A mutant of Bst DNA polymerase and its preparation method and application
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI YINGJI BIOLOGICAL TECH CO LTD
- Filing Date
- 2024-12-23
- Publication Date
- 2026-06-23
Smart Images
Figure CN122256291A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to a Bst DNA polymerase mutant, its preparation method, and its application. Background Technology
[0002] In vitro nucleic acid amplification, as a molecular detection method, has been applied in various fields of life sciences and technology, such as pathogen detection, cancer research, sequencing, genotyping, molecular archaeology, and food testing. Traditional standard nucleic acid amplification is limited by the need for a programmable cyclic heating and cooling device. With the rise of point-of-care testing (POCT), isothermal amplification technology (IAT) in molecular detection has significant application value. This technology not only has similar sensitivity to current RT-qPCR tests, but also has the advantages of rapid detection, high specificity, and applicability to direct reaction analysis with on-site samples without the need for a thermal cycling device.
[0003] For IAT, the core lies in polymerase. Most IAT uses DNA polymerase, and Bst DNA polymerase (BP) is a very important enzyme that has been widely used, such as loop-mediated isothermal amplification (LAMP), strand substitution amplification (SDA), rolling circle amplification (RCA), nicking enzyme amplification reaction (NEAR), linear target isothermal multiple polymerase amplification (LIMA), and recombinase-assisted amplification (RAA).
[0004] In recent years, faster, more sensitive, and more widely applicable detection methods have become the mainstream trend. This has posed new challenges to the performance of Bst DNA polymerase. For example, Bst DNA polymerase generally loses its activity at 70°C. When the sample to be tested is interfered with by inhibitors, higher inhibitor tolerance is required. The timeliness of point-of-care diagnosis requires higher synthesis capacity, etc. However, there are relatively few reports on improving the characteristics of Bst DNA polymerase, and they often only target one aspect of the performance of Bst DNA polymerase. For example, CN117946997A discloses a heat-resistant Bst DNA polymerase mutant and its application. It is generated by mutating amino acids at one or more different sites on the basis of truncated wild-type Bst DNA polymerase, which has higher thermal stability and a Tm value that is significantly higher than that of wild-type Bst DNA polymerase.
[0005] In summary, developing novel Bst DNA polymerase mutants can effectively improve polymerization activity, strand displacement activity, heat resistance, and inhibitor resistance, which is of great significance for the field of in vitro nucleic acid amplification. Summary of the Invention
[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a Bst DNA polymerase mutant, its preparation method, and its applications. A novel Bst DNA polymerase mutant is designed to enhance polymerization activity, strand displacement activity, heat resistance, and inhibitor resistance, providing a new tool for in vitro nucleic acid amplification.
[0007] To achieve this objective, the present invention adopts the following technical solution:
[0008] In a first aspect, the present invention provides a Bst DNA polymerase mutant, wherein the Bst DNA polymerase mutant is a mutant obtained by mutation based on the enzyme with the amino acid sequence shown in SEQ ID No. 1; the mutation site includes at least one selected from K2, T54, M74, S77, C92, D112, D113, K121, E124, A140, Q228, N231, L251, T256, N277, S289, K304, L334, S359, D373, N404, R407, D422, T481, N484, A540, C549 or A579.
[0009] In this invention, the activity and thermostability of wild-type Bst DNA polymerase are calculated using deep learning, and a mutant library is constructed based on this. Through quantitative detection of enzyme activity, thermostability detection, and screening of 11 inhibitors, a series of Bst DNA polymerase mutants are obtained. Compared with wild-type Bst DNA polymerase, the Bst DNA polymerase mutants have improved polymerization activity, strand substitution activity, thermostability, and inhibitor resistance, and have broader application prospects.
[0010] SEQ ID No.1
[0011] .
[0012] Preferably, the mutation includes at least one of K2F, T54W, M74V, S77A, C92K, D112R, D113L, K121L, E124W, A140G, Q228E, N231D, L251I, T256W, N277L, S289R, K304G, L334W, S359D, D373E, N404W, R407C, D422A, T481N, N484D, A540G, C549K, or A579D.
