A mixed enzyme system capable of amplifying circular DNA, and its application and product

Abstract

The application belongs to the technical field of DNA amplification, and has a mixed enzyme system capable of amplifying circular DNA, application and product thereof. The application provides a mixed enzyme system capable of amplifying circular DNA, wherein the mixed enzyme system comprises a polymerase III tau clamp loader and a catalytic core complex. The mixed enzyme system provided by the application is significantly superior to traditional methods in terms of efficiency, cost, safety, applicability and sensitivity, and provides a brand-new solution for efficient preparation of circular DNA, and has a wide application prospect in the field of biotechnology.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of DNA amplification technology, and relates to a mixed enzyme system capable of amplifying circular DNA and application and product thereof. BACKGROUND

[0002] Circular DNA (such as plasmid) is a key tool in the fields of gene cloning, gene editing, vaccine research and development, and biopharmaceuticals, and its efficient preparation technology has always been a research focus in the field of biotechnology.

[0003] Currently, the traditional circular DNA preparation method is highly dependent on the in vivo replication system of host cells (such as Escherichia coli). This process usually requires the introduction of target circular DNA into host cells, proliferation of host cells through fermentation culture, and then amplification of circular DNA by the host's own DNA replication mechanism, followed by cell lysis, nucleic acid extraction and other steps to obtain the target product. However, this preparation method dependent on host cells has many limitations:

[0004] First, the fermentation process of host cells is time-consuming and complicated. The culture of host cells requires strict control of environmental conditions such as temperature, pH value, and nutrient composition. The entire fermentation cycle often takes several hours to several days, which seriously restricts the preparation efficiency of circular DNA. At the same time, large-scale fermentation also faces problems such as high equipment cost and high energy consumption, making it difficult to meet the demand for low-cost and rapid preparation.

[0005] Second, the dependence on host cells limits the application range. Some circular DNA may carry toxic genes, repeated sequences or special promoters, etc., which may cause the host cells to be unable to grow normally or replicate the circular DNA, making it difficult to obtain through traditional methods. In addition, nucleases, methylases and other enzymes in host cells may modify or degrade the circular DNA, affecting its subsequent application effect.

[0006] Third, the traditional method is highly dependent on screening marker genes. In order to screen positive clones successfully introduced with target circular DNA from a large number of host cells, the existing technology usually needs to introduce antibiotic resistance genes and other screening markers into the circular DNA. However, the presence of screening markers may cause biological safety problems (such as horizontal transfer of antibiotic resistance genes), which are strictly limited in fields such as gene therapy and vaccine production that have extremely high safety requirements. At the same time, the introduction of screening markers also increases the complexity of the construction of circular DNA, and may even interfere with its function.

[0007] Fourth, the product purity and uniformity of the traditional method cannot be guaranteed. The host cell's own genomic DNA, RNA, and protein impurities are easily mixed into the product, which needs to be removed through complex purification steps, which not only increases the preparation cost, but also may cause the loss of target circular DNA during the purification process.

[0008] Fifthly, the sensitivity of the traditional method is significantly insufficient, and the response ability to low-abundance templates is weak. The traditional method relies on the replication mechanism of host cells, and the host cells have a higher threshold requirement for the initial template amount. When the initial concentration of the target circular DNA is extremely low (such as a small amount of circular plasmid in a clinical sample or a natural circular DNA molecule), it is difficult to trigger effective replication initiation. The competitive replication resources (such as DNA polymerase, nucleotide substrate, etc.) in the host cell prefer to support the replication of the host genome, resulting in that the amplification efficiency of the low-abundance target circular DNA in the host is severely suppressed, and a large amount of initial template is often required to achieve a detectable level of accumulation, which greatly limits its application in the micro-template scenario (such as the preparation of circular DNA in a clinical biopsy sample). In addition, the low-abundance target circular DNA is easily recognized and degraded by nucleases in the host cell, further exacerbating signal loss, so that the traditional method is prone to false negative results when detecting and amplifying micro-target circular DNA, which seriously restricts its application in high-sensitivity demand fields (such as early disease diagnosis related circular DNA preparation).

[0009] In summary, the existing circular DNA preparation method relying on host cells such as Escherichia coli has significant deficiencies in efficiency, cost, safety, applicability and sensitivity, and cannot meet the demand for rapid, efficient, safe and high-sensitivity preparation of circular DNA in the modern biotechnology field. Therefore, developing a mixed enzyme system that does not rely on host cells, does not require a selection marker, can directly realize efficient amplification of circular DNA in vitro, and has high sensitivity has become a key problem to be solved in the field. SUMMARY

[0010] In view of the above deficiencies, the present application provides a mixed enzyme system capable of amplifying circular DNA, and its application and product.

[0011] The term "dnaX" used herein refers to the gene of the tau subunit of DNA polymerase III (Pol III tau subunit), which can participate in the assembly of Pol III holoenzyme, dimerize the core enzyme, etc., and plays an important role in DNA replication.

[0012] The term "dnaN" used herein refers to the beta subunit of DNA polymerase III (Pol III beta subunit), which forms a sliding clamp that surrounds the DNA and enhances the continuous synthesis ability of the Pol III holoenzyme.

[0013] The term "holC" used herein refers to the chi subunit of DNA polymerase III (Pol III chi subunit), which is a component of the gamma complex (clamp loader).

[0014] As used herein, the term "holD" refers to the ψ subunit of DNA polymerase III (Pol III ψ subunit), which is a component of the γ complex (clamp loader).

[0015] As used herein, the term "holA" refers to the δ subunit of DNA polymerase III (Pol III δ subunit), which is one of the core components of the γ complex (clamp loader) and is responsible for opening the β sliding clamp and loading it onto DNA.

[0016] As used herein, the term "holB" refers to the δ' subunit of DNA polymerase III (Pol III δ' subunit), which is one of the core components of the γ complex (clamp loader) and works in conjunction with the δ subunit.

[0017] As used herein, the term "dnaE" refers to the α subunit of DNA polymerase III (Pol III α subunit), which has DNA polymerase activity and is responsible for chain elongation.

[0018] As used herein, the term "dnaQ" refers to the ε subunit of DNA polymerase III (Pol III ε subunit), which has 3'→5' proofreading exonuclease activity and can proofread DNA replication to improve replication accuracy.

[0019] As used herein, the term "holE" refers to the θ subunit of DNA polymerase III (Pol III θ subunit), which binds to the ε subunit and may play a role in stabilizing the ε subunit or enhancing its proofreading activity.

[0020] As used herein, the term "dnaA protein" refers to a replication initiation factor that recognizes the origin of replication (oriC), promotes DNA unwinding at the initiation stage, and recruits other replication initiation proteins.

[0021] As used herein, the term "dnaC protein" refers to a replication initiation factor that helps the DnaB helicase load onto unwound replication origin DNA as an auxiliary protein.

[0022] As used herein, the term "dnaB protein" refers to the main replication helicase that unwinds DNA double strands at the replication fork to provide a single-stranded template for replication. It is the core engine of the replisome complex.

[0023] As used herein, the term "IhfA" refers to the α subunit of integration host factor (IHF α subunit).

[0024] As used herein, the term "IhfB" refers to the β subunit of integration host factor (IHF β subunit). IhfA and IhfB together form a functional IHF protein.

[0025] As used herein, the term "SSB protein" refers to a Single-Stranded DNA-Binding Protein that binds and stabilizes the single-stranded DNA region at the replication fork, preventing it from reannealing or being degraded by nucleases. Plays a key role in DNA replication, repair and recombination.

[0026] As used herein, the term "gyrB" refers to the B subunit of DNA gyrase (a type II topoisomerase). Has ATPase activity that provides the energy for the process of DNA supercoil introduction (negative supercoiling).

[0027] As used herein, the term "gyrA" refers to the A subunit of DNA gyrase (a type II topoisomerase). Has the activity of cutting and religating the DNA strand, responsible for catalyzing the breakage and reunion of DNA duplexes to achieve the introduction of supercoiling.

[0028] As used herein, the term "parE" refers to the B subunit of topoisomerase IV (a topo IV B subunit). Topoisomerase IV is another key type II topoisomerase, mainly responsible for untying catenated DNA daughter molecules (decatenation) at the end of DNA replication. ParE is its ATPase subunit.

[0029] As used herein, the term "parC" refers to the A subunit of topoisomerase IV (a topo IV A subunit), the catalytic subunit of topoisomerase IV, responsible for cutting and religating DNA to untie DNA catenation.

[0030] As used herein, the term "topB" refers to DNA topoisomerase III (a topo III), a type I topoisomerase, mainly involved in processes such as DNA decatenation, recombination intermediate processing, and preventing replication fork collapse.

[0031] As used herein, the term "RecQ" refers to a RecQ helicase. A DNA helicase that plays an important role in DNA repair, maintaining genome stability.

[0032] As used herein, the term "dnaG" refers to a primase. Synthesizes RNA primers, providing the 3'-OH end required for DNA synthesis by DNA polymerase. Acts at the origin of replication and at the Okazaki fragments initiation.

[0033] As used herein, the term "tus" refers to a Tus protein. A binding protein of the replication termination site (ter site). Binds to a specific ter DNA sequence to form a "replication termination complex", acting as a molecular barrier against the movement of the DnaB helicase, ensuring that replication forks meet and terminate at specific regions.

[0034] The technical scheme of the present application is as follows:

[0035] In one aspect, the present application provides a mixed enzyme system capable of amplifying circular DNA, wherein the mixed enzyme system comprises a polymerase III tau clamp loader and a catalytic core complex, and a preparation method thereof comprises the following steps:

[0036] S1, resuspending strain 1 slurry using resuspension Buffer, diluting the resuspended bacterial liquid with dilution Buffer, taking the supernatant after crushing, precipitating the supernatant with ammonium sulfate, collecting the precipitate by centrifugation, dissolving the precipitate, and obtaining the tau clamp loader primary product; the strain 1 is a strain expressing dnaX, holC, holD, holA and holB; the final concentration of ammonium sulfate in the supernatant is 0.18-0.22 g / mL; the resuspension Buffer contains sucrose, PMSF, DTT and a proteinase inhibitor mixture without EDTA; the dilution Buffer contains sucrose, NaCl, spermidine and EDTA;

[0037] S2, resuspending strain 3 slurry using core resuspension Buffer, taking the supernatant after crushing, precipitating the supernatant with ammonium sulfate, collecting the precipitate by centrifugation, dissolving the precipitate, and obtaining the catalytic core primary product; the strain 3 is a strain expressing dnaE, dnaQ and holE; the final concentration of ammonium sulfate in the supernatant is 0.2-0.25 g / mL; the core resuspension Buffer contains NaCl and spermidine;

[0038] S3, mixing the tau clamp loader primary product after protein electrophoresis and the catalytic core primary product after protein electrophoresis at a molar ratio of 1:2-4 to obtain a mixture;

[0039] S4, eluting the mixture after dialysis to obtain an eluate, precipitating the eluate with ammonium sulfate, collecting the precipitate by centrifugation, and purifying to obtain the product; the final concentration of ammonium sulfate in the eluate is 0.3-0.5 g / mL.

[0040] Specifically, the resuspension Buffer in step S1 is a Buffer containing 8%-12% w / v sucrose, 0.8-1.2 mM PMSF, 0.4-0.6 mM DTT and 1 tablet / 50 mL cOmplete ™ proteinase inhibitor mixture without EDTA.

[0041] Preferably, the resuspension Buffer in step S1 contains 8%-9% w / v, 9%-10% w / v, 10%-11% w / v or 11%-12% w / v sucrose.

[0042] Further preferably, the resuspension Buffer in step S1 contains 10% w / v sucrose.

[0043] Preferably, the resuspension Buffer in step S1 contains 0.8-0.9 mM, 0.9-1.0 mM, 1.0-1.1 mM or 1.1-1.2 mM PMSF.

[0044] Further preferably, the resuspension Buffer in step S1 contains 1 mM PMSF.

[0045] Preferably, the resuspension Buffer in step S1 contains 0.4-0.5 mM or 0.5-0.6 mM DTT.

[0046] Further preferably, the resuspension Buffer in step S1 contains 0.5 mM DTT.

[0047] Preferably, the resuspension Buffer in step S1 contains 1 tablet / 50 mL cOmplete™ Protease Inhibitor Cocktail without EDTA. ™ with catalog number 04693132001.

[0048] Preferably, the buffer includes but is not limited to any one or more of Tris-HCl buffer, PBS buffer, HEPES buffer, TAE buffer, TBE buffer, MES buffer.

[0049] Further preferably, the buffer is Tris-HCl buffer.

[0050] Still further preferably, the buffer is Tris-HCl buffer with pH 7.0-7.1, 7.1-7.2, 7.2-7.3, 7.3-7.4, 7.4-7.5, 7.5-7.6, 7.6-7.7, 7.7-7.8, 7.8-7.9 or 7.9-8.0.

[0051] Still further preferably, the buffer is Tris-HCl buffer with pH 7.5.

[0052] In certain embodiments, the resuspension Buffer is Tris-HCl buffer with pH 7.5 containing 10% w / v sucrose, 1 mM PMSF, 0.5 mM DTT and 1 tablet / 50 mL cOmplete™ Protease Inhibitor Cocktail without EDTA.

[0053] In particular, the dilution Buffer of step S1 is a buffer comprising 8-12% w / v sucrose, 0.05-0.15 M NaCl, 10-20 mM spermidine, 0.8-1.2 mM EDTA.

[0054] Preferably, the dilution Buffer of step S1 comprises 8-9% w / v, 9-10% w / v, 10-11% w / v or 11-12% w / v sucrose.

[0055] Further preferably, the dilution Buffer of step S1 comprises 10% w / v sucrose.

[0056] Preferably, the dilution Buffer of step S1 comprises 0.05-0.06 M, 0.06-0.07 M, 0.07-0.08 M, 0.08-0.09 M, 0.09-0.10 M, 0.10-0.11 M, 0.11-0.12 M, 0.12-0.13 M, 0.13-0.14 M or 0.14-0.15 M NaCl.

[0057] Further preferably, the dilution Buffer of step S1 comprises 0.1 M NaCl.

[0058] Preferably, the dilution Buffer of step S1 comprises 10-11 mM, 11-12 mM, 12-13 mM, 13-14 mM, 14-15 mM, 15-16 mM, 16-17 mM, 17-18 mM, 18-19 mM or 19-20 mM spermidine.

[0059] Further preferably, the dilution Buffer of step S1 comprises 15 mM spermidine.

[0060] Preferably, the dilution Buffer of step S1 comprises 0.8-0.9 mM, 0.9-1.0 mM, 1.0-1.1 mM or 1.1-1.2 mM EDTA.

[0061] Further preferably, the dilution Buffer of step S1 comprises 1 mM EDTA.

[0062] Preferably, the buffer comprises any one or more of, but not limited to, Tris-HCl buffer, PBS buffer, HEPES buffer, TAE buffer, TBE buffer, MES buffer.

[0063] Further preferably, the buffer is Tris-HCl buffer.

[0064] Further preferably, the buffer is a Tris-HCl buffer with a pH of 7.5.

[0065] Further preferably, the buffer is a Tris-HCl buffer with a pH of 7.5.

[0066] In some embodiments, the dilution Buffer is a 50 mM Tris / HCl, pH 7.5 containing 10% w / v sucrose, 0.1 M NaCl, 15 mM spermidine, 1 mM EDTA.

[0067] Specifically, the method for preparing the strain 1 in step S1 comprises: transforming a plasmid expressing dnaX, a plasmid expressing holC and holD, and a plasmid expressing holA and holB into a competent cell.

[0068] Preferably, the dnaX comprises an amino acid sequence as shown in SEQ ID NO. 1; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 1;

[0069] Preferably, the holC comprises an amino acid sequence as shown in SEQ ID NO. 3; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 3;

[0070] Preferably, the holD comprises an amino acid sequence as shown in SEQ ID NO. 4; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 4;

[0071] Preferably, the holA comprises an amino acid sequence as shown in SEQ ID NO. 5; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 5;

[0072] Preferably, the holB comprises an amino acid sequence as shown in SEQ ID NO. 6; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 6.

[0073] Preferably, the competent cells include, but are not limited to, any one or more of DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21 (DE3), BL21 (DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS, Origami (DE3), and Origami (DE3) pLysS.

[0074] Further preferably, the competent cells are BL21 (DE3).

[0075] Preferably, the method for preparing the strain 1 in step S1 includes: transforming pRSFDuet-t, pETDuet-xp, or pCDFDuet-CmR-s's into BL21 (DE3) cells.

[0076] Specifically, the ratio of the strain 1 slurry to the resuspension buffer in step S1 is 1 g of the strain 1 slurry to 0.5-1.5 mL of the resuspension buffer.

[0077] Preferably, the ratio of the strain 1 slurry to the resuspension buffer in step S1 is 1 g of the strain 1 slurry to 1 mL of the resuspension buffer.

[0078] Specifically, the ratio of the strain 1 slurry to the dilution buffer in step S1 is 1 g of the strain 1 slurry to 0.5-1.5 mL of the dilution buffer.

[0079] Preferably, the ratio of the strain 1 slurry to the dilution buffer in step S1 is 1 g of the strain 1 slurry to 1 mL of the dilution buffer.

[0080] Specifically, the final concentration of ammonium sulfate in the supernatant in step S1 is 0.18-0.19 g / mL, 0.19-0.20 g / mL, 0.20-0.21 g / mL, or 0.21-0.22 g / mL.

[0081] Preferably, the final concentration of ammonium sulfate in the supernatant in step S1 is 0.20 g / mL.

[0082] Specifically, the crushing in step S1 is ultrasonic crushing, and the power of the ultrasonic crushing is 50-150 W.

[0083] Preferably, the power of the ultrasonic crushing in step S1 is 50-60 W, 60-70 W, 70-80 W, 80-90 W, 90-100 W, 100-110 W, 110-120 W, 120-130 W, 130-140 W, or 140-150 W.

[0084] In particular, the centrifugation to collect the precipitate in step S1 comprises centrifugation to collect the precipitate, washing, centrifugation again, and collecting the precipitate again.

[0085] Preferably, the centrifugation in step S1 comprises centrifugation at 10000-14000 x g for 5-15 min.

[0086] Preferably, the centrifugation in step S1 comprises centrifugation at 10000-11000 x g, 11000-12000 x g, 12000-13000 x g, or 13000-14000 x g for 5-6 min, 6-7 min, 7-8 min, 8-9 min, 9-10 min, 10-11 min, 11-12 min, 12-13 min, 13-14 min, or 14-15 min.

[0087] Further preferably, the centrifugation in step S1 is centrifugation at 12000 x g for 10 min.

[0088] In particular, the washing agent comprises a buffer containing glycerol, DTT, EDTA, and ammonium sulfate.

[0089] Preferably, the washing agent comprises a buffer containing 15%-25% v / v glycerol, 2.5-7.5 mM DTT, 0.8-1.2 mM EDTA, and 0.15-0.20 g / mL ammonium sulfate.

