Anti-inhibition Taq DNA polymerase and application thereof

Abstract

The invention discloses an anti-inhibition Taq DNA polymerase and application thereof, and relates to the technical field of biology. According to the anti-inhibition Taq DNA polymerase, amino acid mutations of K206R, F667Y and the like are carried out on an amino acid sequence of a wild type Taq DNA polymerase with an amino acid sequence as shown in SEQ ID No.1, so that a certain degree of anti-inhibition property is obtained. In addition, the invention also discloses application of the anti-inhibition Taq DNA polymerase in preparation of reagents or kits, and the like. Compared with a wild type Taq DNA polymerase, the anti-inhibition Taq DNA polymerase can maintain the PCR amplification capability in the presence of an inhibitor such as polysaccharide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and more particularly, to an anti-inhibition Taq DNA polymerase and application thereof. BACKGROUND

[0002] Molecular detection technology has become a core tool for food safety detection due to its high sensitivity and specificity, and is widely used in pathogenic microorganism detection, genetically modified ingredient screening and other fields. PCR (polymerase chain reaction) is the cornerstone of molecular detection technology. PCR technology can realize exponential growth of DNA through cyclic amplification (denaturation, annealing and extension), and the detection limit can be as low as single-digit copies, which greatly improves the detection sensitivity. With the development of related technologies, it also supports simultaneous detection of multiple targets and various complex scene applications, fully demonstrating its efficiency and versatility.

[0003] However, in general, PCR technology has certain requirements for the nucleic acid template of the sample to be tested. The nucleic acid template of the sample to be tested needs to avoid the inhibition of Taq DNA polymerase activity and the interference with primer binding by high-concentration proteins, polysaccharides, polyphenols, organic solvents (such as phenol and ethanol) and ion inhibitors (such as EDTA and hematin). For scenes that do not require rapid detection, the sample to be tested often needs to undergo a targeted, complex and long-periodic pretreatment process to obtain a nucleic acid template that meets the requirements of PCR amplification. However, in the food rapid detection scene, the long pretreatment process obviously does not meet the core requirement of pursuing rapid and efficient detection.

[0004] Although the nucleic acid extraction technology can realize rapid extraction of the nucleic acid template of the sample to be tested, significantly shortening the time, food samples, especially dairy products, are rich in impurities such as polysaccharides and lipids, which interfere with PCR amplification, shield fluorescence signals, and even cause false negatives. SUMMARY

[0005] In view of the problems existing in the prior art, the present application provides an anti-inhibition Taq DNA polymerase, which mainly performs K206R, F667Y and other amino acid mutations on the wild-type Taq DNA polymerase (the amino acid sequence is shown as SEQ ID No. 1) to obtain specific anti-inhibition properties. The present application also provides some applications related to the anti-inhibition Taq DNA polymerase, such as its application in the preparation of reagents or kits and the fusion protein, polynucleotide, biological material, reagent or kit involved therein.

[0006] The first aspect of the present application provides an anti-inhibition Taq DNA polymerase.

[0007] The anti-inhibitory Taq DNA polymerase comprises the following amino acid mutations: K206R and F667Y, compared with the amino acid sequence of the wild-type Taq DNA polymerase; the amino acid sequence of the wild-type Taq DNA polymerase is shown in SEQ ID No. 1; the mutation K206R enhances the positive charge density, neutralizes the electrostatic effect of polysaccharides, and blocks the competition of polysaccharides with the nucleic acid template; and the mutation F667Y increases the rigidity and reduces the allosteric effect of polysaccharide inhibitors.

[0008] Further, compared with the wild-type Taq DNA polymerase with the amino acid sequence shown in SEQ ID No. 1, the anti-inhibitory Taq DNA polymerase can further comprise the following amino acid mutation: Q680R; wherein the mutation Q680R mainly weakens the competition of inhibitors with the nucleic acid template by increasing the positive charge of the DNA binding channel, thereby providing relatively broad anti-inhibitory properties.

[0009] The second aspect of the present application provides a fusion protein containing the domain of the anti-inhibitory Taq DNA polymerase described in the first aspect. The fusion protein further incorporates other functional domains; specifically, a heat-stable domain such as a HhH repeat domain or a DNA binding protein (such as Sso7d protein, etc.).

[0010] The third aspect of the present application provides a polynucleotide encoding the anti-inhibitory Taq DNA polymerase described in the first aspect or the fusion protein described in the second aspect.

[0011] The fourth aspect of the present application provides a biological material containing the anti-inhibitory Taq DNA polymerase described in the first aspect or the fusion protein described in the second aspect or the polynucleotide described in the third aspect.

