Modified-avian myeloblastoma virus reverse transcriptase

Abstract

Provided is an avian myeloblastoma virus (AMV) reverse transcriptase selected from any of (I) to (III) below. (I) an AMV reverse transcriptase having an amino acid sequence described by SEQ ID NO: 6 and containing the amino acid substitution described in (1); (1) a substitution where an amino acid residue corresponding to the leucine residue at position 476 of SEQ ID NO: 6 is substituted with a glutamine residue. (II) An AMV reverse transcriptase exhibiting enzymatic activity and having an amino acid sequence that includes any one or more of a substitution, a deletion, an insertion, and an addition of one or several amino acid residues at one or several positions other than the amino acid substitution indicated in (1) above, the amino acid sequence being amino acids described by SEQ ID NO: 6 and including the amino acid substitution described in (1) above. (III) An AMV reverse transcriptase exhibiting enzymatic activity and having an amino acid sequence described by SEQ ID NO: 6, the amino acid sequence being 70% or more identical to the entire amino acid sequence including the amino acid substitution described in (1) and said amino acid substitution being kept.

Description

Modified avian myeloblastoma virus reverse transcriptase

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

[0002] AMV reverse transcriptase, a type of reverse transcriptase, is used as a genetic engineering reagent and a genetic diagnostic reagent 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 an α chain alone (α form) or a 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 by using a recombinant Escherichia coli into which only a gene encoding the β chain of AMV reverse transcriptase has been introduced, and by degrading the α chain from the β chain expressed in the recombinant Escherichia coli.

[0007] JP 2003-334095 A JP 2013-165669 A JP 2014-209898 A JP 2013-126402 A

[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.

[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 one 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 the amino acid substitution set forth in (1) below; (1) the amino acid residue corresponding to the leucine residue at position 476 of SEQ ID NO: 6 is substituted with a glutamine residue; (II) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution set forth in (1), and which further contains one or more substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions in addition to the amino acid substitution set forth in (1), and which has enzymatic activity; (III) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which has an amino acid sequence that is 70% or more identical to the entire amino acid sequence containing the amino acid substitution set forth in (1), provided that the amino acid substitution is maintained, and which has enzymatic activity. [2] The AMV reverse transcriptase of [1], selected from any one 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 the amino acid substitution of (1) above, and further contains one or more amino acid substitutions selected from the following (2) to (16);(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 550th serine residue in SEQ ID NO:6 is substituted with a threonine residue. (4) The amino acid residue corresponding to the 691st lysine residue in SEQ ID NO:6 is substituted with a glutamic acid residue. (5) The amino acid residue corresponding to the 776th asparagine residue in SEQ ID NO:6 is substituted with a lysine residue. (6) The amino acid residue corresponding to the 332nd isoleucine residue in 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 in SEQ ID NO:6 is substituted with a serine residue. (9) The amino acid residue corresponding to the 797th lysine residue in SEQ ID NO:6 is substituted with an arginine residue. (10) The amino acid residue corresponding to the 850th lysine residue in SEQ ID NO:6 is substituted with an arginine residue. (11) The amino acid residue corresponding to the 61st isoleucine residue in SEQ ID NO:6 is substituted with a valine residue. (12) an amino acid residue corresponding to the valine residue at position 105 of SEQ ID NO: 6 is substituted with an alanine residue; (13) an amino acid residue corresponding to the asparagine residue at position 623 of SEQ ID NO: 6 is substituted with an aspartic acid residue; (14) an amino acid residue corresponding to the threonine residue at position 692 of SEQ ID NO: 6 is substituted with an alanine residue; (15) an amino acid residue corresponding to the glycine residue at position 717 of SEQ ID NO: 6 is substituted with an aspartic acid residue; (16) an amino acid residue corresponding to the lysine residue at position 850 of SEQ ID NO: 6 is substituted with a glutamic acid residue; (ii) an AMV reverse transcriptase having an amino acid sequence set forth in SEQ ID NO: 6, which is an amino acid sequence containing the amino acid substitution of (1) above, and further containing one or more amino acid substitutions selected from (2) to (16), and further containing any one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions in addition to the amino acid substitutions shown in (1) to (16), and having enzymatic activity;(iii) An AMV reverse transcriptase having an amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution of (1) and which further contains one or more amino acid substitutions selected from (2) to (16), and which has an identity of 70% or more to the entire amino acid sequence in which the amino acid substitutions are maintained, and which has enzymatic activity. [3] The AMV reverse transcriptase of [2], wherein the one or more amino acid substitutions selected from (2) to (16) include at least the amino acid substitutions of (2) to (5). [4] The AMV reverse transcriptase of [2], wherein the one or more amino acid substitutions selected from (2) to (16) include at least the amino acid substitutions of (2) to (5) and one or more amino acid substitutions selected from (6), (11) to (16). [5] An AMV reverse transcriptase of [1] selected from any of the following (IV) to (VI): (IV) an AMV reverse transcriptase having the amino acid sequence set forth in any of SEQ ID NOs: 10, 20, 34, 36, 38, and 44; (V) an AMV reverse transcriptase having the amino acid sequence set forth in any of SEQ ID NOs: 10, 20, 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 having 70% or more identity to the amino acid sequence set forth in any of SEQ ID NOs: 10, 20, 34, 36, 38, and 44, and which has enzymatic activity. [6] A polynucleotide encoding the AMV reverse transcriptase of any of [1] to [5]. [7] An expression vector comprising the polynucleotide of [6]. [8] A transformant obtained by transforming a host with the expression vector of [7]. [9] The transformant of [8], wherein the host is Escherichia coli.

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

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

[0011] 1 shows the results of comparing the thermal stability of the amino acid substituted mutants prepared in Example 1 with AMV-RT m4-2 (SEQ ID NO: 6). In this figure, the amount of residual enzyme is shown as a relative value, with the amount of residual enzyme in AMV-RT m4-2 set to 1. This figure shows the results of comparing the purification yield 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 yield is shown as a relative value, with the amount of purification in AMV-RT m4-2 set to 1. This figure shows the results of comparing the thermal stability of the amino acid substituted mutants prepared in Example 9 with AMV-RT m9 (SEQ ID NO: 20). In this figure, the amount of residual enzyme is shown as a relative value, with the amount of residual enzyme in AMV-RT m4-2 set to 1. This figure shows the results of comparing the thermal stability of the amino acid substituted mutants 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. This figure shows the results of comparing the thermal stability of the amino acid substitution mutants 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.

[0012] <1> AMV Reverse Transcriptase The present disclosure provides an avian myeloblastoma virus (AMV) reverse transcriptase having the "specific mutations" described herein. More specifically, the present disclosure provides an avian myeloblastoma virus (AMV) reverse transcriptase selected from the following (I) to (III): (I) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution set forth in (1) below; (1) the amino acid residue corresponding to the leucine residue at position 476 of SEQ ID NO: 6 is substituted with a glutamine residue; (II) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution set forth in (1), and which further contains any one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions in addition to the amino acid substitution set forth in (1), and which has enzymatic activity; (III) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which has an amino acid sequence that is 70% or more identical to the entire amino acid sequence containing the amino acid substitution set forth in (1), with the proviso that the amino acid substitution is maintained, and which has enzymatic activity.