[0090] Further preferably, the washing agent contains 15%-16% v / v, 16%-17% v / v, 17%-18% v / v, 18%-19% v / v, 19%-20% v / v, 20%-21% v / v, 21%-22% v / v, 22%-23% v / v, 23%-24% v / v, or 24%-25% v / v glycerol.

[0091] Still further preferably, the washing agent contains 20% v / v glycerol.

[0092] Further preferably, the washing agent contains 2.5-3.0 mM, 3.0-3.5 mM, 3.5-4.0 mM, 4.0-4.5 mM, 4.5-5.0 mM, 5.0-5.5 mM, 5.5-6.0 mM, 6.0-6.5 mM, 6.5-7.0 mM, or 7.0-7.5 mM DTT.

[0093] Still further preferably, the washing agent contains 5 mM DTT.

[0094] Further preferably, the formulation used for washing contains 0.8-0.9 mM, 0.9-1.0 mM, 1.0-1.1 mM or 1.1-1.2 mM of EDTA.

[0095] Still further preferably, the formulation used for washing contains 1 mM of EDTA.

[0096] Further preferably, the formulation used for washing contains 0.15-0.16 g / mL, 0.16-0.17 g / mL, 0.17-0.18 g / mL, 0.18-0.19 g / mL or 0.19-0.20 g / mL of ammonium sulfate.

[0097] Still further preferably, the formulation used for washing contains 0.15 g / mL of ammonium sulfate.

[0098] Further preferably, the buffer includes but is not limited to any one or more of Tris-HCl buffer, PBS buffer, HEPES buffer, TAE buffer, TBE buffer, MES buffer.

[0099] Still further preferably, the buffer is Tris-HCl buffer.

[0100] Still further preferably, the buffer is Tris-HCl buffer with pH 7.0-7.1, 7.1-7.2, 7.2-7.3, 7.3-7.4, 7.4-7.5, 7.5-7.6, 7.6-7.7, 7.7-7.8, 7.8-7.9 or 7.9-8.0.

[0101] Still further preferably, the buffer is Tris-HCl buffer with pH 7.5.

[0102] In certain embodiments, the formulation used for washing is Wash Buffer-1, which is 50 mM Tris-HCl with pH 7.5 containing 20% v / v glycerol, 5 mM DTT, 1 mM EDTA, 0.15 g / mL ammonium sulfate.

[0103] Specifically, the formulation used for pellet dissolution contains glycerol, DTT, EDTA and NaCl.

[0104] Preferably, the formulation used for pellet dissolution contains 15%-25% v / v glycerol, 2.5-7.5 mM DTT, 0.8-1.2 mM EDTA and 15-25 mM NaCl.

[0105] Further preferably, the formulation used for dissolving the precipitate in step S1 contains 15-16% v / v, 16-17% v / v, 17-18% v / v, 18-19% v / v, 19-20% v / v, 20-21% v / v, 21-22% v / v, 22-23% v / v, 23-24% v / v or 24-25% v / v of glycerol.

[0106] Still further preferably, the formulation used for dissolving the precipitate in step S1 contains 20% v / v of glycerol.

[0107] Further preferably, the formulation used for dissolving the precipitate in step S1 contains 2.5-3.0 mM, 3.0-3.5 mM, 3.5-4.0 mM, 4.0-4.5 mM, 4.5-5.0 mM, 5.0-5.5 mM, 5.5-6.0 mM, 6.0-6.5 mM, 6.5-7.0 mM or 7.0-7.5 mM of DTT.

[0108] Further preferably, the formulation used for dissolving the precipitate in step S1 contains 5 mM of DTT.

[0109] Further preferably, the formulation used for dissolving the precipitate in step S1 contains 0.8-0.9 mM, 0.9-1.0 mM, 1.0-1.1 mM or 1.1-1.2 mM of EDTA.

[0110] Still further preferably, the formulation used for dissolving the precipitate in step S1 contains 1 mM of EDTA.

[0111] Further preferably, the formulation used for dissolving the precipitate in step S1 contains 15-16 mM, 16-17 mM, 17-18 mM, 18-19 mM, 19-20 mM, 20-21 mM, 21-22 mM, 22-23 mM, 23-24 mM or 24-25 mM of NaCl.

[0112] Still further preferably, the formulation used for dissolving the precipitate in step S1 contains 20 mM of NaCl.

[0113] Preferably, the buffer includes but is not limited to any one or more of Tris-HCl buffer, PBS buffer, HEPES buffer, TAE buffer, TBE buffer, MES buffer.

[0114] Further preferably, the buffer is Tris-HCl buffer.

[0115] Further preferably, the buffer is a Tris-HCl buffer at pH 7.5.

[0116] Further preferably, the buffer is a Tris-HCl buffer at pH 7.5.

[0117] In certain embodiments, the formulation used for the resolubilization of the pellet in step S1 is Buffer A-2, which is 50 mM Tris / HCl at pH 7.5 containing 20% v / v glycerol, 5 mM DTT, 1 mM EDTA, 20 Mm NaCl.

[0118] In particular, the core resuspension Buffer in step S2 contains a buffer with 0.05-0.15 M NaCl and 8-12 mM spermidine.

[0119] Preferably, the core resuspension Buffer in step S2 contains 0.05-0.06 M, 0.06-0.07 M, 0.07-0.08 M, 0.08-0.09 M, 0.09-0.10 M, 0.10-0.11 M, 0.11-0.12 M, 0.12-0.13 M, 0.13-0.14 M or 0.14-0.15 M NaCl.

[0120] Further preferably, the core resuspension Buffer in step S2 contains 0.1 M NaCl.

[0121] Preferably, the core resuspension Buffer in step S2 contains 8-9 mM, 9-10 mM, 10-11 mM or 11-12 mM spermidine.

[0122] Further preferably, the core resuspension Buffer in step S2 contains 10 mM spermidine.

[0123] Preferably, the buffer includes, but is not limited to, any one or more of: Tris-HCl buffer, PBS buffer, HEPES buffer, TAE buffer, TBE buffer, MES buffer.

[0124] Further preferably, the buffer is a Tris-HCl buffer.

[0125] Further preferably, the buffer is a Tris-HCl buffer with a pH of 7.5.

[0126] Further preferably, the buffer is a Tris-HCl buffer with a pH of 7.5.

[0127] In some embodiments, the core resuspension buffer in step S2 is a 50 mM Tris-HCl buffer with a pH of 7.5 containing 10 mM spermidine and 0.1 M NaCl.

[0128] Specifically, the method for preparing the strain 3 in step S2 comprises: transforming a plasmid expressing dnaE and a plasmid expressing dnaQ and holE into a competent cell.

[0129] Preferably, the dnaE comprises an amino acid sequence as shown in SEQ ID NO. 7; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 7.

[0130] Preferably, the dnaQ comprises an amino acid sequence as shown in SEQ ID NO. 8; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 8.

[0131] Preferably, the holE comprises an amino acid sequence as shown in SEQ ID NO. 9; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 9.

[0132] Preferably, the competent cell comprises any one or more of, but not limited to: DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21 (DE3), BL21 (DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS, Origami (DE3), Origami (DE3) pLysS.

[0133] Further preferably, the competent cell is: BL21 (DE3).

[0134] Preferably, the method for preparing the strain 1 in step S1 comprises: transforming pRSFDuet-a or pET21a-eo into BL21 (DE3) cells.

[0135] Specifically, the ratio of the strain 3 slurry to the core resuspension buffer in step S2 is: 1 g of strain 1 slurry is added to 4 mL of core resuspension buffer for resuspension.

[0136] Specifically, the final concentration of ammonium sulfate in the supernatant in step S2 is 0.20-0.21 g / mL, 0.21-0.22 g / mL, 0.22-0.23 g / mL, 0.23-0.24 g / mL, or 0.24-0.25 g / mL.

[0137] Preferably, the final concentration of ammonium sulfate in the supernatant in step S2 is 0.221-0.222 g / mL, 0.222-0.223 g / mL, 0.223-0.224 g / mL, 0.224-0.225 g / mL, 0.225-0.226 g / mL, 0.226-0.227 g / mL, 0.227-0.228 g / mL, 0.228-0.229 g / mL, or 0.229-0.230 g / mL.

[0138] Further preferably, the final concentration of ammonium sulfate in the supernatant in step S2 is 0.226 g / mL.

[0139] Specifically, the breaking in step S2 is ultrasonic breaking, and the power of the ultrasonic breaking is 50-150 W.

[0140] Preferably, the power of the ultrasonic breaking in step S2 is 50-60 W, 60-70 W, 70-80 W, 80-90 W, 90-100 W, 100-110 W, 110-120 W, 120-130 W, 130-140 W, or 140-150 W.

[0141] Specifically, the centrifugation in step S2 includes: collecting the precipitate includes: centrifuging to collect the precipitate, washing, centrifuging again, and collecting the precipitate again.

[0142] Preferably, the centrifugation includes 10000-14000 x g centrifugation for 5-15 min.

[0143] Preferably, the centrifugation in step S2 includes 10000-11000 x g, 11000-12000 x g, 12000-13000 x g, or 13000-14000 x g centrifugation for 5-6 min, 6-7 min, 7-8 min, 8-9 min, 9-10 min, 10-11 min, 11-12 min, 12-13 min, 13-14 min, or 14-15 min.

[0144] Further preferably, the centrifugation in step S2 is 12000 x g centrifugation for 10 min.

[0145] In particular, the formulation used for washing is a buffer containing glycerol, DTT, EDTA and ammonium sulfate.

[0146] Preferably, the formulation used for washing is a buffer containing 15-25% v / v glycerol, 2.5-7.5 mM DTT, 0.8-1.2 mM EDTA and 0.15-0.20 g / mL ammonium sulfate.

[0147] Further preferably, the formulation used for washing contains 15-16% v / v, 16-17% v / v, 17-18% v / v, 18-19% v / v, 19-20% v / v, 20-21% v / v, 21-22% v / v, 22-23% v / v, 23-24% v / v or 24-25% v / v glycerol.

[0148] Still further preferably, the formulation used for washing contains 20% v / v glycerol.

[0149] Further preferably, the formulation used for washing contains 2.5-3.0 mM, 3.0-3.5 mM, 3.5-4.0 mM, 4.0-4.5 mM, 4.5-5.0 mM, 5.0-5.5 mM, 5.5-6.0 mM, 6.0-6.5 mM, 6.5-7.0 mM or 7.0-7.5 mM DTT.

[0150] Still further preferably, the formulation used for washing contains 5 mM DTT.

[0151] Further preferably, the formulation used for washing contains 0.8-0.9 mM, 0.9-1.0 mM, 1.0-1.1 mM or 1.1-1.2 mM EDTA.

[0152] Still further preferably, the formulation used for washing contains 1 mM EDTA.

[0153] Further preferably, the formulation used for washing contains 0.15-0.16 g / mL, 0.16-0.17 g / mL, 0.17-0.18 g / mL, 0.18-0.19 g / mL or 0.19-0.20 g / mL ammonium sulfate.

[0154] Still further preferably, the formulation used for washing contains 0.20 g / mL ammonium sulfate.

[0155] Further preferably, the buffer includes, but is not limited to, any one or more of Tris-HCl buffer, PBS buffer, HEPES buffer, TAE buffer, TBE buffer, MES buffer.

[0156] Still further preferably, the buffer is Tris-HCl buffer.

[0157] Yet further preferably, the buffer is Tris-HCl buffer with pH 7.0-7.1, 7.1-7.2, 7.2-7.3, 7.3-7.4, 7.4-7.5, 7.5-7.6, 7.6-7.7, 7.7-7.8, 7.8-7.9 or 7.9-8.0.

[0158] Still further preferably, the buffer is Tris-HCl buffer with pH 7.5.

[0159] In some embodiments, the formulation used for the washing is Wash Buffer-2, which is 50mM Tris-HCl with pH 7.5 containing 20% v / v glycerol, 5mM DTT, 1mM EDTA, 0.2g / mL ammonium sulfate.

[0160] Specifically, the formulation used for the dissolving of the pellet in step S2 is a buffer containing glycerol, DTT, EDTA and NaCl.

[0161] Preferably, the formulation used for the dissolving of the pellet in step S2 is a buffer containing 15%-25% v / v glycerol, 2.5-7.5mM DTT, 0.8-1.2mM EDTA and 15-25mM NaCl.

[0162] Further preferably, the formulation used for the dissolving of the pellet in step S2 contains 15%-16% v / v, 16%-17% v / v, 17%-18% v / v, 18%-19% v / v, 19%-20% v / v, 20%-21% v / v, 21%-22% v / v, 22%-23% v / v, 23%-24% v / v or 24%-25% v / v glycerol.

[0163] Still further preferably, the formulation used for the dissolving of the pellet in step S2 contains 20% v / v glycerol.

[0164] Further preferably, the preparation used for dissolving the precipitate in step S2 contains 2.5-3.0 mM, 3.0-3.5 mM, 3.5-4.0 mM, 4.0-4.5 mM, 4.5-5.0 mM, 5.0-5.5 mM, 5.5-6.0 mM, 6.0-6.5 mM, 6.5-7.0 mM or 7.0-7.5 mM of DTT.

[0165] Further preferably, the preparation used for dissolving the precipitate in step S2 contains 5 mM of DTT.

[0166] Further preferably, the preparation used for dissolving the precipitate in step S2 contains 0.8-0.9 mM, 0.9-1.0 mM, 1.0-1.1 mM or 1.1-1.2 mM of EDTA.

[0167] Further preferably, the preparation used for dissolving the precipitate in step S2 contains 1 mM of EDTA.

[0168] Further preferably, the preparation used for dissolving the precipitate in step S2 contains 15-16 mM, 16-17 mM, 17-18 mM, 18-19 mM, 19-20 mM, 20-21 mM, 21-22 mM, 22-23 mM, 23-24 mM or 24-25 mM of NaCl.

[0169] Further preferably, the preparation used for dissolving the precipitate in step S2 contains 20 mM of NaCl.

[0170] Preferably, the buffer includes but is not limited to any one or more of Tris-HCl buffer, PBS buffer, HEPES buffer, TAE buffer, TBE buffer, MES buffer.

[0171] Further preferably, the buffer is Tris-HCl buffer.

[0172] Further preferably, the buffer is Tris-HCl buffer.

[0173] Further preferably, the buffer is Tris-HCl buffer.

[0174] In certain embodiments, the formulation used for the solubilization of the precipitate in step S2 is Buffer A-2, which is 50 mM Tris / HCl pH 7.5 containing 20% v / v glycerol, 5 mM DTT, 1 mM EDTA, 20 mM NaCl.

[0175] In particular, the molar ratio of the tau-clamp loader primary purified product after the protein electrophoresis and the catalytic core primary purified product after the protein electrophoresis in step S3 comprises: 1 : 2-3 or 1 : 3-4.

[0176] Preferably, the molar ratio of the tau-clamp loader primary purified product after the protein electrophoresis and the catalytic core primary purified product after the protein electrophoresis in step S3 is 1 : 3.

[0177] In particular, the formulation used for the dialysis in step S4 is a buffer containing glycerol, DTT, EDTA and NaCl.

[0178] Preferably, the formulation used for the dialysis in step S4 is a buffer containing 15-25% v / v glycerol, 2.5-7.5 mM DTT, 0.8-1.2 mM EDTA and 15-25 mM NaCl.

[0179] Further preferably, the formulation used for the dialysis in step S4 contains 15-16% v / v, 16-17% v / v, 17-18% v / v, 18-19% v / v, 19-20% v / v, 20-21% v / v, 21-22% v / v, 22-23% v / v, 23-24% v / v or 24-25% v / v of glycerol.

[0180] Still further preferably, the formulation used for the dialysis in step S4 contains 20% v / v of glycerol.

[0181] Further preferably, the formulation used for the dialysis in step S4 contains 2.5-3.0 mM, 3.0-3.5 mM, 3.5-4.0 mM, 4.0-4.5 mM, 4.5-5.0 mM, 5.0-5.5 mM, 5.5-6.0 mM, 6.0-6.5 mM, 6.5-7.0 mM or 7.0-7.5 mM of DTT.

[0182] Further preferably, the formulation used for the dialysis in step S4 contains 5 mM of DTT.

[0183] Further preferably, the formulation used for the dialysis in step S4 contains 0.8-0.9 mM, 0.9-1.0 mM, 1.0-1.1 mM or 1.1-1.2 mM of EDTA.

[0184] Further preferably, the formulation used for dialysis in step S4 contains 1 mM EDTA.

[0185] Further preferably, the formulation used for dialysis in step S4 contains 15-16 mM, 16-17 mM, 17-18 mM, 18-19 mM, 19-20 mM, 20-21 mM, 21-22 mM, 22-23 mM, 23-24 mM or 24-25 mM NaCl.

[0186] Further preferably, the formulation used for dialysis in step S4 contains 20 mM NaCl.

[0187] Preferably, the buffer includes, but is not limited to, any one or more of Tris-HCl buffer, PBS buffer, HEPES buffer, TAE buffer, TBE buffer, MES buffer.

[0188] Further preferably, the buffer is Tris-HCl buffer.

[0189] Further preferably, the buffer is Tris-HCl buffer with pH 7.0-7.1, 7.1-7.2, 7.2-7.3, 7.3-7.4, 7.4-7.5, 7.5-7.6, 7.6-7.7, 7.7-7.8, 7.8-7.9 or 7.9-8.0.

[0190] More preferably, the buffer is Tris-HCl buffer with pH 7.5.

[0191] In certain embodiments, the formulation used for dialysis in step S4 is Buffer A-2, which is 50 mM Tris / HCl with 20% v / v glycerol, 5 mM DTT, 1 mM EDTA, 20 Mm NaCl, pH 7.5.

[0192] Specifically, the formulation used for adjusting conductivity in step S4 is a buffer containing glycerol, DTT and EDTA.

[0193] Preferably, the formulation used for adjusting conductivity in step S4 is a buffer containing 15%-25% v / v glycerol, 2.5-7.5 mM DTT and 0.8-1.2 Mm EDTA.

[0194] Further preferably, the formulation used for dialysis in step S4 contains 15-16% v / v, 16-17% v / v, 17-18% v / v, 18-19% v / v, 19-20% v / v, 20-21% v / v, 21-22% v / v, 22-23% v / v, 23-24% v / v or 24-25% v / v of glycerol.

[0195] Still further preferably, the formulation used for adjusting conductivity in step S4 contains 20% v / v of glycerol.

[0196] Further preferably, the formulation used for adjusting conductivity in step S4 contains 2.5-3.0 mM, 3.0-3.5 mM, 3.5-4.0 mM, 4.0-4.5 mM, 4.5-5.0 mM, 5.0-5.5 mM, 5.5-6.0 mM, 6.0-6.5 mM, 6.5-7.0 mM or 7.0-7.5 mM of DTT.

[0197] Further preferably, the formulation used for adjusting conductivity in step S4 contains 5 mM of DTT.

[0198] Further preferably, the formulation used for adjusting conductivity in step S4 contains 0.8-0.9 mM, 0.9-1.0 mM, 1.0-1.1 mM or 1.1-1.2 mM of EDTA.

