Mutant DNA polymerase, encoding gene, recombinant expression vector, recombinant bacteria and their application

Abstract

The present invention discloses a mutant DNA polymerase, a coding gene, a recombinant expression vector, a recombinant bacterium and its application, and belongs to the technical field of molecular biology. The mutant DNA polymerase of the present invention significantly improves the reverse transcription activity, amplification sensitivity and blood tolerance of the mutant enzyme by introducing one or more amino acid mutations into at least two motifs of motif I, motif II and motif III of the polymerase domain. The experimental results show that the reverse transcription activity of mutants M13, M14, M16, M17, M19, M20 and M22 is significantly higher than that of wild-type Z05 DNA polymerase, and the reverse transcription reaction time is shortened; at the same time, mutants M13, M14, M17, M19 and M22 can amplify low-copy RNA (10 1 copies), which is beneficial to improve the detection sensitivity; in terms of blood tolerance, the blood tolerance of mutants M13, M17 and M19 is significantly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to a mutant DNA polymerase, a coding gene, a recombinant expression vector, a recombinant bacterium and applications thereof. Background Art

[0002] Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. It can be used not only for basic research such as gene isolation, cloning, and nucleic acid sequence analysis, but also for disease diagnosis and sequencing. The basic principle of PCR is designed based on the mechanism of somatic cell replication established by the DNA double helix model and the characteristics of double-stranded DNA replication in vitro. It is similar to the natural replication process of DNA, and its specificity depends on oligonucleotide primers that are complementary to both ends of the target sequence. In general, PCR consists of three basic reaction steps: denaturation-annealing-extension: ① Denaturation of template DNA: After the template DNA is heated or treated with a denaturant for a period of time, the double-stranded DNA dissociates into single strands in order to bind with primers and prepare for the next round of reaction; ② Annealing (renaturation) of template DNA and primers: After the template DNA is denatured into a single strand, the temperature is lowered until the primers and the complementary sequences of the template DNA single strand are paired and combined to form a DNA template-primer complex; ③ Extension of primers: Under the action of DNA polymerase, the DNA template-primer complex undergoes semi-conservative replication according to the principle of base complementary pairing, and synthesizes a new semi-conservative replication chain complementary to the template DNA chain. The above denaturation-annealing-extension steps are repeated to obtain more DNA chains, which are used as templates for the next cycle.

[0003] From the above reaction process, it can be seen that PCR requires at least the participation of template DNA, primers, DNA polymerase and dNTPs (dATP, dCTP, dGTP and dTTP), and amplification is carried out in the presence of polymerase reaction buffer. Among them, DNA polymerase uses dNTPs as reaction substrates, connects the corresponding deoxynucleotides to the 3' end of the newly synthesized DNA chain along the 3'→5' direction of the template DNA, and extends the newly synthesized DNA chain along the 5'→3' direction. In this way, the newly synthesized DNA chain is not only complementary to the sequence of the template DNA chain, but also consistent with the original paired chain sequence of the template DNA chain. All known DNA polymerases synthesize DNA along the 5'→3' direction, so deoxynucleotides can only be added to the 3' terminal hydroxyl group of the existing DNA. It can be seen that in addition to the template as a sequence guide, DNA polymerase also requires primers to initiate synthesis.

[0004] Based on the similarity of amino acid sequences with Escherichia coli DNA polymerases I, II, and III, different DNA polymerases are divided into seven subfamilies: A, B, C, D, E, F, and RT on the basis of crystal structure analysis. Although the DNA polymerases of family A and family B reveal a common structural core of the nucleotide binding site in crystal structure analysis, the sequence motifs that are well conserved within the family are only weakly conserved between families. It is known that DNA polymerases of family A include T7 DNA polymerase, Taq DNA polymerase, Bst DNA polymerase, Tth DNA polymerase, Z05 DNA polymerase, etc. Z05 DNA polymerase was first disclosed in U.S. Pat. No. 5,455,170A (Roche), which is derived from thermophilic bacteria Thermus species Z05 (TZ05), encodes 834 amino acids, the full length of the encoding gene is 2502 bp, and the molecular weight is 94 kDa. Z05 DNA polymerase has 5'→3' polymerase activity and 5'→3' exonuclease activity, and also has reverse transcription activity, but lacks 3'→5' exonuclease activity. In order to improve the polymerase activity and reverse transcription activity, Chinese patent CN101528919B (Roche) discloses a mutant Z05 DNA polymerase, which contains the following motif in the polymerase domain: TGRLSSX b7 -X b8 -PNLQN, where X b7 is amino acid S, X b8It is an amino acid selected from G, T, R, K or L; experimental results show that the introduction of D580G / K / L / R / T mutations in wild-type Z05 DNA polymerase can improve polymerase activity, and the reverse transcription activity of the D580G / K mutant is significantly improved. Chinese patent CN110023493A (Toyobo) discloses a mutant Z05 DNA polymerase, which changes the amino acid selected from at least one of the 509th and 744th positions, and the change is to replace it with any one of histidine, arginine or lysine; experimental results show that compared with the wild-type Z05 DNA polymerase, the amplification rate of the Q509K / R or E744K / R mutant is increased and the tolerance to blood is enhanced, but the reverse transcription activity of the mutant enzyme was not verified in the experiment, and only the reverse transcription reaction (amplification of β-actin) of the TthDNA polymerase mutant (Q509R or E744K) belonging to the A family was verified to be completed in 5 minutes or even less than 1 minute. Chinese patent CN103987843B (Roche) also discloses a mutant Z05 DNA polymerase, corresponding to the amino acid at position 616 in the wild-type Z05 DNA polymerase as methionine (M), and / or corresponding to the amino acid at position 580 in the wild-type Z05 DNA polymerase as glycine (G); experimental results show that compared with the D580G / I709K parent enzyme, the mutant Z05 DNA polymerase (D580G / I709K / I616M) can amplify the target RNA more effectively without compromising the amplification efficiency of the target DNA. Therefore, in order to further improve the performance of Z05 DNA polymerase, it is necessary to design a new mutation combination to simultaneously improve the reverse transcription activity, amplification sensitivity and blood tolerance of the mutant enzyme. Summary of the invention

[0005] The purpose of the present invention is to provide a mutant DNA polymerase to improve the reverse transcription activity, amplification sensitivity and blood tolerance of the enzyme.

[0006] Secondly, the present invention provides a gene for editing mutant DNA polymerase.

[0007] Again, the present invention provides a recombinant expression vector comprising the gene of the above-mentioned edited mutant DNA polymerase.

[0008] At the same time, the present invention provides a recombinant bacterium comprising the above recombinant expression vector.

[0009] Finally, the present invention provides an application of a mutant DNA polymerase in preparing a detection kit.

[0010] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0011] A mutant DNA polymerase comprising at least the following motif in the polymerase domain:

[0012] Motif I: EX 11 -EVFRLAGHPFNLNSRDQLERV-LFDELRLPAX 12 -GKTX 13 -K, where X 11 Is glutamic acid (E) or aliphatic amino acid, X 12 is leucine (L) or basic amino acid, X 13 It is glutamine (Q) or basic amino acid;

[0013] Motif II: LSSSX 21 -PNLX 22 -X 23 -IPIRT, where X 21 is aspartic acid (D) or a basic amino acid, X 22 is glutamine (Q) or basic amino acid, X 23 It is asparagine (N) or aliphatic amino acid;

[0014] Motif III: SQX 31 -LAIPYX 32 -EAVAFX 33 -ERYFQ, where X 31 Is glutamic acid (E) or aromatic amino acid, X 32 is glutamic acid (E) or basic amino acid, X 33 It is isoleucine (I) or sulfur-containing amino acid;

[0015] At least one amino acid mutation exists in at least two of the motifs of motif I, motif II, and motif III;

[0016] The DNA polymerase is Z05 DNA polymerase.