[0013] It is understood that, based on the Bst DNA polymerase mutant designed in this invention, enzymes with similar functions obtained by using genetic modification methods in the art, such as amino acid substitution, deletion, or addition, should all be within the scope of protection of this invention. The number of amino acids substituted, deleted, or added can be any value, such as 1, 5, 10, 15, or more, such that the sequence identity between the changed amino acid sequence and its corresponding original sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. Furthermore, Bst DNA polymerases obtained by conservatively substituting amino acids with similar or comparable properties are also within the scope of protection of this invention.
[0014] In a second aspect, the present invention provides a nucleic acid molecule that encodes the Bst DNA polymerase mutant described in the first aspect.
[0015] In this invention, due to the degeneracy of the genetic code, a large number of nucleic acid molecules that can be used to encode the BstDNA polymerase mutant of this invention can be obtained. Therefore, given the identification of a specific amino acid sequence, those skilled in the art can easily prepare any number of different nucleic acids by changing the sequence of one or more codons without altering the amino acid sequence encoding the protein. More preferred polynucleotides can be selected through codon optimization based on the preferences of the host cells used in the actual preparation process. The nucleic acid molecules can be obtained using conventional methods, such as PCR amplification or artificial synthesis.
[0016] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the second aspect.
[0017] Fourthly, the present invention provides a recombinant cell expressing the Bst DNA polymerase mutant described in the first aspect.
[0018] The recombinant cell expression of the Bst DNA polymerase mutant described in the first aspect of the present invention may contain a nucleic acid molecule encoding the Bst DNA polymerase mutant or a recombinant vector containing said nucleic acid molecule. The host cell may be a prokaryotic cell, a lower eukaryotic cell, or a higher eukaryotic cell. Prokaryotic cells include bacterial cells, lower eukaryotic cells include yeast cells, and higher eukaryotic cells include mammalian cells. Representative examples include Escherichia coli and yeast cells.
[0019] Fifthly, the present invention provides a method for preparing the Bst DNA polymerase mutant described in the first aspect, the method comprising:
[0020] The nucleic acid molecule encoding the Bst DNA polymerase mutant described in the first aspect is inserted into the expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell, cultured, and the product is purified to obtain the Bst DNA polymerase mutant.
[0021] Transformation of the vector into host cells can be performed using conventional methods well known to those skilled in the art. These include methods such as CaCl2 injection, electroporation, calcium phosphate co-precipitation, and conventional mechanical methods like microinjection, electroporation, and liposome packaging. The resulting transformants can be cultured using conventional methods well known to those skilled in the art, and the culture medium can be a standard culture medium. The Bst DNA polymerase mutant generated by the transformants can be separated and purified using physical and chemical methods, such as salting out, centrifugation, cell disruption, and chromatography.
[0022] In a sixth aspect, the present invention provides the application of the Bst DNA polymerase mutant described in the first aspect in nucleic acid amplification.
[0023] This invention develops a Bst DNA polymerase mutant with significantly improved performance in multiple aspects, which can be widely used in nucleic acid amplification reactions.
[0024] In a seventh aspect, the present invention provides a nucleic acid amplification kit, the nucleic acid amplification kit comprising the Bst DNA polymerase mutant described in the first aspect.
[0025] Eighthly, the present invention provides a nucleic acid amplification method, the nucleic acid amplification method comprising:
[0026] The nucleic acid amplification system was prepared using the Bst DNA polymerase mutant described in the first aspect, and the nucleic acid amplification reaction was carried out.
[0027] Preferably, the nucleic acid amplification reaction includes at least one of loop-mediated isothermal amplification reaction, strand displacement amplification reaction, rolling circle amplification reaction, nicking enzyme amplification reaction, linear target isothermal multiple polymerization amplification reaction, or recombinase-assisted amplification reaction.
[0028] It is understood that the Bst DNA polymerase mutant of the present invention is used as the amplification enzyme in the amplification reaction. Other reagents required for the amplification reaction, such as buffer systems, templates, primers, etc., can be selected according to actual needs.