[0013] The present invention may further provide an AMV reverse transcriptase selected from any one of the following (i) to (iii): (i) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which comprises the amino acid substitution of (1) above, and further comprises one or more amino acid substitutions selected from the following (2) to (16); (2) an amino acid residue corresponding to the valine residue at position 377 of SEQ ID NO: 6 is substituted with an isoleucine residue; (3) an amino acid residue corresponding to the serine residue at position 550 of SEQ ID NO: 6 is substituted with a threonine residue; (4) an amino acid residue corresponding to the lysine residue at position 691 of SEQ ID NO: 6 is substituted with a glutamic acid residue; (5) an amino acid residue corresponding to the asparagine residue at position 776 of SEQ ID NO: 6 is substituted with a lysine residue; (6) an amino acid residue corresponding to the isoleucine residue at position 332 of SEQ ID NO: 6 is substituted with a valine residue; (7) an amino acid residue corresponding to the alanine residue at position 474 of SEQ ID NO: 6 is substituted with a threonine residue; (8) an amino acid residue corresponding to the threonine residue at position 716 of SEQ ID NO: 6 is substituted with a serine residue (9) The amino acid residue corresponding to the lysine residue at position 797 in SEQ ID NO:6 is replaced with an arginine residue. (10) The amino acid residue corresponding to the lysine residue at position 850 in SEQ ID NO:6 is replaced with an arginine residue. (11) The amino acid residue corresponding to the isoleucine residue at position 61 in SEQ ID NO:6 is replaced with a valine residue. (12) The amino acid residue corresponding to the valine residue at position 105 in SEQ ID NO:6 is replaced with an alanine residue. (13) The amino acid residue corresponding to the asparagine residue at position 623 in SEQ ID NO:6 is replaced with an aspartic acid residue. (14) The amino acid residue corresponding to the threonine residue at position 692 in SEQ ID NO:6 is replaced with an alanine residue. (15) The amino acid residue corresponding to the glycine residue at position 717 in SEQ ID NO:6 is replaced with an aspartic acid residue. (16) The amino acid residue corresponding to the lysine residue at position 850 in SEQ ID NO:6 is replaced with a glutamic acid residue.(ii) AMV reverse transcriptase having an amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution of (1) above, and further contains one or more amino acid substitutions selected from (2) to (16), 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 set forth in (1) to (16), and which has enzymatic activity; (iii) AMV reverse transcriptase having an amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution of (1), and further contains one or more amino acid substitutions selected from (2) to (16), and which has an identity of 70% or more to the entire amino acid sequence, in which the amino acid substitutions are maintained, and which has enzymatic activity;

[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." That is, 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 the monomer include an α chain or a β chain, and examples of the dimer include a dimer of only an α chain (αα form), a homodimer of only a 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 an αβ form.

[0017] Note that the term "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 to a multimeric AMV reverse transcriptase containing a protein containing that amino acid sequence as a subunit. Typically, the term "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 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 a β chain of AMV reverse transcriptase having an 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 a β chain of AMV reverse transcriptase having an amino acid sequence described herein and an α chain formed from the β chain.

[0019] Hereinafter, the amino acid sequences described in this specification 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. In other words, the AMV reverse transcriptase of the present disclosure is a mutant AMV reverse transcriptase. 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 for distinguishing "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 a mutation 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 referred to as "the wild-type AMV reverse transcriptase corresponding to the certain mutant AMV reverse transcriptase," and the mutant AMV reverse transcriptase is also referred to as "the mutant AMV reverse transcriptase corresponding to the certain wild-type AMV reverse transcriptase."

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

[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 one 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 sequence registered under GenBank No. AAB31929 (SEQ ID NO: 1), or may be a wild-type AMV reverse transcriptase β chain (SEQ ID NO: 2), which is a natural variant of AMV reverse transcriptase having an amino acid sequence in which the methionine residue at position 273 is substituted with an arginine residue, the glutamine residue at position 304 is substituted with an arginine residue, and the glutamic acid residue at position 395 is substituted with an aspartic acid residue in the amino acid sequence of the wild-type AMV reverse transcriptase β chain. Furthermore, the wild-type AMV reverse transcriptase may specifically be an AMV reverse transcriptase having an amino acid sequence (SEQ ID NO: 6) in which, in SEQ ID NO: 2, 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. 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 in which 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 several amino acids at one or several positions in the above 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 may specifically mean, 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 for each other when the substitution site is an aromatic amino acid, Leu, Ile, and Val are substituted for each other when the substitution site is a hydrophobic amino acid, Gln and Asn are substituted for each other when the substitution site is a polar amino acid, Lys, Arg, and His are substituted for each other when the substitution site is a basic amino acid, Asp and Glu are substituted for each other when the substitution site is an acidic amino acid, and Ser and Thr are substituted for 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 substitutions, deletions, insertions or additions also include those resulting from naturally occurring mutations (mutants or variants), such as those based on individual differences in the organism from which the gene is derived.

[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 base sequence (e.g., the base sequence shown in SEQ ID NO: 3), e.g., a sequence complementary to all or part of the above-mentioned base 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. Examples of conditions include 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 such as washing once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for ordinary Southern hybridization, i.e., 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0033] As described 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, 2xSSC, 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 refers to the identity between amino acid sequences calculated by blastp using default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). The "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated by blastn using 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, "enzyme activity" refers to "the enzymatic 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 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 have one or more of RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and RNase H activity. Among these, it is more preferable that the mutant AMV reverse transcriptase has the same enzymatic activity as that possessed by wild-type AMV reverse transcriptase, i.e., all of 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. Furthermore, 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 shown 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 of substitution, deletion, insertion, and addition of one or several amino acid residues at one or several positions in the amino acid sequence shown 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 shown in SEQ ID NO: 6, except for the "specific mutation," and having enzymatic activity.

[0042] More specifically, the mutant AMV reverse transcriptase may be an enzyme selected from any one 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 the amino acid substitution set forth in (1) below; (1) the amino acid residue corresponding to the leucine residue at position 476 of SEQ ID NO: 6 is substituted with a glutamine residue; (II) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution set forth in (1), 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 substitution set forth in (1), and which has enzymatic activity; (III) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which has an amino acid sequence that is 70% or more identical to the entire amino acid sequence containing the amino acid substitution set forth in (1), provided that the amino acid substitution is maintained, and which has enzymatic activity.

[0043] The mutant AMV reverse transcriptase may be an enzyme selected from any one of the following (i) to (iii): (i) an enzyme having the amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution of (1) above and further contains one or more amino acid substitutions selected from the following (2) to (16); (2) an amino acid residue corresponding to the valine residue at position 377 of SEQ ID NO: 6 is substituted with an isoleucine residue; (3) an amino acid residue corresponding to the serine residue at position 550 of SEQ ID NO: 6 is substituted with a threonine residue; (4) an amino acid residue corresponding to the lysine residue at position 691 of SEQ ID NO: 6 is substituted with a glutamic acid residue; (5) an amino acid residue corresponding to the asparagine residue at position 776 of SEQ ID NO: 6 is substituted with a lysine residue; (6) an amino acid residue corresponding to the isoleucine residue at position 332 of SEQ ID NO: 6 is substituted with a valine residue; (7) an amino acid residue corresponding to the alanine residue at position 474 of SEQ ID NO: 6 is substituted with a threonine residue; (8) an amino acid residue corresponding to the threonine residue at position 716 of SEQ ID NO: 6 is substituted with a serine residue. (9) The amino acid residue corresponding to the lysine residue at position 797 in SEQ ID NO:6 is replaced with an arginine residue. (10) The amino acid residue corresponding to the lysine residue at position 850 in SEQ ID NO:6 is replaced with an arginine residue. (11) The amino acid residue corresponding to the isoleucine residue at position 61 in SEQ ID NO:6 is replaced with a valine residue. (12) The amino acid residue corresponding to the valine residue at position 105 in SEQ ID NO:6 is replaced with an alanine residue. (13) The amino acid residue corresponding to the asparagine residue at position 623 in SEQ ID NO:6 is replaced with an aspartic acid residue. (14) The amino acid residue corresponding to the threonine residue at position 692 in SEQ ID NO:6 is replaced with an alanine residue. (15) The amino acid residue corresponding to the glycine residue at position 717 in SEQ ID NO:6 is replaced with an aspartic acid residue. (16) The amino acid residue corresponding to the lysine residue at position 850 in SEQ ID NO:6 is replaced with a glutamic acid residue.(ii) an enzyme having an amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution of (1) above, and further contains one or more amino acid substitutions selected from (2) to (16) above, and 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 set forth in (1) to (16), and which has enzymatic activity; (iii) an enzyme having an amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution of (1) above, and further contains one or more amino acid substitutions selected from (2) to (16), and which has an identity of 70% or more to the entire amino acid sequence containing the amino acid substitution of (1), and further contains one or more amino acid substitutions selected from (2) to (16), provided that the amino acid substitutions are maintained, and which has enzymatic activity;

[0044] The "one or several" in (II) and (ii) will vary 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.