[0199] Still further preferably, the formulation used for adjusting conductivity in step S4 contains 1 mM of EDTA.

[0200] Preferably, the buffer includes but is not limited to any one or more of Tris-HCl buffer, PBS buffer, HEPES buffer, TAE buffer, TBE buffer, MES buffer.

[0201] Further preferably, the buffer is Tris-HCl buffer.

[0202] Still further preferably, the buffer is Tris-HCl buffer with pH 7.0-7.1, 7.1-7.2, 7.2-7.3, 7.3-7.4, 7.4-7.5, 7.5-7.6, 7.6-7.7, 7.7-7.8, 7.8-7.9 or 7.9-8.0.

[0203] Still further preferably, the buffer is Tris-HCl buffer with pH 7.5.

[0204] In particular, step S4 comprises the following steps:

[0205] 1) dialysis of the mixture, adjusting conductivity, to obtain a dialysate;

[0206] 2) chromatographic separation and purification of the dialysate to obtain an eluate;

[0207] 3) adding ammonium sulfate to the eluate, centrifuging after standing, collecting the precipitate, dissolving, dialysis, to obtain a mixed purified solution;

[0208] 4) performing size exclusion chromatography purification on the mixed purified solution, collecting the protein peak, to obtain the purified polymerase III tau clamp loader and catalytic core complex.

[0209] In some embodiments, the formulation used for adjusting conductivity in step 1) is Buffer A, which is 50 mM Tris / HCl, pH 7.5, containing 20% v / v glycerol, 5 mM DTT, 1 mM EDTA.

[0210] Specifically, the chromatographic separation and purification in step 2) comprises: adding the dialysate to a gel column, washing, and eluting to obtain an eluate.

[0211] Further specifically, the formulation used for washing or eluting in step 2) is a buffer containing glycerol, DTT, EDTA and NaCl.

[0212] Preferably, the formulation used for washing or eluting in step 2) is a buffer containing 15%-25% v / v glycerol, 2.5-7.5 mM DTT, 0.8-1.2 mM EDTA and 15-25 mM NaCl.

[0213] Further preferably, the formulation used for washing or eluting in step 2) contains 15%-16% v / v, 16%-17% v / v, 17%-18% v / v, 18%-19% v / v, 19%-20% v / v, 20%-21% v / v, 21%-22% v / v, 22%-23% v / v, 23%-24% v / v or 24%-25% v / v glycerol.

[0214] Still further preferably, the formulation used for washing or eluting in step 2) contains 20% v / v glycerol.

[0215] Further preferably, the formulation used for washing or eluting in step 2) contains 2.5-3.0 mM, 3.0-3.5 mM, 3.5-4.0 mM, 4.0-4.5 mM, 4.5-5.0 mM, 5.0-5.5 mM, 5.5-6.0 mM, 6.0-6.5 mM, 6.5-7.0 mM or 7.0-7.5 mM DTT.

[0216] Further preferably, the formulation used for washing or eluting in step 2) contains 5 mM DTT.

[0217] Further preferably, the formulation used for washing or eluting in step 2) contains 0.8-0.9 mM, 0.9-1.0 mM, 1.0-1.1 mM or 1.1-1.2 mM EDTA.

[0218] Still further preferably, the formulation used for washing or eluting in step 2) contains 1 mM EDTA.

[0219] Further preferably, the formulation used for washing or eluting in step 2) contains 15-16 mM, 16-17 mM, 17-18 mM, 18-19 mM, 19-20 mM, 20-21 mM, 21-22 mM, 22-23 mM, 23-24 mM or 24-25 mM NaCl.

[0220] Still further preferably, the formulation used for washing or eluting in step 2) contains 20 mM NaCl.

[0221] Preferably, the buffer includes but is not limited to any one or more of Tris-HCl buffer, PBS buffer, HEPES buffer, TAE buffer, TBE buffer, MES buffer.

[0222] Further preferably, the buffer is Tris-HCl buffer.

[0223] Still further preferably, the buffer is Tris-HCl buffer with pH 7.0-7.1, 7.1-7.2, 7.2-7.3, 7.3-7.4, 7.4-7.5, 7.5-7.6, 7.6-7.7, 7.7-7.8, 7.8-7.9 or 7.9-8.0.

[0224] Still further preferably, the buffer is Tris-HCl buffer with pH 7.5.

[0225] In certain embodiments, the formulation used for washing or eluting in step 2) is Buffer A-2, which is 50 mM Tris / HCl with 20% v / v glycerol, 5 mM DTT, 1 mM EDTA, 20 Mm NaCl, pH 7.5.

[0226] Further specifically, the solvent used for eluting in step 2) is Buffer A + gradient NaCl (20-350 mM).

[0227] Preferably, the Buffer A is a buffer comprising glycerol, DTT and EDTA.

[0228] Further preferably, the Buffer A is a buffer comprising 15-25% v / v glycerol, 2.5-7.5 mM DTT and 0.8-1.2 mM EDTA.

[0229] Further preferably, the Buffer A comprises 15-16% v / v, 16-17% v / v, 17-18% v / v, 18-19% v / v, 19-20% v / v, 20-21% v / v, 21-22% v / v, 22-23% v / v, 23-24% v / v or 24-25% v / v glycerol.

[0230] Still further preferably, the Buffer A comprises 20% v / v glycerol.

[0231] Further preferably, the Buffer A comprises 2.5-3.0 mM, 3.0-3.5 mM, 3.5-4.0 mM, 4.0-4.5 mM, 4.5-5.0 mM, 5.0-5.5 mM, 5.5-6.0 mM, 6.0-6.5 mM, 6.5-7.0 mM or 7.0-7.5 mM DTT.

[0232] Further preferably, the Buffer A comprises 5 mM DTT.

[0233] Further preferably, the Buffer A comprises 0.8-0.9 mM, 0.9-1.0 mM, 1.0-1.1 mM or 1.1-1.2 mM EDTA.

[0234] Still further preferably, the Buffer A comprises 1 mM EDTA.

[0235] Preferably, the buffer comprises any one or more of Tris-HCl buffer, PBS buffer, HEPES buffer, TAE buffer, TBE buffer, MES buffer, but not limited to.

[0236] Further preferably, the buffer is a Tris-HCl buffer.

[0237] Still further preferably, the buffer is a Tris-HCl buffer having a pH of 7.0-7.1, 7.1-7.2, 7.2-7.3, 7.3-7.4, 7.4-7.5, 7.5-7.6, 7.6-7.7, 7.7-7.8, 7.8-7.9 or 7.9-8.0.

[0238] More preferably, the buffer is Tris-HCl buffer with pH 7.5.

[0239] Specifically, the final concentration of ammonium sulfate in the eluent in step 3) is 0.3-0.5 g / mL.

[0240] Preferably, the final concentration of ammonium sulfate in the eluent in step 3) is 0.3-0.4 g / mL or 0.4-0.5 g / mL.

[0241] Further preferably, the final concentration of ammonium sulfate in the eluent in step 3) is 0.40 g / mL.

[0242] Specifically, the standing in step 3) is standing after ice bath in a shaker.

[0243] Preferably, the standing in step 3) is standing for 8-12 h after ice bath in a shaker at 50-150 rpm for 1-3 h.

[0244] Further preferably, the standing in step 3) is standing for 10 h after ice bath in a shaker at 100 rpm for 2 h.

[0245] Specifically, the centrifugation in step 3) includes centrifugation at 15000-25000 x g for 5-15 min.

[0246] Preferably, the centrifugation in step 3) includes centrifugation at 15000-16000 x g, 16000-17000 x g, 17000-18000 x g, 18000-19000 x g, 19000-20000 x g, 20000-21000 x g, 21000-22000 x g, 22000-23000 x g, 23000-24000 x g or 24000-25000 x g for 5-6 min, 6-7 min, 7-8 min, 8-9 min, 9-10 min, 10-11 min, 11-12 min, 12-13 min, 13-14 min or 14-15 min.

[0247] Further preferably, the centrifugation in step 3) is centrifugation at 20000 x g for 10 min.

[0248] Specifically, the preparation used for dissolving or dialysis in step 3) is a buffer containing glycerol, DTT, EDTA, NaCl, ATP and MgCl2.

[0249] Preferably, the formulation used for the solubilization or dialysis in step 3) is a buffer containing 15-25% v / v glycerol, 2.5-7.5 mM DTT, 0.8-1.2 mM EDTA, 15-25 mM NaCl, 0.2-0.8 mM ATP and 2-8 mM MgCl2.

[0250] Further preferably, the formulation used for the solubilization or dialysis in step 3) contains 15-16% v / v, 16-17% v / v, 17-18% v / v, 18-19% v / v, 19-20% v / v, 20-21% v / v, 21-22% v / v, 22-23% v / v, 23-24% v / v or 24-25% v / v glycerol.

[0251] Still further preferably, the formulation used for the solubilization or dialysis in step 3) contains 20% v / v glycerol.

[0252] Further preferably, the formulation used for the solubilization or dialysis in step 3) contains 2.5-3.0 mM, 3.0-3.5 mM, 3.5-4.0 mM, 4.0-4.5 mM, 4.5-5.0 mM, 5.0-5.5 mM, 5.5-6.0 mM, 6.0-6.5 mM, 6.5-7.0 mM or 7.0-7.5 mM DTT.

[0253] Further preferably, the formulation used for the solubilization or dialysis in step 3) contains 5 mM DTT.

[0254] Further preferably, the formulation used for the solubilization or dialysis in step 3) contains 0.8-0.9 mM, 0.9-1.0 mM, 1.0-1.1 mM or 1.1-1.2 mM EDTA.

[0255] Still further preferably, the formulation used for the solubilization or dialysis in step 3) contains 1 mM EDTA.

[0256] Further preferably, the formulation used for the solubilization or dialysis in step 3) contains 15-16 mM, 16-17 mM, 17-18 mM, 18-19 mM, 19-20 mM, 20-21 mM, 21-22 mM, 22-23 mM, 23-24 mM or 24-25 mM NaCl.

[0257] Still further preferably, the formulation used for the solubilization or dialysis in step 3) contains 20 mM NaCl.

[0258] Further preferably, the formulation used for the dissolving or dialysis in step 3) contains 0.2-0.3 mM, 0.3-0.4 mM, 0.4-0.5 mM, 0.5-0.6 mM, 0.6-0.7 mM or 0.7-0.8 mM ATP.

[0259] Still further preferably, the formulation used for the dissolving or dialysis in step 3) contains 0.5 mM ATP.

[0260] Further preferably, the formulation used for the dissolving or dialysis in step 3) contains 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM or 7-8 mM MgCl2.

[0261] Still further preferably, the formulation used for the dissolving or dialysis in step 3) contains 5 mM MgCl2.

[0262] Preferably, the buffer includes but is not limited to any one or more of Tris-HCl buffer, PBS buffer, HEPES buffer, TAE buffer, TBE buffer, MES buffer.

[0263] Further preferably, the buffer is Tris-HCl buffer.

[0264] Still further preferably, the buffer is Tris-HCl buffer with pH 7.0-7.1, 7.1-7.2, 7.2-7.3, 7.3-7.4, 7.4-7.5, 7.5-7.6, 7.6-7.7, 7.7-7.8, 7.8-7.9 or 7.9-8.0.

[0265] Still further preferably, the buffer is Tris-HCl buffer with pH 7.5.

[0266] In certain embodiments, the formulation used for the dissolving or dialysis in step 3) is Buffer A-3, containing 20% v / v glycerol, 5 mM DTT, 1 mM EDTA, 20 mM NaCl, 0.5 mM ATP and 5 mM MgCl2in 50 mM Tris / HCl, pH 7.5.

[0267] Specifically, the mixed enzyme system further comprises: an E. coli polymerase III beta clamp, an E. coli DnaA protein, an E. coli helicase, an E. coli initiation replication factor IHF2, an E. coli single-stranded binding protein, an E. coli GyrA2B2, an E. coli topoisomerase IV, an E. coli topoisomerase III, an E. coli RecQ protein, an E. coli primase, an E. coli termination site binding protein, a ribonuclease H, an E. coli DNA ligase, and an E. coli DNA polymerase I.

[0268] Specifically, the E. coli polymerase III beta clamp is a purified product of a strain expressing dnaN.

[0269] Preferably, the dnaN comprises an amino acid sequence as shown in SEQ ID NO. 2, or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 2.

[0270] Preferably, the method for preparing the strain expressing dnaN comprises: transforming a plasmid expressing dnaN into a competent cell.

[0271] Further preferably, the plasmid expressing dnaN is: pRSFDuet-beta.

[0272] Preferably, the competent cell comprises, but is not limited to, any one or more of: DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21 (DE3), BL21 (DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS, Origami (DE3), and Origami (DE3) pLysS.

[0273] Further preferably, the competent cell is: Rosetta (DE3) pLysS.

[0274] Specifically, the E. coli DnaA protein is a purified product of a strain expressing dnaA protein.

[0275] Preferably, the dnaA protein comprises an amino acid sequence as shown in SEQ ID NO. 10, or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 10.

[0276] Preferably, the method for preparing the strain expressing dnaA protein comprises: transforming a plasmid expressing dnaA protein into a competent cell.

[0277] Further preferably, the plasmid expressing the dnaA protein is: pETDuet-dnaA.

[0278] Preferably, the competent cell includes but is not limited to any one or more of: DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, Origami(DE3), Origami(DE3)pLysS.

[0279] Further preferably, the competent cell is: BL21(DE3).

[0280] Specifically, the E. coli helicase is a purified product of a strain co-expressing a dnaC protein and a dnaB protein.

[0281] Preferably, the dnaC protein comprises an amino acid sequence as shown in SEQ ID NO. 11; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 11.

[0282] Preferably, the dnaB protein comprises an amino acid sequence as shown in SEQ ID NO. 12; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 12.

[0283] Preferably, the method for preparing the strain co-expressing the dnaC protein and the dnaB protein comprises: transforming a plasmid co-expressing the dnaC protein and the dnaB protein into a competent cell.

[0284] Further preferably, the plasmid co-expressing the dnaC protein and the dnaB protein is: pETDuet-dnaC-dnaB.

[0285] Preferably, the competent cell includes but is not limited to any one or more of: DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, Origami(DE3), Origami(DE3)pLysS.

[0286] Further preferably, the competent cell is: BL21(DE3).

[0287] Specifically, the E. coli initiation replication factor IHF2 is a purified product of a strain co-expressing ihfA protein and ihfB protein.

[0288] Preferably, the ihfA protein comprises an amino acid sequence as shown in SEQ ID NO. 14; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 14.

[0289] Preferably, the ihfB protein comprises an amino acid sequence as shown in SEQ ID NO. 13; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 13.

[0290] Preferably, the method for preparing the strain co-expressing ihfA protein and ihfB protein comprises: transforming a plasmid co-expressing ihfA protein and ihfB protein into a competent cell.

[0291] Further preferably, the plasmid co-expressing ihfA protein and ihfB protein is: pETDuet-ihfB-ihfA.

[0292] Preferably, the competent cell includes, but is not limited to, any one or more of: DH5α, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21 (DE3), BL21 (DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS, Origami (DE3), Origami (DE3) pLysS.

[0293] Further preferably, the competent cell is: BL21 (DE3).

[0294] Specifically, the E. coli single-stranded binding protein is a purified product of a strain expressing SSB protein.

[0295] Preferably, the SSB protein comprises an amino acid sequence as shown in SEQ ID NO. 15; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 15.

[0296] Preferably, the method for preparing the strain expressing SSB protein comprises: transforming a plasmid expressing SSB protein into a competent cell.

[0297] Further preferably, the plasmid expressing SSB protein is: pET21a-SSB.

[0298] Preferably, the competent cell includes but is not limited to any one or more of DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, Origami(DE3), and Origami(DE3)pLysS.

[0299] Further preferably, the competent cell is BL21(DE3).

[0300] Specifically, the E. coli GyrA2B2 is a co-purification product of a gyrB protein-expressing strain and a gyrA protein-expressing strain.

[0301] Preferably, the gyrB protein comprises an amino acid sequence as shown in SEQ ID NO. 16, or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 16.

[0302] Preferably, the gyrA protein comprises an amino acid sequence as shown in SEQ ID NO. 17, or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 17.

[0303] Preferably, the method for preparing the gyrB protein-expressing strain comprises: transforming a gyrB protein-expressing plasmid into a competent cell.

[0304] Further preferably, the gyrB protein-expressing plasmid is pETDuet-his-gyrB.

[0305] Preferably, the competent cell includes but is not limited to any one or more of DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, Origami(DE3), and Origami(DE3)pLysS.

[0306] Further preferably, the competent cell is BL21(DE3).

[0307] Preferably, the method for preparing the gyrA protein-expressing strain comprises: transforming a gyrA protein-expressing plasmid into a competent cell.

[0308] Further preferably, the gyrA protein-expressing plasmid is pET21a-gyrA.

[0309] Preferably, the competent cell includes but is not limited to any one or more of DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, Origami(DE3), and Origami(DE3)pLysS.

[0310] Further preferably, the competent cell is BL21(DE3).

[0311] Specifically, the E. coli topoisomerase IV is a co-purification product of a parE protein-expressing strain and a parC protein-expressing strain.

[0312] Preferably, the parE protein comprises an amino acid sequence as shown in SEQ ID NO. 18, or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 17.

[0313] Preferably, the parC protein comprises an amino acid sequence as shown in SEQ ID NO. 19, or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 19.

[0314] Preferably, the method for preparing the parE protein-expressing strain comprises: transforming a parE protein-expressing plasmid into a competent cell.

[0315] Further preferably, the parE protein-expressing plasmid is pET21a-his-parE.

[0316] Preferably, the competent cell includes but is not limited to any one or more of DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, Origami(DE3), and Origami(DE3)pLysS.

[0317] Further preferably, the competent cell is BL21(DE3).

[0318] Preferably, the method for preparing the gparC protein-expressing strain comprises: transforming a gparC protein-expressing plasmid into a competent cell.

[0319] Further preferably, the plasmid expressing the gparC protein is: pETDuet-parC.

[0320] Preferably, the competent cell includes but is not limited to any one or more of: DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, Origami(DE3), Origami(DE3)pLysS.

[0321] Further preferably, the competent cell is: BL21(DE3).

[0322] Specifically, the E. coli topoisomerase III is a purified product of a topB protein-expressing strain.

[0323] Preferably, the topB protein comprises an amino acid sequence as shown in SEQ ID NO. 20; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 20.

[0324] Preferably, the method for preparing the topB protein-expressing strain comprises: transforming a topB protein-expressing plasmid into a competent cell.

[0325] Further preferably, the topB protein-expressing plasmid is: pETDuet-topB.

[0326] Preferably, the competent cell includes but is not limited to any one or more of: DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, Origami(DE3), Origami(DE3)pLysS.

[0327] Further preferably, the competent cell is: BL21(DE3).

[0328] Specifically, the E. coli RecQ protein is a purified product of a RecQ protein-expressing strain.

