A fusion DNA polymerase and its preparation method and application

Abstract

The present invention discloses a fusion DNA polymerase and a preparation method and application thereof, and relates to the field of biotechnology. The fusion DNA polymerase comprises any one of A1) to A2): A1), the N-terminus to the C-terminus sequentially contain the amino acid sequence shown in SEQ ID NO: 1 at positions 10 to 187 and positions 197 to 748; A2), the amino acid sequence obtained by connecting a tag sequence in the middle and / or N-terminus or / and C-terminus of the amino acid sequence shown in A1). The fusion DNA polymerase can reduce the inhibition of PPi on the reaction, effectively promote the forward progress of the PCR reaction, increase the yield of the PCR product, and has a higher amplification efficiency than the wild-type Taq DNA polymerase or a combination thereof with PPase.

Description

Technical Field

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

[0002] Polymerase chain reaction (PCR) has become an indispensable technical means in modern molecular biology and diagnostics. PCR technology is based on three repeated steps (DNA template denaturation, primer annealing to the denatured DNA template, and polymerase extension of primers to synthesize nucleic acids complementary to the template). The key component of PCR is the thermostable fusion DNA polymerase, which can synthesize new DNA complementary to the DNA template strand at high temperatures. Taq DNA polymerase is the most popular and widely used enzyme in most PCR applications due to its thermostability, high specificity, and simple and economical production methods. However, the amplification efficiency of Taq DNA polymerase is not high, and the yield of PCR products is low. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a fusion DNA polymerase, which has both DNA polymerase activity and inorganic pyrophosphatease (PPase) activity for decomposing inorganic pyrophosphate (PPi), and can significantly improve the amplification efficiency of PCR reaction.

[0004] The present invention also provides biological materials related to the above fusion DNA polymerase.

[0005] The invention also provides an enzyme preparation.

[0006] The present invention also provides a method for preparing the fusion DNA polymerase.

[0007] The invention also provides a method for synthesizing DNA molecules.

[0008] The present invention also provides applications related to the above-mentioned fusion DNA polymerase, biological material or enzyme preparation.

[0009] A fusion DNA polymerase according to the first aspect of the present invention, the fusion DNA polymerase comprises any one of A1) to A2):

[0010] A1), the N-terminus to the C-terminus sequentially comprise the amino acid sequence shown in positions 10 to 187 and positions 197 to 748 of SEQ ID NO: 1;

[0011] A2), an amino acid sequence obtained by connecting a tag sequence in the middle and / or at the N-terminus and / or the C-terminus of the amino acid sequence shown in A1).

[0012] The fusion DNA polymerase according to the embodiment of the present invention has at least the following beneficial effects:

[0013] With the incorporation of dNTPs in the PCR reaction, pyrophosphate gradually accumulates, which will affect the amplification efficiency of the DNA polymerase. The fusion DNA polymerase of the embodiment fuses the DNA polymerase domain, protein linker and thermostable inorganic pyrophosphatase (PPase) of Taq DNA polymerase, and ingeniously fuses PPase at the spatial position of the 5'-3' exonuclease domain of Taq DNA polymerase, so that it can catalyze DNA polymerization reaction while also having PPase activity, catalyzing the synchronous hydrolysis of the byproduct PPi, reducing the inhibition of PPi on the reaction, effectively promoting the forward progress of the PCR reaction, and increasing the yield of the PCR product. Compared with the wild-type Taq DNA polymerase or its combination with PPase, it has a higher amplification efficiency.

[0014] 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 fusion DNA polymerase. It is understandable that the fusion DNA polymerase of the present invention may include 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 fusion DNA polymerase include but are not limited to nus tag sequences or maltose binding protein sequences; tags that facilitate the purification of the fusion DNA polymerase include but are not limited to strep tag sequences, His tag sequences, GST tag sequences, pelB signal sequences or ompA signal sequences; tags that facilitate the detection of the fusion DNA polymerase include but are not limited to horseradish peroxidase (HRP) sequences, β-galactosidase sequences, luciferase sequences, green fluorescent protein (GFP) sequences, HcRed sequences, DsRed sequences or cyan fluorescent protein (CFP) sequences. The tag can specifically be a His tag sequence.

