Preparation method of linear closed DNA (deoxyribonucleic acid) and plasmid used in method

Abstract

The invention provides a preparation method of linear closed DNA (deoxyribonucleic acid) and plasmids used in the method. The plasmid comprises two editing regions of a prokaryotic telomerase target sequence which are connected in series in the same direction, and one or more restriction enzyme cutting sites are independently arranged at the two ends of each editing region. According to the method, high-purity LcDNA can be obtained, and the method can be better applied to clinical research and commercial application.

Description

Method for preparing linear closed DNA and plasmid used in the method

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202210955391.7 filed on August 10, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of genetic engineering, and in particular to a method for preparing linear closed DNA and a plasmid used in the method. Background Art

[0004] Gene therapy uses nucleic acid drugs to repair, replace, or regulate genes to prevent or treat diseases. Delivery vectors used in gene therapy can be broadly categorized as viral and non-viral. Viral vectors are widely used in cell and gene therapy due to their natural ability to infect cells and efficiently deliver genes. However, the use of viral vectors carries varying degrees of risk. Lentivirus and retrovirus vectors are primarily used to integrate target genes into the cell genome, which carries a high risk of genetic mutations. Adeno-associated virus vectors have a lower risk of insertional mutagenesis, but this risk still exists. Adenovirus vectors can exist episomally but may be toxic and immunogenic to cells. Certain viral vectors, such as AdV5 and AAV2, are widely distributed and many people have pre-existing immunity to them, making them unsuitable for gene delivery. All of these viral vector families, even with low seroprevalence in humans, cannot be repeatedly used as gene delivery vehicles due to the development of immunity. Non-viral vectors, however, exhibit significantly lower cytotoxicity and immunogenicity than viral vectors, and are therefore gaining increasing attention in both clinical research and commercial applications of gene and cell therapy.

[0005] Conventional non-viral vectors are mainly plasmids. Plasmid vectors are mainly composed of prokaryotic replicons, resistance genes, target genes and other elements. However, the prokaryotic replicons, resistance genes and CpG sequences on plasmid vectors may cause cells to produce unnecessary immune responses and cell carcinogenesis. Therefore, scientists used the prokaryotic telomerase TelN derived from Escherichia coli phage N15 to invent a method for producing linear closed DNA (LcDNA). LcDNA only carries the target gene and TelN recognition sites at both ends, effectively removing sequences such as the prokaryotic replicon and resistance genes on the plasmid that do not need to be delivered into the cell. Compared with conventional plasmid vectors, LcDNA has comparable or even better expression levels in vitro or in vivo, is safer, and has higher transfection efficiency.

[0006] Small-scale production of LcDNA is not difficult, but large-scale industrial production under GMP conditions faces many challenges. For example, the circular plasmid backbone used to produce LcDNA is too large and not universal; large-scale enzyme digestion is unstable; the purity does not reach clinical level, and organic reagents such as Triton, isopropanol and ethanol may be introduced, with a high risk of residues; the fidelity of in vitro amplification by polymerase Phi29 is lower than that of in vivo replication in E. coli, which may produce unnecessary mutations.

[0007] Summary of the Invention

[0008] The invention relates to a plasmid comprising two editing regions of protelomerase target sequences connected in series in the same direction. Two ends of the editing region are independently provided with one or more restriction endonuclease cutting sites.

[0009] In some embodiments, the length of the plasmid is ≤2500 bp, or ≤2300 bp, or ≤2100 bp. In some preferred embodiments, the length of the plasmid is ≤2100 bp.

[0010] In some embodiments, the plasmid further comprises a prokaryotic replicon and a selection gene.

[0011] In some embodiments, the restriction endonuclease cleavage site at each end is selected from any one or more of the following: the cleavage sites of AccI, AflII, AgeI, ApaI, AscI, AvrII, BamHI, BglI, BglII, BsaI, BspQI, BstBI, BsteII, ClaI, EcoNI, EcoRI, EcoRV, FseI, HindIII, KpnI, MfeI, MluI, NcoI, NdeI, NheI, NotI, PacI, PstI, PvuI, PvuII, SacI, SacII, SalI, ScaI, SmaI, SpeI, StuI, SwaI, XbaI, XhoI and XmaI, preferably selected from any one or more of the cleavage sites of HindIII, SalI, SmaI, BamHI, EcoRV, XbaI, KpnI and EcoRI. In some preferred embodiments, HindIII, SalI, SmaI and BamHI restriction sites are inserted near one end of the editing region, and EcoRV, XbaI, KpnI and EcoRI restriction sites are inserted near the other end of the editing region.

[0012] In some embodiments, the protelomerase target sequence comprises the sequence shown in SEQ ID NO: 1, or a nucleotide sequence that has at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% identity to the sequence shown in SEQ ID NO: 1 and is capable of exerting telomerase function. In some preferred embodiments, the protelomerase target sequence is shown in SEQ ID NO: 1.

[0013] In some embodiments, the plasmid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% identical to the sequence shown in SEQ ID NO: 2. In some preferred embodiments, the sequence of the plasmid is shown in SEQ ID NO: 2.

[0014] The present invention also relates to a recombinant plasmid having a target gene inserted between the two protelomerase target sequences of the plasmid described above.

[0015] The present invention also relates to a method for preparing linear closed DNA, comprising:

[0016] a) using protelomerase to digest a recombinant plasmid containing a target gene to separate the target gene from the miscellaneous nucleic acids in the recombinant plasmid;

[0017] b) digesting the nucleic acid using at least two restriction endonucleases to expose the ends of the heterologous nucleic acid, and removing the heterologous nucleic acid using an exonuclease;

[0018] c) isolating and purifying the linear closed DNA containing the target gene;

[0019] The recombinant plasmid comprises a protelomerase target sequence and a restriction endonuclease cutting site, and a target gene is inserted between the protelomerase target sequences.

[0020] In some embodiments, the recombinant plasmid mentioned in the above preparation method can be a plasmid in which the target gene is inserted between the two protelomerase target sequences of the plasmid described above.

