DNA polymerase with amplification activity at room temperature and application thereof

Abstract

The application discloses a DNA polymerase with amplification activity under room temperature and application thereof. The DNA polymerase is a protein IME199DNAP with an amino acid sequence as shown in SEQ ID NO:2. The protein IME199DNAP can efficiently amplify single-stranded DNA and double-stranded DNA under the condition of room temperature (20-30 DEG C) and pH value 3.5-9.5 with or without primers, and the enzyme has strand displacement activity and can be used for isothermal amplification. The application has important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a DNA polymerase with amplification activity under room temperature conditions and application thereof. BACKGROUND

[0002] Since the polymerase chain reaction (PCR) technology was invented, it has had a profound impact on the development of the entire life science, and has become one of the most commonly used methods for people to obtain target genes in vitro, and has been applied in various fields. However, when using PCR technology for amplification, in addition to DNA polymerase, a large and expensive thermal cycler must also be provided, which greatly limits the application of PCR technology in resource-limited environments and temporary medical detection analysis. Compared with PCR technology which requires complex thermal cycler-mediated denaturation, annealing and extension, isothermal amplification technology can achieve rapid and effective amplification under room temperature or simple conditions (such as water bath), and has become a promising alternative to PCR technology.

[0003] Isothermal amplification technology is an in vitro amplification technology, and the reaction process is always maintained at a constant temperature, and rapid nucleic acid amplification is achieved by adding constant temperature amplification enzyme and specific primers. The currently used constant temperature amplification enzymes (such as Bst polymerase and phi29 polymerase) not only need to be imported, but also are expensive, which greatly limits the application. Therefore, it is of great value to develop new constant temperature amplification enzymes. SUMMARY

[0004] The purpose of the present application is to provide a new DNA polymerase.

[0005] The present application first protects a protein IME199DNAP, which is any one of C1) to C4):

[0006] C1) a protein with an amino acid sequence as shown in SEQ ID NO: 2;

[0007] C2) a fusion protein obtained by connecting a tag to the N terminus or / and C terminus of the protein shown in C1);

[0008] C3) a protein obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the protein shown in C1) or C2) and having DNA polymerase activity, 3'→5' exonuclease activity, strand displacement activity and / or primase activity;

[0009] C4) a protein having 80% or more homology with the amino acid sequence of the protein shown in C1) or C2) and having DNA polymerase activity, 3'→5' exonuclease activity, strand displacement activity and / or primase activity.

[0010] SEQ ID NO: 2 consists of 779 amino acid residues.

[0011] In order to facilitate the purification of the protein in C1), a tag as shown in Table 1 can be linked to the amino terminal or carboxyl terminal of the protein shown in SEQ ID NO: 2.

[0012] Table 1. Sequence of the tag

[0013]

[0014]

[0015] The protein in C3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0016] The protein in C3) above can be artificially synthesized, or the encoding gene thereof can be synthesized first, and then expressed biologically.

[0017] The encoding gene of the protein in C3) above can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 1, and / or performing one or several base pair missense mutations, and / or connecting the encoding sequence of the tag shown in Table 1 to the 5' end and / or 3' end thereof.

[0018] The present application also protects a nucleic acid molecule encoding any of the above-mentioned proteins IME199DNAP.

[0019] The nucleic acid molecule encoding any of the above-mentioned proteins IME199DNAP can be a DNA molecule as shown in e1) or e2) or e3) below:

[0020] e1) the nucleotide sequence is a DNA molecule shown in SEQ ID NO: 1;

[0021] e2) has 75% or more identity with the nucleotide sequence defined in e1), and encodes the protein IME199DNAP;

[0022] e3) hybridizes to the nucleotide sequence defined in e1) or e2) under stringent conditions, and encodes the protein IME199DNAP.

[0023] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

[0024] SEQ ID NO: 1 consists of 2340 nucleotides, and the nucleotides of SEQ ID NO: 1 encode the amino acid sequence shown in SEQ ID NO: 2.