[0029] The nucleic acid amplification method based on Bst DNA polymerase mutants in this invention has broad application prospects and can be used for diagnostic testing as well as non-diagnostic testing, such as sequencing, genotyping, molecular archaeology, food testing, etc.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] This invention uses deep learning to calculate the activity and thermostability of Bst DNA polymerase, and constructs a mutant library based on this. Through quantitative detection of enzyme activity, thermostability detection, and screening of 11 inhibitors, a series of amino acid sites and their mutations that play an important role in improving the activity, strand substitution activity, thermostability, and inhibitor tolerance of Bst DNA polymerase were finally screened, resulting in Bst DNA polymerase mutants with excellent performance, which is beneficial to promoting the development of in vitro nucleic acid amplification detection. Attached Figure Description
[0032] Figure 1 The image shows the results of the LAMP reaction. Detailed Implementation
[0033] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.
[0034] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.
[0035] Example 1
[0036] In this embodiment, Bst DNA polymerase (BP) mutants with different mutations were prepared.
[0037] The Bst DNA polymerase mutant is formed by at least one of the following mutations based on the enzyme sequence shown in SEQ ID No. 1: K2F, T54W, M74V, S77A, C92K, D112R, D113L, K121L, E124W, A140G, Q228E, N231D, L251I, T256W, N277L, S289R, K304G, L334W, S359D, D373E, N40 4W, R407C, D422A, T481N, N484D, A540G, C549K, or A579D; specifically, the combination mutations include the combination of M74V, L251I, and D373E (M74V-L251I-D373E), and the combination of S77A, D112R, Q228E, and C549K (S77A-D112R-Q228E-C549K).
[0038] (1) Cloning of mutant BP gene sequences: Nucleotide sequences encoding different BP mutation sites were synthesized and inserted into the pET-28a vector using seamless cloning technology. The vector was then transformed into BL21(DE3) competent cells and plated on LB (containing kanamycin) solid medium. The cells were incubated overnight at 37°C with the cells inverted. The next day, single colonies were selected, labeled, and transferred to LB (containing kanamycin) liquid medium. The cells were incubated overnight at 37°C and 220 rpm. After centrifugation at 8,000 rpm for 1 min, the supernatant was discarded, and the cells were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were analyzed and the desired mutant BP strains were identified.
[0039] (2) Induction of mutant BP protein expression: The correctly sequenced strains were inoculated into LB (containing kanamycin) liquid medium and cultured at 37°C and 220 rpm until the bacterial culture OD... 600 When the value reaches 0.6-0.8, add an inducer with a final concentration of 0.4 mM IPTG. After incubation at 16°C for 20 h, centrifuge to collect the bacterial pellet. The obtained bacterial pellet contains the mutant BP protein.
[0040] (3) Isolation of mutant BP protein: Add 1×PBS lysis buffer to the bacterial cell precipitate in (2) to obtain bacterial cell suspension, sonicate to break up the bacterial cells, centrifuge to remove the precipitate, and obtain supernatant crude enzyme solution. Then perform nickel ion affinity chromatography, anion exchange chromatography and heparin column chromatography respectively to obtain Bst DNA polymerase mutant.
[0041] Example 2
[0042] Polymerization activity was tested on the BP mutants obtained in Example 1. First, different mutated BP molecules were diluted to 1 ng / μL and kept on ice. The 10× reaction buffer, 10 mM dNTPs, 10 μM reaction substrate nucleic acid, and Nuclease-free H2O were thawed at room temperature beforehand. After thawing, the mixture was vortexed, briefly centrifuged, and placed on ice. Picogreen nucleic acid fluorescent dye was kept at room temperature in the dark. The polymerization activity assay system was prepared on ice according to Table 1 below. After preparation, the mixture was vortexed for 10 s and briefly centrifuged. The 10× reaction buffer and reaction substrate nucleic acid were prepared according to the national standard GB / T 36755-2018 "BST DNA Polymerase". The BP dilution buffer contained: 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT, 50% Glycerol, pH 8.0 (25℃). The 10× reaction buffer contains: 250 mM Tris-Cl, 50 mM (NH4)2SO4, 50 mM KCl, 25 mM MgSO4, 1% TweenX-100, pH 8.5 (25°C). The nucleic acid sequence of the reaction substrate is SEQ ID No. 2: 5'-tagcgaaggatgtgaacctaatccctgctcccgcggccgatctgccggccgcgggagca-3'.
