] [ka]

[0047] The N-linked glycan is added to the consensus sequence of Bst DNA polymerase by producing the Bst DNA polymerase in an in vitro expression system for recombinant proteins using a host capable of glycosylation of proteins. Such hosts are preferably eukaryotes, such as yeast, insect cells, mammalian cells, and insects. Among these, insects and insect cells are preferred, and lepidopteran insects and their insect cells are particularly preferred. Lepidopteran insects suitable for recombinant protein expression systems are known, and examples thereof include the silkworm ( Bombyx mori ), Mulberry butterfly ( Spilosoma imparilis ), Sakusan ( Antheraea pernyi ), Spodoptera frugiperda ( Spodoptera frugiperda ), nettle silver moth (Trichoplusiani ) and the like. Among them, silkworms are particularly preferred. The lepidopteran insect may be any of adults, pupae and larvae, but it is preferable to use pupae from the viewpoint of serine protease activity and susceptibility to baculovirus. The cells of lepidopteran insects are not particularly limited as long as they are cell lines established from the above-mentioned lepidopteran insects, and examples thereof include BmN, BmN4, SpIm, Anpe, Sf9, Sf21, High5 and the like.

[0048] N-linked glycans can be added to Bst DNA polymerase, for example, by a protein expression system using silkworms as a host (hereinafter also referred to as "silkworm expression system"). Generally, a protein containing an N-linked glycan with up to nine mannose residues added can be produced in the endoplasmic reticulum of silkworms. Then, in the Golgi apparatus, N-linked glycans of various structures can be formed by stepwise cleavage of the added mannose residues, addition and cleavage of N-acetyl D-glucosamine residues to the non-reducing end, addition of one or two fucose residues to the core structure, addition of glucose residues to the non-reducing end, and the like. Preferably, at least one non-reducing end of the N-linked glycan is a mannose residue or an N-acetyl D-glucosamine residue.

[0049] An N-linked glycan that may be contained in the polymerase of this embodiment produced in a silkworm expression system is, for example, a glycan having a structure represented by the above formula (I) (hereinafter also referred to as "glycan of formula (I)"). In a silkworm expression system, multiple molecules of the polymerase of this embodiment are usually produced. The structure of the glycan of formula (I) in multiple molecules of the polymerase of this embodiment is not limited to one type. In other words, multiple molecules of the polymerase of this embodiment may contain glycan of formula (I) with two or more types of structures. Examples of the glycan of formula (I) include glycan having the structures represented by the following (IV) to (XI).

[0050] [ka]

[0051] [ka]

[0052] [ka] (In the formula, a1, a2, and a3 are each independently an integer of 0 or more and 2 or less, and the sum of a1, a2, and a3 is 1 or more and 3 or less.)

[0053] [ka] (In the formula, b1, b2, and b3 each independently represent an integer of 0 or more and 2 or less, and the sum of b1, b2, and b3 is 4 or less.)

[0054] [ka] (In the formula, c1 and c2 each independently represent an integer of 0 or more and 2 or less.)

[0055] [ka]

[0056] The N-linked glycan having the structure represented by the above formulas (IV) to (VI) corresponds to the case where S1, S2, S3 and S4 are not present in formula (I). The N-linked glycan having the structure represented by the above formula (IV) corresponds to the case where S1, S2, S3 and S4 are not present in formula (I), m is 1 and n is 0. The N-linked glycan having the structure represented by the above formula (V) corresponds to the case where S1, S2, S3 and S4 are not present in formula (I), m is 0 and n is 1. The N-linked glycan having the structure represented by the above formulas (VII), (VIII) and (X) corresponds to the case where at least one of the glycans S1, S2, S3 and S4 in formula (I) is an N-acetyl-D-glucosamine residue.

[0057] In formula (I), when at least one of the sugar chains S1, S2, S3 and S4 has a structure represented by the above formula (II), examples of the sugar chain of formula (I) include sugar chains having the structures represented by the following (XII) to (XIV).

[0058] [ka] (In the formula, d1 and d2 are each independently 0 or 1, and the sum of d1 and d2 is 0 or 1.)

[0059] As described above, the polymerase of the present embodiment can be produced by an in vitro expression system for recombinant proteins using a host capable of glycosylation of proteins. The method for producing recombinant proteins using such an expression system is itself publicly known. For example, when the host is yeast or cultured cells, the polymerase of the present embodiment can be expressed by transfecting a vector DNA containing a gene encoding the polymerase of the present embodiment into the yeast or cultured cells by a known gene transfer method. The polymerase of the present embodiment can be obtained by solubilizing the yeast or cultured cells expressing the polymerase of the present embodiment in a solution containing a cell lysing agent such as a surfactant.