[0045] Furthermore, the "identity" in (III) and (iii) may refer to an amino acid sequence that has, 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 identity to the entire amino acid sequence.

[0046] An example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to the leucine residue 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 the valine residue at position 377 in SEQ ID NO: 6 has been substituted with an isoleucine residue, the amino acid residue corresponding to the leucine residue at position 476 has been substituted with a glutamine residue, the amino acid residue corresponding to the serine residue at position 550 has been substituted with a threonine residue, the amino acid residue corresponding to the lysine residue at position 691 has been substituted with a glutamic acid residue, and the amino acid residue corresponding to the asparagine residue at position 776 has been substituted with a lysine residue is shown in SEQ ID NO: 20. Furthermore, an example of a mutant AMV reverse transcriptase in which the amino acid residue corresponding to the isoleucine residue at position 61 in 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.

[0047] Specifically, the mutant AMV reverse transcriptase may be an enzyme selected from any of the following (IV) to (VI): (IV) an AMV reverse transcriptase having an amino acid sequence set forth in any of SEQ ID NOs: 10, 20, 34, 36, 38, and 44; (V) an AMV reverse transcriptase having an amino acid sequence set forth in any of SEQ ID NOs: 10, 20, 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, with the proviso that specific mutations are maintained, and which has enzymatic activity; (VI) an AMV reverse transcriptase having an amino acid sequence having 70% or more identity to the amino acid sequence set forth in any of SEQ ID NOs: 10, 20, 34, 36, 38, and 44, with the proviso that specific mutations are maintained, and which has enzymatic activity.

[0048] 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.

[0049] Furthermore, the "identity" in (VI) may be, for example, an amino acid sequence having 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.

[0050] The mutant AMV reverse transcriptase may contain other amino acid sequences in addition to the amino acid sequence of the mutant AMV reverse transcriptase 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. Unless otherwise specified, "the mutant AMV reverse transcriptase contains an additional sequence" or "the mutant AMV reverse transcriptase is a fusion protein with an additional sequence" means that the mutant AMV reverse transcriptase obtained in the end contains the additional sequence. On the other hand, "the mutant AMV reverse transcriptase is expressed in a form containing an additional sequence" or "the mutant AMV reverse transcriptase contains an additional sequence upon expression" means that the mutant AMV reverse transcriptase contains the additional sequence at least upon expression, but does not necessarily mean that the mutant AMV reverse transcriptase obtained in the end contains the additional sequence. In other words, the mutant AMV reverse transcriptase gene may contain a base sequence encoding an additional sequence in addition to the base 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 appropriately selected depending on various conditions, such as the intended use. Examples of the additional sequence include a peptide tag, a signal peptide (also referred to as a signal sequence), and a protease recognition sequence. The additional sequence may be linked, for example, to the N-terminus, the 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.

[0051] 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.

[0052] 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 mutant AMV reverse transcriptase is secreted using a signal peptide, the signal peptide is cleaved during secretion, and the mutant AMV reverse transcriptase without the signal peptide can be secreted outside the bacterial cell. That is, typically, the mutant AMV reverse transcriptase obtained finally does not need to have a signal peptide.

[0053] 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, for example, when the mutant AMV reverse transcriptase is expressed as a fusion protein with a peptide tag, a protease recognition sequence can be introduced into 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 obtaining a mutant AMV reverse transcriptase that does not have a peptide tag.

[0054] 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, when the specific amino acid sequence is aligned with the amino acid sequence set forth in SEQ ID NO: 6. For example, "the amino acid residue corresponding to the leucine residue at position 476 in SEQ ID NO: 6" in a specific amino acid sequence refers to an amino acid residue in the specific amino acid sequence, which is arranged at the same position as the 476th leucine in the amino acid sequence set forth in SEQ ID NO: 6, when the specific amino acid sequence is aligned with the amino acid sequence set forth in SEQ ID NO: 6. Note that "the amino acid residue corresponding to the Xth amino acid in the amino acid sequence set forth in SEQ ID NO: 6" in the amino acid sequence set forth in SEQ ID NO: 6 refers to the Xth amino acid itself in the amino acid sequence set forth in SEQ ID NO: 6. That is, the positions of the 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 set forth in SEQ ID NO: 6. That is, for example, if the protein of the present invention contains an insertion, deletion, or addition of an amino acid residue N-terminally to the positions of the exemplified amino acid substitutions, the absolute positions of the amino acid substitutions may vary accordingly. The positions of the 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 set forth in 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 exemplified amino acid substitutions in any amino acid sequence, such as a variant sequence of the amino acid sequence set forth in 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 (16) 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.

[0055] The mutant AMV reverse transcriptase gene is not particularly limited as long as it encodes the mutant AMV reverse transcriptase described above. In this specification, 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, the "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 above-mentioned descriptions regarding DNA, such as the nucleotide sequence, may be appropriately interpreted to correspond to RNA. The form of the mutant AMV reverse transcriptase gene can be appropriately selected depending on various conditions, such as its mode of use.

[0056] The "specific mutation" will be explained below.

[0057] The term "specific mutation" refers to a mutation that is useful for the reverse transcription reaction, 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 the effect of improving thermostability and / or resistance to reaction inhibition by contaminants.

[0058] "Specific mutations" include one or more amino acid substitutions selected from the following (1) to (16): (1) a substitution of an amino acid residue corresponding to the leucine residue at position 476 of SEQ ID NO:6 with a glutamine residue; (2) a substitution of an amino acid residue corresponding to the valine residue at position 377 of SEQ ID NO:6 with an isoleucine residue; (3) a substitution of an amino acid residue corresponding to the serine residue at position 550 of SEQ ID NO:6 with a threonine residue; (4) a substitution of an amino acid residue corresponding to the lysine residue at position 691 of SEQ ID NO:6 with a glutamic acid residue; (5) a substitution of an amino acid residue corresponding to the asparagine residue at position 776 of SEQ ID NO:6 with a lysine residue; (6) a substitution of an amino acid residue corresponding to the isoleucine residue at position 332 of SEQ ID NO:6 with a valine residue; (7) a substitution of an amino acid residue corresponding to the alanine residue at position 474 of SEQ ID NO:6 with a threonine residue; (8) a substitution of an amino acid residue corresponding to the threonine residue at position 716 of SEQ ID NO:6 with a serine residue; (9) a substitution of an amino acid residue corresponding to the lysine residue at position 797 of SEQ ID NO:6 with an arginine residue. (10) The amino acid residue corresponding to the 850th lysine residue in SEQ ID NO:6 is substituted with an arginine residue. (11) The amino acid residue corresponding to the 61st isoleucine residue in SEQ ID NO:6 is substituted with a valine residue. (12) The amino acid residue corresponding to the 105th valine residue in SEQ ID NO:6 is substituted with an alanine residue. (13) The amino acid residue corresponding to the 623rd asparagine residue in SEQ ID NO:6 is substituted with an aspartic acid residue. (14) The amino acid residue corresponding to the 692nd threonine residue in SEQ ID NO:6 is substituted with an alanine residue. (15) The amino acid residue corresponding to the 717th glycine residue in SEQ ID NO:6 is substituted with aspartic acid. (16) The amino acid residue corresponding to the 850th lysine residue in SEQ ID NO:6 is substituted with a glutamic acid residue.

[0059] Introducing the amino acid substitution (1) into wild-type AMV reverse transcriptase is expected to not only improve the thermal stability of the AMV reverse transcriptase but also improve the purification yield of the AMV reverse transcriptase. Therefore, the "specific mutation" preferably includes the amino acid substitution (1).

[0060] Furthermore, the "specific mutation" may include at least the amino acid substitutions (1) to (5). Furthermore, the "specific mutation" may include at least the amino acid substitutions (1) to (5) and one or more amino acid substitutions selected from (6), (11) to (16).

