Modified-avian myeloblastoma virus reverse transcriptase

Abstract

To provide an avian myeloblastosis virus (AMV) reverse transcriptase with enhanced resistance to thermal stability and / or inhibition of reaction due to impurities.SOLUTION: An AMV reverse transcriptase selected from any of (i) to (iii) below is provided. (i) An AMV reverse transcriptase having an amino acid sequence in which one or more amino acid residues selected from 15 specific amino acid residues of a specific amino acid sequence are substituted with other specific amino acid residues; (ii) An AMV reverse transcriptase comprising amino acids having a specific sequence and, in addition to the amino acid substitution shown in (i), further including one or more of substitution, deletion, insertion, and addition of an amino acid residue at one or several positions, and exhibiting enzymatic activity;(iii) An AMV reverse transcriptase comprising a specific amino acid sequence having 70% or more identity to the entire amino acid sequence including the amino acid substitution of (i), the amino acid substitution being maintained, and exhibiting enzymatic activity.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to modified avian myeloblastoma virus (AMV) reverse transcriptases. [Background technology]

[0002] AMV reverse transcriptase, a type of reverse transcriptase, is used in genetic engineering reagents and genetic diagnostic reagents required for cDNA synthesis, etc. AMV reverse transcriptase is known to have two subunits: an α chain with a molecular weight of approximately 63 kDa and a β chain with a molecular weight of approximately 95 kDa. Of these, the α chain is formed from the β chain by proteolytic processing.

[0003] Patent Document 1 reports that a heterodimer of an α chain and a β chain (αβ form) has higher activity in RNA amplification reactions than either the α chain alone (α form) or the β chain alone (β form), and is useful as a component of reagents such as genetic engineering reagents and genetic diagnostic reagents.

[0004] Patent Document 2 discloses an α-mutant AMV reverse transcriptase with improved thermostability.

[0005] Patent Document 3 discloses a mutant having, for example, S65G, A583T, G626D, and A689V as an AMV reverse transcriptase β chain with improved thermostability.

[0006] Patent document 4 describes a method for producing the αβ form of AMV reverse transcriptase using recombinant Escherichia coli into which only the gene encoding the β chain of AMV reverse transcriptase has been introduced, by degrading the α chain from the β chain expressed within the recombinant Escherichia coli. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2003-334095 [Patent Document 2] Japanese Patent Application Laid-Open No. 2013-165669 [Patent Document 3] Japanese Patent Application Laid-Open No. 2014-209898 [Patent Document 4] Japanese Patent Application Laid-Open No. 2013-126402 Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present disclosure is to provide a modified avian myeloblastoma virus (AMV) reverse transcriptase. Specifically, in one aspect, an objective is to provide an AMV reverse transcriptase with improved thermostability. In another aspect, an objective is to provide an AMV reverse transcriptase with improved resistance to reaction inhibition by contaminants. [Means for solving the problem]

[0009] The present inventors have discovered amino acid mutations that improve the thermostability of AMV reverse transcriptase and / or resistance to reaction inhibition by contaminants.

[0010] [1] An avian myeloblastoma virus (AMV) reverse transcriptase selected from any of the following (i) to (iii): (i) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which contains one or more amino acid substitutions selected from the following (1) to (15): (1) The amino acid residue corresponding to the leucine residue at position 476 of SEQ ID NO: 6 is substituted with a glutamine residue (2) The amino acid residue corresponding to the 377th valine residue of SEQ ID NO: 6 is substituted with an isoleucine residue (3) The amino acid residue corresponding to the serine residue at position 550 of SEQ ID NO: 6 is substituted with a threonine residue (4) The amino acid residue corresponding to the lysine residue at position 691 of SEQ ID NO: 6 is substituted with a glutamic acid residue (5) The amino acid residue corresponding to the asparagine residue at position 776 of SEQ ID NO: 6 is substituted with a lysine residue (6) The amino acid residue corresponding to the isoleucine residue at position 332 of SEQ ID NO: 6 is substituted with a valine residue (7) The amino acid residue corresponding to the 474th alanine residue in SEQ ID NO: 6 is substituted with a threonine residue (8) The amino acid residue corresponding to the 716th threonine residue of SEQ ID NO: 6 is substituted with a serine residue (9) The amino acid residue corresponding to the lysine residue at position 797 of SEQ ID NO: 6 is substituted with an arginine residue (10) The amino acid residue corresponding to the lysine residue at position 850 of SEQ ID NO: 6 is substituted with an arginine residue or a glutamic acid residue (11) The amino acid residue corresponding to the 61st isoleucine residue of SEQ ID NO: 6 is substituted with a valine residue (12) The amino acid residue corresponding to the 105th valine residue of SEQ ID NO: 6 is substituted with an alanine residue (13) The amino acid residue corresponding to the asparagine residue at position 623 of SEQ ID NO: 6 is substituted with an aspartic acid residue (14) The amino acid residue corresponding to the threonine residue at position 692 of SEQ ID NO: 6 is substituted with an alanine residue (15) a substitution of the amino acid residue corresponding to position 717 of glycine in SEQ ID NO: 6 with aspartic acid; (ii) an AMV reverse transcriptase having an amino acid sequence set forth in SEQ ID NO: 6, which contains one or more amino acid substitutions selected from (1) to (15), and which further contains one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions shown in (1) to (15), and which has enzymatic activity; (iii) An AMV reverse transcriptase having an amino acid sequence set forth in SEQ ID NO: 6, which has 70% or more identity to the entire amino acid sequence containing one or more amino acid substitutions selected from (1) to (15), with the proviso that the amino acid sequence maintains the amino acid substitutions and has enzymatic activity. [2] The AMV reverse transcriptase of [1], wherein the one or more amino acid substitutions selected from (1) to (15) include at least the amino acid substitution of (1). [3] The AMV reverse transcriptase of [1], wherein the one or more amino acid substitutions selected from (1) to (15) include at least amino acid substitutions (2) to (4). [4] The AMV reverse transcriptase of [1], wherein the one or more amino acid substitutions selected from (1) to (15) include at least an amino acid substitution selected from (1) to (5) and one or more amino acid substitutions selected from (11) to (15). [5] The AMV reverse transcriptase of [1] is selected from any one of the following (iv) to (vi): (iv) an AMV reverse transcriptase having an amino acid sequence set forth in any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 38, and 44; (v) an AMV reverse transcriptase having an amino acid sequence set forth in any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 38, and 44, which contains any one or more of substitution, deletion, insertion, and addition of one or several amino acid residues at one or several positions, and which has enzymatic activity; (vi) An AMV reverse transcriptase having an amino acid sequence that is 70% or more identical to the amino acid sequence set forth in any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 38, and 44, and having enzymatic activity. [6] A polynucleotide encoding any one of the AMV reverse transcriptases [1] to [5]. [7] [6] An expression vector comprising the polynucleotide of [6]. [8] A transformant obtained by transforming a host with the expression vector [7]. [9] [8] transformants, whose host is E. coli.

[10] A method for producing AMV reverse transcriptase, comprising the steps of: culturing the transformant of [8] or [9] to express AMV reverse transcriptase; and recovering the expressed reverse transcriptase from the resulting culture.

[11] A reagent for amplifying a target nucleic acid, comprising any one of the AMV reverse transcriptases [1] to [5]. [Effects of the Invention]

[0011] According to one embodiment of the present disclosure, an AMV reverse transcriptase with improved thermostability can be provided. Surprisingly, by introducing the amino acid mutations discovered in this disclosure into a mutant of an AMV reverse transcriptase with improved thermostability that has already been reported, an AMV reverse transcriptase with even improved thermostability can be provided. Furthermore, according to one embodiment of the present disclosure, an AMV reverse transcriptase with improved resistance to reaction inhibition by contaminants can be provided. This embodiment is expected to be useful in infectious disease testing of various clinical specimens using nucleic acid amplification methods. [Brief explanation of the drawings]

[0012] [Figure 1] This figure shows the results of comparing the thermal stability of the amino acid substitution mutants prepared in Example 1 with AMV-RT m4-2 (SEQ ID NO: 6). In this figure, the amount of remaining enzyme is shown as a relative value, with the amount of remaining enzyme in AMV-RT m4-2 set to 1. [Figure 2] This figure shows the results of comparing the purification yields of the amino acid-substituted mutants prepared in Examples 5 and 6 with AMV-RT m4-2 (SEQ ID NO: 6). In this figure, the purification yields are shown as relative values, with the purification yield for AMV-RT m4-2 set at 1. [Figure 3] This figure shows the results of comparing the thermal stability of the amino acid substitution mutants prepared in Example 9 with AMV-RT m9 (SEQ ID NO: 20). In this figure, the amount of remaining enzyme is shown as a relative value, with the amount of remaining enzyme in AMV-RT m4-2 set at 1. [Figure 4]This figure shows the results of comparing the thermal stability of the amino acid substitution mutant prepared in Example 12 with AMV-RT m9 (SEQ ID NO: 20) when heat treated at 50° C. In this figure, the residual activity is shown as a relative value, with the residual activity of AMV-RT m9 set to 1. [Figure 5] This figure shows the results of comparing the thermal stability of the amino acid substitution mutant prepared in Example 12 with AMV-RT m9 (SEQ ID NO: 20) when heat treated at 52° C. In this figure, the residual activity is shown as a relative value, with the residual activity of AMV-RT m9 set to 1. DETAILED DESCRIPTION OF THE INVENTION

[0013] <1> AMV reverse transcriptase The present disclosure provides avian myeloblastoma virus (AMV) reverse transcriptases having the "specific mutations" described herein. More particularly, the present disclosure provides an avian myeloblastoma virus (AMV) reverse transcriptase selected from any of the following (i) to (iii): (i) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which contains one or more amino acid substitutions selected from the following (1) to (15): (1) The amino acid residue corresponding to the leucine residue at position 476 of SEQ ID NO: 6 is substituted with a glutamine residue (2) The amino acid residue corresponding to the 377th valine residue of SEQ ID NO: 6 is substituted with an isoleucine residue (3) The amino acid residue corresponding to the serine residue at position 550 of SEQ ID NO: 6 is substituted with a threonine residue (4) The amino acid residue corresponding to the lysine residue at position 691 of SEQ ID NO: 6 is substituted with a glutamic acid residue (5) The amino acid residue corresponding to the asparagine residue at position 776 of SEQ ID NO: 6 is substituted with a lysine residue (6) The amino acid residue corresponding to the isoleucine residue at position 332 of SEQ ID NO: 6 is substituted with a valine residue (7) The amino acid residue corresponding to the 474th alanine residue in SEQ ID NO: 6 is substituted with a threonine residue (8) The amino acid residue corresponding to the 716th threonine residue of SEQ ID NO: 6 is substituted with a serine residue (9) The amino acid residue corresponding to the lysine residue at position 797 of SEQ ID NO: 6 is substituted with an arginine residue (10) The amino acid residue corresponding to the lysine residue at position 850 of SEQ ID NO: 6 is substituted with an arginine residue or a glutamic acid residue (11) The amino acid residue corresponding to the 61st isoleucine residue of SEQ ID NO: 6 is substituted with a valine residue (12) The amino acid residue corresponding to the 105th valine residue of SEQ ID NO: 6 is substituted with an alanine residue (13) The amino acid residue corresponding to the asparagine residue at position 623 of SEQ ID NO: 6 is substituted with an aspartic acid residue (14) The amino acid residue corresponding to the threonine residue at position 692 of SEQ ID NO: 6 is substituted with an alanine residue (15) a substitution of the amino acid residue corresponding to position 717 of glycine in SEQ ID NO: 6 with aspartic acid; (ii) an AMV reverse transcriptase having an amino acid sequence set forth in SEQ ID NO: 6, which contains one or more amino acid substitutions selected from (1) to (15), and which further contains one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions shown in (1) to (15), and which has enzymatic activity; (iii) an amino acid sequence having 70% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 6, which contains one or more amino acid substitutions selected from (1) to (15), and which maintains the amino acid substitutions; and , which has enzymatic activity, AMV reverse transcriptase.

[0014] AMV reverse transcriptase may refer to a reverse transcriptase originating from AMV. Reverse transcriptase may refer to a protein having the activity of catalyzing a reverse transcription reaction that synthesizes DNA using RNA as a template. This activity is also referred to as "reverse transcriptase activity." This activity is also referred to as "RNA-dependent DNA polymerase activity." AMV reverse transcriptase may also have the activity of catalyzing a replication reaction that synthesizes DNA using DNA as a template. This activity is also referred to as "DNA replication activity" or "DNA-dependent DNA polymerase activity." AMV reverse transcriptase may also have the activity of catalyzing a reaction that selectively hydrolyzes RNA hybridized with DNA. This activity is also referred to as "ribonuclease H (RNase H) activity." In other words, AMV reverse transcriptase may have any one or more activities of RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and RNase H activity. In other words, the enzymatic activity of AMV reverse transcriptase includes one or more of RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and RNase H activity. Hereinafter, unless otherwise specified, "enzymatic activity" in this application means the above-mentioned "enzymatic activity of AMV reverse transcriptase." Wild-type AMV reverse transcriptase may typically have RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and RNase H activity.

[0015] The enzymatic activity of the AMV reverse transcriptase preferably includes at least RNA-dependent DNA polymerase activity. Specifically, the enzymatic activity of the AMV reverse transcriptase may preferably be only RNA-dependent DNA polymerase activity, or may be RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity, or may be RNA-dependent DNA polymerase activity and RNase H activity, or may be RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and RNase H activity. Among these, it is more preferable that the AMV reverse transcriptase has all of the enzymatic activities identical to those of wild-type AMV reverse transcriptase, i.e., RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and RNase H activity.

[0016] The term "AMV reverse transcriptase" may refer to either a monomer or a dimer. Examples of monomers include the α chain or the β chain, and examples of dimers include a dimer of only the α chain (αα form), a homodimer of only the β chain (ββ form), and a heterodimer of an α chain and a β chain (αβ form). Furthermore, the AMV reverse transcriptase may be a mixture containing these exemplified monomers and / or dimers in any ratio. From the viewpoint of reverse transcriptase activity, it is preferable that the AMV reverse transcriptase contains at least the αβ form.

[0017] Note that an AMV reverse transcriptase having a certain amino acid sequence may refer to an AMV reverse transcriptase consisting of a protein containing that amino acid sequence, or may refer to a multimeric AMV reverse transcriptase containing a protein containing that amino acid sequence as a subunit. Typically, an AMV reverse transcriptase having a certain amino acid sequence refers to an AMV reverse transcriptase consisting of a protein containing that amino acid sequence, or refers to a dimeric AMV reverse transcriptase containing a protein containing that amino acid sequence as a subunit.

