Taq DNA polymerase mutant

Abstract

The invention provides a Taq DNA (Deoxyribose Nucleic Acid) polymerase mutant. The Taq DNA polymerase mutant provided by the invention solves the problems of slow amplification speed and short extended DNA length of polymerase in the prior art. The Taq DNA polymerase mutant disclosed by the invention has the advantages that the amplification speed and the amplification length are obviously superior to those of a wild type product and a product in the prior art, and a large DNA fragment can be quickly expanded.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Taq DNA polymerase mutant. Background Art

[0002] The polymerase chain reaction (PCR) is a widely used in vitro DNA amplification technique that plays a crucial role in laboratory molecular biology, clinical disease diagnosis, and forensic identification. For example, it can be used to detect gene mutations and microbial or viral infectious agents, or further to detect antibiotic resistance genes and biothreat agents. The Taq DNA polymerase used in PCR belongs to the DNA polymerase I family. Its gene is 2496 bp long, encoding 832 aa, with a molecular weight of 94 kD. It is a heat-resistant polymerase. Researchers first applied Taq to PCR technology, enabling the PCR process to achieve automated, continuous cycling. The optimal temperature for Taq polymerase is 70-75°C (the temperature at which catalytic activity is highest), and its catalytic extension efficiency can reach 150 bp / s.

[0003] With the rapid development of PCR technology, Taq DNA polymerase has become an important molecular biology tool, with increasing demand in biology and medicine. However, this has also exposed its many shortcomings, such as low DNA yield, short DNA extension length, slow polymerase amplification speed, low process fidelity, and poor tolerance.

[0004] Therefore, there is an urgent need in the art to develop a Taq DNA polymerase mutant with faster amplification speed and longer amplification length. Summary of the Invention

[0005] The purpose of the present invention is to provide a Taq DNA polymerase mutant.

[0006] In the first aspect of the present invention, a Taq DNA polymerase mutant is provided, wherein the mutant is selected from any one of the following (a)-(d):

[0007] (a) A mutant obtained by simultaneously performing mutations at both G504K and E507N on the amino acid sequence of SEQ ID NO. 6;

[0008] (b) A mutant obtained by simultaneously performing mutations at both G504R and E507N sites in the amino acid sequence of SEQ ID NO. 6;

[0009] (c) A mutant obtained by simultaneously performing mutations at both G504K and E507P sites in the amino acid sequence of SEQ ID NO. 6;

[0010] (d) A mutant obtained by simultaneously performing mutations at both G504R and E507T on the amino acid sequence shown in SEQ ID NO. 6;

[0011] (e) A mutant obtained by simultaneously performing mutations at both G504R and E507V on the amino acid sequence of SEQ ID NO. 6;

[0012] (f) A mutant obtained by simultaneously performing mutations at both G504A and E507R on the amino acid sequence of SEQ ID NO. 6;

[0013] (g) A mutant obtained by simultaneously performing mutations at both G504H and E507R sites in the amino acid sequence of SEQ ID NO. 6;

[0014] (h) A mutant obtained by simultaneously performing mutations at both G504R and E507P sites in the amino acid sequence of SEQ ID NO. 6;

[0015] (i) A mutant obtained by simultaneously performing mutations at both G504I and E507A on the amino acid sequence shown in SEQ ID NO. 6.

[0016] In another preferred embodiment, the mutant is selected from any one of (a) or (b).

[0017] In another preferred embodiment, the amino acid sequence of the mutant is shown in SEQ ID NO.1.

[0018] In another preferred embodiment, the amplification rate of the mutant is V1, and the amplification rate of the unmutated Taq DNA polymerase is V0, wherein V1 / V0 is ≥5, preferably ≥10, and more preferably ≥15.

[0019] In another preferred embodiment, the amplification speed of the mutant is 50 bp / s-1500 bp / s; preferably 100 bp / s-1500 bp / s; more preferably 200 bp / s-1000 bp / s.

[0020] In another preferred embodiment, the amplification length of the mutant is L1, and the amplification length of the unmutated Taq DNA polymerase is L0, wherein L1 / L0 is ≥1, preferably ≥2, and more preferably ≥5.