[0017] The amino acid sequence of the wild-type Z05 DNA polymerase is shown in SEQ ID NO: 1. 11 Corresponding to the 474th amino acid in the amino acid sequence, X 12 Corresponding to the 505th amino acid in the amino acid sequence, X 13 The corresponding amino acid is the 509th amino acid in the amino acid sequence. 21 Corresponding to the 580th amino acid in the amino acid sequence, X 22Corresponding to the 584th amino acid in the amino acid sequence, X 23 The corresponding amino acid is the 585th amino acid in the amino acid sequence. 31 Corresponding to the 683rd amino acid in the amino acid sequence, X 32 Corresponding to the 689th amino acid in the amino acid sequence, X 33 The corresponding amino acid is at position 695 of the amino acid sequence. In the amino acid sequence shown in SEQ ID NO: 1, one or more amino acids at the above specific positions are mutated, and the mutation is caused by substitution with other amino acids.

[0018] As a preferred embodiment, the mutant DNA polymerase is a mutant Z05 DNA polymerase, and the amino acid sequence of the mutant Z05 DNA polymerase has more than 90% homology with the amino acid sequence of the wild-type Z05 DNA polymerase. That is to say, in addition to one or more amino acid mutations at specific positions of motif I, motif II, and motif III, the mutant Z05 DNA polymerase may also have amino acid mutations and / or deletions at other positions. For example, in order to reduce or remove the 5'→3' exonuclease activity of the enzyme, multiple amino acids at the N-terminus are removed; or, in order to terminate the reaction at low temperature, the 710th glutamic acid (E) in the amino acid sequence is mutated to other amino acids. Further preferably, the amino acid sequence of the mutant Z05 DNA polymerase has more than 96% homology with the amino acid sequence of the wild-type Z05 DNA polymerase. Even more preferably, in the mutant Z05 DNA polymerase, only one or more amino acids at specific positions of motif 1, motif 2, and motif 3 are mutated, and amino acids at other positions are not mutated.

[0019] As a preferred embodiment, the aliphatic amino acid is selected from any one of glycine (G), alanine (A), valine (V), leucine (L), and isoleucine (I), and more preferably glycine (G) or leucine (L).

[0020] As a preferred embodiment, the basic amino acid is selected from any one of arginine (R), lysine (K), and histidine (H), and more preferably arginine (R) or lysine (K).

[0021] As a preferred embodiment, the aromatic amino acid is selected from any one of phenylalanine (F), tyrosine (Y), and tryptophan (W), and more preferably tyrosine (Y).

[0022] As a preferred embodiment, the sulfur-containing amino acid is selected from any one of cysteine ​​(C) and methionine (M), and more preferably methionine (M).

[0023] As a preferred embodiment, in the motif I of the mutant Z05 DNA polymerase, X 11 is glutamate (E) or leucine (L), X 12 is leucine (L) or arginine (R), X 13 It is either glutamine (Q) or arginine (R).

[0024] As a preferred embodiment, in the motif II of the mutant Z05 DNA polymerase, X 21 is aspartic acid (D) or lysine (K), X 22 is glutamine (Q) or lysine (K), X 23 It is either asparagine (N) or glycine (G).

[0025] As a preferred embodiment, in the motif III of the mutant Z05 DNA polymerase, X 31 is glutamate (E) or tyrosine (Y), X 32 is glutamate (E) or arginine (R), X 33 isoleucine (I) or methionine (M).

[0026] As a preferred embodiment, the mutant DNA polymerase comprises at least the following motif in the polymerase domain:

[0027] Motif I:EEEVFRLAGHPFNLNSRDQLE-RVLFDELRLPAX 12 -GKTX 13 -K, where X 12 is leucine (L) or basic amino acid, X 13 It is glutamine (Q) or basic amino acid;

[0028] Motif II: LSSSX 21 -PNLX 22 -NIPIRT, where X 21 is aspartic acid (D) or a basic amino acid, X 22 It is glutamine (Q) or basic amino acid;

[0029] Motif III: SQX 31 -LAIPYEEAVAFIERYFQ, where X 31 It is glutamic acid (E) or aromatic amino acids;

[0030] At least one amino acid mutation exists in at least two of the motifs of motif I, motif II, and motif III;

[0031] The DNA polymerase is Z05 DNA polymerase.

[0032] As a preferred embodiment, in the motif I of the mutant Z05 DNA polymerase, X 13 is arginine (R), X 12 No mutation occurs; in motif II, X 21 is lysine (K), X 22 No mutation occurs; in motif III, X 31 No mutation occurs, that is, the Q509R / D580K mutant M13.

[0033] As a preferred embodiment, in the motif I of the mutant Z05 DNA polymerase, X 12 , X 13 No mutation occurs; in motif II, X 22 is lysine (K), X 21 No mutation occurs; in motif III, X 31 It is tyrosine (Y), that is, the Q584K / E683Y mutant M14.

[0034] As a preferred embodiment, in the motif I of the mutant Z05 DNA polymerase, X 12 is arginine (R), X 13 No mutation occurs; in motif II, X 22 is lysine (K), X 21 No mutation occurs; in motif III, X 31 No mutation occurs, that is, the L505R / Q584K mutant M15.

[0035] As a preferred embodiment, in the motif I of the mutant Z05 DNA polymerase, X 12 is arginine (R), X 13 No mutation occurs; in motif II, X 21 , X 22 No mutation occurs; in motif III, X 31 It is tyrosine (Y), that is, the L505R / E683Y mutant M16.

[0036] As a preferred embodiment, in the motif I of the mutant Z05 DNA polymerase, X 12 is arginine (R), X 13 is arginine (R); in motif II, X 21 is lysine (K), X22 No mutation occurs; in motif III, X 31 No mutation occurs, that is, the L505R / Q509R / D580K mutant M17.

[0037] As a preferred embodiment, in the motif I of the mutant Z05 DNA polymerase, X 13 is arginine (R), X 12 No mutation occurs; in motif II, X 21 is lysine (K), X 22 No mutation occurs; in motif III, X 31 It is tyrosine (Y), that is, the Q509R / D580K / E683Y mutant M18.

[0038] As a preferred embodiment, in the motif I of the mutant Z05 DNA polymerase, X 13 is arginine (R), X 12 No mutation occurs; in motif II, X 22 is lysine (K), X 21 No mutation occurs; in motif III, X 31 It is tyrosine (Y), that is, the Q509R / Q584K / E683Y mutant M19.

[0039] As a preferred embodiment, in the motif I of the mutant Z05 DNA polymerase, X 12 is arginine (R), X 13 No mutation occurs; in motif II, X 22 is lysine (K), X 21 No mutation occurs; in motif III, X 31 It is tyrosine (Y), that is, the L505R / Q584K / E683Y mutant M20.

[0040] As a preferred embodiment, in the motif I of the mutant Z05 DNA polymerase, X 12 is arginine (R), X 13 is arginine (R); in motif II, X 21 , X 22 No mutation occurs; in motif III, X 31 It is tyrosine (Y), that is, the L505R / Q509R / E683Y mutant M21.

[0041] As a preferred embodiment, in the motif I of the mutant Z05 DNA polymerase, X 12 is arginine (R), X 13 No mutation occurs; in motif II, X 21is lysine (K), X 22 No mutation occurs; in motif III, X 31 It is tyrosine (Y), that is, the L505R / D580K / E683Y mutant M22.