[0021] In some embodiments, the exonuclease in the above method includes Exonuclease III and / or T5 Exonuclease. In some embodiments, the amount of the exonuclease added is 3000U / mg to 6000U / mg. In some specific embodiments, the exonuclease cleavage temperature is 34°C to 40°C, and the cleavage time is 2 to 12 hours; preferably, the cleavage time is 3 to 6 hours. In other specific embodiments, after the exonuclease cleavage, EDTA is added to a final concentration of 1mM to 50mM and the temperature is raised to 70°C to 80°C for 5 to 30 minutes. In some specific embodiments, after the protelomerase cleavage is completed and / or the restriction endonuclease cleavage is completed, the temperature is raised to 70°C to 80°C for 5 to 30 minutes.

[0022] In some embodiments, the separation and purification method in the above preparation method comprises one of gel filtration chromatography, anion chromatography, concentration and liquid exchange, and sterile filtration, or a combination thereof. In some preferred embodiments, the separation and purification method comprises gel filtration chromatography and anion chromatography in sequence.

[0023] In some specific embodiments, the filler of the gel filtration chromatography is Bestarose 6FF, and the eluate with ≥50 mAu of the first UV peak is collected.

[0024] In other specific embodiments, the filler material for the anion chromatography is Fractogel EMD DEAE. In some preferred embodiments, the loading conditions for the anion chromatography include: using a loading solution containing 30% to 70% chromatography buffer B, a loading capacity of 0.2 mg / mL to 1.0 mg / mL, and a loading flow rate of ≤120 cm / h; the chromatography buffer B comprises 0.5M to 1.5M NaCl, 5mM to 15mM EDTA, and a buffer substance, with a pH of 7.2 to 7.8. In some preferred embodiments, the elution conditions for the anion chromatography include: elution with 65% to 100% chromatography buffer B, an elution flow rate of 30 cm / h to 120 cm / h, and collecting ≥100 mAu in the eluate.

[0025] The present invention also relates to a kit comprising protelomerase and the plasmid as described above;

[0026] Or comprising protelomerase and the recombinant plasmid as described above.

[0027] In some embodiments, the kit further comprises a restriction endonuclease and / or an exonuclease, wherein the restriction endonuclease is capable of recognizing the restriction endonuclease cleavage site.

[0028] The present invention also provides the linear closed DNA prepared by the above preparation method.

[0029] The present invention uses a plasmid produced by bacterial fermentation as the starting material, rather than obtaining it through polymerase amplification methods such as Phi29, which results in higher fidelity. Moreover, through specific design, the plasmid can be kept small enough, thereby improving the overall yield of LcDNA production from the source. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 is a schematic diagram of the LcDNA production and purification process;

[0032] Figure 2A is a diagram of the universal vector backbone CFL Universal;

[0033] FIG2B is a full sequence map of the vector of FIG2A inserted with the target sequence (GOI); wherein Kan promoter: kanamycin gene promoter, KanR: kanamycin resistance gene, pUC origin: pUC promoter, MCS: multiple cloning site; GOI: target sequence; telR and telL: protelomerase target sequence;

[0034] Figure 3 is agarose gel electrophoresis (AGE) detection of LcDNA, A and B are AGE detection patterns of a batch of 60 mg of LcDNA intermediate samples and finished products after enzyme digestion and purification of circular plasmids containing the target sequence, and C is AGE detection pattern of another batch of 60 mg of LcDNA intermediate samples and finished products after enzyme digestion and purification of circular plasmids containing the target sequence;

[0035] In A to C, lane M1: supercoiled DNA ladder; lane M2: 1 kb DNA ladder;

[0036] A: Lane 1: TelN digestion product; Lane 2: Exonuclease digestion product; Lane 3: GF chromatography product; Lane 4: AEX chromatography product;

[0037] B: Lane 1: LcDNA finished product; Lane 2: 1×TAE buffer;

[0038] C: Lane 1: TelN digestion product; Lane 2: Exonuclease digestion product; Lane 3: GF chromatography product; Lane 4: AEX chromatography product; Lane 5: LcDNA finished product;

[0039] Figures 4A to 4E are LcDNA gel filtration chromatograms; Figures 4A and 4B are respectively LcDNA gel filtration chromatograms of two 60 mg batches of circular plasmids containing the target sequence after enzyme digestion, Figure 4C is a LcDNA gel filtration chromatogram of a 100 mg batch of circular plasmids containing the target sequence after enzyme digestion, and Figures 4D and 4E are LcDNA gel chromatograms of two 150 mg batches of circular plasmids containing the target sequence after enzyme digestion;

[0040] Figures 5A to 5E are LcDNA anion chromatograms. Figures 5A and 5B are the anion chromatograms of two 60 mg batches of circular plasmid LcDNA containing the target sequence, Figure 5C is the anion chromatogram of a 100 mg batch of circular plasmid LcDNA containing the target sequence, and Figures 5D and 5E are the anion chromatograms of two 150 mg batches of circular plasmid LcDNA containing the target sequence, respectively.

[0041] Figures 6A and 6B are graphs showing the optimized loading conditions for LcDNA anion chromatography. Figure 6A shows the loading solution containing 30% chromatography buffer B, and Figure 6B shows the loading solution containing 70% chromatography buffer B.

[0042] Figures 7A and 7B are graphs showing optimization of the sample loading capacity for LcDNA anion chromatography. The sample loading capacity in Figure 7A is 0.25 mg / mL, and the sample loading capacity in Figure 7B is 1.0 mg / mL.

[0043] FIG8 is a graph showing linear elution of LcDNA by anionic chromatography (gradient elution from 0 to 100% of chromatographic buffer B), wherein the maximum UV peak appears at 67.5% of chromatographic buffer B;

[0044] In FIG4 to FIG8 , curve a represents conductivity, and curve b represents ultraviolet peak. DETAILED DESCRIPTION

[0045] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0046] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0047] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0048] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0049] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0050] Concentration values ​​used in this invention include fluctuations within a certain range. For example, fluctuations within a certain precision range are permitted. For example, for 2%, fluctuations within ±0.1% are permitted. For larger values ​​or values ​​that do not require overly precise control, greater fluctuations are permitted. For example, for 100 mM, fluctuations within ±1%, ±2%, ±5%, etc. are permitted. Regarding molecular weight, fluctuations within ±10% are permitted.

