Phi29 DNA polymerase mutants with improved performance, their preparation methods and applications

Abstract

The present invention discloses phi29 DNA polymerase mutants with improved performance, their preparation methods and applications, which relate to the field of biotechnology. Specifically, the present invention discloses phi29 DNA polymerase mutants that contain mutation sites Y163K, F181K, Y224K, W277K / W277P, Y298K, Y369K, Y439K, F526R relative to wild-type phi29 DNA polymerase; the amino acid sequence of the wild-type phi29 DNA polymerase is as shown in SEQ ID NO:1. Compared with the wild-type phi29 DNA polymerase, it has higher specific enzyme activity, stability and higher amplification efficiency, and has good application prospects in the fields of nucleic acid amplification and sequencing, etc.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a phi29 DNA polymerase mutant with improved performance, and a preparation method and application thereof. Background Art

[0002] Phi29 DNA polymerase is encoded by the Bacillus subtilis phage phi29 and is a viral DNA replicase, which can be widely used in nucleic acid amplification, virus detection, and various sequencing technologies, including multiple displacement amplification (MDA), rolling circle amplification (RCA), whole genome amplification (WGA), SNP detection, single polymerase molecule real-time DNA sequencing, DNA nanoball sequencing, and nanopore sequencing using the synthesis sequencing method.

[0003] Studies have shown that the stability of phi29 DNA polymerase is poor. The half-life of wild-type phi29 DNA polymerase at 30°C is 18 min without adding substrates, and only 1 min at 40°C. Moreover, a relatively high salt ion concentration is usually required in its storage buffer to ensure the stability of the enzyme.

[0004] Therefore, it is important to provide a Phi29 DNA polymerase that still has good stability under low salt ion concentration conditions. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides a phi29 DNA polymerase mutant, which still has good stability under low salt ion concentration conditions, and has higher thermal stability and amplification efficiency compared with wild-type phi29 DNA polymerase.

[0006] The present invention also provides a recombinant protein.

[0007] The present invention also provides a biological material related to the above-mentioned phi29 DNA polymerase mutant or recombinant protein.

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

[0009] The present invention also provides a preparation method of the above-mentioned phi29 DNA polymerase mutant.

[0010] The present invention also provides a method for amplifying DNA molecules.

[0011] The present invention also provides an application related to the above-mentioned phi29 DNA polymerase mutant, recombinant protein, biological material or enzyme preparation.

[0012] The present invention also provides a kit for nucleic acid amplification or sequencing.

[0013] A phi29 DNA polymerase mutant according to an embodiment of the first aspect of the present invention. Compared with the wild-type phi29 DNA polymerase, the phi29 DNA polymerase mutant contains any one set of mutation sites from A1) to A2):

[0014] A1), Y163K, F181K, Y224K, W277K, Y298K, Y369K, Y439K, F526R;

[0015] A2), Y163K, F181K, Y224K, W277P, Y298K, Y369K, Y439K, F526R;

[0016] The amino acid sequence of the wild-type phi29 DNA polymerase is shown as SEQ ID NO:1.

[0017] The phi29 DNA polymerase mutant according to the embodiment of the present invention has at least the following beneficial effects:

[0018] By analyzing the molecular structure of the wild-type phi29 DNA polymerase, it is found that there are many hydrophobic groups on its protein surface. This phenomenon may be related to the stability of phi29 DNA polymerase in solution. In the present invention, through protein engineering technology, some surface hydrophobic groups on the wild-type phi29 DNA polymerase are modified into polar groups to enhance the solubility of its protein in the buffer system, thereby improving its performance and stability.

[0019] Compared with the wild-type phi29 DNA polymerase, the phi29 DNA polymerase mutant of the embodiment has a higher specific enzyme activity, which is 2 to 2.7 times that of the wild-type phi29 DNA polymerase; it has a higher genomic amplification efficiency. When single-cell library construction is carried out by the MDA technique, the brightness of the amplified bands is higher than that of the wild-type, and the amplification efficiency is higher; it has higher thermal stability and still has good amplification performance after being treated at 37°C; it has higher stability and solubility in low-salt solutions. It has good application prospects in the fields of nucleic acid amplification and sequencing.

[0020] According to some embodiments of the present invention, the amino acid sequence of the phi29 DNA polymerase mutant is shown as SEQ ID NO:3 or SEQ ID NO:5.

[0021] A recombinant protein according to an embodiment of the second aspect of the present invention, comprising a tag and the phi29 DNA polymerase mutant described in the embodiment of the first aspect of the present invention.