[0043] Table 1
[0044] Components Volume (μL) used in a 20 μL reaction volume 10× reaction buffer 2 10mM dNTPs 0.5 Picogreen 0.5 10 μM reaction substrate nucleic acid 1 BP mutant to be tested 2 <![CDATA[Nuclease-free H2O]]> 14
[0045] Set the qPCR reaction program on the qPCR instrument: SYBR fluorescence acquisition channel, Non-Rox mode, temperature 65℃, reaction time 16s / cycle, 80 cycles. Place the prepared reaction system (Table 1) into the instrument, start the program, and begin the reaction. Use wild-type BP (WT) with the sequence shown in SEQ ID No. 1 as a control. After the reaction is complete, collect data and perform data analysis.
[0046] Under the same amount of BP mutant to be tested, the change in fluorescence value was observed. The higher the fluorescence value, the stronger the polymerization ability of the BP mutant. It is directly proportional to the polymerization ability of the BP mutant. The polymerization ability results of different BP are shown in Table 2. It can be seen that, compared with wild-type BP, the BP mutant designed in this invention can effectively improve the polymerization ability.
[0047] Table 2
[0048]
[0049]
[0050] Example 3
[0051] This embodiment tests the strand substitution activity of the BP mutant obtained in Example 1.
[0052] The principle of the strand substitution activity assay is as follows: A molecular beacon sequence containing only fluorescent groups is designed, and a reverse complementary DNA strand is designed for the longer stem region of the molecular beacon. One end of the complementary strand is modified with a quenching group. The two sequences are annealed to form a complementary double-stranded structure. Because the fluorescent group on the molecular beacon is close to the quenching group on the complementary strand, the fluorescence is quenched. When an enzyme with strand substitution ability is added, the fluorescent group is released as the reaction proceeds. The strand substitution ability of the BP mutant is evaluated by the fluorescence value at the same enzyme input. Since the fluorescence value is directly proportional to the strand substitution ability, the higher the fluorescence value, the stronger the strand substitution ability of the BP mutant. However, because some polymerases have 5'-3' exonuclease activity, the fluorescent group modified at the 5' end of the molecular beacon may be excised, releasing fluorescence and interfering with the detection of the enzyme's strand substitution ability. Therefore, although the molecular beacon in this embodiment has a fluorescent group modified at its 5' end, the tested BP samples do not have 5'-3' exonuclease activity.
[0053] Specifically, the nucleic acid sequences of the substrates for evaluating the strand substitution ability of the BP mutant are as follows: SEQ ID No. 3, a molecular beacon sequence containing only fluorescent groups: 5'-accgagcctagcgaaggatgtgaacctaatccctgctcccgcggccgatctgccggccgcgggagca-3' (5' end is FAM modified); and SEQ ID No. 4, a reverse complementary strand DNA sequence with a quenching group modified at one end: 5'-gggattaggttcacatccttcgctaggctcggt-3' (3' end is Dabcyl modified).
[0054] Experimental Procedure: First, the strand displacement reaction substrate beacon and the reverse complementary DNA were annealed: (a) The dry powders synthesized from SEQ ID No. 3 and SEQ ID No. 4 were diluted to 100 μM stock solutions (actual concentration) using 1×TE buffer; (b) In the same PCR tube, SEQ ID No. 3 (100 μM stock solution) and SEQ ID No. 4 (100 μM stock solution) were prepared into working solutions with a final concentration of 10 μM using 1×TE buffer; (c) The PCR reaction program was set on the PCR instrument as follows: 95℃, 2 min, cooling at 0.1℃ / s, 22℃, 5 min, storage at 4℃; (d) The prepared DNA to be annealed was placed in the instrument, the program was started, and the reaction began. After the reaction was completed, the PCR tube was removed and placed on ice for later use. The specific procedures for subsequent chain displacement activity assays are as follows: Dilute different mutant BPs to 0.125 ng / μL and store on ice. Thaw the 10× reaction buffer and Nuclease-free H2O at room temperature beforehand. After thawing, vortex to mix, briefly centrifuge, and then store on ice. Prepare the chain displacement activity assay system on ice according to Table 3 below. After preparation, vortex to mix for 10 seconds and then briefly centrifuge.