[0015] According to some embodiments of the present invention, the fusion DNA polymerase may also have an amino acid sequence obtained by replacing, deleting or adding one or more residues of the amino acid sequence shown in A1), and the function is the same or similar to the protein function corresponding to A1). The function is PCR amplification performance (such as PCR amplification rate). It has at least 90% homology with the amino acid sequence shown in A1). For example, it can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0016] According to some embodiments of the present invention, the N-terminus to the C-terminus of the fusion DNA polymerase A1) sequentially comprises the amino acid sequence shown in positions 1 to 187 and positions 197 to 748 of SEQ ID NO: 1.

[0017] According to some embodiments of the present invention, the amino acid sequence of the fusion DNA polymerase A1) is shown in SEQ ID NO:1.

[0018] According to some embodiments of the present invention, the amino acid sequence shown at positions 10 to 187 of SEQ ID NO: 1 is connected to the amino acid sequence shown at positions 197 to 748 of SEQ ID NO: 1 via a connecting peptide.

[0019] According to some embodiments of the present invention, the connecting peptide is a flexible connecting peptide.

[0020] According to some embodiments of the present invention, the flexible connecting peptide consists of at least one of amino acids G and S.

[0021] According to some embodiments of the present invention, the length of the flexible connecting peptide is 3 to 15 amino acids, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0022] According to some embodiments of the present invention, the amino acid sequence of the connecting peptide is GSSGGSSGGS (SEQ ID NO: 5), (GGS) n 、(GGGS) n or (GGGGS) n ; where n is 1, 2 or 3.

[0023] According to the second aspect of the present invention, the biological material related to the above-mentioned fusion DNA polymerase is any one of B1) to B4):

[0024] B1), a nucleic acid molecule encoding the fusion DNA polymerase described in the embodiment of the first aspect of the present invention;

[0025] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0026] B3), a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0027] 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).

[0028] According to some embodiments of the present invention, the nucleic acid molecule is any one of B11) to B13):

[0029] B11), a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 2;

[0030] B12), a DNA molecule having 80%, 85% or 90% or more homology with the nucleotide sequence shown in SEQ ID No: 2, and encoding the above-mentioned fusion DNA polymerase;

[0031] B13), a DNA molecule that hybridizes with the nucleotide sequence defined in B11) or B12) under stringent conditions and encodes the above-mentioned fusion DNA polymerase.

[0032] According to some embodiments of the present invention, the expression cassette refers to a DNA capable of expressing the fusion DNA polymerase in a host cell. The DNA may include not only a promoter for initiating transcription of the fusion DNA polymerase gene, but also a terminator for terminating transcription of the protein gene. Further, the expression cassette may also include an enhancer sequence.

[0033] According to some embodiments of the present invention, the vector may be a plasmid, a cosmid, a phage or a viral vector. For example, the vector may be pET-28a(+).

[0034] According to some embodiments of the present invention, the recombinant vector may be a recombinant vector obtained by inserting a nucleic acid molecule encoding the fusion DNA polymerase into the multiple cloning site of the vector.

[0035] 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 JM109. The biological cells do not contain reproductive materials.

[0036] According to some embodiments of the present invention, the recombinant biological cell is a recombinant biological cell obtained by introducing the nucleic acid molecule B1), the expression cassette B2) or the recombinant vector B3) into a biological cell. Specifically, it can be a recombinant Escherichia coli obtained by introducing the recombinant vector into E. coli DH5α or E. coli JM109.

[0037] An enzyme preparation according to an embodiment of the third aspect of the present invention comprises the fusion DNA polymerase described in the embodiment of the first aspect of the present invention.

[0038] According to some embodiments of the present invention, the enzyme preparation also includes a PCR premix.

[0039] According to some embodiments of the present invention, the PCR premix includes Tris-HCl, NH4 + , K +Mg 2+ , Triton X-100, and at least one of dNTPs.

[0040] According to some embodiments of the present invention, the NH4 + The source is an ammonium salt. The ammonium salt includes at least one of an organic ammonium salt (including but not limited to ammonium acetate) and an inorganic ammonium salt (including but not limited to (NH4)2SO4, NH4Cl or a combination thereof).

[0041] According to some embodiments of the present invention, the K + The source includes at least one of potassium chloride, potassium acetate and potassium sulfate.

[0042] According to some embodiments of the present invention, the Mg 2+ The source includes at least one of magnesium chloride, magnesium acetate and magnesium sulfate.