[0018] The α chain of AMV reverse transcriptase can be formed from the β chain via proteolytic processing. The AMV reverse transcriptase of the present disclosure may comprise the β chain of AMV reverse transcriptase having the amino acid sequence described herein, and / or an α chain formed from the β chain. For example, when the AMV reverse transcriptase of the present disclosure comprises an αβ form, the αβ form may be a dimer that is a combination of the β chain of AMV reverse transcriptase having the amino acid sequence described herein and the α chain formed from the β chain.

[0019] Hereinafter, the amino acid sequences described herein may define at least the amino acid sequence of the β chain of AMV reverse transcriptase. In other words, the AMV reverse transcriptase of the present disclosure may include at least the β chain of AMV reverse transcriptase defined by the amino acid sequence of the present disclosure.

[0020] The gene encoding AMV reverse transcriptase is also referred to as the "AMV reverse transcriptase gene."

[0021] The AMV reverse transcriptase of the present disclosure has a "specific mutation." An AMV reverse transcriptase having a "specific mutation" is also referred to as a modified AMV reverse transcriptase or a mutant AMV reverse transcriptase. That is, the AMV reverse transcriptase of the present disclosure is a mutant AMV reverse transcriptase. Furthermore, a gene encoding the mutant AMV reverse transcriptase is also referred to as a "mutant AMV reverse transcriptase gene." As an example, a "mutant AMV reverse transcriptase gene" may refer to a polynucleotide encoding the mutant AMV reverse transcriptase.

[0022] An AMV reverse transcriptase that does not have a "specific mutation" is also referred to as a "wild-type AMV reverse transcriptase." A gene encoding a wild-type AMV reverse transcriptase is also referred to as a "wild-type AMV reverse transcriptase gene." Note that the term "wild-type" used here is a convenient description to distinguish "wild-type" AMV reverse transcriptase from "mutant" AMV reverse transcriptase, and is not limited to those obtained in nature, as long as they do not have a "specific mutation." As long as they do not have a "specific mutation," wild-type AMV reverse transcriptase may or may not have mutations other than the "specific mutation."

[0023] When a certain wild-type AMV reverse transcriptase and a certain mutant AMV reverse transcriptase are identical except for the presence or absence of a "specific mutation," the wild-type AMV reverse transcriptase is also called "a wild-type AMV reverse transcriptase corresponding to a certain mutant AMV reverse transcriptase," and the mutant AMV reverse transcriptase is also called "a mutant AMV reverse transcriptase corresponding to a certain wild-type AMV reverse transcriptase."

[0024] The wild-type AMV reverse transcriptase will be described below.

[0025] The wild-type AMV reverse transcriptase may or may not have enzymatic activity, as long as the corresponding mutant AMV reverse transcriptase has enzymatic activity. The wild-type AMV reverse transcriptase may generally have enzymatic activity. Specifically, the wild-type AMV reverse transcriptase may have reverse transcriptase activity.

[0026] The wild-type AMV reverse transcriptase is not limited to those obtained in nature, as long as it does not have the "specific mutation," and may have a mutation other than the "specific mutation." Here, mutations other than the "specific mutation" include known mutations that have been reported to be introduced into AMV reverse transcriptase. Specifically, mutations other than the "specific mutation" include, for example, mutations described in JP 2014-209898 A.

[0027] Specifically, the wild-type AMV reverse transcriptase may be, for example, the amino acid sequence of the wild-type AMV reverse transcriptase β chain extracted from the registered sequence of GenBank No. AAB31929 (SEQ ID NO: 1), or may be a wild-type AMV reverse transcriptase β chain (SEQ ID NO: 2) that is a natural variant of AMV reverse transcriptase having an amino acid sequence in which the methionine residue at position 273 is replaced with an arginine residue, the glutamine residue at position 304 is replaced with an arginine residue, and the glutamic acid residue at position 395 is replaced with an aspartic acid residue in the amino acid sequence of the wild-type AMV reverse transcriptase β chain. Furthermore, specifically, the wild-type AMV reverse transcriptase may be, for example, an amino acid sequence in which the serine residue at position 65 is replaced with a glycine residue, the alanine residue at position 583 is replaced with a threonine residue, the glycine residue at position 626 is replaced with an aspartic acid residue, and the alanine residue at position 689 is replaced with a valine residue in SEQ ID NO: 2. The wild-type AMV reverse transcriptase gene may be an AMV reverse transcriptase having the sequence (SEQ ID NO: 6). An example of a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6 is shown in SEQ ID NO: 7. The wild-type AMV reverse transcriptase gene may be a gene having the nucleotide sequence shown in SEQ ID NO: 7. Unless otherwise specified, the expression "a gene or protein has a nucleotide sequence or amino acid sequence" may mean that the gene or protein contains the nucleotide sequence or amino acid sequence, and may also include cases where the gene or protein consists of the nucleotide sequence or amino acid sequence.

[0028] The wild-type AMV reverse transcriptase may be a variant of the above-exemplified wild-type AMV reverse transcriptase (e.g., a protein having the amino acid sequence shown in SEQ ID NO: 6), as long as it does not have a "specific mutation." Similarly, the wild-type AMV reverse transcriptase gene may be a variant of the above-exemplified wild-type AMV reverse transcriptase gene (e.g., a gene having the nucleotide sequence shown in SEQ ID NO: 7), as long as the AMV reverse transcriptase it encodes does not have a "specific mutation." That is, the term "wild-type AMV reverse transcriptase" may encompass not only the above-exemplified wild-type AMV reverse transcriptase (e.g., a protein having the amino acid sequence shown in SEQ ID NO: 6) but also variants thereof. Similarly, the term "wild-type AMV reverse transcriptase gene" may encompass not only the above-exemplified wild-type AMV reverse transcriptase gene (e.g., a gene having the nucleotide sequence shown in SEQ ID NO: 7) but also variants thereof. Examples of variants include artificially modified forms of the above-exemplified genes and proteins.

[0029] As long as the AMV reverse transcriptase it encodes does not have a "specific mutation," the wild-type AMV reverse transcriptase gene may encode a protein having an amino acid sequence in which one or more amino acids at one or more positions in the amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 6) have been substituted, deleted, inserted, and / or added. For example, the encoded protein may have its N-terminus and / or C-terminus extended or shortened. Note that the term "one or several" or "one or several" varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, and specifically may be, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0030] The above-mentioned substitution, deletion, insertion, or addition of one or several amino acids is a conservative mutation that maintains the original function of the protein. A typical conservative mutation is a conservative substitution. A conservative substitution is a mutation in which Phe, Trp, and Tyr are substituted with each other when the substitution site is an aromatic amino acid; Leu, Ile, and Val are substituted with each other when the substitution site is a hydrophobic amino acid; Gln and Asn are substituted with each other when the substitution site is a polar amino acid; Lys, Arg, and His are substituted with each other when the substitution site is a basic amino acid; Asp and Glu are substituted with each other when the substitution site is an acidic amino acid; and Ser and Thr are substituted with each other when the substitution site is an amino acid having a hydroxyl group. Specific examples of substitutions that are considered to be conservative substitutions include: These include substitutions of Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr, Ile to Leu, Met , Val or Phe substitution, Leu with Ile, Met, Val or Phe substitution, Lys with Asn, Glu, Gln, His or Arg substitution, Met with Ile, Leu, Val or Phe substitution, Phe with Trp, Tyr, Met, Ile or Leu substitution, Ser with Thr or Ala substitution, Thr with Ser or Ala substitution, Trp with Phe or Tyr substitution, Tyr with His, Phe or Trp substitution, and Val with Met, Ile or Leu substitution. Furthermore, the above-mentioned amino acid substitution, deletion, insertion or addition may be natural, such as when based on individual differences in the organism from which the gene is derived. This also includes those that arise due to naturally occurring mutations (mutants or variants).

[0031] Furthermore, the wild-type AMV reverse transcriptase gene may be a gene encoding a protein having an amino acid sequence that is, for example, 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identical to the entire amino acid sequence described above, as long as the AMV reverse transcriptase it encodes does not have a "specific mutation."

[0032] Furthermore, the wild-type AMV reverse transcriptase gene may be a gene, e.g., DNA, that hybridizes under stringent conditions with a probe that can be prepared from the above-mentioned nucleotide sequence (e.g., the nucleotide sequence shown in SEQ ID NO: 3), such as a sequence complementary to all or part of the above-mentioned nucleotide sequence, as long as the AMV reverse transcriptase it encodes does not have a "specific mutation." "Stringent conditions" may refer to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. One example of such conditions includes conditions under which DNAs with high identity, for example, DNAs with identity of 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more, hybridize with each other, but DNAs with lower identity do not hybridize with each other, or conditions for washing once, preferably two to three times, at a salt concentration and temperature equivalent to those used in standard Southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, and more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0033] As mentioned above, the probe used in the hybridization may be a portion of the complementary sequence of the gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on a known gene sequence as primers and a DNA fragment containing the gene as a template. For example, a DNA fragment of about 300 bp in length can be used as the probe. When a DNA fragment of about 300 bp in length is used as the probe, washing conditions for the hybridization include 50°C, 2×SSC, and 0.1% SDS.

[0034] Furthermore, since codon degeneracy differs depending on the host, the wild-type AMV reverse transcriptase gene may be one in which any codon has been replaced with an equivalent codon. That is, the wild-type AMV reverse transcriptase gene may be a variant of the wild-type AMV reverse transcriptase gene exemplified above due to the degeneracy of the genetic code. For example, the wild-type AMV reverse transcriptase gene may be modified to have optimal codons depending on the codon usage frequency of the host used.

[0035] The "identity" between amino acid sequences was calculated using the default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment) by blastp. "Identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated by blastn using the default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0036] The mutant AMV reverse transcriptase will be explained below.

[0037] The mutant AMV reverse transcriptase has enzymatic activity.

[0038] As described above, the term "enzyme activity" refers to the "enzyme activity possessed by AMV reverse transcriptase." In a preferred example, the enzymatic activity possessed by the mutant AMV reverse transcriptase may be only RNA-dependent DNA polymerase activity, or may be both RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity. and RNase H activity, or may have one or more of RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and RNase H activity. Of these, it is more preferable that the AMV reverse transcriptase has all of the same enzymatic activities as wild-type AMV reverse transcriptase, i.e., RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and RNase H activity.

[0039] The mutant AMV reverse transcriptase has a "specific mutation" in the wild-type AMV reverse transcriptase.

[0040] That is, the mutant AMV reverse transcriptase may be, for example, an enzyme having an amino acid sequence with a "specific mutation" in the amino acid sequence shown in SEQ ID NO: 6. Alternatively, the mutant AMV reverse transcriptase may be, for example, an enzyme having an amino acid sequence with a "specific mutation" in the amino acid sequence shown in SEQ ID NO: 6, and further including any one or more of substitution, deletion, insertion, and addition of one or several amino acid residues at one or several positions, and having enzymatic activity.

[0041] In other words, the mutant AMV reverse transcriptase may be an enzyme having the same amino acid sequence as the wild-type AMV reverse transcriptase, except for the "specific mutation." That is, the mutant AMV reverse transcriptase may be, for example, an enzyme having the amino acid sequence set forth in SEQ ID NO: 6, except for the "specific mutation." The mutant AMV reverse transcriptase may be, for example, an enzyme having an amino acid sequence that includes one or more substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions in the amino acid sequence set forth in SEQ ID NO: 6, except for the "specific mutation," and having enzymatic activity. The mutant AMV reverse transcriptase may be, for example, an enzyme having an amino acid sequence that is 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 6, except for the "specific mutation," and having enzymatic activity.

[0042] More particularly, the mutant AMV reverse transcriptase may be an enzyme selected from any of the following (i) to (iii): (i) an enzyme having the amino acid sequence set forth in SEQ ID NO: 6, which contains one or more amino acid substitutions selected from the following (1) to (15): (1) The amino acid residue corresponding to the leucine residue at position 476 of SEQ ID NO: 6 is substituted with a glutamine residue (2) The amino acid residue corresponding to the 377th valine residue of SEQ ID NO: 6 is substituted with an isoleucine residue (3) The amino acid residue corresponding to the serine residue at position 550 of SEQ ID NO: 6 is substituted with a threonine residue (4) The amino acid residue corresponding to the lysine residue at position 691 of SEQ ID NO: 6 is substituted with a glutamic acid residue (5) The amino acid residue corresponding to the asparagine residue at position 776 of SEQ ID NO: 6 is substituted with a lysine residue (6) The amino acid residue corresponding to the isoleucine residue at position 332 of SEQ ID NO: 6 is substituted with a valine residue (7) The amino acid residue corresponding to the 474th alanine residue in SEQ ID NO: 6 is substituted with a threonine residue (8) The amino acid residue corresponding to the 716th threonine residue of SEQ ID NO: 6 is substituted with a serine residue (9) The amino acid residue corresponding to the lysine residue at position 797 of SEQ ID NO: 6 is substituted with an arginine residue (10) The amino acid residue corresponding to the lysine residue at position 850 of SEQ ID NO: 6 is substituted with an arginine residue or a glutamic acid residue (11) The amino acid residue corresponding to the 61st isoleucine residue of SEQ ID NO: 6 is substituted with a valine residue (12) The amino acid residue corresponding to the 105th valine residue of SEQ ID NO: 6 is substituted with an alanine residue (13) The amino acid residue corresponding to the asparagine residue at position 623 of SEQ ID NO: 6 is substituted with an aspartic acid residue (14) The amino acid residue corresponding to the threonine residue at position 692 of SEQ ID NO: 6 is substituted with an alanine residue (15) a substitution of the amino acid residue corresponding to position 717 of glycine in SEQ ID NO: 6 with aspartic acid; (ii) an enzyme having an amino acid sequence set forth in SEQ ID NO: 6, which contains one or more amino acid substitutions selected from (1) to (15), and which further contains one or more of substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions shown in (1) to (15), and which has enzymatic activity; (iii) An enzyme having an amino acid sequence set forth in SEQ ID NO: 6, which has 70% or more identity to the entire amino acid sequence containing one or more amino acid substitutions selected from (1) to (15), with the proviso that the amino acid sequence maintains the amino acid substitutions, and which has enzymatic activity.

[0043] The term "one or several" in (ii) varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically may be, for example, 1 to 100, 1 to 90, 1 to 85, 1 to 80, 1 to 70, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0044] Furthermore, the "identity" in (iii) may be, for example, an amino acid sequence that has 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity to the entire amino acid sequence.