[0012] Further, the biological material can refer to recombinant DNA, an expression cassette, a plasmid vector, a viral vector, a transposon vector, an engineered bacterium, or an engineered cell.

[0013] The fifth aspect of the present application provides a reagent or kit containing the anti-inhibitory Taq DNA polymerase described in the first aspect or the fusion protein described in the second aspect or the polynucleotide described in the third aspect.

[0014] Further, the reagent or kit can be used for PCR reaction.

[0015] Still further, the reagent or kit can comprise the anti-inhibitory Taq DNA polymerase described in the first aspect, a reaction buffer, and a dNTP mixture.

[0016] The sixth aspect of the present application provides a use of the anti-inhibited Taq DNA polymerase of the first aspect or the fusion protein of the second aspect or the polynucleotide of the third aspect in the preparation of a reagent or a kit.

[0017] The present application provides an anti-inhibited Taq DNA polymerase and its use, specifically discloses that the anti-inhibited Taq DNA polymerase is obtained by performing K206R, F667Y and other amino acid mutations on the amino acid sequence of the wild-type Taq DNA polymerase shown in SEQ ID No. 1, and has polysaccharide-related anti-inhibition properties, and discloses its use in the preparation of a reagent or a kit and the fusion protein, polynucleotide, biological material, reagent or kit involved therein, which overall improves the anti-inhibition of related PCR amplification to nucleic acid template impurities, and particularly strengthens the anti-inhibition to polysaccharides, lipids and other impurities in food samples. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 SDS-PAGE electrophoretogram of wild-type Taq DNA polymerase and anti-inhibited Taq DNA polymerase mutants KF and KFQ;

[0019] Figure 2 Fluorescent quantitative PCR amplification curve of wild-type Taq DNA polymerase and its mutants KF and KFQ in the absence of polysaccharide inhibition and in the presence of polysaccharide inhibition. DETAILED DESCRIPTION

[0020] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987 and periodic updates thereto); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0021] The present application will be illustrated hereinafter in the manner of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present application. All other examples that can be obtained by those of ordinary skill in the art based on the examples in the present application without creative efforts, fall within the scope of the present application. The methods and materials used in the examples are conventional in the art, unless otherwise specified.

[0022] Examples

[0023] I. Example 1: Preparation of Anti-inhibitory Taq DNA Polymerase

[0024] 1. Design of Anti-inhibitory Taq DNA Polymerase

[0025] The amino acid sequence of wild-type Taq DNA polymerase is shown in SEQ ID No. 1;

[0026] Amino acid sequence of wild-type Taq DNA polymerase (SEQ ID No. 1):

[0027] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAK APSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRIL TADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLE EWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFG SLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKE ARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANL WGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNS RDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRT GRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENL IRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYF QSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAM VKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE.

[0028] Based on this, two multi-site mutants were designed as follows:

[0029] Mutant KF: K206R, F667Y;

[0030] Mutant KFQ: K206R, F667Y, Q680R.

[0031] Amino acid mutation primer design:

[0032] K206R:

[0033] GCGCAGGCTGCTGGAAGAATGGG (SEQ ID No. 2);

[0034] GCAGCCTGCGCGCGGTTTTTTCGC (SEQ ID No. 3);

[0035] F667Y:

[0036] CCATTAACTATGGCGTGCTGTATGGC (SEQ ID No. 4);

[0037] GCCATAGTTAATGGTTTTCGCC (SEQ ID No. 5);

[0038] Q680R:

[0039] TGAGCCGGGAACTGGCGATTCC (SEQ ID No. 6);

[0040] AGTTCCCGGCTCAGGCGATGCGCGCT (SEQ ID No. 7).

[0041] 2. Reagent preparation

[0042] IPTG solution (1 mol / L): Take 2.38 g IPTG (isopropyl-β-D-thiogalactoside) and add to 10 mL enzyme-free water to dissolve, filter through a 0.22 μm filter membrane, store at -20 °C for standby;

[0043] Lysis solution (50 mmol / L): 50 mmol / L Tris HCl (pH 8.0), 500 mmol / L NaCl, 0.1% NP-40, 0.1% Triton X-100;

[0044] Imidazole storage solution (2 mol / L): Take 1.36 g imidazole and add to 10 mL enzyme-free water to dissolve, adjust the pH value to 8.0 with HCl, store at 4 °C for standby;

[0045] Elution solution:

[0046] 10 mmol / L elution solution: 50 mmol / L Tris HCl (pH 8.0), 100 mmol / L NaCl, 0.05% NP-40, 5% glycerol, 5 mmol / L β-mercaptoethanol, 1 mmol / L benzamidine, 10 mmol / L imidazole;

[0047] 20 mM elution buffer: 50 mM Tris HCl (pH 8.0), 100 mM NaCl, 0.05% NP-40, 5% glycerol, 5 mM β-mercaptoethanol, 1 mM benzamidine, 20 mM imidazole;

[0048] 50 mM elution buffer: 50 mM Tris HCl (pH 8.0), 100 mM NaCl, 0.05% NP-40, 5% glycerol, 5 mM β-mercaptoethanol, 1 mM benzamidine, 50 mM imidazole;

[0049] 300 mM elution buffer: 50 mM Tris HCl (pH 8.0), 100 mM NaCl, 0.05% NP-40, 5% glycerol, 5 mM β-mercaptoethanol, 1 mM benzamidine, 300 mM imidazole;

[0050] Dialysis buffer / protein storage buffer: 50 mM Tris HCl (pH 8.0), 100 mM NaCl.

[0051] 2. Construction of plasmid vector

[0052] Based on pET-28a plasmid, wild type and mutant expression plasmids were constructed. According to the mutation site, primers were designed, and the target fragment containing the mutation site was amplified, and the target fragment was ligated into the BamHI and HindIII sites of the vector to complete the plasmid construction.

[0053] 3. Induced expression of protein

[0054] The plasmids encoding wild type Taq DNA polymerase and mutants KF, KFQ were transformed into BL21 (DE3) competent cells, respectively. The transformed bacterial liquid was spread on solid LB culture plates containing kanamycin and incubated at 37°C overnight. Single colonies were picked and inoculated into 200 mL of LB medium containing 50 μg / mL kanamycin and incubated at 37°C, 220 rpm until the OD value reached 0.8-1.0. IPTG solution was added to the culture medium to a final concentration of 1 mmol / L. The bacterial liquid was induced to culture at 37°C for 2 h, and the bacterial cells were collected by centrifugation. 600

[0055] 4. Protein extraction and purification

[0056] ​The bacterial cells were washed twice with PBS, centrifuged to discard the supernatant, 4 mL lysis solution was added to the bacterial cell precipitate, resuspended thoroughly, 20 μL 100 mg / mL lysozyme solution was added, and mixed uniformly. The mixture was placed in a four-dimensional rotary mixer, and rotated at room temperature for 15 min. The enzyme-degraded bacterial cell suspension was placed in a 75°C water bath for heating for 1 h, centrifuged at 12000 rpm (4°C) for 10 min, the supernatant was collected, filtered using a 0.22 μm filter membrane, 400 μL glycerol and 1.4 μL 100 mmol / L benzamidine were added to the filtrate, mixed uniformly, and stored at -80°C for standby use.

[0057] Nickel column (Ni-NTA) purification: the nickel column was equilibrated with lysis solution, 20 μL imidazole stock solution (2 mol / L) was added to the cell lysate, mixed uniformly, and then combined with the nickel column. The target protein was eluted in turn using gradient elution buffer with increasing concentration.

[0058] The eluted components were subjected to SDS-PAGE electrophoresis. The results are shown in Figure 1 , where the leftmost lane is Marker, and the other lanes from left to right are wild-type Taq DNA polymerase, mutant KF, and mutant KFQ.

[0059] II. Example 2: Anti-inhibition performance test of wild-type Taq DNA polymerase and its mutants KF and KFQ

[0060] Considering that the polysaccharide component in dairy products is mainly lactose, which has less impact on PCR amplification, and that the metabolic product of lactic acid bacteria fermentation, lactic acid bacteria exopolysaccharide (EPS), contains carboxyl / phosphoryl groups and other anionic groups, which can interfere with the binding of Taq DNA polymerase and DNA through electrostatic interaction, thereby inhibiting PCR amplification. Therefore, in the following, by comparing the addition of lactic acid bacteria exopolysaccharide (EPS) as an inhibitor, the anti-polysaccharide inhibition performance of wild-type Taq DNA polymerase and its mutants KF and KFQ in qPCR will be compared.

