Taq DNA polymerase as well as preparation method and application thereof

Abstract

The invention provides Taq DNA polymerase as well as a preparation method and application thereof. The Taq DNA polymerase has an amino acid sequence as shown in SEQ ID NO. 1. According to the invention, the amino acid sequence of the Taq DNA polymerase is improved, and the obtained polymerase has better thermal stability; according to the present invention, the polymerase purification process is improved, such that the obtained polymerase has characteristics of low nucleic acid residue and high purity, the PCR amplification efficiency can be improved, and the method can be widely used for the nucleic acid detection platform.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a Taq DNA polymerase and a preparation method and application thereof. Background Art

[0002] DNA polymerases that can be used in PCR can be roughly divided into two types. One type is the polymerase isolated from the hyperthermophilic Archaea, represented by KOD DNA polymerase (hereinafter referred to as KOD), which is classified as family B (α type). The other type is the polymerase isolated from the aquatic thermophilic bacterium Thermus aquaticus, represented by Taq DNA polymerase (hereinafter referred to as Taq), which is classified as family A (Pol I type). Because this bacterium lives in hot springs at 70-75℃, Taq DNA polymerase has extremely high thermal stability and can withstand the thermal denaturation step of PCR. In addition, it has other enzymatic characteristics: (1) 5'-3' polymerase activity; (2) 5'-3' nuclease activity; (3) A is added to the 3' end of some PCR products; (4) no 3'-5' nuclease activity; (5) an isoelectric point of approximately 6.0 and an overall average hydrophilic index of -0.282.

[0003] The widely used Taq DNA polymerase is mainly constructed on an expression vector in the form of gene cloning, and then introduced into common engineering strains such as Escherichia coli. After induction to express the target protein, foreign proteins, nucleic acid substances and environmental pollution sources are removed through various purification processes. The current purification steps can remove most impurities, such as nucleic acids, nucleases, etc., but in order to apply Taq DNA polymerase to actual nucleic acid detection kit products, higher requirements are placed on the comprehensive performance of the enzyme, such as the purity of the enzyme (such as the residual amount of nucleases and nucleic acids) and the thermal stability of the enzyme. At the same time, it is also necessary to meet the requirements that Taq DNA polymerase can be produced in large quantities and have stable quality.

[0004] CN114891762A discloses a method for rapidly preparing Taq DNA polymerase. High salt and ultrasonic disruption are added during the crude purification process to break up nucleic acid fragments without attaching them to proteins. However, the addition of Triton X-114 to the buffer system may affect different types of PCR amplification.

[0005] CN115678870A discloses a reagent and method for preparing Taq DNA polymerase with low host DNA content. After the Ni affinity chromatography step, heat-sensitive dsDNase is added to degrade nucleic acids, and then the dsDNase is inactivated by high temperature. However, this method is prone to contamination by exogenous nucleases and is not suitable for large-scale production.

[0006] Therefore, it is necessary to further develop new Taq DNA polymerases and explore the impact of the preparation process on them, so as to improve the comprehensive performance such as protein purity and thermal stability, and also realize industrial production. Summary of the Invention

[0007] In view of the above technical problems, the present invention provides a method for preparing Taq DNA polymerase. The Taq DNA polymerase provided by the present invention can effectively remove host DNA residues, improve protein purity and thermal stability, and is expected to achieve industrial production, thereby being widely used in nucleic acid detection platforms.

[0008] In order to achieve the above technical objectives, the technical solutions adopted in this application are as follows:

[0009] In a first aspect, the present invention provides a Taq DNA polymerase having an amino acid sequence as shown in SEQ ID NO.1.

[0010] In a second aspect, the present invention provides a polynucleotide sequence encoding the Taq DNA polymerase.

[0011] Preferably, the polynucleotide sequence has the nucleotide sequence shown in SEQ ID NO.2.

[0012] In a third aspect, the present invention provides a polynucleotide construct comprising the polynucleotide sequence; preferably, the polynucleotide construct is a polynucleotide construct in which the DNA fragment between the BamHI and HindIII recognition sequences of pET-28a is replaced with the polynucleotide sequence.

[0013] In a fourth aspect, the present invention provides a host cell comprising the polynucleotide sequence or the polynucleotide construct.

[0014] In a fifth aspect, the present invention provides use of the Taq DNA polymerase, the polynucleotide sequence, the polynucleotide construct, or the host cell in nucleic acid amplification or preparation of a nucleic acid amplification product.