[0021] In another preferred embodiment, the amplification speed of the mutant is 50 bp / s-1000 bp / s; preferably 200 bp / s; more preferably 1000 bp / s.

[0022] In the second aspect of the present invention, a nucleic acid molecule is provided, which encodes the Taq DNA polymerase mutant as described in the first aspect of the present invention.

[0023] In the third aspect of the present invention, a vector is provided, wherein the vector contains the nucleic acid molecule according to the second aspect of the present invention.

[0024] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector as described in the third aspect of the present invention, or an exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated into the chromosome.

[0025] In a fifth aspect of the present invention, a method for preparing the Taq DNA polymerase mutant according to the first aspect of the present invention is provided, comprising the following steps:

[0026] (1) operably connecting the coding gene of the Taq DNA polymerase mutant to an expression regulatory element to construct a recombinant expression vector;

[0027] (2) transforming the recombinant expression vector into host cells, culturing the host cells, inducing expression of the recombinant protein, and purifying the recombinant protein to obtain the Taq DNA polymerase mutant.

[0028] In a fifth aspect of the present invention, there is provided use of the Taq DNA polymerase mutant described in the first aspect of the present invention as Taq DNA polymerase in gene amplification.

[0029] In a sixth aspect of the present invention, a kit is provided, comprising the Taq DNA polymerase mutant according to the first aspect of the present invention.

[0030] In another preferred embodiment, the kit further comprises dNTPs, a buffer and primers.

[0031] In a seventh aspect of the present invention, a method for amplifying a nucleic acid molecule is provided.

[0032] The method comprises:

[0033] mixing at least one nucleic acid template with a Taq DNA polymerase mutant to obtain a mixture, wherein the DNA polymerase is any one of the Taq DNA polymerase mutants described in the first aspect of the present invention;

[0034] The mixture is subjected to an amplification treatment to obtain a nucleic acid molecule that is fully or partially complementary to the at least one nucleic acid template.

[0035] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a Taq wild-type and mutant qPCR amplification curve, showing mutants with Ct values ​​lower than the wild-type.

[0037] Figure 2 This is a diagram of the purification of the G504K / E507N mutant protein, which shows the size of the G504K / E507N mutant protein and the purification effect.

[0038] Figure 3 This is a graph showing the amplification rate of the G504K / E507N mutant.

[0039] Figure 4 Comparison of the amplicon length plots of G504K / E507N and another mutant, G504R / E507N. DETAILED DESCRIPTION

[0040] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that, through extensive screening, mutations at specific sites in Taq DNA polymerase, G504 and E507, yielded Taq DNA polymerase mutants that significantly improved Taq DNA polymerase performance, such as accelerating amplification speed and extending amplification length. This discovery led to the completion of the present invention.

[0041] Specifically,

[0042] The present invention obtains a new Taq DNA polymerase mutant through research, and the specific process is as follows:

[0043] 1) Mutation site selection;

[0044] 2) Construction of a saturation mutation library: Using a circular plasmid containing Taq DNA polymerase as a template, PCR amplification was performed to construct a Taq DNA polymerase site-directed saturation mutation plasmid library;

[0045] 3) Inducing expression of the mutant library: The site-directed saturation mutation plasmid obtained in step 2) is transformed into Escherichia coli cells for induced expression to obtain a bacterial suspension;

[0046] 4) The suspension obtained in step 3) was disrupted with lysozyme and incubated at 75° C. for 40 min, followed by centrifugation to obtain the supernatant;

[0047] 5) The supernatant is Taq crude enzyme, which is used to prepare a qPCR system and screen mutants with higher activity by running qPCR;

[0048] 6) Purifying the high-activity mutant obtained in step 5);

[0049] 7) The mutant protein obtained in step 6) is subjected to enzyme activity detection and amplification performance detection.

[0050] the term

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0052] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0053] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0054] Taq DNA polymerase mutants

[0055] As used herein, "Taq DNA polymerase mutant of the present invention," "Taq DNA enzyme mutant of the present invention," or "mutant of the present invention" are used interchangeably to refer to the Taq DNA polymerase mutant described in the first aspect of the present invention. In particular, the Taq DNA polymerase double-site mutant of the present invention has enhanced amplification activity, exhibiting faster amplification speeds and longer amplification lengths.