[0045] An example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to valine at position 377 in SEQ ID NO: 6 has been substituted with an isoleucine residue is shown in SEQ ID NO: 8. Furthermore, an example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to leucine at position 476 in SEQ ID NO: 6 has been substituted with a glutamine residue is shown in SEQ ID NO: 10. Furthermore, an example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to serine at position 550 in SEQ ID NO: 6 has been substituted with a threonine residue is shown in SEQ ID NO: 12. Furthermore, an example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to lysine at position 691 in SEQ ID NO: 6 has been substituted with a glutamic acid residue is shown in SEQ ID NO: 14. Furthermore, an example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to asparagine at position 776 in SEQ ID NO: 6 has been substituted with a lysine residue is shown in SEQ ID NO: 16. An example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to valine residue at position 377 of SEQ ID NO: 6 has been substituted with an isoleucine residue, the amino acid residue corresponding to serine residue at position 550 has been substituted with a threonine residue, and the amino acid residue corresponding to lysine residue at position 691 has been substituted with a glutamic acid residue is shown in SEQ ID NO: 18. An example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to valine residue at position 377 of SEQ ID NO: 6 has been substituted with an isoleucine residue, the amino acid residue corresponding to leucine residue at position 476 has been substituted with a glutamine residue, the amino acid residue corresponding to serine residue at position 550 has been substituted with a threonine residue, the amino acid residue corresponding to lysine residue at position 691 has been substituted with a glutamic acid residue, and the amino acid residue corresponding to asparagine residue at position 776 has been substituted with a lysine residue is shown in SEQ ID NO: 20. An example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to the isoleucine residue at position 332 of SEQ ID NO: 6 has been substituted with a valine residue is shown in SEQ ID NO: 26. Furthermore, an example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to the alanine residue at position 474 of SEQ ID NO: 6 has been substituted with a threonine residue is shown in SEQ ID NO: 28. Furthermore, an example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to the threonine residue at position 716 of SEQ ID NO: 6 has been substituted with a serine residue is shown in SEQ ID NO: 30. Furthermore, an example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to the lysine residue at position 797 of SEQ ID NO: 6 has been substituted with an arginine residue, and the amino acid residue corresponding to the lysine residue at position 850 has been substituted with an arginine residue is shown in SEQ ID NO: 32. Furthermore, an example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to the isoleucine residue at position 61 of SEQ ID NO: 20 has been substituted with a valine residue is shown in SEQ ID NO: 34. An example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to valine at position 105 in SEQ ID NO: 20 has been substituted with an alanine residue and the amino acid residue corresponding to lysine at position 850 has been substituted with a glutamic acid residue is shown in SEQ ID NO: 36. An example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to asparagine at position 623 in SEQ ID NO: 20 has been substituted with an aspartic acid residue, the amino acid residue corresponding to threonine at position 692 has been substituted with an alanine residue, the amino acid residue corresponding to glycine at position 717 has been substituted with an aspartic acid residue, and the amino acid residue corresponding to leucine at position 856 has been substituted with a proline residue is shown in SEQ ID NO: 38. An example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to isoleucine at position 332 in SEQ ID NO: 36 has been substituted with a valine residue is shown in SEQ ID NO: 44.

[0046] Specifically, the mutant AMV reverse transcriptase may be an enzyme selected from any one of the following (iv) to (vi): (iv) an enzyme having an amino acid sequence set forth in any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 38, and 44; (v) an enzyme having an amino acid sequence set forth in any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 38, and 44, which contains one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, with the proviso that specific mutations are maintained, and which has enzymatic activity; (vi) An enzyme having an amino acid sequence that is 70% or more identical to the amino acid sequence set forth in any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 38, and 44, with certain mutations maintained, and having enzymatic activity.

[0047] The term "one or several" in (v) varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically may be, for example, 1 to 100, 1 to 90, 1 to 85, 1 to 80, 1 to 70, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0048] Furthermore, the "identity" in (vi) may be, for example, an amino acid sequence that has 50% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity to the entire amino acid sequence.

[0049] The mutant AMV reverse transcriptase may contain other amino acid sequences in addition to the amino acid sequence of the mutant AMV reverse transcriptase as exemplified above. Such other amino acid sequences are also referred to as "additional sequences." That is, the mutant AMV reverse transcriptase may contain a fusion protein with an additional sequence. Furthermore, the mutant AMV reverse transcriptase may be expressed, for example, in a form containing an additional sequence (i.e., as a fusion protein with an additional sequence), and may ultimately lose some or all of the additional sequence. The terms "mutant AMV reverse transcriptase contains an additional sequence" or "mutant AMV reverse transcriptase" are used interchangeably. "The heterologous AMV reverse transcriptase is a fusion protein with an additional sequence" means that the mutant AMV reverse transcriptase finally obtained contains the additional sequence, unless otherwise specified. On the other hand, "the mutant AMV reverse transcriptase is expressed in a form containing the additional sequence" or "the mutant AMV reverse transcriptase contains the additional sequence upon expression" means that the mutant AMV reverse transcriptase at least contains the additional sequence upon expression, unless otherwise specified, and does not necessarily mean that the mutant AMV reverse transcriptase finally obtained contains the additional sequence. In other words, the mutant AMV reverse transcriptase gene may contain a nucleotide sequence encoding an additional sequence in addition to the nucleotide sequence of the mutant AMV reverse transcriptase gene as exemplified above. The same applies to wild-type AMV reverse transcriptase and wild-type AMV reverse transcriptase gene. The additional sequence is not particularly limited as long as the mutant AMV reverse transcriptase has enzymatic activity. The additional sequence can be selected appropriately depending on various conditions, such as the intended use. Examples of additional sequences include peptide tags, signal peptides (also called signal sequences), and protease recognition sequences. The additional sequence may be linked, for example, to the N-terminus, C-terminus, or both, of the mutant AMV reverse transcriptase. As the additional sequence, one type of amino acid sequence may be used, or two or more types of amino acid sequences may be used in combination.

[0050] Specific examples of peptide tags include His tags, FLAG tags, GST tags, Myc tags, maltose binding protein (MBP), cellulose binding protein (CBP), thioredoxin (TRX), green fluorescent protein (GFP), horseradish peroxidase (HRP), alkaline phosphatase (ALP), and antibody Fc regions. Examples of His tags include 6xHis tags. Peptide tags can be used, for example, to detect and purify expressed mutant AMV reverse transcriptase.

[0051] The signal peptide is not particularly limited as long as it functions in a host in which the mutant AMV reverse transcriptase is expressed. Examples of signal peptides include signal peptides recognized by the Sec secretory pathway and signal peptides recognized by the Tat secretory pathway. The signal peptide can be used, for example, for the secretory production of the mutant AMV reverse transcriptase. When the signal peptide is used to secrete and produce the mutant AMV reverse transcriptase, the signal peptide is cleaved during secretion, and the mutant AMV reverse transcriptase without the signal peptide can be secreted outside the bacterial cell. In other words, the mutant AMV reverse transcriptase obtained finally does not typically have a signal peptide.

[0052] Specific examples of protease recognition sequences include the recognition sequence for Factor Xa protease and the recognition sequence for proTEV protease. Protease recognition sequences can be used, for example, to cleave the expressed mutant AMV reverse transcriptase. Specifically, when the mutant AMV reverse transcriptase is expressed as a fusion protein with a peptide tag, a protease recognition sequence can be introduced at the junction between the mutant AMV reverse transcriptase and the peptide tag, allowing the peptide tag to be cleaved from the expressed mutant AMV reverse transcriptase using the protease, thereby yielding a mutant AMV reverse transcriptase without the peptide tag.

[0053] As used herein, "the Xth amino acid in the amino acid sequence set forth in SEQ ID NO: 6" refers to the amino acid located at position X counting from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 6. In a specific amino acid sequence, "the amino acid residue corresponding to the Xth amino acid in the amino acid sequence set forth in SEQ ID NO: 6" refers to an amino acid residue in the specific amino acid sequence, which is arranged at the same position as the Xth amino acid in the amino acid sequence set forth in SEQ ID NO: 6 in an alignment of the specific amino acid sequence with the amino acid sequence set forth in SEQ ID NO: 6. For example, in a specific amino acid sequence, "the amino acid residue corresponding to the leucine residue at position 476 in SEQ ID NO: 6" refers to an amino acid residue in the specific amino acid sequence, which is arranged at the same position as the 476th amino acid in the amino acid sequence set forth in SEQ ID NO: 6 in an alignment of the specific amino acid sequence with the amino acid sequence set forth in SEQ ID NO: 6. The term "amino acid substitution" refers to the amino acid residue located at the same position as leucine in SEQ ID NO: 6. In the amino acid sequence of SEQ ID NO: 6, "an amino acid residue corresponding to the Xth amino acid in the amino acid sequence of SEQ ID NO: 6" refers to the Xth amino acid in the amino acid sequence of SEQ ID NO: 6 itself. In other words, the positions of the above-exemplified amino acid substitutions (i.e., the amino acid substitutions at the specific positions and, optionally, other amino acid substitutions) do not necessarily indicate absolute positions in the protein of the present invention, but indicate relative positions based on the amino acid sequence of SEQ ID NO: 6. For example, if the protein of the present invention contains an insertion, deletion, or addition of an amino acid residue N-terminally to the position of the above-exemplified amino acid substitution, the absolute position of the amino acid substitution may vary accordingly. The positions of the above-exemplified amino acid substitutions in the protein of the present invention can be identified, for example, by aligning the amino acid sequence of the protein of the present invention with the amino acid sequence of SEQ ID NO: 6. The alignment can be performed, for example, using an alignment program such as BLAST or FASTA. The same applies to the positions of the above-exemplified amino acid substitutions in any amino acid sequence, such as a variant of the amino acid sequence of SEQ ID NO: 6. Furthermore, the amino acid residues before the amino acid substitutions in the AMV reverse transcriptase exemplified in the present invention (for example, the amino acid substitutions shown in (1) to (15) above) indicate the types of amino acid residues before the substitution in the amino acid sequence set forth in SEQ ID NO: 6, and may or may not be conserved in unmodified amino acid sequences other than the amino acid sequence set forth in SEQ ID NO: 6.

[0054] The mutant AMV reverse transcriptase gene is not particularly limited as long as it encodes the mutant AMV reverse transcriptase described above. As used herein, the term "gene" is not limited to DNA and may encompass any polynucleotide as long as it encodes a protein of interest. In other words, "mutant AMV reverse transcriptase gene" may refer to any polynucleotide encoding a mutant AMV reverse transcriptase. The mutant AMV reverse transcriptase gene may be DNA, RNA, or a combination thereof. The mutant AMV reverse transcriptase gene may be single-stranded or double-stranded. The mutant AMV reverse transcriptase gene may be single-stranded DNA or single-stranded RNA. The mutant AMV reverse transcriptase gene may be double-stranded DNA, double-stranded RNA, or a hybrid strand consisting of a DNA strand and an RNA strand. The mutant AMV reverse transcriptase gene may contain both DNA residues and RNA residues in a single polynucleotide strand. When the mutant AMV reverse transcriptase gene contains RNA, the descriptions regarding DNA, such as the nucleotide sequences exemplified above, may be interpreted appropriately to refer to RNA. The form of the mutant AMV reverse transcriptase gene can be appropriately selected depending on various conditions such as the mode of use.

[0055] The "specific mutation" will be explained below.

[0056] The term "specific mutation" refers to a mutation that is useful for reverse transcription, and may particularly refer to a mutation that is useful for improving thermostability and / or resistance to reaction inhibition by contaminants. Specifically, the term "specific mutation" may refer to a mutation that, when introduced into a wild-type AMV reverse transcriptase, confers on the wild-type AMV reverse transcriptase improved thermostability and / or resistance to reaction inhibition by contaminants.

[0057] The "specific mutation" includes one or more amino acid substitutions selected from the following (1) to (15): (1) The amino acid residue corresponding to the leucine residue at position 476 of SEQ ID NO: 6 is substituted with a glutamine residue (2) The amino acid residue corresponding to the 377th valine residue of SEQ ID NO: 6 is substituted with an isoleucine residue (3) The amino acid residue corresponding to the serine residue at position 550 of SEQ ID NO: 6 is substituted with a threonine residue (4) The amino acid residue corresponding to the lysine residue at position 691 of SEQ ID NO: 6 is substituted with a glutamic acid residue (5) The amino acid residue corresponding to the asparagine residue at position 776 of SEQ ID NO: 6 is substituted with a lysine residue (6) The amino acid residue corresponding to the isoleucine residue at position 332 of SEQ ID NO: 6 is substituted with a valine residue (7) The amino acid residue corresponding to the 474th alanine residue in SEQ ID NO: 6 is substituted with a threonine residue (8) The amino acid residue corresponding to the 716th threonine residue of SEQ ID NO: 6 is substituted with a serine residue (9) The amino acid residue corresponding to the lysine residue at position 797 of SEQ ID NO: 6 is substituted with an arginine residue (10) The amino acid residue corresponding to the lysine residue at position 850 of SEQ ID NO: 6 is substituted with an arginine residue or a glutamic acid residue (11) The amino acid residue corresponding to the 61st isoleucine residue of SEQ ID NO: 6 is substituted with a valine residue (12) The amino acid residue corresponding to the 105th valine residue of SEQ ID NO: 6 is substituted with an alanine residue (13) The amino acid residue corresponding to the asparagine residue at position 623 of SEQ ID NO: 6 is substituted with an aspartic acid residue (14) The amino acid residue corresponding to the threonine residue at position 692 of SEQ ID NO: 6 is substituted with an alanine residue (15) The amino acid residue corresponding to the glycine residue at position 717 of SEQ ID NO: 6 is substituted with aspartic acid.

[0058] Furthermore, by introducing the amino acid substitution (1) into wild-type AMV reverse transcriptase, not only is the thermal stability of the AMV reverse transcriptase improved, but the purification yield of the AMV reverse transcriptase can also be expected to be improved. Therefore, the "specific mutation" preferably includes the amino acid substitution (1).

[0059] Furthermore, the "specific mutation" may include at least (2) to (4) amino acid substitutions. Furthermore, the "specific mutation" may include at least the amino acid substitutions (1) to (5) and one or more amino acid substitutions selected from (11) to (15).

[0060] The "specific mutation" improves the thermal stability and / or resistance to reaction inhibition by contaminants of the mutant AMV reverse transcriptase compared to the wild-type AMV reverse transcriptase. The mutant AMV reverse transcriptase may have improved thermal stability compared to the wild-type AMV reverse transcriptase, for example, by having one or more amino acid substitutions selected from (1) to (15) above. Furthermore, the mutant AMV reverse transcriptase may have improved resistance to reaction inhibition by contaminants compared to the wild-type AMV reverse transcriptase, for example, by having one or more amino acid substitutions selected from (1), (2), (3), (4), (5), (6), (10), and (12) above.

