A mutant DNA polymerase and its preparation method and application

Abstract

The application relates to the technical field of molecular biology, and discloses a mutant DNA polymerase as well as a preparation method and application thereof, wherein the amino acid sequence of the mutant DNA polymerase is shown as SEQ ID NO. 4, the amino acid sequence is obtained by mutation on the basis of a wild-type FWT polymerase, and the mutation sites include that alanine at the 80th position is mutated into arginine, asparagine at the 107th position is mutated into aspartic acid, glutamic acid at the 172nd position is mutated into arginine, asparagine at the 325th position is mutated into aspartic acid, aspartic acid at the 423rd position is mutated into alanine, serine at the 490th position is mutated into alanine, and phenylalanine at the 491st position is mutated into alanine. The mutant DNA polymerase has better continuous synthesis, strand displacement and high-salt resistance than the wild type under room temperature conditions, and is an ideal tool for rolling circle amplification and multiple displacement amplification.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to a mutant DNA polymerase and a preparation method and application thereof. BACKGROUND

[0002] DNA polymerase is responsible for the replication and maintenance of the genome, which is essential for accurately transmitting genetic information from generation to generation. Chain displacement refers to the ability of polymerase to displace, rather than degrade, the downstream DNA encountered during DNA synthesis. During chain displacement replication, only one DNA strand is replicated at a time. Chain displacement synthesis releases single-stranded DNA, which is then copied into double-stranded DNA. The commonly used polymerase that can perform isothermal amplification at room temperature is PHI29 polymerase, but because it has exonuclease activity, its application in nanopore sequencing is limited.

[0003] In the related art, FWT polymerase is a member of polymerase family A, and wild-type FWT polymerase is derived from Psychrobacilus sp. FJAT-21963poly A0A0Q3VXV0, which can be used for sequencing, but it does not have obvious chain displacement ability, and only has high activity at low temperature, cannot effectively perform isothermal amplification at room temperature, and cannot tolerate high salt. Therefore, it is necessary to continuously improve the properties of wild-type FWT polymerase to obtain FWT polymerase with better performance.

[0004] To this end, the present application provides a salt-tolerant mutant DNA polymerase and a preparation method and application thereof. The polymerase is obtained by mutation screening based on wild-type FWT polymerase, and has better continuous synthesis, chain displacement and high salt tolerance than wild-type FWT polymerase at room temperature, and is an ideal tool for rolling circle amplification and multiple displacement amplification. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a mutant DNA polymerase and a preparation method and application thereof. The mutant DNA polymerase is obtained by mutation at a specific site based on wild-type FWT polymerase, and still has excellent rolling displacement replication capacity under high salt conditions (300mM KCl).

[0006] The present application also provides a nucleic acid molecule.

[0007] The present application also provides a biological material related to the above-mentioned mutant DNA polymerase.

[0008] The present application also provides an enzyme preparation.

[0009] The present application also provides a preparation method of a mutant DNA polymerase.

[0010] The application further provides a method for amplifying a DNA template.

[0011] The application further provides application of the mutant DNA polymerase, the nucleic acid molecule, the biological material or the enzyme preparation in preparation of a salt-tolerant nucleic acid amplification related product.

[0012] In a first aspect, the application provides a mutant DNA polymerase, wherein the amino acid sequence of the mutant DNA polymerase is shown as SEQ ID NO. 4.

[0013] The amino acid sequence shown as SEQ ID NO. 4 is as follows:

[0014] MSEVTFEILDEINASILKDKMAIHLEMFNEQYHTSELLGIALYDGEQSYFVTGSVAFESA

[0015] GFCKWLEDESRIKYLVDSKRTQAVSRKHNVSLLGADFDLVLAAYIVDPSISSDDVSTMAKEF

[0016] GNYDVLTDENVYGKGAKKATPPLDKLAEHAIRKAQAVWKLKPILEKKLERNEQYALYKDI

[0017] ELPLASILGTMESDGVKVDVHVLKEMGIDLNQKLRAIEKEIYTLAGQEFNINSPKQLGVILF

[0018] EKIGLTPLKKTKTGYSTAADVLEKLEGEHEIISHILNYRQLGKLQSTYIEGLTKEIHGEDQKIH

[0019] TRYQQALTSTGRLSSIDPNLQNIPIRLEEGRKIRKAFVPSQPDWIMFAADYSQIELRVLAHMC

[0020] EDEKLVDAFNHDLDIHTKTAMDVFHVGIDEVTSDMRRAAKAVNFGIVYGISAYGLSQSLNI

[0021] TRKEAATFIDQYLNSFPGVKDYMDDIVREAKQTGYVTTILKRRRYLPDITSSNFNLRAAAER

[0022] TAMNTPIQGSAADIIKKAMIDMAKRLKDENLQTKMLLQVHDELIFEAPKEEIAILEKIVPEV

[0023] MEGAIKLIVPLKVEYAYGSSWYDTK.

[0024] The mutant DNA polymerase according to the embodiment of the present application has at least the following beneficial effects: the mutant DNA polymerase is obtained by mutation screening based on a wild type FWT polymerase (FWT-WT polymerase), and has better continuous synthesis, strand displacement and high salt tolerance than the wild type FWT polymerase at room temperature, and is an ideal tool for rolling circle amplification and multiple displacement amplification.

[0025] The amino acid sequence of the mutant DNA polymerase of the present application is obtained by mutation based on the wild type FWT polymerase (SEQ ID NO. 3), and the mutation sites include: alanine at position 80 is mutated to arginine (A80R), asparagine at position 107 is mutated to aspartic acid (N107D), glutamic acid at position 172 is mutated to arginine (E172R), asparagine at position 325 is mutated to aspartic acid (N325D), aspartic acid at position 423 is mutated to alanine (D423A), serine at position 490 is mutated to alanine (S490A), and phenylalanine at position 491 is mutated to alanine (F491A).

[0026] The amino acid sequence represented by SEQ ID NO. 3 is as follows:

[0027] MSEVTFEILDEINASILKDKMAIHLEMFNEQYHTSELLGIALYDGEQSYFVTGSVAFESA

[0028] GFCKWLEDESRIKYLVDSKATQAVSRKHNVSLLGADFDLVLAAYIVNPSISSDDVSTMAKEF

[0029] GNYDVLTDENVYGKGAKKATPPLDKLAEHAIRKAQAVWKLKPILEKKLEENEQYALYKDI

[0030] ELPLASILGTMESDGVKVDVHVLKEMGIDLNQKLRAIEKEIYTLAGQEFNINSPKQLGVILF

[0031] EKIGLTPLKKTKTGYSTAADVLEKLEGEHEIISHILNYRQLGKLQSTYIEGLTKEIHGEDQKIH

[0032] TRYQQALTSTGRLSSINPNLQNIPIRLEEGRKIRKAFVPSQPDWIMFAADYSQIELRVLAHMC

[0033] EDEKLVDAFNHDLDIHTKTAMDVFHVGIDEVTSDMRRAAKAVNFGIVYGISDYGLSQSLNI

[0034] TRKEAATFIDQYLNSFPGVKDYMDDIVREAKQTGYVTTILKRRRYLPDITSSNFNLRSFAERT

[0035] AMNTPIQGSAADIIKKAMIDMAKRLKDENLQTKMLLQVHDELIFEAPKEEIAILEKIVPEVM

[0036] EGAIKLIVPLKVEYAYGSSWYDTK.

[0037] The second aspect of the present application provides a nucleic acid molecule, which is:

[0038] A1) a nucleotide sequence encoding the mutant DNA polymerase described above; or

[0039] A2) a nucleotide sequence complementary to the nucleotide sequence described in A1).

[0040] In some embodiments of the present application, the sequence of the nucleic acid molecule is as shown in SEQ ID NO. 2.

[0041] The third aspect of the present application provides a biological material, which is any one of B1) to B3):

[0042] B1) an expression cassette containing the nucleic acid molecule described above;

[0043] B2) a recombinant vector containing the nucleic acid molecule described above or the expression cassette described in B1);

[0044] B3) a recombinant biological cell containing the nucleic acid molecule described above, the expression cassette described in B1) or the recombinant vector described in B2).