[0056] As a specific embodiment of the present invention, the present invention provides a Taq DNA polymerase double-site mutant with improved amplification activity, wherein the mutant is selected from any one of the following (a)-(d):

[0057] (a) A mutant obtained by simultaneously performing mutations at both G504K and E507N on the amino acid sequence of SEQ ID NO. 6;

[0058] (b) A mutant obtained by simultaneously performing mutations at both G504R and E507N sites in the amino acid sequence of SEQ ID NO. 6;

[0059] (c) A mutant obtained by simultaneously performing mutations at both G504K and E507P sites in the amino acid sequence of SEQ ID NO. 6;

[0060] (d) A mutant obtained by simultaneously performing mutations at both G504R and E507T on the amino acid sequence shown in SEQ ID NO. 6;

[0061] (e) A mutant obtained by simultaneously performing mutations at both G504R and E507V on the amino acid sequence of SEQ ID NO. 6;

[0062] (f) A mutant obtained by simultaneously performing mutations at both G504A and E507R on the amino acid sequence of SEQ ID NO. 6;

[0063] (g) A mutant obtained by simultaneously performing mutations at both G504H and E507R sites in the amino acid sequence of SEQ ID NO. 6;

[0064] (h) A mutant obtained by simultaneously performing mutations at both G504R and E507P sites in the amino acid sequence of SEQ ID NO. 6;

[0065] (i) A mutant obtained by simultaneously performing mutations at both G504I and E507A on the amino acid sequence shown in SEQ ID NO. 6.

[0066] The terms "mutation" and "mutant" have their ordinary meanings herein and refer to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, and their meanings are the same as those generally understood by those skilled in the art.

[0067] The point mutation "G504K" of the present invention indicates that the 504th amino acid of the Taq DNA polymerase (wild type) with the amino acid sequence shown in SEQ ID NO.6 is mutated from glycine (G) to lysine (K); the expressions of the remaining point mutations of the present invention are similar.

[0068] The multi-site mutants "G504K and E507N" of the present invention represent that the 504th amino acid of the Taq DNA polymerase (wild type) with the amino acid sequence shown in SEQ ID NO.6 is mutated from glycine (G) to lysine (K) and the 507th amino acid is mutated from glycine (G) to asparagine (N); the expression of the multi-site mutations of the remaining amino acids of the present invention is similar.

[0069] The present invention also includes preferred active derivatives, which are polypeptides formed by replacing no more than three, preferably no more than two, and more preferably no more than one amino acid with similar or similar amino acids compared to the amino acid sequences of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0070] Table A

[0071] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile;Leu;Met;Phe;Ala Leu

[0072] The present invention also provides analogs of the mutants of the present invention. These analogs may differ from the polypeptides of the present invention in terms of amino acid sequence, in terms of modifications that do not affect the sequence, or in terms of both. Analogs also include analogs having residues other than naturally occurring L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β- and γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0073] In addition, the mutants of the present invention may also be modified. Modifications (generally without altering the primary structure) include chemical derivatization of the polypeptide in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation during polypeptide synthesis and processing or in further processing steps. Such modifications can be accomplished by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modifications also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or optimize their solubility.

[0074] The term "polynucleotide encoding the mutant of the present invention" may include a polynucleotide encoding the mutant of the present invention, and may also include additional coding and / or non-coding sequences.

[0075] The present invention also relates to variants of the aforementioned polynucleotides, including fragments, analogs, and derivatives encoding polypeptides or mutants having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may contain one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the encoded variant.

[0076] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%.

[0077] The mutants and polynucleotides of the present invention are preferably provided in isolated form, and more preferably, purified to homogeneity.

[0078] The full-length sequences of the polynucleotides of the present invention can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. For long sequences, two or more PCR amplifications are often required, followed by splicing the fragments amplified in the correct order.