[0060] In a preferred embodiment, the polymerase of the present embodiment is expressed by infecting a baculovirus recombinant with a vector DNA into a lepidopteran insect or cultured cells of the insect. When a lepidopteran insect is used, the polymerase of the present embodiment can be obtained by collecting a body fluid or by disrupting the insect to prepare a homogenate. If necessary, the polymerase of the present embodiment may be separated and purified from the homogenate by a known method such as centrifugation, salting out, or chromatography. When cultured cells of a lepidopteran insect are used, the polymerase of the present embodiment can be obtained in the same manner as when yeast or cultured cells are used. Since the polymerase of the present embodiment is a heat-resistant DNA polymerase, the baculovirus may be inactivated by heating a solution containing the polymerase of the present embodiment to 60°C to 70°C.

[0061] When the polymerase of this embodiment is a Bst DNA polymerase comprising the amino acid sequence of SEQ ID NO:1, the gene encoding the polymerase has the nucleotide sequence shown in SEQ ID NO:3, for example.

[0062] The vector DNA may have a promoter that enables gene expression in vitro in a host capable of glycosylation of a protein, and may be DNA into which a gene encoding the polymerase of the present embodiment can be inserted downstream of the promoter. Such vector DNAs themselves are known, and vector DNAs for protein expression according to the host are commercially available. If necessary, a gene encoding a tag peptide may be inserted upstream or downstream of the gene encoding the polymerase of the present embodiment. This makes it possible to obtain the polymerase of the present embodiment fused with the tag peptide. The tag peptide is preferably a tag for protein purification, and examples thereof include His tag, glutathione-S-transferase (GST) tag, and maltose-binding protein (MBP) tag.

[0063] A preferred vector DNA is a transfer vector capable of producing a recombinant baculovirus into which the above-mentioned gene is inserted by homologous recombination with baculovirus DNA. Such transfer vectors themselves are known, and examples thereof include pM02, pYNG, pBM030, pBM050, and pVL1392. The above-mentioned promoter can be appropriately selected from known promoters such as polyhedrin promoter, p10 promoter, and silkworm actin promoter. The method of recombining a baculovirus with a DNA having a desired nucleotide sequence is known. For example, first, a baculovirus DNA linearized with a restriction enzyme or the like and a transfer vector into which a gene encoding the polymerase of the present embodiment is incorporated are cotransfected into cultured cells of a lepidopteran insect. Then, a recombinant baculovirus can be obtained by screening the infected cells.

[0064] The type of baculovirus is not particularly limited as long as it is a virus capable of infecting the above-mentioned lepidopteran insects or their insect cells. A preferred baculovirus is a nuclear polyhedrosis virus (NPV) or a modified virus thereof. Examples of such viruses include BmNPV, HycuNPV, AnpeNPV, AcNPV, and the silkworm virus of the Bombycidae family ( Bombyx mori ) and the noctuid moth Autographa californica ( Autographa californica ) (see Japanese Patent Application Laid-Open No. 2003-052371). Preferably, a cysteine ​​protease-deficient (CPd) baculovirus is used (see Japanese Patent Application Laid-Open No. 7-303488).

[0065] The means for infecting a lepidopteran insect or its insect cells with the recombinant baculovirus is not particularly limited and can be appropriately selected from known methods. For example, when infecting a lepidopteran insect, a liquid containing the recombinant baculovirus can be injected into the insect. When infecting a cultured cell, a liquid containing the recombinant baculovirus can be added to a culture medium. Five to eight days after infecting a lepidopteran insect or its insect cells with the virus, the polymerase of the present embodiment is expressed in the lepidopteran insect or its insect cells.

[0066] The present invention also includes a reagent composition for use in a nucleic acid amplification method, which contains the polymerase of this embodiment (hereinafter, also referred to as the "reagent composition of this embodiment"). The reagent composition of this embodiment may contain a component for stably storing the polymerase. Examples of such components include a buffer solution (e.g., Tris-HCl), an antifreeze solution (e.g., glycerol), inorganic salts (KCl, NaCl, etc.), a reducing agent (e.g., dithiothreitol (DTT)), a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)), and a surfactant (e.g., Triton (trademark) X-100). The reagent composition of this embodiment may be in a solid (e.g., powder) state or in a solution state. The reagent composition of this embodiment may be contained in a container and provided to a user. The reagent composition of this embodiment contained in a container may be further packaged in a box and provided to a user.