[0043] According to some embodiments of the present invention, the PCR premix includes 100-150 mmol / L Tris-HCl, 15-25 mmol / L K + 、4~6mmol / L NH4 + 1.5~2.5mmol / L Mg 2+ , 0.02-0.03% (v / v) Triton X-100, 150-250 μmol / L dNTPs. For example, the PCR premix may include 120 mmol / L Tris-HCl, 20 mmol / L KCl, 5 mmol / L (NH4)2SO4, 2 mmol / L MgCl2, 0.025% (v / v) Triton X-100, and 200 μmol / L dNTPs.

[0044] According to some embodiments of the present invention, the pH of the PCR premix is ​​8.5-9.2.

[0045] It is understood that the reaction buffer should preferably not affect the activity of the fusion DNA polymerase.

[0046] According to the fourth aspect of the present invention, the method for preparing the fusion DNA polymerase described in the first aspect of the present invention comprises the following steps:

[0047] The coding gene of the fusion DNA polymerase described in the embodiment of the first aspect of the present invention is expressed in a biological cell to obtain the fusion DNA polymerase.

[0048] According to some embodiments of the present invention, the biological cells include prokaryotic cells and eukaryotic cells.

[0049] According to some embodiments of the present invention, the prokaryotic cell includes bacteria or algae. Specifically, the biological cell may be Escherichia coli (eg, E. coli JM109).

[0050] According to some embodiments of the present invention, the eukaryotic cells include fungi (such as yeast), mammalian cells (such as HEK293 cells) or insect cells.

[0051] According to some embodiments of the present invention, the preparation method further comprises a purification process, which comprises at least one of a nickel ion affinity chromatography purification process, a strong anion exchange chromatography purification process, and a heparin column affinity chromatography purification process.

[0052] According to some embodiments of the present invention, the binding buffer in the purification process (including nickel ion affinity chromatography purification process, strong anion exchange chromatography purification process, and heparin column affinity chromatography purification process) includes: 30-50mM Tris-HCl, 0.08-0.15mM EDTA, 100-120mM KCl, 0.2-0.3% Tween-20, pH7.5-7.8.

[0053] According to some embodiments of the present invention, the elution buffer in the strong anion exchange chromatography or heparin column affinity chromatography comprises: 30-50 mmol / L Tris-HCl, 600-800 mM KCl, 0.2-0.3% Tween-20, pH 7.5-7.8.

[0054] According to some embodiments of the present invention, the storage solution for storing the fusion DNA polymerase includes: 10-30mM Tris-HCl, 0.08-0.15mM EDTA, 100-120mM KCl, 0.45-0.6% Tween-20, 1-1.2mM DTT, 50-53% glycerol, pH 7.5-7.8.

[0055] A method for synthesizing a DNA molecule according to a fifth aspect of the present invention comprises:

[0056] The fusion DNA polymerase described in the embodiment of the first aspect of the present invention is used for PCR amplification.

[0057] According to some embodiments of the present invention, the following steps may be specifically included:

[0058] The fusion DNA polymerase is mixed with the template DNA, primers and PCR premix solution to react.

[0059] Applications related to the above-mentioned fusion DNA polymerase, biomaterial or enzyme preparation according to the sixth aspect of the present invention.

[0060] According to some embodiments of the present invention, the application is the application of the biomaterial described in the embodiment of the second aspect of the present invention in the preparation of a fusion DNA polymerase, an enzyme preparation, a nucleic acid amplification product or a sequencing product.

[0061] According to some embodiments of the present invention, the application is the application of C1) or C2) in any one of D1) to D4), C1), the fusion DNA polymerase described in the embodiment of the first aspect of the present invention;

[0062] C2), the enzyme preparation described in the embodiment of the third aspect of the present invention;

[0063] D1), nucleic acid amplification;

[0064] D2), preparing nucleic acid amplification products;

[0065] D3), ​​sequencing;

[0066] D4) Prepare sequencing products.

[0067] According to some embodiments of the present invention, the nucleic acid amplification includes polymerase chain reaction (such as conventional PCR, fluorescent quantitative PCR, allele-specific PCR, multiplex PCR, etc.).

[0068] According to some embodiments of the present invention, the nucleic acid amplification product includes a nucleic acid amplification kit. The sequencing product includes a sequencing kit.

[0069] A kit according to a seventh aspect of the present invention, comprising at least one of C1) to C3);

[0070] C1), the fusion DNA polymerase described in the embodiment of the first aspect of the present invention;

[0071] C2), the biomaterial described in the embodiment of the second aspect of the present invention;

[0072] C3), the enzyme preparation described in the embodiment of the third aspect of the present invention.