[0079] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0080] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0081] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0082] Methods using PCR techniques to amplify DNA / RNA are preferably used to obtain the polynucleotides of the present invention. In particular, when full-length cDNA is difficult to obtain from a library, the RACE method (RACE - rapid amplification of cDNA ends) is preferably used. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods, such as gel electrophoresis.

[0083] expression vector

[0084] The present invention also relates to vectors comprising the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors of the present invention or the mutant coding sequences of the present invention, and methods for producing the polypeptides of the present invention by recombinant technology.

[0085] The polynucleotide sequences of the present invention can be used to express or produce recombinant mutants using conventional recombinant DNA techniques. Generally, the following steps are involved:

[0086] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a mutant of the present invention, or a recombinant expression vector containing the polynucleotide;

[0087] (2) Host cells cultured in a suitable culture medium;

[0088] (3) Isolate and purify proteins from culture medium or cells.

[0089] In the present invention, the polynucleotide sequence encoding the mutant can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it generally contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0090] Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a mutant of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operatively linked to an appropriate promoter within the expression vector to direct mRNA synthesis. Representative examples of such promoters include the lac or trp promoters of Escherichia coli; the lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoter, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0091] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0092] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0093] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, and plant cells (such as ginseng cells).

[0094] When the polynucleotides of the present invention are expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting DNA factors, typically about 10 to 300 base pairs in length, that act on promoters to increase gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs on the late replication origin side), the polyoma enhancer on the late replication origin side, and adenovirus enhancers.

[0095] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0096] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0097] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0098] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0099] The main advantages of the present invention include

[0100] 1) The amplification speed of the Taq DNA polymerase mutant was increased to 1000 bp / s compared to 150 bp / s of the wild type.

[0101] 2) The amplified length of the Taq DNA polymerase mutant was increased to 12 kb compared to 7.6 kb of the wild type.

[0102] 3) The Taq DNA polymerase mutant of the present invention solves the problems of slow amplification speed and short extended DNA length of the wild-type polymerase. Its amplification speed and amplification length are significantly better than those of the wild-type and commercially available products, and it can quickly amplify long DNA fragments.

[0103] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0104] Example 1 Construction of Taq DNA polymerase mutants

[0105] 1. Mutation site selection

[0106] The present inventors, through protein structure simulation, predicted that G504 and E507 are the active sites of Taq DNA polymerase. Based on the spatial distance between the amino acids, they determined that hydrogen bonding exists between G504 and E507. Research targeting different mutation sites revealed that mutating both G504 and E507 impairs Taq DNA polymerase function.

[0107] 2. Construction of saturation mutation library

[0108] In this experiment, pPAP-Taq DNA polymerase, the wild-type enzyme (SEQ ID NO. 6) derived from the circular plasmid of Thermus aquaticus, was used as a template, and saturation mutagenesis primers were designed for PCR amplification.

[0109] Wild-type enzyme of pPAP-Taq DNA polymerase SEQ ID NO: 6

[0110] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAI G KT E KTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATwATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE

[0111] The specific sequences of the mutant primers are as follows:

[0112] 504 / 507-F: GGCTTCCCGCCATCNNKAAGACGNNKAAGACCGGCAAGCGCTCCACC(SEQ IDNO.2)

[0113] 504 / 507-R:CGCTTGCCGGTCTTMNNCGTCTTMNNGATGGCGGGAAGCCCTAGCTC(SEQ IDNO.3)

[0114] Circular plasmid PCR system:

[0115] Table 1

[0116]

[0117]

[0118] Use a pipette to gently pipette to mix thoroughly and centrifuge briefly to remove the reaction solution to the bottom of the tube. Place the sample in a PCR instrument and set the reaction program as follows:

[0119] Table 2

[0120]

[0121] After the reaction, the amplified product was digested with Dpn I and the concentration was detected by Qubit nucleic acid detector.

[0122] 3. Saturation mutation library induced expression

[0123] The digestion product was cleaned and recovered by the Tiangen kit and then electroporated into the Escherichia coli SS320 strain. Single clones were selected into a 96-well plate, and 1 ml of corresponding resistance liquid culture medium was added to each well. The plates were cultured on a shaker at 37°C and 220 rpm until the OD600 reached 0.5. Arabinose with a final concentration of 0.2 ppm was added to each well, and the plates were induced at 37°C for 3 h and centrifuged at 4000 rpm for 20 min to collect the bacteria.