[0051] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0052] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0053] In the present invention, "preferably", "better", "more preferably", and "suitably" are merely descriptions of preferred implementation methods or examples, and should be understood to not limit the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, to being selected from either of the two parallel options of "with" or "without". If multiple "options" appear in a technical solution, unless otherwise specified and without contradiction or mutual restriction, each "optional" is independent.

[0054] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this invention are cited with all their contents and all their purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description in this application.

[0055] The first aspect of the present invention relates to a plasmid comprising two editing regions of protelomerase target sequences connected in series in the same direction, wherein one or more restriction endonuclease cutting sites are independently provided at both ends of the editing region.

[0056] In some embodiments, the length of the plasmid is ≤2500 bp, such as 2400 bp, 2300 bp, 2200 bp, 2100 bp, 2050 bp, 2000 bp, 1950 bp, 1900 bp, 1850 bp, 1800 bp or less.

[0057] In some embodiments, the plasmid further comprises a prokaryotic replicon and a selection gene.

[0058] Typical examples of screening genes include antibiotic resistance genes, such as kanamycin resistance gene (KanR), tetracycline resistance gene (tetR), neomycin phosphotransferase gene (npt), etc. Screening genes can also be certain compound detoxification enzyme genes, carbohydrate metabolism enzyme selection marker genes, etc.

[0059] In some embodiments, the number of restriction endonuclease cleavage sites at each end is independently selected from 1, 2, 3, 4, 5, 6 or more, and preferably the types of restriction endonuclease cleavage sites located at the same end are different.

[0060] In a more preferred embodiment, the restriction endonuclease cleavage sites located at the same end of the editing region overlap.

[0061] In a more preferred embodiment, all restriction endonuclease cutting sites are different.

[0062] The restriction endonuclease cleavage site at each end can be selected from any one or more of the following: the cleavage sites of AccI, AflII, AgeI, ApaI, AscI, AvrII, BamHI, BglI, BglII, BsaI, BspQI, BstBI, BsteII, ClaI, EcoNI, EcoRI, EcoRV, FseI, HindIII, KpnI, MfeI, MluI, NcoI, NdeI, NheI, NotI, PacI, PstI, PvuI, PvuII, SacI, SacII, SalI, ScaI, SmaI, SpeI, StuI, SwaI, XbaI, XhoI and XmaI, preferably selected from any one or more of the cleavage sites of HindIII, SalI, SmaI, BamHI, EcoRV, XbaI, KpnI and EcoRI. In one embodiment, HindIII, SalI, SmaI, and BamHI recognition sites are inserted near one end of the editing region, and EcoRV, XbaI, KpnI, and EcoRI recognition sites are inserted near the other end of the editing region, thereby providing 16 groups of enzyme digestion combinations to choose from. This improves practicality and broad spectrum.

[0063] According to the present invention, closed linear DNA molecules are generated by the action of protelomerase on DNA amplified from a DNA template containing at least one protelomerase target sequence. A protelomerase target sequence is any DNA sequence present in the DNA template that enables its conversion into closed linear DNA by the enzymatic activity of protelomerase. In other words, the protelomerase target sequence is essential for the cleavage and religation of double-stranded DNA by protelomerase to form covalently closed linear DNA.

[0064] Protelomerase and its target sequence are well known to those skilled in the art, and its description is for example described in CN102301010A disclosed on 2011-12-28. Typically, the protelomerase target sequence includes any complete palindrome, i.e., any double-stranded DNA sequence with dual rotational symmetry. In Borrelia burgdorferi, the length of the complete inversion repeat sequence is 14 base pairs. In various normal temperature phages, the length of the complete inversion repeat sequence is 22 base pairs or longer. In addition, in some cases, such as Escherichia coli N15, the central complete inversion palindrome sequence is flanked by inversion repeat sequences, i.e., forming a part of a larger incomplete inversion palindrome sequence. In some embodiments, the protelomerase target sequence is derived from Escherichia coli N15 phage, Klebsiella Phi K02 phage, Yersinia Py54 phage, Halomonas Phi HAP phage, Vibrio VP882 phage, or Borrelia burgdorferi lpB31.16, or a variant thereof. Preferably, the protelomerase target sequence is derived from phage N15 TelN or a variant thereof. In some specific embodiments, the protelomerase target sequence comprises the sequence shown in SEQ ID NO: 1, or a nucleotide sequence that has at least 80% identity to the sequence shown in SEQ ID NO: 1 and can exert telomerase function, for example, a nucleotide sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, for example, one or more nucleotides are truncated, inserted, substituted, and / or deleted.

[0065] The plasmid can be transformed from a conventional cloning vector, such as a prokaryotic vector, preferably an Escherichia coli plasmid vector, and more preferably a plasmid of less than 2000bp. In some specific embodiments, it is transformed from pUC57-Kan-mini (1824bp). In some embodiments, the plasmid comprises a nucleotide sequence having at least 80% identity to the sequence shown in SEQ ID NO: 2, for example, a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, for example, one or more nucleotides are truncated, inserted, substituted, and / or deleted. In some specific embodiments, the plasmid comprises the nucleotide sequence shown in SEQ ID NO: 2.

[0066] In the editing region, in some embodiments, two tandem protelomerase target sequences are directly connected; in some embodiments, the two tandem protelomerase target sequences further contain less than 30 bp, or less than 20 bp, or less than 10 bp of nucleotide sequences, and these nucleotide sequences are preferably sites or related sequences that are conducive to the insertion of the target gene.

[0067] The second aspect of the present invention relates to a recombinant plasmid having a target gene inserted between the two protelomerase target sequences of the plasmid described above.

[0068] In some embodiments, the expression product of the target gene is interfering RNA or an aptamer.

[0069] The interfering RNA may be selected from, for example, siRNA, shRNA or miRNA.

[0070] In some embodiments, the expression product of the target gene is mRNA, and such mRNA is preferably capable of expressing a protein, for example, a protein that imparts certain desired characteristics to the target cell, such as a fluorescent protein that allows cell tracking, provides an active enzyme that is lost or altered in the target cell, and the like.