[0329] Preferably, the RecQ protein comprises an amino acid sequence as shown in SEQ ID NO. 21; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 21.

[0330] Preferably, the method for preparing the strain expressing the RecQ protein comprises: transforming a plasmid expressing the RecQ protein into a competent cell.

[0331] Further preferably, the plasmid expressing the RecQ protein is: pETDuet-RecQ.

[0332] Preferably, the competent cell comprises any one or more of: DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, Origami(DE3), and Origami(DE3)pLysS.

[0333] Further preferably, the competent cell is: BL21(DE3).

[0334] In particular, the E. coli primase is a purified product of a strain expressing a dnaG protein.

[0335] Preferably, the dnaG protein comprises an amino acid sequence as shown in SEQ ID NO. 22; or an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 22.

[0336] Preferably, the method for preparing the strain expressing the dnaG protein comprises: transforming a plasmid expressing the dnaG protein into a competent cell.

[0337] Further preferably, the plasmid expressing the dnaG protein is: pRSFDuet-dnaG.

[0338] Preferably, the competent cell comprises any one or more of: DH5a, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, Origami(DE3), and Origami(DE3)pLysS.

[0339] Further preferably, the competent cell is: BL21(DE3).

[0340] In particular, the E. coli terminator-binding protein is a purified product of a strain expressing a tus protein.

[0341] Preferably, the tus protein comprises an amino acid sequence as shown in SEQ ID NO. 23; or comprises an amino acid sequence having at least 50% homology with the amino acid sequence as shown in SEQ ID NO. 23.

[0342] Preferably, the method for preparing the strain expressing the tus protein comprises: transforming a plasmid expressing the tus protein into a competent cell.

[0343] Further preferably, the plasmid expressing the tus protein is: pET21a-his-tus.

[0344] Preferably, the competent cell comprises any one or more of, but not limited to: DH5α, TOP10, JM109, DH10B, Stbl3, XL1-Blue, BL21 (DE3), BL21 (DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS, Origami (DE3), Origami (DE3) pLysS.

[0345] Further preferably, the competent cell is: BL21 (DE3) pLysS.

[0346] Specifically, the molar ratio of the polymerase III τ clamp loader and catalytic core complex, E. coli polymerase III β clamp, E. coli DnaA protein, E. coli helicase, E. coli initiation replication factor IHF2, E. coli single-stranded binding protein, E. coli GyrA2B2, E. coli topoisomerase IV, E. coli topoisomerase III, E. coli RecQ protein, E. coli primase, E. coli termination site binding protein, ribonuclease H, E. coli DNA ligase and E. coli DNA polymerase I in the mixed enzyme system is 2.5-7.5: 30-50: 80-120: 10-30: 30-50: 350-450: 40-60: 2.5-7.5: 40-60: 40-60: 350-450: 1-3: 5-15: 40-60: 40-60.

[0347] Preferably, the molar ratio of the polymerase III tau clamp loader and catalytic core complex, E. coli polymerase III beta clamp, E. coli DnaA protein, E. coli helicase, E. coli initiation replication factor IHF2, E. coli single-stranded binding protein, E. coli GyrA2B2, E. coli topoisomerase IV, E. coli topoisomerase III, E. coli RecQ protein, E. coli primase, E. coli termination site binding protein, RNase H, E. coli DNA ligase, and E. coli DNA polymerase I in the mixed enzyme system is 5:40:100:20:40:400:50:5:50:50:400:2:10:50:50.

[0348] In another aspect, the present application provides a method for preparing the mixed enzyme system of any one of the above, comprising mixing the polymerase III tau clamp loader and catalytic core complex, E. coli polymerase III beta clamp, E. coli DnaA protein, E. coli helicase, E. coli initiation replication factor IHF2, E. coli single-stranded binding protein, E. coli GyrA2B2, E. coli topoisomerase IV, E. coli topoisomerase III, E. coli RecQ protein, E. coli primase, E. coli termination site binding protein, RNase H, E. coli DNA ligase, and E. coli DNA polymerase I in a molar ratio to obtain the mixed enzyme system.

[0349] In another aspect, the present application provides a mixed enzyme buffer comprising the mixed enzyme system of any one of the above.

[0350] Specifically, the mixed enzyme buffer further comprises a reaction buffer.

[0351] Preferably, the reaction buffer is a buffer comprising DTT, potassium glutamate, magnesium acetate, creatine phosphate, NTPs, dNTPs, NAD + , ammonium sulfate, tRNA, BSA, and creatine kinase.

[0352] Further preferably, the reaction buffer is a 100 mM Tris-HCl buffer, pH 8.0, comprising 40 mM DTT, 750 mM potassium glutamate, 50 mM magnesium acetate, 20 mM creatine phosphate, 5 mM NTPs, 0.5 mM dNTPs, 1 mM NAD + , 50 mM ammonium sulfate, 250 μg / mL tRNA, 0.5 mg / mL BSA, and 100 μg / mL creatine kinase.

[0353] In yet another aspect, the present application provides use of the mixed enzyme system or the mixed enzyme buffer of any one of the above in the preparation of a circular DNA amplification product.

[0354] Preferably, the product comprises any one or more of a reagent, a kit, a chip.

[0355] In yet another aspect, the present application provides a product capable of amplifying circular DNA, the product comprising the mixed enzyme system or the mixed enzyme buffer of any one of the above.

[0356] Preferably, the product comprises any one or more of a reagent, a kit, a chip.

[0357] In yet another aspect, the present application provides a method for amplifying circular DNA, the method comprising using the mixed enzyme system, the mixed enzyme buffer or the product of any one of the above.

[0358] Preferably, the method comprises mixing the circular DNA with the mixed enzyme system, the mixed enzyme buffer or the product of any one of the above.

[0359] The present application has the following beneficial effects:

[0360] 1. The mixed enzyme system provided by the present application can directly realize efficient amplification of circular DNA in vitro, without relying on in vivo replication system of host cells such as Escherichia coli, greatly improving the preparation efficiency of circular DNA. At the same time, without large-scale fermentation equipment, the device cost and energy consumption are reduced, meeting the needs of low-cost and rapid preparation.

[0361] 2. In the traditional method, part of the circular DNA will cause the host cells to be unable to grow or replicate normally due to carrying toxic genes, repeated sequences or special promoters, etc., so it is difficult to obtain. The mixed enzyme system of the present application does not rely on host cells and can effectively amplify such special circular DNA.

[0362] 3. The mixed enzyme system of the present application can realize amplification of circular DNA without selection marker, fundamentally solving the biological safety hidden danger brought by the selection marker, reducing the construction complexity of circular DNA, and avoiding the interference of the selection marker on the function of circular DNA.

[0363] 4. The mixed enzyme system of the present application amplifies circular DNA in an in vitro reaction system, reduces the impurity pollution from host cells, and has higher product purity; and the reaction condition is controllable, the uniformity of the amplification product is better, and the subsequent purification difficulty and cost are reduced.

[0364] 5、The mixed enzyme system of the present application has very high sensitivity to low-abundance circular DNA templates, can efficiently amplify as low as 25fg / μL of circular DNA templates, and can meet the application requirements of micro-amount template scenes such as micro-amount circular plasmid and natural circular DNA molecules in clinical samples, and has important value in high-sensitivity requirement fields such as early disease diagnosis.

[0365] 6、The present application optimizes the composition of the mixed enzyme system and the preparation method of each component, ensures the efficiency and stability of the system. Compared with the comparative example, the mixed enzyme system of the present application has better amplification effect on different concentrations of templates, especially lower detection limit for low concentration templates, which fully proves its superiority in circular DNA amplification. BRIEF DESCRIPTION OF DRAWINGS

[0366] Figure 1 Figure 4 is the amplification result of the mixed enzyme system of the present application on M13ms9 bacteriophage plasmid; in the figure, "-" represents 0.5ng of unreacted template; "1" represents 0.25ng / μL of plasmid template; "2" represents 25pg / μL of plasmid template; "3" represents 2.5pg / μL of plasmid template; "4" represents 250fg / μL of plasmid template; "5" represents 25fg / μL of plasmid template.

[0367] Figure 2 Figure 5 is the synthesis result of the mixed enzyme system of the present application on lenticrispr plasmid; in the figure, A is the electrophoresis result after amplification; B is the BamH I enzyme digestion result.

[0368] Figure 3 Figure 6 is the synthesis result of the mixed enzyme system of the present application on pMD and pRL plasmid; in the figure, A is the electrophoresis result after amplification; B is the BamH I enzyme digestion result. DETAILED DESCRIPTION

[0369] The following non-limiting examples can enable those of ordinary skill in the art to more fully understand the present application, but in no way limit the present application. The following content is only an exemplary description of the scope of the present application, and those skilled in the art can make various changes and modifications to the present application according to the disclosed content, and it should also belong to the scope of the present application.

[0370] The present application is further described below in the form of specific examples. The various instruments, devices, equipment, reagents, products, etc. used in the examples of the present application are obtained through conventional commercial channels unless otherwise specified. The ribonuclease H of the present application is purchased from takara, item number 2151; the E. coli DNA ligase is purchased from takara, item number 2161; the E. coli DNA polymerase I is purchased from takara, item number 2130A.

[0371] Example 1

[0372] 1. Sequence information

[0373] The amino acid sequence of the dnaX of the present application is shown as SEQ ID NO. 1, and SEQ ID NO. 1:

[0374] MSYQVLARKWRPQTFADVVGQEHVLTALANGLSLGRIHHAYLFSGTRGVGKTSIARLLAKGLNCETGITATPCGVCDNCREIEQGRFVDLIEIDAASRTKVEDTRDLLDNVQYAPARGRFKVYLIDEVHMLSRHSFNALLKTLEEPPEHVKFLLATTDPQKLPVTILSRCLQFHLKALDVEQIRHQLEHILNEEHIAHEPRALQLLARAAEGSLRDALSLTDQAIASGDGQVSTQAVSAMLGTLDDDQALSLVEAMVEANGERVMALINEAAARGIEWEALLVEMLGLLHRIAMVQLSPAALGNDMAAIELRMRELARTIPPTDIQLYYQTLLIGRKELPYAPDRRMGVEMTLLRALAFHPRMPLPEPEVPRQSFAPVAPTAVMTPTQVPPQPQSAPQQAPTVPLPETTSQVLAARQQLQRVQGATKAKKSEPAAATRARPVNNAALERLASVTDRVQARPVPSALEKAPAKKEAYRWKATTPVMQQKEVVATPKALKKALEHEKTPELAAKLAAEAIERDPWAAQVSQLSLPKLVEQVALNAWKEESDNAVCLHLRSSQRHLNNRGAQQKLAEALSMLKGSTVELTIVEDDNPAVRTPLEWRQAIYEEKLAQARESIIADNNIQTLRRFFDAELDEESIRPI.

[0375] The amino acid sequence of the dnaN of the present application is shown as SEQ ID NO. 2, and SEQ ID NO. 2:

[0376] MKFTVEREHLLKPLQQVSGPLGGRPTLPILGNLLLQVADGTLSLTGTDLEMEMVARVALVQPHEPGATTVPARKFFDICRGLPEGAEIAVQLEGERMLVRSGRSRFSLSTLPAADFPNLDDWQSEVEFTLPQATMKRLIEATQFSMAHQDVRYYLNGMLFETEGEELRTVATDGHRLAVCSMPIGQSLPSHSVIVPRKGVIELMRMLDGGDNPLRVQIGSNNIRAHVGDFIFTSKLVDGRFPDYRRVLPKNPDKHLEAGCDLLKQAFARAAILSNEKFRGVRLYVSENQLKITANNPEQEEAEEILDVTYSGAEMEIGFNVSYVLDVLNALKCENVRMMLTDSVSSVQIEDAASQSAAYVVMPMRL.

[0377] The amino acid sequence of the holC of the present application is shown as SEQ ID NO. 3, SEQ ID NO. 3:

[0378] MKNATFYLLDNDTTVDGLSAVEQLVCEIAAERWRSGKRVLIACEDEKQAYRLDEALWARPAESFVPHNLAGEGPRGGAPVEIAWPQKRSSSRRDILISLRTSFADFATAFTEVVDFVPYEDSLKQLARERYKAYRVAGFNLNTATWK.

[0379] The amino acid sequence of the holD of the present application is shown as SEQ ID NO. 4, SEQ ID NO. 4:

[0380] MTSRRDWQLQQLGITQWSLRRPGALQGEIAIAIPAHVRLVMVANDLPALTDPLVSDVLRALTVSPDQVLQLTPEKIAMLPQGSHCNSWRLGTDEPLSLEGAQVASPALTDLRANPTARAALWQQICTYEHDFFPRND.

[0381] The amino acid sequence of the holA of the present application is shown as SEQ ID NO. 5, SEQ ID NO. 5:

[0382] MIRLYPEQLRAQLNEGLRAAYLLLGNDPLLLQESQDAVRQVAAAQGFEEHHTFSIDPNTDWNAIFSLCQAMSLFASRQTLLLLLPENGPNAAINEQLLTLTGLLHDDLLLIVRGNKLSKAQENAAWFTALANRSVQVTCQTPEQAQLPRWVAARAKQLNLELDDAANQVLCYCYEGNLLALAQALERLSLLWPDGKLTLPRVEQAVNDAAHFTPFHWVDALLMGKSKRALHILQQLRLEGSEPVILLRTLQRELLLLVNLKRQSAHTPLRALFDKHRVWQNRRGMMGEALNRLSQTQLRQAVQLLTRTELTLKQDYGQSVWAELEGLSLLLCHKPLADVFIDG.

[0383] The amino acid sequence of the holB of the application is shown as SEQ ID NO. 6, and SEQ ID NO. 6:

[0384] MRWYPWLRPDFEKLVASYQAGRGHHALLIQALPGMGDDALIYALSRYLLCQQPQGHKSCGHCRGCQLMQAGTHPDYYTLAPEKGKNTLGVDAVREVTEKLNEHARLGGAKVVWVTDAALLTDAAANALLKTLEEPPAETWFFLATREPERLLATLRSRCRLHYLAPPPEQYAVTWLSREVTMSQDALLAALRLSAGSPGAALALFQGDNWQARETLCQALAYSVPSGDWYSLLAALNHEQAPARLHWLATLLMDALKRHHGAAQVTNVDVPGLVAELANHLSPSRLQAILGDVCHIREQLMSVTGINRELLITDLLLRIEHYLQPGVVLPVPHL.

[0385] The amino acid sequence of the dnaE of the application is shown as SEQ ID NO. 7, and SEQ ID NO. 7:

[0386]

[0387] The amino acid sequence of the dnaQ of the present application is shown as SEQ ID NO. 8, SEQ ID NO. 8:

[0388] MSTAITRQIVLDTETTGMNQIGAHYEGHKIIEIGAVEVVNRRLTGNNFHVYLKPDRLVDPEAFGVHGIADEFLLDKPTFAEVADEFMDYIRGAELVIHNAAFDIGFMDYEFSLLKRDIPKTNTFCKVTDSLAVARKMFPGKRNSLDALCARYEIDNSKRTLHGALLDAQILAEVYLAMTGGQTSMAFAMEGETQQQQGEATIQRIVRQASKLRVVFATDEEIAAHEARLDLVQKKGGSCLWRA.

[0389] The amino acid sequence of the holE of the present application is shown as SEQ ID NO. 9, SEQ ID NO. 9:

[0390] MLKNLAKLDQTEMDKVNVDLAAAGVAFKERYNMPVIAEAVEREQPEHLRSWFRERLIAHRLASVNLSRLPYEPKLK.

[0391] The amino acid sequence of the dnaA protein of the present application is shown as SEQ ID NO. 10, SEQ ID NO. 10:

[0392] MSLSLWQQCLARLQDELPATEFSMWIRPLQAELSDNTLALYAPNRFVLDWVRDKYLNNINGLLTSFCGADAPQLRFEVGTKPVTQTPQAAVTSNVAAPAQVAQTQPQRAAPSTRSGWDNVPAPAEPTYRSNVNVKHTFDNFVEGKSNQLARAAARQVADNPGGAYNPLFLYGGTGLGKTHLLHAVGNGIMARKPNAKVVYMHSERFVQDMVKALQNNAIEEFKRYYRSVDALLIDDIQFFANKERSQEEFFHTFNALLEGNQQIILTSDRYPKEINGVEDRLKSRFGWGLTVAIEPPELETRVAILMKKADENDIRLPGEVAFFIAKRLRSNVRELEGALNRVIANANFTGRAITIDFVREALRDLLALQEKLVTIDNIQKTVAEYYKIKVADLLSKRRSRSVARPRQMAMALAKELTNHSLPEIGDAFGGRDHTTVLHACRKIEQLREESHDIKEDFSNLIRTLSS.

[0393] The amino acid sequence of the dnaC protein of the present application is shown as SEQ ID NO. 11, and SEQ ID NO. 11:

[0394] MKNVGDLMQRLQKMMPAHIKPAFKTGEELLAWQKEQGAIRSAALERENRAMKMQRTFNRSGIRPLHQNCSFENYRVECEGQMNALSKARQYVEEFDGNIASFIFSGKPGTGKNHLAAAICNELLLRGKSVLIITVADIMSAMKDTFRNSGTSEEQLLNDLSNVDLLVIDEIGVQTESKYEKVIINQIVDRRSSSKRPTGMLTNSNMEEMTKLLGERVMDRMRLGNSLWVIFNWDSYRSRVTGKEY.

[0395] The amino acid sequence of the dnaB protein of the present application is shown as SEQ ID NO. 12, and SEQ ID NO. 12:

[0396] MAGNKPFNKQQAEPRERDPQVAGLKVPPHSIEAEQSVLGGLMLDNERWDDVAERVVADDFYTRPHRHIFTEMARLQESGSPIDLITLAESLERQGQLDSVGGFAYLAELSKNTPSAANISAYADIVRERAVVREMISVANEIAEAGFDPQGRTSEDLLDLAESRVFKIAESRANKDEGPKNIADVLDATVARIEQLFQQPHDGVTGVNTGYDDLNKKTAGLQPSDLIIVAARPSMGKTTFAMNLVENAAMLQDKPVLIFSLEMPSEQIMMRSLASLSRVDQTKIRTGQLDDEDWARISGTMGILLEKRNIYIDDSSGLTPTEVRSRARRIAREHGGIGLIMIDYLQLMRVPALSDNRTLEIAEISRSLKALAKELNVPVVALSQLNRSLEQRADKRPVNSDLRESGSIEQDADLIMFIYRDEVYHENSDLKGIAEIIIGKQRNGPIGTVRLTFNGQWSRFDNYAGPQYDDE.

[0397] The amino acid sequence of the IhfB protein of the application is shown as SEQ ID NO. 13, and SEQ ID NO. 13:

[0398] MTKSELIERLATQQSHIPAKTVEDAVKEMLEHMASTLAQGERIEIRGFGSFSLHYRAPRTGRNPKTGDKVELEGKYVPHFKPGKELRDRANIYG.