[0061] 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 (16). 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), (12), and (16).

[0062] Improved thermostability may be demonstrated, for example, by improved residual activity after heat treatment, an increased 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 to 60°C for 1 to 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 compared to wild-type AMV reverse transcriptase. As another example, improved thermostability may be demonstrated by heating a mutant AMV reverse transcriptase at 50 to 60°C for 1 to 60 minutes, followed by a reverse transcription reaction, and then detecting an increased amount of DNA generated 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 to 60°C for 1 to 60 minutes, followed by an increased amount of enzyme remaining without heat denaturation compared to wild-type AMV reverse transcriptase. As yet another example, improved thermostability may be demonstrated by using a mutant AMV reverse transcriptase to perform an isothermal gene amplification reaction involving a reverse transcription reaction at a high reaction temperature, and the time until the generated amplification product is detected is shorter compared to that of wild-type AMV reverse transcriptase, or by the generation of an amplification product being detected without being heat-inactivated at a higher reaction temperature than that of the wild-type AMV reverse transcriptase.

[0063] 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." While improved resistance to reaction inhibition by contaminants may be demonstrated under high-temperature conditions, for example, this is not limiting 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 fluid (pleural effusion, ascites, etc.), and nasal / pharyngeal mucus, as well as contaminants derived from natural components, such as soil and environmental water (river water, seawater, lake water). Other examples of contaminants include components of reagents (such as virus transport solutions and transport media, blood preservation solutions, fecal preservation solutions, buffer solutions, and physiological saline solutions) 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, and purify the target nucleic acid from the specimen or measurement sample, as well as viruses, bacteria, and cells containing the target nucleic acid contained therein. The term "in the presence of contaminants" may refer, for example, to conditions in which the contaminants or purified products thereof are present in the reaction solution. Specifically, improved resistance to reaction inhibition by contaminants may be demonstrated, for example, by a shorter time required to detect the generated amplification product when an isothermal gene amplification reaction involving reverse transcription is performed using a mutant AMV reverse transcriptase in a reaction solution containing a saliva or urine purified product, compared to a wild-type AMV reverse transcriptase. The saliva or urine purified product may contain components of the aforementioned reagents, such as reagents for purifying saliva or urine.

[0064] 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), (12), and (16), or has the amino acid substitution (6) in addition to these mutations.

[0065] <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 having the gene.

[0066] The production of mutant AMV reverse transcriptase using a host carrying the mutant AMV reverse transcriptase gene will be described in detail below.

[0067] <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."

[0068] 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 particularly include microorganisms. Examples of the microorganisms include bacteria and yeast. Examples of the microorganisms particularly include bacteria.

[0069] Examples of bacteria include bacteria belonging to the family Enterobacteriaceae, coryneform bacteria, and Bacillus bacteria.

[0070] Examples of bacteria belonging to the Enterobacteriaceae family include bacteria belonging to genera such as Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Photorhabdus, Providencia, Salmonella, and Morganella. Specifically, bacteria classified as Enterobacteriaceae according to the classification used in the NCBI (National Center for Biotechnology Information) database (http: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=91347) can be used. Examples of bacteria belonging to 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, D.C.). Examples of Escherichia bacteria include Escherichia coli, i.e., Escherichia coli.Examples of Escherichia coli include Escherichia coli B strains such as the BL21(DE3) strain; Escherichia coli K-12 strains such as the JM109 strain (ATCC 53323), the HB101 strain (ATCC 33694), the W3110 strain (ATCC 27325), and the MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); and derivatives thereof. 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 planticola.

[0071] Examples of coryneform bacteria include bacteria belonging to genera such as Corynebacterium, Brevibacterium, and Microbacterium.

[0072] 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)). Corynebacterium stationis also includes bacteria that were previously classified as Corynebacterium ammoniagenes but have been reclassified as Corynebacterium stationis based on 16S rRNA sequence analysis or the like (Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)).

[0073] Examples of Bacillus bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, and Bacillus 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).

[0074] 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.

[0075] 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 is assigned a corresponding accession number, and can be obtained using this accession number (see http: / / www.atcc.org / ). The accession numbers corresponding to each strain are listed in the catalog of the American Type Culture Collection. These strains can also be obtained, for example, from the depository institution where they were deposited.

[0076] 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.

[0077] 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, HA Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)).

[0078] 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 in the same manner as the method described in detail below in the section "Method for introducing a gene."

[0079] Furthermore, when a host already has an AMV reverse transcriptase gene such as a wild-type AMV reverse transcriptase gene in a chromosome or the like, the AMV reverse transcriptase gene can be modified to encode a mutant AMV reverse transcriptase, thereby modifying the host to have a mutant AMV reverse transcriptase gene. The AMV reverse transcriptase gene present in a chromosome or the like can be modified, 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.

[0080] The host may have any properties as long as it is capable of producing the mutant AMV reverse transcriptase.

[0081] <2-2> Method for Introducing Genes The following describes the method for introducing genes into a host.

[0082] 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). Gene introduction methods using homologous recombination include, for example, 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 has multiple copies present on the chromosome. Examples of sequences present in multiple copies on a chromosome include repetitive DNA sequences and inverted repeats at both ends of a transposon. Homologous recombination may also be performed by targeting an appropriate sequence on a chromosome, such as a gene that is not necessary for the production of AMV reverse transcriptase. 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 EP805867B1).

[0083] 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.

[0084] 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. A vector capable of autonomous replication within host cells can be used. The vector may be a multicopy vector. Furthermore, the vector may have a marker such as an antibiotic resistance gene to select transformants. The vector may also have 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 bacteria of the Enterobacteriaceae family, such as Escherichia coli, include pUC19, pUC18, pHSG299, pHSG398, pBR322, pSTV29, and pCold-series vectors (all available from Takara Bio Inc.), pACYC177, pACYC184, and pMW219 (Nippon Gene Co., Ltd.), pTrc99A (Pharmacia), pET-series vectors (Merck & Co.), and pQE-series vectors (Qiagen).

[0085] When a gene is introduced, the gene only needs to be expressible by the host. Specifically, the gene only needs to be 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 refer to 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 the T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Bifidobacterium-derived Pm1 promoter, PR promoter, PL promoter, P4 promoter, and P8 promoter.

[0086] 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 of another gene. Specific examples of terminators include the T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

[0087] <2-3> Culturing the host The mutant AMV reverse transcriptase can be expressed by culturing a host having a 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.

[0088] The medium used is not particularly limited as long as it allows the host to grow and a functional mutant AMV reverse transcriptase to be expressed. For example, a conventional medium used for culturing microorganisms such as bacteria and yeast can be used as the medium. 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.

[0089] 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, for example, 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, hemicellulose in plant biomass may be hydrolyzed in advance to liberate pentoses, and then cellulose may be hydrolyzed to produce hexoses. Xylose may also be supplied by converting hexoses such as glucose into xylose, 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, or two or more types of carbon sources may be used in combination.

[0090] 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 %. Furthermore, the carbon source may be additionally supplied to the medium as appropriate. For example, the carbon source may be additionally supplied to the medium in response to a decrease or depletion of the carbon source as the culture progresses. As long as the mutant AMV reverse transcriptase is ultimately produced, the carbon source may be temporarily depleted; however, it may be preferable to carry out the culture so that the carbon source does not become depleted or the depletion state of the carbon source does not continue.

[0091] 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 is used for pH adjustment, may also be used as the 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.

[0092] 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.

[0093] 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.

[0094] Specific examples of other various organic components 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 acid, yeast extract, and soy protein hydrolysate. As other various organic components and inorganic components, one type of component may be used, or two or more types of components may be used in combination.

[0095] 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.

[0096] The culture conditions are not particularly limited as long as the host can grow and a functional mutant AMV reverse transcriptase can be expressed. The culture can be performed under normal conditions used for culturing microorganisms such as bacteria and yeast. The culture conditions can be appropriately set depending on various conditions such as the type of host. Furthermore, the expression of the mutant AMV reverse transcriptase gene can be induced as necessary.