[0015] In a sixth aspect, the present invention provides a method for preparing the Taq DNA polymerase, which comprises the following steps: culturing the host cell; and then isolating the Taq DNA polymerase from the cell culture.

[0016] Preferably, the host cell is obtained by transforming the polynucleotide sequence shown in SEQ ID NO: 2 into competent Escherichia coli cells.

[0017] Preferably, the separation of Taq DNA polymerase from the culture comprises the following steps:

[0018] 1) The collected cell culture is sequentially disrupted, heat-treated, and precipitated to obtain a crude Taq DNA polymerase;

[0019] 2) Redissolve the crude Taq DNA polymerase and purify it using NI affinity chromatography;

[0020] Further preferably, the loading buffer system of the NI affinity chromatography is 20-25 mM Tris buffer at pH = 8.0 containing 50-100 mM KCl, the washing buffer system is 20-25 mM Tris buffer at pH = 8.0 containing 50-100 mM KCl and 30 mM imidazole, and the elution buffer system is 20 mM Tris buffer at pH = 8.0 containing 50-100 mM KCl and 300 mM imidazole;

[0021] 3) The protein solution purified by NI affinity chromatography is further purified by cation exchange column chromatography;

[0022] Further preferably, the loading buffer system of the cation exchange column chromatography is 20-25 mM Tris buffer with a pH of 7.2;

[0023] 4) further purifying the protein solution purified by anion exchange column chromatography by cation exchange column chromatography;

[0024] Further preferably, the loading buffer system of the anion exchange column chromatography is 20-25 mM Tris buffer at pH = 7.8, and the elution buffer system is 20-25 mM Tris buffer at pH = 7.8 containing 150-200 mM KCl;

[0025] 5) the protein solution purified by cation exchange column chromatography is further purified by heparin affinity chromatography;

[0026] Further preferably, the loading buffer system of the heparin affinity chromatography is 20-25 mM Tris buffer at pH = 8.0, and the elution buffer system is 20-25 mM Tris buffer at pH = 8.0 containing 200-300 mM KCl;

[0027] 6) dialyzing the protein solution purified by heparin affinity chromatography;

[0028] Further preferably, the dialysate is a 20-25 mM PBS buffer solution with a pH of 8.0 containing 50-100 mM KCl. Preferably, the collected cell culture is subjected to a disrupting, heat treatment, and precipitation treatment in sequence, including the following:

[0029] 1) mixing 20-25 mM Tris buffer (pH 8.0) containing 50-100 mM KCl with the cell culture at a ratio of buffer volume to cell culture weight of 20:1, and then performing high-pressure homogenization; more preferably, the homogenization temperature is -2°C to 2°C, and the homogenization pressure is 550-700 Barg;

[0030] 2) Treat the disrupted bacterial solution at 70-75°C for 30-40 minutes to separate the solids and retain the liquid;

[0031] 3) Adding polyethyleneimine according to the volume of liquid obtained in step 2), adding nuclease to the supernatant after centrifugation and treating at 35-40° C. for 20-40 minutes, more preferably at 37° C. for 30 minutes; and adding ammonium sulfate to precipitate the Taq DNA polymerase therein.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention improves the amino acid sequence of DNA polymerase, resulting in a polymerase with better thermal stability; and improves the purification process of the polymerase, resulting in a polymerase with low nucleic acid residues and high polymerase purity, which can improve the amplification efficiency and fidelity of PCR (lower tailing rate) and can be widely used in nucleic acid detection platforms, for example, in both fluorescent PCR and digital PCR platforms. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the electrophoresis result of Taq DNA polymerase obtained in Example 1 of the present invention.

[0035] Figure 2 Figure 1 shows the changes in total enzyme activity and specific activity of the protein solution after different separation and purification steps and the final Taq DNA polymerase product in Example 1 of the present invention, wherein Figure (a) shows the changes in total enzyme activity, and Figure (b) shows the changes in specific activity.

[0036] Figure 3 The performance of the Taq DNA polymerase obtained in the different separation and purification steps in Example 1 of the present invention for use in the novel coronavirus 2019-nCoV nucleic acid detection kit, wherein Figures (a) to (b) respectively represent the results of the Taq DNA polymerase obtained after heat treatment, PEI precipitation, anion chromatography, and heparin chromatography for use in the novel coronavirus 2019-nCoV nucleic acid detection kit.

[0037] Figure 4 These are the test results of the residual DNA in the Taq DNA polymerase product obtained in Example 1 of the present invention, wherein (a) is the test result of the fluorescent PCR platform, and (b) is the test result of the digital PCR platform.