[0067] The reagent composition of this embodiment can be used in a nucleic acid amplification method carried out under substantially isothermal conditions. The substantially isothermal conditions are as described above. Nucleic acid amplification methods carried out under substantially isothermal conditions are known, and examples thereof include the loop-mediated isothermal amplification (LAMP) method, the rolling circle amplification (RCA) method, the nucleic acid sequence-based amplification (NASBA) method, the strand displacement amplification (SDA) method, the transcription reverse-transcription concerted reaction (TRC) method, and the isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) method. For example, see Japanese Patent No. 3,313,358 for the LAMP method, U.S. Pat. No. 6,291,187 for the RCA method, U.S. Pat. No. 2,650,159 for the NASBA method, U.S. Pat. No. 5,270,184 for the SDA method, U.S. Pat. No. 4,438,110 for the TRC method, and U.S. Pat. No. 3,433,929 for the ICAN method.

[0068] The present invention also includes a reagent kit for use in a nucleic acid amplification method. The reagent kit includes the reagent composition of this embodiment. The reagent composition of this embodiment is as described above. The reagent kit may further include dNTPs and / or a buffer solution. "dNTPs" refers to a mixture containing deoxyadenosine triphosphate (dATP), deoxyacitidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP). dNTPs are usually provided in the form of a solution or a frozen product thereof. The reagent kit can be used in a nucleic acid amplification method carried out under substantially isothermal conditions. The nucleic acid amplification method carried out under substantially isothermal conditions is as described below.

[0069] An example of the reagent kit is shown in Fig. 1. In Fig. 1, 10 indicates the reagent kit, 11 indicates a first container containing the reagent composition of the present embodiment, 12 indicates a packaging box, and 13 indicates an attached document. The attached document may describe a method for using the reagent composition, a method for storing the reagent composition, and the like.

[0070] Another example of the reagent kit is shown in Fig. 2. In Fig. 2, 20 indicates the reagent kit, 21 indicates a first container containing the reagent composition of this embodiment, 22 indicates a second container containing dNTPs, 23 indicates a packaging box, and 24 indicates an attached document. The attached document may describe how to use and store the various reagents.

[0071] The present invention also includes a method for amplifying a nucleic acid (hereinafter also referred to as the "method of the present embodiment"), which comprises amplifying a target nucleic acid using the polymerase of the present embodiment. The target nucleic acid is any nucleic acid that is desired to be amplified. Preferably, the target nucleic acid is single-stranded or double-stranded DNA. The target nucleic acid may be cDNA produced from RNA by reverse transcription reaction.

[0072] The origin of the target nucleic acid is not particularly limited, and it may be a nucleic acid of a virus, a prokaryote, or a eukaryote. Examples of viruses include coronaviruses (MERS-CoV, SARS-CoV, SARS-CoV-2, etc.), influenza viruses, poxviruses, noroviruses, respiratory syncytial viruses, adenoviruses, human immunodeficiency viruses, human papillomaviruses, and hepatitis viruses. Examples of prokaryotes include Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Campylobacter, Listeria, Legionella, Mycobacterium tuberculosis, Mycoplasma, and Bordetella pertussis. Examples of eukaryotes include Cryptosporidium, Giardia, Plasmodium, fungi, birds, and mammals. A preferred mammal is human. The nucleic acid of a prokaryote or eukaryote may be a nucleic acid of a cultured cell. In the method of this embodiment, a sample containing the above-mentioned nucleic acid is subjected to amplification. In the case of a sample containing RNA, the sample after reverse transcription is subjected to amplification. Examples of samples to be subjected to amplification include samples or excrement collected from a living body, washings of a living body, food products, solubilized products thereof, and extracts thereof. Examples of samples collected from a living body include organs, tissues, cells, body fluids, and sputum. Examples of body fluids include blood, plasma, serum, lymph, and saliva. Examples of excrement include urine and feces. Examples of washings of a living body include body cavity washings. Examples of food products include food and beverages. In addition to the above, sewage, river water, seawater, soil, hot spring water, bath water, tap water, solubilized products thereof, and extracts thereof may also be used as samples.

[0073] In the method of the present embodiment, an appropriate primer is used depending on the type of target nucleic acid and the type of nucleic acid amplification method. The primer may contain a conventionally known artificial nucleic acid such as a bridged nucleic acid (BNA), a phosphorothioate (PS)-oligonucleotide, a peptide nucleic acid (PNA), a morpholino oligonucleotide, or a 2'-O-substituted RNA, as long as the primer can extend the 3' end by a nucleic acid amplification reaction.

[0074] The nucleotide sequence of the primer may be a nucleotide sequence that is completely complementary to the nucleotide sequence of the region where the primer hybridizes to the target nucleic acid. Alternatively, the nucleotide sequence of the primer may contain bases that generate mismatch sites as long as the primer hybridizes to the target nucleic acid. In this case, the nucleotide sequence of the primer has, for example, 80% or more, preferably 90% or more, more preferably 95% or more identity to the nucleotide sequence of the region where the primer hybridizes to the target nucleic acid.