[0097] 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. It is sufficient that the mutant AMV reverse transcriptase is expressed at least in the main culture. The amount of the host contained in the medium at the start of culture is not particularly limited. For example, a seed culture solution with an OD660 of 4 to 100 may be added at the start of culture in an amount of 0.1% by mass to 100% by mass, preferably 1% by mass to 50% by mass, relative to the medium for main culture.

[0098] 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 culture system (e.g., a fermenter) in fed-batch culture or continuous culture is also called the "fed-batch medium." Supplying a fed-batch medium to a culture system in fed-batch culture or continuous culture is also called "fed-batch." When cultivation is carried out separately into a seed culture and a main culture, the culture 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 culture or continuous culture.

[0099] 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. All of these components may be supplied once or 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.

[0100] Cultivation can be carried out, for example, under aerobic conditions. "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, for example, to about 1 to 100% of the saturated oxygen concentration, preferably about 20 to 100%. Cultivation can be carried out, for example, by aeration culture or shaking culture. The pH of the medium may be, for example, 3 to 10, preferably 4.0 to 9.5. During cultivation, 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.

[0101] By culturing the host in this manner, a culture containing the mutant AMV reverse transcriptase can be obtained. The mutant AMV reverse transcriptase can be accumulated, 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 design of the host and / or the mutant AMV reverse transcriptase gene used, it may be possible to accumulate the mutant AMV reverse transcriptase in the periplasm or to secrete and produce the mutant AMV reverse transcriptase outside the bacterial cells.

[0102] 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, in the form of such a fraction (i.e., in the form contained in such a fraction), or in a combination thereof. Such fractions are not particularly limited, as long as the mutant AMV reverse transcriptase is contained in such a fraction so that it can act on its substrate. Such fractions include cultures of hosts harboring a mutant AMV reverse transcriptase gene (i.e., hosts harboring a mutant AMV reverse transcriptase), bacterial cells recovered from the cultures, culture supernatants recovered from the cultures, processed products thereof (e.g., processed bacterial cells such as bacterial cell disruptants, bacterial cell lysates, bacterial cell extracts, and other products described below), partially purified products thereof (i.e., crudely purified products), and combinations thereof. Note that the term "purified mutant AMV reverse transcriptase" may also include 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. Furthermore, the manner in which the mutant AMV reverse transcriptase is recovered may be appropriately determined depending on the application.

[0103] 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.

[0104] 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, for example, by using a surfactant or organic solvent. These treatments may be used alone or in appropriate combination.

[0105] 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 purification using hydrophobic interaction chromatography with a polypropylene glycol group introduced therein, followed by further purification using ion exchange chromatography using a phosphocellulose carrier.

[0106] The mutant AMV reverse transcriptase may be produced alone or in combination with other proteins.

[0107] <2-4> Production of mutant AMV reverse transcriptase The mutant AMV reverse transcriptase may be produced by the method described above.

[0108] For example, the mutant AMV reverse transcriptase may be produced by a method comprising the steps of expressing the 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 AMV reverse transcriptase."

[0109] The method for producing AMV reverse transcriptase may preferably include the steps of: transforming a host with an expression vector containing a mutant AMV reverse transcriptase gene, and culturing the resulting transformant to express AMV reverse transcriptase; and recovering the expressed enzyme from the resulting culture.

[0110] 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.

[0111] The host and its culture are as described above.

[0112] The expressed mutant AMV reverse transcriptase can be recovered as described above. The mutant AMV reverse transcriptase may be purified during the recovery process from the culture. The purification of the enzyme is also as described above.

[0113] <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 known step as another 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.

[0114] 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 using AMV reverse transcriptase and a polymerase chain reaction using a 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, the TMA (Transcription-Mediated Amplification) method described in Japanese Patent Publication No. 4-500759, or the TRC (Transcription Reverse-transcription Concerted) method described in Japanese Patent Application Laid-Open No. 2000-14400. Because mutant AMV reverse transcriptases have excellent thermostability, they can treat RNA 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 due to misannealing. Therefore, they are considered suitable for the amplification of target nucleic acids involving a reverse transcription reaction. Therefore, in one embodiment of the present disclosure, amplification of a target nucleic acid may be performed at a high temperature. 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 a high temperature, or an RNA treatment in a previous step may be performed at a high temperature. Note that "high temperature" here specifically refers to, for example, heating at 50 to 60°C for 1 to 60 minutes.

[0115] 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.

[0116] 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.

[0117] 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-mentioned (1), (2), (3), (4), (5), (6), (12), and (16). Preferably, the mutant AMV reverse transcriptase has all of the amino acid substitutions (1), (2), (3), (4), (5), (12), and (16), or a mutant AMV reverse transcriptase having an amino acid substitution (6) 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 aspect 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 aspect 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.

[0118] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to these examples.

[0119] Example 1: Introduction of Mutations into Avian Myeloblastoma Virus (AMV) Reverse Transcriptase Gene (1) A polynucleotide was synthesized in which an oligonucleotide (SEQ ID NO: 7) 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 added to the 5'-end of the polynucleotide (SEQ ID NO: 7) encoding the AMV reverse transcriptase, AMV-RT m4-2 (disclosed in JP 2014-209898 A; SEQ ID NO: 6), and 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 3'-end of the polynucleotide. AMV-RT m4-2 (SEQ ID NO: 6) is a polypeptide in which the amino acid sequence of the wild-type AMV reverse transcriptase (AMV-RT wild) β-chain (SEQ ID NO: 2) 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 hereinafter), 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 transformed into 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 obtain the AMV reverse transcriptase expression plasmid pTrc99A-His-AMVRTm4. (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).Specifically, the polynucleotides shown in <1> to <5> below were prepared: <1> A polynucleotide in which guanine (G) at position 1129 of SEQ ID NO: 7 is substituted with adenine (A) (SEQ ID NO: 9); <2> A polynucleotide in which thymine (T) at position 1427 of SEQ ID NO: 7 is substituted with adenine (A) (SEQ ID NO: 11); <3> A polynucleotide in which thymine (T) at position 1648 of SEQ ID NO: 7 is substituted with adenine (A) (SEQ ID NO: 13); <4> A polynucleotide in which adenine (A) at position 2071 of SEQ ID NO: 7 is substituted with guanine (G) (SEQ ID NO: 15); <5> A polynucleotide in which cytosine (C) at position 2328 of SEQ ID NO: 7 is substituted with guanine (G) (SEQ ID NO: 17). The nucleotide substitutions shown in <1> to <5> are expressed as amino acid substitutions in the translated AMV reverse transcriptase as follows: <1> corresponds to the substitution of valine (V) at position 377 of SEQ ID NO: 6 with isoleucine (I), <2> corresponds to the substitution of leucine (L) at position 476 of SEQ ID NO: 6 with glutamine (Q), <3> corresponds to the substitution of serine (S) at position 550 of SEQ ID NO: 6 with threonine (T), <4> corresponds to the substitution of lysine (K) at position 691 of SEQ ID NO: 6 with glutamic acid (E), and <5> corresponds to the substitution of asparagine (N) at position 776 of SEQ ID NO: 6 with lysine (K). (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 in any of them.