[0038] Figure 5 The results of protein purity changes after different purification sequences are shown in the figure. The purification sequence of Figures (a) to (f) is explained in detail.

[0039] Figure 6 1 and 2 are the amplification performance results of the fluorescent PCR method in Example 4 of the present invention, wherein FIG. 1 (a) is the FAM channel and FIG. 1 (b) is the VIC channel. DETAILED DESCRIPTION

[0040] The NI nucleophilic chromatography column used in the embodiments of the present invention was purchased from Cytiva, Ni Sepharose 6 fast flow; the anion exchange column was purchased from Cytiva, Capto S; the cation exchange column was purchased from Cytiva, Capto Q; the heparin nucleophilic chromatography column was purchased from Cytiva, and the HiPrep Heparin FF dialysis bag was purchased from Bio-Gene, with a dialysis bag D16 mm and a dialyzable molecular weight of 12 kDa to 14 kDa.

[0041] The novel coronavirus 2019-nCoV nucleic acid detection kit used in the embodiments of the present invention was purchased from Mike Biotech.

[0042] In the description of the present invention, it should be noted that if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0044] In an embodiment of the present invention, the host DNA residue detection scheme is:

[0045] (1) Use the fluorescent dye method to detect the residual nucleic acid obtained in each purification step. Use TE buffer to prepare lambda DNA standards into 200 ng / mL, 75 ng / mL, 30 ng / mL, 8 ng / mL, 2.5 ng / mL, 0.75 ng / mL, 0.4 ng / mL, and 0 ng / mL standard solutions. After precipitating the DNA, add 250 μL of the newly prepared double-stranded DNA fluorescent dye, mix well, and place at room temperature in the dark for 5 minutes. Take 240 μL of the reaction solution in a 96-well black enzyme-labeled plate, and use a fluorescence enzyme-labeled instrument to measure the fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 525 nm, and then prepare a standard curve. The fluorescence intensity of the sample to be tested is measured using a fluorescence enzyme-labeled instrument, and then the DNA content result is obtained according to the standard curve;

[0046] (2) The nucleic acid amplification method was used to detect the residual nucleic acid obtained in each purification step. The amplification primers were: probe: 5'FAM-CGGTGCTGCGACGGCGGAGT-TAMRA3' (SEQ ID NO.3), forward primer: 5'-GAAAGTAACACCAGCGTGCG-3' (SEQ ID NO.4), reverse primer: 5'-CCAATGCATTAACGCTGGCA-3' (SEQID NO.5), and the amplification program was: stage 1, 95℃ for 10min, stage 2, 95℃ for 10s, 60℃ for 1min, and 40 cycles.

[0047] In the embodiment of the present invention, the enzyme activity detection scheme is: using the national standard method (standard number: GB / T 35542-2017; Chinese standard name: Taq DNA polymerase; instrument: fully automatic medical PCR analysis system SLAN-96P) to measure the enzyme activity of each purification step, the hairpin primer is: 5′-tagcgaaggatgtgaacctaatcccTGCTCCCGCGGCCGatctgcCGGCCGCGGGAGCA-3′ (SEQ ID NO. 6), the amplification program is: 74°C 16s, 99 cycles, fluorescence acquisition channel: SYBR, and the total enzyme activity and specific activity of each purification step are calculated.

[0048] In an embodiment of the present invention, a thermal stability detection scheme is used, wherein Taq enzyme is packaged in PCR tubes and placed under temperature conditions of 94°C for 1 hour, 37°C for 7 days, and 4°C for 7 days, respectively, to detect changes in enzyme activity before and after treatment.

[0049] Example 1

[0050] This example provides a method for preparing Taq DNA polymerase, and the specific process is as follows:

[0051] ① Using genetic engineering technology to construct the optimized Taq DNA polymerase nucleotide sequence (as shown in SEQ ID NO.2) into the pET28a expression vector through BamHI and HindIII;

[0052] ② The expression vector was transformed into E. coli competent cells BL21 (DE3) pLysS. When the cells reached OD600 = 0.9-1.9, the inducer IPTG was added to a final concentration of 1 mM for induction. After induction at 37°C for 4-6 hours, the recombinant bacteria were collected.

[0053] ③ Resuspend the cells in a disruption buffer (20 mM Tris 100 mM KCl pH 8.0) at a ratio of 20:1 (volume:cell weight (v / w)). Then, homogenize the cells under high pressure at a temperature of -2°C to 2°C and a homogenization pressure of 550 to 700 barg. Heat treat for 30-40 minutes and centrifuge to remove impurities.