[0055] Table 3
[0056] Components Volume (μL) used in a 20 μL reaction volume 10× reaction buffer 2 10mM dNTPs 0.5 Annealed DNA product (10 μM) 1 BP under test 2 <![CDATA[Nuclease-free H2O]]> 14.5
[0057] Set the qPCR reaction program on the qPCR instrument: FAM fluorescence acquisition channel, Non-Quencher, Non-Rox mode, temperature 65℃, reaction time 60s / cycle, 80 cycles. Place the prepared reaction system (Table 3) into the instrument, start the program, and begin the reaction. Use wild-type BP (WT) with the sequence shown in SEQ ID No. 1 as a control. After the reaction is complete, collect data and perform data analysis.
[0058] Under the same amount of BP to be tested, the change in fluorescence value was observed. The higher the fluorescence value, the stronger the substitution ability of BP. It is directly proportional to the chain substitution ability of BP. The chain substitution ability results of different BP are shown in Table 4. It can be seen that the BP mutant designed in this invention can effectively improve the chain substitution ability.
[0059] Table 4
[0060]
[0061] Example 4
[0062] In this embodiment, the BP mutant obtained in Example 1 is subjected to a heat resistance test to evaluate its thermal stability.
[0063] The enzymes to be tested were homogenized to 1 ng / μL beforehand and incubated at 75°C for 30 min. The enzyme activity detection method described in Example 2 was then used for screening. After completion, fluorescence data from the 30th cycle were collected and analyzed. The control group consisted of the corresponding enzymes that were not incubated at 75°C for 30 min but were stored at -20°C.
[0064] Under the same input amount of BP, the changes in fluorescence difference (net fluorescence difference between the enzyme incubated at 75℃ and the enzyme not incubated at 75℃) were observed. The larger the net fluorescence difference, the weaker the thermostability of BP. The thermostability results of different BPs are shown in Table 5. As can be seen from the table, the thermostability of the mutants is better than that of the wild-type WT.
[0065] Table 5
[0066]
[0067]
[0068] Example 5
[0069] In this embodiment, the BP mutant obtained in Example 1 was subjected to inhibitor stress testing.
[0070] The detection system was prepared according to the reaction system for detecting polymerization activity in Example 2, and the inhibitors to be tested were added, with only one inhibitor added per test. The inhibitors to be tested were: NaCl, EDTA, dUTP, ethanol, SDS, humic acid, sodium citrate, sodium heparin, vitamin C, hemoglobin, and bilirubin. The control group did not contain the corresponding inhibitors to be tested, and the experiment was screened according to the enzyme activity detection method in Example 2. After completion, the fluorescence data from the 30th cycle were collected and analyzed.
[0071] Under the same amount of BP to be tested, the change in fluorescence difference (net fluorescence difference between test samples with added NaCl and those without added NaCl) was observed. The larger the net fluorescence difference, the weaker the tolerance of BP to 100mM NaCl. The results of the NaCl tolerance of different BPs are shown in Table 6.
[0072] Table 6
[0073]
[0074]
[0075] Under the same amount of BP to be tested, the change in fluorescence difference (net fluorescence difference between test samples with and without EDTA) was observed. The larger the net fluorescence difference, the weaker the tolerance of BP to 1.5mM EDTA. The results of the EDTA tolerance of different BP are shown in Table 7.
[0076] Table 7
[0077]
[0078] Under the same amount of BP to be tested, the change in fluorescence difference (net fluorescence difference between test samples with and without dUTP) was observed. The larger the net fluorescence difference, the weaker the tolerance of BP to 1.25mM dUTP. The results of the tolerance of different BP to dUTP are shown in Table 8.
