CN117701525A

CN117701525A - Enzyme digestion system for preparing linear covalent closed DNA microcarrier and application thereof

Info

Publication number: CN117701525A
Application number: CN202311654775.6A
Authority: CN
Inventors: 张平静; 邵梅琪; 刘韬; 孙艳; 钱其军
Original assignee: Shanghai Cell Therapy Group Co Ltd; Maxirna Shanghai Pharmaceutical Co Ltd; Maxirna Zhejiang Technology Co Ltd
Current assignee: Shanghai Cell Therapy Group Co Ltd; Maxirna Shanghai Pharmaceutical Co Ltd; Maxirna Zhejiang Technology Co Ltd
Priority date: 2023-12-04
Filing date: 2023-12-04
Publication date: 2024-03-15

Abstract

The invention discloses an enzyme digestion system for preparing a linear covalent closed DNA microcarrier and application thereof. The cleavage system comprises a prokaryotic telomerase and the cleavage system further comprises ATP and/or a divalent metal ion. The invention also discloses a kit containing the enzyme digestion system and application thereof in preparing linear covalent closed DNA microcarriers. The invention also discloses a preparation method of the linear covalent closed DNA microcarrier, which comprises in vitro enzyme cutting of target DNA comprising at least 1 recognition site of prokaryotic telomerase by using the enzyme cutting system or the kit. When the enzyme cutting system is used for preparing the linear covalent closed DNA microcarrier, the enzyme cutting efficiency is high, the yield is high, the steps are simple, the in-vitro DNA polymerase is not needed, the fidelity is high, and the problem of non-specific degradation is avoided.

Description

Enzyme digestion system for preparing linear covalent closed DNA microcarrier and application thereof

Technical Field

The invention belongs to the field of plasmid microcarrier preparation, and in particular relates to an enzyme digestion system for preparing a linear covalent closed DNA microcarrier and application thereof.

Background

The gene therapy mainly adopts two vector forms of virus and non-virus, wherein the virus vector mainly comprises adeno-associated virus, slow virus, adenovirus and retrovirus, herpes simplex virus vector, vaccinia virus vector, oncolytic virus vector and the like; non-viral vectors refer mainly to plasmids or DNA fragments of different forms, etc. Viral vectors are used in current cellular and gene therapy programs in amounts of about 60-70%, and non-viral vectors are used in amounts of about 30-40% of the total number of programs. In the long term, the non-viral vector has the advantages of low immunogenicity, low cost, easy scale and the like, so that the non-viral vector has better clinical application prospect, but has some unresolved problems.

Traditional non-viral gene delivery DNA vectors are often referred to as plasmid DNA, which is typically circular in structure with no free ends, and is not susceptible to exonuclease degradation. Circular plasmid DNA, although safer than viral vectors, still has safety implications. For example, traditional plasmid vectors typically contain, in addition to therapeutic genes, unwanted prokaryotic sequences, antibiotic resistance genes, cpG motifs, and bacterial origins of replication, which DNA components may lead to stimulation of unwanted immune responses; the conventional DNA plasmid vector has larger molecular weight and lower bioavailability due to the inclusion of unnecessary sequences for gene therapy; the circular nature of conventional DNA plasmid vectors confers chromosomal integration, potentially leading to insertional mutagenesis, and the risk of tumor.

The non-viral gene delivery DNA vector also comprises micro-ring DNA and linear covalent closure DNA microcarriers, the novel DNA structure is smaller, the novel DNA structure does not contain non-therapeutic essential sequences in traditional plasmids, and the safety of the novel DNA vector is obviously improved. The concept of using micro-ring DNA or linear covalently closed DNA microcarriers as advanced drugs is attracting increasing attention, showing great potential in the development of gene therapy breakthrough therapy methods, and has been widely applied in the field of gene therapy such as DNA vaccines, induction of pluripotent stem cells, expression of therapeutic transgenes, production of therapeutic viruses, etc. (Yin H, kanasty RL, eltoukhy AA, et al, no-viral vectors for gene-based therapy, nat Rev gene 2014;15 (8): 541-55).

Micro-circular DNA is a non-viral, episomal, covalently closed circular gene expression vector, typically biosynthesized in recombinant bacteria, and research has shown that micro-circular DNA is indeed a successful concept in reducing vector size and removing bacterial units from non-viral vectors, as well as a promising approach to develop more efficient gene-based therapies. However, research has also demonstrated that micro-circular DNA technology is being moved from the research stage to production applications, and that various challenges such as low yields during production scale-up, difficulty in purification of various circular DNA components contained in bacterial lysates in addition to micro-circular DNA, have to be overcome (V-tor, gaspar, duarte, et al Minicircle DNA vectors for gene therapy: advances and applications [ J ]. Expert Opinion on Biological Therapy,2014;Maniar LEG,Maniar JM,Chen Z-Y, et al Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level. Mol Ther 2013;21 (1): 131-8;Grund M,Schleef M.Minicircle patents:a short IP overview of optimizing nonviral DNA vectors.Minicircle and miniplasmid DNA vectors:the future of nonviral and viral gene transfer.Wiley-Blackwell; weinheim, germany: 2013.p.1-6).

The linear covalent closed DNA is a linear gene expression vector which is not covalently closed by viruses, and the closed linear DNA can be more stable in vivo and is a very potential gene therapy DNA vector, unlike the non-closed linear DNA which is easily degraded by exonucleases. In most prokaryotes, the problem of end replication is solved by using circular DNA molecules as chromosomes. However, some phages and bacteria also store genetic information in linear DNA structures, the ends of which often contain inner rotor or hairpin telomeres. Wherein the formation of hairpin telomeres is catalyzed by prokaryotic telomerase, a unique protein that modifies DNA into hairpin structured telomeres by forming covalent protein binding intermediates that mediate two-step transesterification reactions.

The first prokaryotic telomerase was found in 1964 when V.Ravin isolated E.coli phage N15, and the genetic material of the phage was found to be capable of processing into a linear closed structure by atypical cleavage and telomerase. Since the discovery of N15 telomerase, the scientific community has identified a variety of cognate telomerases in Klebsiella oxytoca phage ΦKO2, yersinia enterocolitica phage PY54, vibrio parahaemolyticus phage VP882 and algal monocyte phage ΦHAP-1. In addition to phage, some bacteria contain similarly functional telomerase. Recent studies have shown that TelA in Agrobacterium tumefaciens C58 belongs to a prokaryotic linear telomerase (Knott S E, milsom S A, rothwell P.J.th Unusual Linear Plasmid Generating Systems of Prokaryotes [ M ]// Bacteriophages-Biology and applications.2019) from ResT from B.hermii, B.park, B.recurrentis, B.turrica and B.anserin purified telomerase.