[0054] ④ Add polyethyleneimine (PEI) according to the volume of the solution, centrifuge and discard the precipitate, add nuclease to the supernatant and treat at 37°C for 30 minutes, and then recover the Taq enzyme by ammonium sulfate precipitation;

[0055] ⑤ The Taq enzyme was redissolved and purified by NI affinity chromatography column. The loading buffer system was 20mM Tris 100mM KCl pH = 8.0, the washing buffer system was 20mM Tris 100mM KCl 30mM imidazole pH = 8.0, and the elution buffer system was 20mMTris 100mM KCl 300mM imidazole pH = 8.0.

[0056] ⑥ The solution obtained in step ⑤ was further purified using a cation exchange column with a loading buffer of 20 mM Tris pH = 7.8 and an elution buffer of 20 mM Tris 200 mM KCl pH = 7.8;

[0057] ⑦ The solution obtained in step ⑥ was further purified using an anion exchange column with a loading buffer of 20 mM Tris pH = 7.2, and the flow-through was collected;

[0058] ⑧ The flow-through obtained in step ⑦ was further purified using a heparin affinity chromatography column, the loading buffer system was 20mM Tris pH=8.0, and the elution buffer system was 20mM Tris 300mM KCl pH=8.0;

[0059] ⑨ The protein solution obtained in step ⑧ was dialyzed and eluted with 20mM PBS 100mM KCl pH = 8.0 to obtain pure Taq DNA polymerase. The purity of Taq DNA polymerase was determined by KTApure Convenience instrument and was 99.43%. The electrophoresis results were as follows: Figure 1 As shown, it was also confirmed that the obtained Taq DNA polymerase had high purity.