[0061] Improved thermostability may be demonstrated, for example, as improved residual activity after heat treatment, an increase in the amount of enzyme remaining after heat treatment, or reactivity at high temperatures. Specifically, improved thermostability may be demonstrated by heating a mutant AMV reverse transcriptase at 50-60°C for 1-60 minutes, followed by an isothermal gene amplification reaction involving a reverse transcription reaction, and then detecting the generated amplification product in a shorter time than with wild-type AMV reverse transcriptase. As another example, improved thermostability may be demonstrated by heating a mutant AMV reverse transcriptase at 50-60°C for 1-60 minutes, followed by a reverse transcription reaction, and then detecting an increased amount of DNA generated in a reverse transcription reaction compared to wild-type AMV reverse transcriptase. As yet another example, improved thermostability may be demonstrated by heating a mutant AMV reverse transcriptase at 50-60°C for 1-60 minutes, followed by an isothermal gene amplification reaction involving a reverse transcription reaction, and then detecting an increased amount of DNA generated in a reverse transcription reaction compared to wild-type AMV reverse transcriptase. Improved thermostability may be demonstrated by an increase in the amount of enzyme remaining without heat denaturation compared to wild-type AMV reverse transcriptase after heating for 1 to 60 minutes at 0°C. As yet another example, improved thermostability may be demonstrated by performing an isothermal gene amplification reaction involving reverse transcription at a high reaction temperature using a mutant AMV reverse transcriptase, and the time until the generated amplification product is detected is shorter compared to wild-type AMV reverse transcriptase, or by detecting the generated amplification product without being heat-inactivated up to a higher reaction temperature than with wild-type AMV reverse transcriptase.

[0062] Improved resistance to reaction inhibition by contaminants may be demonstrated, for example, by superior activity in the presence of contaminants compared to AMV reverse transcriptase lacking the "specific mutation." Improved resistance to reaction inhibition by contaminants may be demonstrated, for example, under high-temperature conditions, but is not limited thereto and may be demonstrated regardless of temperature conditions. Contaminants include, but are not limited to, components of specimens and measurement samples containing target nucleic acids, such as contaminants derived from living organisms, such as saliva, urine, feces, nasal discharge, sputum, blood, puncture fluids (e.g., pleural effusion and ascites), and nasal and pharyngeal mucus, as well as contaminants derived from natural sources, such as soil and environmental water (river water, seawater, and lake water). Other examples of contaminants include components of reagents (e.g., virus transport solution, transport medium, blood preservation solution, fecal preservation solution, buffer solution, and physiological saline) used to collect, store, and transport the specimens and measurement samples. Further examples of contaminants include components of reagents (e.g., alcohols such as ethanol, surfactants, guanidine salts, buffers, organic solvents) used to extract, separate, or purify the target nucleic acid from the specimen or measurement sample, as well as viruses, bacteria, or cells containing the target nucleic acid contained therein. The term "in the presence of contaminants" refers to conditions in which the above-mentioned contaminants or purified products thereof are present in the reaction solution. Specifically, improved resistance to reaction inhibition by contaminants can be demonstrated by, for example, performing an isothermal gene amplification reaction involving reverse transcription using a mutant AMV reverse transcriptase in a reaction solution containing a saliva or urine purified product, and shortening the time required to detect the generated amplification product compared to wild-type AMV reverse transcriptase. The saliva or urine purified product may contain components of the above-mentioned reagents, such as reagents for purifying saliva or urine.

[0063] From the viewpoint of resistance to reaction inhibition by contaminants, it is preferable that the mutant AMV reverse transcriptase has all of the amino acid substitutions (1), (2), (3), (4), (5), (6), and (10), or has the amino acid substitution (12) in addition to these mutations.

[0064] <2> Production of mutant AMV reverse transcriptase The mutant AMV reverse transcriptase can be produced, for example, by expressing the mutant AMV reverse transcriptase gene in a host carrying the gene.

[0065] The production of mutant AMV reverse transcriptase using a host carrying a mutant AMV reverse transcriptase gene will be described in detail below.

[0066] <2-1>Host A host having a mutant AMV reverse transcriptase gene can be obtained by introducing the mutant AMV reverse transcriptase gene into a suitable host. "Introducing a mutant AMV reverse transcriptase gene into a host" may also include modifying a host into which a wild-type AMV reverse transcriptase gene or the like has been introduced so that the wild-type AMV reverse transcriptase gene encodes a mutant AMV reverse transcriptase. "Having a mutant AMV reverse transcriptase gene" is also referred to as "having a mutant AMV reverse transcriptase."

[0067] The host is not particularly limited as long as it can express a functional mutant AMV reverse transcriptase. Examples of the host include microorganisms, plant cells, insect cells, and animal cells. Examples of the host include microorganisms, such as bacteria and yeast. Examples of the microorganism include: , in particular bacteria.

[0068] Examples of bacteria include bacteria belonging to the family Enterobacteriaceae, coryneform bacteria, and Bacillus bacteria.

[0069] Bacteria belonging to the Enterobacteriaceae family include the genus Escherichia, Enterobacter Enterobacter, Pantoea, Klebsiella, and Serratia, Erwinia, Photorhabdus Examples of such bacteria include those belonging to the genera Salmonella, Morganella, and the like. Bacteria classified as Enterobacteriaceae according to the taxonomy used in the National Institute for Biotechnology Information database (http: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=91347) can be used. Bacteria of the genus Escherichia include, but are not limited to, bacteria classified as Escherichia according to classifications known to microbiologists. Examples of Escherichia bacteria include those described in the book by Neidhardt et al. (Backmann, BJ 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, pp. 2460-2488. Table 1. In F.D. Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology / Second Edition, American Society for Microbiology Press, Washington, DC). Examples of Escherichia bacteria include Escherichia coli. Examples of Escherichia coli include Escherichia coli B strains such as BL21(DE3), Escherichia coli K-12 strains such as JM109 (ATCC 53323), HB101 (ATCC 33694), W3110 (ATCC 27325), and MG1655 (ATCC 47076), Escherichia coli K5 (ATCC 23506), and their derivatives. Examples of Enterobacter bacteria include Enterobacter agglomerans and Enterobacter aerogenes.Examples of bacteria of the genus Pantoea include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Examples of bacteria of the genus Erwinia include Erwinia amylovora and Erwinia carotovora. Examples of bacteria of the genus Klebsiella include Klebsiella pneumoniae. Examples include Klebsiella planticola.

[0070] Corynebacterium species include the genus Corynebacterium, Brevibacterium, and Examples of bacteria include those belonging to the genera Brevibacterium and Microbacterium.

[0071] The genus Corynebacterium also includes bacteria that were previously classified as Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). This also includes bacteria that were previously classified as Corynebacterium ammoniagenes but were reclassified as Corynebacterium stationis based on 16S rRNA sequence analysis and other factors (Int. J. Syst. Evol. Microbiol., 60, 874-879(2010)).

[0072] Examples of Bacillus bacteria include Bacillus subtilis, Bacillus Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, Bacillus stearothermophilus (B acillus stearothermophilus). Specific examples of Bacillus subtilis include Bacillus subtilis 168 Marburg strain (ATCC 6051) and Bacillus subtilis PY79 strain (Plasmid, 1984, 12, 1-9). Specific examples of Bacillus amyloliquefaciens include Bacillus amyloliquefaciens T strain (ATCC 23842) and Bacillus amyloliquefaciens N strain (ATCC 23845).

[0073] Examples of yeast include yeasts belonging to the genus Saccharomyces, such as Saccharomyces cerevisiae, the genus Candida, such as Candida utilis, the genus Pichia, such as Pichia pastoris, the genus Hansenula, such as Hansenula polymorpha, and the genus Schizosaccharomyces, such as Schizosaccharomyces pombe.

[0074] These strains can be obtained, for example, from the American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Maryland 20852, PO Box 1549, Manassas, VA 20108, United States of America). Each strain has been assigned a registration number, which can be used to obtain a sample (see http: / / www.atcc.org / ). The registration number for each strain is listed in the catalog of the American Type Culture Collection. These strains can also be obtained from, for example, the depository institution where they were deposited.

[0075] A mutant AMV reverse transcriptase gene can be obtained, for example, by modifying a wild-type AMV reverse transcriptase gene so that the encoded AMV reverse transcriptase has a "specific mutation." The wild-type AMV reverse transcriptase gene to be modified can be obtained, for example, by cloning from a virus having the wild-type AMV reverse transcriptase gene or by chemical synthesis. Alternatively, the wild-type AMV reverse transcriptase gene to be modified can be obtained, for example, by artificially modifying a natural AMV reverse transcriptase gene obtained by cloning from a virus. Alternatively, a mutant AMV reverse transcriptase gene can be obtained without the intervention of a wild-type AMV reverse transcriptase gene. A mutant AMV reverse transcriptase gene can be obtained directly, for example, by chemical synthesis. The obtained mutant AMV reverse transcriptase gene can be used as is or after further modification. For example, a mutant AMV reverse transcriptase gene of one embodiment can be modified to obtain a mutant AMV reverse transcriptase gene of another embodiment.

[0076] Genetic modification can be performed using known techniques. For example, site-directed mutagenesis can be used to introduce a desired mutation into a target site in DNA. That is, for example, site-directed mutagenesis can be used to modify the coding region of a gene so that the encoded protein contains substitutions, deletions, insertions, and / or additions of amino acid residues at specific sites. Examples of site-directed mutagenesis include PCR-based methods (Higuchi, R., 61, in PCR technology, Erlich, H.A. Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, H.J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T.A. et al., Meth. in Enzymol., 154, 367 (1987)).

[0077] The method for introducing the mutant AMV reverse transcriptase gene into the host is not particularly limited, as long as the mutant AMV reverse transcriptase gene is retained in the host in an expressible manner. The mutant AMV reverse transcriptase gene can be introduced into the host using the same method as described in detail below in the section "Method for introducing a gene."

[0078] In addition, when the host already has an AMV reverse transcriptase gene such as a wild-type AMV reverse transcriptase gene in its chromosome, the AMV reverse transcriptase gene is transformed into a gene encoding a mutant AMV reverse transcriptase. The host can also be modified to have a mutant AMV reverse transcriptase gene by modifying the host so that it has a mutant AMV reverse transcriptase gene. Modification of the AMV reverse transcriptase gene present in a chromosome or the like can be carried out, for example, by natural mutation, mutation treatment, or genetic engineering. Note that a host having an AMV reverse transcriptase gene in a chromosome or the like may be obtained, for example, by previously introducing an AMV reverse transcriptase gene such as a wild-type AMV reverse transcriptase gene into the chromosome or the like of the host.

[0079] The host may have any properties as long as it is capable of producing the mutant AMV reverse transcriptase.

[0080] <2-2> Methods for introducing genes The following describes the method for introducing a gene into a host.

[0081] Introduction of a gene into a host can be achieved by introducing the gene into the host's chromosome. Introduction of a gene into a chromosome can be achieved, for example, by homologous recombination (Miller, JH, Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods that utilize homologous recombination include methods using linear DNA, such as Red-driven integration (Datsenko, K. A., and Wanner, BL, Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)), methods using plasmids containing a temperature-sensitive replication origin, methods using conjugatively transferable plasmids, methods using suicide vectors lacking a replication origin that functions in the host, and transduction methods using phages. Only one copy of a gene may be introduced, or two or more copies may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination targeting a sequence that exists in multiple copies on a chromosome. Examples of sequences present in multiple copies on a chromosome include repetitive DNA sequences and inverted repeats at both ends of transposons. Homologous recombination can also be performed by targeting appropriate sequences on a chromosome, such as genes not required for AMV reverse transcriptase production. Genes can also be randomly introduced into a chromosome using transposons or Mini-Mu (see JP-A-2-109985, U.S. Pat. No. 5,882,888, and EP 805867 B1).

[0082] The introduction of the target gene into the chromosome can be confirmed by Southern hybridization using a probe having a sequence complementary to all or part of the gene, or by PCR using primers prepared based on the sequence of the gene.

[0083] Introduction of a gene into a host can also be achieved by introducing a vector containing the gene into the host. For example, a DNA fragment containing a target gene can be ligated to a vector that functions in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to introduce the gene into the host. A host transformed with an expression vector is also called a transformant. A DNA fragment containing a target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism containing the target gene as a template. Vectors that can autonomously replicate within host cells can be used. The vector may be a multicopy vector. Furthermore, the vector may contain a marker such as an antibiotic resistance gene to select transformants. The vector may also contain a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. Specific examples of vectors capable of autonomous replication in Enterobacteriaceae bacteria such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG398, pBR322, pSTV29, pCold series vectors (all available from Takara Bio Inc.), pACYC177, pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pET series vectors (Merck), and pQE series vectors (Qiagen).

[0084] When a gene is introduced, it is sufficient that the gene can be expressed by the host. Specifically, it is sufficient that the gene is maintained so that it is expressed under the control of a promoter that functions in the host. A "promoter that functions in the host" may mean a promoter that has promoter activity in the host. The promoter may be a promoter derived from the host or a heterologous promoter. The promoter may be a promoter native to the gene to be introduced or a promoter of another gene. Specific examples of promoters include T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Pm1 promoter derived from Bifidobacterium, PR promoter, PL promoter, P 4 promoter, and the P8 promoter.

[0085] A terminator for terminating transcription can be placed downstream of the gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a terminator derived from the host or a heterologous terminator. The terminator may be a terminator inherent to the gene to be introduced or a terminator from another gene. Specific examples of terminators include the T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

[0086] <2-3> Host culture The mutant AMV reverse transcriptase can be expressed by culturing a host harboring the mutant AMV reverse transcriptase gene. For example, the mutant AMV reverse transcriptase may be expressed by transforming a host with an expression vector containing the mutant AMV reverse transcriptase and culturing the resulting transformant.

[0087] The medium used is not particularly limited as long as it allows the host to grow and expresses a functional mutant AMV reverse transcriptase. For example, a conventional medium used for culturing microorganisms such as bacteria and yeast can be used. The medium may contain medium components such as a carbon source, a nitrogen source, a phosphate source, a sulfur source, and various other organic and inorganic components as needed. The types and concentrations of medium components may be appropriately determined depending on various conditions, such as the type of host.