[0061] 1. Main reagents

[0062] DNA template: pET28a-Taq recombinant plasmid (containing full-length Taq gene), concentration 10 ng / μL;

[0063] Taq enzyme: wild-type Taq DNA polymerase (abbreviated as wild-type Taq enzyme), mutant KF (abbreviated as mutant KF), and mutant KFQ (abbreviated as mutant KFQ);

[0064] Simulated inhibitor: lactic acid bacteria exopolysaccharide (EPS) freeze-dried powder (Lactobacillus paracasei, purity > 90%);

[0065] Primer / probe: TaqMan probe (FAM-BHQ1), primer targets the conserved region of Taq gene (the nucleotide sequence is shown as SEQ ID No. 8, SEQ ID No. 9);

[0066] Compensation reagent: BSA (0.5 mg / mL), MgCl2 (25 mmol / L), betaine (5 mol / L);

[0067] The primer sequence is specifically as follows:

[0068] CGTATCGCACCTTTCATGCG (SEQ ID No. 8);

[0069] TCAGCCAATCTTCGCCAATGC (SEQ ID No. 9).

[0070] 2. qPCR reaction system

[0071] The qPCR reaction system (20 μL) used in the embodiment is as follows:

[0072] 2 × TaqMan Fast MasterMix, 10.0 μL

[0073] Primer (10 μmol / L), 0.4 μL each;

[0074] TaqMan probe (5 μmol / L), 0.2 μL;

[0075] DNA template, 1.0 μL;

[0076] Simulated inhibitor, 0, 2 mg / L, respectively;

[0077] Nuclease-free water, supplemented to 20 μL.

[0078] 3. qPCR reaction condition setting

[0079] Pre-denaturation: 95℃, 3 min;

[0080] Cycle (40 ×):

[0081] Denaturation: 95℃, 10 s;

[0082] Annealing / extension: 60℃, 30 s (FAM channel acquisition).

[0083] According to the above experimental conditions, qPCR was carried out in groups, and the results are shown in Table 1 and Figure 2 .

[0084] Table 1 Anti-polysaccharide inhibition performance of wild-type Taq DNA polymerase and mutants KF and KFQ in qPCR

[0085] Taq enzyme type EPS concentration (mg / mL) Ct (control) Ct (inhibition) ACt Wild-type Taq enzyme 0 22.57 － 0 2.0 － Amplification failure >4.00 Mutant KF 0 21.81 － 0 2.0 － 23.06 1.25 Mutant KFQ 0 20.73 － 0 2.0 － 21.49 0.76

[0086] Note: ΔCt = Ct(inhibition) - Ct(control).

[0087] From Table 1 and Figure 2 It can be seen that in the group without adding lactic acid bacteria exopolysaccharide (EPS) inhibitor, wild type Taq DNA polymerase and its mutants KF, mutant KFQ can play a key role in qPCR, ensuring the smooth progress of PCR amplification; when the lactic acid bacteria exopolysaccharide (EPS) inhibitor is 2.0 mg / mL, the catalytic PCR amplification function of wild type Taq DNA polymerase is inhibited, while its mutants KF, mutant KFQ have a certain degree of tolerance to lactic acid bacteria exopolysaccharide (EPS) inhibitor.

Claims

1. An anti-suppressive Taq DNA polymerase, characterized by, compared with the amino acid sequence of wild-type Taq DNA polymerase, the anti-suppression Taq DNA polymerase comprises the following amino acid mutations: K206R, and F667Y; the amino acid sequence of the wild-type Taq DNA polymerase is shown as SEQ ID No.

1.

2. The anti-suppression Taq DNA polymerase of claim 1, wherein, the anti-suppression Taq DNA polymerase further comprises the following amino acid mutation: Q680R.

3. A fusion protein comprising the domain of the anti-suppression Taq DNA polymerase of any one of claims 1-2.

4. A polynucleotide encoding the anti-suppression Taq DNA polymerase of any one of claims 1-2 or the fusion protein of claim 3.

5. A biological material comprising the anti-suppression Taq DNA polymerase of any one of claims 1-2 or the fusion protein of claim 3 or the polynucleotide of claim 4.

6. The biological material of claim 5, wherein, the biological material refers to recombinant DNA, expression cassette, plasmid vector, viral vector, transposon vector, engineered bacteria or engineered cells.

7. A reagent or kit comprising the anti-suppression Taq DNA polymerase of any one of claims 1-2 or the fusion protein of claim 3 or the polynucleotide of claim 4.

8. The reagent or kit of claim 7, wherein, the reagent or kit is used for PCR reaction.

9. The reagent or kit of claim 8, wherein, the reagent or kit comprises: the anti-suppression Taq DNA polymerase of any one of claims 1-2; reaction buffer; and dNTP mixture.

10. Use of the anti-suppression Taq DNA polymerase of any one of claims 1-2 or the fusion protein of claim 3 or the polynucleotide of claim 4 in the preparation of a reagent or kit.

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