[0060] The obtained Taq DNA polymerase base sequence: SEQ ID NO.2

[0061] ATGTCGCGTGGCATGCTGCCGCTGTTTGAACCGAAAGGTCGCGTGCTGCTGGTG

[0062] GATGGCCATCATCTGGCCTATCGTACCTTTCATGCACTGAAAGGCCTGACCACCA

[0063] GCCGCGGCGAACCGGTTCAGGCCGTGTATGGCTTTGCAAAAAGTCTGCTGAAA

[0064] GCCCTGAAAGAAGATGGCGATGCAGTTATTGTGGTTTTTGATGCCAAAGCCCCG

[0065] AGCTTTCGTCATGAAGCCTATGGTGGCTATAAAGCAGGTCGTGCCCCGACCCCG

[0066] GAAGATTTTCCGCGTCAGCTGGCACTGATTAAGGAACTGGTTGATCTGCTGGGC

[0067] CTGGCACGTCTGGAAGTGCCGGGTTATGAAGCAGATGATGTGCTGGCCAGTCTG

[0068] GCAAAAAAGGCAGAAAAAGAAGGTTATGAAGTTCGTATTCTGACCGCCGATAA

[0069] AGATCTGTATCAGCTGCTGAGCGATCGTATTCATGTTCTGCATCCGGAAGGCTAT

[0070] CTGATTACCCCGGCCTGGCTGTGGGAAAAATATGGCCTGCGCCCGGATCAGTGG

[0071] GCCGATTATCGCGCACTGACCGGTGACGAAAGCGATAATCTGCCGGGTGTTAAA

[0072] GGTATTGGTGAAAAAACCGCACGTAAACTGCTGGAAGAATGGGGTAGTCTGGA

[0073] AGCCCTGCTGAAAAATCTGGATCGTCTGAAACCGGCAATTCGCGAAAAAATTCT

[0074] GGCACACATGGATGATCTGAAACTGAGCTGGGATCTGGCCAAAGTGCGTACCGA

[0075] TCTGCCGCTGGAAGTGGATTTTGCAAAACGCCGTGAACCGGATCGTGAACGTCT

[0076] GCGTGCCTTTCTGGAACGCCTGGAATTTGGTAGCCTGCTGCATGAATTTGGCCT

[0077] GCTGGCAAGCCCGAAAGCACTGGAAGAAGCACCGTGGCCGCCGCCGGAAGGT

[0078] GCATTTGTGGGTTTTGTGCTGAGTCGTAAAGAACCGATGTGGGCAGATCTGCTG

[0079] GCACTGGCCGCAGCCCGCGGTGGTAGAGTTCATCGTGCCCCGGAACCGTATAAA

[0080] GCACTGCGTGATCTGAAAGAAGCACGTGGCCTGCTGGCAAAAGATCTGAGCGT

[0081] TCTGGCACTGCGTGAAGGCCTGGGCCTGCCGCCTGGTGACGATCCTATGCTGCT

[0082] GGCCTATCTGCTGGACCCTAGCAATACCACCCCGGAAGGCGTGGCACGCCGTTA

[0083] TGGTGGTGAATGGACCGAAGAAGCCGGCGAACGCGCCGCCCTGAGCGAAAGA

[0084] CTGTTTGCCAATCTGTGGGGTCGTCTGGAAGGTGAAGAACGTCTGCTGTGGCTG

[0085] TATCGCGAAGTTGAACGCCCGCTGAGTGCAGTTCTGGCCCACATGGAAGCCACC

[0086] GGCGTGCGTCTGGATGTTGCCTATCTGCGTGCACTGAGCCTGGAAGTTGCCGAA

[0087] GAAATTGCCCGTCTGGAAGCCGAAGTGTTTCGTCTGGCCGGTCATCCGTTTAAT

[0088] CTGAATAGTCGCGATCAGCTGGAACGCGTGCTGTTTGATGAACTGGGTCTGCCG

[0089] GCAATTGGCAAAACCGAAAAAACCGGTAAACGTAGCACCAGTGCCGCAGTTCT

[0090] GGAAGCACTGCGCGAAGCACATCCGATTGTTGAAAAAATTTTACAGTACCGCGA

[0091] ACTGACCAAACTGAAAAGTACCTATATTGATCCGCTGCCGGATCTGATTCATCCG

[0092] CGCACCGGTCGTCTGCATACCCGCTTTAATCAGACCGCAACCGCAACCGGCCGT

[0093] CTGAGCAGTAGTGATCCGAATCTGCAGAATATTCCGGTTCGTACCCCGCTGGGTC

[0094] AGCGTATTCGTCGCGCCTTTATTGCCGAAGAAGGTTGGCTGCTGGTTGCCCTGG

[0095] ATTATAGCCAGATTGAACTGCGCGTGCTGGCACATCTGAGCGGCGATGAAAATC

[0096] TGATTCGTGTGTTTCAGGAAGGCCGTGATATTCATACCGAAACCGCCAGCTGGA

[0097] TGTTTGGCGTGCCGCGTGAAGCAGTGGACCCTCTGATGCGCCGTGCAGCAAAA

[0098] ACCATTAATTTTGGCGTTCTGTATGGTATGAGCGCACATCGCCTGAGCCAGGAAC

[0099] TGGCAATTCCGTATGAAGAAGCACAGGCCTTTATTGAACGTTATTTTCAGAGCTT

[0100] TCCGAAAGTGCGCGCATGGATTGAAAAAACCCTGGAAGAAGGCCGTCGCCGCG

[0101] GTTATGTGGAAACCCTGTTTGGCCGCCGTCGCTATGTGCCGGATCTGGAAGCCC

[0102] GTGTTAAAAGTGTTCGCGAAGCAGCAGAACGTATGGCATTCAATATGCCGGTTC

[0103] AGGGTACCGCCGCCGATCTGATGAAACTGGCAATGGTTAAACTGTTTCCGCGTC

[0104] TGGAAGAAATGGGTGCCCGTATGCTGCTGCAGGTGCATGATGAACTGGTGCTGG

[0105] AAGCCCCGAAAGAACGTGCAGAAGCCGTGGCCCGCCTGGCAAAAGAAGTGAT

[0106] GGAAGGTGTGTATCCGCTGGCAGTTCCGCTGGAAGTTGAAGTGGGCATTGGTG

[0107] AAGATTGGCTGAGCGCCAAAGAATAATaq DNA polymerase amino acid sequence: SEQ ID NO.1

[0108] MSRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKA

[0109] LKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLAR

[0110] LEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPA

[0111] WLVEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKN

[0112] LDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRRREPDRERRLRAFLERL

[0113] EFGSLLHEFGLLASPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGR

[0114] VHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTT

[0115] PEGVARRYGGEWTEEAGERALSERLFANLWGRLEGEERLLWLYRVERPLSAVL

[0116] AHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDE

[0117] LGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPR

[0118] TGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQI

[0119] ELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVL

[0120] YGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGR

[0121] RRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE*

[0122] This example also measured the total enzyme activity and specific activity of the protein solution obtained in each step. The results are shown in Table 1 and Figure 2 As shown, Figure 2 It is a curve graph obtained based on the data in Table 1.

[0123] Table 1 Changes in total enzyme activity and specific activity after different purification steps

[0124]

[0125]

[0126] Note: "Ratio (%)" in Table 1 is the ratio of the specific activity value of a certain step to the specific activity value of the heat treatment step × 100%.