[0071] The target gene may include, for example, a gene (nucleotide sequence) encoding a defective or missing protein in a recipient individual or target cell; a gene encoding a protein with a desired biological or therapeutic effect (e.g., antibacterial, antiviral or antitumor / anticancer function); a nucleotide sequence encoding an RNA that inhibits or reduces the production of harmful or otherwise undesirable proteins (e.g., a nucleotide sequence encoding an RNA interfering agent as defined above); and / or a nucleotide sequence encoding an antigenic protein.

[0072] Suitable genes of interest include, but are not limited to, nucleic acids encoding proteins for treating endocrine, metabolic, hematological, cardiovascular, neurological, musculoskeletal, urological, pulmonary, and immune disorders, including, for example, cancer, inflammatory disorders, immune disorders, chronic and infectious disorders. Cancers such as tumors arising from lesions in any of the bones, bone connections, muscles, lungs, trachea, heart, spleen, arteries, veins, blood, capillaries, lymph nodes, lymphatic vessels, lymph fluid, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testicles, vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid gland, parathyroid gland, adrenal gland, pituitary gland, pineal gland, pancreatic islets, thymus gland, gonads, sublingual gland, and parotid gland; immune disorders such as systemic lupus erythematosus, multiple sclerosis, type I diabetes, psoriasis The infectious disease may be any one of viral, bacterial, fungal and parasitic infections, or a combination thereof.

[0073] Suitable genes of interest include, but are not limited to, genes encoding at least one of the following proteins: interferons (e.g., IFN-γ, IFN-α, IFN-β, IFN-ω, IFN-τ); insulin (e.g., Novolin, Humulin, Humalog, Humalog, Lantus, etc.); erythropoietin ("EPO"; e.g., epoetin-α, darbepoetin-α, epoetin-β; etc.); antibodies (e.g., monoclonal antibodies, such as rituximab, infliximab, trastuzumab, Humira™ (adalimumab), omalizumab, tositumomab, Rapone tiva™ (efalizumab), Erbitux™ (cetuximab), bevacizumab; etc.); antigen-binding fragments including monoclonal antibodies (e.g., ranibizumab); blood factors (e.g., alteplase, tissue plasminogen activator protein, recombinant human factor VIIa, factor VIIa, factor VIII, factor IX, beta-globulin, hemoglobin; etc.); colony stimulating factors (e.g., (filgrastim; G-CSF), Neulasta (pegylated filgrastim), granulocyte colony stimulating factor (G-CSF), granulocyte monocyte colony stimulating factor, macrophage colony stimulating factor, megakaryocyte colony-stimulating factor; etc.); growth hormones (e.g., somatotropin, somatotropin; etc.); interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9; etc.); growth factors (e.g., (beclapermin; PDGF), (trifermin; bFGF), (anciplostatin; stem cell factor), keratinocyte growth factor, acidic fibroblast growth factor, stem cell factor, basic fibroblast growth factor, hepatocyte growth factor; etc.); soluble receptors (e.g., TNF-α binding soluble receptors, such as (etanercept), soluble VEGF receptors, soluble interleukin receptors, soluble gamma / delta T cell receptors; etc.); enzymes (e.g., α-glucosidase, β-glucocerebrosidase, alglucosidase), enzyme activators (e.g., tissue plasminogen activator); chemokines (e.g., IP-10, Mig, Groα / IL-8, RANTES, MIP-1α, MIP-1β, MCP-1, PF-4; etc.); angiogenic agents (e.g., vascular endothelial growth factor (VEGF); anti-angiogenic agents (e.g., soluble VEGF receptor); protein vaccines;Neuroactive peptides, such as bradykinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin-releasing hormone, β-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, cerebellofloxacin, dynorphin, neurotensin, motilin, thyroid-stimulating hormone, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, sleep peptide, etc.; other proteins, such as thrombolytics, atrial natriuretic peptide, bone morphogenetic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle-stimulating hormone, human α -1 antitrypsin, leukemia inhibitory factor, transforming growth factor, insulin-like growth factor, luteinizing hormone, macrophage activating factor, tumor necrosis factor, neutrophil chemotactic factor, nerve growth factor, tissue inhibitor of metalloproteinases; vasoactive intestinal peptide, angiopoietin, angiotropin, fibrinogen; hirudin; leukemia inhibitory factor; IL-1 receptor antagonist (e.g., (anakinra)); ion channels, such as cystic fibrosis transmembrane conductance regulator (CFTR); dystrophin; utrophin (a tumor suppressor); lysosomal enzyme acid α-glucosidase (GAA); etc. Suitable target genes also include those encoding functional fragments of any of the above proteins; and nucleic acids encoding functional variants of any of the above proteins. ;

[0074] In some embodiments, the gene of interest can be inserted into the plasmid in the form of an expression cassette.

[0075] It is easy to understand that the plasmid / recombinant plasmid may further comprise at least one of the following elements: a promoter, an enhancer, an internal ribosome entry site, a reporter gene, a coupled marker gene, and a transcription termination signal. These elements are preferably located in the expression cassette of the target gene.

[0076] A promoter is a nucleotide sequence that initiates and regulates the transcription of a polynucleotide. Promoters include inducible promoters (the expression of the polynucleotide sequence operably connected to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressive promoters (the expression of the polynucleotide sequence operably connected to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The term "promoter" is intended to include full-length promoter regions and functional (e.g., controlling transcription or translation) fragments of these regions. Promoters preferably include promoters that originate from prokaryotic expression. However, when a target gene is present, a promoter for controlling the expression of the target gene may also be included, may be a cell-free specific promoter, or may be a "cell-specific," "cell type-specific," "cell lineage-specific," or "tissue-specific" promoter, which allows expression in cells and tissue types of limited species, respectively.

[0077] Typical examples of reporter genes include fluorescent protein genes, luciferase genes, and LacZ. Both fluorescent proteins and luciferases are luminescent proteins, and fluorescence expression can be detected using a camera or similar device. Fluorescent proteins work by absorbing light of one color (excitation) and then emitting lower-energy light of a different color (emission). In contrast, luciferase (and other bioluminescent enzymes) emit light by catalyzing a chemical reaction with a substrate (i.e., luciferin). Unlike the two markers mentioned above, LacZ does not emit light. The product of the LacZ gene, β-galactosidase, can catalyze the conversion of X-gal into an opaque blue compound similar to indigo. Fluorescent proteins include green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein. Green fluorescent protein can be the common GFP, or a modified GFP gene, such as the enhanced GFP gene EGFP; blue fluorescent protein can be selected from EBFP, Azuritc, TagBFP, etc.; yellow fluorescent protein can be selected from EYFP, Ypct, PhiYFP, etc.; orange fluorescent protein can be selected from mKO, mOrange, mBanana, etc.; red fluorescent protein can be selected from TagRFP, mRuby, mCherry, mKatc, etc.