[0022] According to some embodiments of the present invention, the tag is connected to the middle and / or N-terminus and / or C-terminus of the phi29 DNA polymerase mutant.

[0023] According to some embodiments of the present invention, the tag sequence includes at least one of the tag sequences that facilitate the dissolution, purification, and detection of the phi29 DNA polymerase mutant. It can be understood that the phi29 DNA polymerase mutant of the present invention may contain one or more tag sequences; multiple tag sequences may include a combination of multiple identical tag sequences or a combination of multiple different tag sequences. For example: tags that facilitate the dissolution of the phi29 DNA polymerase mutant include, but are not limited to, the Nus tag sequence or the maltose-binding protein tag sequence; tags that facilitate the purification of the phi29 DNA polymerase mutant include, but are not limited to, the strep tag sequence, His tag sequence, GST tag sequence, pelB signal tag sequence, or ompA signal tag sequence; tags that facilitate the detection of the phi29 DNA polymerase mutant include, but are not limited to, the horseradish peroxidase (HRP) tag sequence, β-galactosidase tag sequence, luciferase tag sequence, green fluorescent protein (GFP) tag sequence, HcRed tag sequence, DsRed tag sequence, or cyan fluorescent protein (CFP) tag sequence. The tag may specifically be the His tag sequence.

[0024] Biological materials related to the phi29 DNA polymerase mutant described in the first aspect embodiment of the present invention or the recombinant protein described in the second aspect embodiment of the present invention according to the third aspect embodiment of the present invention, wherein the biological material is any one of B1) to B4):

[0025] B1), a nucleic acid molecule encoding the phi29 DNA polymerase mutant described in the first aspect embodiment of the present invention or the recombinant protein described in the second aspect embodiment of the present invention;

[0026] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0027] B3), a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0028] B4), a recombinant biological cell containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0029] According to some embodiments of the present invention, the nucleic acid molecule has any one of B11) to B14):

[0030] B11), a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 4;

[0031] B12), a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 6;

[0032] B13), a DNA molecule having a homology of 80%, 85% or more than 90% with the nucleotide sequence shown in any one of B11) to B12) and encoding the phi29 DNA polymerase mutant;

[0033] B14), a DNA molecule that hybridizes with the nucleotide sequence defined in any one of B11) to B13) under stringent conditions and encodes the phi29 DNA polymerase mutant.

[0034] According to some embodiments of the present invention, the stringent conditions can be hybridization and membrane washing twice in a solution of 2×SSC, 0.1% SDS at 68°C for 5 minutes each time; or hybridization and membrane washing twice in a solution of 0.5×SSC, 0.1% SDS at 68°C for 15 minutes each time.

[0035] According to some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the phi29 DNA polymerase mutant in a host cell. This DNA may not only include a promoter that initiates the transcription of the phi29 DNA polymerase mutant gene, but also include a terminator that terminates the transcription of the phi29 DNA polymerase mutant gene. Further, the expression cassette may also include an enhancer sequence.

[0036] According to some embodiments of the present invention, the vector can be a plasmid, cosmid, phage or viral vector. For example: it can be a PET-28a vector.

[0037] According to some embodiments of the present invention, the recombinant vector can be a recombinant vector obtained by inserting a DNA molecule encoding the phi29 DNA polymerase mutant into the multiple cloning site of the vector.

[0038] According to some embodiments of the present invention, biological cells include prokaryotic cells and eukaryotic cells. The prokaryotic cells include bacteria or algae. The eukaryotic cells include fungi, mammalian cells or insect cells. Among them, the bacteria can be Escherichia coli, such as E.coli DH5α or E.coli BL21. The recombinant biological cells do not contain reproductive materials.

[0039] According to some embodiments of the present invention, the recombinant biological cells are recombinant biological cells obtained by introducing the nucleic acid molecule in B1), the expression cassette in B2) or the recombinant vector in B3) into biological cells. Specifically, it can be E.coli DH5α or E.coli recombinant Escherichia coli obtained by introducing a recombinant vector into BL21.

[0040] An enzyme preparation according to an embodiment of the fourth aspect of the present invention includes the phi29 DNA polymerase mutant described in the embodiment of the first aspect of the present invention or the recombinant protein described in the embodiment of the second aspect of the present invention.

[0041] According to some embodiments of the present invention, the enzyme preparation further includes a reaction premix.