Research shows that the prokaryotic telomerase proteins and the mechanisms of the DNA hairpin structures generated by the prokaryotic telomerase proteins have important application values in the production of DNA vectors in the biotechnology industry, and some scientists are motivated to develop a series of methods for producing linear covalent closed DNA microcarriers. For example, nafissi N et al from Mediphage, inc. developed in 2012 a linear covalently-blocked DNA plasmid production system that utilized phage-derived recombinase Tel (PY 54), telN (N15) or Cre, in E.coli, to recombine natural parent plasmids to form two linear covalently-blocked DNAs, one of which contained a resistance gene and plasmid replicon sequence (PL) and the other contained a gene therapy DNA sequence (ML). The method solves the safety problem of microcarrier plasmid to a certain extent, but the production and purification efficiency still has certain problems (low enzyme digestion efficiency, low yield, complex fermentation program control, inconsistent stability among fermentation batches and the like). For example, natural Parent Plasmid (PP) in production systems cannot be completely digested into PL and ML, and the products contain PP, PL and ML at the same time, and it is difficult to purify high-standard ML for gene therapy (Nafissi N, slavcev R.construction and characterization of an in-vivo linear covalently closed DNA vector production system. Microb Cell face. 2012Dec 6;11:154;Nafissi N,Sum CH,Wettig S,Slavcev RA.Optimization of aone-step heat-inducible in vivo mini DNA vector production system. PLoS one.2014Feb20;9 (2): e89345; wong S, lam P, nafissi N, dennis S, slavcev R.production of Double-stranded DNA Ministungs Exp.2016Feb 29; 108): 53177;Nafissi N,Alqawlaq S,Lee EA,Foldvari M,Spagnuolo PA,Slavcev RA.DNA ministrings:highly safe and effective gene delivery vectors.Mol Ther Nucleic Acids.2014May 27;3 (6): e 165).

Touchlight Genetics, alacris Theranostics et al developed a method for preparing linear closed DNA by in vitro amplification enzyme catalysis based on the principle of procaryotic telomerase. The method is a pure in vitro DNA production process, and the cell-free technology uses phage DNA polymerase from Phi29, so that a large amount of concatemers of target DNA can be amplified from a small amount of initial plasmid DNA templates. The DNA concatemers are subjected to catalytic processing and shearing by the prokaryotic telomerase to obtain a large number of linear covalent closed DNA microcarriers. Such linear covalently closed DNA microcarriers have been used in influenza DNA vaccines, HPV16DNA vaccines, lentiviral vectors, recombinant adeno-associated virus (AAV), cellular immunotherapeutic transposon integration studies. The above methods, while showing unusually broad application potential, have problems associated with scale-up production. For example, in vitro amplification reactions with DNA polymerase are required, while in vitro multicycle amplification steps using phi29 DNA polymerase are complex and have DNA sequence fidelity problems, and more specific instrumentation and program control are required for process scale up; the method also adopts the escherichia coli exonuclease III to carry out enzyme digestion on redundant DNA fragments except microcarrier DNA, and non-integral amplification DNA products possibly generate non-integral units of gene therapy DNA target sequences in enzyme digestion process, and the non-integral unit DNA is non-uniform DNA fragments, if the exonuclease is additionally used for digestion, the problem that the non-specific degradation is caused by linear covalent closure of the DNA target sequence units for gene therapy exists, so that the yield is greatly limited and affects the amplification production and stability of the process (Scott VL, patel A, villarral DO, hensley SE, ragwan E, yan J, et al.Novel synthetic plasmid and doggybone DNA Vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice.Human Vaccines & immunothers.20158; 11 (8): 1972-1982;Allen A,Wang C,Caproni LJ,Sugiyarto G,Harden E,Douglas LR,et al.Linear doggybone DNA vaccine induces similar immunological responses to conventional plasmid DNA independently of immune recognition by TLR9 in apre-clinical image, cancer immuno2018; 67 (4): 627 638;Karbowniczek K,Rothwell P,Extance J,Milsom S,Lukashchuk V,Bowes K,et al.DoggyboneTMDNA:An advanced platform for AAV production.Cell&Gene Therapy Insights.2017:731-738;Karda R,Counsell JR,Karbowniczek K,Caproni LJ,Tite JP,Waddington SN.Production of lentiviral vectors using novel,enzymatically produced,linear DNA.Gene Therapy.2019;26:86-92;Bishop DC,Caproni L,Gowrishankar K,Legiewicz M,Karbowniczek K,Tite J,Gottlieb DJ,Micklethwaite KP.CAR T Cell Generation by piggyBac Transposition from Linear Doggybone DNA Vectors Requires Transposon DNA-Flcartridge region 20223:102368B-359:102368B CN104911177A, CN109844134A, US20190185924A1, US20180037943A1, US20030096246A1, US20120282283A1, WO2014020154 A1).

The commercial cloning vector pJAZZ was developed by Lucigen on the basis of the linear N15 phage genome telomerase replication mechanism. The pJAZZ vector simultaneously encodes replication protein RepA and prokaryotic telomerase TelN and has the functions of replication and telomere analysis. Such vectors are optimized for many advantages, such as allowing for the insertion of large cDNAs or operons, complex structures and/or extremely unstable specific sequences. However, the linear covalently closed DNA still contains replicon and resistant DNA sequences, the sequences are generally more than 10kb in length and do not belong to the field of linear covalently closed DNA microcarriers, and are difficult to directly apply to the field of gene therapy (Godista R, mead D, dhodda V, wu C, hochstein R, karsi A, et al, linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coll.nucleic Acids research.2010;38 (6): e 88).

In summary, prokaryotic telomerase and its covalently closed linear plasmid terminal end-pellet structure can protect genetic material from degradation and provide a new solution for nucleic acid synthesis biology, with great development potential in the production of DNA microcarriers for gene therapy, but the early inventions still have problems or drawbacks. Thus, there remains a need to develop new methods for preparing linear covalently closed DNA microcarriers.

Disclosure of Invention

The invention aims to overcome the defects of low enzyme digestion efficiency, low yield, complex steps, inconsistent batch-to-batch stability, low fidelity, non-specific degradation problem and the like in the preparation of a linear covalent closed DNA microcarrier in the prior art.

The inventor finds that most of circular plasmids in bacteria are in a supercoiled state in an in-vitro enzyme digestion test process, and prokaryotic telomerase has poor enzyme digestion effect on supercoiled plasmids, generally lower than 50%, and can not be completely digested to obtain linear covalent closed DNA microcarriers. This is very disadvantageous for the development of production scale-up and post-purification processes. The inventors found that after linearizing the plasmid with backbone sequence specific restriction sites on the circular plasmid, the efficiency of telomerase cleavage can reach 100% under optimized cleavage buffer conditions, allowing 100% complete conversion of the plasmid into DNA microcarriers.