[0045] In some embodiments of the present application, the expression cassette refers to DNA capable of expressing the mutant DNA polymerase in a host cell. The DNA includes not only a promoter capable of initiating transcription of the mutant DNA polymerase gene, but also a terminator capable of terminating transcription of the protein gene.

[0046] Further, the expression cassette can also include an enhancer sequence.

[0047] In some embodiments of the present application, the recombinant vector can be a recombinant vector obtained by inserting a nucleic acid molecule encoding the mutant DNA polymerase into a multiple cloning site of the vector.

[0048] Specifically, the recombinant vector is a PET-21a vector.

[0049] In some embodiments of the present application, the recombinant biological cell is a non-reproductive material.

[0050] In some embodiments of the present application, the biological cell includes prokaryotic cells and eukaryotic cells.

[0051] Further, the prokaryotic cell includes bacteria or algae;

[0052] Specifically, the bacteria can be Escherichia coli, such as Escherichia coli BL21 (DE3).

[0053] Further, the eukaryotic cell includes fungi, mammalian cells or insect cells.

[0054] In some embodiments of the present application, the recombinant biological cell is a recombinant biological cell obtained by introducing the nucleic acid molecule, B1) the expression cassette or B2) the recombinant vector into a biological cell. Specifically, it can be a recombinant Escherichia coli obtained by introducing a recombinant vector into Escherichia coli BL21 (DE3).

[0055] In a fourth aspect of the present application, an enzyme preparation is provided, which includes the above-mentioned mutant DNA polymerase.

[0056] In some embodiments of the present application, the enzyme preparation further includes at least one of dNTP and a reaction buffer. It can be understood that the reaction buffer does not affect the activity of the mutant DNA polymerase.

[0057] In some embodiments of the present application, the enzyme preparation can be used for in vitro DNA synthesis, DNA amplification or DNA sequencing.

[0058] In a fifth aspect of the present application, a preparation method of the above-mentioned mutant DNA polymerase is provided, which includes:

[0059] The coding gene of the mutant DNA polymerase is introduced into a biological cell, and the coding gene is expressed to obtain the mutant DNA polymerase.

[0060] In some embodiments of the present application, the biological cell includes prokaryotic cells and eukaryotic cells.

[0061] In some embodiments of the present application, the prokaryotic cell includes bacteria or algae. The bacteria can be Escherichia coli, such as Escherichia coli BL21 (DE3).

[0062] In some embodiments of the present application, the eukaryotic cell includes fungi, mammalian cells or insect cells.

[0063] In some embodiments of the present application, the preparation method of the mutant DNA polymerase specifically includes the following steps:

[0064] Step S1, the coding gene of the mutant DNA polymerase is connected to a gene expression vector pET-21a, and is transformed into a host cell Escherichia coli BL21 to obtain a recombinant cell;

[0065] Step S2, the recombinant cell is induced for expression, and the bacterial body is collected. Then, the bacterial solution is resuspended and ultrasonically broken, and the supernatant is collected;

[0066] Step S3, the supernatant is purified by using a Ni2+ affinity column, and the purified protein solution is collected;

[0067] Step S4, the protein solution is concentrated by using an ultrafiltration column to obtain the mutant DNA polymerase.

[0068] In a sixth aspect of the present application, a method for amplifying a DNA template is provided, which includes:

[0069] The mutant DNA polymerase is mixed with a template DNA and a reaction reagent to perform amplification. The reaction reagent includes a reaction buffer, a primer and a nucleotide substrate.

[0070] In some embodiments of the present application, the salt concentration of the reaction reagent is 0-400 mM, and the reaction temperature after mixing is 25-40℃.

[0071] In some embodiments of the present application, the primer refers to a natural or artificially synthesized oligonucleotide, which can be extended as a starting point of nucleic acid synthesis under appropriate conditions (for example, providing reaction raw materials such as nucleotide substrates, polymerases in a buffer, and providing specific temperature conditions), so that the nucleotide substrate is incorporated into the DNA template to synthesize the complementary strand of the DNA template.

[0072] In some embodiments of the present application, the nucleotide substrate is at least one of a nucleotide, a nucleotide analogue.