[0127] This example also uses the 2019-nCoV nucleic acid detection kit (fluorescence PCR method) to evaluate the amplification performance of the protein solution obtained in each step after heat treatment, PEI treatment, ion chromatography, and heparin affinity chromatography column purification. The sample type is internal control extract (200 copies / mL, pseudovirus containing 2 new coronavirus genes). The operation process is carried out according to the kit instructions. The results are as follows: Figure 3 As shown, green is the internal standard gene, and red and blue represent two new coronavirus genes, with four replicates for each. The results show that after heparin treatment, the signal value is significantly improved, that is, the Taq enzyme treated with heparin has higher amplification performance (the green is the internal standard gene, which has a high content itself, so the amplification performance improvement is not obvious under the Taq enzyme conditions in different steps).

[0128] This example also uses a fluorescent dye method to detect the residual DNA in the protein solution after the bacterium disruption, heat treatment, PEI treatment, ammonium sulfate precipitation, and each purification step in steps ① to ⑨ above. At the same time, the DNA content of the finished product of the present invention and a commercially available reference substance was detected. The results are shown in Table 2:

[0129] Table 2 DNA content results detected by fluorescent dye method

[0130]

[0131] In Table 2, "finished product" refers to Taq DNA polymerase treated with heparin and dialyzed, diluted to 5 U / μL (hereinafter referred to as "finished product"); "control substance" refers to commercially available Taq DNA polymerase (5 U / μL, hereinafter referred to as "control substance"). As can be seen from Table 2, the Taq DNA polymerase of the present invention has a lower host DNA concentration and a higher purity.

[0132] This embodiment also uses a nucleic acid amplification method to detect the residual nucleic acid in the finished Taq DNA polymerase; a fluorescent quantitative PCR instrument and a digital PCR instrument are used for detection respectively. Among them, the PCR amplification system includes: finished Taq DNA polymerase, amplification reagents (dNTPs, and some substances that promote PCR reactions, such as KCl, MgCl2, Tris-HCl, trehalose, (NH4)2SO4, etc.), probes, primers, and nuclease-free water; the probe and primer sequences for PCR amplification are shown in SEQ ID NO.3 to SEQ ID NO.5 respectively; the PCR amplification procedure is: stage one 95℃10min, stage two 95℃10s, 60℃1min, 40cycles. The test results are as follows Figure 4 As shown, (a) shows a negative result after fluorescence PCR, and (b) shows a negative result after digital PCR. This further demonstrates that the Taq DNA polymerase obtained in this example contains little residual DNA and is of high purity, thus avoiding the problem of interference with experimental results due to trace amounts of DNA carried by the Taq enzyme as template amplification.

[0133] Example 2

[0134] This example is a condition exploration experiment for the purification process (the order of using anion exchange column, cation exchange column, and heparin nucleophilic chromatography column). Steps ① to ⑤ and ⑨ are the same as those in Example 1. The differences in other processes are as follows:

[0135] 1) The Taq DNA polymerase obtained was first subjected to a heparin nucleophilic chromatography column, then an anion exchange column, and finally a cation exchange column. The purity of the obtained Taq DNA polymerase was determined to be 86.19% by using a KTA pure Convenience instrument.

[0136] 2) The Taq DNA polymerase obtained was first subjected to a heparin nucleophilic chromatography column, then to a cation exchange column, and finally to an anion exchange column. The purity of the obtained Taq DNA polymerase was determined to be 86.36% by using a KTA pure Convenience instrument.

[0137] 3) First, an anion exchange column was used, then a cation exchange column was used, and finally a heparin nucleophilic chromatography column was used. The purity of the obtained Taq DNA polymerase was determined to be 97.4% by KTA pure Convenience instrument.

[0138] 4) First, an anion exchange column was used, then a heparin nucleophilic chromatography column was used, and finally a cation exchange column was used. The purity of the obtained Taq DNA polymerase was determined by KTA pure Convenience instrument to be 97.49%.

[0139] 5) First, a cation exchange column was used, then a heparin nucleophilic chromatography column was used, and finally an anion exchange column was used. The purity of the obtained Taq DNA polymerase was determined by KTA pure Convenience instrument to be 98.88%.

[0140] 6) Example 1: First, a cation exchange column was used, then an anion exchange column was used, and finally a heparin nucleophilic chromatography column was used. The purity of Taq DNA polymerase was determined to be 99.43% by KTA pure Convenience instrument.