[0088] Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, blackstrap molasses, starch hydrolysates, and biomass hydrolysates; organic acids such as acetic acid, citric acid, succinic acid, and gluconic acid; alcohols such as ethanol, glycerol, and crude glycerol; and fatty acids. Plant-derived materials are preferably used as carbon sources. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Examples of plant-derived materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plants containing these, and decomposition products of these plant organs. The form of use of plant-derived materials is not particularly limited, and they can be used in any form, such as raw products, squeezed juice, pulverized products, or purified products. Furthermore, pentoses such as xylose, hexoses such as glucose, or mixtures thereof can be obtained from plant biomass and used. Specifically, these sugars can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis with enzymes such as cellulase, and alkali treatment. Since hemicellulose is generally more easily hydrolyzed than cellulose, the hemicellulose in the plant biomass may be hydrolyzed in advance to liberate pentoses, and then the cellulose may be hydrolyzed to produce hexoses. Furthermore, xylose may be supplied by conversion from hexoses such as glucose, for example, by providing the host with a pathway for converting hexoses to xylose. As the carbon source, one type of carbon source may be used. Two or more carbon sources may be used in combination.

[0089] The concentration of the carbon source in the medium is not particularly limited, as long as the host can grow and a functional mutant AMV reverse transcriptase is expressed. The concentration of the carbon source in the medium may be as high as possible, for example, within a range that does not inhibit the production of the mutant AMV reverse transcriptase. The initial concentration of the carbon source in the medium may be, for example, typically 5 to 30 w / v%, preferably 10 to 20 w / v%. Additionally, the carbon source may be additionally supplied to the medium as appropriate. For example, the carbon source may be additionally supplied in response to a decrease or depletion of the carbon source as the culture progresses. The carbon source may be temporarily depleted as long as the mutant AMV reverse transcriptase is ultimately produced; however, it may be preferable to carry out the culture so that the carbon source does not become depleted or does not remain depleted.

[0090] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen sources such as peptone, yeast extract, meat extract, and soy protein hydrolysate; ammonia; and urea. Ammonia gas or aqueous ammonia, which are used for pH adjustment, may also be used as a nitrogen source. As the nitrogen source, one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

[0091] Specific examples of the phosphate source include phosphate salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. As the phosphate source, one type of phosphate source may be used, or two or more types of phosphate sources may be used in combination.

[0092] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one type of sulfur source may be used, or two or more types of sulfur sources may be used in combination.

[0093] Specific examples of other various organic and inorganic components include inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acids, yeast extract, and soy protein hydrolysate. As other various organic and inorganic components, one type of component may be used, or two or more types of components may be used in combination.

[0094] When an auxotrophic mutant strain that requires nutrients such as amino acids for growth is used, it is preferable to supplement the medium with such required nutrients.

[0095] The culture conditions are not particularly limited as long as the host can grow and a functional mutant AMV reverse transcriptase can be expressed. Culture can be performed under normal conditions used for culturing microorganisms such as bacteria and yeast. Culture conditions can be appropriately set depending on various conditions, such as the type of host. Furthermore, expression of the mutant AMV reverse transcriptase gene can be induced as necessary.

[0096] Culturing can be carried out using a liquid medium. For example, the host may be cultured in a solid medium such as an agar medium and then directly inoculated into the liquid medium, or the host may be seed cultured in a liquid medium and then inoculated into the liquid medium for main culture. That is, the culture may be divided into a seed culture and a main culture. In this case, the culture conditions for the seed culture and the main culture may or may not be the same. The mutant AMV reverse transcriptase only needs to be expressed in at least the main culture. The amount of the host contained in the medium at the start of the culture is not particularly limited. For example, a seed culture solution with an OD660 of 4 to 100 may be added at 0.1% by mass to 1% by mass of the medium for main culture at the start of the culture. 00% by mass, preferably 1% to 50% by mass.

[0097] Cultivation can be carried out by batch culture, fed-batch culture, continuous culture, or a combination of these. The medium at the start of cultivation is also called the "initial medium." The medium supplied to a cultivation system (e.g., a fermenter) in fed-batch or continuous cultivation is also called the "fed-batch medium." Supplying a fed-batch medium to a cultivation system in fed-batch or continuous cultivation is also called "fed-batch." When cultivation is carried out separately into a seed culture and a main culture, the cultivation forms of the seed culture and the main culture may or may not be the same. For example, both the seed culture and the main culture may be carried out by batch culture, or the seed culture may be carried out by batch culture and the main culture may be carried out by fed-batch or continuous culture.

[0098] Various components such as a carbon source may be contained in the initial medium, the feed medium, or both. That is, various components such as a carbon source may be additionally supplied to the medium during the culture process, either alone or in any combination. These components may be supplied once, multiple times, or continuously. The types of components contained in the initial medium may or may not be the same as the types of components contained in the feed medium. Furthermore, the concentrations of each component contained in the initial medium may or may not be the same as the concentrations of each component contained in the feed medium. Furthermore, two or more feed media containing different types and / or concentrations of components may be used. For example, when multiple feeds are performed intermittently, the types and / or concentrations of components contained in each feed medium may or may not be the same.

[0099] The culture can be carried out under aerobic conditions, for example. "Aerobic conditions" may mean that the dissolved oxygen concentration in the medium is 0.33 ppm or higher, preferably 1.5 ppm or higher. Specifically, the oxygen concentration may be controlled to, for example, 1 to 100% of the saturated oxygen concentration, preferably about 20 to 100%. The culture can be carried out, for example, by aerobic culture or shaking culture. The pH of the medium may be, for example, 3 to 10, preferably 4.0 to 9.5. During culture, the pH of the medium can be adjusted as needed. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or an aqueous phosphoric acid solution. The culture temperature may be, for example, 20 to 45°C, preferably 25 to 37°C. The culture period may be, for example, 10 to 120 hours. Cultivation may be continued, for example, until the carbon source in the medium is consumed or until the activity of the host is lost.

[0100] Culturing the host in this manner yields a culture containing mutant AMV reverse transcriptase. The mutant AMV reverse transcriptase can accumulate, for example, within the host's bacterial cells. "Bacterial cells" may be interpreted as "cells" as appropriate depending on the type of host. Depending on the host and / or the design of the mutant AMV reverse transcriptase gene used, it may also be possible to cause the mutant AMV reverse transcriptase to accumulate in the periplasm or to produce and secrete the mutant AMV reverse transcriptase outside the bacterial cells.

[0101] The mutant AMV reverse transcriptase may be recovered while still contained in the culture (specifically, the medium or bacterial cells), or may be recovered from the culture (specifically, the medium or bacterial cells). The mutant AMV reverse transcriptase may be purified during the process of recovering it from the culture. Purification can be carried out to a desired extent. That is, the mutant AMV reverse transcriptase includes purified mutant AMV reverse transcriptase and fractions containing mutant AMV reverse transcriptase. In other words, the mutant AMV reverse transcriptase may be recovered in the form of a purified enzyme, or in the form of such a fraction (i.e., in the form contained in such a fraction), The fraction may be recovered in the form of a combination thereof. Such fractions are not particularly limited, as long as they contain the mutant AMV reverse transcriptase so that it can act on its substrate. Examples of such fractions include cultures of hosts harboring the mutant AMV reverse transcriptase gene (i.e., hosts harboring the mutant AMV reverse transcriptase), bacterial cells recovered from the cultures, culture supernatants recovered from the cultures, processed products thereof (e.g., bacterial cell disruption, bacterial cell lysates, bacterial cell extracts, and other processed bacterial products, such as those described below), partially purified products thereof (i.e., crudely purified products), and combinations thereof. Note that the term "purified mutant AMV reverse transcriptase" also encompasses crudely purified products. These fractions can be recovered alone or in appropriate combinations. Mutant AMV reverse transcriptase recovered in any manner, including these, can be used for any application, including the "amplification of target nucleic acids" described below, and may also be used as a "reagent for amplifying target nucleic acids" described below. The manner in which the mutant AMV reverse transcriptase is recovered may be appropriately determined depending on the application.

[0102] For example, the mutant AMV reverse transcriptase may be recovered in a form contained in bacterial cells. The method for recovering bacterial cells from the culture medium is not particularly limited, and known methods can be used, for example. Such methods include, for example, natural sedimentation, centrifugation, and filtration. A flocculant may also be used. These methods can be used alone or in appropriate combination. The recovered bacterial cells can be washed appropriately using an appropriate medium. The recovered bacterial cells can also be resuspended appropriately using an appropriate medium. Examples of media that can be used for washing and suspension include aqueous media (aqueous solvents) such as water and aqueous buffer solutions.

[0103] As another example, the mutant AMV reverse transcriptase may be recovered from the bacterial cells by subjecting the bacterial cells to appropriate treatment. Examples of bacterial cell treatment include immobilization onto a carrier such as acrylamide or carrageenan, freeze-thawing, treatment to increase membrane permeability, and physical disruption using ultrasonic disruption or a pressure homogenizer. Membrane permeability can be increased by using, for example, a surfactant or organic solvent. These treatments can be used alone or in appropriate combination.

[0104] Furthermore, the recovered mutant AMV reverse transcriptase may be purified to a higher purity by applying various types of chromatography, such as ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and affinity chromatography, either alone or in combination. The purification may enable the separation and purification of the α-chain, β-chain, and αβ-form of AMV reverse transcriptase. One example of chromatographic purification is the separation and purification of the α-chain, β-chain, and αβ-form of AMV reverse transcriptase by hydrophobic interaction chromatography incorporating polypropylene glycol groups, followed by further purification using ion exchange chromatography using a phosphocellulose support.

[0105] The mutant AMV reverse transcriptase may be produced alone or in combination with other proteins.

[0106] <2-4> Production of mutant AMV reverse transcriptase The mutant AMV reverse transcriptase may be produced by the method described above.

[0107] For example, a mutant AMV reverse transcriptase may be produced by a method comprising the steps of expressing a mutant AMV reverse transcriptase gene in a host harboring the gene and recovering the expressed enzyme. Such a method is also referred to as a "method for producing an AMV reverse transcriptase."

[0108] The method for producing AMV reverse transcriptase preferably comprises: a step of transforming a host with an expression vector containing a mutant AMV reverse transcriptase gene and culturing the resulting transformant to express the AMV reverse transcriptase; recovering the expressed enzyme from the resulting culture; The method may include:

[0109] The expression vector is as described above. The expression vector having a mutant AMV reverse transcriptase gene may be an expression vector containing a nucleotide encoding the mutant AMV reverse transcriptase.

[0110] The host and its culture are as described above.

[0111] The expressed mutant AMV reverse transcriptase is recovered as described above. The mutant AMV reverse transcriptase may be purified during the recovery process from the culture. The enzyme purification is also as described above.

[0112] <3> Use of mutant AMV reverse transcriptase The use of the mutant AMV reverse transcriptase is not particularly limited. The mutant AMV reverse transcriptase can be used, for example, for amplifying a target nucleic acid. Specifically, the mutant AMV reverse transcriptase can be used, for example, for amplifying a target nucleic acid involving a reverse transcription reaction. The amplification of the target nucleic acid may specifically include a step of performing a reverse transcription reaction using the mutant AMV reverse transcriptase to synthesize DNA using RNA as a template. The amplification of the target nucleic acid may include any other known step, and typically may include, for example, a step of performing a polymerase chain reaction using a DNA-dependent DNA polymerase to synthesize DNA using DNA as a template.

[0113] In the amplification of a target nucleic acid, the target nucleic acid may be RNA. Specifically, the amplification of the target nucleic acid may be carried out, for example, by using the target nucleic acid, RNA, as a template and amplifying it as DNA through a reverse transcription reaction by AMV reverse transcriptase and a polymerase chain reaction by DNA-dependent DNA polymerase. Furthermore, the amplification of the target nucleic acid may be carried out, for example, by the NASBA (Nucleic Acid Sequence Based Amplification) method described in Japanese Patent No. 2650159, This may be carried out by the TMA (Transcription-Mediated Amplification) method described in Japanese Patent Application Laid-Open No. 4-500759 or the TRC (Transcription Reverse-transcription Concerted) method described in Japanese Patent Application Laid-Open No. 2000-14400. The mutant AMV reverse transcriptase is thermostable. Because of its excellent thermal stability, RNA can be treated at higher temperatures during or prior to the reverse transcription reaction, which is expected to suppress the formation of secondary structures in RNA and the generation of by-products caused by misannealing, making it suitable for amplification of target nucleic acids involving reverse transcription. Thus, in one embodiment of the present disclosure, amplification of target nucleic acids may be performed at high temperatures. Specifically, for example, a step of performing a reverse transcription reaction using a mutant AMV reverse transcriptase to synthesize DNA using RNA as a template may be performed at high temperatures, and RNA treatment in a step prior to this may be performed at high temperatures. Note that "high temperature" here specifically refers to, for example, heating at 50 to 60°C for 1 to 60 minutes.

[0114] The reagent used for amplifying the target nucleic acid contains a mutant AMV reverse transcriptase. The reagent used for amplifying the target nucleic acid is also referred to as a target nucleic acid amplification reagent. The mutant AMV reverse transcriptase contained in the target nucleic acid amplification reagent may be a mutant AMV reverse transcriptase recovered in any manner, including the manner described above. The target nucleic acid amplification reagent may contain any known component necessary for a reverse transcription reaction other than the mutant AMV reverse transcriptase. Furthermore, since the target nucleic acid may be amplified by a polymerase chain reaction using a DNA-dependent DNA polymerase, the reagent used for amplifying the target nucleic acid may contain any known component necessary for the polymerase chain reaction, including a DNA-dependent DNA polymerase.

[0115] Furthermore, the target nucleic acid amplification reagent may include a primer set consisting of a first oligonucleotide having a sequence complementary to a portion of a specific base sequence of the target nucleic acid and a second oligonucleotide having a sequence homologous to a portion of the specific base sequence (provided that a promoter sequence capable of initiating transcription by RNA polymerase is added to the 5' end of at least one of the first and second oligonucleotides), an enzyme having RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, an enzyme having RNase H activity, and an RNA polymerase. Such a reagent may be used, for example, in the TRC method described above. In other words, the target nucleic acid amplification reagent also includes, for example, a target nucleic acid amplification reagent using the TRC method.

[0116] In one embodiment, the mutant AMV reverse transcriptase may have improved resistance to reaction inhibition by contaminants. Therefore, in one embodiment, the mutant AMV reverse transcriptase may be used for amplifying a target nucleic acid in the presence of contaminants. Examples of mutant AMV reverse transcriptases with improved resistance to reaction inhibition by contaminants include mutant AMV reverse transcriptases having one or more amino acid substitutions selected from the above-described (1), (2), (3), (4), (5), (6), (10), and (12). Preferably, the mutant AMV reverse transcriptase has all of the amino acid substitutions (1), (2), (3), (4), (5), (6), and (10), or a mutant AMV reverse transcriptase having an amino acid substitution (12) in addition to these amino acid substitutions. "In the presence of contaminants" refers to the same as described above. In one embodiment, amplification of a target nucleic acid may be performed in the presence of contaminants. Specifically, for example, amplification of a target nucleic acid may include a step of adding a substance containing a contaminant to a reaction solution. Examples of the substance containing a contaminant include purified saliva and purified urine. Thus, amplification of a target nucleic acid may include a step of adding purified saliva and purified urine to a reaction solution. Furthermore, amplification of a target nucleic acid may be performed to amplify a target nucleic acid in a substance containing a contaminant, for example, to amplify a target nucleic acid in purified saliva and purified urine. In one embodiment of the present disclosure, for example, a mutant AMV reverse transcriptase may be used for amplifying such a target nucleic acid. In other words, one embodiment of the present disclosure may relate to a mutant AMV reverse transcriptase for amplifying a target nucleic acid in a substance containing a contaminant, specifically, for example, a mutant AMV reverse transcriptase for amplifying a target nucleic acid in purified saliva and purified urine. [Example]

[0117] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to these examples.