[0078] The coupled marker gene is typically a gene capable of expressing biotin or a derivative thereof, wherein the derivative of biotin is any one of D-biotin, activated biotin, biocytin, ethylenediamine biotin, cadaverine biotin and desthiobiotin.

[0079] An internal ribosome entry site (IRES) is a genetic engineering element hundreds of base pairs (bp) long. IRES were originally discovered in viruses and later found on some eukaryotic mRNAs. IRES guides ribosomes into this sequence to translate the 3' open reading frame. Inserting an IRES sequence between two open reading frames (ORFs) within the same transcript allows for the production of two independent proteins from a single transcript.

[0080] The transcription termination signal refers to the stem-loop structure formed by a sequence of RNA produced during the transcription process, which can be specifically recognized by the RNA polymerase transcription complex to terminate transcription.

[0081] In some embodiments, the target gene is a nucleic acid vaccine.

[0082] A third aspect of the present invention relates to a method for preparing linear closed DNA, comprising:

[0083] a) using protelomerase to digest a recombinant plasmid containing a target gene to separate the target gene from the miscellaneous nucleic acids in the recombinant plasmid;

[0084] b) digesting the nucleic acid using at least two restriction endonucleases to expose the ends of the heterologous nucleic acid, and removing the heterologous nucleic acid using an exonuclease;

[0085] c) isolating and purifying the linear closed DNA containing the target gene;

[0086] The recombinant plasmid comprises a protelomerase target sequence and a restriction endonuclease cutting site, and a target gene is inserted between the protelomerase target sequences.

[0087] Trapped nucleic acids refer to unnecessary or additional sequences other than the target gene. These unnecessary or additional sequences typically include various elements in the plasmid, such as replicons and selection genes, as described above. However, it will be readily understood that the tramp nucleic acids herein refer to most, but not all, of these unnecessary or additional sequences. For example, at least the protelomerase target sequences (e.g., telL and telR) directly linked to the target gene cannot be removed.

[0088] In some embodiments, the recombinant plasmid is the recombinant plasmid defined above, in which case the contents defined in the first and second aspects of the present invention also apply to the third aspect.

[0089] In some embodiments, the added amount of protelomerase is 8000 U / mg to 15000 U / mg, for example, 9000 U / mg, 10000 U / mg, 11000 U / mg, 12000 U / mg, 13000 U / mg, or 14000 U / mg.

[0090] In some embodiments, the enzymatic cleavage temperature of the protelomerase is 25°C to 35°C, preferably 27°C to 33°C.

[0091] In some embodiments, the enzymatic digestion time of the protelomerase is 1 to 8 hours, for example, 2, 3, 4, 5, 6, or 7 hours.

[0092] In some embodiments, the added amount of each restriction endonuclease is independently selected from 1000 U / mg to 6000 U / mg, such as 2000 U / mg, 3000 U / mg, 4000 U / mg, and 5000 U / mg.

[0093] In some embodiments, the cleavage temperature of the restriction endonuclease is 32°C to 42°C, preferably 34°C to 40°C, such as 35°C, 36°C, 37°C, 38°C, or 39°C.

[0094] In some embodiments, the restriction endonuclease digestion time is 1 to 4 hours, such as 2 hours or 3 hours.

[0095] In some embodiments, the exonuclease comprises Exonuclease III and / or T5 Exonuclease.

[0096] In some embodiments, the added amount of the nuclease is 3000 U / mg to 6000 U / mg, for example, 3500 U / mg, 4000 U / mg, 4500 U / mg, 5000 U / mg, or 5500 U / mg.

[0097] In some embodiments, the exonuclease cleavage temperature is 34°C to 40°C, for example, 35°C, 36°C, 37°C, 38°C, or 39°C.

[0098] In some embodiments, the exonuclease cleavage time is 2 to 12 hours, such as 3, 4, 5, 6, 7, 8, 9, 10, or 11 hours, and preferably 3 to 6 hours.

[0099] In some embodiments, after exonuclease cleavage, EDTA is added to a final concentration of 1 mM to 50 mM, for example, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, or 45 mM.

[0100] In some embodiments, after exonuclease cleavage, EDTA is added and the temperature is raised to 70° C. to 80° C. for 5 to 30 minutes, such as 10, 15, 20, or 25 minutes.

[0101] After exonuclease cleavage, EDTA and high temperature are used to inactivate the enzymes, effectively inhibiting the residual activity of endonucleases and exonucleases in the system and greatly improving the stability of large-scale production.

[0102] In some embodiments, after the protelomerase digestion and / or restriction endonuclease digestion is completed, the temperature is raised to 70° C. to 80° C. for 5 to 30 minutes, such as 10, 15, 20, or 25 minutes.

[0103] In some embodiments, the separation and purification method comprises one of gel filtration chromatography, anion chromatography, concentration and liquid exchange, and sterilizing filtration, or a combination thereof.

[0104] In some embodiments, the separation and purification method comprises gel filtration chromatography and anion chromatography in sequence. In other embodiments, the separation and purification method comprises gel filtration chromatography, anion chromatography and concentration and liquid exchange in sequence.

[0105] The process of purifying LcDNA by combining gel filtration chromatography and anion chromatography not only improves the purity and quality of LcDNA products, but also makes them more compliant. In addition, this method can significantly increase product yield while ensuring purity, making it more suitable for commercial use.

[0106] In some embodiments, the filler of the gel filtration chromatography is Bestarose 6FF, and the eluate with ≥50 mAu of the first UV peak is collected.

[0107] In some embodiments, the filler material for the anion chromatography is Fractogel EMD DEAE.