[0141] The results are as follows Figure 5 As shown, it is shown that high-purity Taq DNA polymerase can be obtained by adopting the purification method of Example 1.

[0142] Example 3

[0143] The finished Taq DNA polymerase obtained in Example 1 was subjected to a thermal stability test. The experimental conditions are shown in Table 3.

[0144] Table 3 Thermal stability of Taq DNA polymerase finished product

[0145]

[0146]

[0147] Note: The calculation formula for retention rate is current sample enzyme activity ÷ original sample enzyme activity × 100%.

[0148] The results in Table 3 show that the finished Taq DNA polymerase obtained in Example 1 has better thermal stability than the commercially available Taq enzyme.

[0149] Example 4

[0150] The performance of the Taq DNA polymerase obtained using the method of Example 1 was verified using a fluorescent PCR platform, using a Hepatitis B Virus Nucleic Acid Detection Kit (Mike Biotech), amplification kit batch number: PR100801, and a nucleic acid (DNA) extraction kit (Mike Biotech, magnetic bead method), extraction kit batch number: 0623041. The specific fluorescent PCR operation method was carried out according to the aforementioned kit instructions.

[0151] (1) Amplification efficiency detection:

[0152] ① Polymerase: Three batches of Taq DNA polymerase prepared by the method of Example 1 were used, and a commercially available product was used as a control; wherein the yield of the three batches of Taq DNA polymerase was 20 million U (typically 200,000 U in the laboratory), and the enzyme activity was comparable to that of the commercially available control;

[0153] ②Test object: Hepatitis B weak positive quality control product (containing hepatitis B nucleic acid and internal standard)

[0154] The results are shown in Tables 4, 5 and Figure 6 As shown; among them, the FAM channel is the hepatitis B nucleic acid test result, and the VIC channel is the internal standard test result; the Ct mean value is derived from the Ct mean value obtained after repeated testing of the weak positive quality control product for each sample 20 times.

[0155] Table 4 Amplification efficiency of FAM channel

[0156]

[0157]

[0158] Table 5 Amplification efficiency of VIC channel

[0159]

[0160] From Table 4, Table 5 and Figure 6 It can be seen that when testing a weak-positive hepatitis B control sample, the Ct values ​​of all three batches of finished products were lower than those of the commercially available control product, demonstrating that the Taq enzyme of the present invention can improve amplification efficiency. Furthermore, each batch of mass-produced finished product can achieve an earlier Ct value, thereby improving amplification efficiency. Furthermore, the yield of the three batches of Taq DNA polymerase finished product was 20 million U (typically 200,000 U in a laboratory), and its enzyme activity was comparable to that of the commercially available control product (all 5 U / μL), demonstrating that the present invention has promising prospects for industrialization.

[0161] (2) Thermal stability test:

[0162] ① Polymerase: Use the finished Taq DNA polymerase obtained in Example 1, and a commercially available product as a control. Both polymerases are at 5 U / μL. Test the two polymerases after standing at room temperature (25°C) for 0 h and 8 h, respectively.

[0163] ②Hepatitis B weak positive quality control product (containing hepatitis B nucleic acid and internal standard, produced by Mike)

[0164] The results are shown in Tables 6 and 7; among them, the FAM channel is the hepatitis B nucleic acid test result, and the VIC channel is the internal standard test result; the Ct mean value is derived from the Ct mean value obtained after repeated testing of the weak positive quality control product 20 times for each sample.

[0165] Table 6 Room temperature (25°C) stability of FAM channels

[0166]

[0167] Table 7 Room temperature (25°C) stability of VIC channel

[0168]

[0169]

[0170] As shown in Tables 6 and 7, the Taq enzyme of the present invention exhibited a smaller difference in Ct values ​​(ΔCt) after 8 hours of aging, indicating that the Taq enzyme of the present invention possesses higher thermal stability. The Ct value differences (ΔCt) for the VIC channel were similar, primarily due to the relatively high internal standard content in the VIC assay, which resulted in a smaller ΔCt difference.

[0171] (3) Tail rate detection:

[0172] ① Polymerase: Taq DNA polymerase obtained by the method of Example 1, and a commercially available product as a control;

[0173] ②Hepatitis B negative quality control product (contains internal standard, but no hepatitis B nucleic acid, produced by Mike)

[0174] The results are shown in Table 8; the FAM channel is the hepatitis B nucleic acid test result, and the VIC channel is the internal standard test result; the Ct mean value is derived from the Ct mean value obtained after repeated testing of the hepatitis B negative quality control product for each sample 192 times.