[0399] The amino acid sequence of the IhfA protein of the application is shown as SEQ ID NO. 14, and SEQ ID NO. 14:

[0400] MALTKAEMSEYLFDKLGLSKRDAKELVELFFEEIRRALENGEQVKLSGFGNFDLRDKNQRPGRNPKTGEDIPITARRVVTFRPGQKLKSRVENASPKDE.

[0401] The amino acid sequence of the SSB protein of the application is shown as SEQ ID NO. 15, and SEQ ID NO. 15:

[0402] MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPF.

[0403] The amino acid sequence of the gyrB protein of the application is shown as SEQ ID NO. 16, and SEQ ID NO. 16:

[0404] MSNSYDSSSIKVLKGLDAVRKRPGMYIGDTDDGTGLHHMVFEVVDNAIDEALAGHCKEI IVTIHADNSVSVQDDGRGIPTGIHPEEGVSAAEVIMTVLHAGGKFDDNSYKVSGGLHGV GVS VNAL S QKLELVIQREGKIHRQIYEHGVPQAPLAVTGETEKTGTMVRFWPSLETFTN VTEFEYEILAKRLRELSFLNSGVSIRLRDKRDGKEDHFHYEGGIKAFVEYLNKNKTPIHPN I FYFSTEKDGIGVEVALQWNDGFQENIYCFTNNIPQRDGGTHLAGFRAAMTRTLNAYMDK EGYSKKAKVSATGDDAREGLIAVVSVKVPDPKFSSQTKDKLVSSEVKSAVEQQMNELL AEYLLENPTDAKIVVGKIIDAARAREAARRAREMTRRKGALDLAGLPGKLADCQERDP ALSELYLVEGDSAGGSAKQGRNRKNQAILPLKGKILNVEKARFDKMLSSQEVATLITAL GCGIGRDEYNPDKLRYHSIIIMTDADVDGSHIRTLLLTFFYRQMPEIVERGHVYIAQPP LYKVKKGKQEQYIKDDEAMDQYQISIALDGATLHTNASAPALAGEALEKLVSEYNATQ KMINRMERRYPKAMLKELIYQPTLTEADLSDEQTVTRWVNALVSELNDKEQHGSQWKF DVHTNAEQNLFEPIVRVRTHGVDTDYPLDHEFITGGEYRRICTLGEKLRGLLEEDAFIE RGERRQPVASFEQALDWLVKESRRGLSIQRYKGLGEMNPEQLWETTMDPESRRMLRV TVKDAIAADQLFTTLMGDAVEPRRAFIEENALKAANIDI.

[0405] The amino acid sequence of the gyrA protein of the application is shown in SEQ ID NO. 17, SEQ ID NO. 17:

[0406] MSDLAREITPVNIEEELKSSYLDYAMSVIVGRALPDVRDGLKPVHRRVLYAMNVLGNDWNKAYKKSARVVGDVIGKYHPHGDSAVYDTIVRMAQPFSLRYMLVDGQGNFGSIDGDSAAAMRYTEIRLAKIAHELMADLEKETVDFVDNYDGTEKIPDVMPTKIPNLLVNGSSGIAVGMATNIPPHNLTEVINGCLAYIDDEDISIEGLMEHIPGPDFPTAAIINGRRGIEEAYRTGRGKVYIRARAEVEVDAKTGRETIIVHEIPYQVNKARLIEKIAELVKEKRVEGISALRDESDKDGMRIVIEVKRDAVGEVVLNNLYSQTQLQVSFGINMVALHHGQPKIMNLKDIIAAFVRHRREVVTRRTIFELRKARDRAHILEALAVALANIDPIIELIRHAPTPAEAKTALVANPWQLGNVAAMLERAGDDAARPEWLEPEFGVRDGLYYLTEQQAQAILDLRLQKLTGLEHEKLLDEYKELLDQIAELLRILGSADRLMEVIREELELVREQFGDKRRTEITANSADINLEDLITQEDVVVTLSHQGYVKYQPLSEYEAQRRGGKGKSAARIKEEDFIDRLLVANTHDHILCFSSRGRVYSMKVYQLPEATRGARGRPIVNLLPLEQDERITAILPVTEFEEGVKVFMATANGTVKKTVLTEFNRLRTAGKVAIKLVDGDELIGVDLTSGEDEVMLFSAEGKVVRFKESSVRAMGCNTTGVRGIRLGEGDKVVSLIVPRGDGAILTATQNGYGKRTAVAEYPTKSRATKGVISIKVTERNGLVVGAVQVDDCDQIMMITDAGTLVRTRVSEISIVGRNTQGVILIRTAEDENVVGLQRVAEPVDEEDLDTIDGSAAEGDDEIAPEVDVDDEPEEE.

[0407] The amino acid sequence of the parE protein of the present application is shown in SEQ ID NO. 18, and SEQ ID NO. 18:

[0408] MTQTYNADAIEVLTGLEPVRRRPGMYTDTTRPNHLGQEVIDNSVDEALAGHAKRVDVILHADQSLEVIDDGRGMPVDIHPEEGVPAVELILCRLHAGGKFSNKNYQFSGGLHGVGISVVNALSKRVEVNVRRDGQVYNIAFENGEKVQDLQVVGTCGKRNTGTSVHFWPDETFFDSPRFSVSRLTHVLKAKAVLCPGVEITFKDEINNTEQRWCYQDGLNDYLAEAVNGLPTLPEKPFIGNFAGDTEAVDWALLWLPEGGELLTESYVNLIPTMQGGTHVNGLRQGLLDAMREFCEYRNILPRGVKLSAEDIWDRCAYVLSVKMQDPQFAGQTKERLSSRQCAAFVSGVVKDAFILWLNQNVQAAELLAEMAISSAQRRMRAAKKVVRKKLTSGPALPGKLADCTAQDLNRTELFLVEGDSAGGSAKQARDREYQAIMPLKGKILNTWEVSSDEVLASQEVHDISVAIGIDPDSDDLSQLRYGKICILADADSDGLHIATLLCALFVKHFRALVKHGHVYVALPPLYRIDLGKEVYYALTEEEKEGVLEQLKRKKGKPNVQRFKGLGEMNPMQLRETTLDPNTRRLVQLTIDDEDDQRTDAMMDMLLAKKRSEDRRNWLQEKGDMAEIEV.

[0409] The amino acid sequence of the parC protein of the present application is shown in SEQ ID NO. 19, and SEQ ID NO. 19:

[0410] MSDMAERLALHEFTENAYLNYSMYVIMDRALPFIGDGLKPVQRRIVYAMSELGLNASAKFKKSARTVGDVLGKYHPHGDSACYEAMVLMAQPFSYRYPLVDGQGNWGAPDDPKSFAAMRYTESRLSKYSELLLSELGQGTADWVPNFDGTLQEPKMLPARLPNILLNGTTGIAVGMATDIPPHNLREVAQAAIALIDQPKTTLDQLLDIVQGPDYPTEAEIITSRAEIRKIYENGRGSVRMRAVWKKEDGAVVISALPHQVSGARVLEQIAAQMRNKKLPMVDDLRDESDHENPTRLVIVPRSNRVDMDQVMNHLFATTDLEKSYRINLNMIGLDGRPAVKNLLEILSEWLVFRRDTVRRRLNYRLEKVLKRLHILEGLLVAFLNIDEVIEIIRNEDEPKPALMSRFGLTETQAEAILELKLRHLAKLEEMKIRGEQSELEKERDQLQGILASERKMNNLLKKELQADAQAYGDDRRSPLQEREEAKAMSEHDMLPSEPVTIVLSQMGWVRSAKGHDIDAPGLNYKAGDSFKAAVKGKSNQPVVFVDSTGRSYAIDPITLPSARGQGEPLTGKLTLPPGATVDHMLMESDDQKLLMASDAGYGFVCTFNDLVARNRAGKALITLPENAHVMPPVVIEDASDMLLAITQAGRMLMFPVSDLPQLSKGKGNKIINIPSAEAARGEDGLAQLYVLPPQSTLTIHVGKRKIKLRPEELQKVTGERGRRGTLMRGLQRIDRVEIDSPRRASSGDSEE.

[0411] The amino acid sequence of the topB protein of the present application is shown in SEQ ID NO. 20, and SEQ ID NO. 20:

[0412] MRLFIAEKPSLARAIADVLPKPHRKGDGFIECGNGQVVTWCIGHLLEQAQPDAYDSRYARWNLADLPIVPEKWQLQPRPSVTKQLNVIKRFLHEASEIVHAGDPDREGQLLVDEVLDYLQLAPEKRQQVQRCLINDLNPQAVERAIDRLRSNSEFVPLCVSALARARADWLYGINMTRAYTILGRNAGYQGVLSVGRVQTPVLGLVVRRDEEIENFVAKDFFEVKAHIVTPADERFTAIWQPSEACEPYQDEEGRLLHRPLAEHVVNRISGQPAIVTSYNDKRESESAPLPFSLSALQIEAAKRFGLSAQNVLDICQKLYETHKLITYPRSDCRYLPEEHFAGRHAVMNAISVHAPDLLPQPVVDPDIRNRCWDDKKVDAHHAIIPTARSSAINLTENEAKVYNLIARQYLMQFCPDAVFRKCVIELDIAKGKFVAKARFLAEAGWRTLLGSKERDEENDGTPLPVVAKGDELLCEKGEVVERQTQPPRHFTDATLLSAMTGIARFVQDKDLKKILRATDGLGTEATRAGIIELLFKRGFLTKKGRYIHSTDAGKALFHSLPEMATRPDMTAHWESVLTQISEKQCRYQDFMQPLVGTLYQLIDQAKRTPVRQFRGIVAPGSGGSADKKKAAPRKRSAKKSPPADEVGSGAIA.

[0413] The amino acid sequence of the RecQ protein of the present application is shown in SEQ ID NO. 21, and SEQ ID NO. 21:

[0414] MAQAEVLNLESGAKQVLQETFGYQQFRPGQEEIIDTVLSGRDCLVVMPTGGGKSLCYQIP ALLLNGLTVVVSPLISLMKDQVDQLQANGVAAACLNSTQTREQQLEVMTGCRTGQIRLLYI APERLMLDNFLEHLAHWNPVLLAVDEAHCISQWGHDFRPEYAALGQLRQRFPTLPFMAL TATADDTTRQDIVRLLGLNDPLIQISSFDRPNIRYMLMEKFKPLDQLMRYVQEQRGKSGI IYCNSRAKVEDTAARLQSKGISAAAYHAGLENNVRADVQEKFQRDDLQIVVATVAFGMG INKPNVRFVVHFDIPRNIESYYQETGRAGRDGLPAEAMLFYDPADMAWLRRCLEEKPQG QLQDIERHKLNAMGAFAEAQTCRRLVLLNYFGEGRQEPCGNCDICLDPPKQYDGSTDAQ IALSTIGRVNQRFGMGYVVEVIRGANNQRIRDYGHDKLKVYGMGRDKSHEHWVSVIRQ LIHLGLVTQNIAQHSALQLTEAARPVLRGESSLQLAVPRIVALKPKAMQKSFGGNYDRK LFAKLRKLRKSIADESNVPPYVVFNDATLIEMAEQMPITASEMLSVNGVGMRKLERFG KPFMALIRAHVDGDDEE.

[0415] The amino acid sequence of the dnaG protein of the present application is shown as SEQ ID NO. 22, and SEQ ID NO. 22:

[0416] MAGRIPRVFINDLLARTDIVDLIDARVKLKKQGKNFHACCPFHNEKTPSFTVNGEKQFYHCFGCGAHGNAIDFLMNYDKLEFVETVEELAAMHNLEVPFEAGSGPSQIERHQRQTLYQLMDGLNTFYQQSLQQPVATSARQYLEKRGLSHEVIARFAIGFAPPGWDNVLKRFGGNPENRQSLIDAGMLVTNDQGRSYDRFRERVMFPIRDKRGRVIGFGGRVLGNDTPKYLNSPETDIFHKGRQLYGLYEAQQDNAEPNRLLVVEGYMDVVALAQYGINYAVASLGTSTTADHIQLLFRATNNVICCYDGDRAGRDAAWRALETALPYMTDGRQLRFMFLPDGEDPDTLVRKEGKEAFEARMEQAMPLSAFLFNSLMPQVDLSTPDGRARLSTLALPLISQVPGETLRIYLRQELGNKLGILDDSQLERLMPKAAESGVSRPVPQLKRTTMRILIGLLVQNPELATLVPPLENLDENKLPGLGLFRELVNTCLSQPGLTTGQLLEHYRGTNNAATLEKLSMWDDIADKNIAEQTFTDSLNHMFDSLLELRQEELIARERTHGLSNEERLELWTLNQELAKK.

[0417] The amino acid sequence of the tus protein of the present application is shown in SEQ ID NO. 23, and SEQ ID NO. 23:

[0418] MARYDLVDRLNTTFRQMEQELAIFAAHLEQHKLLVARVFSLPEVKKEDEHNPLNRIEVKQHLGNDAQSLALRHFRHLFIQQQSENRSSKAAVRLPGVLCYQVDNLSQAALVSHIQHINKLKTTFEHIVTVESELPTAARFEWVHRHLPGLITLNAYRTLTVLHDPATLRFGWANKHIIKNLHRDEVLAQLEKSLKSPRSVAPWTREEWQRKLEREYQDIAALPQNAKLKIKRPVKVQPIARVWYKGDQKQVQHACPTPLIALINRDNGAGVPDVGELLNYDADNVQHRYKPQAQPLRLIIPRLHLYVAD.

[0419] 2. Plasmid construction

[0420] 2.1 pRSFDuet-τ

[0421] pRSFDuet-τ was constructed according to the method in the literature of Andrea Bogutzki, Natalie Naue, Lidia Litz, Andreas Pich and Ute Curth. E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication. Sci Rep. 2019 Oct 8;9(1):14460. and entrusted to General Biosystems for gene synthesis.

[0422] 2.2 pETDuet-χψ

[0423] pETDuet-χψ was constructed according to the method in the literature of Andrea Bogutzki, Natalie Naue, Lidia Litz, Andreas Pich and Ute Curth. E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication. Sci Rep. 2019 Oct 8;9(1):14460. and entrusted to General Biosystems for gene synthesis.

[0424] 2.3 pCDFDuet-CmR-δ′δ

[0425] pCDFDuet-CmR-δ′δ was constructed according to the method in the literature of Andrea Bogutzki, Natalie Naue, Lidia Litz, Andreas Pich and Ute Curth. E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication. Sci Rep. 2019 Oct 8;9(1):14460. and entrusted to General Biosystems for gene synthesis.

[0426] 2.4 pRSFDuet-β

[0427] The method was constructed according to the method in the literature of Andrea Bogutzki, Natalie Naue, Lidia Litz, Andreas Pich and Ute Curth. E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication. Sci Rep. 2019 Oct 8;9(1):14460. and entrusted to General Biology for gene synthesis.

[0428] 2.5 pRSFDuet-α

[0429] pRSFDuet-1 was linearized by double digestion of pRSFDuet-1 with Nde I and Kpn I, and dnaE The gene was cloned into the linearized pRSFDuet-1 by primer dnaE-F (SEQ ID NO. 24) and dnaE-R (SEQ ID NO. 25) to obtain the pRSFDuet-α plasmid.

[0430] dnaE-F (SEQ ID NO. 24):

[0431] GAAGGAGATATACATATGTCTGAACCACGTTTCGT.

[0432] dnaE-R (SEQ ID NO. 25):

[0433] AGACTCGAGGGTACCTTAGTCAAACTCCAGTTCCAC.

[0434] 2.6 pET21a-εθ

[0435] The construction method of pETcore in reference to Masayuki Su'etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011525-11534. was followed, with the omission of the dnaE The construction was carried out after the construction step of the gene sequence, and the gene synthesis was entrusted to General Biosystems.

[0436] 2.7 pETDuet-dnaA

[0437] pETDuet-1 was obtained by double enzyme digestion of pETDuet-1 with Hind III and Not I, and the linearized pETDuet-1 was obtained by digestion with Hind III and Not I. dnaA The gene was cloned into the linearized pETDuet-1 by primer dnaA-F (SEQ ID NO. 26) and dnaA-R (SEQ ID NO. 27) to obtain the pETDuet-dnaA plasmid.

[0438] dnaA-F (SEQ ID NO. 26):

[0439] CCTGCAGGTCGACAAGCTTATGTCACTTTCGCTTTGGCA.

[0440] dnaA-R (SEQ ID NO. 27):

[0441] TAAGCATTATGCGGCCGCTTACGATGACAATGTTCTGA.

[0442] 2.8 pETDuet-dnaC-dnaB

[0443] pET-dnaC-dnaB was constructed by the method described in Masayuki Su’etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011 525-11534. and the gene synthesis was entrusted to General Biosystems.

[0444] 2.9 pETDuet-ihfB-ihfA

[0445] pET-ihfB-ihfA was constructed by the method described in Masayuki Su’etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011 525-11534. and the gene synthesis was entrusted to General Biosystems.

[0446] 2.10 pET21a-SSB

[0447] pET-ssb was constructed by the method described in Masayuki Su’etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011 525-11534. and the gene synthesis was entrusted to General Biosystems.

[0448] 2.11 pETDuet-his-gyrB

[0449] pET-his-gyrB was constructed by the method described in Masayuki Su’etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011525-11534. and the gene synthesis was entrusted to General Biosystems.

[0450] 2.12 pET21a-gyrA

[0451] pET-gyrA was constructed by the method described in Masayuki Su’etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011525-11534. and the gene synthesis was entrusted to General Biosystems.

[0452] 2.13 pET21a-his-parE

[0453] pET-his-parE was constructed by the method described in Masayuki Su’etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011525-11534. and the gene synthesis was entrusted to General Biosystems.

[0454] 2.14 PETDuet-parC

[0455] pET-parC was constructed by the method described in Masayuki Su’etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011525-11534. and the gene synthesis was commissioned to GenScript.

[0456] 2.15 pETDuet-topB

[0457] pET-his-topB was constructed by the method described in Masayuki Su’etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011525-11534. and the gene synthesis was commissioned to GenScript.

[0458] 2.16 pETDuet-RecQ

[0459] pET-his-recQ was constructed by the method described in Masayuki Su’etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011525-11534. and the gene synthesis was commissioned to GenScript.

[0460] 2.17 pRSFDuet-dnaG

[0461] pRSFDuet-1 was linearized by double digestion of pRSFDuet-1 with Nde I and Kpn I, and dnaG The gene was cloned into the linearized pRSFDuet-1 by primer dnaG-F (SEQ ID NO. 28) and dnaG-R (SEQ ID NO. 29) to obtain pRSFDuet-dnaG plasmid.

[0462] The plasmid vector was linearized by double digestion with Nde I and Kpn I.

[0463] dnaG-F (SEQ ID NO. 28):

[0464] GAAGGAGATATACATATGATGGCTGGACGAATCCCACG.