[0120] 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 2×YT 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 at 1,000 rpm with shaking. (3) A portion of the preculture solution (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 (2) was inoculated into a 96-well deep well plate containing 475 μL of 2xPNa 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 solution from (5) was centrifuged at 4°C and 3000 rpm for 30 minutes, and the supernatant was discarded to recover the bacterial cells. The recovered bacterial cells were stored at -30°C. (7) 100 μL of extraction buffer containing BugBuster reagent (Merck) was added to the bacterial cells recovered in (6), and the cells were shaken at 25°C and 1000 rpm for 1 hour. After centrifugation at 4°C and 3000 rpm for 30 minutes, the supernatant was recovered to prepare a bacterial cell extract.(8) The bacterial cell extract from (7) diluted 100-fold with TBS buffer (20 mmol / L Tris-HCl, 150 mmol / L sodium chloride) (pH 7.5) was dispensed into a 96-well PCR plate at 120 μL / well, heated at 55°C for 10 minutes using a thermal cycler, and then cooled to 4°C and maintained. (9) The amount of AMV reverse transcriptase contained in the extract from (7) (without heat treatment) and the amount of AMV reverse transcriptase contained in the extract heat-treated in (8) (with heat treatment) were evaluated by the following enzyme-linked immunosorbent assay (ELISA). (9-1) The extract (without heat treatment and with heat treatment) was dispensed into a 96-well microplate and incubated at 30°C for 1 hour to immobilize the proteins in the extract to 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 a 1% (w / v) bovine serum albumin (BSA) solution (manufactured by Merck) was added and incubated at 30°C for 1 hour to perform blocking. (9-3) After blocking, the plate was washed with TBS-T buffer, and then a mouse anti-AMV reverse transcriptase monoclonal antibody was added and incubated at 30°C for 1 hour to perform a primary antibody reaction. (9-4) After the primary antibody reaction, the plate was washed with TBS-T buffer, and then a Horse Radish Peroxidase-labeled anti-mouse IgG antibody (manufactured by Bethyl Laboratories) was added and incubated at 30°C for 1 hour to perform a secondary antibody reaction. (9-5) After completion of the secondary antibody reaction, the sample was washed with TBS-T buffer, and TMB Peroxidase Substrate (KPL) was added and incubated at room temperature for 5 minutes. (9-6) An equal volume of 1 mol / L aqueous phosphoric acid solution was added to terminate the reaction, 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.

[0121] 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 in 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 higher remaining enzyme amounts than AMV-RT m4-2 (SEQ ID NO: 6). This demonstrates that introducing at least one amino acid substitution from V377I, L476Q, S550T, K691E, and N776K into AMV-RT m4-2 improves its thermal stability compared to AMV-RT m4-2.

[0122] 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 then the Escherichia coli JM109 strain (Takara Bio Inc.) was transformed with the vector. (3) A recombinant plasmid was extracted from the transformant obtained in (2) using a QIAprep Spin Miniprep Kit (Qiagen) to prepare the protease expression plasmid pSTV_p15. (4) 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 an 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 E. 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 at 37°C and 100 rpm for 8 hours with shaking. (7) IPTG was added to a final concentration of 5 mmol / L, and cultured at 25°C and 100 rpm for an additional 3 days. (8) The culture solution from (7) was centrifuged at 4°C and 8000 rpm for 20 minutes, and the supernatant was discarded to recover wet bacterial cells. The recovered bacterial cells were stored at -30°C.

[0123] Example 4: Evaluation of AMV reverse transcriptase heat resistance (part 2) (1) 5 mL of extraction buffer containing BugBuster reagent (Merck) was added per gram of wet bacterial cells recovered in Example 3, followed by stirring for 10 minutes 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. The solution 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, then cooled to 4°C and maintained. (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. Measurement was performed by monitoring changes in fluorescence intensity using an 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.

[0124] 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.

[0125]

[0126] Example 5 Preparation of AMV reverse transcriptase expression vector without 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 the AMV reverse transcriptase consisting of any one of the amino acid sequences of SEQ ID NOs: 8, 10, 12, 14, and 16 prepared in Example 1(4). (2) The synthesized polynucleotides were inserted between the restriction enzyme EcoRI and KpnI cleavage sites of the plasmid pTrc99A, and the vector was used to transform Escherichia coli JM109 strain (Takara Bio Inc.) to produce transformants. (3) From the transformant prepared in (2), a recombinant plasmid was extracted using QIAprep Spin Miniprep Kit (Qiagen) to prepare an AMV reverse transcriptase expression vector without a histidine tag.

[0127] Example 6 Accumulation of Amino Acid Substitutions The amino acid substitutions found to be involved in improving the thermostability of AMV reverse transcriptase in Examples 2 and 4 were accumulated in AMV-RT m4-2 (SEQ ID NO: 6) to further improve thermostability. Specifically, polynucleotides were synthesized in which nucleotide substitutions corresponding to the amino acid substitutions shown in (a) or (b) below were introduced into predetermined positions in the polynucleotide encoding AMV-RT m4-2 (SEQ ID NO: 7), and AMV reverse transcriptase expression vectors were then 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)

[0128] 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. It has been confirmed that the AMV reverse transcriptase can be similarly expressed when SEQ ID NO: 47 is used as the nucleotide sequence of the polynucleotide encoding AMV-RT m7 and SEQ ID NO: 48 is used as the nucleotide sequence of the polynucleotide encoding AMV-RT m9.

[0129] Example 7 Evaluation of Heat Resistance of AMV Reverse Transcriptase (Part 3) (1) The 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: 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 in 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 antibiotic, to obtain a strain producing each AMV reverse transcriptase. (3) Using each production strain obtained in (2), a purified AMV reverse transcriptase was obtained by culturing in a fermenter and purifying by column chromatography according to the method described in JP 2014-209898 A. The solution was diluted 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, then cooled to 4°C and maintained therein. (5) RNA detection was performed using each AMV reverse transcriptase in the same manner as in Example 4(5), and the detection time was determined.

[0130] The results are shown in Table 2. The introduction of any of the amino acid substitutions (V377I, L476Q, S550T, K691E, N776K) found to be involved in improving the thermostability of AMV reverse transcriptase in Examples 2 and 4 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 the three amino acid substitutions (V377I, S550T, and K691E) further improved thermostability.

[0131]

[0132] 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.

[0133] 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 showed increased purification yields compared to AMV-RT m4-2 (SEQ ID NO: 6) without the substitution. These results demonstrate that the L476Q amino acid substitution improves both thermostability and productivity.

[0134] Example 9 Introduction of Mutations into Avian Myeloblastoma Virus (AMV) Reverse Transcriptase Gene (Part 2) (1) In pTrc99A-His-AMVRTm4 obtained in Example 1(3), nucleotide substitutions were introduced into predetermined positions in the polynucleotide (SEQ ID NO: 7) encoding AMV-RT m4-2 (SEQ ID NO: 6). Specifically, the polynucleotides shown in <6> to <9> below were prepared: <6> A polynucleotide (SEQ ID NO: 27) in which adenine (A) at position 994 of SEQ ID NO: 7 was substituted with guanine (G); <7> A polynucleotide (SEQ ID NO: 29) in which guanine (G) at position 1420 of SEQ ID NO: 7 was substituted with adenine (A); <8> A polynucleotide (SEQ ID NO: 31) in which cytosine (C) at position 2147 of SEQ ID NO: 7 was substituted with guanine (G); <9> A polynucleotide (SEQ ID NO: 33) in which adenine (A) at position 2389 of SEQ ID NO: 7 was substituted with cytosine (C), adenine (A) at position 2390 of SEQ ID NO: 2391 of SEQ ID NO: 2391 was substituted with thymine (T), adenine (A) at position 2548 of SEQ ID NO: 2549 of SEQ ID NO: 2549 of SEQ ID NO: 2550 of SEQ ID NO: 2550 of SEQ ID NO: 2550 The nucleotide substitutions shown in <6> to <9> are expressed as amino acid substitutions in the translated AMV reverse transcriptase, where <6> corresponds to the substitution of isoleucine (I) at position 332 in SEQ ID NO: 6 with valine (V), <7> corresponds to the substitution of alanine (A) at position 474 in SEQ ID NO: 6 with threonine (T), <8> corresponds to the substitution of threonine (T) at position 716 in SEQ ID NO: 6 with serine (S), and <9> corresponds to the substitution of lysine (K) at position 797 with arginine (R) and the substitution of lysine (K) at position 850 with arginine (R) in SEQ ID NO: 6. (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 in any of them.