[0079] Table 8
[0080]
[0081]
[0082] Under the same amount of BP to be tested, the change in fluorescence difference (net fluorescence difference between test samples with and without added ethanol) was observed. The larger the net fluorescence difference, the weaker the tolerance of BP to 12% ethanol. The results of the ethanol tolerance of different BPs are shown in Table 9.
[0083] Table 9
[0084]
[0085] Under the same amount of BP to be tested, the change in fluorescence difference (net fluorescence difference between test samples with and without added SDS) was observed. The larger the net fluorescence difference, the weaker the tolerance of BP to 0.125% SDS. The results of the SDS tolerance of different BPs are shown in Table 10.
[0086] Table 10
[0087]
[0088] Under the same amount of BP to be tested, the change in fluorescence difference (net fluorescence difference between test samples with and without added humic acid) was observed. The larger the net fluorescence difference, the weaker the tolerance of BP to 2.22 μM humic acid. The results of the humic acid tolerance of different BPs are shown in Table 11.
[0089] Table 11
[0090]
[0091]
[0092] Under the same amount of BP to be tested, the change in fluorescence difference (net fluorescence difference between test samples with added sodium citrate and those without added sodium citrate) was observed. The larger the net fluorescence difference, the weaker the tolerance of BP to 5.1mM sodium citrate. The results of the sodium citrate tolerance of different BPs are shown in Table 12.
[0093] Table 12
[0094]
[0095] Under the same amount of BP to be tested, the change in fluorescence difference (net fluorescence difference between test samples with added heparin sodium and those without added heparin sodium) was observed. The larger the net fluorescence difference, the weaker the tolerance of BP to 0.65U heparin sodium. The results of the heparin sodium tolerance of different BPs are shown in Table 13.
[0096] Table 13
[0097]
[0098]
[0099] Under the same amount of BP to be tested, the change in fluorescence difference (net fluorescence difference between test samples with added L-ascorbic acid and those without added L-ascorbic acid) was observed. The larger the net fluorescence difference, the weaker the tolerance of BP to 1.5 μg / μL L-ascorbic acid. The results of the L-ascorbic acid tolerance of different BPs are shown in Table 14.
[0100] Table 14
[0101]
[0102] Under the same amount of BP to be tested, the change in fluorescence difference (net fluorescence difference between test samples with added hemoglobin and those without added hemoglobin) was observed. The larger the net fluorescence difference, the weaker the tolerance of BP to 0.25 μg / μL hemoglobin. The results of the hemoglobin tolerance of different BPs are shown in Table 15.
[0103] Table 15
[0104]
[0105] Under the same amount of BP to be tested, the change in fluorescence difference (net fluorescence difference between test samples with and without added bilirubin) was observed. The larger the net fluorescence difference, the weaker the tolerance of BP to 0.05 μg / μL bilirubin. The results of the bilirubin tolerance of different BPs are shown in Table 16.
[0106] Table 16
[0107]
[0108]
[0109] The results above show that the BP mutant designed in this invention can improve resistance to inhibitors.
[0110] Example 6
[0111] This embodiment uses the BP mutant (M74V-L251I-D373E) obtained in Example 1 as an example to perform LAMP assays to verify its performance difference with commercially available Bst DNA polymerases in practical applications. Commercially available control enzymes are Bst 2.0 DNA polymerase (NEB M0573) and Bst 3.0 DNA polymerase (NEB M0374). The specific reaction system is shown in Table 17 below. The DNA substrate sample used for detection was a plasmid sample containing African swine fever ASFV P72, and the detection primers were as follows:
[0112] ASF-F3: 5'-GCCCTCTAAAGGTGTTTGGT-3';
[0113] ASF-B3: 5'-CCCACCTTAGGAAACAAGCT-3';
[0114] ASF-FIP:
[0115] 5'-TCAGGGCACGTCCCAGATGGTTTTCCCAGTCATATCCGTTGCG-3';
[0116] ASF-FIB:
[0117] 5'-GGAGCATCCTGCCAGGATGAATTTTTTTCCCCAGTACGGAGACTT-3';
[0118] ASF-LF: 5'-AACGTTTGAAGCTGCCCATGGGCC-3';
[0119] ASF-LB: 5'-GCACCCAATATATGATGGCCCACC-3'.