[0108] In some embodiments, the loading conditions of the anion chromatography include:

[0109] Use a loading solution containing 30% to 70% chromatography buffer B (e.g., 40%, 50%, 60%, by volume), a loading capacity of 0.20 mg / mL to 1.0 mg / mL (e.g., 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL), and a loading flow rate ≤ 120 cm / h;

[0110] The chromatography solution B contains 0.5M-1.5M NaCl, 5mM-15mM EDTA and a buffer substance, and has a pH of 7.2-7.8.

[0111] As used herein, the term "buffer substance" refers to an aqueous solution or composition that resists changes in pH when an acid or base is added to the solution or composition. This resistance to pH changes is due to the buffering properties of such solutions. Therefore, solutions or compositions that demonstrate buffering activity are referred to as buffers or buffered solutions. The buffer that can be used in the method of the present invention is preferably selected from phosphate buffer, phosphate buffered saline buffer (PBS), 2-amino-2 hydroxymethyl-1,3-propylene glycol (TRIS) buffer, TRIS buffered saline solution (TBS) and TRIS / EDTA (TE) etc.

[0112] In some preferred embodiments, the loading conditions of the anion chromatography include:

[0113] Use a loading solution containing 50% chromatography buffer B (volume percentage), a loading capacity of 0.20 mg / mL to 1.0 mg / mL (e.g., 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL), and a loading flow rate of ≤120 cm / h;

[0114] The chromatography solution B contains 0.5M-1.5M NaCl, 5mM-15mM EDTA and 50mM-150mM Tris-HCl, with a pH of 7.2-7.8.

[0115] More preferably, the chromatography solution B comprises 1 M NaCl, 10 mM EDTA, 100 mM Tris-HCl, and has a pH of 7.5.

[0116] In some embodiments, the elution conditions of the anion chromatography include:

[0117] Elution was performed with 65% to 100% chromatography fluid B (e.g., 70%, 80%, 90%, by volume), at an elution flow rate of 30 cm / h to 120 cm / h (e.g., 40 cm / h, 50 cm / h, 60 cm / h, 70 cm / h, 80 cm / h, 90 cm / h, 100 cm / h, 110 cm / h), and ≥100 mA of the eluate was collected.

[0118] In some preferred embodiments, the elution conditions of the anion chromatography include:

[0119] Elution was performed with 80% chromatography fluid B (volume percentage), at an elution flow rate of 30 cm / h to 120 cm / h (e.g., 40 cm / h, 50 cm / h, 60 cm / h, 70 cm / h, 80 cm / h, 90 cm / h, 100 cm / h, 110 cm / h), and ≥100 mAu of the eluate was collected.

[0120] In some embodiments, the concentration and liquid replacement method is tangential flow filtration.

[0121] A fourth aspect of the present invention relates to a kit comprising protelomerase and the plasmid as described above;

[0122] Or comprising protelomerase and the recombinant plasmid as described above.

[0123] In some embodiments, the kit further comprises a restriction endonuclease and / or an exonuclease, wherein the restriction endonuclease can recognize the restriction endonuclease cleavage site.

[0124] The kit may also include conventional components such as nuclease-free water and instructions for use.

[0125] The fifth aspect of the present invention provides a linear closed DNA obtained by the above preparation method. The linear closed DNA contains a target gene.

[0126] The present invention also provides a composition comprising the above-mentioned linear closed DNA. In some embodiments, the composition comprises the above-mentioned linear closed DNA at a purity of at least 90%. In some preferred embodiments, the composition comprises the above-mentioned linear closed DNA at a purity of at least 99%.

[0127] The description of the first and second aspects of the present invention also applies to the fourth and fifth aspects.

[0128] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0129] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0130] Example 1 Design of the universal circular plasmid vector CFL universal for LcDNA production

[0131] By designing a universal LcDNA template plasmid backbone, CFL Universal, the size of the template plasmid backbone is minimized, thereby improving the theoretical recovery rate of LcDNA from the source. The specific design method first inserts two TelN enzyme recognition sequences, TelRL (56 bp, sequence information see SEQ ID NO: 1), into pUC57-Kan-mini (1824 bp). Then, HindIII, SalI, SmaI, and BamHI recognition sites are sequentially inserted near the protelomerase TelN recognition site at one end, and EcoRV, XbaI, KpnI, and EcoRI recognition sites are inserted near the TelN recognition site at the other end. This provides 16 groups of restriction enzyme digestion combinations for the second step, greatly improving the practicality and broad spectrum of CFL Universal. The modified CFL Universal is 2017 bp in length. The target sequence to be inserted only needs to be designed between the two TelN recognition sites, TelRL, of CFL Universal. The map is shown in Figure 2A (sequence information is shown in SEQ ID NO: 2), in which the GOI target gene is inserted as an example, with a length of 3169bp, as shown in Figure 2B. The designed circular plasmid sequence was commissioned to Nanjing GenScript Biotechnology Co., Ltd. for regulatory synthesis. It then underwent a series of steps, including E. coli library construction, high-density fermentation, cell lysis, clarification and filtration, concentration and incubation, chromatography purification, concentration and liquid exchange, sterile filtration, and filling, to obtain a high-purity, high-quality circular plasmid as the starting material for LcDNA production.

[0132] Example 2 LcDNA digestion and purification

[0133] (1) LcDNA digestion

[0134] The three-step enzymatic digestion of LcDNA involves TelN digestion, exonuclease digestion, and endonuclease digestion. When using the CFL universal vector as the starting circular plasmid backbone, the TelN digestion process parameters are: TelN enzyme dosage 10,000 U / mg, digestion temperature 30°C, digestion time 3 hours, end temperature 75°C, and treatment time 15 minutes. Endonuclease digestion process parameters are: select endonucleases without corresponding recognition sites based on the inserted target sequence, such as HindIII, SalI, SmaI, BamHI, EcoRV, XbaI, KpnI, and EcoRI. Add two endonucleases at 3,000 U / mg each, digestion temperature 37°C, digestion time 2 hours, end temperature 75°C, and treatment time 15 minutes. The exonuclease digestion process parameters are: Exo III or T5 Exo, added at 4500 U / mg, digestion temperature at 37°C, and digestion time for 4 hours. To terminate the digestion, add EDTA to a final concentration of 15 mM, at 75°C, for 10-20 minutes.