[0073] Specifically, the nucleotide substrate refers to a nucleotide unit formed by a pentose, a phosphate and a nitrogen-containing heterocyclic base, or a modified product of the nucleotide unit. The modified product specifically refers to a modified label of the nucleotide, so that the size, mass, charge amount, etc. of different types of nucleotides (including deoxyadenosine, deoxythymidine, deoxycytidine, deoxyguanosine, deoxyuridine) are obviously distinguished, thereby improving the discrimination between different types of nucleotides in the detection process. On the one hand, the nucleotide analogue can be a nucleotide containing multiple phosphate groups, specifically a diphosphate nucleotide, a triphosphate nucleotide, a tetraphosphate nucleotide, a pentaphosphate nucleotide, a hexaphosphate nucleotide, etc. On the other hand, in order to enhance the identification of the nucleotide analogue, different lengths / masses of labels can be further introduced on the phosphate groups of the polyphosphate nucleotide for different bases. The specific labels can be optional polymer linkers such as polyethylene glycol or its derivatives, and dye molecules such as coumarin, etc.

[0074] In a seventh aspect of the present application, the use of the mutant DNA polymerase, the nucleic acid molecule, the biological material or the enzyme preparation described above in the preparation of a salt-tolerant nucleic acid amplification-related product is provided.

[0075] In some embodiments of the present application, the product includes a reagent or a kit.

[0076] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0077] The present application will be further described below in conjunction with the accompanying drawings and examples, in which:

[0078] Figure 1 SDS-PAGE electrophoretogram of the DNA polymerase of the present application;

[0079] Figure 2 Detection results of the salt tolerance of the DNA polymerase of the present application. DETAILED DESCRIPTION

[0080] The concept and technical effects of the present application will be described below in conjunction with the examples, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0081] In the description of the present application, the description of the terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example", or "some examples" means that the specific feature, structure, material, or characteristic being described is included in at least one embodiment or example of the present application. The illustrative appearances of the above-mentioned terms in this specification are not necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics can be combined in any suitable manner in one or more embodiments or examples.

[0082] In the description of the present application, the term "nucleotide" generally refers to a compound in which a nucleoside is linked to an acidic molecule or group by an ester bond. For example, a phosphate ester of a nucleoside, typically having one, two, or three phosphate groups covalently linked to the 5-position of the sugar group of the nucleoside. In some cases, the definition of nucleotide also includes homologs or analogs of typical nucleotides.

[0083] The term "amino acid" refers to the basic unit that constitutes a protein, confers a specific molecular structural form to the protein, and makes his molecule have biochemical activity. For example, the "amino acid" used in the present application includes the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), lysine (Lys or K), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), and tyrosine (Tyr or Y).

[0084] The term "amplification" refers to the process in which the number of a target nucleic acid fragment is increased under the action of a nucleic acid polymerase.

[0085] Unless specifically defined otherwise, all scientific or technical terms used in this patent have the same meaning as commonly understood by one of ordinary skill in the art to which this patent belongs.

[0086] Unless otherwise specified, the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market.

[0087] Example 1 Obtaining of FWT polymerase mutant sequence

[0088] As a member of the polymerase family A, the previous study of the present application found that the mutation of specific sites (such as A80R, N107D, E172R, N325D, D423A, S490A, F491A) in wild-type FWT polymerase has a certain influence on improving the salt tolerance of the polymerase and enhancing its strand displacement ability. In the present application, the mutant FWT polymerase mutant is based on the nucleotide sequence of wild-type FWT, and a gene fragment containing 7 mutation sites (A80R, N107D, E172R, N325D, D423A, S490A, F491A) is obtained by whole gene synthesis.