[0120] After preparing the LAMP reaction system according to Table 17, it was reacted at 65°C for 1 hour in a BIO-RAD CFX96 System, and the fluorescence was detected every 1 minute.
[0121] Table 17
[0122]
[0123]
[0124] Specific results are as follows Figure 1As shown, the black curve represents the BP mutant designed in this invention; the red curve represents Bst 3.0 DNA polymerase (NEB M0374); the green curve represents Bst 2.0 DNA polymerase (NEB M0573); and the blue curve represents NTC. (From...) Figure 1 It can be seen that the Ct value of the BP mutant designed in this invention is significantly lower than that of the other two commercially available Bst DNA polymerase products. Its performance in this LAMP test is significantly better than Bst3.0 and even better than Bst2.0. This indicates that the present invention has designed a BP mutant with improved performance and can be effectively applied to in vitro nucleic acid amplification detection (such as LAMP).
[0125] Example 7
[0126] This embodiment provides a nucleic acid amplification kit.
[0127] Based on the significantly improved performance of the Bst DNA polymerase mutant, a nucleic acid amplification kit can be further constructed. The kit may also include commonly used reagents for amplification, such as reaction buffer, magnesium ions, dNTPs, etc.
[0128] In summary, this invention uses deep learning to calculate the activity and thermostability of Bst DNA polymerase, and constructs a mutant library based on this. Through quantitative detection of enzyme activity, thermostability detection, and screening of 11 inhibitors, a series of amino acid sites and their mutations that play an important role in improving BP polymerase activity, strand substitution activity, thermostability, and inhibitor tolerance were finally screened out, resulting in BP mutants with excellent performance, which is beneficial to promoting the development of in vitro nucleic acid amplification detection.
[0129] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.
Claims
1. A Bst DNA polymerase mutant, characterized in that, The Bst DNA polymerase mutant is a mutant obtained by mutating the enzyme based on the amino acid sequence shown in SEQ ID No. 1; The mutation sites include at least one of K2, T54, M74, S77, C92, D112, D113, K121, E124, A140, Q228, N231, L251, T256, N277, S289, K304, L334, S359, D373, N404, R407, D422, T481, N484, A540, C549, or A579.
2. The Bst DNA polymerase mutant according to claim 1, characterized in that, The mutations include at least one of K2F, T54W, M74V, S77A, C92K, D112R, D113L, K121L, E124W, A140G, Q228E, N231D, L251I, T256W, N277L, S289R, K304G, L334W, S359D, D373E, N404W, R407C, D422A, T481N, N484D, A540G, C549K, or A579D.
3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the Bst DNA polymerase mutant as described in claim 1 or 2.
4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 3.
5. A recombinant cell, characterized in that, The recombinant cells express the Bst DNA polymerase mutant as described in claim 1 or 2.
6. A method for preparing the Bst DNA polymerase mutant according to claim 1 or 2, characterized in that, The preparation method includes: A nucleic acid molecule encoding the Bst DNA polymerase mutant of claim 1 or 2 is inserted into an expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell, cultured, and the product is purified to obtain the Bst DNA polymerase mutant.
7. The application of the Bst DNA polymerase mutant according to claim 1 or 2 in nucleic acid amplification.
8. A nucleic acid amplification kit, characterized in that, The nucleic acid amplification kit includes the Bst DNA polymerase mutant as described in claim 1 or 2.
9. A method for nucleic acid amplification, characterized in that, The nucleic acid amplification method includes: A nucleic acid amplification system was prepared using the Bst DNA polymerase mutant as described in claim 1 or 2, and a nucleic acid amplification reaction was performed.
10. The nucleic acid amplification method according to claim 9, characterized in that, The nucleic acid amplification reaction includes at least one of the following: loop-mediated isothermal amplification reaction, strand displacement amplification reaction, rolling circle amplification reaction, nicking enzyme amplification reaction, linear target isothermal multiple polymerization amplification reaction, or recombinase-assisted amplification reaction.