[0135] (2) LcDNA gel filtration chromatography (GF)

[0136] After the three-step enzymatic digestion of LcDNA, the digestion reaction solution was centrifuged or filtered, and then subjected to gel filtration chromatography. Gel filtration chromatography parameters were as follows: Bestarose 6FF filler, column height ≥ 20 cm, symmetry factor 0.8–1.8, and plate number > 2000. The loading volume was ≤ 0.3 column volumes, the loading flow rate was 60 cm / h, and the elution flow rate was ≤ 120 cm / h. The equilibration and elution buffer used was GF Buffer A (0.5 M NaCl, 5 mM EDTA, 50 mM Tris-HCl). The eluate containing ≥ 50 mA of Au in the first UV peak was collected.

[0137] (3) LcDNA anion exchange chromatography (AEX)

[0138] After gel filtration chromatography, the collected eluate was subjected to anion chromatography. The anion chromatography process parameters were: Fractogel EMD DEAE filler, column height ≤ 20 cm, symmetry factor 0.8-1.8, and plate number > 2000. The loading conditions were a sample solution containing 30-70% chromatography buffer B (volume ratio), a sample load of 0.25-1.0 mg / mL, a loading flow rate ≤ 120 cm / h, and a retention time ≥ 6 minutes. The elution flow rate was 30-120 cm / h, and ≥ 100 mA of eluate was collected. Buffer A consisted of 10mM EDTA, 100mM Tris-HCl, pH 7.5, and Buffer B consisted of 1M NaCl, 10mM EDTA, 100mM Tris-HCl, pH 7.5. Equilibration was performed with 100% Buffer A, impurities were washed with 58% Buffer B, and elution was performed with 65%-100% Buffer B. Optimization of elution conditions is shown in Figure 8 , where a gradient elution from 0% to 100% Buffer B was used. The maximum UV peak was observed at 67.5% Buffer B. Optimization data for the anion chromatography process at 80% Buffer B are shown in Figure 1A and Table 1B, and the corresponding elution curves are shown in Figures 6A, 6B, 7A, and 7B.

[0139] Table 1 Summary of LcDNA anion chromatography optimization data

[0140] Table 1A Optimization of loading conditions

[0141] Table 1B Sample loading capacity optimization

[0142] (4) LcDNA concentration and medium replacement

[0143] After the anion chromatography, the collected eluate is concentrated by tangential flow filtration to a dialysis point of 1.0 mg / mL ± 0.3 mg / mL, and then the final storage buffer such as Tris-EDTA is used to replace the solution by 10 ± 1 times the volume. The pore size of the tangential flow filtration membrane is 10 kD and the size is 50 cm. 2 , 0.01m 2 , 0.1m 2 , 0.5m 2 , you can choose the membrane package of appropriate specifications according to the production scale.

[0144] (5) Sterile filtration of LcDNA

[0145] After concentrating and replacing the liquid, sterilize and filter the intermediate cDNA sample. Use a sterilizing filter with a pore size of 0.22 μm, a syringe filter, a 1 L filter cup, or a capsule filter. Select the appropriate filter size based on the volume of the sample after concentration and replacement.

[0146] (6) LcDNA filling and storage

[0147] After sterilization and filtration, the LcDNA samples are filled according to the project packaging specifications in a clean bench or isolator. The finished LcDNA products are stored in an ultra-low temperature freezer at -70±10℃.

[0148] Example 3 LcDNA quality detection

[0149] The length of the LcDNA sample is 3169 bp. The full sequence of the circular plasmid into which the LcDNA was inserted is shown in Figure 2B. The quality of the LcDNA intermediate samples and finished products was tested. The test methods and corresponding results are as follows:

[0150] 3.1 Agarose gel electrophoresis (AGE) detection of LcDNA intermediate samples and finished products

[0151] Intermediate and finished LcDNA samples were run on 0.7% agarose gels using 1× TAE as the electrophoresis buffer. The electrophoresis voltage was 150 V for 30 minutes. The gels were then imaged using Image Lab software. The AGE test results are shown in Figure 3 and Table 2.

[0152] Table 2 Summary of LcDNA production data

[0153] Note: Total yield = LcDNA finished product amount / enzyme-digested circular plasmid sample amount × 100%

[0154] Compared to small-scale production methods, circular plasmids undergo TelN digestion, agarose gel electrophoresis (AGE) separation, gel excision, and purification using the Axygen Gel Recovery Kit (AXYGEN, Catalog No. UE-GX-50). The LcDNA yield is low, less than 10%, and the quality does not meet clinical or commercial requirements. Fermentation using engineered bacteria capable of producing LcDNA, followed by one-step Q anion chromatography (NanoGel-50Q, Suzhou Nanovitamin Technology Co., Ltd., Catalog No. 04064-050200-2100) purification also results in a low LcDNA yield of less than 10%, and the LcDNA purity is not high, not meeting clinical or commercial requirements.

[0155] 3.2 High-performance liquid chromatography (IEC-HPLC) detection of LcDNA purity

[0156] The purity of the LcDNA product was tested using an Agilent 1260 high performance liquid chromatograph. The test results are summarized in Table 3.

[0157] 3.3 Sanger sequencing verification of L cDNA

[0158] Appropriate sequencing primers were designed based on the inserted target sequence, and the LcDNA samples were verified by Sanger whole-plasmid sequencing. The sequencing results are summarized in Table 3.

[0159] 3.4 Nanorange fluorescence detection of total protein residues in LcDNA

[0160] The total protein residue in the L cDNA product was detected using the NanoOrange kit (ThermoFisher, Cat. No. N10271) and read using a microplate reader. The test results are summarized in Table 3.

[0161] 3.5 Detection of host DNA (E. coli origin) residues in LcDNA by fluorescence quantitative PCR (qPCR)

[0162] A real-time fluorescence quantitative PCR instrument (ThermoFisher, model: 7500) and an E. coli residual DNA detection kit (Shenke Bio, catalog number: SK030202E100) were used to detect the residual host DNA (E. coli source) in LcDNA. The test results are summarized in Table 3.

[0163] 3.6 Detection of bacterial endotoxins in LcDNA by LAL gel method

[0164] Bacterial endotoxins in LcDNA were detected by gel analysis using Limulus amebocyte lysate (LA-lysate) reagent. The test results are summarized in Table 3.