[0089] The nucleotide sequence of the wild-type FWT is as follows:

[0090] ATGAGTGAAGTTACCTTTGAAATCCTGGATGAAATTAATGCGAGCATTCTGAAAGAT

[0091] AAAATGGCGATTCATCTGGAAATGTTTAATGAACAGTATCATACAAGCGAACTGCTGGGT

[0092] ATTGCGCTGTATGATGGCGAACAGAGCTATTTTGTTACCGGTAGCGTTGCCTTTGAAAGC

[0093] GCGGGTTTTTGTAAATGGCTGGAAGATGAAAGCCGTATTAAATATCTGGTTGATAGTAAA

[0094] GCAACCCAGGCAGTTAGCCGTAAACATAATGTTAGCCTGCTGGGTGCAGATTTTGATCTG

[0095] GTTCTGGCCGCGTATATTGTTAATCCAAGCATTAGCAGCGATGATGTTAGCACAATGGCA

[0096] AAAGAATTTGGTAATTATGATGTGCTGACCGATGAAAACGTTTATGGTAAAGGTGCAAA

[0097] GAAGGCAACACCGCCGCTGGATAAACTGGCAGAACATGCAATTCGTAAAGCACAGGCA

[0098] GTTAAAGTGGATGTGCATGTTCTGAAAGAAATGGGTATTGATCTGAATCAGAAACTGCG

[0099] TGTATAAAGATATCGAACTGCCGCTGGCAAGCATTCTGGGTACCATGGAAAGCGATGGT

[0100] GTTAAAGTGGATGTGCATGTTCTGAAAGAAATGGGTATTGATCTGAATCAGAAACTGCG

[0101] TGCCATCGAAAAGGAAATTTATACCCTGGCAGGCCAGGAATTTAATATTAATAGCCCGAA

[0102] ACAGCTGGGTGTTATTCTGTTTGAAAAGATTGGTCTGACCCCTCTGAAGAAGACCAAAA

[0103] CCGGCTATAGCACCGCAGCCGATGTTCTGGAAAAGCTGGAAGGTGAACATGAAATTATT

[0104] AGCCATATTCTGAACTATCGTCAGCTGGGCAAACTGCAGTCAACTTATATCGAAGGCCTG

[0105] ACAAAAGAAATCCATGGTGAAGATCAGAAAATCCATACCCGTTATCAGCAGGCGCTGAC

[0106] CTCAACCGGCCGTCTGAGTTCAATTAATCCGAATCTGCAGAACATTCCGATTCGTCTGGA

[0107] AGAAGGTCGTAAAATTCGTAAAGCCTTTGTTCCATCTCAGCCGGATTGGATTATGTTTGC

[0108] AGCAGATTATAGCCAGATCGAACTGCGTGTTCTGGCACACATGTGTGAAGATGAAAAGC

[0109] TGGTTGATGCATTTAATCATGATCTGGATATTCATACCAAAACCGCCATGGATGTTTTCCA

[0110] TGTTGGTATTGATGAAGTGACCAGCGATATGCGTCGTGCAGCCAAAGCAGTAAATTTTGG

[0111] TATTGTTTATGGCATTAGCGATTATGGCCTGAGCCAGAGCCTGAATATTACCCGTAAAGAA

[0112] GCAGCAACGTTTATTGATCAGTATCTGAATAGCTTTCCGGGCGTTAAAGATTATATGGATG

[0113] ATATTGTTCGCGAAGCAAAACAGACCGGTTATGTTACCACCATTCTGAAACGTCGTCGTT

[0114] ATCTGCCGGATATTACCAGCTCAAATTTTAATCTGCGTAGTTTTGCAGAACGCACCGCCA

[0115] TGAATACACCGATTCAGGGTAGCGCAGCCGATATTATCAAGAAGGCCATGATTGATATGG

[0116] CCAAACGTCTGAAAGATGAAAACCTGCAGACCAAAATGCTGCTGCAGGTTCATGATGA

[0117] ACTGATTTTCGAAGCACCGAAAGAAGAAATTGCAATTCTGGAAAAGATCGTGCCGGAA

[0118] GTTATGGAAGGTGCAATTAAACTGATTGTTCCGCTGAAAGTTGAATATGCGTATGGTAGC

[0119] TCTTGGTATGATACCAAA(SEQ ID NO. 1).

[0120] On the basis of the wild type FWT polymerase described above, the key mutation sites were designed, and the expression gene of the mutant FWT polymerase was synthesized by Genescript Biotech Co., Ltd.