[0073] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 It is a schematic diagram of the three-dimensional modeling structure of the fusion DNA polymerase Taq-PPase;

[0075] Figure 2This is the result of expression and purification of fusion DNA polymerase Taq-PPase; lane M is a protein marker (10-180 kDa), lanes 1-13 correspond to samples 1-13 respectively; the black arrow marks the target band corresponding to the fusion DNA polymerase Taq-PPase;

[0076] Figure 3 It is the result of the DNA polymerase activity concentration test of the fusion DNA polymerase Taq-PPase;

[0077] Figure 4 It is the test result of PPi hydrolysis activity concentration of commercial PPase and fusion DNA polymerase Taq-PPase; wherein A is the test result of commercial PPase, and B is the test result of fusion DNA polymerase Taq-PPase;

[0078] Figure 5 It is the test result of PCR amplification performance of fusion DNA polymerase Taq-PPase; among them, lanes 1-2 and 3-4 are the test results of 1.25U wild-type Taq DNA polymerase and 3.75U wild-type Taq DNA polymerase, respectively; lanes 5-6 and 7-8 are the test results of 1.25U fusion DNA polymerase Taq-PPase and 3.75U fusion DNA polymerase Taq-PPase, respectively; lanes 9-10 and 11-12 are the test results of 1.25U wild-type Taq DNA polymerase + 2.5U thermostable PPase and 3.75U wild-type Taq DNA polymerase + 2.5U thermostable PPase, respectively; lane M is DNA Marker (GL DNA Marker10000). DETAILED DESCRIPTION

[0079] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0080] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0081] In the description of the present invention, the terms "comprises" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method or product comprising a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods or products.

[0082] Unless otherwise defined, all scientific or technical terms used in the present invention are consistent with the common understanding of most ordinary persons in the art.

[0083] The term "amino acid" refers to the basic unit that constitutes protein, which gives protein a specific molecular structure and makes its molecule have biochemical activity. For example, the "amino acid" used in the present invention includes 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).

[0084] In a specific embodiment of the present invention, 2×ApexHF FS PCR Master Mix was purchased from Hunan Aikerui Biotechnology Co., Ltd., catalog number AG12202;

[0085] 2.5×OK Clon Master Mix was purchased from Hunan Aikerui Bioengineering Co., Ltd., catalog number AG11807;

[0086] Commercial PPase was purchased from Xinhai Gene Detection Co., Ltd., catalog number C5007;

[0087] Commercial DNA polymerase (amino acid sequence is the same as wild-type Taq DNA polymerase) was purchased from NEB, catalog number M0273V.

[0088] The present invention transforms the wild-type Taq DNA polymerase (GenBank: BAA06775.1) and obtains a Taq DNA polymerase truncate by screening the sequence. A fusion DNA polymerase Taq-PPase is provided, whose amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence encoding it is shown in SEQ ID NO: 2. The nucleotide sequence is obtained by codon optimization according to the characteristics of the Escherichia coli expression system, and can significantly improve its expression efficiency in Escherichia coli. The schematic diagram of the three-dimensional modeling structure of the fusion DNA polymerase Taq-PPase is shown in Figure 1 shown.

[0089] (SEQ ID NO:1);

[0090]

[0091] The invention is designed based on PPase DNA (amino acid sequence is shown in SEQ ID NO: 3) and Taq DNA polymerase truncate (amino acid sequence is shown in SEQ ID NO: 4), and GSSGGSSGGS (SEQ ID NO: 5) is a connecting peptide.

[0092] MNPFHDLEPGPNVPEVVYALIEIPKGSRNKYELDKETGLLKLDRVLYTPFHYPVDYGIIPRTWYEDGDPFDIMVIMREPTYPLTIIEARPIGLFKMIDSGDKDYKVLAVPVEDPYFKDWKDISDVPKAFLDEIAHFFKRYKELEGKEIIVEGWEGAEAAKREILRAIEMYKEKFGKKE(SEQ ID NO:3);

[0093] (SEQ ID NO:4).