[0042] According to some embodiments of the present invention, the reaction premix includes at least one of Tris-HCl, Mg 2+ , NH4 + , DTT, dNTPs, BSA.

[0043] According to some embodiments of the present invention, the source of the NH4 + is an ammonium salt. The ammonium salt includes at least one of an organic ammonium salt (including but not limited to ammonium acetate), an inorganic ammonium salt (including but not limited to (NH4)2SO4, NH4Cl, or a combination thereof).

[0044] According to some embodiments of the present invention, the source of the Mg 2+ includes at least one of magnesium chloride, magnesium acetate, and magnesium sulfate.

[0045] According to some embodiments of the present invention, the reaction premix includes 20-100 mmol / L Tris-HCl, 9-12 mmol / L Mg 2+ , 9-12 mmol / L NH4 + , 4-6 mmol / L DTT, 120-140 μmol / L dNTPs, 100-120 ng / μL BSA. For example: the reaction premix may include 50 mmol / L Tris-HCl, 10 mmol / L Mg 2+ , 10 mmol / L NH4 + , 4 mmol / L DTT, 125 μmol / L dNTPs, 100 ng / μL BSA.

[0046] According to some embodiments of the present invention, the pH of the reaction premix is 7.2-7.8.

[0047] It can be understood that the reaction premix is preferably such that it does not affect the activity of the phi29 DNA polymerase mutant.

[0048] A method for preparing the phi29 DNA polymerase mutant described in the embodiment of the first aspect of the present invention or the recombinant protein described in the embodiment of the second aspect of the present invention according to an embodiment of the fifth aspect of the present invention includes:

[0049] Introduce the coding gene of the phi29 DNA polymerase mutant described in the embodiments of the first aspect of the present invention or the recombinant protein described in the embodiments of the second aspect of the present invention into a biological cell, so that the coding gene is expressed to obtain the phi29 DNA polymerase mutant or the recombinant protein.

[0050] According to some embodiments of the present invention, the biological cell includes a prokaryotic cell and a eukaryotic cell.

[0051] According to some embodiments of the present invention, the prokaryotic cell includes a bacterium or an alga. Among them, the bacterium can be Escherichia coli (such as: E.coli BL21).

[0052] According to some embodiments of the present invention, the eukaryotic cell includes a fungus (such as yeast), a mammalian cell (such as HEK293 cell) or an insect cell.

[0053] A method for amplifying a DNA molecule according to the embodiments of the sixth aspect of the present invention includes the following steps:

[0054] Use the phi29 DNA polymerase mutant described in the embodiments of the first aspect of the present invention, or the recombinant protein described in the embodiments of the second aspect of the present invention, or the enzyme preparation described in the embodiments of the fourth aspect of the present invention to amplify the DNA molecule.

[0055] According to some embodiments of the present invention, it may specifically include the following steps:

[0056] Mix the phi29 DNA polymerase mutant / recombinant protein / enzyme preparation, the DNA molecule (template DNA), primers and a reaction premix, and react.

[0057] Use of any one of C1) to C4) according to the embodiments of the seventh aspect of the present invention in the preparation of nucleic acid amplification or sequencing products;

[0058] C1), the phi29 DNA polymerase mutant described in the embodiments of the first aspect of the present invention;

[0059] C2), the recombinant protein described in the embodiments of the second aspect of the present invention;

[0060] C3), the biological material described in the embodiments of the third aspect of the present invention;

[0061] C4), the enzyme preparation described in the embodiments of the fourth aspect of the present invention.

[0062] According to some embodiments of the present invention, the product includes a kit.

[0063] The application of the phi29 DNA polymerase mutant described in the embodiment of the first aspect of the present invention, the recombinant protein described in the embodiment of the second aspect of the present invention, or the enzyme preparation described in the embodiment of the third aspect of the present invention in nucleic acid amplification or sequencing.

[0064] According to some embodiments of the present invention, the nucleic acid amplification includes, but is not limited to, rolling circle amplification or multiple strand displacement amplification.

[0065] According to some embodiments of the present invention, the sequencing includes DNA sequencing. It includes, but is not limited to, single cell sequencing (such as being used as a DNA polymerase for single cell library construction), single polymerase molecule real-time DNA sequencing, DNA nanoball sequencing, or nanopore sequencing.

[0066] A kit for nucleic acid amplification or sequencing according to an embodiment of the ninth aspect of the present invention, comprising the above-mentioned phi29 DNA polymerase mutant, the above-mentioned recombinant protein, or the above-mentioned enzyme preparation.