[0075] As used herein, "hybridize" refers to the formation of a double strand with all or a part of a given polynucleotide through hydrogen bonds with all or a part of another polynucleotide under stringent conditions. "Stringent conditions" may be conditions generally used by those skilled in the art when hybridizing polynucleotides. For example, conditions under which one polynucleotide molecule can specifically hybridize to another polynucleotide molecule when there is at least 50%, preferably at least 75%, more preferably at least 90% identity between two polynucleotide molecules. It is known that the stringency of hybridization is a function of temperature, salt concentration, base length and GC content of the oligonucleotide, and the concentration of the chaotropic agent contained in the hybridization buffer. Examples of stringent conditions include those described in Sambrook, J. et al., 1998, Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York.

[0076] As used herein, the term "fully complementary nucleotide sequence" refers to a nucleotide sequence that forms complementary base pairs in the Watson-Crick model with all bases in the entire or partial nucleotide sequence of a given polynucleotide. As used herein, the term "mismatch site" refers to a site where, when two polynucleotide molecules are hybridized, a base that is not complementary to a given base in the nucleotide sequence of one polynucleotide molecule is present at a corresponding position in the nucleotide sequence of the other polynucleotide molecule, and thus cannot form complementary base pairs in the Watson-Crick model.

[0077] The polymerase of this embodiment can be used in the same manner as the conventional Bst DNA polymerase. That is, the conditions of the amplification reaction in the method of this embodiment can be equivalent to those of the conventional nucleic acid amplification method using Bst DNA polymerase. In addition, since the polymerase of this embodiment has strand displacement activity, the amplification reaction of the target nucleic acid can be carried out under substantially isothermal conditions. The substantially isothermal conditions and the nucleic acid amplification method carried out under those conditions are as described above.

[0078] When performing a quantitative amplification reaction such as the real-time LAMP method, a fluorescent intercalator (e.g., SYTO (registered trademark)-16, SYBR (registered trademark) Green, etc.) or a fluorescently labeled probe (e.g., TaqMan (trademark) probe, etc.) whose 5' end is modified with a fluorescent substance and whose 3' end is modified with a quencher substance may be further added. The amplification product can be quantitatively detected by detecting the fluorescence generated from the intercalator or probe incorporated into the amplification product. In addition, in the LAMP method, insoluble magnesium pyrophosphate is generated as a by-product with the increase in the amplification product. As this magnesium pyrophosphate increases, the reaction solution becomes cloudy. Therefore, the amount of target nucleic acid can be calculated based on the measured value of the turbidity or absorbance of the reaction solution, or the time until these values ​​reach a predetermined reference value.

[0079] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0080] Example 1: Construction of recombinant Bst DNA polymerase A recombinant baculovirus containing a gene encoding Bst DNA polymerase (SEQ ID NO: 3) and a gene encoding a His tag on the 5' side of the gene encoding Bst DNA polymerase was transformed into a recombinant baculovirus by incubation in a silkworm ( Bombyx mori ) male pupae. The virus-inoculated pupae were stored at room temperature for 7 days, and then the infected pupae were frozen at -70°C or lower. The frozen infected pupae were ground in grinding buffer (50 mM Tris-HCl, pH 7.5; 300 mM NaCl; 5 mM EDTA; 5 mM benzamidine hydrochloride; 0.5 mM PMSF; 0.5 mM DTT; 0.1% 1-phenyl-2-thiourea). The ground material was centrifuged at low speed to collect the homogenate. The homogenate was heated at 65°C for 20 minutes, then centrifuged to collect the supernatant. 50% ammonium sulfate was added to the supernatant, and the precipitate was collected. The precipitate was dissolved in purification buffer (50 mM Tris-HCl, pH 7.5; 300 mM NaCl; 20 mM imidazole). CHAPS was added to the resulting solution to a final concentration of 1% to solubilize it. The solution was passed through a Ni Sepharose column, and then an His-tag fused Bst DNA polymerase derived from silkworm (hereinafter also referred to as "polymerase of Example 1") was obtained using an elution buffer (50 mM Tris-HCl, pH 8.5; 300 mM NaCl; 250 mM imidazole). The solution containing the polymerase of Example 1 is an example of the reagent composition of the present invention.