[0165] 3.7 Direct inoculation method to detect the sterility of LcDNA

[0166] Use a sterile disposable syringe or a sterilized pipette to draw up the LcDNA sample and inoculate it into 100 mL of thioglycollate FTM medium. Incubate at 30-35°C for 14 days to test the LcDNA sample for sterility. The test results are summarized in Table 3.

[0167] Table 3 Summary of LcDNA finished product test results

[0168] in conclusion

[0169] 1) This LcDNA production process is very stable. The scale of enzymatically digested circular plasmids was gradually scaled up from 60 mg to 100 mg and then to 150 mg, without any abnormal deviations during the production process. This process can also meet the requirements for LcDNA production at scales exceeding 150 mg of circular plasmids.

[0170] 2) This LcDNA production process has a high total yield. A total yield of 25%-33% was achieved across five batches of production using three scales of circular plasmid digestion.

[0171] 3) The purity and quality of the LcDNA products produced by this process can meet the requirements of the pharmacopoeia and can be used for clinical research and commercial purposes in fields such as gene therapy.

[0172] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. A plasmid comprising two editing regions of protelomerase target sequences connected in series in the same direction, wherein one or more restriction endonuclease cleavage sites are independently provided at both ends of the editing region.

2. The plasmid according to claim 1, wherein the length is ≤2500 bp, or ≤2300 bp, or ≤2100 bp. The plasmid according to claim 1 , further comprising a prokaryotic replicon and a selection gene.

4. The plasmid according to claim 3, wherein the restriction endonuclease cleavage site at each end is selected from any one or more of the following: the cleavage sites of AccI, AflII, AgeI, ApaI, AscI, AvrII, BamHI, BglI, BglII, BsaI, BspQI, BstBI, BsteII, ClaI, EcoNI, EcoRI, EcoRV, FseI, HindIII, KpnI, MfeI, MluI, NcoI, NdeI, NheI, NotI, PacI, PstI, PvuI, PvuII, SacI, SacII, SalI, ScaI, SmaI, SpeI, StuI, SwaI, XbaI, XhoI and XmaI, preferably selected from any one or more of the cleavage sites of HindIII, SalI, SmaI, BamHI, EcoRV, XbaI, KpnI and EcoRI.

5. The plasmid according to claim 4, wherein HindIII, SalI, SmaI and BamHI restriction sites are inserted near one end of the editing region, and EcoRV, XbaI, KpnI and EcoRI restriction sites are inserted near the other end of the editing region.

6. The plasmid according to any one of claims 1 to 5, wherein the protelomerase target sequence comprises the sequence shown in SEQ ID NO: 1, or a nucleotide sequence that has at least 80% identity with the sequence shown in SEQ ID NO: 1 and can exert telomerase function. 7 . The plasmid according to claim 1 , comprising a nucleotide sequence having at least 80% identity with the sequence shown in SEQ ID NO:

2.

8. A recombinant plasmid comprising a target gene inserted between two protelomerase target sequences of the plasmid according to any one of claims 1 to 7.

9. A method for preparing linear closed DNA, comprising: a) using protelomerase to digest a recombinant plasmid containing a target gene to separate the target gene from the miscellaneous nucleic acids in the recombinant plasmid; b) digesting the nucleic acid using at least two restriction endonucleases to expose the ends of the heterologous nucleic acid, and removing the heterologous nucleic acid using an exonuclease; c) isolating and purifying the linear closed DNA containing the target gene; The recombinant plasmid comprises a protelomerase target sequence and a restriction endonuclease cutting site, and a target gene is inserted between the protelomerase target sequences.

10. The method according to claim 9, wherein the recombinant plasmid is the recombinant plasmid according to claim 8. The method according to claim 9 or 10, wherein the exonuclease comprises Exonuclease III and / or T5 Exonuclease. 12 . The method according to claim 11 , wherein the amount of the exonuclease added is 3000 U / mg to 6000 U / mg. 13 . The method according to claim 12 , wherein the exonuclease cleavage temperature is 34° C. to 40° C., and the cleavage time is 2 to 12 hours; preferably, the cleavage time is 3 to 6 hours.

14. The method according to claim 9 or 10, wherein after exonuclease cleavage, EDTA is added to a final concentration of 1 mM to 50 mM and the temperature is raised to 70°C to 80°C for 5 to 30 minutes. 15 . The method according to claim 9 , wherein after the protelomerase digestion and / or restriction enzyme digestion is completed, the temperature is raised to 70° C. to 80° C. for 5 to 30 minutes.

16. The method according to any one of claims 9 to 15, wherein the separation and purification method comprises one of gel filtration chromatography, anion chromatography, concentration and liquid exchange, and sterilizing filtration, or a combination thereof.

17. The method according to claim 16, wherein the separation and purification method comprises gel filtration chromatography and anion chromatography in sequence.

18. The method according to claim 17, wherein the filler of the gel filtration chromatography is Bestarose 6FF, and the eluate with ≥50 mAu of the first UV peak is collected.

19. The method according to claim 18, wherein the filler material for the anion chromatography is Fractogel EMD DEAE.

20. The method according to claim 19, wherein the loading conditions of the anion chromatography include: Use a loading solution containing 30% to 70% chromatography buffer B, a loading capacity of 0.2 mg / mL to 1.0 mg / mL, and a loading flow rate of ≤120 cm / h; The chromatography solution B contains 0.5M-1.5M NaCl, 5mM-15mM EDTA and a buffer substance, and has a pH of 7.2-7.

8.

21. The method according to claim 20, wherein the elution conditions of the anion chromatography comprise: Elution was performed with 65% to 100% of chromatography fluid B at a flow rate of 30 cm / h to 120 cm / h, and ≥100 mA of eluate was collected.

22. A kit comprising protelomerase and the plasmid according to any one of claims 1 to 7; or comprising protelomerase and the recombinant plasmid according to claim 8.

23. The kit according to claim 22, further comprising a restriction endonuclease and / or an exonuclease, wherein the restriction endonuclease is capable of recognizing the restriction endonuclease cleavage site.

24. The linear closed DNA prepared by the method according to any one of claims 9 to 21.

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