[0175] Table 8 Results of the warping rate of the two channels

[0176]

[0177] The formula for calculating the lift rate is (number of groups exceeding the Ct threshold of 38 / total number of groups) × 100%. Since the test subjects are negative hepatitis B quality controls, a lower lift rate in the FAM channel indicates a greater number of groups exceeding the Ct threshold, meaning less nonspecific amplification and a lower likelihood of false positives. VIC is the internal standard channel, and all Ct values ​​are less than 38, well within the threshold range.

[0178] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A Taq DNA polymerase, characterized in that It has the amino acid sequence shown in SEQ ID NO.

1.

2. A polynucleotide sequence encoding the Taq DNA polymerase according to claim 1.

3. The polynucleotide sequence according to claim 2, characterized in that The polynucleotide sequence has the nucleotide sequence shown in SEQ ID NO.

2.

4. A polynucleotide construct, characterized in that Comprising the polynucleotide sequence of claim 2 or 3; preferably, the polynucleotide construct is a polynucleotide construct in which the DNA fragment between the BamHI and HindIII recognition sequences of pET-28a is replaced with the polynucleotide sequence of claim 2 or 3.

5. A host cell, characterized in that: Comprising the polynucleotide sequence of claim 2 or 3 or the polynucleotide construct of claim 4.

6. Use of the Taq DNA polymerase according to claim 1, the polynucleotide sequence according to claim 2 or 3, the polynucleotide construct according to claim 4, or the host cell according to claim 5 in nucleic acid amplification or preparation of a nucleic acid amplification product.

7. The method for preparing Taq DNA polymerase according to claim 1, characterized in that: The method comprises the following steps: culturing the host cell according to claim 5; and then isolating Taq DNA polymerase from the cell culture.

8. The preparation method according to claim 7, characterized in that The host cell is obtained by transforming the polynucleotide sequence shown in SEQ ID NO: 2 into Escherichia coli competent cells.

9. The preparation method according to claim 7 or 8, characterized in that The method of separating Taq DNA polymerase from the culture comprises the following steps: 1) The collected cell culture is sequentially disrupted, heat-treated, and precipitated to obtain a crude Taq DNA polymerase; 2) Redissolve the crude Taq DNA polymerase and purify it using NI affinity chromatography; Preferably, the loading buffer system of the NI affinity chromatography is 20-25 mM Tris buffer at pH 8.0 containing 50-100 mM KCl, the washing buffer system is 20-25 mM Tris buffer at pH 8.0 containing 50-100 mM KCl and 30 mM imidazole, and the elution buffer system is 20 mM Tris buffer at pH 8.0 containing 50-100 mM KCl and 300 mM imidazole; 3) The protein solution purified by NI affinity chromatography is further purified by cation exchange column chromatography; Preferably, the loading buffer system of the cation exchange column chromatography is 20-25 mM Tris buffer at pH = 7.2; 4) further purifying the protein solution purified by anion exchange column chromatography by cation exchange column chromatography; Preferably, the loading buffer system of the anion exchange column chromatography is 20-25 mM Tris buffer at pH = 7.8, and the elution buffer system is 20-25 mM Tris buffer at pH = 7.8 containing 150-200 mM KCl; 5) the protein solution purified by cation exchange column chromatography is further purified by heparin affinity chromatography; Preferably, the loading buffer system of the heparin affinity chromatography is 20-25 mM Tris buffer at pH 8.0, and the elution buffer system is 20-25 mM Tris buffer at pH 8.0 containing 200-300 mM KCl; 6) dialyzing the protein solution purified by heparin affinity chromatography; Preferably, the dialysate is 20-25 mM PBS buffer with a pH of 8.0 and containing 50-100 mM KCl.

10. The preparation method according to claim 9, characterized in that The collected cell culture is sequentially subjected to crushing, heat treatment, and precipitation treatment, including the following: 1) mixing 20-25 mM Tris buffer (pH 8.0) containing 50-100 mM KCl with the cell culture at a ratio of buffer volume to cell culture weight of 20:1, and then performing high-pressure homogenization; preferably, the homogenization temperature is -2°C to 2°C, and the homogenization pressure is 550-700 barg; 2) Treat the disrupted bacterial solution at 70-75°C for 30-40 minutes to separate the solids and retain the liquid; 3) Adding polyethyleneimine according to the volume of liquid obtained in step 2), adding nuclease to the supernatant after centrifugation and treating at 35-40° C. for 20-40 min, preferably at 37° C. for 30 min; and adding ammonium sulfate to precipitate the Taq DNA polymerase therein.

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