[0465] dnaG-R (SEQ ID NO. 29):

[0466] AGACTCGAGGGTACCTTAGTCAAACTCCAGTTCCAC.

[0467] 2.18 pET21a-his-tus

[0468] Reference Masayuki Su’etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011525-11534. The construction method of PET-his-tus in the literature was constructed, and the gene synthesis was entrusted to general biology.

[0469] 3, Strain construction

[0470] The information of the strains 1-14 of the application is shown in Table 1:

[0471] Table 1

[0472]

[0473] Note: Amp in the table represents ampicillin; Cm represents chloramphenicol; Kan represents kanamycin.

[0474] 3.1 Construction of strain 1

[0475] The pRSFDuet-t plasmid, pETDuet-xi plasmid and pCDFDuet-CmR-δ'δ plasmid were overexpressed in BL21 (DE3) competent cells. The specific steps were as follows: 100 μL of BL21 (DE3) competent cells were added with 5 μL of pRSFDuet-t plasmid and 5 μL of pETDuet-xi plasmid for double transformation, and then the LB plate containing 100 μg / mL of ampicillin and 15 μg / mL of kanamycin was used for antibiotic screening and PCR verification to obtain the double plasmid strain. After the strain was prepared into competent cells, 100 μL of the double resistant competent cells were added with 2 mL of pCDFDuet-CmR-δ'δ plasmid for transformation, and then the LB plate containing 100 μg / mL of ampicillin, 15 μg / mL of kanamycin and 30 μg / mL of chloramphenicol was used for antibiotic screening and PCR verification to obtain strain 1.

[0476] 3.2 Construction of strain 2

[0477] The pRSFDuet-β plasmid was overexpressed in Rosetta (DE3) pLysS competent cells. 100 μL of Rosetta (DE3) pLysS competent cells were added with 2 μL of pRSFDuet-β plasmid for transformation, and then the LB plate containing 15 μg / mL of kanamycin was used for antibiotic screening and PCR verification to obtain strain 2.

[0478] 3.3 Construction of strain 3

[0479] The pRSFDuet-α plasmid and pET21a-εθ plasmid were overexpressed in BL21 (DE3) competent cells. 100 μL of BL21 (DE3) competent cells were added with 5 μL of pRSFDuet-α plasmid and 5 μL of pET21a-εθ plasmid for double transformation, and then the LB plate containing 100 μg / mL of ampicillin and 15 μg / mL of kanamycin was used for antibiotic screening and PCR verification to obtain strain 3.

[0480] 3.4 Construction of strain 4

[0481] The pETDuet-dnaA plasmid was overexpressed in BL21 (DE3) competent cells. 100 μL of BL21 (DE3) competent cells were added with 2 μL of pETDuet-dnaA plasmid for transformation, and then the LB plate containing 100 μg / mL of ampicillin was used for antibiotic screening and PCR verification to obtain strain 4.

[0482] 3.5 Construction of strain 5

[0483] The pETDuet-dnaC-dnaB plasmid was overexpressed in BL21 (DE3) competent cells. 100 μL of BL21 (DE3) competent cells were added to 2 μL of the pETDuet-dnaC-dnaB plasmid for transformation, and the bacterial liquid was spread on an LB plate containing 100 μg / mL ampicillin. After antibiotic screening and PCR verification, strain 5 was obtained.

[0484] 3.6 Construction of strain 6

[0485] The pETDuet-ihfB-ihfA plasmid was overexpressed in BL21 (DE3) competent cells. 100 μL of BL21 (DE3) competent cells were added to 2 μL of the pETDuet-ihfB-ihfA plasmid for transformation, and the bacterial liquid was spread on an LB plate containing 100 μg / mL ampicillin. After antibiotic screening and PCR verification, strain 6 was obtained.

[0486] 3.7 Construction of strain 7

[0487] The pET21a-SSB plasmid was overexpressed in BL21 (DE3) competent cells. 100 μL of BL21 (DE3) competent cells were added to 2 μL of the pET21a-SSB plasmid for transformation, and the bacterial liquid was spread on an LB plate containing 100 μg / mL ampicillin. After antibiotic screening and PCR verification, strain 7 was obtained.

[0488] 3.8 Construction of strain 8

[0489] The pETDuet-his-gyrB plasmid was overexpressed in BL21 (DE3) competent cells. 100 μL of BL21 (DE3) competent cells were added to 2 μL of the pETDuet-his-gyrB plasmid for transformation, and the bacterial liquid was spread on an LB plate containing 100 μg / mL ampicillin. After antibiotic screening and PCR verification, strain 8 was obtained.

[0490] 3.9 Construction of strain 9

[0491] The pET21a-gyrA plasmid was overexpressed in BL21 (DE3) competent cells. 100 μL of BL21 (DE3) competent cells were added to 2 μL of the pET21a-gyrA plasmid for transformation, and the bacterial liquid was spread on an LB plate containing 100 μg / mL ampicillin. After antibiotic screening and PCR verification, strain 9 was obtained.

[0492] 3.10 Construction of strain 10

[0493] The pET21a-his-parE plasmid was overexpressed in BL21 (DE3) competent cells. 100 μL of BL21 (DE3) competent cells were added to 2 μL of the pET21a-his-parE plasmid for transformation, and the bacterial liquid was spread on an LB plate containing 100 μg / mL ampicillin. After antibiotic screening and PCR verification, strain 10 was obtained.

[0494] 3.11 Construction of strain 11

[0495] The pETDuet-parC plasmid was overexpressed in BL21 (DE3) competent cells. 100 μL of BL21 (DE3) competent cells were added to 2 μL of the pETDuet-parC plasmid for transformation, and the bacterial liquid was spread on an LB plate containing 100 μg / mL ampicillin. After antibiotic screening and PCR verification, strain 11 was obtained.

[0496] 3.12 Construction of strain 12

[0497] The pETDuet-topB plasmid was overexpressed in BL21 (DE3) competent cells. 100 μL of BL21 (DE3) competent cells were added to 2 μL of the pETDuet-topB plasmid for transformation, and the bacterial liquid was spread on an LB plate containing 100 μg / mL ampicillin. After antibiotic screening and PCR verification, strain 12 was obtained.

[0498] 3.13 Construction of strain 13

[0499] The pETDuet-RecQ plasmid was overexpressed in BL21 (DE3) competent cells. 100 μL of BL21 (DE3) competent cells were added to 2 μL of the pETDuet-RecQ plasmid for transformation, and the bacterial liquid was spread on an LB plate containing 100 μg / mL ampicillin. After antibiotic screening and PCR verification, strain 13 was obtained.

[0500] 3.14 Construction of strain 14

[0501] The pRSFDuet-dnaG plasmid was overexpressed in BL21 (DE3) competent cells. 100 μL of BL21 (DE3) competent cells were added to 2 μL of the pRSFDuet-dnaG plasmid for transformation, and the bacterial liquid was spread on an LB plate containing 15 μg / mL kanamycin. After antibiotic screening and PCR verification, strain 14 was obtained.

[0502] 3.15 Construction of strain 15

[0503] The pET21a-his-tus plasmid was overexpressed in BL21 (DE3) pLysS competent cells. 100 μL BL21 (DE3) pLysS competent cells were added with 2 μL pET21a-his-tus plasmid for transformation, and the bacterial liquid was spread on LB plate containing 100 μg / mL ampicillin for antibiotic selection and PCR verification, and strain 15 was obtained.

[0504] All successfully constructed strains should be stored at -80°C in LB glycerol containing the corresponding antibiotics for standby use.

[0505] Example 2

[0506] 1. Solution preparation

[0507] (1) Resuspension Buffer: 50 mM Tris / HCl, pH 7.5, containing 10% w / v sucrose, 1 mM PMSF, 0.5 mM DTT and 1 tablet / 50 mL EDTA-free cOmplete ™ Protease Inhibitor Cocktail (Roche Diagnostics, Germany, Cat. No. 04693132001).

[0508] (2) Core Resuspension Buffer: 50 mM Tris-HCl, pH 7.5, containing 10 mM spermidine and 0.1 M NaCl.

[0509] (3) Dilution Buffer: 50 mM Tris / HCl, pH 7.5, containing 10% w / v sucrose, 0.1 M NaCl, 15 mM spermidine, 1 mM EDTA.

[0510] (4) Wash Buffer-1: 50 mM Tris-HCl, pH 7.5, containing 20% v / v glycerol, 5 mM DTT, 1 mM EDTA, 0.15 g / mL ammonium sulfate.

[0511] (5) Wash Buffer-2: 50 mM Tris-HCl, pH 7.5, containing 20% v / v glycerol, 5 mM DTT, 1 mM EDTA, 0.2 g / mL ammonium sulfate.

[0512] (6) Buffer A: 50 mM Tris / HCl, pH 7.5, containing 20% v / v glycerol, 5 mM DTT, 1 mM EDTA.

[0513] (7) Buffer A-2: Buffer A containing 20 mM NaCl.

[0514] (8) Buffer A-3: Buffer A containing 20 mM NaCl, 0.5 mM ATP and 5 mM MgCl2.

[0515] (9) Buffer B: 50 mM Tris-HCl, pH 7.5, containing 10% v / v glycerol, 0.1 mM EDTA, 5 mM DTT, 100 mM NaCl, 5 mM MgCl2and 0.4 mM ATP.

[0516] (10) Lysis buffer: 50 mM Tris-HCl, pH 8.0, containing 10% w / v sucrose, 250 mM NaCl, 10 mM spermidine, 2 mM DTT, 20 mM EDTA, 0.3 mg / mL lysozyme and 1 mM PMSF.

[0517] (11) Column buffer: 50 mM Tris-HCl, pH 7.5, containing 20% v / v glycerol, 5 mM DTT, 2 mM EDTA.

[0518] (12) ATP column buffer: 50 mM Tris-HCl, pH 7.5, containing 20% v / v glycerol, 5 mM DTT, 5 mM MgCl2and 0.1 mM ATP.

[0519] (13) Ni column buffer: 20 mM Tris-HCl, pH 7.5, containing 10 v / v glycerol, 500 mM NaCl, 2 mM 2-mercaptoethanol and 0.2 mM EDTA.

[0520] (14) Buffer C: 50 mM HEPES.KOH, pH 7.6, containing 20% w / v sucrose, 2 mM DTT, 1 mM EDTA.

[0521] (15) Buffer D: 50 mM HEPES.KOH, pH 7.6, containing 20% w / v sucrose, 2 mM DTT, 0.1 mM EDTA, 10 mM magnesium acetate, 0.2 M ammonium sulfate.

[0522] (16) IHF2 lysis buffer: 25 mM Tris-HCl, pH 7.4, containing 1 mM EDTA, 3 mM β-mercaptoethanol and 50 mM NaCl.

[0523] 2. Cultivation of strains

[0524] (1) 5 μL of each preserved bacterial solution was inoculated into 5 mL of LB medium containing the corresponding antibiotic, and incubated at 37°C overnight to obtain the cultured bacterial solution;

[0525] (2) Take 500-1000 μL of the cultured bacteria liquid and transfer it to 500 mL of LB medium containing corresponding antibiotics, and incubate at 37°C for 3-5 h.

[0526] (3) When the OD value of the bacteria liquid in the medium reaches 0.6-1.5, add IPTG to a final concentration of 0.4-1 mM, and incubate at 37°C for 2 h. For the strains other than No. 3, add 0.5 mM of IPTG after the OD value reaches about 0.3, and incubate at 13°C overnight.

[0527] (4) After the incubation, centrifuge to collect the bacteria liquid, freeze it in liquid nitrogen, and store it at -80°C for subsequent purification.

[0528] 3. Purification of strains

[0529] 3.1 Preliminary purification of E. coli polymerase III τ clamp loader and purification of catalytic core

[0530] 3.1.1 Preliminary purification of E. coli polymerase III τ clamp loader

[0531] Weigh the bacteria mud of the cultured strain 1, resuspend the bacteria mud with 1 mL of resuspension Buffer according to 1 g of bacteria mud weight, and dilute the resuspended bacteria liquid with an equal volume of dilution Buffer; after ultrasonic crushing (100 W), centrifuge at 12000 x g for 10 min, take the supernatant, slowly add ammonium sulfate to the supernatant to a final concentration of 0.2 g / mL for precipitation, centrifuge at 12000 x g for 10 min, discard the supernatant, and wash the precipitate with a washing Buffer-1 of equal weight as the bacteria mud, centrifuge at 12000 x g for 10 min, collect the precipitate, and dissolve the precipitate with 1000 μL of Buffer A-2, to obtain the preliminary purified product of τ clamp loader.

[0532] 3.1.2 Preliminary purification of E. coli polymerase III catalytic core

[0533] Weigh the bacteria mud of the cultured strain 3, resuspend the bacteria mud with 4 mL of core resuspension Buffer according to 1 g of bacteria mud weight, crush the cells by ultrasonic crushing (100 W), centrifuge the crushing product at 12000 x g for 10 min, take the supernatant, slowly add ammonium sulfate to the supernatant to a final concentration of 0.226 g / mL for precipitation, centrifuge at 12000 x g for 10 min, discard the supernatant, wash the precipitate with a washing Buffer-2 of equal weight as the bacteria mud, centrifuge at 12000 x g for 10 min, collect the precipitate, dissolve the precipitate with 1000 μL of Buffer A-2, and obtain the preliminary purified product of catalytic core.

[0534] 3.1.3 Heparin Sepharose column purification

[0535] (1) Protein electrophoresis of the tau clamp loader initial purification product and catalytic core initial purification product, mixing the protein electrophoresis products of the tau clamp loader initial purification product and the catalytic core initial purification product at a molar ratio of 1:3 to obtain a mixture;

[0536] (2) 2000 μL of the mixture was placed in 1 L of Buffer A-2 for overnight dialysis, and after dialysis, the conductivity of the dialyzed sample was adjusted using Buffer A to equal that of Buffer A-2 to obtain a dialysate;

[0537] (3) The dialysate was added to a heparin sepharose gel column equilibrated with Buffer A-2, washed with 5 column volumes of Buffer-A 2, and then eluted with 10 column volumes of Buffer A + gradient NaCl (20-350 mM) to obtain an eluate;

[0538] (4) Ammonium sulfate was slowly added to the eluate to a final concentration of 0.4 g / mL, and after being placed in an ice bath at 100 rpm for 2 h, it was allowed to stand for 10 h, and after centrifugation at 20000 x g for 30 min, the supernatant was discarded, the precipitate was dissolved in 3 mL of Buffer A-3, and dialyzed with 2 L of Buffer A-3 to obtain a mixed purification solution;

[0539] (5) Size exclusion chromatography purification (SEC): the mixed purification solution was subjected to SEC purification using a Superose 6 column equilibrated with Buffer B. The protein peak was collected according to the instrument display to obtain the purified polymerase III tau clamp loader and catalytic core complex.

[0540] 3.2 Purification of E. coli polymerase III beta clamp

[0541] The bacterial slurry of the cultured strain 2 was weighed, 5 mL of lysis buffer was added per 1 g of bacterial slurry to resuspend the bacterial slurry, and the cells were broken using ultrasonic crushing (100 W). The broken product was centrifuged at 12000 x g for 10 min, and the supernatant was taken. Ammonium sulfate was slowly added to the supernatant to a final concentration of 0.28 g / mL for precipitation, and the precipitate was resuspended in 5 mL of column buffer after centrifugation at 12000 x g for 10 min and discarding the supernatant. The sample was subjected to heparin sepharose column purification, washed with 5 column volumes of column buffer, and eluted with 5 column volumes of linear gradient column buffer containing 50-300 mM NaCl. The protein peak was collected according to the instrument display to obtain the purified E. coli polymerase III beta clamp.

[0542] 3.3 Purification of E. coli DnaA protein

[0543] The bacterial slurry of strain 4 after culture was weighed, 5 mL of Buffer C containing 20 mM spermidine was added to resuspend the bacterial slurry according to 1 g bacterial slurry weight, and the cells were broken by ultrasonic crushing (100 W). The broken product was centrifuged at 12000 x g for 10 min, and the supernatant was taken. Ammonium sulfate was slowly added to the supernatant to a final concentration of 0.28 g / mL for precipitation, and the precipitate was centrifuged at 12000 x g for 10 min. The supernatant was discarded, 0.2 g of the precipitate was added to 5 mL of Buffer C, and the precipitate was dialyzed overnight with 2 L of Buffer C. After dialysis, the precipitate was centrifuged at 12000 x g for 10 min, the supernatant was discarded, and 0.05 g of the precipitate was added to 2 mL of Buffer C containing 4 M guanidine hydrochloride, 10 mM magnesium acetate, and 0.6 M ammonium sulfate to dissolve the precipitate. The dissolved sample was purified using a Superose-12 column equilibrated with Buffer D, and the protein peak was collected according to the instrument display to obtain the purified E. coli DnaA protein.

[0544] 3.4 Purification of E. coli helicase

[0545] The bacterial slurry of strain 5 after culture was weighed, 5 mL of Buffer C containing 20 mM spermidine was added to resuspend the bacterial slurry according to 1 g bacterial slurry weight, and the cells were broken by ultrasonic crushing (100 W). The broken product was centrifuged at 12000 x g for 10 min, and the supernatant was taken. Ammonium sulfate was slowly added to the supernatant to a final concentration of 0.28 g / mL for precipitation, and the precipitate was centrifuged at 12000 x g for 10 min. The supernatant was discarded, 0.2 g of the precipitate was added to 5 mL of Buffer C, and the precipitate was dialyzed overnight with 2 L of Buffer C. After dialysis, the precipitate was centrifuged at 12000 x g for 10 min, the supernatant was discarded, and 0.05 g of the precipitate was added to 2 mL of Buffer C containing 4 M guanidine hydrochloride, 10 mM magnesium acetate, and 0.6 M ammonium sulfate to dissolve the precipitate. The dissolved sample was purified using a Superose-12 column equilibrated with Buffer D, and the protein peak was collected according to the instrument display to obtain the purified E. coli DnaA protein.

[0546] 3.5 Purification of E. coli initiation replication factor IHF2

[0547] The bacterial slurry of the cultured strain 6 was weighed, 5 mL of IHF2 lysis buffer was added according to 1 g of the bacterial slurry, and the cells were broken by ultrasonic crushing (100 W). The broken product was centrifuged at 12,000 x g for 10 min, and the supernatant was taken. Ammonium sulfate was slowly added to the supernatant to a final concentration of 0.334 g / mL for precipitation. The precipitate was removed by centrifugation at 12,000 x g for 10 min. Ammonium sulfate was continuously slowly added to the supernatant to a final concentration of 0.564 g / mL, and the supernatant was removed by centrifugation at 12,000 x g for 10 min. 0.2 g of the precipitate was added with 5 mL of IHF2 lysis buffer, and dialysis was performed in 2 L of the same buffer at 4°C overnight. The dialyzed sample was added to a heparin agarose column equilibrated with the IHF2 lysis buffer. After washing with the IHF2 lysis buffer, elution was performed using 10 column volumes of a linear gradient of the IHF2 lysis buffer containing 0.1-1.7 M NaCl. The protein peak at a concentration of 0.72-1.06 M NaCl was collected, and the purified E. coli initiation replication factor IHF2 was obtained.