[0121] The nucleotide sequence of the mutant FWT polymerase expression gene is as follows:

[0122] ATGAGCGAAGTTACGTTTGAAATTCTGGATGAAATTAACGCCTCTATTCTGAAAGAT

[0123] AAAATGGCCATTCATCTGGAAATGTTTAATGAACAGTATCATACAAGCGAACTGCTGGGT

[0124] ATTGCACTGTATGATGGTGAACAGAGCTATTTTGTTACCGGTTCCGTCGCATTTGAAAGC

[0125] GCCGGTTTTTGTAAATGGCTGGAAGATGAAAGCCGTATTAAATATCTGGTTGATAGTAAA

[0126] CGTACCCAGGCAGTTAGCCGTAAACATAATGTTAGCCTGCTGGGTGCAGATTTTGATCTG

[0127] GTTCTGGCGGCGTATATTGTTGATCCGTCAATTAGCAGCGATGATGTTAGCACAATGGCA

[0128] AAAGAATTTGGTAATTATGATGTGCTGACCGATGAAAACGTTTATGGTAAAGGTGCAAA

[0129] GAAGGCAACACCGCCGCTGGATAAACTGGCAGAACATGCAATTCGTAAAGCACAGGCA

[0130] GTGTGGAAACTGAAACCGATTCTGGAAAAGAAGCTGGAACGTAACGAACAGTATGCAC

[0131] TGTATAAAGATATCGAACTGCCGCTGGCAAGCATTCTGGGTACCATGGAAAGCGATGGT

[0132] GTGAAAGTTGATGTGCATGTTCTGAAAGAAATGGGTATTGATCTGAATCAGAAACTGCG

[0133] TGCAATTGAAAAGGAAATTTATACCCTGGCAGGCCAGGAATTTAATATTAATAGCCCGAA

[0134] ACAGCTGGGTGTGATCCTGTTTGAAAAGATTGGTCTGACCCCGCTGAAGAAGACCAAA

[0135] ACCGGTTATAGCACAGCAGCAGATGTGCTGGAAAAGCTGGAAGGTGAACATGAAATTAT

[0136] TAGCCATATTCTGAACTATCGCCAGCTGGGCAAACTGCAGTCAACCTATATTGAAGGTCT

[0137] GACCAAAGAAATTCATGGTGAAGATCAGAAAATCCATACCCGTTATCAGCAGGCCCTGA

[0138] CCTCCACCGGTCGTCTGAGTTCAATTGATCCGAACCTGCAGAACATTCCGATTCGTCTGG

[0139] AAGAAGGTCGTAAAATTCGTAAAGCCTTTGTTCCTTCACAGCCGGATTGGATTATGTTTG

[0140] CCGCAGATTATTCCCAGATCGAACTGCGTGTGCTGGCCCACATGTGTGAAGATGAAAAG

[0141] CTGGTTGATGCATTTAATCATGATCTGGATATTCATACCAAAACCGCCATGGATGTGTTTC

[0142] ATGTTGGCATTGATGAAGTGACCTCAGATATGCGTCGTGCAGCCAAAGCAGTTAATTTTG

[0143] GTATTGTTTATGGCATTAGCGCCTATGGCCTGAGCCAGAGCCTGAACATTACGCGTAAAG

[0144] AAGCAGCAACCTTTATTGATCAGTATCTGAATTCTTTTCCGGGTGTTAAAGATTATATGGA

[0145] TGATATTGTGCGCGAAGCAAAACAGACCGGTTATGTTACCACCATTCTGAAACGTCGTC

[0146] GTTATCTGCCGGATATTACCAGCTCAAACTTTAATCTGCGTGCCGCCGCGGAACGTACCG

[0147] CAATGAATACCCCGATTCAGGGTAGCGCAGCCGATATTATCAAGAAGGCCATGATTGATA

[0148] TGGCCAAACGTCTGAAAGATGAAAATCTGCAGACCAAAATGCTGCTGCAGGTTCATGAT

[0149] GAACTGATTTTCGAAGCACCGAAAGAAGAAATTGCAATTCTGGAAAAGATCGTTCCGG

[0150] AAGTTATGGAAGGTGCAATTAAACTGATTGTACCGCTGAAAGTTGAATATGCGTATGGTA

[0151] GCTCCTGGTATGATACCAAA(SEQ ID NO.2)。

[0152] Preparation of mutant FWT polymerase

[0153] (1) Preparation of FWT polymerase

[0154] The mutant FWT polymerase expression gene sequence and the wild type FWT polymerase expression gene sequence described above were respectively connected to the gene expression vector pET-21a, and then respectively transformed into the E. coli competent cell BL21 (Shanghai Tuoluogang Biotechnology Co., Ltd., Catalog No. CC96107).