[0094] Expression and purification of fusion DNA polymerase Taq-PPase

[0095] 1. Construction of recombinant expression vector:

[0096] PCR amplification was performed using the gene-synthesized DNA molecule encoding PPase and the pET-28a(+) vector plasmid inserted with the DNA molecule encoding wild-type Taq DNA polymerase (the 5' end was connected to a 6× histidine tag sequence via a connecting peptide sequence shown in SEQ ID NO: 5) as templates to obtain the target fragment (PPase DNA gene fragment or vector fragment containing a truncated wild-type Taq DNA polymerase). The PCR amplification system is as follows:

[0097]

[0098] The PCR amplification program for the PPase DNA gene fragment was: 94°C for 30 s; 98°C for 10 s, 55°C for 15 s, 72°C for 30 s, for a total of 25 cycles; 72°C for 5 min. The upstream primer and downstream primer used were PP-FP and PP-RP, respectively, and the nucleotide sequences were as follows.

[0099] PP-FP: ATCACCATCACCATGGCAGCATGAACCCGTTCCATGAT (SEQ ID NO: 6);

[0100] PP-RP: CTGCTGCCGCCGCTCGAGCCTTCTTTTTTGCCGAATTTTT (SEQ ID NO: 7).

[0101] The PCR amplification program for the vector fragment containing the wild-type Taq DNA polymerase truncated form was: 94°C for 30 s; 98°C for 10 s, 55°C for 15 s, 72°C for 3 min, for a total of 25 cycles; 72°C for 5 min. The upstream primer and downstream primer used were Taq-FP and Taq-RP, respectively, and the nucleotide sequences were as follows.

[0102] Taq-FP: GGCTCGAGCGGCGGCAGCAGCGGCGGCAGCCTGCTGCATGAATTC (SEQ ID NO: 8);

[0103] Taq-RP: GGAACGGTTCATGCTGCCATGGTGATGGTGATGATGCAT (SEQ ID NO: 9).

[0104] The PPase DNA gene fragment of about 0.5 kb in length and the pET-28a(+) vector fragment containing the wild-type Taq DNA polymerase truncation of about 6.6 kb in length were recovered by agarose gel electrophoresis. After the recovered products were connected by homologous recombination, the connected products were transformed into E. coli DH5α competent cells. The homologous recombination system is as follows:

[0105] 2.5×OK Clon Master Mix 4μL;

[0106] 50 ng of each product was recovered;

[0107] Make up to 10 μL with water.

[0108] The reaction conditions for homologous recombination were: incubation at 50°C for 10 min.

[0109] Single colonies were picked for colony PCR identification, and positive single clones were sequenced for verification. Correct competent cells were cultured and verified, and plasmids were extracted. The obtained plasmid was a recombinant expression vector containing a DNA molecule encoding a fusion DNA polymerase Taq-PPase (the nucleotide sequence is shown in SEQ ID NO: 2).

[0110] 2. Expression of fusion DNA polymerase Taq-PPase:

[0111] The recombinant expression vector encoding the fusion DNA polymerase Taq-PPase was transformed into the host cell E. coli JM109, and a single colony was picked and inoculated into 100 mL of LB liquid culture medium containing 50 μg / mL ampicillin, and cultured in a shaker at 30°C overnight, and inoculated into 2 L of LB liquid culture medium containing 50 μg / mL ampicillin at a volume ratio of 1:100, and cultured in a shaker at 30°C until OD600 was 0.6-0.8; IPTG was added to a final concentration of 0.4 mmol / L, and the shaking induction was continued at 30°C for 10h-12h; the induced bacteria (sample 1) were collected by centrifugation at 4°C and 12000rpm and weighed, the wet weight of the bacteria was recorded, and stored at -20°C.

[0112] 3. Purification of fusion DNA polymerase Taq-PPase:

[0113] (1) Add 3 mL of lysis buffer (100 mmol / L Tris-HCl, 0.2 mM EDTA, pH 7.5) to each gram (wet weight) of bacterial cells, and lyse the bacterial cells using a high-pressure crusher (crushing conditions: 800 bar). Repeat the crushing for three rounds to obtain a crushing liquid (sample 2); centrifuge the crushing liquid at 12000 rpm at 4°C for 30 min to obtain a supernatant (sample 3) and a precipitate (sample 4); incubate sample 3 in a 75°C water bath for 20 min, and then centrifuge it at 12000 rpm at 4°C for 30 min to obtain a supernatant (sample 5) and a precipitate (sample 6); filter sample 5 through a 0.22 μm microporous filter membrane to obtain a filtrate (sample 7).