[0118] Example 1 Introduction of a mutation into the avian myeloblastoma virus (AMV) reverse transcriptase gene (1) A polynucleotide encoding AMV-RT m4-2 (disclosed in JP 2014-209898 A, SEQ ID NO: 6), an AMV reverse transcriptase with improved thermostability compared to wild-type AMV reverse transcriptase, was synthesized by adding an oligonucleotide (SEQ ID NO: 25) encoding a histidine tag consisting of the amino acid sequence set forth in SEQ ID NO: 24 to the 5' end and a termination codon (TAA) to the 3' end of the polynucleotide (SEQ ID NO: 7). AMV-RT m4-2 (SEQ ID NO: 6) is a polypeptide with the amino acid sequence of the wild-type AMV reverse transcriptase (AMV-RT wild) β chain (SEQ ID NO: 2) that has the following amino acid substitutions in the amino acid sequence: S65G (this notation indicates that the amino acid residue corresponding to serine at position 65 in SEQ ID NO: 2 (or SEQ ID NO: 6) has been substituted with glycine; the same applies below), A583T, G626D, and A689V. (2) The synthesized polynucleotide was inserted between the restriction enzyme NcoI / KpnI cleavage sites of the plasmid pTrc99A to prepare a recombinant plasmid, which was then used to transform Escherichia coli JM109 strain (Takara Bio Inc.) to produce a transformant. (3) From the transformants prepared in (2), recombinant plasmids were extracted using a QIAprep Spin Miniprep Kit (Qiagen), and the AMV reverse transcriptase expression plasmids were then inserted. The plasmid pTrc99A-His-AMVRTm4 was obtained. (4) In the pTrc99A-His-AMVRTm4 obtained in (3), nucleotide substitutions were introduced at predetermined positions in the polynucleotide (SEQ ID NO: 7) encoding AMV-RT m4-2 (SEQ ID NO: 6). <1> from <5> The polynucleotide shown in was prepared; <1> A polynucleotide (SEQ ID NO: 9) in which the 1129th guanine (G) of SEQ ID NO: 7 is substituted with adenine (A). <2> A polynucleotide (SEQ ID NO: 11) in which thymine (T) at position 1427 of SEQ ID NO: 7 is substituted with adenine (A). <3> A polynucleotide (SEQ ID NO: 13) in which thymine (T) at position 1648 of SEQ ID NO: 7 is substituted with adenine (A). <4> a polynucleotide (SEQ ID NO: 15) in which the adenine (A) at position 2071 of SEQ ID NO: 7 is substituted with guanine (G); <5> A polynucleotide (SEQ ID NO: 17) in which the cytosine (C) at position 2328 of SEQ ID NO: 7 is substituted with guanine (G). In addition, <1> from <5> The nucleotide substitutions shown in are expressed as amino acid substitutions in the translated AMV reverse transcriptase: The aforementioned <1> is a substitution of valine (V) at position 377 of SEQ ID NO: 6 with isoleucine (I), <2> is a substitution of glutamine (Q) for leucine (L) at position 476 of SEQ ID NO: 6, <3> represents a substitution of serine (S) at position 550 of SEQ ID NO: 6 with threonine (T), The aforementioned <4> The substitution of lysine (K) at position 691 of SEQ ID NO: 6 with glutamic acid (E) results in the <5> and correspond to a substitution of asparagine (N) at position 776 of SEQ ID NO:6 with lysine (K), respectively. (5) The base sequences of the five polynucleotides prepared in (4) were analyzed using a capillary sequencer, and it was confirmed that there were no problems with any of them.

[0119] Example 2: Evaluation of AMV reverse transcriptase heat resistance (part 1) (1) An AMV reverse transcriptase-producing strain (transformant) was prepared by transforming Escherichia coli HB101 (Takara Bio Inc.) with the AMV reverse transcriptase expression plasmid containing pTrc99A-His-AMVRTm4 prepared in Example 1(3) or the polynucleotide set forth in SEQ ID NO: 9, 11, 13, 15, or 17 prepared in Example 1(4). (2) The AMV reverse transcriptase-producing strain prepared in (1) was inoculated into a 96-well deep-well plate containing 200 μL of 2xYT medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride) containing an appropriate amount of antibiotics, and pre-cultured overnight at 37°C and 1000 rpm with shaking. (3) A portion of the preculture medium from (2) was collected and centrifuged at 4°C and 3000 rpm for 30 minutes, and the supernatant was removed to recover the bacterial cells. The recovered bacterial cells were stored at -30°C. (4) The preculture solution from (2) was inoculated into a 96-well deep-well plate containing 475 μL of 2×PNa medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, 14.5 g / L sodium dihydrogen phosphate dihydrate, 2.5 g / L disodium hydrogen phosphate dodecahydrate) (pH 6.0) containing an appropriate amount of antibiotics at 20 μL / well, and then cultured with shaking at 37°C and 1000 rpm for 4.5 hours. (5) IPTG (isopropyl-β-thiogalactopyranoside) was added to each well to a final concentration of 5 mmol / L, and the cells were cultured at 25°C and 1000 rpm for an additional 3 days. (6) The culture medium from (5) was centrifuged at 4°C and 3000 rpm for 30 minutes, and the supernatant was discarded to collect the bacterial cells, which were then stored at -30°C. (7) 100 μL of extraction buffer containing BugBuster Reagent (Merck) was added to each of the cells collected in (6), and the mixture was shaken at 25°C and 1000 rpm for 1 hour. After centrifugation at 4°C and 3000 rpm for 30 minutes, the supernatant was collected to prepare a cell extract. did. (8) The bacterial cell extract from (7) was diluted 100-fold with TBS buffer (20 mmol / L Tris-HCl, 150 mmol / L sodium chloride) (pH 7.5) and dispensed into a 96-well PCR plate at 120 μL / well. The plate was heated at 55°C for 10 minutes using a thermal cycler, then cooled to 4°C and maintained there. (9) The amount of AMV reverse transcriptase contained in the extract of (7) (without heat treatment) and the amount of AMV reverse transcriptase contained in the extract of (8) (with heat treatment) were evaluated using the following enzyme-linked immunosorbent assay (ELISA). (9-1) The extract (with and without heat treatment) was dispensed into a 96-well microplate and incubated at 30°C for 1 hour to immobilize the proteins in the extract onto the microplate. (9-2) After immobilization, the plate was washed with TBS-T buffer (TBS buffer containing 0.05% (v / v) Tween (registered trademark) 20), and then blocked by adding 1% (w / v) bovine serum albumin (BSA) solution (Merck) and incubating at 30°C for 1 hour. (9-3) After blocking, the plate was washed with TBS-T buffer, and a mouse anti-AMV reverse transcriptase monoclonal antibody was added and incubated at 30°C for 1 hour to carry out a primary antibody reaction. (9-4) After the primary antibody reaction, wash with TBS-T buffer and A peroxidase-labeled anti-mouse IgG antibody (Bethyl Laboratories) was added and incubated at 30°C for 1 hour to carry out a secondary antibody reaction. (9-5) After the secondary antibody reaction, the mixture was washed with TBS-T buffer, and TMB Peroxidase Substrate (KPL) was added and incubated at room temperature for 5 minutes. (9-6) The reaction was stopped by adding an equal volume of 1 mol / L aqueous phosphoric acid solution, and the amount of AMV reverse transcriptase was evaluated by measuring the absorbance at 450 nm. (10) The amount of AMV reverse transcriptase in the heat-treated extract (absorbance at 450 nm in ELISA measurement) was divided by the amount of AMV reverse transcriptase in the corresponding extract without heat treatment to calculate the remaining enzyme amount.

[0120] The results are shown in Figure 1. In Figure 1, the remaining enzyme amount is expressed as a relative value to the remaining enzyme amount in AMV-RT m4-2 (relative remaining enzyme amount). AMV reverse transcriptase with the amino acid substitutions V377I (SEQ ID NO: 8), L476Q (SEQ ID NO: 10), S550T (SEQ ID NO: 12), K691E (SEQ ID NO: 14), or N776K (SEQ ID NO: 16) introduced into AMV-RT m4-2 all had higher remaining enzyme amounts than AMV-RT m4-2 (SEQ ID NO: 6). This indicates that introducing at least one of the amino acid substitutions V377I, L476Q, S550T, K691E, and N776K into AMV-RT m4-2 improves its thermostability compared to AMV-RT m4-2.

[0121] Example 3: Mass production of AMV reverse transcriptase (part 1) (1) A polynucleotide was synthesized by adding an initiation codon (ATG) to the 5' end and a termination codon (TAA) to the 3' end of a polynucleotide (SEQ ID NO: 23) encoding AMVp15 protease, which consists of the amino acid sequence from leucine 74 to leucine 204 of the amino acid sequence set forth in SEQ ID NO: 22 (GenBank No. AAB21262.1). (2) The polynucleotide synthesized in (1) was inserted downstream of the lac promoter of the pSTV28 plasmid vector, and the vector was then used to transform Escherichia coli JM109 strain (Takara Bio Inc.). (3) From the transformants obtained in (2), QIAprep Spin Miniprep The recombinant plasmid was extracted using p Kit (Qiagen) to prepare the protease expression plasmid pSTV_p15. (4) The Escherichia coli JM109 strain was transformed with pSTV_p15 prepared in (3). The transformant was then transformed with pTrc99A-His-AMVRTm4 prepared in Example 1(3) or the AMV reverse transcriptase expression plasmid containing the polynucleotide set forth in SEQ ID NO: 9, 11, 13, 15, or 17 prepared in Example 1(4), thereby producing protease-coexpressing AMV reverse transcriptase-producing Escherichia coli. (5) The AMV reverse transcriptase-producing E. coli prepared in (4) was inoculated into 20 mL of 2xYT medium containing an appropriate amount of antibiotics dispensed into a 100 mL baffled flask, and pre-cultured overnight at 37°C and 130 rpm with shaking. (6) The preculture solution from (5) was inoculated into 1 L of 2xYT medium containing an appropriate amount of antibiotics dispensed into a 5 L baffled flask, and cultured with shaking at 37°C and 100 rpm for 8 hours. (7) IPTG was added to a final concentration of 5 mmol / L, and the cells were cultured at 25°C and 100 rpm for an additional 3 days. (8) The culture medium (7) was centrifuged at 4°C and 8000 rpm for 20 minutes, and the supernatant was discarded to recover the wet bacterial cells. The recovered bacterial cells were stored at -30°C.

[0122] Example 4 Evaluation of AMV reverse transcriptase heat resistance (part 2) (1) 5 mL of extraction buffer containing BugBuster reagent (Merck) was added per 1 g of wet bacterial cells recovered in Example 3, and the mixture was stirred for 10 minutes while cooling on ice to prepare a bacterial cell extract. (2) The extract prepared in (1) was added to 1 mL of TALON (Cobalt) resin (Takara Bio Inc.), washed with 25 mL of buffer containing 10 mmol / L imidazole, and then eluted with 3 mL of buffer containing 200 mmol / L imidazole. (3) The eluate from (2) was desalted and concentrated using an Amicon Ultra ultrafiltration filter (Merck) to obtain a purified AMV reverse transcriptase solution, which was diluted to a concentration of 1 mg / mL. (4) The purified AMV reverse transcriptase solution obtained in (3) was heated at 54°C for 4 minutes using a thermal cycler, and then cooled to 4°C and maintained there. (5) Of the components of the Mycobacterium tuberculosis complex rRNA detection reagent TRCReady MTB (manufactured by Tosoh Corporation), only the AMV reverse transcriptase was replaced with the AMV reverse transcriptase (heat-treated) heat-treated in (4), and the positive standard RNA (RNA to be detected with a known concentration) included in the reagent was measured. Measurements were performed by monitoring changes in fluorescence intensity using the automated genetic testing device TRCReady-80 (manufactured by Tosoh Corporation), and the detection time was defined as the time when the fluorescence measurement value reached 1.2 times the initial fluorescence value.

[0123] The results are shown in Table 1. In Table 1, "detection time" refers to the relative value obtained by dividing the detection time using each heat-treated AMV reverse transcriptase by the detection time using heat-treated AMV-RT m4-2 (SEQ ID NO: 6). It can be seen that the AMV reverse transcriptases into which the amino acid substitutions V377I (SEQ ID NO: 8), L476Q (SEQ ID NO: 10), S550T (SEQ ID NO: 12), K691E (SEQ ID NO: 14), or N776K (SEQ ID NO: 16) were introduced into AMV-RT m4-2 all had shorter detection times than AMV-RT m4-2 (SEQ ID NO: 6). These results also demonstrate that introducing at least one of the amino acid substitutions V377I, L476Q, S550T, K691E, and N776K into AMV-RT m4-2 improves its thermal stability compared to AMV-RT m4-2.

[0124] [Table 1]

[0125] Example 5 Preparation of an AMV reverse transcriptase expression vector without a histidine tag (1) Polynucleotides were synthesized by adding a restriction enzyme EcoRI cleavage site (GAATTC) to the 5' end and a stop codon and a restriction enzyme KpnI cleavage site (GGTACC) to the 3' end of the polynucleotide (SEQ ID NO: 7) encoding AMV-RT m4-2 (SEQ ID NO: 6) prepared in Example 1(1), and the polynucleotide (SEQ ID NOs: 9, 11, 13, 15, and 17) encoding AMV reverse transcriptase consisting of any of the amino acid sequences of SEQ ID NOs: 8, 10, 12, 14, and 16 prepared in Example 1(4). (2) The synthesized polynucleotide was inserted between the restriction enzyme EcoRI / KpnI cleavage sites of the plasmid pTrc99A, and the vector was used to transform Escherichia coli JM109 strain (Takara Bio Inc.) to produce a transformant. (3) From the transformant prepared in (2), a recombinant plasmid was extracted using a QIAprep Spin Miniprep Kit (Qiagen) to prepare an AMV reverse transcriptase expression vector without a histidine tag.