[0067] Other features and advantages of the present invention will be described in the subsequent specification, and in part, will become apparent from the specification, or will be understood by implementing the present invention. Brief Description of the Drawings

[0068] Figure 1 It is a figure of the expression and purification results of phi29 DNA polymerase; among them, Figure A is phi29-wt, Figure B is phi29-E1, Figure C is phi29-E2, M in the figure is a protein marker, and lanes 1-10 respectively refer to samples 1-10;

[0069] Figure 2 It is a figure of the detection results of the genomic amplification performance of wild-type phi29 DNA polymerase, phi29 DNA polymerase mutants phi29-E1 and phi29-E2; in the figure, M is a DNA marker ( GL DNA Marker 10000), 1-2 are experimental groups with a template amount of 10 ng, and 3-6 are experimental groups with a template amount of 0.1 ng;

[0070] Figure 3 It is a figure of the detection results of the stability of wild-type phi29 DNA polymerase, phi29 DNA polymerase mutants phi29-E1 and phi29-E2 in a low-salt system;

[0071] Figure 4 It is a figure of the detection results of the genomic amplification performance of phi29 DNA polymerase after different temperature treatments; in the figure, the marker used for wild-type phi29 DNA polymerase is GLDNA Marker 10000, the marker used for phi29 DNA polymerase mutants phi29-E1 and phi29-E2 is GL DNA Marker 15000. In each figure, lanes 1-2 are the untreated group, lanes 3-4 are the group heated at 37 °C for 30 min, lanes 5-6 are the group heated at 42 °C for 30 min, and lanes 7-8 are the group heated at 45 °C for 30 min;

[0072] Figure 5 It is the differential scanning fluorimetry (DSF) test result graph of wild-type phi29 DNA polymerase;

[0073] Figure 6 It is the DSF test result graph of phi29 DNA polymerase mutants phi29-E1 and phi29-E2. Detailed implementation manners

[0074] The following will clearly and completely describe the concept of the present invention and the technical effects generated in combination with the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts all fall within the scope of protection of the present invention.

[0075] For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained by commercial purchase.

[0076] In the description of the present invention, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method or product comprising a series of steps or units does not necessarily have to be limited to those clearly listed steps or units, but may include other steps or units not clearly listed or inherent to these processes, methods or products.

[0077] Unless otherwise defined, all scientific or technical professional terms in this patent are consistent with the common understanding of most ordinary people in the field.

[0078] The following amino acid sequences are all in the order from the N-terminus to the C-terminus; the nucleotide sequences are all in the order from the 5'-end to the 3'-end.

[0079] The term "amino acid" refers to the basic unit that constitutes proteins, endowing proteins with specific molecular structural forms and making their molecules biochemically active. For example, the "amino acids" used in the present invention include the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), lysine (Lys or K), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), and tyrosine (Tyr or Y).

[0080] The term "amplification" refers to the process in which the number of target nucleic acid fragments increases under the action of a nucleic acid polymerase.

[0081] To obtain phi29 DNA polymerase with good thermal stability and amplification performance, the wild-type phi29 DNA polymerase was mutated and subjected to extensive screening to obtain the phi29 DNA polymerase mutants phi29-E1 and phi29-E2.

[0082] The amino acid sequence of wild-type phi29 DNA polymerase phi29-wt is shown in SEQ ID NO:1. MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK (SEQ ID NO:1).

[0083] The coding nucleotide sequence of the amino acid sequence of wild-type phi29 DNA polymerase is shown in SEQ ID NO:2:

[0084]

[0085] The phi29 DNA polymerase mutant phi29-E1 is formed by mutating based on the amino acids shown in SEQ ID NO:1. The specific amino acid mutation modes are Y163K, F181K, Y224K, W277K, Y298K, Y369K, Y439K, and F526R. The amino acid sequence is specifically as shown in SEQ ID NO:3.

[0086] MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEKAYIKNDIQIIAEALLIQKKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVKDEDYPLHIQHIRCEFELKEGKIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTKIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARKTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKRSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK (SEQ ID NO:3).

[0087] The coding nucleotide sequence of the amino acid sequence of the phi29 DNA polymerase mutant phi29-E1 is as shown in SEQ ID NO:4:

[0088]

[0089] The phi29 DNA polymerase mutant phi29-E2 is formed by mutating based on the amino acids shown in SEQ ID NO:1. The specific amino acid mutation modes are Y163K, F181K, Y224K, W277P, Y298K, Y369K, Y439K, and F526R. The amino acid sequence is specifically as shown in SEQ ID NO:5.