[0114] (2) Nickel ion affinity chromatography purification: Sample 7 was loaded onto a chromatography column Ni-NTA Purose 6 Fast Flow 6 mL (purchased from Jiaxing Qianchun Biotechnology Co., Ltd.) that had been equilibrated with buffer A (50 mM Tris-HCl, 0.1 mM EDTA, 100 mM KCl, 0.2% Tween-20, pH 7.5), and then gradient eluted with buffer B (50 mM Tris-HCl, 0.1 mM EDTA, 100 mM KCl, 700 mM imidazole, 0.2% Tween-20, pH 7.5) at a rate of 0% to 100%, and the eluate (sample 8) was collected, and the eluate was dialyzed into buffer A, and the dialyzate (sample 9) was collected.

[0115] (3) Purification by strong anion exchange chromatography: The dialysate obtained in step (2) was loaded onto a chromatography column Q Purose 6 Fast Flow 6 mL (purchased from Jiaxing Qianchun Biotechnology Co., Ltd.) that had been equilibrated with buffer A. The column was rinsed with buffer A, and then gradient eluted with buffer C (50 mM Tris-HCl, 0.1 mM EDTA, 700 mM KCl, 0.2% Tween-20, pH 7.5) at a rate of 0% to 100%. The eluate (sample 10) was collected, and the eluate was dialyzed into buffer A, and the dialysate (sample 11) was collected.

[0116] (4) Heparin column affinity chromatography purification: The eluate obtained in step (3) was loaded onto a chromatography column Heparin Sepharose6 Fast Flow 6 mL (purchased from GE Healthcare) that had been equilibrated with buffer A. The chromatography column was rinsed with buffer A, and then gradient eluted with buffer C from 0% to 100%. The eluate was collected (sample 12), and the eluate was dialyzed into enzyme preservation solution (20 mM Tris-HCl, 0.1 mM EDTA, 100 mM KCl, 0.5% (v / v) Tween-20, 1 mM DTT, 50% (v / v) glycerol, pH 7.5) for storage (sample 13).

[0117] (5) Detect the expression of target protein (fusion DNA polymerase Taq-PPase) by SDS-PAGE electrophoresis.

[0118] The fusion DNA polymerase Taq-PPase was efficiently expressed. The purification results are as follows Figure 2 shown.

[0119] Test Example 1

[0120] This test example detects the DNA polymerase activity of the purified fusion DNA polymerase Taq-PPase. SYBR Green I is a highly sensitive fluorescent dye that binds to double-stranded DNA and can generate a fluorescent signal by binding to double-stranded DNA. The intensity of the fluorescent signal is positively correlated with the concentration of double-stranded DNA. The test method is as follows:

[0121] (1) Design a hairpin oligonucleotide sequence as a template for enzyme activity detection reaction Test 1 (nucleotide sequence: tagcgaaggatgtgaacctaatcccTGCTCCCGCGGCCGatctgcCGGCCGCGGGAGCA). According to the brightness of the protein liquid band finally obtained in the SDS-PAGE electrophoresis gel image of the purified protein, the polymerase activity of the purified fusion DNA polymerase Taq-PPase was calculated in advance according to a certain ratio to obtain an estimated polymerase activity value, and the enzyme preservation solution was used to dilute the polymerase activity value to an estimated polymerase activity value of 5U / μL, and then the commercial DNA polymerase (control) and the fusion DNA polymerase Taq-PPase were diluted at different multiples using the enzyme preservation solution to obtain the enzyme solution to be tested. To 15 μL of reaction premix (25 mmol / L Tris-HCl, 50 mmol / L KCl, 5 mmol / L (NH4)2SO4, 2.5 mmol / L MgCl2, 0.1% (v / v) TritonX-100, 0.25 mmol / L dNTPs, 0.5 μmol / L Test 1, pH 8.5), add 5 μL of the enzyme solution to be tested, and react at 74°C for 5 min; after the reaction, immediately place it on ice for cooling, and add EDTA (final concentration of 8 mM) to terminate the reaction; add 1×SYBR solution (SYBR Green I (10000×, purchased from Thermo Fisher) diluted with 1×TE solution (10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH 8.5)) at a volume ratio of 1:30, and use a microplate reader for detection, and the detection conditions are: excitation wavelength 485 nm, emission wavelength 520 nm. At least 2 parallels were prepared for each dilution factor to reduce experimental errors; at the same time, 2 blank controls were prepared for each reaction to deduct the fluorescence background.