[0155] After resistance screening and sequencing verification, a single colony was picked and inoculated in 5 mL LB liquid medium at 37°C for overnight shaking culture, then transferred to 200 mL LB liquid medium, and after 5 h shaking culture at 37°C, the OD 600= 1, the bacterial liquid was taken out and cooled at 4°C, and then IPTG was added to a final concentration of 0.6 mM for induction, and the induction was continued for 24 hours at 20°C. The culture liquid was centrifuged, and the bacterial bodies were collected and lysed by an ultrasonic cell pulverizer, wherein the power of the ultrasonic wave was 70 W, the ultrasonic wave was applied for 10 s, the interval was 10 s, and the total duration was 30 min. Then, the fusion type polymerase was purified by Ni column affinity chromatography, and the mutant FWT polymerase and the wild type FWT polymerase were concentrated by using an ultrafiltration column AMICON ULTRA 15 mL 50K (Millipore Corporation, catalog number: UFC905024), and subsequent detection was performed.

[0156] (2) Purity and concentration detection of the FWT polymerase

[0157] The mutant FWT polymerase and the wild type FWT polymerase obtained above were subjected to SDS-PAGE protein electrophoresis detection to identify the purity of the DNA polymerase protein.

[0158] The SDS-PAGE protein electrophoresis results are shown in Figure 1 , wherein lanes 1 / 2 / 3 / 4 / 5 are 160 ng / 320 ng / 480 ng / 640 ng / 800 ng of BSA, lane 8 is the wild type FWT polymerase (FWT-WT DNA polymerase), and lane 9 is the mutant FWT polymerase (FWT DNA polymerase), and the molecular weight of the polymerase is 65.4 kD. As can be seen from Figure 1 , the mutant FWT polymerase protein obtained in the application has high purity.

[0159] The amino acid sequence of the wild type FWT is as follows:

[0160] MSEVTFEILDEINASILKDKMAIHLEMFNEQYHTSELLGIALYDGEQSYFVTGSVAFESA

[0161] GFCKWLEDESRIKYLVDSKATQAVSRKHNVSLLGADFDLVLAAYIVNPSISSDDVSTMAKEF

[0162] GNYDVLTDENVYGKGAKKATPPLDKLAEHAIRKAQAVWKLKPILEKKLEENEQYALYKDI

[0163] ELPLASILGTMESDGVKVDVHVLKEMGIDLNQKLRAIEKEIYTLAGQEFNINSPKQLGVILF

[0164] EKIGLTPLKKTKTGYSTAADVLEKLEGEHEIISHILNYRQLGKLQSTYIEGLTKEIHGEDQKIH

[0165] TRYQQALTSTGRLSSINPNLQNIPIRLEEGRKIRKAFVPSQPDWIMFAADYSQIELRVLAHMC

[0166] EDEKLVDAFNHDLDIHTKTAMDVFHVGIDEVTSDMRRAAKAVNFGIVYGISDYGLSQSLNI

[0167] TRKEAATFIDQYLNSFPGVKDYMDDIVREAKQTGYVTTILKRRRYLPDITSSNFNLRSFAERT

[0168] AMNTPIQGSAADIIKKAMIDMAKRLKDENLQTKMLLQVHDELIFEAPKEEIAILEKIVPEVM

[0169] EGAIKLIVPLKVEYAYGSSWYDTK (SEQ ID NO. 3).

[0170] The mutant FWT polymerase mutant amino acid sequence is as follows:

[0171]

[0172]

[0173] The black bold underlined part in SEQ ID NO. 4 is the mutation site.

[0174] Detection of the enzyme activity of the FWT polymerase

[0175] The exonuclease activity of the mutant FWT polymerase and the wild type FWT polymerase and the polymerase activity under high salt conditions were detected. The reaction system involved in the detection is shown in Table 1.