[0042] As a preferred embodiment, the mutant Z05 DNA polymerase may include other non-substitution modifications. For example, a heat-reversibly inactivated chemical modification, the modified mutant Z05 DNA polymerase loses polymerase activity at low temperatures, but the chemical modification is destroyed at high temperatures, and the polymerase activity is reactivated, thereby being suitable for hot-start PCR reactions. Further preferably, the heat-reversibly inactivated chemically modified mutant Z05 DNA polymerase is prepared by reacting the mutant Z05 DNA polymerase with a dicarboxylic anhydride having the general formula (1) or (2) at an alkaline pH value and below 25°C (see patent CN1282741C, Roche):

[0043]

[0044] In formula I, R1 and R2 are selected from hydrogen or an organic group, which may be connected;

[0045] In formula II, R1 and R2 are selected from organic groups, they may be linked together, and hydrogen is cis.

[0046] As a preferred embodiment, the mutant Z05 DNA polymerase can be fused with Sso7d, PCNA, or with a protein tag such as a His tag, a GST tag, etc. to obtain a fusion protein with improved enzyme activity.

[0047] A method for preparing a mutant DNA polymerase comprises: introducing a mutation into a gene encoding a wild-type Z05 DNA polymerase, and then using a protein engineering method to prepare a mutant Z05 DNA polymerase with improved performance.

[0048] As a preferred embodiment, the introduced mutation can use the site-directed mutagenesis method based on SOE PCR (overlap extension PCR) technology, which uses primers with complementary ends to form overlapping chains in the PCR products. In the subsequent amplification reaction, the amplified fragments from different sources are overlapped and spliced ​​together by extending the overlapping chains. Among them, the design of overlapping complementary primers is the key to the success of the overlap extension PCR technology. The overlapping region between the connected primers is preferably 15-20 bases, and AT-rich sequences are avoided as much as possible in the overlapping region, because mismatches are very likely to occur in the AT-rich region of the joint region. At the same time, considering the amplification efficiency and mismatch rate of DNA polymerase, the number of cycles of each round of reaction is preferably controlled at 20-25.

[0049] As a preferred embodiment, the introduced mutation can use a site-directed mutagenesis method based on the Inverse PCR technology. For example, using KOD-Plus-Mutagenesis Kit (Toyobo), using a plasmid as a template, using a reverse primer to perform PCR, amplifying the full length of the plasmid, and introducing a mutation at a specific position by adding a primer of a replacement sequence. Specifically, the following steps are included: (1) denaturing the plasmid into which the target gene is inserted, annealing the mutation primer to the plasmid, and then using KOD DNA polymerase for an extension reaction; (2) repeating step (1) 15 times in a cycle; (3) selectively cutting the template plasmid using the restriction endonuclease DpnI; (4) phosphorylating the resynthesized gene, and cyclizing it after ligation; (5) transforming the cyclized gene into a host cell such as Escherichia coli, thereby obtaining a transformant carrying a plasmid into which the target mutation is introduced.

[0050] As a preferred embodiment, the mutant Z05 DNA polymerase has a significantly increased reverse transcription activity and a significantly shortened reverse transcription reaction time compared to the wild-type Z05 DNA polymerase or the mutant Z05 DNA polymerase containing only one amino acid mutation at the above-mentioned specific position. For example, the reverse transcription reaction can be completed in 5 minutes, preferably in 3 minutes, or even less than 1 minute. For example, the reverse transcription activity can be determined by detecting the hepatitis C virus (HCV) transcription product generated from the first 800 bases of the HCV genotype Ib 5'NTR of pSP64 poly (A) (Promega) by real-time RT-PCR reaction (see patent CN103987843B, Roche). Compared with the wild-type Z05 DNA polymerase or the mutant Z05 DNA polymerase containing only one amino acid mutation at the above-mentioned specific position, the mutant Z05 DNA polymerase having at least one amino acid mutation in at least two motifs of motif I, motif II, and motif III will amplify the transcription product with higher efficiency, or will require a smaller number of PCR cycles to amplify the transcription product (i.e., showing a lower Ct value). In addition, the mutant Z05 DNA polymerase has enhanced tolerance to blood, for example, it can tolerate 10 μL plasma / 25 μL reaction system. Meanwhile, the mutant Z05 DNA polymerase with improved reverse transcription activity also has improved long-chain RNA template replication ability.

[0051] A gene encoding the mutant DNA polymerase. Specifically, a gene encoding the mutant Z05 DNA polymerase L505R / Q584K, L505R / E683Y, Q509R / D580K, Q584K / E683Y, L505R / Q509R / D580K, L505R / Q509R / E683Y, L505R / D580K / E683Y, L505R / Q584K / E683Y, Q509R / D580K / E683Y, Q509R / Q584K / E683Y.

[0052] A recombinant expression vector comprising the gene encoding the mutant DNA polymerase. Specifically, the preparation method of the recombinant expression vector is: introducing the gene encoding the mutant DNA polymerase into the expression vector, and screening to obtain the recombinant expression vector.

[0053] A recombinant bacterium comprising the above recombinant expression vector. Specifically, the preparation method of the recombinant bacterium is: transforming the recombinant expression vector comprising the gene encoding the mutant DNA polymerase into a host bacterium, and screening to obtain the recombinant bacterium.

[0054] As a preferred embodiment, the preparation of the recombinant bacteria may include the following steps: transforming the recombinant expression vector into a host such as Escherichia coli, and then coating it on an agar medium containing drugs such as ampicillin, and after the colonies are formed, inoculating the colonies on LB medium, 2×YT medium, and culturing them at 37°C for 12-20 hours, breaking the bacteria (for example, using physical crushing methods such as ultrasonic treatment, French press, glass bead crushing, or lysozyme lysis), and extracting a crude enzyme solution; heat-treating the crude enzyme solution (for example, treating it at 80°C for 30 minutes), then using PEI to remove nucleic acids, using ammonium sulfate precipitation to remove some impurities, recovering the DNA polymerase fraction, and then using Sephadex G-25 gel filtration and other methods for desalting, and then using a heparin agarose column for separation and purification to obtain a purified enzyme sample.

[0055] A mutant DNA polymerase is used in the preparation of a detection kit. Specifically, the detection kit can implement a method for nucleic acid amplification, comprising: contacting the mutant DNA polymerase with a primer, a polynucleotide template (i.e., a target nucleic acid), and nucleoside triphosphates under conditions suitable for primer extension, thereby generating an extension product.

[0056] As a preferred embodiment, the polynucleotide template can be an RNA template or a DNA template. The polynucleotide template can come from any type of biological sample.

[0057] As a preferred embodiment, the nucleoside triphosphates include conventional deoxyribonucleoside triphosphates (such as dATP, dGTP, dTTP, dCTP and dUTP) and unconventional ribonucleotides or labeled nucleotides.

[0058] As a preferred embodiment, the primer or polynucleotide template may contain one or more nucleotide analogs.

[0059] As a preferred embodiment, the nucleic acid amplification method can adopt conventional polymerase chain reaction (PCR), isothermal amplification reaction (such as MDA, HDA, SDA, LAMP, etc.), sequencing reaction (such as Illumina sequencing, Sanger sequencing, 454 sequencing reaction), etc.

[0060] As a preferred embodiment, the conditions suitable for primer extension include Mg 2+ and / or Mn 2+ , and the temperature and pH value suitable for the mutant DNA polymerase to exert the best 5'→3' polymerase activity. In the nucleic acid amplification method, the amplification temperature, time, cycle number and other conditions can be determined according to the type / length and base sequence of the polynucleotide template to be amplified.

[0061] A detection kit comprises: at least one container, wherein the container contains the mutant DNA polymerase.