[0548] 3.6 Purification of E. coli single-stranded binding protein

[0549] The bacterial slurry of the cultured strain 7 was weighed, and 5 mL of lysis buffer was added according to 1 g of the bacterial slurry for lysis, and a cell lysate was obtained. Ammonium sulfate was added to the cell lysate to a final concentration of 0.2 g / mL for precipitation. The precipitate was collected after backwashing the column buffer containing 0.16 g / mL of ammonium sulfate.

[0550] According to 1 g of the precipitate, 5 mL of column buffer was added to resuspend the precipitate, and the sample was loaded into a HiTrap Q column (GE Healthcare). After the washing step, the SSB peak component eluted by a linear gradient of 100-300 mM NaCl at 5 column volumes was collected.

[0551] Ammonium sulfate was added to the SSB peak component to a final concentration of 0.2 g / mL for precipitation. The precipitate was collected, 1 mL of column buffer was added according to 1 g of the precipitate to resuspend the precipitate, and the sample was loaded into a Superdex 200 gel filtration column (GE Healthcare) equilibrated with the column buffer containing 300 mM NaCl. The SSB peak component was collected, and the E. coli single-stranded binding protein was obtained.

[0552] 3.7 Purification of E. coli gyrase B subunit and A subunit

[0553] The bacterial slurry of the cultured strain 8 and the bacterial slurry of the cultured strain 9 were weighed, respectively, and mixed according to a weight ratio of 1:1.5 to obtain a mixed bacterial slurry. The mixed bacterial slurry was lysed by adding 5 mL of lysis buffer according to 1 g of the mixed bacterial slurry, and a cell lysate was obtained.

[0554] Ammonium sulfate was added to the cell lysate to a final concentration of 0.28 g / mL for precipitation. 0.2 g of the precipitate was resuspended in 1 mL of Ni column buffer containing 30 mM imidazole and loaded onto a HisTrap HP column (GE Healthcare). After a washing step, the GyrA2B2 peak fraction eluted in the column buffer with a linear concentration gradient of imidazole from 30 to 300 mM was collected.

[0555] The GyrA2B2 peak fraction was precipitated by adding ammonium sulfate to a final concentration of 0.28 g / mL. The precipitate was collected, 0.01 g of the precipitate was dissolved in 2 mL of column buffer and loaded onto a Superose 6 gel filtration column (GE Healthcare) equilibrated with column buffer containing 150 mM NaCl. The GyrA2B2 peak fraction was collected to obtain purified E. coli GyrA2B2.

[0556] 3.9 Purification of E. coli Topoisomerase IV B subunit and A subunit

[0557] The bacterial slurry of the cultured strain 10 and strain 11 was weighed respectively, mixed according to a weight ratio of 1:1.3 to obtain a mixed bacterial slurry. 5 mL of lysis buffer (100 W) was added according to 1 g of the mixed bacterial slurry weight for lysis to obtain a cell lysate;

[0558] Ammonium sulfate was added to the cell lysate to a final concentration of 0.3 g / mL for precipitation. 0.2 g of the precipitate was resuspended in 1 mL of Ni column buffer containing 30 mM imidazole and loaded onto a HisTrap HP column (GE Healthcare). After a washing step, the Topo IV peak fraction eluted in the column buffer with a linear concentration gradient of imidazole from 30 to 700 mM was collected.

[0559] The Topo IV peak fraction was precipitated by adding ammonium sulfate to a final concentration of 0.3 g / mL. The precipitate was collected, 0.01 g of the precipitate was dissolved in 2 mL of column buffer and loaded onto a Superose 6 gel filtration column (GE Healthcare) equilibrated with column buffer containing 150 mM NaCl. The Topo IV peak fraction was collected to obtain purified E. coli Topoisomerase IV.

[0560] 3.10 Purification of E. coli Topoisomerase III

[0561] The bacterial slurry of the cultured strain 12 was weighed, 5 mL of lysis buffer (100 W) was added according to 1 g of the bacterial slurry weight for lysis to obtain a cell lysate;

[0562] Ammonium sulfate was added to the cell lysate to a final concentration of 0.26 g / mL for precipitation. 0.2 g of the precipitate was resuspended in 2 mL of Ni column buffer containing 10 mM imidazole and loaded onto a HisTrap HP column (GE Healthcare). After a washing step, the Topo III peak fraction eluted with a 10-300 mM linear gradient of imidazole in the column buffer was collected.

[0563] The Topo III peak fraction was precipitated by adding ammonium sulfate to a final concentration of 0.26 g / mL. 0.01 g of the precipitate was resuspended in 2 mL of column buffer and loaded onto a Superdex 200 gel filtration column (GE Healthcare) equilibrated with column buffer containing 300 mM NaCl. The Topo III peak fraction was collected to obtain purified E. coli Topoisomerase III.

[0564] 3.11 Purification of E. coli RecQ protein

[0565] The bacterial slurry of strain 13 after cultivation was weighed and lysed by adding 5 mL of lysis buffer (100 W) per 1 g of bacterial slurry weight to obtain a cell lysate;

[0566] Ammonium sulfate was added to the cell lysate to a final concentration of 0.22 g / mL for precipitation. 0.2 g of the precipitate was resuspended in 2 mL of Ni column buffer containing 10 mM imidazole and loaded onto a HisTrap HP column (GE Healthcare). After a washing step, the RecQ peak fraction eluted with a 10-300 mM linear gradient of imidazole in the column buffer was collected.

[0567] The RecQ peak fraction was precipitated by adding ammonium sulfate to a final concentration of 0.22 g / mL. 0.01 g of the precipitate was resuspended in 2 mL of column buffer and loaded onto a Superdex 200 gel filtration column (GE Healthcare) equilibrated with column buffer containing 300 mM NaCl. The RecQ peak fraction was collected to obtain purified E. coli RecQ protein.

[0568] 3.12 Purification of E. coli primase

[0569] The bacterial slurry of strain 14 after cultivation was weighed and lysed by adding 5 mL of lysis buffer (100 W) per 1 g of bacterial slurry weight to obtain a cell lysate;

[0570] Ammonium sulfate was added to the cell lysate to a final concentration of 0.22 g / mL for precipitation. 0.2 g of the precipitate was resuspended in 5 mL column buffer and loaded onto a HiTrap Q column (GE Healthcare). After a washing step, the DnaG peak fractions were collected in column buffer eluted with a 10-300 mM linear NaCl gradient.

[0571] Further loading was performed onto a heparin sepharose column (GE Healthcare). After a washing step, the DnaG peak fractions were collected in column buffer eluted with a 10-500 mM NaCl linear concentration gradient, resulting in purified E. coli primase.

[0572] 3.13 Purification of E. coli terminator binding protein

[0573] The bacterial slurry of strain 14 after cultivation was weighed and 5 mL of lysis buffer without EDTA (100 W) was added to the 1 g of bacterial slurry for lysis, resulting in a cell lysate;

[0574] The cell lysate was loaded onto a HisTrap HP column (GE Healthcare) equilibrated with Ni column buffer. After a washing step, the Tus peak fractions were collected in Ni column buffer eluted with a 30-500 mM linear concentration imidazole in column buffer.

[0575] Ammonium sulfate was added to the Tus peak fractions to a final concentration of 0.4 g / mL for precipitation. 0.01 g of the precipitate was resuspended in 2 mL column buffer and loaded onto a Superdex 200 gel filtration column (GE Healthcare) equilibrated with column buffer containing 150 mM NaCl. The Tus peak fractions were collected, resulting in purified E. coli terminator binding protein.

[0576] Example 3

[0577] The polymerase III tau clamp loader and catalytic core complex purified from the above Example 2, E. coli polymerase III beta clamp, E. coli DnaA protein, E. coli helicase, E. coli initiation replication factor IHF2, E. coli single-stranded binding protein, E. coli GyrA2B2, E. coli topoisomerase IV, E. coli topoisomerase III, E. coli RecQ protein, E. coli primase, E. coli termination site binding protein, and ribonuclease H, E. coli DNA ligase, E. coli DNA polymerase I were mixed to obtain a mixed enzyme system, the mixed enzyme system was added to 5x reaction buffer (100 mM Tris-HCl buffer, pH 8.0, containing 40 mM DTT, 750 mM potassium glutamate, 50 mM magnesium acetate, 20 mM phosphocreatine, 5 mM NTPs, 0.5 mM dNTPs, 1 mM NAD + , 50 mM ammonium sulfate, 250 μg / mL tRNA, 0.5 mg / mL BSA, and 100 μg / mL creatine kinase) to obtain a mixed enzyme buffer, and the working concentrations of the components of the mixed enzyme system in the mixed enzyme buffer are shown in Table 2. Amplification tests were performed on different plasmid templates or ligation products.

[0578] Table 2

[0579]

[0580] 1. Plasmid template synthesis test

[0581] The M13ms9 phage plasmid was constructed according to the template construction method of Masayuki Su’etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. xponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011525-11534., and different concentrations of plasmid templates (0.25 ng / μL, 25 pg / μL, 2.5 pg / μL, 250 fg / μL, 25 fg / μL) were added to the system and incubated for 4 h (1 μL of sample volume), and the results are shown in Figure 1 .

[0582] The results show that the mixed enzyme system of the present application can efficiently amplify a circular DNA template as low as 25 fg / μL, and has a lower requirement for the amount of starting template, and has good sensitivity.

[0583] 2. Synthesis of ligation product

[0584] 2.1 lenticrispr plasmid template

[0585] The lenticrispr plasmid (disclosed in Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014 Jan 3;343(6166):84-87., and presented by Hangzhou University) template was digested with EcoRI, and after recovering the digestion product, Gibson assembly was used to carry and circularize the oriC sequence, obtaining the ligation product. 1 ng / μL ligation product was added to the mixed enzyme system for synthetic amplification. Gel electrophoresis was used to determine the amplification results, DL15000 was used as a molecular weight marker (DNA Ladder) to estimate the size of the amplification product (the maximum band was 15,000 bp), and whether the oriC sequence was successfully carried was verified. The determination results are shown in Figure 2 .

[0586] OriC sequence (SEQ ID NO. 30):

[0587] CTGCTCTGATGCCGCATAGtatgttgtaactaaagatctactgtggataactctgtcaggaagcttggatcaaccggtagttatccaaagaacaactgttgttcagtttttgagttgtgtataacccctcattctgatcccagcttatacggtccaggatcaccgatcattcacagttaatgatcctttccaggttgttgatcttaaaagccggatccttgttatccacagggcagtgcgatcctaataagagatcacaatagaacagatctctaaataaatagatcttctttttaatactttagttacaacatactGTTAAGCCAGCCCCGACAC.

[0588] 2.2 Other circular plasmid templates

[0589] (1) Construction of pRL-xAmp:

[0590] PCR amplification was performed on the pRL plasmid template to remove the Amp resistance gene sequence, and a circular plasmid pRL-xAmp was constructed. The primers used are shown in Table 3 below:

[0591] Table 3

[0592]

[0593] (2) Construction of pMD-Accl-BsrGl:

[0594] The pMD plasmid template was linearized by AccI and BsrGI digestion and removal of the penicillin resistance gene, electrophoresis and gel recovery, and the recovered digestion product was used for Gibson assembly to carry and circularize the oriC sequence to obtain the ligation product. The circular plasmid pMD-Accl-BsrGl was constructed. The primers used are shown in Table 4:

[0595] Table 4

[0596]

[0597] (3) Construction of pRL-oriC:

[0598] The pRL plasmid template was PCR amplified by pRL-F and pRL-R to construct the circular plasmid pRL-oriC. The primers used are shown in Table 5:

[0599] Table 5

[0600]

[0601] (4) Amplification determination

[0602] The above circular plasmid templates were subjected to Gibson assembly to carry and circularize the oriC sequence, respectively, to obtain ligation products; 1 ng / μL of the ligation product was added to the mixed enzyme system for synthesis amplification. Gel electrophoresis was used to determine the amplification results, and DL15000 was used as a molecular weight marker (DNA Ladder) to estimate the size of the amplification product (the largest band was 15,000 bp) and verify whether the oriC sequence was successfully carried. The determination results are shown in Figure 3 The determination results show that the supercoiled band (the smallest band in terms of molecular weight) is about half the size of the linear band, and the single enzyme digestion enzyme digestion map is correct, proving that the ligation and synthesis are successful.

[0603] Comparative Example 1

[0604]

[0605] ​Comparative Example 1 differs from Example 2 only in that Step "3.1 E. coli polymerase III tau-clamp loader preliminary purification and purification of catalytic core" is different. The specific steps are as follows:

[0606] 3.1.1 Preliminary purification of E. coli polymerase III tau-clamp loader and catalytic core

[0607] Weigh the strain 1 slurry after culture and the strain 3 slurry after culture, mix the strain 1 slurry and the strain 3 slurry according to a weight ratio of 1:3, resuspend the slurry by adding 6 mL of resuspension Buffer per 1 g of slurry, use ultrasonic crushing (100 W), then centrifuge at 12000 x g for 10 min, take the supernatant, slowly add ammonium sulfate to the supernatant to a final concentration of 0.226 g / mL for precipitation, centrifuge at 12000 x g for 10 min, discard the supernatant, wash the precipitate with a weight of wash Buffer-2 equal to that of the slurry, centrifuge at 12000 x g for 10 min, then collect the precipitate, 0.2 g of precipitate, dissolve the precipitate with 1000 μL of Buffer A-2, and obtain the polymerase III tau-clamp loader and catalytic core complex.

[0608] 3.1.2 Heparin Sepharose column purification

[0609] (1) Place 2000 μL of the polymerase III tau-clamp loader and catalytic core complex after protein electrophoresis in 1 L of Buffer A-2 for overnight dialysis, after dialysis, adjust the conductivity of the dialyzed sample using Buffer A to a conductivity equal to that of Buffer A-2, and obtain the dialysate;

[0610] (2) Add the dialysate to a heparin Sepharose gel column equilibrated with Buffer A-2, wash with 5 column volumes of Buffer-A 2, then elute with 10 column volumes of Buffer A + gradient NaCl (20-350 mM), and obtain the eluate;

[0611] (3) Slowly add ammonium sulfate to the eluate to a final concentration of 0.4 g / mL, place in an ice bath on a 100 rpm shaker for 2 h, then stand for 10 h, centrifuge at 20000 x g for 30 min, discard the supernatant, dissolve the precipitate with 3 mL of Buffer A-3, and dialyze with 2 L of Buffer A-3, and obtain the mixed purified liquid;

[0612] (4) Size exclusion chromatography purification (SEC): use a Superose 6 column equilibrated with Buffer B to purify the mixed purified liquid by SEC. Collect the protein peak according to the instrument display, and obtain the purified polymerase III tau-clamp loader and catalytic core complex.

[0613] Comparative Example 2

[0614] Comparative Example 1 and Example 2 differ only in the steps "3.1.1 E. coli polymerase III tau-clamp loader preliminary purification" and "3.1.2 E. coli polymerase III catalytic core preliminary purification". The specific steps are as follows:

[0615] 3.1.1 E. coli polymerase III tau-clamp loader preliminary purification

[0616] Take the bacterial slurry of the cultured strain 1, resuspend the bacterial slurry according to 1 g of bacterial slurry weight with 4 mL of core resuspension Buffer, use ultrasonic crushing (100 W), then centrifuge at 12000 x g for 10 min, take the supernatant, slowly add ammonium sulfate to the supernatant to a final concentration of 0.2 g / mL for precipitation, centrifuge at 12000 x g for 10 min, discard the supernatant, wash the precipitate with a buffer-1 of the same weight as the bacterial slurry, centrifuge at 12000 x g for 10 min, collect the precipitate, 0.2 g of precipitate, dissolve the precipitate with 1000 μL of Buffer A-2, and obtain the tau-clamp loader preliminary product.

[0617] 3.1.2 E. coli polymerase III catalytic core preliminary purification

[0618] Take the bacterial slurry of the cultured strain 3, resuspend the bacterial slurry according to 1 g of bacterial slurry weight with 1 mL of resuspension Buffer, and dilute the resuspended bacterial liquid with an equal volume of dilution Buffer; use ultrasonic crushing (100 W) to break the cells, centrifuge the broken product at 12000 x g for 10 min, take the supernatant, slowly add ammonium sulfate to the supernatant to a final concentration of 0.226 g / mL for precipitation, centrifuge at 12000 x g for 10 min, discard the supernatant, wash the precipitate with a buffer-2 of the same weight as the bacterial slurry, centrifuge at 12000 x g for 10 min, collect the precipitate, 0.2 g of precipitate, dissolve the precipitate with 1000 μL of Buffer A-2, and obtain the catalytic core preliminary product.

[0619] Comparative Example 3

[0620] Comparative Example 3 and Example 2 differ only in the steps "3.1 E. coli polymerase III tau-clamp loader preliminary purification and catalytic core purification". The specific steps are as follows:

[0621] 3.1.1 E. coli polymerase III tau-clamp loader preliminary purification

[0622] Take the cultured strain 1 slurry, resuspend the slurry according to 1 g slurry weight add 1 mL resuspension Buffer, and dilute the resuspended slurry with an equal volume of dilution Buffer; after using ultrasonic crushing (100 W), centrifuge at 12000 x g for 10 min, take the supernatant, slowly add ammonium sulfate to the supernatant to a final ammonium sulfate concentration of 0.15 g / mL for precipitation, centrifuge at 12000 x g for 10 min, discard the supernatant, wash the precipitate with a Buffer-1 equal in weight to the slurry, centrifuge at 12000 x g for 10 min, collect the precipitate, 0.2 g of precipitate, use 1000 μL Buffer A-2 to dissolve the precipitate, and obtain the tau clip loader initial purification product.

[0623] 3.1.2 E. coli polymerase III catalytic core preliminary purification

[0624] Take the cultured strain 3 slurry, resuspend the slurry according to 1 g slurry weight add 4 mL core resuspension Buffer, crush the cells using ultrasonic crushing (100 W), centrifuge the crushing product at 12000 x g for 10 min, take the supernatant, slowly add ammonium sulfate to the supernatant to a final ammonium sulfate concentration of 0.3 g / mL for precipitation, centrifuge at 12000 x g for 10 min, discard the supernatant, wash the precipitate with a Buffer-2 equal in weight to the slurry, centrifuge at 12000 x g for 10 min, collect the precipitate, 0.2 g of precipitate, use 1000 μL Buffer A-2 to dissolve the precipitate, and obtain the catalytic core initial purification product.