[0090] MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEKAYIKNDIQIIAEALLIQKKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVPDEDYPLHIQHIRCEFELKEGKIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTKIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARKTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKRSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK (SEQ ID NO:5).

[0091] The coding nucleotide sequence of the amino acid sequence of the phi29 DNA polymerase mutant phi29-E2 is as shown in SEQ ID NO:6:

[0092]

[0093] Example 1

[0094] 1. Construction of expression vectors:

[0095] Entrust Hunan Aikery Bioengineering Co., Ltd. to design a nucleotide sequence containing a coding histidine tag (HHHHHHHH) and a linker peptide (GS) at its 3' end based on the coding nucleotide sequence information of phi29-wt, phi29-E1, and phi29-E2, i.e., ATGCATCACCACCATCACCATCACCACGGTAGC (SEQ ID NO:7), and synthesize the corresponding DNA molecules. Then, the DNA molecules are respectively ligated to the PET-28a vector through overlap PCR to obtain recombinant expression vectors PET-28a / phi29-wt, PET-28a / phi29-E1, and PET-28a / phi29-E2.

[0096] Furthermore, using the above recombinant expression vectors as templates respectively, perform PCR amplification, and then use Steady PurePCR reaction solution purification kit (Hunan Aikery Bioengineering Co., Ltd., product number AG21003) to purify and recover the obtained PCR products. The recovered products are transformed into DH5α competent cells, and positive monoclonal colonies are screened for sequencing verification. After verification, the results show consistency with the expectation.

[0097] 2. Expression:

[0098] Transform the recombinant plasmids obtained in step 1 into the host cell E.coli BL21 respectively. Pick single colonies and inoculate them into 100 mL LB medium containing 50 μg / mL ampicillin. Place them in a shaker at 37°C and shake overnight. Then inoculate them into 2 L LB medium containing 50 μg / mL ampicillin at a volume ratio of 1:100, and shake culture in a shaker at 37°C until the OD 600 reaches 0.6 - 0.8; add IPTG to a final concentration of 0.4 mmol / L, reduce the temperature to 16°C, and continue shaking induction for 16 h - 18 h; centrifuge to collect the induced bacteria and weigh them, record the wet weight of the bacteria, and store them at -80°C.

[0099] 3. Purification:

[0100] Take the induced-expression cells stored at -80 °C, resuspend the cells by adding 3 mL of lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 300 mM NaCl, 1 mM DTT, pH 7.5) per gram of wet weight of the cells, and lyse the cells with a high-pressure cell disruptor. The high-pressure disruption conditions are 700 bar, and the cells are disrupted in three cycles. Centrifuge the lysed cells at 4 °C and 12,000 rpm for 30 min, transfer the supernatant A (Sample 1) to a 200 mL sterilized beaker, and discard the pellet A (Sample 2). Add 10% PEI to the supernatant A until the final concentration of PEI is 0.6%, stir gently at low speed for 30 min to 60 min, then centrifuge at 4 °C and 12,000 rpm for 30 min. Transfer the supernatant B (Sample 3) to a new 200 mL sterilized beaker, and discard the pellet B (Sample 4). Add (NH4)2SO4 to the supernatant B until the final concentration of (NH4)2SO4 is 65%, then centrifuge at 4 °C and 12,000 rpm for 30 min. Collect the pellet C (Sample 5), and discard the supernatant C. Dissolve the pellet C in buffer A (50 mM Tris-HCl, 1 mM EDTA, 300 mM NaCl, 1 mM DTT, 20 mM imidazole, pH 7.5) to obtain the protein solution to be purified (Sample 6).

[0101] After equilibrating the Ni-NTA Purose 6 Fast Flow chromatography column (purchased from Jiaxing Qianchun Biotechnology Co., Ltd.) with buffer A, load the protein solution to be purified onto the chromatography column. First, wash the column with buffer A, and then perform a gradient elution from 0% to 100% with buffer B (50 mM Tris-HCl, 1 mM EDTA, 300 mM NaCl, 1 mM DTT, 700 mM imidazole, pH 7.5). Detect the eluted fractions by SDS-PAGE protein electrophoresis. According to the detection results, collect the eluate A containing the target protein (Sample 7), and dialyze the eluate A into buffer A to collect the dialysis solution A (Sample 8). Then, perform secondary nickel ion affinity chromatography purification on the dialysis solution A using the same procedure as above. Collect the eluate B containing the target protein (Sample 9), and dialyze the eluate B into the enzyme storage solution (10 mM Tris-HCl, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% Glycerol, 0.5% Tween20, 0.5% Nonidet P-40, pH 7.5) to collect the dialysis solution B (Sample 10) for standby.