[0124] 4. Bacteria rupture

[0125] The bacterial cells obtained in step 2) were resuspended with lysozyme and broken in a 37°C water bath for 30 minutes, and incubated at 75°C for 40 minutes, and then centrifuged to obtain the supernatant.

[0126] 5. Mutant activity screening

[0127] The fluorescent signal in real-time quantitative PCR (qPCR) is generated by a fluorescent probe or fluorescent DNA-binding dye (such as SYBR Green), and its intensity is proportional to the number of PCR product molecules (amplicons). If a specific sequence (DNA or RNA) is abundant in the sample, amplification is observed in earlier cycles, resulting in a smaller Ct value; if the sequence is scarce, amplification is observed in later cycles, resulting in a larger Ct value. Taq DNA polymerase has 5′→3′ polymerase activity, which we use in qPCR amplification. Because Taq DNA polymerase mutants have different amplification activities, the Ct values ​​of their amplified products vary. Under the same amplification conditions, a smaller Ct value indicates a higher mutant activity, and the final fluorescent signal intensity can reflect the amount of amplified product.

[0128] Therefore, the activity of Taq DNA polymerase mutants can be screened by qPCR. The mutant with a small Ct value and a strong fluorescence signal is the target mutant.

[0129] qPCR primers were designed as follows:

[0130] OYXV-F:CCAGTCCAGGGAATCTGTGT(SEQ ID NO.4)

[0131] OYXV-R:GAAGATGAGGCCGAGAGTGA(SEQ ID NO.5)

[0132] The supernatant is the crude Taq enzyme, which is used to prepare the qPCR system as follows:

[0133] Table 3

[0134] Components Addition amount 10x KTQ Buffer 1 μl OYXV-F (10 μM) 0.5 μl OYXV-R (10 μM) 0.5 μl Protein supernatant 10 μl dNTPs (10 μM) 0.5 μl 20x SYBR Green I 0.6 μl OYXV plasmid 0.01ng ddH2O To 20μl

[0135] In the PCR instrument, the reaction program is set as follows:

[0136] Table 4

[0137]

[0138] After the reaction is completed, if Figure 1 As shown, the red curve is the wild-type Taq enzyme, and the blue curve is the mutant. The mutants with low Ct values ​​and strong fluorescence signals were selected for subsequent testing.

[0139] In this example, more than 30 96-well plates and more than 3,000 monoclonal samples were screened, and 9 mutants (mutant G504K / E507N, mutant G504R / E507N, mutant G504K / E507P, mutant G504R / E507T, mutant G504R / E507V, mutant G504A / E507R, mutant G504H / E507R, mutant G504R / E507P, and mutant G504I / E507A) were finally selected for subsequent testing.

[0140] 6. Mutant Sequencing

[0141] The amino acid sequence of the most active mutant G504K / E507N is as follows:

[0142] Mutant G504K / E507N (SEQ ID NO: 1)

[0143] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDA

[0144] KAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTA

[0145] DKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSL

[0146] EALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGL

[0147] LESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLA

[0148] LRELGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREV

[0149] ERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAI K KT

[0150] N KTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATwATGRLSSSD

[0151] PNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPR

[0152] EAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLF

[0153] GRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAE

[0154] AVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE

[0155] Example 2 Performance Test of Taq DNA Polymerase Mutants

[0156] The G504K / E507N mutant was tested for amplification speed and length. The mutant G504K / E507N was expressed and purified (eg Figure 2 After the enzyme activity was adjusted to 5 U / μl, PCR amplification verification was performed.

[0157] The amplification rate is Figure 3 As shown, the amplified fragment size is 4 kb, and the delay time is 4 s. The G504K / E507N mutant can amplify a 4 kb target band in 4 s (amplification speed is 1000 bp / s). The control Taq enzyme is Nearshore Taq DNA Polymerase Product No. E001, enzyme activity 5 U / μl, which can amplify an 8 kb target band (amplification speed is 200 bp / s).