[0025] The nucleotide sequence encoding any of the above-mentioned protein IME199 DNAP of the present application can be easily mutated by a person of ordinary skill in the art using known methods, such as methods of directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity with the nucleotide sequence of any of the above-mentioned protein IME199 DNAP isolated from the present application, as long as they encode any of the above-mentioned protein IME199 DNAP, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application.

[0026] The term "identity" as used herein refers to sequence similarity with the natural nucleic acid sequence. The "identity" includes nucleotide sequences having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein IME199 DNAP of the present application which encodes the amino acid sequence shown in SEQ ID NO: 2. The identity can be evaluated by naked eyes or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.

[0027] The expression cassette, the recombinant vector or the recombinant microorganism containing any of the above-mentioned nucleic acid molecules also belong to the protection scope of the present application.

[0028] The recombinant vector containing any of the above-mentioned nucleic acid molecules can be a recombinant plasmid obtained by inserting the DNA molecule shown in SEQ ID NO: 1 or the DNA molecule shown in SEQ ID NO: 1 from the 1st to the 2337th nucleotide from the 5' end (i.e. without the terminator) into the multiple cloning site of the expression vector.

[0029] The recombinant vector can be specifically the recombinant plasmid pET28a-IME199 DNAP. The recombinant plasmid pET28a-IME199 DNAP can be a recombinant plasmid obtained by replacing the small DNA fragment between the restriction enzymes BamH I and Xho I of the pET28a plasmid with the DNA molecule shown in SEQ ID NO: 1 from the 1st to the 2337th nucleotide from the 5' end (i.e. without the terminator).

[0030] The recombinant microorganism containing any of the above-mentioned nucleic acid molecules can be a recombinant bacterium obtained by introducing the recombinant vector containing any of the above-mentioned nucleic acid molecules into a starting microorganism.

[0031] The starting microorganism can be Escherichia coli. The Escherichia coli can be Escherichia coli BL21 (DE3).

[0032] The recombinant microorganism containing any of the above nucleic acid molecules can be a recombinant Escherichia coli. The recombinant Escherichia coli can be a recombinant bacterium obtained by transforming the recombinant plasmid pET28a-IME199DNAP into Escherichia coli BL21 (DE3).

[0033] The application also protects the use of any of the above-mentioned protein IME199DNAP, any of the above-mentioned nucleic acid molecules, or an expression cassette, a recombinant vector or a recombinant microorganism containing any of the above-mentioned nucleic acid molecules in the preparation of a DNA polymerase or as a DNA polymerase.

[0034] Both temperature and pH value have important effects on the DNA polymerase activity of any of the above-mentioned protein IME199DNAP. The protein IME199DNAP has good DNA polymerase activity at a temperature in the range of 20-30℃ (such as 20℃ or 30℃). The protein IME199DNAP has good DNA polymerase activity at a pH value in the range of pH 3.5-9.5 (such as pH 3.5, pH 4.5, pH 5.5, pH 6.5, pH 7.5, pH 8.5 or pH 9.5).

[0035] In the above-mentioned application, the DNA polymerase has 3'→5' exonuclease activity and / or strand displacement activity.

[0036] The application also protects the use of any of the above-mentioned protein IME199DNAP, any of the above-mentioned nucleic acid molecules, or an expression cassette, a recombinant vector or a recombinant microorganism containing any of the above-mentioned nucleic acid molecules in the preparation of a primer enzyme or as a primer enzyme.

[0037] The use of any of the above-mentioned protein IME199DNAP, any of the above-mentioned nucleic acid molecules, or an expression cassette, a recombinant vector or a recombinant microorganism containing any of the above-mentioned nucleic acid molecules in the amplification of DNA also falls within the protection scope of the application.

[0038] In the above-mentioned application, the DNA can be single-stranded DNA or double-stranded DNA.