[0102] Take 30 μL from each of Samples 1 to 10, and analyze the purification results by SDS-PAGE electrophoresis.

[0103] The results are as Figure 1as shown

[0104] Detection Example 1

[0105] In this detection example, the enzyme activity of the obtained phi29 DNA polymerase was detected. Within a certain period of time, a control curve was established based on the amount of products obtained with different enzyme addition amounts of the commercial DNA polymerase, and at the same time, a curve of the DNA polymerase to be tested was established. By calculating the ratio of the slopes of the two, it can be used to determine the enzyme activity of the DNA polymerase to be tested. A hairpin-shaped oligonucleotide sequence was designed. Under the catalysis of DNA polymerase, dNTPs gradually infiltrated, and the hairpin-shaped oligonucleotide sequence would eventually form a double-stranded DNA product. SYBR Green I is a highly sensitive fluorescent dye that binds to double-stranded DNA and can bind to double-stranded DNA to produce a fluorescent signal, and the intensity of its fluorescent signal is positively correlated with the concentration of double-stranded DNA. By measuring the fluorescent signal value within a certain period of time, the enzyme activity value of the DNA polymerase to be tested can be calculated.

[0106] Enzyme activity determination: Design a hairpin-shaped oligonucleotide sequence (Test 1: 5'-tagcgaaggatgtgaacctaatcccTGCTCCCGCGGCCGatctgcCGGCCGCGGGAGCA-3'). According to the brightness of the protein solution band finally obtained in the SDS-PAGE gel of the purified protein, the enzyme activities of the purified wild-type and mutant phi29 DNA polymerases were pre-calculated in a certain proportion to obtain the estimated enzyme activity value, which was diluted to the estimated 10 U / uL with the enzyme storage solution. Then, the commercial DNA polymerase (purchased from Novoprotein, catalog number E013-02A), the wild-type phi29 DNA polymerase solution, and the phi29 DNA polymerase mutant solution were respectively diluted by different multiples with the enzyme storage solution to obtain the diluted enzyme solutions. Add 5 μL of the diluted enzyme solution to a 20 μL reaction system (25 mmol / L Tris-HC1, 50 mmol / L KCl, 5 mmol / L (NH4)2SO4, 2.5 mmol / L MgC12, 0.1% TritonX-100, 0.25 mmol / L dNTPs, 0.5 μmol / L Test 1, pH 8.5), react at 30 °C for 5 min, immediately place on ice to cool, and add EDTA to a final concentration of 10 mM to terminate the reaction; add 1×SYBR dye solution at a volume ratio of 1:30 and detect with a microplate reader. Note that 2 parallels are prepared for each dilution factor to reduce experimental errors, and at the same time, 2 blank controls are prepared for each reaction to deduct the fluorescence background. Export the original fluorescence signal value data of the detection, deduct the fluorescence background signal value, and calculate the slope value of the fluorescence signal value and the enzyme amount.

[0107] The detection results of the enzyme activity values of wild-type phi29 and mutants phi29-E1 and phi29-E2 are shown in Table 1.

[0108] Table 1

[0109]

[0110] The results showed that the specific enzyme activities of the purified phi29 DNA polymerase mutants phi29-E1 and phi29-E2 were 2-2.7 times that of the wild-type phi29 DNA polymerase, with higher specific enzyme activities.

[0111] Detection Example 2

[0112] This detection example detected the amplification performance of the phi29 DNA polymerase mutant in single-cell library construction. Using human genomic DNA as a template, single-cell library construction amplification reaction was carried out on it with wild-type phi29 DNA polymerase or phi29 DNA polymerase mutant through MDA technology, and the amplification efficiency was determined by the brightness of the overall electrophoresis band obtained after the amplification reaction. The steps are as follows:

[0113] A 20 μL reaction system containing 10 ng or 0.1 ng of template (50 mmol / L Tris-HCl, 10 mmol / L MgCl2, 10 mmol / L (NH4)2SO4, 4 mmol / L DTT, 125 μmol / L dNTPs, 25 μmol / L Random Primer 6 (purchased from NEB, catalog number S1230S), 100 ng / μL BSA, 10 U of enzyme, pH 7.5) was incubated at 30 °C for 2 h, then heat-incubated at 65 °C for 10 min, and the products were detected by electrophoresis using 1% agarose gel.