[0062] As a preferred embodiment, the detection kit may further include one or more of the following containers:

[0063] (a) a container comprising a primer that hybridizes to a predetermined polynucleotide template under conditions suitable for primer extension;

[0064] (b) a container comprising nucleoside triphosphates;

[0065] (c) A container containing a buffer solution suitable for primer extension.

[0066] Beneficial effects of the present invention:

[0067] The present invention provides a mutant Z05 DNA polymerase, which can significantly improve the reverse transcription activity, amplification sensitivity and blood tolerance of the mutant enzyme by introducing one or more amino acid mutations in at least two motifs of motif I, motif II and motif III of the polymerase domain. Experimental results show that compared with the wild-type Z05 DNA polymerase, the reverse transcription activity of mutants M13, M14, M16, M17, M19, M20 and M22 is significantly improved, and the reverse transcription reaction time is shortened; at the same time, mutants M13, M14, M17, M19 and M22 can amplify low-copy RNA (101 copies), especially mutants M13, M17, M19 and M22 have stronger ability to amplify low-copy RNA (amplification of 10 0 copies), which is beneficial to improve the detection sensitivity; in terms of blood tolerance, mutants M13, M17 and M19 have good blood tolerance, especially mutants M17 and M19 can tolerate 10μL plasma / 25μL reaction system.

[0068] The present invention also provides a gene for editing the mutant Z05 DNA polymerase, a recombinant expression vector and a recombinant bacterium, and the use of the mutant Z05 DNA polymerase or the heat-reversibly inactivated chemically modified mutant Z05 DNA polymerase in the preparation of a detection kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The RT-PCR results of the mutant enzyme in Experimental Example 1 of the present invention;

[0070] Figure 2 This is the reverse transcription activity detection result of the mutant enzyme in Experimental Example 2 of the present invention. DETAILED DESCRIPTION

[0071] The following examples are only used to further illustrate the present invention, but do not constitute any limitation to the present invention.

[0072] In order to facilitate understanding of the technical solution of the present invention, some terms are defined below.

[0073] The term "gene" refers to a DNA sequence containing the control and coding sequences necessary for the production of a recoverable biologically active polypeptide or precursor.

[0074] The term "wild type" refers to a gene or gene product isolated from a natural source. The term also refers to a recombinant form of a natural protein prepared by molecular biology techniques, whose amino acid sequence is identical to that of the natural protein.

[0075] The term "mutant" refers to a gene whose nucleotide sequence has been altered or a gene product whose amino acid sequence has been altered, resulting in a gene product having functional properties different from those of the native or wild-type gene or gene product.

[0076] The term "mutation" refers to a single amino acid substitution, and point mutations are preferably introduced into the amino acid sequence by changing the appropriate codons in the encoding DNA. The amino acid positions are numbered according to the full-length sequence of the protein, and the regions containing the mutations are from the full-length sequence. Nucleotides and point mutations in DNA sequences are represented similarly.

[0077] The term "primer" refers to a natural or synthetic oligonucleotide that can serve as a starting point for synthesis when placed under conditions that initiate primer extension. The primer is preferably a single-stranded deoxyribonucleotide, and the appropriate length of the primer depends on the intended use of the primer. The primer does not need to have the exact sequence of the reaction template, but must be sufficiently complementary to hybridize with the polynucleotide template.

[0078] The term "amplification reaction system" refers to an aqueous solution containing various reagents for amplifying a polynucleotide template, wherein the reagents include a polynucleotide template, an enzyme, a primer, a salt, a nucleoside triphosphate, and an aqueous buffer. In different cases, the amplification reaction system can be a complete or incomplete reaction system. The amplification reaction in the invention is mainly a polymerase chain reaction (PCR), and the amplification reaction system also refers to a PCR system or a PCR mixture. The amplification reaction system can be included in a reaction system for amplifying RNA in a coupled reverse transcription / amplification reaction.

[0079] The term "reverse transcription reaction system" refers to an aqueous solution containing various reagents for reverse transcription of target RNA, including RNA template, enzyme, primer, salt, nucleoside triphosphate and aqueous buffer. In different cases, the reverse transcription reaction system can be a complete or incomplete reaction system.

[0080] The term "buffer" refers to a solution containing a buffering agent or a buffer mixture, optionally containing divalent cations and monovalent cations.

[0081] In the invention, the conventional techniques of molecular biology and nucleic acid chemistry involved are all prior art and are described in detail in the literature, see Molecular Cloning Laboratory Manual, Sambrook et al., 1989; Oligonucleotide Synthesis, MJ Gait et al., 1984; Nudeic Acid Hybridization, BD Hames and SJ Higgins, 1984; Basic Methods in Molecular Biology, Elsevier; Current Protocols in Molecular Biology, John Wiley and Sons; Methods in Enzymology, American Academic Press.

[0082] The specific embodiments of the present invention are further described in detail below in conjunction with the examples. It should be noted that the detailed description of the following examples is only used to illustrate the technical solution of the present invention, and is not used to limit the scope of protection of the present invention, that is, the present invention is not limited to the specific embodiments described in the examples, and especially covers any modification, replacement and improvement of raw materials and means without departing from the spirit of the present invention. In the absence of conflict, the features in the embodiments and the examples can be combined with each other. The raw materials, instruments, etc. used in the following examples, comparative examples and experimental examples are all commercially available products.

[0083] Example 1

[0084] The mutant Z05 DNA polymerase of this example (L505R / E683Y, i.e., mutant M16, with an amino acid sequence as shown in SEQ ID NO: 32) is prepared in the following steps:

[0085] (1) Construction of recombinant strains

[0086] ① Obtain the target gene

[0087] The plasmid containing the wild-type Z05 DNA polymerase gene synthesized by Bio-Industry (as shown in SEQ ID NO: 2) was used as a template, and primer pairs NZ-F / EZ-R, Z2-R / Z2-F, and Z8-R / Z8-F (see Table 4) were used for PCR amplification, and the amplification reaction system was shown in Table 1. The PCR program was as follows: first, a pre-reaction was performed at 95°C for 5 min, and then 95°C, 15s→65°C, 15s→72°C, 1 kb / min were repeated for 30 cycles to obtain small fragments of the target gene with mutation sites, and then these small fragments were used as templates at a molar ratio of 1:1:1, and overlap PCR was performed with primer pair NZ-F / EZ-R (reaction system and procedure as above) to obtain the target gene with the corresponding mutation site.

[0088] The sequences of the primer pair NZ-F / EZ-R are as follows:

[0089] NZ-F: 5'-GGAATTCCATATGAAAGCGATGCTGCCACTCTTCG-3' (SEQ ID NO: 3);

[0090] EZ-R: 5'-CGGAATTCTTAGCCCTTCGCGCTGAGCCAGTC-3' (SEQ ID NO: 4).

[0091] Table 1 Amplification reaction system

[0092] Components Addition volume μL Template DNA 0.5 10 μM Primer F 1 10 μM Primer R 1 PrimeSTAR 0.5 dNTP(10mM) 1 10×PS buffer 10 water 36

[0093] ② Restriction digestion and ligation

[0094] The amplified target gene sequence with mutation sites and expression vector pET-30a were digested with restriction endonucleases NdeI and EcoRI at 37°C for 2 h, respectively. The digested fragments were recovered and ligated with T4 DNA ligase. The transformation was then performed at 16°C for 2 h.

[0095] ③Conversion

[0096] The ligation product was transformed into the expression host BL21 (DE3), plated and screened by colony PCR to obtain appropriate positive clones, and sequencing was performed to obtain recombinant strains containing the corresponding mutation sites. The PCR screening system is shown in Table 2 below. 14 μL was dispensed into a tube and 1 μL of bacterial solution was added for detection. The program selection was: first, a pre-reaction at 95°C for 5 min was performed, and then 95°C, 30 s→65°C, 30 s→72°C, 2 min 50 s was repeated for 28 cycles. After the reaction, agarose gel detection was performed to screen out positive clones.