[0039] In the above-mentioned application, the amplification of DNA can use primers or can not use primers.

[0040] The protein IME199DNAP provided by the present application has DNA polymerase activity, 3'→5' exonuclease activity and strand displacement activity, and can exponentially amplify single-stranded DNA and double-stranded DNA by using random primers or fixed primers at room temperature (20-30℃) and pH value of 3.5-9.5; meanwhile, the protein IME199DNAP also has primase activity, and can amplify single-stranded DNA and double-stranded DNA without primers. Therefore, the protein IME199DNAP can efficiently amplify single-stranded DNA and double-stranded DNA by using primers or without primers at room temperature (20-30℃), and the enzyme has strand displacement activity, and can be used for isothermal amplification. The present application has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 SDS-PAGE result of step two in Example 1.

[0042] Figure 2 DNA polymerase activity detection of the protein IME199DNAP.

[0043] Figure 3 3'→5' exonuclease activity detection of the protein IME199DNAP.

[0044] Figure 4 Effect of temperature on the DNA polymerase activity of the protein IME199DNAP.

[0045] Figure 5 Effect of pH value on the DNA polymerase activity of the protein IME199DNAP.

[0046] Figure 6 Detection result of the protein IME199DNAP amplifying single-stranded DNA by using primers.

[0047] Figure 7 Detection result of the protein IME199DNAP amplifying double-stranded DNA by using primers.

[0048] Figure 8 Detection result of the protein IME199DNAP amplifying single-stranded DNA and double-stranded DNA without primers. DETAILED DESCRIPTION

[0049] The present application will be further described in detail below in combination with specific embodiments, and the examples given are only for illustrating the present application, but not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0050] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0051] Example 1, Expression and purification of protein IME199DNAP

[0052] I. Construction of recombinant plasmid pET28a-IME199DNAP

[0053] 1. Using the genome of Enterococcus faecium phage IME199 (Sequence Accession No. NC_049931.1) as the template, the PCR amplification was carried out with the primer 199DNAP-F: 5'-cagcaaatgggtcgc ggatcc ATGAAATTCGTACAATGGTTAGACAAT-3' (underlined is the recognition site of restriction enzyme BamH I) and the primer 199DNAP-R: 5'-gtggtggtggtggtg ctcgag AGAAATATTATTTCTAATATCTCCTGAATCG-3' (underlined is the recognition site of restriction enzyme Xho I).

[0054] Reaction conditions: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 60 °C annealing for 15 s, 72 °C extension for 60 s, 35 cycles; 72 °C extension for 5 min.

[0055] 2. The pET28a plasmid (recorded in the following literature: Ping Li, Hong Lin, Zhiqiang Mi, Shaozhen Xing, Yigang Tong, Jingxue Wang. Screening of Polyvalent Phage-Resistant Escherichia coli Strains Based on Phage Receptor Analysis. Front. Microbiol., 18 April 2019. The name of the plasmid in the literature is pET-28a) was double-digested with restriction enzymes BamH I and Xho I, and a vector backbone of about 5.3 kb was recovered.

[0056] 3. The PCR amplification product recovered in step 1 and the vector backbone recovered in step 2 were mixed, and then homologous recombination was carried out using a homologous recombination enzyme (Nanjing Novozyme), to obtain the recombinant plasmid pET28a-IME199DNAP.

[0057] Recombination condition: 37℃ for 30min, and cooling on ice.

[0058] The recombinant plasmid pET28a-IME199DNAP was sequenced. According to the sequencing result, the structure of the recombinant plasmid pET28a-IME199DNAP was described as follows: the DNA fragment between the restriction endonuclease BamH I and Xho I of the pET28a plasmid was replaced by the DNA molecule shown in SEQ ID NO: 1 from the 1st to the 2337th position at the 5' end, and the obtained recombinant plasmid was pET28a-IME199DNAP.

[0059] The recombinant plasmid pET28a-IME199DNAP expressed the protein IME199DNAP, and the amino acid sequence of the protein IME199DNAP was shown in SEQ ID NO: 2.