[0114] The electrophoresis results are as Figure 2 shown, and the concentration measurement results are shown in Table 2 below.

[0115] Table 2

[0116]

[0117] Under the same reaction system and reaction conditions, the band brightness amplified by phi29 DNA polymerase mutants phi29-E1 and phi29-E2 is much brighter than that of wild-type phi29 DNA polymerase. Moreover, when the template amount is reduced to 0.1 ng / uL, both phi29 DNA polymerase mutants phi29-E1 and phi29-E2 can detect amplified bands, while wild-type phi29 DNA polymerase can no longer amplify bands. This indicates that the amplification efficiency of phi29 DNA polymerase mutants phi29-E1 and phi29-E2 for single-cell library construction is superior to that of wild-type phi29 DNA polymerase.

[0118] Detection Example 3

[0119] This detection example detects the stability of phi29 DNA polymerase mutants in a low-salt buffer system.

[0120] The wild-type phi29 DNA polymerase and each phi29 DNA polymerase mutant were respectively diluted to the same protein concentration (1.5 mg / mL) using an enzyme preservation solution, and then diluted 2-fold and 3-fold with deionized water and mixed well. The buffer components of each protein stock solution were the same as those of the enzyme preservation solution. After dilution, the concentrations of each component decreased accordingly, forming a low-salt buffer system. After placing each diluted sample at 4°C for 12 h, it was centrifuged at 12000 rpm for 10 min to detect the precipitation of proteins.

[0121] The experimental results are as Figure 3 shown.

[0122] The results showed that after dilution, there were no obvious changes in the protein solutions of phi29 DNA polymerase mutants phi29-E1 and phi29-E2; while for wild-type phi29 DNA polymerase, obvious protein precipitation occurred at both 2-fold and 3-fold dilution multiples. This indicates that the solubility of phi29 DNA polymerase mutants phi29-E1 and phi29-E2 has increased, and their stability in the low-salt buffer system is superior to that of wild-type phi29 DNA polymerase.

[0123] Detection Example 4

[0124] This detection example detects the thermal stability of phi29 DNA polymerase mutants.

[0125] 1. Changes in the amplification performance of phi29 DNA polymerase mutants after treatment at different temperatures for 30 min for single-cell library construction.

[0126] Take the wild-type phi29 DNA polymerase and the phi29 DNA polymerase mutants phi29-E1 and phi29-E2 with the same volume obtained in Example 1, and heat-treat them at 37 °C, 42 °C, and 45 °C for 30 min respectively and simultaneously, and then detect the changes in the amplification performance of each sample for single-cell library construction. Using the same performance measurement method as in Detection Example 2, adjust the template addition amount to 10 ng and detect the amplification performance.

[0127] The experimental results are as Figure 4 shown, and the measurement results of the recovered product concentration are shown in Table 3 below.

[0128] Table 3

[0129]

[0130] Note: Groups 1 and 2 are not heated, 3 and 4 are treated at 37 °C for 30 min, 5 and 6 are treated at 42 °C for 30 min, and 7 and 8 are treated at 42 °C for 30 min.

[0131] The results show that under the same reaction system, the overall band brightness of the wild-type phi29 DNA polymerase is weak, and there are no amplification bands after heat treatment at 3 temperatures; both phi29-E1 and phi29-E2 have amplification bands brighter than those of the wild-type phi29 DNA polymerase, and there are amplification bands in the group treated at 37 °C for 30 min. This shows that the thermal stabilities of the two phi29 DNA polymerase mutants obtained in the present invention have both been improved.

[0132] 2. Detect the Tm value of the phi29 DNA polymerase mutant by differential scanning fluorimetry (DSF).

[0133] DSF refers to slowly heating the sample on a fluorescence quantitative PCR instrument, and detecting the amount of the fluorescent dye combined with the protein with a changed structure during the heating process to evaluate the thermal stability of the protein. DSF can monitor the conformational change of the protein during the heating process through the fluorescent dye or the endogenous fluorescence signal of the protein and calculate its melting temperature Tm (the temperature when the folded protein and the unfolded protein are equal). Due to the strong hydrophobicity of the phi29 DNA polymerase, the fluorescent dye Basic Yellow 1 is selected as the detection dye in this experiment. When the temperature rises, the protein unfolds, the hydrophobic part is exposed, and the hydrophobic dye Basic Yellow 1 (purchased from TCI, product number T0558) specifically binds to the hydrophobic part of the protein, and the fluorescence increases. The higher the measured Tm value, the stronger the thermal stability of the protein.