Further, the efficient in-vitro enzymatic linear covalent closed DNA microcarrier production process is obtained by optimizing the buffer composition and adjusting the structure type of the DNA substrate, so that the method can be applied to the field of gene therapy.

The invention also discovers that under the optimized condition, the prokaryotic telomerase can directly digest and digest the product DNA by restriction endonuclease without purification, thereby avoiding the loss of the DNA product caused by secondary purification.

DNA microcarriers are widely used for overexpression of therapeutic genes, as gene knock-in DNA donors, etc. The invention prepares the linear covalent closed DNA microcarrier by utilizing the technology of preparing the linear covalent closed DNA in telomerase cells, taking linear or annular plasmid as a substrate, combining a continuous reaction system for preparing the DNA microcarrier by in vitro telomerase secondary enzyme digestion, and preparing the linear covalent closed DNA microcarrier by in vitro restriction DNA endonuclease and telomerase continuous enzyme digestion technology.

The use of the optimized cleavage reaction system according to the invention, in particular the use of ATP and divalent metal ions, and the switching of the buffer system for the continuous cleavage of restriction endonucleases has not been disclosed in such detail.

Within the scope of the present invention, the linear covalently closed DNA microcarriers prepared may be used as over-expression therapeutic gene DNA vectors and/or as transposon DNA donors, including but not limited to piggybac transposons, sleep beauty transposons, etc.

In order to solve the technical problems of the invention, one of the technical schemes provided by the invention is as follows: an enzyme cleavage system for preparing a linear covalently closed DNA microcarrier, said enzyme cleavage system comprising a prokaryotic telomerase and said enzyme cleavage system further comprising ATP and/or divalent metal ions.

In a specific embodiment of the invention, the prokaryotic telomerase is TelA, telN, PY, phiKO2, phiHAP-1 or ResT.

In the present invention, the term "prokaryotic telomerase" refers generally to telomerase in prokaryotes, which is capable of catalyzing the formation of hairpin telomeres in prokaryotes to form a covalently closed linear plasmid terminal telomere structure that protects genetic material from DNase degradation.

Within the scope of the present invention, other prokaryotic telomerases, including but not limited to TelA and TelN, may be selected by those skilled in the art for similar DNA microcarrier production purposes, such as PY54, phiKO2, phiHAP-1, resT, etc., in the enzymatic cleavage system for preparing linear covalently closed DNA microcarriers.

In a specific embodiment of the invention, the amino acid sequence of TelA is shown as SEQ ID NO. 3, the amino acid sequence of TelN is shown as SEQ ID NO. 1, the amino acid sequence of PY54 is shown as SEQ ID NO. 14, the amino acid sequence of phiKO2 is shown as SEQ ID NO. 15, the amino acid sequence of phiHAP-1 is shown as SEQ ID NO. 16 or the amino acid sequence of ResT is shown as SEQ ID NO. 17.

In a specific embodiment of the invention, the nucleotide sequence encoding TelA is shown as SEQ ID NO. 4 or the nucleotide sequence encoding TelN is shown as SEQ ID NO. 2.

In a specific embodiment of the invention, the ATP concentration is 0.1 to 1.2mM, preferably 0.5 to 1mM; and/or the divalent metal ion concentration is 1 to 25mM, preferably 5 to 20mM, for example 10mM.

In a specific embodiment of the present invention, the divalent metal ion comprises a metal selected from the group consisting of Mg ²⁺ 、Ca ²⁺ And Mn of ²⁺ One or more of the following; preferably comprises Mg ²⁺ And/or Ca ²⁺ 。

In a specific embodiment of the invention, the cleavage system further comprises a base cleavage buffer.

In a specific embodiment of the present invention, the base digestion buffer comprises 18 to 22mM Tris-HCl, pH7.0 to 8.0, 48 to 52mM potassium glutamate, 0.8 to 1.2mM DTT, and 0.08 to 0.12mM EDTA; for example, the base cleavage buffer comprises 20mM Tris-HCl, 50mM potassium glutamate, 1mM DTT and 0.1mM EDTA, pH 7.5. The concentration of the basic cleavage buffer is 1×the concentration of the basic cleavage buffer (i.e., the working concentration).

In a specific embodiment of the invention, the cleavage system further comprises a restriction endonuclease and/or a restriction endonuclease buffer.

In a specific embodiment of the invention, the restriction endonuclease buffer comprises 45 to 55mM potassium acetate, 15 to 25mM Tris acetate, 5 to 15mM magnesium acetate and 90 to 110. Mu.g/ml BSA, pH7.5 to 8.0; for example, the restriction endonuclease buffer contained 50mM potassium acetate, 20mM Tris acetate, 10mM magnesium acetate and 100. Mu.g/ml BSA, pH7.9. The concentration of the restriction endonuclease buffer was 1×the concentration of the restriction endonuclease buffer (i.e., working concentration).

In the invention, the concentration of each component in the enzyme digestion system is the concentration of each component, ATP and divalent salt ion of the enzyme digestion system before the reaction occurs in the whole system.

In order to solve the technical problem of the invention, the second technical scheme provided by the invention is as follows: a kit comprising the cleavage system according to one of the embodiments of the present invention.

In order to solve the technical problem of the invention, the third technical scheme provided by the invention is as follows: the application of the enzyme digestion system according to one of the technical schemes of the invention or the kit according to the second technical scheme of the invention in preparing linear covalent closed DNA microcarrier.

In order to solve the technical problems of the invention, the technical scheme provided by the invention is as follows: a method for preparing a linear covalently closed DNA microcarrier comprising the steps of: in vitro digestion of target DNA comprising at least 1 recognition site for a prokaryotic telomerase is performed using the digestion system according to one of the embodiments of the present invention or the kit according to a second embodiment of the present invention.

In the invention, the in vitro digestion refers to digestion of prokaryotic telomerase, and the target DNA can realize the digestion reaction as long as the target DNA at least comprises 1 recognition site of the prokaryotic telomerase; when the target DNA is a linear plasmid, if redundant sequences such as a replicon resistance gene to be excised and the like are all concentrated on the same side, the target DNA containing only one recognition site can be prepared into a target linear closed microcarrier.

In a specific embodiment of the invention, the mass ratio of the target DNA to the prokaryotic telomerase is 3 (0.25-3.75), e.g., 3 (0.5-1), 3 (0.25-1), 0.8-4): 1 or 0.8-1.2): 1.