[0097] Table 2 PCR screening system

[0098] Components Addition volume μL water 20 10uM Primer NZ-F 1 10uM Primer EZ-R 1 2×Taq Mix 25

[0099] (2) Expression and purification of recombinant proteins

[0100] The recombinant strain with correct sequencing was inoculated for fermentation to prepare bacteria. The inoculation volume was 1% in LB medium and cultured at 37°C with shaking until OD 600 =0.6-0.8, add 1 mM IPTG for induction and continue culturing for 4 h, then collect the bacteria.

[0101] The bacteria were resuspended in 50mM Tris-HCl (pH 7.5), 0.3M NaCl, 1mM EDTA, and 1mM DTT buffer, and then the cells were broken by ultrasonic treatment and the supernatant was collected by centrifugation; PEI was then used to remove nucleic acids, and ammonium sulfate precipitation was used to remove some impurities to obtain a crude sample; the crude sample was purified in sequence through a Butyl chromatography column, a Q chromatography column, an SP chromatography column, and a heparin chromatography column to obtain a qualified polymerase sample for mutant screening.

[0102] Example 2

[0103] According to the same method as in Example 1, mutants shown in Table 3 were constructed respectively. The primers corresponding to the mutation sites are shown in Table 4. The primers correspond to SEQ ID NOs: 5-28 in the sequence table respectively.

[0104] Table 3 Constructed Z05 DNA polymerase mutants

[0105]

[0106]

[0107] Table 4 Primers corresponding to each mutation site

[0108]

[0109]

[0110] Experimental Example 1

[0111] Reverse transcription / amplification efficiency detection: Using the mutant Z05 DNA polymerase prepared in Example 1, a one-step RT-PCR starting from RNA was performed to test the mutation effect. Using the reaction buffer attached to Tth DNA polymerase (Roche), MS2 RNA (Roche, 800 ng / μL) was used as a template, and the probe method was used for detection to compare the differences between the wild-type Z05 DNA polymerase and each mutant. The reaction system is shown in Table 5. The reaction procedure is: first, reverse transcription reaction is performed at 60°C for 5 min, then pre-reaction is performed at 95°C for 5 min, 95°C for 15 s and 60°C for 30 s are repeated for 50 cycles, and real-time amplification is performed using a fluorescent quantitative PCR instrument.

[0112] Table 5 RT-PCR reaction system

[0113]

[0114]

[0115] The sequences of primer pair RT1F / RT1R and probe RT1 are as follows:

[0116] RT1F: 5'-GGTTCACCTTCAAGAGTTTC-3' (SEQ ID NO: 29);

[0117] RT1R: 5'-TCCTGACATGTAGGAGCATC-3' (SEQ ID NO: 30);

[0118] RT1: 5'-TGAGAGCCGTACGTCAGGTCG-3' (SEQ ID NO: 31).

[0119] According to the experimental design, the single point mutation effect was first detected, and the comparative analysis results are shown in Table 6 below.

[0120] Table 6 Experimental results of single point mutation

[0121] Mutant naming Mutation site Mutation significance M1 474 - M2 505 + M3 509 + M4 580 ++ M5 584 + M6 585 - M7 616 - M8 683 ++ M9 689 - M10 695 - M11 709 - M12 744 ＝

[0122] Note: “+” indicates a positive mutation, which can improve the efficiency of RT-PCR reaction; “++” indicates a better improvement than “+”; “-” indicates a reverse mutation, which will reduce the efficiency of RT-PCR reaction; “=” indicates no effect, which is consistent with the effect of the wild-type enzyme.

[0123] As can be seen from Table 6, most mutations have positive significance. Then, based on the results of single-point mutations, multiple-point mutations were combined to further improve the mutation effect. The experimental results are shown in Figure 1 shown.

[0124] from Figure 1 It can be seen that the effects of most combined mutations are better than those of wild-type Z05 DNA polymerase and single-point mutant Z05 DNA polymerase, among which mutants M13, M14, M16, M17, M19, M20 and M22 have the best effects. These seven mutants will be used as the objects to study their reverse transcription activity, plasma tolerance and amplification ability of low-copy templates.

[0125] Experimental Example 2

[0126] Reverse transcription activity detection: Using the RT-PCR reaction buffer attached to Tth DNA polymerase (Roche), with MS2 RNA (Roche, 800 ng / μL) as the template, the wild-type Z05 DNA polymerase and mutants M13, M14, M16, M17, M19, M20, and M22 were tested for reverse transcription activity. The reaction system is shown in Table 7 below. The reaction extent is: first, reverse transcription is performed at 60°C for 5 min, and then PCR amplification is performed using cDNA as a template to detect the number of template copies, thereby determining the reverse transcription activity.

[0127] Table 7 Reverse transcription reaction system

[0128] Components Addition volume μL MS2 RNA 0.5 10 μM RT1F 0.75 10 μM RT1R 0.75 Enzyme (0.01 mg / mL) 2 dNTPs (10 mM each) 1 5×RT-PCR buffer 5 25mM MnOAc 2.5 water 12.5

[0129] The PCR amplification system is shown in Table 8 below. In this reaction system, a DNA polymerase without reverse transcription activity should be used to eliminate the interference of residual RNA. After comprehensive analysis, PrimeSTAR was selected as the amplification enzyme for detection. The PCR procedure is: first, a pre-reaction at 95°C for 5 minutes is performed, and then 95°C for 15 seconds and 60°C for 30 seconds are repeated for 40 cycles, and real-time detection is performed using a fluorescent PCR instrument. The analysis results are summarized as follows: Figure 2 shown.

[0130] Table 8 PCR amplification reaction system

[0131]

[0132]

[0133] from Figure 2 It can be seen that the reverse transcription activity of each mutant is improved, among which the effect of combined mutation is better than that of wild-type Z05 DNA polymerase and single-point mutation Z05 DNA polymerase.

[0134] Comparing the results of Experimental Example 1 and Experimental Example 2, it can be seen that the detection results of reverse transcription activity are basically consistent with the RT-PCR results, so it can be inferred that the decrease in the Ct value of the RT-PCR reaction is caused by the increase in reverse transcription activity.

[0135] Experimental Example 3

[0136] Low-copy template RNA amplification ability test: In order to test the low-copy RNA amplification ability, each mutant was used to amplify RNA with different copy numbers to observe the amplification results. The reaction system and procedure were as shown in Experimental Example 1. The RNA copy numbers in the 25 μL reaction system reached 10 5 , 10 4 , 10 3 , 10 2 , 10 1 and 10 0 , the results are shown in Table 9 below.

[0137] Table 9 Amplification ability of mutants for low copy RNA

[0138] Copy number WT M13 M14 M17 M19 M20 M22 <![CDATA[10 5 ]]> 27.7 24.7 26.28 23.11 24.96 27.4 27.96 <![CDATA[10 4 ]]> 30.8 29.63 29.49 26.35 28.18 30.89 29.18 <![CDATA[10 3 ]]> 35.58 34.58 34.03 30.76 32.05 36.88 32.05 <![CDATA[10 2 ]]> 40.2 37.21 37.39 34 36.49 44.59 36.49 <![CDATA[10 1 ]]> / 39.49 39.12 35.68 38.11 / 40.11 <![CDATA[10 0 ]]> / 40.25 / 36.72 40.11 / 41.31 NTC / / / / / / /

[0139] Note: “ / ” means not detected.

[0140] As can be seen from Table 9, mutants M13, M14, M17, M19 and M22 can all be used to amplify low-copy RNA, especially mutants M13, M17, M19 and M22, which have stronger ability to amplify low-copy RNA.