[0122] (2) Data processing: The original fluorescence signal value data obtained by the detection was exported, and the fluorescence background signal value was deducted. The fluorescence signal value and enzyme amount curves of the commercial DNA polymerase and the fusion DNA polymerase Taq-PPase were drawn respectively, and the K value of the regression equation in the two curves was converted into the enzyme activity value to calculate the enzyme activity concentration of the fusion DNA polymerase Taq-PPase.

[0123] The results are as follows Figure 3 shown.

[0124] The polymerase activity concentration of the purified fusion DNA polymerase Taq-PPase is 15.4 U / μL.

[0125] Test Example 2

[0126] This test example uses sodium pyrophosphate as a substrate to test the PPi hydrolysis activity of the purified fusion DNA polymerase Taq-PPase. The test method is as follows:

[0127] (1) Preparation of standard curve: 0 μL, 7.5 μL, 12.5 μL, 17.5 μL, 22.5 μL, 27.5 μL, 30 μL, 37.5 μL, and 42.5 μL of Na2HPO4 solution (0.1 mol / L) were taken respectively and the volume was fixed to 1.25 mL to obtain diluted Na2HPO4 solution; 120 μL of the diluted Na2HPO4 solution was taken and added to 1 mL of AAM (0.125 mL of 20 mmol / L ammonium molybdate, 0.3125 mL of 2 mol / L sulfuric acid, 0.5 mL of acetone, and 0.0625 mL of ddH2O), and the solution was allowed to stand for 3 min to develop color. The phosphorus content standard curve was prepared by colorimetric determination at 420 nm.

[0128] (2) Determination of PPi hydrolysis activity: According to the brightness of the protein liquid band finally obtained in the SDS-PAGE electrophoresis gel image of the purified protein, the PPi hydrolysis activity of the purified fusion DNA polymerase Taq-PPase was calculated in advance according to a certain ratio to obtain an estimated PPi hydrolysis activity value, and the enzyme preservation solution was used to dilute the PPi hydrolysis activity value to an estimated value of 2U / μL, and then the commercial PPase (control) and fusion DNA polymerase Taq-PPase were diluted with the enzyme preservation solution at different times to obtain the enzyme solution to be tested. 0.25mL P Buffer (50mmol / L Tris-HCl, 1mmol / L MgSO4, pH8.0), 0.01mL sodium pyrophosphate solution (final concentration of sodium pyrophosphate is 17mmol / L), 0.01mL enzyme solution to be tested, and 0.05mL ddH20 were added to the centrifuge tube in sequence, and after incubation at 72℃ for 10min, 0.1mL 0.1mol / L citric acid solution was added to terminate the reaction. After the reaction solution is cooled to room temperature, take 0.12 mL of the reaction solution and add 1 mL of AAM. Let it stand for 3 minutes to develop color. Perform colorimetric determination at 420 nm and record the results. Prepare at least 2 parallels for each dilution factor to reduce experimental errors. Prepare 2 blank controls for each reaction to deduct the background. Calculate the PPi hydrolase activity of the fusion DNA polymerase Taq-PPase.

[0129] The results are as follows Figure 4 shown.

[0130] The PPi hydrolysis activity concentration of the purified fusion DNA polymerase Taq-PPase is 31.2 U / μL.

[0131] Test Example 3

[0132] This test example tests the PCR amplification performance of the purified fusion DNA polymerase Taq-PPase. The test method is as follows:

[0133] Using HL60 DNA as a template, amplification was performed using fusion DNA polymerase Taq-PPase (experimental group), wild-type Taq DNA polymerase (control group 1), and wild-type Taq DNA polymerase + commercial PPase (control group 2). The target fragment size was 1kb, and the upstream and downstream primers were H-FP-1kb and H-RP-1kb, respectively. The reaction procedure was: 94℃30s; 98℃10s, 55℃30s, 72℃1min, a total of 30 cycles; 72℃1min. After the reaction was completed, the results were detected by electrophoresis on 1% agarose gel (sample volume 5μL). The nucleotide sequences of H-FP-1kb and H-RP-1kb are as follows:

[0134] H-FP-1kb:TTTACCCTCTTCATCATCTTCC (SEQ ID NO:10);

[0135] H-RP-1kb: CAAGCCAGAAGGAGGAACTG (SEQ ID NO: 11).