In a specific embodiment of the invention, the in vitro cleavage time is 4 to 24 hours and/or the in vitro cleavage temperature is 30 ℃.

In a specific embodiment of the invention, the target DNA is synthesized artificially or the target DNA is extracted or amplified in a prokaryote.

In a specific embodiment of the invention, the target DNA comprises a sequence of interest and a redundant sequence; both ends of the sequence of interest comprise recognition sites for the prokaryotic telomerase.

In the present invention, the sequence of interest comprises a promoter sequence, a sequence of a coding region for a protein of interest and a terminator sequence, preferably further comprises a gene function regulatory sequence and/or a transposase recognition sequence; the redundant sequences comprise replicon sequences and/or resistance gene sequences.

In a specific embodiment of the invention, the transposase recognition sequence is a piggybac transposase recognition DNA sequence ITR; and/or, the gene function regulatory sequence is polyA.

When the target DNA is a linear plasmid, the recognition sites of the prokaryotic telomerase at the two ends of the linear plasmid and the recognition sites of the prokaryotic telomerase at the two ends of the target sequence are the recognition sites of different types of prokaryotic telomerase; the backbone of the linear plasmid is, for example, the pJAZZ-OK plasmid.

Within the scope of the present invention, a DNA target sequence unit for gene therapy is constructed into a commercial pJazz linear covalent closed DNA plasmid of Lucigen company, which is prepared by mass purification by fermentation in E.coli; and then carrying out in vitro enzyme digestion and connection by using procaryotease on the linear DNA plasmid substrates to obtain a large number of uniform linear covalent closed DNA microcarriers. The inventor surprisingly found that the novel scheme can save the linearization step of the circular plasmid, namely, the uniform linear covalent closed DNA microcarrier can be obtained by one-step efficient and massive enzyme digestion, and the problem that the DNA sequence can be stably amplified in an escherichia coli organism is solved.

When the target DNA is a circular plasmid, the circular plasmid preferably comprises at least 1 restriction DNA endonuclease site, and the sequence of interest does not comprise the restriction DNA endonuclease site; the backbone of the circular plasmid is for example puc57.

In the present invention, the circular plasmid contains at least one restriction endonuclease site, i.e., can be linearized by a restriction endonuclease.

In a specific embodiment of the invention, the target DNA is a circular plasmid comprising at least 1 restriction endonuclease site and no restriction endonuclease site is included in the sequence of interest, and the method of preparation further comprises linearizing the circular plasmid with a restriction endonuclease prior to the in vitro cleavage.

When using a circular plasmid amplified in a bacterial cell as a substrate for digestion, it is necessary to cleave the circular plasmid into linear DNA with a restriction enzyme that allows only the circular plasmid backbone sequence to appear before digestion with in vitro telomerase, and redundant sequences refer to sequences necessary for the plasmid to maintain replication and amplification in bacterial cells, and include, but are not limited to, replicon, resistance gene DNA sequences.

In the context of the present invention, the linearized plasmid product digested by the first restriction endonuclease can be used directly as a substrate for the digestion of the second prokaryotic telomerase without any purification. Wherein the first step of restriction endonuclease digestion buffer composition can be: 50mM potassium acetate, 20mM Tris acetic acid, 10mM magnesium acetate, 100. Mu.g/ml BSA, pH7.9. Wherein the second step of prokaryotic telomerase digestion is performed, preferably the substrate DNA is added in a volume of less than 60% of the total volume of the final reaction, preferably the linear DNA substrate endpoint concentration is less than 2mg/mL.

In a specific embodiment of the invention, the time for linearization of the restriction endonuclease is 4 to 16 hours; and/or, the temperature at which the restriction endonuclease linearizes is 37 ℃.

The present invention actually provides a continuous production process of linear covalent closed DNA microcarriers, comprising the following steps: 1. respectively adding a prokaryotic telomerase recognition sequence at two sides of a DNA target sequence unit for gene therapy, and cloning the prokaryotic telomerase recognition sequence into a commercial linear plasmid, wherein the DNA target sequence unit for gene therapy comprises, but is not limited to, a promoter sequence, a terminator sequence, a protein coding region sequence, a gene function regulating sequence and a transposase recognition sequence ITR;2. e.coli fermentation large-scale amplification to produce linear plasmid and purification; 3. the linear covalent closed DNA microcarrier is obtained by cutting the telomerase and purified.

The invention also provides another continuous production process of the linear covalent closed DNA microcarrier, which comprises the following steps: 1. adding a prokaryotic telomerase recognition sequence to each side of a DNA target sequence unit for gene therapy, and then cloning the DNA target sequence unit into a circular plasmid, wherein the DNA target sequence unit for gene therapy comprises, but is not limited to, a promoter sequence, a terminator sequence, a protein coding region sequence, a gene function regulating sequence and a transposase recognition sequence ITR, the non-gene therapy DNA target sequence in the circular plasmid at least comprises 1 restriction endonuclease site, and the gene therapy DNA target sequence unit does not comprise the restriction endonuclease sites; 2. e.coli fermentation large-scale amplification to produce circular plasmid and purification; 3. sequentially carrying out enzyme cutting on the linear circular plasmid by using a restriction DNA endonuclease, and then carrying out enzyme cutting on the linear circular plasmid by using a prokaryotic telomerase to obtain a linear covalent closed DNA microcarrier and purifying.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

the invention adopts a combined method of combining the amplification of DNA plasmids in bacterial cells and the digestion of telomerase in vitro to produce the linear covalent closed DNA microcarrier, thereby realizing the production process capable of stably amplifying the scale and improving the production efficiency of the DNA microcarrier.

According to the preparation method of the linear covalent closed DNA microcarrier, the used enzyme digestion substrate is directly amplified in DNA in escherichia coli cells, in-vitro polymerase amplification is not needed, and industrial fermentation and amplification production are easy to realize; the in vitro enzyme digestion production method can realize complete conversion of substrate DNA into DNA microcarrier, and does not generate incomplete DNA microcarrier units; the linear DNA plasmid is used as a substrate to realize the preparation of the DNA microcarrier by a one-step method, and the production purpose is realized simply and economically.

When the enzyme cutting system is used for preparing the linear covalent closed DNA microcarrier, the enzyme cutting efficiency is high, the yield is high, the steps are simple, the in-vitro DNA polymerase is not needed, the fidelity is high, and the problem of non-specific degradation is avoided.

Drawings

FIG. 1 shows an electrophoretogram of SDS-PAGE to identify purified TelN and TelA prokaryotic telomerase proteins.

FIG. 2 shows a schematic representation of the production of linear covalently closed DNA microcarriers by in vitro cleavage of supercoiled plasmids.