[0126] Example 6. Amino acid substitution accumulation The amino acid substitutions that were found to be involved in improving the thermostability of AMV reverse transcriptase in Examples 2 and 4 were integrated into AMV-RT m4-2 (SEQ ID NO: 6) to further improve thermostability. Specifically, a polynucleotide was synthesized in which a nucleotide substitution corresponding to the amino acid substitution shown in (a) or (b) below was introduced into a predetermined position of the polynucleotide encoding AMV-RT m4-2 (sequence number 7), and then an AMV reverse transcriptase expression vector was prepared in the same manner as in Example 5. (a) V377I, S550T, and K691E (designated AMV-RT m7) (b) V377I, L476Q, S550T, K691E, and N776K (designated AMV-RT m9)

[0127] The amino acid sequence of AMV-RT m7 is shown in SEQ ID NO: 18, the nucleotide sequence of the polynucleotide encoding AMV-RT m7 is shown in SEQ ID NO: 19, the amino acid sequence of AMV-RT m9 is shown in SEQ ID NO: 20, and the nucleotide sequence of the polynucleotide encoding AMV-RT m9 is shown in SEQ ID NO: 21.

[0128] Example 7 Evaluation of the heat resistance of AMV reverse transcriptase (part 3) (1) The Escherichia coli W3110 strain was transformed according to standard methods with a vector capable of expressing the histidine-tagged AMV reverse transcriptase consisting of the amino acid sequence set forth in SEQ ID NO: 6 (AMV-RT m4-2), 8, 10, 12, 14, or 16 prepared in Example 5, or a vector capable of expressing SEQ ID NO: 18 (AMV-RT m7) or 20 (AMV-RT m9) prepared in Example 6. (2) Each of the resulting transformants was cultured overnight at 37°C on LB agar medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L purified agar) containing an appropriate amount of antibiotics to obtain strains producing each AMV reverse transcriptase. (3) Each of the production strains obtained in (2) was cultured in a fermenter and purified by column chromatography according to the method described in JP 2014-209898 A. The purified AMV reverse transcriptase was obtained by diluting the solution to a concentration of 1 mg / mL. (4) Each purified AMV reverse transcriptase obtained in (3) was heated at 58°C for 5 minutes using a thermal cycler, and then cooled to 4°C and maintained there. (5) RNA detection was carried out using each AMV reverse transcriptase in the same manner as in Example 4(5), and the detection time was determined.

[0129] The results are shown in Table 2. The introduction of any of the amino acid substitutions (V377I, L476Q, S550T, K691E, and N776K) found in Examples 2 and 4 to be involved in improving the thermostability of AMV reverse transcriptase shortened the detection time compared to AMV reverse transcriptase (AMV-RT m4-2) without these amino acid substitutions, demonstrating that the introduction of these amino acid substitutions improved thermostability. Furthermore, the AMV reverse transcriptase (SEQ ID NOs: 18 / 20) incorporating at least the V377I, S550T, and K691E amino acid substitutions further shortened the detection time compared to AMV reverse transcriptase (SEQ ID NOs: 8 / 10 / 12 / 14 / 16) incorporating only one amino acid substitution, demonstrating that the incorporation of these three amino acid substitutions (V377I, S550T, and K691E) further improved thermostability.

[0130] [Table 2]

[0131] Example 8: Evaluation of AMV reverse transcriptase productivity Each purified AMV reverse transcriptase obtained in Example 7(3) was subjected to size exclusion chromatography using a TSKgel UP-SW Aggregate column (manufactured by Tosoh Corporation), and the purification yield of AMV reverse transcriptase was quantified.

[0132] The results are shown in Figure 2. In Figure 2, the purification yield of each mutant is expressed as a relative value, with the purification yield of AMV-RT m4-2 set to 1. The AMV reverse transcriptases (SEQ ID NO: 10 / 20) into which the L476Q amino acid substitution was introduced all had increased purification yields compared to AMV-RT m4-2 (SEQ ID NO: 6) without this substitution. These results demonstrate that the L476Q amino acid substitution improves both thermostability and productivity.

[0133] Example 9 Introduction of mutations into the avian myeloblastoma virus (AMV) reverse transcriptase gene (part 2) (1) In pTrc99A-His-AMVRTm4 obtained in Example 1(3), nucleotide substitutions were introduced into the polynucleotide (SEQ ID NO: 7) encoding AMV-RT m4-2 (SEQ ID NO: 6) at predetermined positions. <6> from <9> The polynucleotide shown in was prepared; <6> A polynucleotide (SEQ ID NO: 27) in which the 994th adenine (A) of SEQ ID NO: 7 is substituted with guanine (G) <7> A polynucleotide (SEQ ID NO: 29) in which the 1420th guanine (G) of SEQ ID NO: 7 is substituted with adenine (A). <8> A polynucleotide (SEQ ID NO: 31) in which the 2147th cytosine (C) of SEQ ID NO: 7 is substituted with guanine (G) <9> A polynucleotide in which the adenine (A) at position 2389 of SEQ ID NO: 7 is substituted with cytosine (C), the adenine (A) at position 2390 with guanine (G), the adenine (A) at position 2391 with thymine (T), the adenine (A) at position 2548 with cytosine (C), the adenine (A) at position 2549 with guanine (G), and the guanine (G) at position 2550 with thymine (T) (SEQ ID NO: 33). In addition, <6> from <9> The nucleotide substitutions shown in are expressed as amino acid substitutions in the translated AMV reverse transcriptase: The aforementioned <6> isoleucine (I) at position 332 of SEQ ID NO: 6 is substituted with valine (V), <7> is a substitution of alanine (A) at position 474 of SEQ ID NO: 6 with threonine (T), <8> represents a substitution of threonine (T) at position 716 of SEQ ID NO: 6 with serine (S), The aforementioned <9> represents a substitution of lysine (K) at position 797 with arginine (R) and a substitution of lysine (K) at position 850 with arginine (R) in SEQ ID NO: 6, Each corresponds to. (2) The base sequences of the four polynucleotides prepared in (1) were analyzed using a capillary sequencer, and it was confirmed that there were no problems with any of them.

[0134] Example 10: Evaluation of the heat resistance of AMV reverse transcriptase (part 4) (1) An AMV reverse transcriptase-producing strain (transformant) was prepared by transforming Escherichia coli HB101 (Takara Bio Inc.) with the AMV reverse transcriptase expression plasmid containing pTrc99A-His-AMVRTm4 prepared in Example 1(3) or the polynucleotide set forth in SEQ ID NO: 27, 29, 31, or 33 prepared in Example 9(1). (2) The AMV reverse transcriptase-producing strain prepared in (1) was cultured in the same manner as in Example 2(2) to (10), and the residual AMV reverse transcriptase activity in the extract was evaluated by ELISA. The results are shown in Figure 3. In Figure 3, the remaining enzyme amount is expressed as a relative value to the remaining enzyme amount in AMV-RT m4-2 (relative remaining enzyme amount). AMV reverse transcriptase in which the amino acid substitutions I332V (SEQ ID NO: 26), A474T (SEQ ID NO: 28), T716S (SEQ ID NO: 30), or K797R and K850R (SEQ ID NO: 32) were introduced into AMV-RT m4-2 all had higher remaining enzyme amounts than AMV-RT m4-2 (SEQ ID NO: 6). This indicates that introducing at least one of the amino acid substitutions I332V, A474T, T716S, K797R, and K850R into AMV-RT m4-2 improves its thermostability compared to AMV-RT m4-2.

[0135] Example 11 Evaluation of heat resistance of AMV reverse transcriptase (part 5) (1) Among the AMV reverse transcriptase-producing strains prepared in Example 10(1), a transformant (producing AMV reverse transcriptase with the I332V amino acid substitution shown in SEQ ID NO: 26) prepared using an AMV reverse transcriptase expression plasmid containing the polynucleotide shown in SEQ ID NO: 27 was selected, and the transformant was inoculated into 3 mL of 2xYT medium containing an appropriate amount of antibiotic, followed by shaking overnight at 37°C and 160 rpm for pre-culture. (2) The preculture solution from (1) was inoculated into 100 mL of SOC medium (20 g / L tryptone, 5 g / L yeast extract, 0.5 g / L sodium chloride, 0.186 g / L potassium chloride, 3.6 g / L glucose, 2.46 g / L magnesium sulfate heptahydrate, 2.03 g / L magnesium chloride hexahydrate) containing an appropriate amount of antibiotics, which had been dispensed into a 500 mL baffled flask, and the mixture was cultured at 37°C and 130 rpm for 4 hours with shaking. (3) IPTG was added to a final concentration of 0.5 mmol / L, and the cells were cultured at 25°C and 130 rpm for an additional 3 days. (4) The culture medium from (3) was centrifuged at 10,000 rpm at 4°C for 20 minutes, and the supernatant was discarded to recover the wet bacterial cells. The recovered bacterial cells were stored at -30°C. (5) The wet cells recovered in (4) were added to 50 mmol / L Tris-HCl buffer (pH 7.2) containing 150 mmol / L sodium chloride, 2.4 mmol / L magnesium sulfate, 3000 Unit / L Benzonase (Merck), 0.006% (w / v) lysozyme, 2 mmol / L Phenylmethylsulfonyl fluoride, and 0.6% (w / v) Triton X-100 (trade name), stirred at room temperature for 1 hour, and then centrifuged at 4°C and 15,000 rpm for 60 minutes to express the nucleotides. An extract containing AMV reverse transcriptase was obtained. (6) The extract prepared in (5) was applied to TALON (Cobalt) resin (Takara Bio The column was washed with 8 mL of a buffer containing 10 mmol / L imidazole, and then eluted with 0.8 mL of a buffer containing 200 mmol / L imidazole. (7) The purified AMV reverse transcriptase solution obtained in (6) was heated at 52°C or 53°C for 5 minutes, and then cooled to 4°C and maintained there. (8) Of the components of the Mycobacterium tuberculosis complex rRNA detection reagent TRCReady MTB (manufactured by Tosoh Corporation), only the AMV reverse transcriptase was replaced with the AMV reverse transcriptase (heat-treated) heat-treated in (7), and the positive standard RNA (RNA to be detected with a known concentration) included in the reagent was measured. Measurements were performed by monitoring changes in fluorescence intensity using the automated genetic testing device TRCReady-80 (manufactured by Tosoh Corporation), and the detection time was defined as the time when the measured fluorescence value reached 1.2 times the initial fluorescence value. The results are shown in Table 3. In Table 3, "detection time" refers to the relative value obtained by dividing the detection time using each heat-treated AMV reverse transcriptase by the detection time using heat-treated AMV-RT m4-2 (SEQ ID NO: 6). It can be seen that the AMV reverse transcriptase in which the amino acid substitution I332V (SEQ ID NO: 26) was introduced into AMV-RT m4-2 had a shorter detection time than AMV-RT m4-2 (SEQ ID NO: 6). These results demonstrate that introducing the amino acid mutation I332V into AMV-RT m4-2 improves its thermal stability compared to AMV-RT m4-2.

[0136] [Table 3]

[0137] Example 12: Introduction of mutations into the avian myeloblastoma virus (AMV) reverse transcriptase gene (part 3) (1) A polynucleotide was synthesized in which an oligonucleotide (SEQ ID NO: 25) encoding a histidine tag consisting of the amino acid sequence set forth in SEQ ID NO: 24 was added to the 5' end of a polynucleotide (SEQ ID NO: 21) encoding AMV-RT m9 (SEQ ID NO: 20), and a stop codon (TAA) was added to the 3' end. (2) The synthesized polynucleotide was inserted between the restriction enzyme NcoI / KpnI cleavage sites of the plasmid pTrc99A to prepare a recombinant plasmid, which was then used to transform Escherichia coli JM109 strain (Takara Bio Inc.) to produce a transformant. (3) A recombinant plasmid was extracted from the transformant prepared in (2) using a QIAprep Spin Miniprep Kit (Qiagen) to obtain the AMV reverse transcriptase expression plasmid pTrc99A-His-AMVRTm9. (4) In the pTrc99A-His-AMVRTm9 obtained in (3), nucleotide substitutions were introduced into the polynucleotide (SEQ ID NO: 21) encoding AMV-RT m9 (SEQ ID NO: 20) at predetermined positions. <10> from <12> Polynuclear shown Reotide was prepared; <10> A polynucleotide (SEQ ID NO: 35) in which the 181st adenine (A) of SEQ ID NO: 21 is substituted with guanine (G) <11> A polynucleotide (SEQ ID NO: 37) in which thymine (T) at position 314 of SEQ ID NO: 21 is replaced with cytosine (C) and adenine (A) at position 2548 is replaced with guanine (G). <12> A polynucleotide in which the adenine (A) at position 1867 of SEQ ID NO: 21 is substituted with guanine (G), the adenine (A) at position 2074 is substituted with guanine (G), the guanine (G) at position 2150 is substituted with adenine (A), and the thymine (T) at position 2567 is substituted with cytosine (C) (SEQ ID NO: 39). In addition, <10> from <12> The nucleotide substitutions shown in are expressed as amino acid substitutions in the translated AMV reverse transcriptase: The aforementioned <10> isoleucine (I) at position 61 of SEQ ID NO: 20 is replaced with valine (V), The aforementioned <11> represents a substitution of valine (V) at position 105 of SEQ ID NO: 20 with alanine (A) and a substitution of lysine (K) at position 850 with glutamic acid (E), The aforementioned <12> correspond to a substitution of asparagine (N) at position 623 with aspartic acid (D), a substitution of threonine (T) at position 692 with alanine (A), a substitution of glycine (G) at position 717 with aspartic acid (D), and a substitution of leucine (L) at position 856 with proline (P) in SEQ ID NO: 20, respectively. <10> The AMV reverse transcriptase having the amino acid substitutions is AMV-RT m10, <11> The AMV reverse transcriptase having the amino acid substitutions is AMV-RT m11, <12> The AMV reverse transcriptase having the above amino acid substitutions was designated AMV-RT m13. (5) The base sequences of the three polynucleotides prepared in (4) were analyzed using a capillary sequencer, and it was confirmed that there were no problems with any of them.