[0141] Experimental Example 4

[0142] Plasma tolerance test: The plasma tolerance of mutants was tested by directly adding different amounts of plasma to the reaction system. The RT-PCR reaction system and procedure were as shown in Experimental Example 1. 0, 1, 3, 5, 7.5 and 10 μL of plasma were added to the 25 μL reaction system to test the tolerance of each mutant. The real-time amplification results are summarized in Table 10 below.

[0143] Table 10 Plasma tolerance of mutants

[0144] Plasma dosage WT M13 M17 M19 M22 0μL 22.66 19.58 15.51 16.06 21.38 1μL 40.87 36.22 34.86 35.18 38.22 3μL / 40.37 35.31 34.56 40.37 5μL / 42.53 35.45 37.23 / 7.5μL / / 36.17 36.64 / 10μL / / 43.28 44.03 /

[0145] Note: “ / ” means not detected.

[0146] As can be seen from Table 10, the four selected mutants all have a certain degree of plasma tolerance, among which mutants M13, M17 and M19 have better plasma tolerance, especially mutants M17 and M19 can tolerate 10 μL plasma / 25 μL reaction system.

[0147] It can be seen from the above embodiments and experimental examples that the mutant Z05 DNA polymerase of the present invention significantly improves the reverse transcription activity, amplification sensitivity and blood tolerance of the mutant enzyme by introducing one or more amino acid mutations in at least two of the motifs I, motif II and motif III of the polymerase domain. The experimental results show that the reverse transcription activity of the mutants M13, M14, M16, M17, M19, M20 and M22 is significantly higher than that of the wild-type Z05 DNA polymerase; at the same time, the mutants M13, M14, M17, M19 and M22 can be used to amplify low-copy RNA (10 1 copies), especially mutants M13, M17, M19 and M22 have strong ability to amplify low-copy RNA (amplification of 10 0 copies), which is beneficial to improve the detection sensitivity; in terms of blood tolerance, mutants M13, M17 and M19 have good blood tolerance, especially mutants M17 and M19 can tolerate 10μL plasma / 25μL reaction system.