FIG. 3 shows the effect of varying amounts of prokaryotic telomerase TelA on the digestion of supercoiled plasmids.

FIG. 4 shows the effect of different ATP doses on the prokaryotic telomerase TelA digested supercoiled plasmid.

FIG. 5 shows the effect of varying calcium ion doses on prokaryotic telomerase TelA digested supercoiled plasmid.

FIG. 6 shows the effect of different divalent metal ions on telomerase TelA digested supercoiled plasmids.

FIG. 7A shows the effect of different buffer compositions on telomerase TelA digested supercoiled plasmid.

FIG. 7B shows the effect of different buffer compositions on telomerase TelN digested supercoiled plasmid.

FIGS. 8A and 8B show a flow chart of a process for preparing a linear covalently closed DNA microcarrier by continuous digestion of circular plasmids.

FIG. 9 shows a graph of the results of the preparation of linear covalently closed DNA microcarriers (TelA) by continuous digestion of circular plasmids.

FIG. 10 shows a graph of the results of the preparation of linear covalently closed DNA microcarriers (TelN) by continuous digestion of circular plasmids.

FIG. 11 shows the identification of successive enzyme digestion results for telomerase at different starting DNA concentrations and volumes.

FIG. 12 shows a schematic representation of the preparation of DNA microcarriers by means of a linear plasmid production system in combination with in vitro telomerase cleavage.

FIG. 13 shows an identification of linear covalently closed DNA microcarriers prepared from telomerase tangential plasmid preparation.

FIG. 14 shows the green fluorescence positivity of eGFP at various times for CHO cell electrotransfer verified DNA microcarriers or circular plasmids.

FIG. 15 shows the fluorescence integration expression positive rate of CHO cell electrotransfer verified DNA microcarriers or circular plasmid eGFP.

FIG. 16 shows a map of pJAZZ-TelA-ITR-CopGFP plasmid.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

EXAMPLE 1 expression and purification of the prokaryotic telomerase TelN and TelA proteins

In order to be able to overexpress the prokaryotic telomerase TelN and TelA respectively in E.coli, the synthetic DNA sequences (SEQ ID NO:2, 4) are designed optimally according to the amino acid sequences (SEQ ID NO:1, 3) corresponding to the above-mentioned prokaryotic telomerase respectively and cloned into the commercial prokaryotic expression vector of pET28a (Novagen company), a DNA sequence (SEQ ID NO: 6) encoding the amino acid sequence of the protein Tag (SEQ ID NO: 5) is added to the amino terminus (N terminus) of the 2 enzyme proteins respectively for the purpose of purification, these tags including Strep-Tag II, his-Tag and TEV protease specific recognition amino acid sequences.

The plasmids were transformed into E.coli BL21 (DE 3) expressing bacteria according to the instructions of commercial expression vectors of Novagen, single colony LB was selected and cultured at 37℃to logarithmic growth phase, and induction culture was continued at 25℃for 16 hours with the addition of IPTG inducer at a final concentration of 1mM. After collection of the inducer, all the enzyme proteins were purified using two standard purification steps, ni affinity chromatography, capto Q ImpRes (Cytiva) ion exchange chromatography to obtain the final enzyme proteins. The finally purified enzyme proteins were subjected to SDS-PAGE (polyacrylamide gel electrophoresis) to identify the molecular weight and purity of the proteins, respectively. As shown in FIG. 1, the purified TelN and TelA prokaryotic telomerase proteins with higher purity are obtained respectively, and the molecular weights respectively accord with the expected sizes of 78.3kD and 56.5 kD.

EXAMPLE 2 conditional screening of prokaryotic telomerase in vitro restriction supercoiled plasmid DNA

The puc57-Kan-TelA-BamH2-CopGFP and puc57-Kan-TelN-BamH2-CopGFP plasmids were constructed, the plasmid sequences were shown in SEQ ID NO:7 and SEQ ID NO:8, two prokaryotic telomerase TelA and/or TelN specific recognition sequences (SEQ ID NO:9, SEQ ID NO: 10) were contained in the plasmids, two BamHI restriction enzyme sites were contained in the plasmid DNA backbone sequences, and the puc57-Kan-TelA-BamH2-CopGFP plasmids were used as cleavage substrates for prokaryotic telomerase TelA, and the most suitable cleavage conditions for telomerase A were selected. The 10 times basic enzyme digestion buffer solution of the prokaryotic telomerase is prepared by the following components: 200mM Tris-HCl (pH 7.5) 500mM potassium glutamate, 10mM DTT,1mM EDTA. The effect of ATP and divalent metal ions on the efficiency of producing DNA microcarriers by enzyme-cutting supercoiled plasmid by the dosage of procaryotic telomerase is studied respectively. The complete cleavage process is shown in FIG. 2, which results in two linear covalently closed DNA fragments of about 2.4kb in length, one of which is a DNA microcarrier containing the GFP and purine resistance gene expression cassette, and the other of which is a plasmid backbone microcarrier containing the prokaryotic resistance gene and plasmid replicon DNA sequence. If the TelA recognition site is not completely digested, a linear covalently closed DNA fragment of about 4.8kb is produced. As described in tables 1-4, the optimum conditions for cleavage of circular plasmids by telomerase were studied by adjusting the different components in the telomerase reaction system.

TABLE 1 grouping information of the different TelA enzyme usage

As shown in Table 1 and FIG. 3, on the one hand, the increase in telomerase concentration is beneficial to the digestion of supercoiled plasmid containing its recognition site sequence, but cannot completely cleave supercoiled plasmid into the final DNA microcarrier; on the other hand, when the amount of telomerase added is excessive, the residual DNase in the enzyme protein sample may partially degrade the DNA component, resulting in a decrease in yield. The results indicated that the optimal amount of telomerase addition to 3. Mu.g of circular DNA plasmid substrate was about 0.5-1. Mu.g. Under optimal conditions, circular plasmids are not completely transformed into linear covalently closed DNA microcarriers.

TABLE 2 grouping information with different ATP usage

As shown in Table 2 and FIG. 4, the ATP component in the reaction system is advantageous for cleavage of the supercoiled DNA substrate by telomerase, and the final ATP concentration in the reaction system is preferably 0.5 to 1mM. The presence of ATP also prevents to some extent plasmid digestion by telomerase protein non-specific DNase. Under optimal conditions, circular plasmids are not completely transformed into linear covalently closed DNA microcarriers.

TABLE 3 grouping information of calcium ion concentration

As shown in Table 3 and FIG. 5, the calcium ion component in the reaction system is advantageous for cleavage of the supercoiled DNA substrate by telomerase, and preferably the final concentration of calcium ion in the reaction system is 5 to 20mM, and most preferably the final concentration of calcium ion is 10mM. Under optimal conditions, circular plasmids are not completely transformed into linear covalently closed DNA microcarriers.