[0081] Example 2: Glycoanalysis It is generally known that no sugar chain is added to a protein produced in an E. coli expression system. On the other hand, a protein produced in a silkworm-baculovirus expression system can be sugar-modified. Since the amino acid sequence (SEQ ID NO: 1) of the polymerase of Example 1 has a consensus sequence (Asn-Ile-Thr) for N-linked sugar chain modification at positions 437 to 439, a sugar chain can be added to the polymerase of Example 1. In Example 2, sugar chain analysis was performed to examine whether a sugar chain was added to the polymerase of Example 1 and a commercially available Bst DNA polymerase (comparative example) produced by the E. coli expression system. Furthermore, the N-linked sugar chain of the polymerase of Example 1 was released by a glycosidase that cleaves the bond between GlcNAc and an aspartic acid residue of the N-linked sugar chain, and the structure of the sugar chain was analyzed.

[0082] (1) Detection of the presence or absence of glycans The presence or absence of sugar chain addition was confirmed for the polymerase of Example 1 and the polymerase of Comparative Example by lectin blotting using concanavalin A (ConA). Specifically, the sample separated by SDS-PAGE was transferred to a PVDF membrane and treated with ConA-biotin overnight at 4°C. Then, HRP-labeled streptavidin was added for enzyme labeling. Furthermore, DAB (3,3'-diaminobenzidine) was added as a substrate for HRP to perform an enzyme reaction and detect the product. The results are shown in Figure 3. In Figure 3, lane 1 is a marker, lanes 2 to 7 are the polymerase of Example 1, lane 8 is the polymerase of Comparative Example (New England Biolabs), and lane 9 is alkaline phosphatase derived from calf small intestine (Oriental Yeast) (positive control). Lanes 2 to 4 are polymerases purified from a sample of mixed male and female silkworm pupae, and lanes 5 to 7 are polymerases purified from male silkworm pupae. As can be seen from FIG. 3, sugar chains were detected in the polymerase of Example 1, but not in the polymerase of the comparative example.

[0083] (2) Analysis of glycan structure (2.1) Preparation of measurement samples The polymerase of Example 1 was desalted and lyophilized, and then redissolved in water (22.8 μL). A 5% solution (6 μL) of RapiGest™ SF (Waters) was added to this solution, and the mixture was reacted at 90° C. for 3 minutes, and then left to stand at room temperature for 3 minutes. A solution (1.2 μL) of Rapid™ PNGase F (New England Biolabs) was added to this solution, and the mixture was reacted at 50° C. for 5 minutes, and then left to stand at room temperature for 3 minutes. A glycan labeling reagent (12 μL) contained in the GlycoWorks™ RapiFluor-MS™ N-Glycan Kit (Waters) was added to this solution, and the mixture was left to stand at room temperature for 5 minutes. Acetonitrile (358 μL) was added to this solution to dilute it. RFMS-labeled glycans were extracted in solid phase using a GlycoWorks HILIC μElution Plate (Waters). The extract was lyophilized, and then redissolved in a solvent to obtain a measurement sample.

[0084] (2.2) Measurement by liquid chromatography / mass spectrometry (LC / MS) method The measurement sample (10 μL) obtained in 1) above was subjected to LC measurement using a Waters H-Class system (Waters). The measurement was performed as follows. The measurement sample was loaded onto an ACQUITY UPLC (trademark) Glycan BEH Amide 130 A, 1.7 μm, 2.1 x 150 mm column equilibrated with a mobile phase (A: 50 mM ammonium formate solution (pH 4.4), B: 100% acetonitrile; A / B = 25 / 75) at 0.4 mL / min and a column temperature of 60.0°C. Then, measurements were performed under gradient conditions of A / B = 25 / 75 (0 min, 0.4 mL / min) - 46 / 54 (35 min, 0.4 mL / min) -100 / 0 (36.5 min, 0.2 mL / min) -100 / 0 (39.5 min, 0.2 mL / min) - 25 / 75 (43.1 min, 0.2 mL / min) -25 / 75 (47.6 min, 0.4 mL / min) -25 / 75 (55 min, 0.4 mL / min) with detection wavelengths of Ex. 265 nm and Em. 425 nm.

[0085] MS measurements were performed using a Waters Xevo-G2XS QTOF (Waters). The MS measurement conditions were: polarity: ES+, analytical section: sensitivity mode, capillary voltage (kV): 3.00, sampling cone voltage (V): 80.0, source temperature (℃): 120, source offset voltage (V): 60, desolvation temperature (℃): 350, cone gas flow rate (L / Hr): 50.0, desolvation gas flow rate (L / Hr): 800.0.

[0086] (3) Results The results of the analysis are shown in Table 1. In the table, in the column for predicted glycan structure, a circle represents a mannose residue, a square represents an N-acetyl-D-glucosamine residue, a triangle represents a fucose residue, and an open circle represents a glucose residue. In the table, in the column for glycan type, "1" refers to a glycan represented by the following formula (1), "2" refers to a glycan represented by the following formula (2), "3" refers to a glycan represented by the following formula (3), "4" refers to a glycan represented by the following formula (4), and "5" refers to a glycan represented by the following formula (5). As can be seen from Table 1, the polymerase of Example 1 was shown to have a glycan.