[0136] The reaction system (50 μL) of the experimental group and the control group 1 is as follows:

[0137] 120mmol / L Tris-HCl, 20mmol / L KCl, 5mmol / L (NH4)2SO4, 2mmol / L MgCl2, 0.025% (v / v) Triton X-100, 200μmol / L dNTPs, 0.2μmol / L H-FP-1kb, 0.2μmol / L H-RP-1kb, 100ngHL60DNA, 1.25U / 3.75U enzyme (fusion DNA polymerase Taq-PPase or wild-type Taq DNA polymerase, the enzymatic activities of both enzymes are measured as DNA polymerase activity), pH 7.8.

[0138] The reaction system (50 μL) of control group 2 is as follows:

[0139] 120mmol / L Tris-HCl, 20mmol / L KC1, 5mmol / L (NH4)2SO4, 2mmol / L MgCl2, 0.025% (v / v) Triton X-100, 200μmol / L dNTPs, 0.2μmol / L H-FP-1kb, 0.2μmol / L H-RP-1kb, 1.25U / 3.75U wild-type Taq DNA polymerase, 2.5U thermostable PPase, pH 7.8.

[0140] The results are as follows Figure 5 shown.

[0141] In the same reaction system, the fusion DNA polymerase Taq-PPase can amplify a large number of target fragments, and the band brightness is higher than that of wild-type Taq DNA polymerase and wild-type Taq DNA polymerase + thermostable PPase. This shows that the amplification performance of the fusion DNA polymerase Taq-PPase is significantly better than that of wild-type Taq DNA or wild-type Taq DNA polymerase + thermostable PPase.

[0142] In summary, the fusion DNA polymerase Taq-PPase retains the original DNA polymerase activity and increases the PPi hydrolysis activity of PPase, which can promote the forward progress of the PCR reaction and effectively improve the amplification performance of Taq DNA polymerase.

[0143] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A fusion DNA polymerase, characterized in that The fusion DNA polymerase is any one of A1) to A2): A1), the amino acid sequence shown at positions 10 to 187 of SEQ ID NO: 1, a connecting peptide, and the amino acid sequence shown at positions 197 to 748, from the N-terminus to the C-terminus; A2), an amino acid sequence obtained by connecting a tag sequence to the N-terminus and / or C-terminus of the amino acid sequence shown in A1).

2. The biomaterial related to the fusion DNA polymerase according to claim 1, characterized in that: The biological material is any one of B1) to B4): B1), a nucleic acid molecule encoding the fusion DNA polymerase according to claim 1; 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).

3. The biomaterial according to claim 2, characterized in that The nucleic acid molecule is any one of B11) to B13): B11), a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 2; B12), a DNA molecule having 80%, 85% or 90% homology with the nucleotide sequence shown in SEQ ID No: 2 and encoding the fusion DNA polymerase; B13), a DNA molecule that hybridizes with the nucleotide sequence defined in B11) or B12) under stringent conditions and encodes the fusion DNA polymerase.

4. An enzyme preparation, characterized in that Comprising the fusion DNA polymerase according to claim 1.

5. The enzyme preparation according to claim 4, characterized in that The enzyme preparation also includes a PCR premix; the PCR premix includes Tris-HCl, K + NH4 + Mg 2+ , Triton X-100, and at least one of dNTPs.

6. A method for preparing the fusion DNA polymerase according to claim 1, characterized in that: The following steps are involved: The coding gene of the fusion DNA polymerase is expressed in biological cells to obtain the fusion DNA polymerase.

7. A method for synthesizing a DNA molecule, characterized in that: The following steps are involved: The fusion DNA polymerase according to claim 1 is used for PCR amplification.

8. Use of the biomaterial according to claim 2 or 3 in the preparation of fusion DNA polymerase, enzyme preparation, nucleic acid amplification product or sequencing product.

9. The application of C1) or C2) to any of D1) to D4), C1), the fusion DNA polymerase according to claim 1; C2), the enzyme preparation according to claim 4 or 5; D1), nucleic acid amplification; D2) Preparation of nucleic acid amplification related products; D3), ​​sequencing; D4) Prepare sequencing related products.

10. A kit, characterized in that: It comprises at least one of the fusion DNA polymerase according to claim 1, the biological material according to claim 2 or 3, and the enzyme preparation according to claim 4 or 5.

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