TABLE 4 grouping information for different divalent metal ion concentrations

As shown in table 4 and fig. 6, the divalent metal ions in the reaction system may be magnesium ions and manganese ions other than calcium ions, wherein the magnesium ions are superior to the manganese ions. Under optimal conditions, circular plasmids are not completely transformed into linear covalently closed DNA microcarriers.

As shown in FIG. 7A, further increases in the amount of prokaryotic telomerase A used did not allow complete cleavage of circular DNA plasmids into linear covalently closed circular DNA microcarriers. Most preferably, the reaction system conditions are 1.5. Mu.g of enzyme, 10mM Ca ions, 1mM ATP digestion target plasmid 6. Mu.g. Under optimal conditions, circular plasmids are not completely transformed into linear covalently closed DNA microcarriers.

As shown in FIG. 7B, the N-cleavage of its target DNA plasmid by prokaryotic telomerase also does not completely cleave the circular DNA plasmid to obtain a linear covalently closed circular DNA microcarrier. Most preferably, the reaction system conditions are 0.5. Mu.g of enzyme, 20mM Ca ions, 1mM ATP digestion target plasmid 3. Mu.g. Under optimal conditions, circular plasmids are not completely transformed into linear covalently closed DNA microcarriers.

EXAMPLE 3 preparation of Linear covalently closed DNA microcarriers by in vitro continuous enzymatic cleavage of circular DNA plasmids with procaryotic telomerase

The plasmids puc57-Kan-TelA-BamH2-ITR-CopGFP and puc57-Kan-TelN-BamH2-ITR-CopGFP are constructed, the plasmid sequences are shown in SEQ ID NO. 11 and SEQ ID NO. 12, the plasmid contains two specific recognition sequences of procaryotease TelA or TelN, the plasmid DNA framework sequence contains two BamHI restriction endonuclease sites, and the CopGFP gene expression frame contains functional elements such as a promoter, a CopGFP expression gene, a puromycin resistance gene, a polyA, piggybac transposase recognition DNA sequence ITR and the like. The puc57-Kan-TelA-BamH2-ITR-CopGFP or puc57-Kan-TelN-BamH2-ITR-CopGFP plasmid is used as a substrate for enzyme digestion of procaryotease TelA or TelN, and a continuous enzyme digestion process program (see FIG. 8A and FIG. 8B) for preparing linear covalent closed DNA microcarrier is formulated, wherein restriction enzymes are used for linearizing circular plasmids, and skeleton plasmids can be digested into DNA fragments with larger difference from target DNA microcarriers, so that the downstream purification process is facilitated.

1) Large amount of plasmid DNA is extracted from colibacillus fermentation culture amplified plasmid, silica gel film or commercial kit of anion centrifugal column method;

2) The circular DNA plasmid was digested with BamHI, a restriction endonuclease, into two linear non-closed DNA fragments of 750bp and 5.3kb in size, respectively, according to the reaction system shown in Table 5, wherein the 1 Xrestriction endonuclease buffer composition was: 50mM potassium acetate, 20mM Tris acetic acid, 10mM magnesium acetate, 100. Mu.g/ml BSA, pH7.9;

TABLE 5 restriction endonuclease cleavage of circular plasmid reaction System

3) The restriction enzyme product is not required to be purified, and the prokaryotic telomerase is cut continuously according to a reaction system shown in the table 6 to obtain a mixture of a linear covalent closed DNA microcarrier and three redundant DNA fragments, wherein the size of the linear covalent closed DNA microcarrier is 3.7kb, and the sizes of the redundant DNA fragments are 500bp, 750bp and 1.1kb respectively;

TABLE 6 reaction system for preparing linear covalent closed DNA microcarrier by procaryotease cleavage

4) The 3.7kb linear covalently closed DNA microcarrier was obtained by purification using commercial DNA gel recovery kits or chromatography.

As a result, as shown in FIGS. 9 and 10, a large amount of plasmids were obtained by E.coli fermentation amplification, and after linearizing the plasmids in vitro, 3.7kb linear covalently closed DNA microcarriers were obtained with telomerase TelA or TelN. Under optimal conditions, the circular plasmid is completely transformed into a linear covalently closed DNA microcarrier and no incomplete DNA microcarrier units are produced. The experimental optimization result also shows that under the optimization condition, the prokaryotic telomerase can directly digest restriction endonuclease digestion product DNA which is not purified. As shown in Table 7 and FIG. 11, when the substrate was digested with BamHI, the unpurified linear DNA product obtained by digestion of the circular plasmid was less than 60% of the total volume of substrate DNA, and when the final substrate DNA concentration was less than 2mg/mL, 100% digestion of the DNA substrate containing the two specific DNA recognition sequence sites by the prokaryote telomerase was not affected.

TABLE 7 continuous enzyme cleavage sets of telomerase at different initial DNA concentrations and volumes

EXAMPLE 4 preparation of Linear covalently closed DNA microcarriers by in vitro continuous digestion of Linear DNA plasmids with procaryotic telomerase

A linear pJAZZ-OK plasmid vector of Lucigen company is purchased, and pJAZZ-TelA-ITR-CopGFP plasmid is constructed according to the specification of the product, wherein the sequence of the TelA-ITR-CopGFP fragment is shown in SEQ ID NO. 13, two prokaryotic telomerase TelA specific recognition sequences are contained in the plasmid, and a CopGFP gene expression cassette comprises but is not limited to a promoter, copGFP expression gene, puromycin resistance gene, polyA, piggybac transposase recognition DNA sequence ITR and other gene functional elements (pJAZZ-TelA-ITR-CopGFP plasmid map is shown in FIG. 16). The plasmid with the TelA specific recognition sequence replaced by the TelN specific recognition sequence SEQ ID NO. 10 in pJAZZ-TelA-ITR-CopGFP was designated pJAZZ-TelN-ITR-CopGFP. Linear covalent closed DNA microcarriers were prepared using pJAZZ-TelA-ITR-CopGFP or pJAZZ-TelN-ITR-CopGFP linear plasmids as substrates for cleavage of the procaryote telomerase TelA, as shown in FIG. 12 for a schematic process sequence.

2) Continuing to carry out prokaryotic telomerase shearing according to a reaction system shown in Table 8 to obtain a mixture of a linear covalent closed DNA microcarrier and 2 redundant DNA fragments, wherein the size of the linear covalent closed DNA microcarrier is 3.7kb, and the sizes of the redundant DNA fragments are respectively 10.2kb and 2.2kb;

3) The 3.7kb linear covalently closed DNA microcarrier was obtained by purification using commercial DNA gel recovery kits or chromatography.