[0087] [Table 1]

[0088] [ka]

[0089] [ka]

[0090] [ka] (In the formula, a1, a2, and a3 are each independently an integer of 0 or more and 2 or less, and the sum of a1, a2, and a3 is 1 or more and 3 or less.)

[0091] [ka] (In the formula, b1, b2, and b3 each independently represent an integer of 0 or more and 2 or less, and the sum of b1, b2, and b3 is 4 or less.)

[0092] [ka]

[0093] Example 3: Confirmation of effects of interfering substances We investigated the influence of interfering substances on the nucleic acid amplification reaction using the polymerase of Example 1. We also investigated the nucleic acid amplification reaction using the polymerase of Comparative Example. Interfering substances are substances known to cause inhibition of nucleic acid amplification reaction, reduction in reaction rate, etc.

[0094] (1) Preparation of template DNA sample A sample containing human CK19 DNA was prepared as a template DNA sample in a nucleic acid amplification reaction using Bst DNA polymerase as follows. First, human CK19 DNA was synthesized by PCR using human CK19 plasmid DNA cloned with pBluescript (Agilent), Taq PCR master mix and RNase-free water included in a Taq PCR master mix kit (QIAGEN), and a primer set designed based on the base sequence of human CK19. The PCR product containing human CK19 DNA was purified with a QIAquick PCR purification kit (QIAGEN) to obtain a human CK19 DNA solution. The DNA concentration of the obtained solution was calculated by measuring the absorbance at 260 nm. Based on the calculated value, the copy number of human CK19 DNA in the solution was calculated. The human CK19 DNA solution was diluted with 50 ng / μL yeast RNA (Thermo Fisher Scientific) so that the copy number of human CK19 DNA was 250,000. The obtained solution was used as a template DNA sample in the subsequent experiments.

[0095] (2) Calculation of unit value of Bst DNA polymerase The enzyme amount (unit value) and concentration based on activity of the polymerase of the comparative example were known. Based on the measured values ​​and unit values ​​of the nucleic acid amplification reaction using the polymerase of the comparative example, the unit value of the polymerase of Example 1 was determined as follows. First, the polymerase of the comparative example was diluted with a glycerol solution (50 mM Tris-HCl, pH 7.5; 50% glycerol; 1 mM DTT; 50 mM KCl; 0.1 mM EDTA; 0.1% Triton (trademark) X-100) to different concentrations to prepare three enzyme solutions. Human CK19 DNA in the template DNA sample was measured using the enzyme solutions of each concentration and a CK19 primer solution contained in the gene amplification detection reagent Linoamp (trademark) CK19 (Sysmex Corporation). For the measurement, a gene amplification detection device RD-200 (Sysmex Corporation) was used. A calibration curve was created from the measured values ​​and the unit value of the polymerase of the comparative example after dilution. The polymerase of Example 1 was diluted with a glycerol solution so as to fall within the range in which the calibration curve can be applied, and human CK19 DNA in the template DNA sample was measured in the same manner as described above. The measurement results were applied to the calibration curve to determine the unit value of the polymerase of Example 1. The polymerase of Example 1 was then diluted with a glycerol solution so as to be equivalent to 12 units, to obtain an enzyme solution.

[0096] (3) Measurement using real-time LAMP (RT-LAMP) method A reaction buffer containing the following six primers was prepared. The nucleotide sequences of the primers are shown below. The composition of the reaction buffer is shown in Table 2.

[0097] FA primer: 5'- ggagttctcaatggtggcaccaactactacacgaccatcca -3' (SEQ ID NO: 4) RA primer: 5'- gtcctgcagatcgacaacgcctccgtctcaaacttggttcg -3' (SEQ ID NO: 5) F3 primer: 5'- tggtaccagaagcagggg -3' (SEQ ID NO: 6) R3 primer: 5'- gttgatgtcggcctccacg -3' (SEQ ID NO: 7) Loop primer F: 5'- agaatcttgtcccgcagg -3' (SEQ ID NO: 8) Loop primer R: 5'- cgtctggctgcagatga -3' (SEQ ID NO: 9)

[0098] [Table 2]

[0099] The above reaction buffer (14 μL), 12 units of the polymerase of the comparative example (3 μL) or 12 units of the polymerase of Example 1 (3 μL), 50 μM of fluorescent dye SYTO-16 (1 μL), 31250 copies of template DNA sample (2 μL), and each interfering substance (5 μL) shown in Table 3 were mixed to prepare a reaction solution (25 μL). For comparison, nuclease-free water (5 μL) was added instead of the interfering substance to prepare a reaction solution that did not contain the interfering substance. Each reaction solution was heated at 65° C. for 1 hour to perform RT-LAMP. The measurement device used was CFX96 (BioRad). The increase in fluorescence intensity at 450 to 490 nm was measured over time using the CFX96.