[0625] 3.1.3 Heparin Sepharose column purification

[0626] (1) Perform protein electrophoresis on the tau clip loader initial purification product and the catalytic core initial purification product, mix the protein electrophoresis products of the tau clip loader initial purification product and the catalytic core initial purification product at a molar ratio of 1:3, and obtain a mixture;

[0627] (2) Place 2000 μL of the mixture in 1 L of Buffer A-2 for overnight dialysis, after dialysis, use Buffer A to adjust the conductivity of the dialyzed sample to equal that of Buffer A-2, and obtain a dialysate;

[0628] (3) Add the dialysate to a heparin Sepharose gel column equilibrated with Buffer A-2, wash with 5 column volumes of Buffer-A 2, then elute with 10 column volumes of Buffer A + gradient NaCl (20-350 mM), and obtain an eluate;

[0629] (4) Slowly add ammonium sulfate to the eluate to a final concentration of 0.45 g / mL, and then place the solution in an ice bath on a 100 rpm shaker for 2 h. After standing for 10 h, centrifuge the solution at 20,000 x g for 30 min, discard the supernatant, and dissolve the pellet in 3 mL of Buffer A-3. Then dialyze the solution against 2 L of Buffer A-3 to obtain a mixed purified solution.

[0630] (5) Size-exclusion chromatography (SEC): The mixed purified solution is subjected to SEC purification using a Superose 6 column equilibrated with Buffer B. The protein peak is collected according to the display of the instrument to obtain the purified polymerase III tau clamp loader and catalytic core complex.

[0631] Comparative Example 4

[0632] Comparative Example 4 differs from Example 2 only in that the resuspension Buffer, the core resuspension Buffer, and the dilution Buffer in Step "1. Solution preparation" are different. The specific steps are as follows:

[0633] (1) Resuspension Buffer: 50 mM Tris / HCl, pH 7.5, containing 10% v / v glycerol, 1 mM PMSF, 0.5 mM DTT, and 1 tablet / 50 mL of cOmplete ™ Protease Inhibitor Cocktail (Roche Diagnostics, Germany, Cat. No. 04693132001).

[0634] (2) Core resuspension Buffer: 50 mM Tris-HCl, pH 7.5, containing 10 mM spermidine and 0.1 M NaCl.

[0635] (3) Dilution Buffer: 50 mM Tris / HCl, pH 7.5, containing 10% v / v glycerol, 0.1 M NaCl, 15 mM spermidine, and 1 mM EDTA.

[0636] Comparative Example 5

[0637] Comparative Example 5 differs from Example 2 only in that Step "3.1. E. coli polymerase III tau clamp loader preliminary purification and catalytic core purification" is different. The specific steps are as follows:

[0638] 3.1.1 E. coli polymerase III tau clamp loader preliminary purification

[0639] Take the cultured strain 1 slurry, resuspend the slurry according to 1 g slurry weight with 4 mL core resuspension Buffer, use ultrasonic disruption (100 W), then centrifuge at 12000 x g for 10 min, take the supernatant, slowly add ammonium sulfate to the supernatant to a final concentration of 0.15 g / mL for precipitation, centrifuge at 12000 x g for 10 min, discard the supernatant, use and wash Buffer-1 of the same weight as the slurry to wash the precipitate, centrifuge at 12000 x g for 10 min, collect the precipitate, 0.2 g of precipitate, use 1000 μL Buffer A-2 to dissolve the precipitate, and obtain the tau clamp loader initial purification product.

[0640] 3.1.2 E. coli polymerase III catalytic core preliminary purification

[0641] Take the cultured strain 3 slurry, resuspend the slurry according to 1 g slurry weight with 1 mL resuspension Buffer, and dilute the resuspended slurry with an equal volume of dilution Buffer; use ultrasonic disruption (100 W) to disrupt the cells, centrifuge the disruption product at 12000 x g for 10 min, take the supernatant, slowly add ammonium sulfate to the supernatant to a final concentration of 0.3 g / mL for precipitation, centrifuge at 12000 x g for 10 min, discard the supernatant, use and wash Buffer-2 of the same weight as the slurry to wash the precipitate, centrifuge at 12000 x g for 10 min, collect the precipitate, 0.2 g of precipitate, use 1000 μL Buffer A-2 to dissolve the precipitate, and obtain the catalytic core initial purification product.

[0642] 3.1.3 Heparin Sepharose column purification

[0643] (1) Perform protein electrophoresis on the tau clamp loader initial purification product and the catalytic core initial purification product, mix the protein electrophoresis products of the tau clamp loader initial purification product and the catalytic core initial purification product at a molar ratio of 1:3, and obtain a mixture;

[0644] (2) Place 2000 μL of the mixture in 1 L of Buffer A-2 for overnight dialysis, after dialysis, use Buffer A to adjust the conductivity of the dialyzed sample to equal that of Buffer A-2, and obtain a dialysate;

[0645] (3) Add the dialysate to a heparin Sepharose gel column equilibrated with Buffer A-2, wash with 5 column volumes of Buffer-A 2, then elute with 10 column volumes of Buffer A + gradient NaCl (20-350 mM), and obtain an eluate;

[0646] (4) Slowly add ammonium sulfate to the eluate to a final concentration of 0.45 g / mL, and then place the mixture in an ice bath on a 100 rpm shaker for 2 h. After standing for 10 h, centrifuge the mixture at 20,000 x g for 30 min. Discard the supernatant, and dissolve the precipitate in 3 mL of Buffer A-3. Dialyze the solution against 2 L of Buffer A-3 to obtain a mixed purified solution;

[0647] (5) Size exclusion chromatography (SEC) purification: Purify the mixed purified solution using a Superose 6 column equilibrated with Buffer B. Collect the protein peak according to the instrument display to obtain the purified polymerase III τ clamp loader and catalytic core complex.

[0648] Comparative Example 6

[0649] Comparative Example 1 and Example 2 differ only in the step of "3.1.3 Heparin Sepharose column purification". The specific steps are as follows:

[0650] 3.1.3 Heparin Sepharose column purification

[0651] (1) Perform protein electrophoresis on the τ clamp loader primary purification product and the catalytic core primary purification product. Mix the protein electrophoresis products of the τ clamp loader and the catalytic core at a molar ratio of 1:1 to obtain a mixture;

[0652] (2) Dialyze 2000 μL of the mixture against 1 L of Buffer A-2 overnight. After dialysis, adjust the conductivity of the dialyzed sample using Buffer A to a conductivity equal to that of Buffer A-2 to obtain a dialyzed solution;

[0653] (3) Add the dialyzed solution to a heparin Sepharose gel column equilibrated with Buffer A-2. Wash the column with 5 column volumes of Buffer A-2, and then elute the column with 10 column volumes of Buffer A + gradient NaCl (20-350 mM) to obtain an eluate;

[0654] (4) Slowly add ammonium sulfate to the eluate to a final concentration of 0.45 g / mL, and then place the mixture in an ice bath on a 100 rpm shaker for 2 h. After standing for 10 h, centrifuge the mixture at 20,000 x g for 30 min. Discard the supernatant, and dissolve the precipitate in 3 mL of Buffer A-3. Dialyze the solution against 2 L of Buffer A-3 to obtain a mixed purified solution;

[0655] (5) Size exclusion chromatography (SEC) purification: Purify the mixed purified solution using a Superose 6 column equilibrated with Buffer B. Collect the protein peak according to the instrument display to obtain the purified polymerase III τ clamp loader and catalytic core complex.

[0656] Comparative Example 7

[0657] Comparative Example 7 differs from Example 2 only in the step of "3.1.3 Heparin Sepharose column purification". The specific steps are as follows:

[0658] 3.1.3 Heparin Sepharose column purification

[0659] (1) Protein electrophoresis was performed on the tau clip loader initial purification product and the catalytic core initial purification product. The tau clip loader initial purification product and the catalytic core initial purification product after protein electrophoresis were mixed at a molar ratio of 3:1 to obtain a mixture;

[0660] (2) 2000 μL of the mixture was placed in 1 L of Buffer A-2 for overnight dialysis. After dialysis, the sample after dialysis was adjusted in conductivity using Buffer A to a conductivity equal to Buffer A-2 to obtain a dialysate;

[0661] (3) The dialysate was added to a heparin Sepharose gel column equilibrated with Buffer A-2. After washing with 5 column volumes of Buffer-A 2, elution was performed using 10 column volumes of Buffer A + gradient NaCl (20-350 mM) to obtain an eluate;

[0662] (4) Ammonium sulfate was slowly added to the eluate to a final concentration of 0.4 g / mL. After being placed in an ice bath at 100 rpm for 2 h, the sample was allowed to stand for 10 h. After centrifugation at 20000 x g for 30 min, the supernatant was discarded. The precipitate was dissolved using 3 mL of Buffer A-3 and dialyzed using 2 L of Buffer A-3 to obtain a mixed purification solution;

[0663] (5) Size exclusion chromatography purification (SEC): The mixed purification solution was subjected to SEC purification using a Superose 6 column equilibrated with Buffer B. Protein peaks were collected according to the instrument display to obtain the purified polymerase III tau clip loader and catalytic core complex.

[0664] Experimental Example 1

[0665] The amplification effect of the mixed enzyme system of Comparative Examples 1-7 was verified according to the method described in Example 3.

[0666] M13ms9 phage plasmid was constructed according to the template construction method of Masayuki Su'etsugu, Hiraku Takada, Tsutomu Katayama and Hiroko Tsujimoto. Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Nucleic Acids Research, 2017, Vol. 45, No. 2011525-11534. and was added to the mixed enzyme system of Comparative Examples 1-7 at different concentrations of plasmid templates (0.25 ng / μL, 25 pg / μL, 2.5 pg / μL, 250 fg / μL, 25 fg / μL) for incubation for 4 h (1 μL of sample volume), and the determination results are shown in Table 6.

[0667] Table 6

[0668]

[0669] The above results show that there is a significant difference in the detection limit of low-concentration templates in the mixed enzyme systems of different comparative examples, and the amplification effect of Comparative Examples 1-7 is obviously poorer than that of Invention Example 2.

[0670] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A mixed enzyme system that can amplify circular DNA, characterized by, The application relates to a mixed enzyme system, a preparation method of the mixed enzyme system and an application of the mixed enzyme system. The preparation method of the polymerase III tau clamp loader and catalytic core complex comprises the following steps: S1, resuspending the strain 1 slurry with resuspension Buffer, diluting the resuspended bacterial solution with dilution Buffer, taking the supernatant after crushing; adding ammonium sulfate to the supernatant for precipitation, collecting the precipitate by centrifugation, dissolving the precipitate to obtain the initial purification product of the tau clamp loader; the strain 1 is a strain expressing dnaX, holC, holD, holA and holB; the final concentration of ammonium sulfate in the supernatant is 0.20 g / mL; the resuspension Buffer is 8%-12% w / v sucrose, 0.8-1.2 mM PMSF, 0.4-0.6 mM DTT and 1 piece / 50 mL EDTA-free cOmplete ™ protease inhibitor cocktail; the dilution Buffer is a buffer containing 8%-12% w / v sucrose, 0.05-0.15 M NaCl, 10-20 mM spermidine, 0.8-1.2 mM EDTA; S2, resuspending the strain 3 slurry by using a core resuspension buffer, taking the supernatant after crushing, adding ammonium sulfate to the supernatant for precipitation, collecting the precipitate by centrifugation, dissolving the precipitate, and obtaining a catalytic core primary product; the strain 3 is a strain for expressing dnaE, dnaQ and holE; the final concentration of the ammonium sulfate in the supernatant is 0.2-0.226 g / mL; the core resuspension buffer is a buffer containing 0.05-0.15 M NaCl and 8-12 mM spermidine; S3, mixing the tau clamp loader primary product after protein electrophoresis and the catalytic core primary product after protein electrophoresis in a molar ratio of 1:2-4 to obtain a mixture; S4, eluting the mixture after dialysis to obtain an eluate, adding ammonium sulfate to the eluate for precipitation, collecting the precipitate by centrifugation, and purifying to obtain the mixed enzyme system. The final concentration of the ammonium sulfate in the eluate is 0.4 g / mL. The mixed enzyme system further comprises: an E. coli polymerase III beta clamp, an E. coli DnaA protein, an E. coli helicase, an E. coli initiation replication factor IHF2, an E. coli single-strand binding protein, an E. coli GyrA2B2, an E. coli topoisomerase IV, an E. coli topoisomerase III, an E. coli RecQ protein, an E. coli primase, an E. coli termination site binding protein, an E. coli RNase H, an E. coli DNA ligase and an E. coli DNA polymerase I. The molar ratio of the polymerase III tau clamp loader and catalytic core complex, the E. coli polymerase III beta clamp, the E. coli DnaA protein, the E. coli helicase, the E. coli initiation replication factor IHF2, the E. coli single-strand binding protein, the E. coli GyrA2B2, the E. coli topoisomerase IV, the E. coli topoisomerase III, the E. coli RecQ protein, the E. coli primase, the E. coli termination site binding protein, the E. coli RNase H, the E. coli DNA ligase and the E. coli DNA polymerase I in the mixed enzyme system is 5:40:100:20:40:400:50:5:50:50:400:2:10:50:

50.

2. The mixed enzyme system according to claim 1, characterized in that, The resuspension Buffer described in step S1 is a buffer containing 10% w / v sucrose, 1 mM PMSF, 0.5 mM DTT and 1 tablet / 50 mL cOmplete ™ Protease inhibitor cocktail buffer; The dilution buffer in step S1 is a buffer containing 10% w / v sucrose, 0.1 M NaCl, 15 mM spermidine and 0.8-1.2 mM EDTA. The core resuspension buffer in step S2 is a buffer containing 0.1 M NaCl and 10 mM spermidine.

3. The mixed enzyme system of claim 1, wherein, The ratio of the strain 1 slurry to the resuspension buffer in step S1 is: 1 g of the strain 1 slurry is resuspended in 0.5-1.5 mL of the resuspension buffer; the ratio of the strain 1 slurry to the dilution buffer in step S1 is: 1 g of the strain 1 slurry is diluted in 0.5-1.5 mL of the dilution buffer.

4. The mixed enzyme system of claim 1, wherein, The ratio of the strain 3 slurry to the core resuspension buffer in step S2 is: 1g of strain 1 slurry is added to 3-5mL of core resuspension buffer for resuspension.

5. The mixed enzyme system of claim 1, wherein, The preparation method of the strain 1 in step S1 comprises: transforming a plasmid expressing dnaX, a plasmid expressing holC and holD, and a plasmid expressing holA and holB into a competent cell; The preparation method of the strain 3 in step S2 comprises: transforming a plasmid expressing dnaE and a plasmid expressing dnaQ and holE into a competent cell.

6. The mixed enzyme system of claim 5, wherein, The dnaX comprises an amino acid sequence as shown in SEQ ID NO. 1; the holC comprises an amino acid sequence as shown in SEQ ID NO. 3; the holD comprises an amino acid sequence as shown in SEQ ID NO. 4; the holA comprises an amino acid sequence as shown in SEQ ID NO. 5; and the holB comprises an amino acid sequence as shown in SEQ ID NO.

6.

7. The mixed enzyme system of claim 5, wherein, The dnaE comprises an amino acid sequence as shown in SEQ ID NO. 7; the dnaQ comprises an amino acid sequence as shown in SEQ ID NO. 8; and the holE comprises an amino acid sequence as shown in SEQ ID NO.

9.

8. The mixed enzyme system of claim 5, wherein, The competent cell comprises any one or more of: BL21 (DE3), BL21 (DE3) pLysS, and Rosetta (DE3) pLysS.

9. The mixed enzyme system of claim 1, wherein, The E. coli polymerase III beta clamp is a purified product of a strain expressing dnaN; The E. coli DnaA protein is a purified product of a strain expressing dnaA protein; The E. coli helicase is a purified product of a strain co-expressing dnaC protein and dnaB protein; The E. coli initiation replication factor IHF2 is a purified product of a strain co-expressing ihfA protein and ihfB protein; The E. coli single-strand binding protein is a purified product of a strain expressing SSB protein; The E. coli GyrA2B2 is a co-purified product of a strain expressing gyrB protein and a strain expressing gyrA protein; The E. coli topoisomerase IV is a co-purified product of a strain expressing parE protein and a strain expressing parC protein; The E. coli topoisomerase III is a purified product of a strain expressing topB protein; The E. coli RecQ protein is a purified product of a strain expressing RecQ protein; The E. coli primase is a purified product of a strain expressing dnaG protein; The E. coli termination site binding protein is a purified product of a strain expressing tus protein.

10. The mixed enzyme system of claim 9, wherein, The dnaN comprises an amino acid sequence as shown in SEQ ID NO. 2; the dnaA protein comprises an amino acid sequence as shown in SEQ ID NO. 10; the dnaC protein comprises an amino acid sequence as shown in SEQ ID NO. 11; the dnaB protein comprises an amino acid sequence as shown in SEQ ID NO. 12; the ihfA protein comprises an amino acid sequence as shown in SEQ ID NO. 14; the ihfB protein comprises an amino acid sequence as shown in SEQ ID NO. 13; the SSB protein comprises an amino acid sequence as shown in SEQ ID NO. 15; the gyrB protein comprises an amino acid sequence as shown in SEQ ID NO. 16; the gyrA protein comprises an amino acid sequence as shown in SEQ ID NO. 17; the parE protein comprises an amino acid sequence as shown in SEQ ID NO. 18; the parC protein comprises an amino acid sequence as shown in SEQ ID NO. 19; the topB protein comprises an amino acid sequence as shown in SEQ ID NO. 20; the RecQ protein comprises an amino acid sequence as shown in SEQ ID NO. 21; the dnaG protein comprises an amino acid sequence as shown in SEQ ID NO. 22; and the tus protein comprises an amino acid sequence as shown in SEQ ID NO.

23.

11. A process for the preparation of the mixed enzyme system according to any one of claims 1 to 10, characterized in that, The preparation method comprises mixing the polymerase III tau clamp loader and catalytic core complex, the E. coli polymerase III beta clamp, the E. coli DnaA protein, the E. coli helicase, the E. coli initiation replication factor IHF2, the E. coli single-stranded binding protein, the E. coli GyrA2B2, the E. coli topoisomerase IV, the E. coli topoisomerase III, the E. coli RecQ protein, the E. coli primase, the E. coli termination site binding protein, the E. coli RNase H, the E. coli DNA ligase and the E. coli DNA polymerase I in a molar ratio to obtain a mixed enzyme system.

12. A cocktail enzyme buffer, characterized in that, The mixed enzyme buffer comprises the mixed enzyme system according to any one of claims 1-10.

13. Use of the mixed enzyme system according to any one of claims 1-10 or the mixed enzyme buffer according to claim 12 in the preparation of a circular DNA amplification product.

14. Use according to claim 13, characterized in that, The product comprises any one or more of a reagent, a kit, a chip.

15. A product for amplifying circular DNA, characterized by, The product comprises the mixed enzyme system according to any one of claims 1-10 or the mixed enzyme buffer according to claim 12.

16. A method of amplifying circular DNA, characterized by, The method comprises using the mixed enzyme system according to any one of claims 1-10, the mixed enzyme buffer according to claim 12 or the product according to claim 15.

17. The method of claim 16, wherein, The method comprises mixing a circular DNA template with the mixed enzyme system according to any one of claims 1-10, the mixed enzyme buffer according to claim 12 or the product according to claim 15.

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