[0148] The above are only specific embodiments of the present invention and do not limit the protection scope of the present invention. Without departing from the technical concept of the present invention, it is clear to those skilled in the art that the implementation of the present invention may be subject to various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. Sequence Listing <110> Zhengzhou Sikun Bioengineering Co., Ltd. <120> Mutant DNA polymerase, encoding gene, recombinant expression vector, recombinant bacteria and their application <160> 32 <170> SIPOSequenceListing 1.0 <210> 1 <211> 834 <212> PRT <213> Thermus species Z05 <400> 1 Met Leu Ala Met Leu Pro Leu Pro Gly Pro Leu Gly Ala Val Leu Leu 1 5 10 15 Val Ala Gly His His Leu Ala Thr Ala Thr Pro Pro Ala Leu Leu Gly 20 25 30 Leu Thr Thr Ser Ala Gly Gly Pro Val Gly Ala Val Thr Gly Pro Ala 35 40 45 Leu Ser Leu Leu Leu Ala Leu Leu Gly Ala Gly Thr Leu Ala Val Pro 50 55 60 Val Val Pro Ala Ala Leu Ala Pro Ser Pro Ala His Gly Ala Thr Gly 65 70 75 80 Path Thr Leu Path Gly Path Path Pro Thr Pro Gly Path Pro Pro Path Gly 85 90 95 Leu Ala Leu Ile Leu Gly Leu Val Ala Leu Leu Gly Pro Thr Ala Leu 100 105 110 Gly Val Pro Gly Pro Gly Ala Ala Ala Val Leu Ala Thr Leu Ala Leu 115 120 125 Leu Ala Gly Ala Gly Gly Thr Gly Val Ala Ile Leu Thr Ala Ala Ala 130 135 140 Ala Leu Thr Gly Leu Val Ser Ala Ala Val Ala Val Leu His Pro Gly 145 150 155 160 Gly His Leu Ile Thr Pro Gly Thr Leu Thr Gly Leu Thr Gly Leu Leu 165 170 175 Pro Gly Gly Thr Val Path Pro Path Path Leu Val Gly Path Pro Ser Path 180 185 190 Ala Leu Pro Gly Val Leu Gly Ile Gly Gly Leu Thr Ala Leu Leu Leu 195 200 205 Leu Leu Gly Thr Gly Ser Leu Gly Ala Ile Leu Leu Ala Leu Ala Ala 210 215 220 Val Leu Pro Gly Ser Val Ala Gly Ala Ile Leu Ala His Leu Gly Ala 225 230 235 240 Leu Leu Leu Ser Leu Gly Leu Ser Ala Val Ala Ser Ala Leu Pro Leu 245 250 255 Gly Val Ala Pro Ala Ala Ala Gly Pro Ala Ala Gly Gly Leu Ala 260 265 270 Path Pro Leu Gly Path Leu Gly Pro Gly Ser Leu Leu His Gly Pro Gly 275 280 285 Leu Leu Gly Path Pro Path Pro Leu Gly Gly Path Pro Thr Pro Pro Pro 290 295 300 Gly Gly Path Pro Val Gly Pro Val Leu Ser Path Pro Gly Pro Met Thr 305 310 315 320 Ala Gly Leu Leu Ala Leu Ala Ala Cys Leu Gly Gly Ala Val His Ala 325 330 335 Way Leu Way Pro Leu Way Gly Leu Leu Way Leu Leu Gly Val Way Gly 340 345 350 Leu Leu Ala Leu Ala Leu Ala Val Leu Ala Leu Ala Gly Gly Leu Ala 355 360 365 Leu Ala Pro Ser Ala Ala Pro Met Leu Leu Ala Thr Leu Leu Ala Pro 370 375 380 Ser Ala Thr Thr Pro Gly Gly Val Ala Ala Ala Thr Gly Gly Gly Thr 385 390 395 400 Thr Gly Ala Ala Ala His Ala Ala Leu Leu Ala Gly Ala Leu Gly Gly 405 410 415 Way Leu Leu Gly Way Leu Leu Gly Gly Gly Leu Leu Leu Thr Leu Thr 420 425 430 Gly Gly Val Gly Leu Pro Leu Ser Ala Val Leu Ala His Met Gly Ala 435 440 445 Thr Gly Val Ala Leu Ala Val Ala Thr Leu Leu Ala Leu Ser Leu Gly 450 455 460 Leu Ala Gly Gly Ile Ala Ala Leu Gly Gly Gly Val Pro Ala Leu Ala 465 470 475 480 Gly His Pro Pro Path Leu Path Ser Path Path Gly Leu Gly Path Val Leu 485 490 495 Pro Ala Gly Leu Ala Leu Pro Ala Leu Gly Leu Thr Gly Leu Thr Gly 500 505 510 Leu Ala Ser Thr Ser Ala Ala Val Leu Gly Ala Leu Ala Gly Ala His 515 520 525 Pro Ile Val Gly Leu Ile Leu Gly His Ala Gly Leu Thr Leu Leu Leu 530 535 540 Ala Thr Thr Val Ala Pro Leu Pro Gly Leu Val His Pro Ala Thr Gly 545 550 555 560 Ala Leu His Thr Ala Pro Ala Gly Thr Ala Thr Ala Thr Gly Ala Leu 565 570 575 Ser Ser Ser Ala Pro Ala Leu Gly Ala Ile Pro Ile Ala Thr Pro Leu 580 585 590 Gly Gly Ala Ile Ala Ala Ala Pro Val Ala Gly Ala Gly Thr Ala Leu 595 600 605 Val Ala Leu Ala Thr Ser Gly Ile Gly Leu Ala Val Leu Ala His Leu 610 615 620 Ser Gly Ala Gly Ala Leu Ile Ala Val Pro Gly Gly Gly Leu Ala Ile 625 630 635 640 His Thr Gly Thr Ala Ser Thr Met Pro Gly Val Ser Pro Gly Ala Val 645 650 655 Path Pro Leu Met Path Path Path Path Leu Thr Val Path Pro Gly Val Leu 660 665 670 Thr Gly Met Ser Ala His Ala Leu Ser Gly Gly Leu Ala Ile Pro Thr 675 680 685 Gly Gly Ala Val Ala Pro Ile Gly Ala Thr Pro Gly Ser Pro Pro Leu 690 695 700 Val Ala Ala Thr Ile Gly Leu Thr Leu Gly Gly Gly Ala Leu Ala Gly 705 710 715 720 Thr Val Gly Thr Leu Pro Gly Ala Ala Ala Thr Val Pro Ala Leu Ala 725 730 735 Ala Ala Val Leu Ser Val Ala Gly Ala Ala Gly Ala Met Ala Pro Ala 740 745 750 Met Pro Val Gly Gly Thr Ala Ala Ala Leu Met Leu Leu Ala Met Val 755 760 765 Leu Leu Pro Pro His Leu Ala Gly Met Gly Ala Ala Met Leu Leu Gly 770 775 780 Val His Ala Gly Leu Leu Leu Gly Ala Pro Gly Ala Ala Ala Gly Gly 785,790,795,800 Val Ala Ala Leu Ala Leu Gly Ala Met Gly Leu Ala Thr Pro Leu Ala 805 810 815 Val Pro Leu Gly Val Gly Val Gly Ile Gly Gly Ala Thr Leu Ser Ala 820 825 830 Leu Gly <210> 2 <211> 2502 <212> DNA <213> Thermus species Z05 <400> 2 atgaaagcga tgctgccact cttcgagcca aaaggccgcg ttctgctggt tgatggtcac 60 caccgcct accgcacctt ctttgccctc aaaggtctga cgacgagccg tggcgaaccg 120 gttcaagccg tttatggctt cgcgaaaagt ctgctcaagg cgctgaagga agacggttac 180 aaggccgtgt tcgtggtttt cgacgccaag gccccgagct ttcgccatga agcctacgag 240 gcgtataaag ccggtcgcgc cccaacgcca gaagattttc cacgccagct ggcgctgatc 300 aaagagctcg ttgatctgct cggcttcacc cgtctggaag tgccgggctt tgaagccgat 360 gatgtgctgg ccacgctggc caagaaggcg gaacgcgaag gttatgaggt gcgcattctg 420 acggccgacc gcgatctcta ccaactcgtg agcgatcgcg ttgcggttct gcacccagaa 480 ggccatctga ttacgccgga atggctgtgg gagaagtatg gtctgaaacc ggaacagtgg 540 gtggactttc gtgcgctggt tggtgatccg agtgacaatc tgccgggtgt taaaggcatc 600 ggcgaaaaaa ccgccctcaa actgctcaag gagtggggca gcctcgaaaa catcctcaag 660 aacctcgacc gcgtgaaacc ggagagcgtg cgtgagcgca ttaaggccca tctggaggat 720 ctgaaactga gcctcgaact gagccgtgtt cgcagcgatc tgccactgga ggttgatttc 780 gcccgtcgcc gtgaaccaga ccgtgaaggt ctgcgcgcct ttctggagcg cctcgagttt 840 ggtagtctgc tgcacgagtt cggtctgctc gaagccccag cgccactgga agaagcccca 900 tggccgccac cagaaggtgc cttcgtgggt tttgttctca gtcgcccgga accaatgtgg 960 gcggagctca aagcgctcgc cgcgtgcaaa gaaggtcgcg tgcatcgtgc caaggacccg 1020 ctggcgggtc tgaaagatct gaaggaagtg cgcggtctgc tggcgaaaga cctcgcggtt 1080 ctggcgctgc gcgaaggtct ggatctggcg ccgagtgatg atccgatgct gctggcgtat 1140 ctgctcgacc cgagcaatac caccccggaa ggtgttgccc gccgttacgg tggcgaatgg 1200 acggaagatg cggcccatcg tgcgctgctg gcggaacgcc tccagcagaa tctgctggaa 1260 cgcctcaaag gtgaggagaa actgctgtgg ctgtaccaag aagttgaaaa gccactcagc 1320 cgcgttctgg cccacatgga agcgaccggt gttcgtctgg atgttgccta cctcaaagcg 1380 ctgagtctgg aactggccga agaaatccgc cgcctcgaag aagaagtgtt ccgtctggcg 1440 ggtcatccgt tcaacctcaa tagccgcgac caactcgaac gcgtgctgtt tgatgagctg 1500 cgcctcccag cgctgggtaa aacgcagaaa acgggcaagc gtagcacgag tgccgcggtt 1560 ctcgaagccc tccgtgaagc gcatccgatc gtggagaaga ttctgcagca ccgtgaactg 1620 accaagctga agaacacgta cgttgaccca ctgccgggcc tcgttcaccc acgcacgggt 1680 cgtctgcaca cccgttttaa ccagacggcc accgccaccg gtcgtctgag tagcagtgac 1740 ccgaatctgc agaacatccc aatccgcacg ccactcggtc agcgcattcg ccgtgcgttt 1800 gttgccgaag cgggctgggc gctggtggcg ctggattaca gccagatcga actgcgtgtt 1860 ctggcccacc tcagcggcga tgaaaacctc atccgcgtgt tccaagaagg caaagatatc 1920 cacacgcaga cggccagctg gatgttcggt gtgagcccgg aagcggtgga tccactgatg 1980 cgccgtgccg cgaaaaccgt gaactttggc gtgctctacg gcatgagcgc ccatcgtctg 2040 agccaagagc tcgcgattcc gtacgaggaa gcggtggcct ttatcgaacg ctacttccag 2100 agcttcccaa aagttcgcgc gtggattgag aagaccctcg aagagggtcg taagcgcggt 2160 tacgttgaaa cgctgtttgg tcgccgtcgc tatgtgccgg atctgaacgc ccgcgttaaa 2220 agtgtgcgcg aagccgccga acgcatggcc ttcaacatgc cggtgcaagg cacggccgcg 2280 gatctgatga aactggcgat ggtgaagctg ttcccacatc tgcgtgagat gggcgcccgc 2340 atgctgctgc aagttcatga tgaactgctg ctggaagcgc caaagcccg tgcggaagaa 2400 gttgcggcgc tcgccaaaga agccatggaa aaagcgtatc cgctcgcggt tccgctggaa 2460 gttgaagttg gcattggcga agactggctc agcgcgaagg gc 2502 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <400> 3 ggaattccat atgaaagcga tgctgccact cttcg 35 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <400> 4 cggaattctt agcccttcgc gctgagccag tc 32 <210> 5 <211> 36 <212> DNA <213> Artificial Sequence <400> 5 gaaatccgcc gcctcgaact ggaagtgttc cgtctg 36 <210> 6 <211> 36 <212> DNA <213> Artificial Sequence <400> 6 cagacggaac acttccagtt cgaggcggcg gatttc 36 <210> 7 <211> 36 <212> DNA <213> Artificial Sequence <400> 7 gctgcgcctc ccagcgcgcg gtaaaacgca gaaaac 36 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <400> 8 gttttctgcg ttttaccgcg cgctgggagg cgcagc 36 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <400> 9 cagcgctggg taaaacgcgc aaaacgggca agcgtagcac 40 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <400> 10 gtgctacgct tgcccgtttt gcgcgtttta cccagcgctg 40 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <400> 11 gtcgtctgag tagcagtaaa ccgaatctgc agaac 35 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <400> 12 gttctgcaga ttcggtttac tgctactcag acgac 35 <210> 13 <211> 34 <212> DNA <213> Artificial Sequence <400> 13 cagtgacccg aatctgaaaa acatcccaat ccgc 34 <210> 14 <211> 34 <212> DNA <213> Artificial Sequence <400> 14 gcggattggg atgtttttca gattcgggtc actg 34 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <400> 15 gacccgaatc tgcagggcat cccaatccgc acg 33 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <400> 16 cgtgcggatt gggatgccct gcagattcgg gtc 33 <210> 17 <211> 38 <212> DNA <213> Artificial Sequence <400> 17 gctggattac agccagatgg aactgcgtgt tctggccc 38 <210> 18 <211> 38 <212> DNA <213> Artificial Sequence <400> 18 gggccagaac acgcagttcc atctggctgt aatccagc 38 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <400> 19 cccatcgtct gagccaatat ctcgcgattc cgtac 35 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <400> 20 gtacggaatc gcgagatatt ggctcagacg atggg 35 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <400> 21 ctcgcgattc cgtaccgcga agcggtggcc tttatc 36 <210> 22 <211> 36 <212> DNA <213> Artificial Sequence <400> 22 gataaaggcc accgcttcgc ggtacggaat cgcgag 36 <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <400> 23 gaagcggtgg cctttatgga acgctacttc cag 33 <210> 24 <211> 33 <212> DNA <213> Artificial Sequence <400> 24 ctggaagtag cgttccataa aggccaccgc ttc 33 <210> 25 <211> 36 <212> DNA <213> Artificial Sequence <400> 25 caaaagttcg cgcgtggaaa gagaagaccc tcgaag 36 <210> 26 <211> 36 <212> DNA <213> Artificial Sequence <400> 26 cttcgagggt cttctctttc cacgcgcgaa cttttg 36 <210> 27 <211> 33 <212> DNA <213> Artificial Sequence <400> 27 gttaaaagtg tgcgcaaagc cgccgaacgc atg 33 <210> 28 <211> 33 <212> DNA <213> Artificial Sequence <400> 28 catgcgttcg gcggctttgc gcacactttt aac 33 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <400> 29 ggttcacctt caagagtttc 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <400> 30 tcctgacatg taggagcatc 20 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <400> 31 tgagagccgt acgtcaggtc g 21 <210> 32 <211> 834 <212> PRT <213> Artificial Sequence <400> 32 Met Lys Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe 50 55 60 Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu 65 70 75 80 Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln 85 90 95 Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu 100 105 110 Glu Val Pro Gly Phe Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys 115 120 125 Lys Ala Glu Arg Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg 130 135 140 Asp Leu Tyr Gln Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu 145 150 155 160 Gly His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Lys 165 170 175 Pro Glu Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp 180 185 190 Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu 195 200 205 Leu Lys Glu Trp Gly Ser Leu Glu Asn Ile Leu Lys Asn Leu Asp Arg 210 215 220 Val Lys Pro Glu Ser Val Arg Glu Arg Ile Lys Ala His Leu Glu Asp 225 230 235 240 Leu Lys Leu Ser Leu Glu Leu Ser Arg Val Arg Ser Asp Leu Pro Leu 245 250 255 Glu Val Asp Phe Ala Arg Arg Arg Glu Pro Asp Arg Glu Gly Leu Arg 260 265 270 Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly 275 280 285 Leu Leu Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro 290 295 300 Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp 305 310 315 320 Ala Glu Leu Lys Ala Leu Ala Ala Cys Lys Glu Gly Arg Val His Arg 325 330 335 Ala Lys Asp Pro Leu Ala Gly Leu Lys Asp Leu Lys Glu Val Arg Gly 340 345 350 Leu Leu Ala Lys Asp Leu Ala Val Leu Ala Leu Arg Glu Gly Leu Asp 355 360 365 Leu Ala Pro Ser Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro 370 375 380 Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp 385 390 395 400 Thr Glu Asp Ala Ala His Arg Ala Leu Leu Ala Glu Arg Leu Gln Gln 405 410 415 Asn Leu Leu Glu Arg Leu Lys Gly Glu Glu Lys Leu Leu Trp Leu Tyr 420 425 430 Gln Glu Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala 435 440 445 Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Lys Ala Leu Ser Leu Glu 450 455 460 Leu Ala Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala 465 470 475 480 Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu 485 490 495 Phe Asp Glu Leu Arg Leu Pro Ala Arg Gly Lys Thr Gln Lys Thr Gly 500 505 510 Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His 515 520 525 Pro Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys 530 535 540 Asn Thr Tyr Val Asp Pro Leu Pro Gly Leu Val His Pro Arg Thr Gly 545 550 555 560 Arg Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu 565 570 575 Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Ile Arg Thr Pro Leu 580 585 590 Gly Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu 595 600 605 Val Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu 610 615 620 Ser Gly Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Lys Asp Ile 625 630 635 640 His Thr Gln Thr Ala Ser Trp Met Phe Gly Val Ser Pro Glu Ala Val 645 650 655 Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu 660 665 670 Tyr Gly Met Ser Ala His Arg Leu Ser Gln Tyr Leu Ala Ile Pro Tyr 675 680 685 Glu Glu Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys 690 695 700 Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly 705 710 715 720 Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn 725 730 735 Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn 740 745 750 Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val 755 760 765 Lys Leu Phe Pro His Leu Arg Glu With Gly Ala Arg With Leu Leu Gln 770 775 780 Val His Asp Glu Leu Leu Leu Glu Ala Pro Gln Ala Arg Ala Glu Glu 785 790 795 800 Val Ala Ala Leu Ala Lys Glu Ala With Glu Lys Ala Tyr Pro Leu Ala 805 810 815 Val Pro Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala 820 825 830 Lys Gly