[0135] Example 10 Evaluation of Heat Resistance of AMV Reverse Transcriptase (Part 4) (1) Escherichia coli HB101 (Takara Bio Inc.) was 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: 27, 29, 31, or 33 prepared in Example 9(1) to prepare an AMV reverse transcriptase-producing strain (transformant). (2) The AMV reverse transcriptase-producing strain prepared in (1) was cultured in the same manner as in Examples 2(2) to (10), and the residual activity of AMV reverse transcriptase in the extract was evaluated by ELISA. The results are shown in Figure 3. In Figure 3, the residual enzyme amount is expressed as a relative value to the residual enzyme amount in AMV-RT m4-2 (relative residual enzyme amount). AMV reverse transcriptases into 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) had been introduced into AMV-RT m4-2 all had higher residual enzyme amounts than AMV-RT m4-2 (SEQ ID NO: 6). This demonstrates that introducing at least one amino acid substitution from I332V, A474T, T716S, K797R, and K850R into AMV-RT m4-2 improves its thermal stability compared to AMV-RT m4-2.

[0136] 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 into which the amino acid substitution I332V shown in SEQ ID NO:26 has been introduced) 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 culture overnight at 37°C at 160 rpm, thereby carrying out 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 dispensed into a 500 mL baffled flask, and 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 culture was further cultured at 25°C and 130 rpm for 3 days. (4) The culture solution from (3) was centrifuged at 4°C and 10,000 rpm for 20 minutes, and the supernatant was discarded to recover wet bacterial cells. The recovered bacterial cells were stored at -30°C. (5) To the wet cells recovered in (4), 50 mmol / L of Tris-HCl buffer (pH 7.2) containing 150 mmol / L sodium chloride, 2.4 mmol / L magnesium sulfate, 3000 Units / L of Benzonase (Merck), 0.006% (w / v) lysozyme, 2 mmol / L of phenylmethylsulfonyl fluoride, and 0.6% (w / v) of Triton X-100 (trade name) was added, and the mixture was stirred at room temperature for 1 hour and then centrifuged at 4°C and 15,000 rpm for 60 minutes to obtain an extract containing the expressed AMV reverse transcriptase. (6) The extract prepared in (5) was added to 0.8 mL of TALON (Cobalt) resin (Takara Bio Inc.), washed with 8 mL of a buffer containing 10 mmol / L of imidazole, and then eluted with 0.8 mL of a buffer containing 200 mmol / L of imidazole. (7) The purified AMV reverse transcriptase solution obtained in (6) was heated at 52°C or 53°C for 5 minutes, then cooled to 4°C and maintained.(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 as described 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 an 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, the "detection time" refers to the relative value obtained by dividing the detection time using each AMV reverse transcriptase after heat treatment by the detection time using AMV-RT m4-2 (SEQ ID NO: 6) after heat treatment. 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.

[0137]

[0138] Example 12 Introduction of Mutations into 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) A recombinant plasmid was prepared by inserting the synthesized polynucleotide between the restriction enzyme NcoI / KpnI cleavage sites of the plasmid pTrc99A, and then the Escherichia coli JM109 strain (Takara Bio Inc.) was transformed with the plasmid 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 predetermined positions of the polynucleotide (SEQ ID NO: 21) encoding AMV-RT m9 (SEQ ID NO: 20).Specifically, the polynucleotides shown in <10> to <12> below were prepared: <10> A polynucleotide (SEQ ID NO: 35) in which adenine (A) at position 181 of SEQ ID NO: 21 was substituted with guanine (G); <11> A polynucleotide (SEQ ID NO: 37) in which thymine (T) at position 314 of SEQ ID NO: 21 was substituted with cytosine (C) and adenine (A) at position 2548 of SEQ ID NO: 21 was substituted with guanine (G); <12> A polynucleotide (SEQ ID NO: 39) in which adenine (A) at position 1867 of SEQ ID NO: 21 was substituted with guanine (G), adenine (A) at position 2074 of SEQ ID NO: 21 was substituted with guanine (G), guanine (G) at position 2150 of SEQ ID NO: 21 was substituted with adenine (A), and thymine (T) at position 2567 of SEQ ID NO: 21 was substituted with cytosine (C). The nucleotide substitutions shown in <10> to <12> can be expressed as amino acid substitutions in the translated AMV reverse transcriptase, as follows: The <10> corresponds to a substitution of isoleucine (I) at position 61 in SEQ ID NO: 20 with valine (V); the <11> corresponds to a substitution of valine (V) at position 105 with alanine (A) and a substitution of lysine (K) at position 850 with glutamic acid (E) in SEQ ID NO: 20; and the <12> corresponds 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. The AMV reverse transcriptase having the amino acid substitution at <10> was named AMV-RT m10, the AMV reverse transcriptase having the amino acid substitution at <11> was named AMV-RT m11, and the AMV reverse transcriptase having the amino acid substitution at <12> was named AMV-RT m13. It has been confirmed that the AMV reverse transcriptase can be expressed in the same manner when SEQ ID NO: 49 is used as the nucleotide sequence of the polynucleotide encoding AMV-RT m10, SEQ ID NO: 50 is used as the nucleotide sequence of the polynucleotide encoding AMV-RT m11, or SEQ ID NO: 51 is used as the nucleotide sequence of the polynucleotide encoding AMV-RT m13. (5) The nucleotide 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.

[0139] 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 the 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 recovered. The recovered 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, followed by incubation at 4°C. (5) 20 μL of the reverse transcription reaction solution listed in Table 4 was prepared in a PCR tube and reacted at 46°C for 30 minutes. The standard RNA used was the sequence of carbonic anhydrase derived from sulfate-reducing bacteria listed in SEQ ID NO: 40, optimized for Escherichia coli codons. The primer was designed to complementarily bind to the 3' end of the standard RNA as listed in SEQ ID NO: 41. (6) 20 μL of the PCR reaction solution listed in Table 5 was prepared in a PCR tube and incubated at 95°C for 2 minutes, followed by 20 thermal cycles of 95°C for 30 seconds, 65°C for 30 seconds, and 72°C for 1 minute. The primer pair listed in SEQ ID NO: 42 and SEQ ID NO: 43 was used for PCR. (7) The resulting amplification products were separated by electrophoresis using a 1.0% by mass agarose gel, stained with SYBR Gold nucleic acid gel stain (Thermo Fisher Scientific), and then photographed with a Printgraph CMOS I (Atto Corporation). The brightness of each band was quantitatively analyzed using image analysis software ImageQuant TL (Cytiva). The residual activity was calculated by dividing the band brightness obtained using each AMV reverse transcriptase after heat treatment by the band brightness obtained using each AMV reverse transcriptase before heat treatment.

[0140]

[0141]

[0142] The results for heat treatment at 50°C are shown in Fig. 4, and the results for heat treatment at 52°C are shown in Fig. 5. In Fig. 4 and Fig. 5, the residual activity is expressed as a relative value to the residual activity of AMV-RT m9 (relative residual activity value). The residual activity after heat treatment was significantly increased for AMV reverse transcriptase (AMV-RT m10) in which the amino acid substitutions I61V (SEQ ID NO: 34) had been introduced into AMV-RT m9, AMV reverse transcriptase (AMV-RT m11) in which the amino acid substitutions V105A and K850E (SEQ ID NO: 36) had been introduced into AMV-RT m9, and AMV reverse transcriptase (AMV-RT m13) in which the amino acid substitutions N623D, T692A, G717D, ​​and L856P (SEQ ID NO: 38) had been introduced into AMV-RT m9 compared to AMV-RT m9 (SEQ ID NO: 20) without the above substitutions. These results demonstrate that introducing any of the amino acid substitutions I61V, V105A, K850E, N623D, T692A, G717D, ​​and L856P into AMV-RT m9 improves thermostability compared to AMV-RT m9.

[0143] Example 14 Preparation of AMV reverse transcriptase expression vector without histidine tag (part 2) AMV-RT m11 was selected from the AMV reverse transcriptase mutants whose improved thermostability was confirmed in Example 13, and an AMV reverse transcriptase expression vector without a histidine tag was prepared. 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, thereby preparing an AMV reverse transcriptase expression vector without a histidine tag.