[0176] Table 1 Enzyme activity detection reaction system

[0177] Reagents Final concentration BSA 0.5 μg / μL 10x reaction buffer 1× Template (M13mp18) 5 nM Primer 5 nM dNTP (10 mM) 200 nM KCl 0 mM / 150 mM / 300 mM Polymerase 200 nM

[0178] The primer nucleotide sequence is 5'-CGCCAGGGTTTTCCCAGTCACGAC-3' (SEQ ID NO. 5), the reaction buffer is composed of 50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4 and 4 mM DTT, and the pH of the reaction buffer is 7.5 at 25°C. The reaction system is reacted at 30°C for 3 h to obtain a reaction product.

[0179] The reaction product is added to 6×DNA Loading and then loaded into a 0.6% alkaline agarose gel; the electrophoresis condition is 100 v for 90 min. After electrophoresis, the PAGE gel is soaked in SYBR Gold dye solution and slowly shaken horizontally for about 20 min. Then the brightness of the product chain is detected by using Azure Biosystems imaging, and the strand displacement ability of the polymerase is judged according to the brightness.

[0180] The results are shown in Figure 2 The first / second / third lane and the fourth / fifth / sixth lane in the figure are the rolling circle replication of wild-type FWT polymerase and mutant FWT polymerase under the conditions of 0 mM, 150 mM and 300 mM KCl respectively. It can be seen from the figure that the wild-type FWT polymerase has strand displacement ability under the condition of 30°C and 0 mM KCl, but it cannot perform rolling circle replication under the conditions of 150 mM and 300 mM KCl, while the mutant FWT polymerase still has strong strand displacement ability under the high-salt conditions of 150 mM and 300 mM KCl.

[0181] The above has made a detailed description of the embodiments of the present application, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge range possessed by those skilled in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A mutant DNA polymerase, characterized in that, The amino acid sequence of the mutant DNA polymerase is shown as SEQ ID NO.

4.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the mutant DNA polymerase of claim 1.

3. The nucleic acid molecule of claim 2, wherein, The sequence of the nucleic acid molecule is shown as SEQ ID NO.

2.

4. Biomaterial, characterized in that, The biological material is any one of B1) to B3): B1), an expression cassette containing the nucleic acid molecule of any one of claims 2 to 3; B2), a recombinant vector containing the nucleic acid molecule of any one of claims 2 to 3 or the expression cassette of B1); B3), a recombinant biological cell containing the nucleic acid molecule of any one of claims 2 to 3, the expression cassette of B1) or the recombinant vector of B2).

5. An enzyme preparation, characterized in that, The mutant DNA polymerase of claim 1.

6. The enzyme preparation of claim 5, characterized in that, The enzyme preparation further comprises at least one of dNTP and reaction buffer.

7. A method for producing the mutant DNA polymerase according to claim 1, characterized by, The enzyme preparation further comprises at least one of dNTP and reaction buffer. The coding gene of the mutant DNA polymerase of claim 1 is introduced into a biological cell, and the coding gene is expressed to obtain the mutant DNA polymerase.

8. A method of amplifying a DNA template, characterized by, The coding gene of the mutant DNA polymerase of claim 1 is introduced into a biological cell, and the coding gene is expressed to obtain the mutant DNA polymerase. The mutant DNA polymerase of claim 1 is mixed with template DNA and reaction reagents to perform amplification; wherein the reaction reagents comprise reaction buffer, primers and nucleotide substrates.

9. The method of claim 8, wherein, The salt concentration of the reaction reagents is 0-400 mM.

10. Use of the mutant DNA polymerase of claim 1 in the preparation of a salt-tolerant nucleic acid amplification-related product.

11. Use of the nucleic acid molecule of any one of claims 2 to 3 in the preparation of a salt-tolerant nucleic acid amplification-related product.

12. Use of the biological material of claim 4 in the preparation of a salt-tolerant nucleic acid amplification-related product.

13. Use of the enzyme preparation of any one of claims 5 to 6 in the preparation of a salt-tolerant nucleic acid amplification-related product.

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