[0138] Example 13 Evaluation of heat resistance of AMV reverse transcriptase (part 6) (1) Escherichia coli HB101 strain (Takara Bio Inc.) was transformed with pTrc99A-His-AMVRTm9 prepared in Example 12(3) or an AMV reverse transcriptase expression plasmid containing the polynucleotide set forth in SEQ ID NO: 35, 37, or 39 prepared in Example 12(4) to prepare an AMV reverse transcriptase-producing strain (transformant). (2) The AMV reverse transcriptase-producing E. coli prepared in (1) was cultured in the same manner as in Example 11 (1) to (4), and wet cells were collected. The collected cells were stored at -30°C. (3) AMV reverse transcriptase was prepared in the same manner as in Example 13(5) to (6). (4) The purified AMV reverse transcriptase solution obtained in (3) was heated at 50°C or 52°C for 5 minutes using a thermal cycler, and then kept at 4°C. (5) 20 μL of the reverse transcription reaction solution shown in Table 4 was prepared in a PCR tube and reacted at 46°C for 30 minutes. The standard RNA used was a sequence optimized for Escherichia coli codons from the sequence of carbonic anhydrase derived from sulfate-reducing bacteria shown in SEQ ID NO: 40. The primer was designed to bind complementarily to the 3' end of the standard RNA as shown in SEQ ID NO: 41. (6) 20 μL of the PCR reaction solution shown in Table 5 was prepared in a PCR tube, and after incubation at 95°C for 2 minutes, 20 thermal cycles of 95°C for 30 seconds, 65°C for 30 seconds, and 72°C for 1 minute were repeated. The primer pair shown in SEQ ID NO: 42 and SEQ ID NO: 43 was used as the PCR primer pair. (7) The resulting amplification products were separated by electrophoresis using a 1.0% agarose gel, stained with SYBR Gold nucleic acid gel stain (Thermo Fisher Scientific), and photographed with a Printgraph CMOS I (Atto Corporation). The intensity of each band was quantitatively analyzed using image analysis software ImageQuant TL (Cytiva). The residual activity was calculated by dividing the band intensity obtained using each AMV reverse transcriptase after heat treatment by the band intensity obtained using each AMV reverse transcriptase before heat treatment.

[0139] [Table 4]

[0140] [Table 5]

[0141] The results for heat treatment at 50°C are shown in Figure 4, and the results for heat treatment at 52°C are shown in Figure 5. In Figures 4 and 5, the residual activity is expressed as a relative value to the residual activity of AMV-RT m9 (relative residual activity value). AMV reverse transcriptase (AMV-RT m10) in which the amino acid substitution of I61V (SEQ ID NO: 34) was introduced into AMV-RT m9, and AMV reverse transcriptase (AMV-RT m10) in which the amino acid substitutions of V105A and K850E (SEQ ID NO: 36) were introduced into AMV-RT m9 were also shown. The AMV reverse transcriptase (AMV-RT m13) into which the amino acid substitutions I61V, V105A, K850E, N623D, T692A, G717D, and L856P (SEQ ID NO: 38) were introduced showed significantly increased residual activity after heat treatment compared to AMV-RT m9 (SEQ ID NO: 20) without any of the above substitutions. These results suggest that introducing any of the amino acid substitutions I61V, V105A, K850E, N623D, T692A, G717D, and L856P into AMV-RT m9 is effective in reducing the residual activity of the AMV reverse transcriptase. This indicates that the thermal stability is improved compared to AMV-RT m9.

[0142] Example 14 Preparation of AMV reverse transcriptase expression vector without histidine tag (part 2) Among the AMV reverse transcriptase mutants whose improved thermostability was confirmed in Example 13, AMV-RT m11 was selected to prepare an AMV reverse transcriptase expression vector without a histidine tag. Specifically, the polynucleotide (SEQ ID NO: 37) encoding AMV-RT m11 (SEQ ID NO: 36) prepared in Example 12(4) was inserted into the plasmid pTrc99A in the same manner as in Example 5 to prepare an AMV reverse transcriptase expression vector without a histidine tag.

[0143] Example 15: Accumulation of Amino Acid Substitutions (Part 2) The I332V amino acid substitution was selected from the amino acid substitutions found to be involved in improving thermal stability in Examples 10 and 11, and was integrated into AMV-RT m11 (sequence number 36) without a histidine tag, prepared in Example 14 (designated AMV-RT m12). Specifically, a polynucleotide was synthesized in which a nucleotide substitution corresponding to the amino acid substitution of I332V was introduced into a predetermined position of the polynucleotide encoding AMV-RT m11 (sequence number 37), and then an AMV reverse transcriptase expression vector was prepared in the same manner as in Example 5. The amino acid sequence of AMV-RT m12 is shown in SEQ ID NO: 44, and the nucleotide sequence of the polynucleotide encoding AMV-RT m12 is shown in SEQ ID NO: 45.

[0144] Example 16: Evaluation of heat resistance of AMV reverse transcriptase (part 7) (1) Escherichia coli W3110 strain was transformed according to a standard method with a vector capable of expressing the histidine-tagged AMV reverse transcriptase consisting of the amino acid sequence set forth in SEQ ID NO: 36 (AMV-RT m11) prepared in Example 14, or a vector capable of expressing the histidine-tagged AMV reverse transcriptase consisting of the amino acid sequence set forth in SEQ ID NO: 44 (AMV-RT m12) prepared in Example 15. (2) Using each of the obtained transformants, cultivation in a fermenter and purification by column chromatography were carried out in the same manner as in Example 7(2) and (3), to obtain purified AMV reverse transcriptase. (3) The concentration of the purified AMV enzyme was quantified by size exclusion chromatography using a TSKgel UP-SW Aggregate column (Tosoh Corporation). (4) Composition of the Mycobacterium tuberculosis complex rRNA detection reagent TRCReady MTB (manufactured by Tosoh Corporation) In these experiments, the AMV reverse transcriptase was replaced with the purified AMV reverse transcriptase (AMV-RT m11 or AMV-RT m12) obtained in (2) or the purified AMV reverse transcriptase (AMV-RT m4-2) obtained in Example 7(3), and the T7 RNA polymerase was replaced with the thermostable T7 RNA polymerase consisting of the amino acid sequence set forth in SEQ ID NO: 46. A positive control RNA (target RNA with a known concentration) included in the reagent was measured at various reaction temperatures. Measurements were performed by monitoring changes in fluorescence intensity using an automated genetic testing system, TRCReady-80 (Tosoh Corporation). The detection time was defined as the time when the measured fluorescence value reached 1.2 times the initial fluorescence value.

[0145] The results are shown in Table 6. When AMV-RT m4-2 (SEQ ID NO: 6) was used, the detection time was delayed to 6.9 minutes when the reaction temperature was raised to 53°C, and when it was raised to 54°C, the enzyme was thermally inactivated and no detection was possible. On the other hand, when an AMV reverse transcriptase (AMV-RT m11, SEQ ID NO: 36) in which the amino acid substitutions V105A, V377I, L476Q, S550T, K691E, N776K, and K850E had been introduced into AMV-RT m4-2, or an AMV reverse transcriptase (AMV-RT m12, SEQ ID NO: 44) in which the amino acid substitution I332V had been introduced was used, the detection time was maintained at approximately 4 minutes even when the reaction temperature was raised to 53°C, and even when it was raised to 54°C, the positive label remained. These results indicate that introducing one or more amino acid substitutions selected from V105A, V377I, L476Q, S550T, K691E, N776K, K850E, and I332V into AMV-RT m4-2 improves thermostability compared to AMV-RT m4-2, enabling amplification of target nucleic acids at higher temperatures.

[0146] [Table 6]

[0147] Example 17 Evaluation of AMV reverse transcriptase resistance to inhibition (1) A purified saliva product was prepared according to the following procedure. (1-1) Saliva from a healthy subject was suspended in a four-fold volume of PBS buffer solution (Nacalai Tesque) and centrifuged at 10,000×G for 1 minute. (1-2) 100 μL of the centrifuged supernatant of (1-1) was added to the denaturing reagent of a TRCR nucleic acid purification kit (manufactured by Tosoh Corporation), and purified according to the standard method of the purification kit to prepare a purified saliva product. (2) A purified urine product was prepared according to the following procedure. (2-1) 500 μL of urine from a healthy subject was added to the denaturing reagent of a TRCR nucleic acid purification kit (manufactured by Tosoh Corporation), and the mixture was centrifuged at 10,000×G for 3 minutes. (2-2) The entire volume of the centrifuged supernatant of (2-1) was transferred to a TRCR transfer tube (manufactured by Tosoh Corporation) and purified according to the standard method of the purification kit to prepare a purified urine product. (3) The positive standard RNA (RNA to be detected with a known concentration) included in the Mycobacterium tuberculosis complex rRNA detection reagent TRCReady MTB (manufactured by Tosoh Corporation) was added to the purified saliva prepared in (1) or the purified urine prepared in (2) to prepare measurement samples containing various impurities. (4) Among the components of the TRCReady MTB reagent, the AMV reverse transcriptase was replaced with the purified AMV reverse transcriptase (AMV-RT m11 or AMV-RT m12) obtained in Example 16(2) or the purified AMV reverse transcriptase (AMV-RT m4-2) obtained in Example 7(3), and the T7 RNA polymerase was replaced with the heat-stable T7 RNA polymerase consisting of the amino acid sequence set forth in SEQ ID NO: 46. The measurement samples containing various impurities prepared in (3) were measured at a reaction temperature of 51°C.

[0148] The results are shown in Table 7. When AMV-RT m4-2 (SEQ ID NO: 6) was used, the reaction was inhibited in measurement samples containing various contaminants (saliva purified products or urine purified products), and the detection time was significantly delayed. On the other hand, when using AMV reverse transcriptase (AMV-RT m11, SEQ ID NO: 36) in which the amino acid substitutions V105A, V377I, L476Q, S550T, K691E, N776K, and K850E were introduced into AMV-RT m4-2, or AMV reverse transcriptase (AMV-RT m12, SEQ ID NO: 44) in which the amino acid substitution I332V was further introduced, detection was possible in a shorter time than when using AMV-RT m4-2. These results indicate that performing amplification reactions of target nucleic acids using AMV reverse transcriptase mutants in which one or more amino acid substitutions selected from V105A, V377I, L476Q, S550T, K691E, N776K, K850E, and I332V have been introduced into AMV-RT m4-2, for example under high-temperature conditions, improves detection performance in the presence of various contaminants, and is useful for infectious disease testing of various clinical samples using nucleic acid amplification methods.

[0149] [Table 7] [Industrial Applicability]

[0150] The modified avian myeloblastoma virus (AMV) reverse transcriptase of the present disclosure has improved thermostability and / or resistance to reaction inhibition by contaminants compared to conventional AMV reverse transcriptases. Therefore, by replacing the AMV reverse transcriptase contained in a target nucleic acid amplification reagent with the enzyme of the present disclosure, the performance of the amplification reagent is expected to improve.

Claims

1. An avian myeloblastoma virus (AMV) reverse transcriptase selected from any of the following (i) to (iii): (i) AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which contains one or more amino acid substitutions selected from the following (1) to (15): (1) The amino acid residue corresponding to the leucine residue at position 476 of SEQ ID NO: 6 is substituted with a glutamine residue (2) The amino acid residue corresponding to the 377th valine residue in SEQ ID NO: 6 is substituted with an isoleucine residue (3) The amino acid residue corresponding to the serine residue at position 550 of SEQ ID NO: 6 is substituted with a threonine residue (4) The amino acid residue corresponding to the lysine residue at position 691 of SEQ ID NO: 6 is substituted with a glutamic acid residue (5) The amino acid residue corresponding to the asparagine residue at position 776 of SEQ ID NO: 6 is substituted with a lysine residue. (6) The amino acid residue corresponding to the isoleucine residue at position 332 of SEQ ID NO: 6 is substituted with a valine residue (7) The amino acid residue corresponding to the 474th alanine residue in SEQ ID NO: 6 is substituted with a threonine residue (8) The amino acid residue corresponding to the 716th threonine residue of SEQ ID NO: 6 is substituted with a serine residue (9) The amino acid residue corresponding to the lysine residue at position 797 of SEQ ID NO: 6 is substituted with an arginine residue. (10) The amino acid residue corresponding to the lysine residue at position 850 of SEQ ID NO: 6 is substituted with an arginine residue or a glutamic acid residue. (11) The amino acid residue corresponding to the 61st isoleucine residue of SEQ ID NO: 6 is substituted with a valine residue (12) The amino acid residue corresponding to the 105th valine residue of SEQ ID NO: 6 is substituted with an alanine residue (13) The amino acid residue corresponding to the asparagine residue at position 623 of SEQ ID NO: 6 is substituted with an aspartic acid residue (14) The amino acid residue corresponding to the 692nd threonine residue of SEQ ID NO: 6 is substituted with an alanine residue (15) the amino acid residue corresponding to the glycine residue at position 717 of SEQ ID NO: 6 is replaced with aspartic acid; (ii) an AMV reverse transcriptase having an amino acid sequence set forth in SEQ ID NO: 6, which contains one or more amino acid substitutions selected from (1) to (15), and which further contains any one or more of substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions shown in (1) to (15), and which has enzymatic activity; (iii) AMV reverse transcriptase, which is an amino acid sequence set forth in SEQ ID NO: 6, having 70% or more identity to the entire amino acid sequence containing one or more amino acid substitutions selected from (1) to (15), with the proviso that the amino acid substitutions are maintained, and which has enzymatic activity.

2. The AMV reverse transcriptase according to claim 1, wherein the one or more amino acid substitutions selected from (1) to (15) include at least the amino acid substitution of (1).

3. The one or more amino acid substitutions selected from (1) to (15) above are at least (2) to The AMV reverse transcriptase of claim 1, comprising the amino acid substitution (4).

4. The AMV reverse transcriptase according to claim 1, wherein the one or more amino acid substitutions selected from (1) to (15) include at least an amino acid substitution selected from (1) to (5) and one or more amino acid substitutions selected from (11) to (15).

5. The AMV reverse transcriptase of claim 1, selected from any of the following (iv) to (vi): (iv) an AMV reverse transcriptase having an amino acid sequence set forth in any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 38, and 44; (v) an AMV reverse transcriptase having an amino acid sequence set forth in any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 38, and 44, which contains any one or more of substitution, deletion, insertion, and addition of one or several amino acid residues at one or several positions, and which has enzymatic activity; (vi) An AMV reverse transcriptase having an amino acid sequence that is 70% or more identical to the amino acid sequence set forth in any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36, 38, and 44, and having enzymatic activity.

6. A polynucleotide encoding the AMV reverse transcriptase of any one of claims 1 to 5.

7. An expression vector comprising the polynucleotide of claim 6.

8. A transformant obtained by transforming a host with the expression vector according to claim 7.

9. The transformant according to claim 8, wherein the host is Escherichia coli.

10. A method for producing AMV reverse transcriptase, comprising the steps of: culturing the transformant described in claim 8 to express AMV reverse transcriptase; and recovering the expressed reverse transcriptase from the resulting culture.

11. A reagent for amplifying a target nucleic acid, comprising the AMV reverse transcriptase according to any one of claims 1 to 5.

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