[0060] II. Expression and purification of the protein IME199DNAP

[0061] 1. The recombinant plasmid pET28a-IME199DNAP was transformed into Escherichia coli BL21 (DE3) (Beijing Zixingjin Bioengineering) to obtain a recombinant Escherichia coli.

[0062] 2. A single colony of the recombinant Escherichia coli was inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37℃ overnight to obtain culture liquid 1. Then, 2 mL of the culture liquid 1 was inoculated into 200 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37℃ to obtain culture liquid 2 with OD 600nm of about 0.8.

[0063] 3. The culture liquid 2 was cooled on ice for 30 min, and then IPTG was added to make the concentration in the system 0.1 mM. The culture was induced at 16℃ for 16-20 h to obtain culture liquid 3.

[0064] 4. The culture liquid 3 was centrifuged at 4℃ and 10000 x g for 15 min, and the bacterial cells were collected.

[0065] 5. The bacterial cells collected in step 4 were resuspended with LB solution (20 mM Tris, 500 mM NaCl, 30 mM imidazole and 10% glycerol in water) to obtain a bacterial suspension.

[0066] 6. The bacterial suspension obtained in step 5 was crushed with a low-temperature high-pressure cell crusher at 4℃, and then centrifuged at 4℃ and 7000 x g for 1 h. The supernatant was collected.

[0067] 7. The supernatant collected in step 6 is slowly passed through the nickel column (to adsorb the protein IME199DNAP with His tag); then the nickel column is eluted with WB solution (20 mM Tris, 500 mM NaCl, 50 mM imidazole and 10% glycerol in water) to remove the unbound protein IME199DNAP; then the nickel column is eluted with EB solution (20 mM Tris, 500 mM NaCl, 500 mM imidazole and 10% glycerol in water) (to wash away the protein IME199DNAP with His tag), and the eluate is collected; finally, the nickel column is washed with RB solution (20 mM Tris, 500 mM NaCl, 700 mM imidazole and 10% glycerol in water).

[0068] 8. The eluate collected in step 7 is concentrated to 2 mL with a 30 KD ultrafiltration tube, and then is injected into a molecular sieve chromatography column for separation, and the eluate collected at the peak is concentrated to 1 mL with a 30 KD ultrafiltration tube to obtain a protein concentrate.

[0069] The protein concentrate is added with 50% (v / v) glycerol solution, and is stored at -80°C.

[0070] 9. The total protein of the recombinant E. coli is extracted. The total protein of the culture solution 2 is extracted. Then the total protein of the recombinant E. coli, the total protein of the culture solution 2, the supernatant collected in step 6, the eluate collected in step 7 and the protein concentrate are subjected to SDS-PAGE.

[0071] The detection results are shown in Figure 1 (Marker is protein Marker, A is the total protein of the recombinant E. coli, B is the total protein of the culture solution 2, C is the supernatant, D is the eluate, and E is the protein concentrate). The results show that the supernatant collected in step 6, the eluate collected in step 7 and the protein concentrate all contain the protein IME199DNAP, which is completely consistent with the expected size.

[0072] Example 2, Detection of DNA polymerase activity and 3'→5' exonuclease activity of the protein IME199DNAP

[0073] I. DNA polymerase activity of the protein IME199DNAP

[0074] 1. Preparation of substrate

[0075] The sequence A: 5' FAM-tcctaacgagattagttttgctgt-3' and the sequence B: 5'-cccatacaaataaaccaaaaaacaatacagcaaaactaatctcgttagga-3' are annealed to obtain the substrate.

[0076] Annealing program: 95°C for 3 min, then natural cooling to room temperature.