[0144] Example 15: Accumulation of Amino Acid Substitutions (Part 2) From the amino acid substitutions found to be involved in improved thermostability in Examples 10 and 11, the I332V amino acid substitution was selected and accumulated in the histidine-tagged AMV-RT m11 (SEQ ID NO: 36) prepared in Example 14 (designated AMV-RT m12). Specifically, a polynucleotide was synthesized in which a nucleotide substitution corresponding to the I332V amino acid substitution was introduced into a predetermined position in the polynucleotide encoding AMV-RT m11 (SEQ ID NO: 37), and 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. It has been confirmed that AMV-RT m12 can be similarly expressed when SEQ ID NO: 52 is used as the nucleotide sequence of the polynucleotide encoding AMV-RT m12.

[0145] Example 16 Evaluation of Heat Resistance of AMV Reverse Transcriptase (Part 7) (1) E. 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 resulting transformants, fermentation was performed in a fermenter and purification by column chromatography was performed in the same manner as in Example 7(2) and (3), to obtain purified AMV reverse transcriptase. (3) The concentration of the obtained purified AMV enzyme was quantified by size exclusion chromatography using a TSKgel UP-SW Aggregate column (Tosoh Corporation). (4) Among the components of the Mycobacterium tuberculosis complex rRNA detection reagent TRCReady MTB (manufactured by Tosoh Corporation), 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 a heat-stable T7 RNA polymerase consisting of the amino acid sequence set forth in SEQ ID NO: 46. A positive standard RNA (RNA to be detected 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 (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.

[0146] 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 the temperature was raised to 54°C, the enzyme was thermally inactivated and detection became impossible. On the other hand, when an AMV reverse transcriptase in which the amino acid substitutions V105A, V377I, L476Q, S550T, K691E, N776K, and K850E were introduced into AMV-RT m4-2 (AMV-RT m11, SEQ ID NO: 36) or an AMV reverse transcriptase in which the amino acid substitution I332V was further introduced (AMV-RT m12, SEQ ID NO: 44) was used, the detection time was maintained at approximately 4 minutes even when the reaction temperature was raised to 53°C, and positive control RNA could be detected even when the temperature was raised to 54°C. These results demonstrate 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 reactions of target nucleic acids to be carried out at higher temperatures.

[0147]

[0148] Example 17 Evaluation of AMV Reverse Transcriptase Inhibition Resistance (1) A saliva purified product was prepared by the following procedure. (1-1) Saliva from a healthy subject was suspended in a 4-fold volume of PBS buffer (manufactured by Nacalai Tesque) and centrifuged at 10,000 × G for 1 minute. (1-2) 100 μL of the supernatant from the centrifugation 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, thereby preparing a saliva purified product. (2) A urine purified product was prepared by 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 centrifuged at 10,000 × G for 3 minutes. (2-2) The entire supernatant of the centrifugation in (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) To the purified saliva product prepared in (1) or the purified urine product prepared in (2), 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 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 a thermostable 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 then measured at a reaction temperature of 51°C.

[0149] 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 (purified saliva or purified urine), resulting in a significant delay in detection time. 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 had been introduced into AMV-RT m4-2, or AMV reverse transcriptase (AMV-RT m12, SEQ ID NO: 44) in which the amino acid substitution I332V had been further introduced, detection was possible in a shorter time than when AMV-RT m4-2 was used. These results demonstrate that by performing an amplification reaction of a target nucleic acid, for example, under high temperature conditions, using an AMV reverse transcriptase mutant 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, detection performance in the presence of various contaminants is improved, and this can be said to be useful in infectious disease testing of various clinical specimens using nucleic acid amplification methods.

[0150]

[0151] This application claims priority based on a Japanese patent application filed on February 6, 2024 (Patent Application No. 2024-016673), a Japanese patent application filed on June 18, 2024 (Patent Application No. 2024-097920), and a Japanese patent application filed on October 16, 2024 (Patent Application No. 2024-181275), the entire contents of which are incorporated by reference and incorporated as the disclosure of the specification of the present disclosure.

[0152] According to one aspect of the present disclosure, an AMV reverse transcriptase with improved thermostability can be provided. Surprisingly, by introducing the amino acid mutations discovered in the present 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 aspect of the present disclosure, an AMV reverse transcriptase with improved resistance to reaction inhibition by contaminants can be provided. This aspect is expected to be useful in infectious disease testing of various clinical specimens using nucleic acid amplification methods.

[0153] 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) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution set forth in (1) below; (1) the amino acid residue corresponding to the leucine residue at position 476 of SEQ ID NO: 6 is substituted with a glutamine residue; (II) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution set forth in (1) above, 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 substitution set forth in (1), and which has enzymatic activity; (III) an AMV reverse transcriptase having the amino acid sequence set forth in SEQ ID NO: 6, which has an amino acid sequence that is 70% or more identical to the entire amino acid sequence containing the amino acid substitution set forth in (1) above, provided that the amino acid substitution is maintained, and which has enzymatic activity.

2. The AMV reverse transcriptase according to claim 1, selected from any one 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 the amino acid substitution of (1) above and further contains one or more amino acid substitutions selected from the following (2) to (16); (2) an amino acid residue corresponding to the valine residue at position 377 of SEQ ID NO: 6 is substituted with an isoleucine residue; (3) an amino acid residue corresponding to the serine residue at position 550 of SEQ ID NO: 6 is substituted with a threonine residue; (4) an amino acid residue corresponding to the lysine residue at position 691 of SEQ ID NO: 6 is substituted with a glutamic acid residue; (5) an amino acid residue corresponding to the asparagine residue at position 776 of SEQ ID NO: 6 is substituted with a lysine residue; (6) an amino acid residue corresponding to the isoleucine residue at position 332 of SEQ ID NO: 6 is substituted with a valine residue; (7) an amino acid residue corresponding to the alanine residue at position 474 of SEQ ID NO: 6 is substituted with a threonine residue; (8) an amino acid residue corresponding to the threonine residue at position 716 of SEQ ID NO: 6 is substituted with a serine residue (9) The amino acid residue corresponding to the lysine residue at position 797 in SEQ ID NO:6 is replaced with an arginine residue. (10) The amino acid residue corresponding to the lysine residue at position 850 in SEQ ID NO:6 is replaced with an arginine residue. (11) The amino acid residue corresponding to the isoleucine residue at position 61 in SEQ ID NO:6 is replaced with a valine residue. (12) The amino acid residue corresponding to the valine residue at position 105 in SEQ ID NO:6 is replaced with an alanine residue. (13) The amino acid residue corresponding to the asparagine residue at position 623 in SEQ ID NO:6 is replaced with an aspartic acid residue. (14) The amino acid residue corresponding to the threonine residue at position 692 in SEQ ID NO:6 is replaced with an alanine residue. (15) The amino acid residue corresponding to the glycine residue at position 717 in SEQ ID NO:6 is replaced with an aspartic acid residue. (16) The amino acid residue corresponding to the lysine residue at position 850 in SEQ ID NO:6 is replaced with a glutamic acid residue.(ii) AMV reverse transcriptase having an amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution of (1) above, and further contains one or more amino acid substitutions selected from (2) to (16), 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 set forth in (1) to (16), and which has enzymatic activity; (iii) AMV reverse transcriptase having an amino acid sequence set forth in SEQ ID NO: 6, which contains the amino acid substitution of (1), and further contains one or more amino acid substitutions selected from (2) to (16), and which has an identity of 70% or more to the entire amino acid sequence, in which the amino acid substitutions are maintained, and which has enzymatic activity; 3. The AMV reverse transcriptase according to claim 2, wherein the one or more amino acid substitutions selected from (2) to (16) include at least the amino acid substitutions of (2) to (5).

4. The AMV reverse transcriptase according to claim 2, wherein the one or more amino acid substitutions selected from (2) to (16) include at least an amino acid substitution of (2) to (5) and one or more amino acid substitutions selected from (6) and (11) to (16).

5. The AMV reverse transcriptase according to claim 1, 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: 10, 20, 34, 36, 38 and 44; (V) an AMV reverse transcriptase having an amino acid sequence set forth in any one of SEQ ID NOs: 10, 20, 34, 36, 38 and 44, which contains one or more substitutions, deletions, insertions and additions 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: 10, 20, 34, 36, 38 and 44, and which has 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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