[0100] [Table 3]

[0101] (4) Results Amplification curves were obtained from the measurement results for each of the polymerases of the comparative example and the polymerase of Example 1. For each polymerase, a Ct (threshold cycle) value was obtained, which is the intersection point between the amplification curves in the presence and absence of interfering substances and the threshold value of the fluorescence intensity. The threshold value of the fluorescence intensity was automatically set by CFX96. Using the Ct values ​​of each polymerase in the presence and absence of interfering substances, the difference (ΔCt) in the rate of change in nucleic acid amplification between the polymerase of the comparative example and the polymerase of Example 1 was calculated according to the following formula.

[0102]

number

[0103] In the above formula, Ct (E. coli w / ) was the Ct value of the amplification by the polymerase of the comparative example in the presence of an interfering substance. Ct (E. coli w / o) was the Ct value of the amplification by the polymerase of the comparative example in the absence of an interfering substance. Ct (Ex1 w / ) was the Ct value of the amplification by the polymerase of Example 1 in the presence of an interfering substance. Ct (Ex1 w / o) was the Ct value of the amplification by the polymerase of Example 1 in the absence of an interfering substance. When the value of ΔCt (%) is positive, it indicates that the nucleic acid amplification change rate due to the influence of the interfering substance is lower for the polymerase of Example 1 than for the polymerase of the comparative example. That is, it indicates that the influence of the interfering substance is reduced in the nucleic acid amplification reaction using the polymerase of Example 1 compared to the case where the polymerase of the comparative example is used. FIG. 4 shows ΔCt for each interfering substance.

[0104] As can be seen from FIG. 4, urea, EDTA, SDS, glycerol, DMSO, xylan and Zn 2+ It was shown that, regardless of which of the above was added, when the polymerase of Example 1 was used, the effect of interfering substances in the nucleic acid amplification reaction was reduced compared to when the polymerase of the comparative example was used.

[0105] Example 4: Confirmation of specificity of reaction temperature The optimum reaction temperature for Bst DNA polymerase is said to be around 60 to 65°C. It is generally known that in a nucleic acid amplification reaction, enzyme activity at a temperature lower than the optimum reaction temperature can be a factor in causing non-specific nucleic acid amplification. RT-LAMP was performed at a temperature lower than 60°C using the polymerase of Example 1, and the extent to which nucleic acid amplification occurs was examined. Similarly, the polymerase of the comparative example was also examined.

[0106] (1) Samples and reagents The template DNA sample, primers, and reagents used for preparing the reaction solution were the same as those in Example 2. The reaction solution was prepared in the same manner as the reaction solution without interfering substances in Example 2.

[0107] (2) Measurement by RT-LAMP method The prepared reaction solution was heated at 58.0, 57.6, 56.7, 55.1, 53.2, 51.6, 50.5, 50.0, 48.8 or 47.4°C for 1 hour to carry out RT-LAMP. Measurements were carried out in the same manner as in Example 2 using a CFX96 (BioRad) measuring device. For comparison, nucleic acid amplification reactions were also carried out at 65°C, which is the optimal reaction temperature.

[0108] (3) Results Amplification curves were obtained from the measurement results for each of the polymerases of the comparative example and the polymerase of Example 1. For each polymerase, the Ct' value, which is the intersection point between the amplification curve and the fluorescence intensity threshold, was obtained. The fluorescence intensity threshold was automatically set by CFX96. Using the Ct' value of each polymerase, the nucleic acid amplification change rate (ΔCt') for each polymerase with a change in reaction temperature was calculated from the following formula.

[0109]