[0158] Figure 3 , the lane between Taq and G504K / E507N is G504R / E507N

[0159] Amplification length test such as Figure 4 As shown, the G504R / E507N mutant can amplify fragments up to 8 kb, while the G504K / E507N mutant can amplify fragments up to 12 kb, significantly outperforming the control Taq enzyme. Therefore, the G504K / E507N mutant has higher activity and better performance.

[0160] Comparative Example

[0161] The remaining mutants were tested for amplification speed and length using the method described in Example 2. The amplification performance test results of the remaining mutants are shown in Table 5.

[0162] Table 5 Performance of different mutants of G504 / E507

[0163] serial number mutant Amplification speed Amplification length 1 G504K / E507N 1000bp / s 12kb 2 G504R / E507N 200bp / s 8kb 3 G504K / E507P 100bp / s 4kb 4 G504R / E507T 50bp / s 4kb 5 G504R / E507V 100bp / s 4kb 6 G504A / E507R 50bp / s 4kb 7 G504H / E507R 50bp / s 4kb 8 G504R / E507P 200bp / s 4kb 9 G504I / E507A 200bp / s 4kb

[0164] The results showed that G504K / E507N had a faster amplification speed and longer length than other mutants, making it the optimal mutant (the amplification speed of the wild type Taq DNA Polymerase is 150bp / s).

[0165] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A Taq DNA polymerase mutant, characterized in that The mutant is selected from any one of the following (a)-(d): (a) A mutant obtained by simultaneously performing mutations at both G504K and E507N on the amino acid sequence of SEQ ID NO. 6; (b) A mutant obtained by simultaneously performing mutations at both G504R and E507N sites in the amino acid sequence of SEQ ID NO. 6; (c) A mutant obtained by simultaneously performing mutations at both G504K and E507P sites in the amino acid sequence of SEQ ID NO. 6; (d) A mutant obtained by simultaneously performing mutations at both G504R and E507T on the amino acid sequence shown in SEQ ID NO. 6; (e) A mutant obtained by simultaneously performing mutations at both G504R and E507V on the amino acid sequence of SEQ ID NO. 6; (f) A mutant obtained by simultaneously performing mutations at both G504A and E507R on the amino acid sequence of SEQ ID NO. 6; (g) A mutant obtained by simultaneously performing mutations at both G504H and E507R sites in the amino acid sequence of SEQ ID NO. 6; (h) A mutant obtained by simultaneously performing mutations at both G504R and E507P sites in the amino acid sequence of SEQ ID NO. 6; (i) A mutant obtained by simultaneously performing mutations at both G504I and E507A on the amino acid sequence shown in SEQ ID NO.

6.

2. The Taq DNA polymerase mutant according to claim 1, wherein The mutant is selected from any one of (a) or (b).

3. The Taq DNA polymerase mutant according to claim 1, wherein The amino acid sequence of the mutant is shown in SEQ ID NO.

1.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Taq DNA polymerase mutant according to claim 1.

5. A carrier, characterized in that The vector contains the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that The host cell contains the vector according to claim 5, or the exogenous nucleic acid molecule according to claim 2 is integrated into the chromosome.

7. A method for preparing the Taq DNA polymerase mutant according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) operably connecting the coding gene of the Taq DNA polymerase mutant to an expression regulatory element to construct a recombinant expression vector; (2) transforming the recombinant expression vector into host cells, culturing the host cells, inducing expression of the recombinant protein, and purifying the recombinant protein to obtain the Taq DNA polymerase mutant.

8. Use of the Taq DNA polymerase mutant according to any one of claims 1 to 3 as Taq DNA polymerase in gene amplification.

9. A kit, characterized in that The method comprises the Taq DNA polymerase mutant according to any one of claims 1 to 3.

10. A method for amplifying a nucleic acid molecule, characterized in that: The method comprises: mixing at least one nucleic acid template with a Taq DNA polymerase mutant to obtain a mixture, wherein the DNA polymerase is the Taq DNA polymerase mutant according to any one of claims 1 to 3; The mixture is subjected to an amplification treatment to obtain a nucleic acid molecule that is fully or partially complementary to the at least one nucleic acid template.

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