[0077] 2. Preparation of reaction system. The reaction system was 20 μL, which was composed of substrate, Tris-HCl buffer solution with pH 7.5, MgCl2, (NH4)2SO4, DTT, dNTPs and protein IME199 DNAP (added in the form of protein concentrate obtained in Example 1). In the reaction system, the concentration of substrate was 0.4 μM, the concentration of MgCl2 was 10 mM, the concentration of (NH4)2SO4 was 10 mM, the concentration of DTT was 4 mM, the concentration of dNTPs was 50 μM, and the concentration of protein IME199 DNAP was 500, 250, 100, 50, 25 or 10 nM.

[0078] 3. Taking the reaction system prepared in step 2, 10 μL of the reaction system was subjected to 20% polyacrylamide gel electrophoresis.

[0079] The detection results are shown in Table 1. Figure 2 (M is a fluorescently labeled sequence of different lengths). The results show that protein IME199 DNAP can synthesize a complete DNA strand from an incomplete DNA strand, i.e. protein IME199 DNAP has DNA polymerase activity, and 100 nM of protein IME199 DNAP exhibits good DNA polymerase activity.

[0080] II. 3'→5' exonuclease activity of protein IME199 DNAP

[0081] 1. Preparation of substrate

[0082] The same as 1 in step I.

[0083] 2. Preparation of reaction system. The reaction system was 20 μL, which was composed of substrate, Tris-HCl buffer solution with pH 7.5, MgCl2, (NH4)2SO4, DTT and protein IME199 DNAP (added in the form of protein concentrate obtained in Example 1). In the reaction system, the concentration of substrate was 0.4 μM, the concentration of MgCl2 was 10 mM, the concentration of (NH4)2SO4 was 10 mM, the concentration of DTT was 4 mM, and the concentration of protein IME199 DNAP was 0, 25, 50 or 100 nM.

[0084] 3. Taking the reaction system prepared in step 2, 10 μL of the reaction system was subjected to 20% polyacrylamide gel electrophoresis.

[0085] The detection results are shown in Table 2.Figure 3 (M is a fluorescently labeled sequence of different lengths). The results show that protein IME199 DNAP can degrade the substrate in the reaction system without dNTPs. Thus, protein IME199 DNAP has 3'→5' exonuclease activity.

[0086] III. Effect of temperature on the DNA polymerase activity of protein IME199 DNAP

[0087] 1. Preparation of substrate

[0088] 1 in step I.

[0089] 2. Preparation of reaction system. The reaction system is 20 μL, which is composed of substrate, Tris-HCl buffer with pH 7.5, MgCl2, (NH4)2SO4, DTT, dNTPs and protein IME199 DNAP (added in the form of protein concentrate obtained in Example 1). In the reaction system, the concentration of substrate is 0.4 μM, the concentration of MgCl2 is 10 mM, the concentration of (NH4)2SO4 is 10 mM, the concentration of DTT is 4 mM, the concentration of dNTPs is 50 μM, and the concentration of protein IME199 DNAP is 100 nM.

[0090] 3. Take the reaction system prepared in step 2, and react at 20°C, 30°C, 37°C, 45°C, 55°C, 65°C or 75°C for 15 min to obtain a reaction solution. Then take 10 μL of the reaction solution for 20% polyacrylamide gel electrophoresis.

[0091] The detection results are shown in Figure 4 (M is a fluorescently labeled sequence of different lengths). The results show that temperature has an important effect on the DNA polymerase activity of protein IME199 DNAP, and protein IME199 DNAP has good DNA polymerase activity in the temperature range of 20-30°C.

[0092] IV. Effect of pH value on the DNA polymerase activity of protein IME199 DNAP

[0093] 1. Preparation of substrate

[0094] 1 in step I.