number

[0110] In the above formula, Ct'(Temp.) was the Ct' value of amplification by each polymerase at an arbitrary reaction temperature. Ct'(65°C) was the Ct' value of amplification by each polymerase at 65°C, which is the optimal reaction temperature. In other words, a polymerase with a lower nucleic acid amplification change rate (ΔCt') value at an arbitrary temperature shows that non-specific amplification reaction is suppressed. The calculated ΔCt'(%) value is shown as the nucleic acid amplification change rate in FIG. 5. In the figure, "Example 1" refers to the polymerase of Example 1, and "Comparative Example" refers to the polymerase of the comparative example. As can be seen from FIG. 5, at temperatures lower than 65°C, when the polymerase of Example 1 was used, the Ct value was lower than when the polymerase of the comparative example was used. In other words, it was shown that at temperatures lower than 65°C, when the polymerase of Example 1 was used, nucleic acid amplification was suppressed more than when the polymerase of the comparative example was used. For example, at 48.8°C, the difference in Ct value between the polymerase of Example 1 and the polymerase of Comparative Example was about 10%. In addition, at 47.4°C, the polymerase of Comparative Example still had enzymatic activity, but the polymerase of Example 1 did not. As shown in FIG. 5, it was suggested that the use of the polymerase of Example 1 suppressed the amplification reaction at a temperature lower than the optimal reaction temperature, compared to the use of the polymerase of Comparative Example. For example, it is considered that unintended amplification reactions that may produce non-specific amplification products can be reduced before the optimal reaction temperature is reached when the temperature of the reaction solution is raised. [Explanation of symbols]

[0111] 10, 20 Reagent Kit 11, 21 1st container 12, 23 Packing box 13, 24 Attached document 22 Second container

Claims

1. (1) the amino acid sequence shown in SEQ ID NO:1, or (2) An amino acid sequence that contains the consensus sequence Asn-X-(Ser / Thr) (wherein X is any amino acid residue other than a proline residue) and has an identity of 80% or more with the amino acid sequence shown in SEQ ID NO:

1. and has 5'→3' DNA polymerase activity and strand displacement activity, The consensus sequence includes an N-linked glycan, and the N-linked glycan includes three or more mannose residues. Bst DNA polymerase.

2. The Bst DNA polymerase according to claim 1, which comprises the consensus sequence in its finger domain.

3. The Bst DNA polymerase according to claim 1, wherein the N-linked sugar chain contains 9 or less mannose residues.

4. The Bst DNA polymerase according to claim 3 , wherein the N-linked sugar chain has a structure represented by the following formula (I): 【Chemistry 1】 wherein Asn is an asparagine residue in the consensus sequence; Man is a mannose residue, GlcNAc is an N-acetyl-D-glucosamine residue, and Fuc is a fucose residue. m and n each independently represent 0 or 1; S1, S2, S3 and S4 are each independently a glycan that is absent or contains 1 to 5 monosaccharide residues.

5. 5. The Bst DNA polymerase according to claim 4, wherein said S1 comprises at least one monosaccharide residue selected from the group consisting of a mannose residue, an N-acetyl-D-glucosamine residue, and a glucose residue.

6. 5. The Bst DNA polymerase according to claim 4, wherein said S2 comprises at least one monosaccharide residue selected from the group consisting of a mannose residue, an N-acetyl-D-glucosamine residue, and a glucose residue.

7. 5. The Bst DNA polymerase according to claim 4, wherein said S3 comprises at least one monosaccharide residue selected from the group consisting of a mannose residue, an N-acetyl-D-glucosamine residue, and a glucose residue.

8. 5. The Bst DNA polymerase according to claim 4, wherein said S4 comprises at least one monosaccharide residue selected from the group consisting of a mannose residue, an N-acetyl-D-glucosamine residue, and a glucose residue.

9. The Bst DNA polymerase according to claim 4, wherein S1, S2, S3 and S4 are absent.

10. 5. The Bst DNA polymerase according to claim 4, wherein at least one of the sugar chains S1, S2, S3 and S4 is an N-acetyl-D-glucosamine residue.

11. 5. The Bst DNA polymerase according to claim 4, wherein at least one of the sugar chains S1, S2, S3 and S4 has a structure represented by the following formula (II): 【Chemistry 2】 (In the formula, S5 and S6 are each independently a sugar chain that is absent or contains 1 to 3 mannose residues.)

12. 2. The Bst DNA polymerase according to claim 1, wherein at least one non-reducing end of the N-linked sugar chain is a mannose residue or an N-acetyl-D-glucosamine residue.

13. 10. The Bst DNA polymerase according to claim 9, wherein m is 1 and n is 0, or m is 0 and n is 1.

14. A reagent composition for use in a method for amplifying nucleic acid, comprising the Bst DNA polymerase according to any one of claims 1 to 13.

15. The reagent composition of claim 14, wherein the nucleic acid amplification method is carried out under substantially isothermal conditions.

16. The reagent composition according to claim 15, wherein the nucleic acid amplification method is a LAMP method, an RCA method, a NASBA method, an SDA method, a TRC method or an ICAN method.

17. A method for amplifying a target nucleic acid, comprising amplifying a target nucleic acid using the Bst DNA polymerase according to any one of claims 1 to 13.

18. The method for amplifying nucleic acid according to claim 17, wherein the amplification reaction of the target nucleic acid is carried out under substantially isothermal conditions.

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