[0134] Put a 20 μL reaction system (20 mmol / L HEPES-KOH, 150 mmol / L KCl, 1 mg enzyme, 20×BasicYellow 1 fluorescent dye, pH 8.0) into a real-time fluorescence quantitative PCR instrument, adjust the excitation and emission wavelengths to the corresponding wavelengths of the fluorescent dye respectively, set the heating rate to 1℃ / 10 s, the temperature change range from 25℃ to 99℃, and measure the fluorescence intensity every 1℃ - 3℃. After the reaction, use the data processing software Protein Thermal Shift Software to plot the curve of fluorescence intensity versus temperature, and calculate the Tm value and related parameters of the protein sample.

[0135] The results are as Figure 5 and Figure 6 shown.

[0136] The Tm value of phi29-wt is 33.4, the Tm value of phi29-E1 is 36.3, and the Tm value of phi29-E2 is 37.3. This indicates that the thermal stabilities of the phi29 DNA polymerase mutants phi29-E1 and phi29-E2 are both higher than that of phi29-wt.

[0137] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A phi29 DNA polymerase mutant, characterized in that: Compared to the wild-type phi29 DNA polymerase, the mutation site of the phi29 DNA polymerase mutant is any one of the groups A1) to A2): A1), Y163K, F181K, Y224K, W277K, Y298K, Y369K, Y439K, F526R; A2), Y163K, F181K, Y224K, W277P, Y298K, Y369K, Y439K, F526R; The amino acid sequence of the wild-type phi29 DNA polymerase is shown in SEQ ID NO:

1.

2. A recombinant protein, characterized in that: It comprises a tag and the phi29 DNA polymerase mutant according to claim 1.

3. A biological material related to the phi29 DNA polymerase mutant of claim 1 or the recombinant protein of claim 2, characterized in that: The biological material is any one of B1) to B4): B1), a nucleic acid molecule encoding the phi29 DNA polymerase mutant according to claim 1 or the recombinant protein according to claim 2; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) A recombinant biological cell containing the nucleic acid molecule described in B1), the expression cassette described in B2) or the recombinant vector described in B3).

4. The biomaterial according to claim 3, characterized in that The nucleic acid molecule has any one of B11) to B14): B11), a DNA molecule having a nucleotide sequence as shown in SEQ ID NO:4; B12), a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 6; B13), a DNA molecule having more than 80% homology with the nucleotide sequence shown in any one of B11) to B12), and encoding the phi29 DNA polymerase mutant; B14), a DNA molecule that hybridizes with the nucleotide sequence defined in any one of B11) to B13) under stringent conditions and encodes the phi29 DNA polymerase mutant; The stringent conditions are hybridization in a 2×SSC, 0.1% SDS solution at 68°C and washing the membrane twice, each time for 5 min; or hybridization in a 0.5×SSC, 0.1% SDS solution at 68°C and washing the membrane twice, each time for 15 min.

5. An enzyme preparation, characterized in that It includes the phi29 DNA polymerase mutant described in claim 1 or the recombinant protein described in claim 2.

6. A method for preparing the phi29 DNA polymerase mutant according to claim 1 or the recombinant protein according to claim 2, characterized in that: include: The coding gene of the phi29 DNA polymerase mutant described in claim 1 or the recombinant protein described in claim 2 is introduced into a biological cell to express the coding gene, thereby obtaining the phi29 DNA polymerase mutant or the recombinant protein.

7. A method for amplifying a DNA molecule, characterized in that: include: The DNA molecule is amplified using the phi29 DNA polymerase mutant described in claim 1, the recombinant protein described in claim 2 or the enzyme preparation described in claim 5.

8. Use of any one of C1) to C4) in the preparation of nucleic acid amplification or sequencing products; C1), the phi29 DNA polymerase mutant according to claim 1; C2), the recombinant protein according to claim 2; C3), the biological material according to claim 3 or 4; C4) and the enzyme preparation according to claim 5.

9. Use of the phi29 DNA polymerase mutant according to claim 1, the recombinant protein according to claim 2 or the enzyme preparation according to claim 5 in nucleic acid amplification or sequencing.

10. A kit for nucleic acid amplification or sequencing, characterized in that: It includes the phi29 DNA polymerase mutant according to claim 1, the recombinant protein according to claim 2 or the enzyme preparation according to claim 5.

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