[0095] 2. Preparation of reaction system. The reaction system was 20 μL, consisting of substrate, Tris-HCl buffer, MgCl2, (NH4)2SO4, DTT, dNTPs and protein IME199 DNAP (added in the form of protein concentrate obtained in Example 1). In the reaction system, the concentration of substrate was 0.4 μM, the concentration of MgCl2 was 10 mM, the concentration of (NH4)2SO4 was 10 mM, the concentration of DTT was 4 mM, the concentration of dNTPs was 50 μM, the concentration of protein IME199 DNAP was 100 nM, and the Tris-HCl buffer was pH 1.5, 50 mM Tris-HCl buffer, pH 2.5, 50 mM Tris-HCl buffer, pH 3.5, 50 mM Tris-HCl buffer, pH 4.5, 50 mM Tris-HCl buffer, pH 5.5, 50 mM Tris-HCl buffer, pH 6.5, 50 mM Tris-HCl buffer, pH 7.5, 50 mM Tris-HCl buffer, pH 8.5, 50 mM Tris-HCl buffer, pH 9.5, 50 mM Tris-HCl buffer, pH 10.5, 50 mM Tris-HCl buffer or pH 11.5, 50 mM Tris-HCl buffer.

[0096] 3. Taking the reaction system prepared in step 2, the reaction was carried out at 30°C for 15 min to obtain a reaction solution. Then 10 μL of the reaction solution was subjected to 20% polyacrylamide gel electrophoresis.

[0097] The detection results are shown in Table 1. Figure 5 (M is a fluorescently labeled sequence of different lengths). The results show that the pH value has an important influence on the DNA polymerase activity of protein IME199 DNAP, and protein IME199 DNAP has good DNA polymerase activity in the pH range of pH 3.5-9.5.

[0098] Example 3. Strand displacement activity of protein IME199 DNAP

[0099] M13mp18 single-stranded DNA is a product of the U.S. NEB company, which is single-stranded and circular DNA with a size of 7.25 kb.

[0100] Plasmid pUC19 is a product of the U.S. NEB company, which is double-stranded and circular DNA with a size of 2686 bp.

[0101] I. Amplification of single-stranded DNA by protein IME199 DNAP using primers

[0102] 1. Preparation of reaction system

[0103] The reaction system was 25 μL, and was composed of M13mp18 single-stranded DNA (as a substrate), Tris-HCl buffer solution (pH 7.5, 50 mM), primer (nucleotide sequence was 5'-TCGTAATCATGGTCATAGCTGTTTCCTG-3'), MgCl2, (NH4)2SO4, DTT, dNTPs and protein IME199 DNAP (added in the form of protein concentrate obtained in Example 1). In the reaction system, the concentration of M13mp18 single-stranded DNA was 10 ng / μL, the concentration of primer was 10 μM, the concentration of MgCl2 was 10 mM, the concentration of (NH4)2SO4 was 10 mM, the concentration of DTT was 4 mM, the concentration of dNTPs was 50 μM, and the concentration of protein IME199 DNAP was 100 nM.

[0104] 2. The reaction system prepared in step 1 was reacted at 20°C for 20 min, 40 min, 60 min or 120 min to obtain a reaction solution. Then 10 μL of the reaction solution was subjected to 1% agarose gel electrophoresis together with M13mp18 single-stranded DNA.

[0105] The detection results are shown in Fig. 2. Figure 6 (M is 15K Maker, 1 is M13mp18 single-stranded DNA, 2 is reaction for 20 min, 3 is reaction for 40 min, 4 is reaction for 60 min, and 5 is reaction for 120 min). The results showed that protein IME199 DNAP could replicate the DNA sequence of M13mp18 single-stranded DNA at 20°C, and the replication product (more than 15Kb) was larger than the full length of M13mp18 single-stranded DNA, indicating that protein IME199 DNAP had strand displacement activity.