TABLE 8 reaction system for preparing linear covalent closed DNA microcarrier by procaryotease cleavage

As shown in FIG. 13, in an in vitro TelA or TelN telomerase cleavage reaction system, 100% cleavage can be achieved to obtain linear covalently closed DNA microcarriers, respectively obtaining DNA fragments of the expected size. Thus, the natural linear plasmid DNA substrate is more advantageous than the circular plasmid DNA substrate, and the DNA microcarrier can be obtained by one-step enzyme catalysis without pre-linearization.

Example 5 Linear covalently closed DNA microcarriers for transient and Integrated expression of genes

The specific steps of electrotransfection are as follows: centrifuging cultured CHO cells, removing culture medium, adding 1×PBS, washing, adding Lonza corporation electrotransport buffer solution, and mixing according to 5×10 ⁶ The cells were added with 4. Mu.g of DNA microcarriers containing transposon ITR and eGFP expression cassettes and/or circular master plasmid DNA to obtain 100. Mu.l of electrotransfer mix containing plasmid cells, which were subjected to the subsequent electrotransfer steps according to the Lonza Co electrotransfer kit instructions, with 2-3 parallel test wells per group. Wherein the DNA microcarrier and/or circular master plasmid DNA is purified from example 2 and/or example 3, respectively; wherein the transient expression test group only electrotransduces the DNA microcarrier or the circular master plasmid alone; wherein the integrated expression test group co-transfects the piggybac transposase expression plasmid PB200A-1 of SBI company with the DNA microcarrier or the circular master plasmid. After electrotransfection, the cells were transferred to 6-well plates containing 2ml of medium, and GFP positive rate and fluorescence intensity were measured by flow cytometry on days 2, 4, 6, 8, 10, and 13 after transfection.

TABLE 9 transient expression Effect of eGFP expression element-containing DNA microcarriers and circular plasmids in CHO cells

As shown in Table 9 and FIG. 14, the transient expression effect of the eGFP expression element-containing DNA microcarrier and the circular master plasmid DNA in CHO cells is similar, no obvious difference exists, and the duration of expression can reach 13 days. The results of linear covalently closed DNA microcarriers as transposon donor DNA integration expression are shown in table 10 and fig. 15, and can reach the equivalent eGFP green fluorescence integration expression level of traditional circular plasmid DNA in CHO cells.

TABLE 10 Integrated expression Effect of eGFP expression element-containing DNA microcarriers and circular plasmids in CHO cells

The sequences used in the parts of the invention:

SEQ ID NO. 1TelN amino acid sequence

MSKVKIGELINTLVNEVEAIDASDRPQGDKTKRIKAAAARYKNALFNDKRKFRGKGLQKRITANTFNAYMSRARKRFDDKLHHSFDKNINKLSEKYPLYSEELSSWLSMPTANIRQHMSSLQSKLKEIMPLAEELSNVRIGSKGSDAKIARLIKKYPDWSFALSDLNSDDWKERRDYLYKLFQQGSALLEELHQLKVNHEVLYHLQLSPAERTSIQQRWADVLREKKRNVVVIDYPTYMQSIYDILNNPATLFSLNTRSGMAPLAFALAAVSGRRMIEIMFQGEFAVSGKYTVNFSGQAKKRSEDKSVTRTIYTLCEAKLFVELLTELRSCSAASDFDEVVKGYGKDDTRSENGRINAILAKAFNPWVKSFFGDDRRVYKDSRAIYARIAYEMFFRVDPRWKNVDEDVFFMEILGHDDENTQLHYKQFKLANFSRTWRPEVGDENTRLVALQKLDDEMPGFARGDAGVRLHETVKQLVEQDPSAKITNSTLRAFKFSPTMISRYLEFAADALGQFVGENGQWQLKIETPAIVLPDEESVETIDEPDDESQDDELDEDEIELDEGGGDEPTEEEGPEEHQPTALKPVFKPAKNNGDGTYKIEFEYDGKHYAWSGPADSPMAAMRSAWETYYS*

SEQ ID NO. 3TelA amino acid sequence

MLAAKRKTKTPVLVERIDQFVGQIKEAMKSDDASRNRKIRDLWDAEVRYHFDNGRTEKTLELYIMKYRNALKAEFGPKSTPLAICNMKKLRERLNTYIARGDYPKTGVATSIVEKIERAEFNTAGRKPTVLLRIADFIAAMNGMDAKQDMQALWDAEIAIMNGRAQTTIISYITKYRNAIREAFGDDHPMLKIATGDAAMYDEARRVKMEKIANKHGALITFENYRQVLKICEDCLKSSDPLMIGIGLIGMTGRRPYEVFTQAEFSPAPYGKGVSKWSILFNGQAKTKQGEGTKFGITYEIPVLTRSETVLAAYKRLRESGQGKLWHGMSIDDFSSETRLLLRDTVFNLFEDVWPKEELPKPYGLRHLYAEVAYHNFAPPHVTKNSYFAAILGHNNNDLETSLSYMTYTLPEDRDNALARLKRTNERTLQQMATIAPVSRKG*

SEQ ID NO. 5 protein tag amino acid sequence

MKSSWSHPQFEKGAMTGWSHPQFEKRSAGSWSHPQFEKHHHHHHENLYFQS

SEQ ID NO. 9: prokaryotic telomerase TelA specific recognition sequence

GCGATCGATCATAATAACAATATCATGATATTGTTATTGTAATCGATCGC

SEQ ID NO. 10: prokaryotic telomerase TelN specific recognition sequence

TATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGTGCTGATA

SEQ ID NO. 14:PY54 telomerase amino acid sequence

MKIHFRDLVSGLVKEIDEIEKSDRAQGDKTRRYQGAARKFKNAVFMDKRKYRGNGMKNRISLTTFNKYLSRARSRFEERLHHSFPQSIATISNKYPAFSEIIKDLDNRPAHEVRIKLKELITHLESGVNLLEKIGSLGKIKPSTAKKIVSLKKMYPSWANDLDTLISTEDATELQQKLEQGTDLLNALHSLKVNHEVMYALTMQPSDRAALKARHDAALHFKKRNIVPIDYPGYMQRMTDILHLPDIAFEDSMASLAPLAFALAAASGRRQIEILITGEFDAKNKSIIKFSGQAKKRMAVSGGHYEIYSLIDSELFIQRLEFLRSHSSILRLQNLEIAHDEHRTELSVINGFVAKPLNDAAKQFFVDDRRVFKDTRAIYARIAYEKWFRTDPRWAKCDEDVFFSELLGHDDPDTQLAYKQFKLVNFNPKWTPNISDENPRLAALQELDNDMPGLARGDAAVRIHEWVKEQLAQNPAAKITAYQIKKNLNCRNDLASRYMAWCADALGVVIGDDGQARPEELPPSLVLDINADDTDAEEDEIEEDFTDEEIDDTEFDVSDNASDEDKPEDKPRFAAPIRRSEDSWLIKFEFAGKQYSWEGNAESVIDAMKQAWTENME*