Claims

1. A mutant DNA polymerase, characterized in that: Compared with the amino acid sequence of wild-type Z05 DNA polymerase, the mutant DNA polymerase has the following mutations at the amino acid sites: L505R; or, L505R and E683Y; Or, L505R, Q509R and D580K; or, L505R, Q584K, and E683Y; Or, L505R, D580K and E683Y; The nucleotide sequence of the wild-type Z05 DNA polymerase is shown in SEQ ID NO:

2.

2. The mutant DNA polymerase according to claim 1, characterized in that: The mutant DNA polymerase includes a chemical modification that is thermally reversibly inactivated.

3. A gene encoding the mutant DNA polymerase according to any one of claims 1 to 2.

4. A recombinant expression vector comprising the gene according to claim 3. A recombinant bacterium comprising the recombinant expression vector according to claim 4 .

6. Use of the mutant DNA polymerase according to any one of claims 1 to 2 in preparing a detection kit.

7. The use according to claim 6, characterized in that: The detection kit is used to implement a method for nucleic acid amplification, comprising: contacting a mutant DNA polymerase with a primer, a polynucleotide template, and nucleoside triphosphates under conditions suitable for primer extension to produce an extension product; The nucleic acid amplification method adopts polymerase chain reaction, isothermal amplification reaction or sequencing reaction; The polynucleotide template is an RNA template or a DNA template, and the polynucleotide template is from any type of biological sample.

8. The use according to claim 6, characterized in that: The detection kit comprises: at least one container containing a mutant DNA polymerase.

9. The use according to claim 8, characterized in that: The detection kit may further comprise one or more of the following containers: (a) a container comprising a primer that hybridizes to a predetermined polynucleotide template under conditions suitable for primer extension; (b) a container comprising nucleoside triphosphates; (c) A container containing a buffer solution suitable for primer extension.

Images