[0106] II. Amplification of double-stranded DNA by protein IME199 DNAP using a primer

[0107] 1. Preparation of reaction system

[0108] The reaction system was 25 μL, and was composed of plasmid pUC19 (as a substrate), Tris-HCl buffer solution with pH 7.5, random primer 6bp (product of NEB, USA), MgCl2, (NH4)2SO4, DTT, dNTPs and protein IME199 DNAP (added in the form of protein concentrate obtained in Example 1). In the reaction system, the concentration of plasmid pUC19 was 4 ng / μL, the concentration of primer was 25 μM, the concentration of MgCl2 was 10 mM, the concentration of (NH4)2SO4 was 10 mM, the concentration of DTT was 4 mM, the concentration of dNTPs was 50 μM, and the concentration of protein IME199 DNAP was 100 nM. It should be noted that plasmid pUC19 and random primer were added first, and the reaction system was heated at 95 °C for 3 min and then cooled to room temperature, and then other substances were added.

[0109] 2. The reaction system prepared in step 1 was taken, and the reaction was carried out at 20 °C for 10 min, 20 min, 40 min, 60 min or 120 min to obtain a reaction solution. Then 10 μL of the reaction solution and plasmid pUC19 were subjected to 1% agarose gel electrophoresis.

[0110] The detection results are shown in Figure 2. Figure 7 (M is 15K Maker, 1 is plasmid pUC19, 2 is reaction for 10 min, 3 is reaction for 20 min, 4 is reaction for 40 min, 5 is reaction for 60 min, and 6 is reaction for 120 min). The results show that protein IME199 DNAP can replicate the DNA sequence of plasmid pUC19 under the condition of 20 °C, and the replication product (more than 15Kb) is much larger than pUC19, indicating that protein IME199 DNAP has strand displacement activity.

[0111] The above results show that protein IME199 DNAP has DNA polymerase activity, 3'→5' exonuclease activity and strand displacement activity, and can amplify single-stranded DNA and double-stranded DNA using primers (random primers or fixed primers).

[0112] III. Amplification of single-stranded DNA and double-stranded DNA by protein IME199 DNAP without primers

[0113] 1. Preparation of reaction system

[0114] The reaction system was 20 μL, which was composed of substrate (plasmid pUC19 or M13mp18 single-stranded DNA), Tris-HCl buffer solution with pH 7.5, MgCl2, (NH4)2SO4, DTT, dNTPs and protein IME199 DNA polymerase (added in the form of protein concentrate obtained in Example 1). In the reaction system, the concentration of substrate was 5 ng / μL, the concentration of MgCl2 was 10 mM, the concentration of (NH4)2SO4 was 10 mM, the concentration of DTT was 4 mM, the concentration of dNTPs was 50 μM, and the concentration of protein IME199 DNA polymerase was 100 nM.

[0115] 2. The reaction system prepared in step 1 was reacted at 20°C for 3 h. During the reaction, samples were taken at 0 h, 1 h, 2 h and 3 h, respectively, and the DNA content was detected by Qubit.

[0116] The detection results are shown in Table 1. Figure 8 The results show that protein IME199 DNA polymerase can replicate the DNA sequence of M13mp18 single-stranded DNA and the DNA sequence of plasmid pUC19 without primer at room temperature (20°C).

[0117] It can be seen that protein IME199 DNA polymerase has primase activity and can amplify single-stranded DNA and double-stranded DNA without primer.

[0118] The above has described the present application in detail. For those skilled in the art, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present application and without performing unnecessary experiments. Although the present application gives special examples, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, the present application is intended to include any changes, uses or improvements of the present application, including changes made by using conventional techniques known in the art, which are out of the scope disclosed in the present application. Some basic features can be applied according to the scope of the following attached claims.

Claims

1. Application of protein IME199DNAP in the preparation of primases or as a primase; The protein IME199DNAP is either C1 or C2. C1) The protein with the amino acid sequence shown in SEQ ID NO: 2; C2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in C1).

2. The application of nucleic acid molecules encoding the protein IME199DNAP, or expression cassettes, recombinant vectors, or recombinant microorganisms containing said nucleic acid molecules, in the preparation of primers and enzymes; The protein IME199DNAP is either C1 or C2. C1) The protein with the amino acid sequence shown in SEQ ID NO: 2; C2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in C1).

3. The application according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 1.

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