SEQ ID NO. 15:phiKO2 telomerase amino acid sequence

MRKVKIGELINSLVSEVEAIDASDRPQGDKTKKIKAAALKYKNALFNDKRKFRGKGLEKRISANTFNSYMSRARKRFDDRLHHNFEKNVIKLSEKYPLYSEELSSWLSMPAASIRQHMSRLQAKLKEIMPLAEDLSNIKlGTKNSEAKINKLANKVPEWQFAISDLNSEDWKDKRDYLYKLFQQGSSLLEDLNNLKVNHEVLYHLQLSSAERTSIQQRWANVLSEKKRNVVVIDYPRYMQAIYDIINKPIVSFDLTTRRGMAPLAFALAALSGRRMIEIMLQGEFSVAGKYTVTFLGQAKKRSEDKGISRKIYTLCDATLFVSLVNELRSCPAAADFDEVIKGYGENDTRSENGRINAILATAFNPWVKTFLGDDRRVYKDSRAIYARIAYEMFFPVDPRWKNVDEDVFFMEILGHDDENTQLHYKQFKLANFSRTWRPNVGEENARLAALQKLDSMMPDFARGDAGVRIHETVKQLVEQDPSIKITNSTLRPFNFSTRLIPRYLEFAADALGQFVGENGQWQLKDEAPAIVLPDEEILEPMDDVDLDDENHDDETLDDDEIEVDESEGEELEEAGDAEEAEVAEQEEKHPGKPNFKAPRDNGDGTYMVEFEFGGRHYAWSGAAGNRVEAMQSAWSAYFK*

phiHAP-1(SEQ ID NO:16)

MSGESRRKVDLAELIEWLLSEIKEIDADDEMPRKEKTKRMARLARSFKTRLHDDKRRKDSERIAVTTFRRYMTEARKAVTAQNWRHHSFDQQIERLASRYPAYASKLEALGKLTDISAIRMAHRELLDQIRNDDDAYEDIRAMKLDHEIMRHLTLSSAQKSTLAEEASETLEERAVNTVEINYHWLMETVYELLSNRERMVDGEYRGFFSYLALGLALATGRRSIEVLKTGRITKVGEYELEFSGQAKKRGGVDYSEAYHIYTLVKADLVIEAWDELRSLPEAAELQGMDNSDVNRRTAKTLNTLTKRIFNNDERVFKDSRAIWARLVFELHFSRDKRWKKVTEDVFWREMLGHEDMDTQRSYRAFKIDYDEPDQADQEDYEHASRLAALQALDGHEQLESSDAQARVHAWVKAQIEQEPDAKITQSLISRELGVYRPAIKAYLELAREALDAPNVDLDKVAAAVPKEVAEAKPRLNAHPQGDGRWVGVASINGVEVARVGNQAGRIEAMKAAYKAAGGR*

ResT(SEQ ID NO:17)

MPPKVKIKNDFEIFRKELEILYKKYLNNELSYLKLKEKLKILAENHKAILFRKDKFTNRSIILNLSKTRKIIKEYINLSVIERIRRDNTFLFFWKSRRIKELKNIGIKDRKKIEELIFSNQMNDEKSYFQYFIDLFVTPKWLNDYAHKYKIEKINSYRKEQIFVKINLNTYIEIIKLLLNQSRDIRLKFYGVLMAIGRRPVEVMKLSQFYIADKNHIRMEFIAKKRENNIVNEVVFPVFADPELIINSIKEIRYMEQTENLTKEIISSNLAYSYNRLFRQIFNNIFAPEESVYFCRAIYCKFSYLAFAPKNMEMNYWITKVLGHEPNDITTAFHYNRYVLDNLDDKADNSLLTLLNQRIYTYVRRKATYSTLTMDRLESLIKEHHIFDDNYIKTLIVIKNLMLKDNLETLAMVRGLNVKIRKAFKATYGYNYNYIKLTEYLSIIFNYKL*

Claims

1. An enzyme digestion system for preparing a linear covalently closed DNA microcarrier, characterized in that the enzyme digestion system comprises a prokaryotic telomerase and the enzyme digestion system further comprises ATP and/or divalent metal ions.

2. The cleavage system of claim 1, wherein the prokaryotic telomerase is TelA, telN, PY, phiKO2, phiHAP-1 or ResT;

preferably, the amino acid sequence of TelA is shown as SEQ ID NO. 3, the amino acid sequence of TelN is shown as SEQ ID NO. 1, the amino acid sequence of PY54 is shown as SEQ ID NO. 14, the amino acid sequence of phiKO2 is shown as SEQ ID NO. 15, the amino acid sequence of phiHAP-1 is shown as SEQ ID NO. 16 or the amino acid sequence of ResT is shown as SEQ ID NO. 17;

more preferably, the nucleotide sequence encoding TelA is shown as SEQ ID NO. 4 or the nucleotide sequence encoding TelN is shown as SEQ ID NO. 2.

3. The cleavage system according to claim 1, characterized in that the concentration of ATP is between 0.1 and 1.2mM, preferably between 0.5 and 1mM; and/or the divalent metal ion concentration is 1 to 25mM, preferably 5 to 20mM, for example 10mM;

preferably, the divalent metal ion comprises a metal selected from the group consisting of Mg ²⁺ 、Ca ²⁺ And Mn of ²⁺ One or more of the following; preferably comprises Mg ²⁺ And/or Ca ²⁺ 。

4. The cleavage system according to claim 1, characterized in that the cleavage system further comprises a base cleavage buffer;

preferably, the base digestion buffer comprises 18-22 mM Tris-HCl, pH 7.0-8.0, 48-52 mM potassium glutamate, 0.8-1.2 mM DTT, and 0.08-0.12 mM EDTA; for example, the base cleavage buffer comprises 20mM Tris-HCl, 50mM potassium glutamate, 1mM DTT and 0.1mM EDTA, pH 7.5.

5. The cleavage system according to any one of claims 1 to 4, characterized in that the cleavage system further comprises a restriction endonuclease and/or a restriction endonuclease buffer;

preferably, the restriction endonuclease buffer comprises 45-55 mM potassium acetate, 15-25 mM Tris acetate, 5-15 mM magnesium acetate and 90-110. Mu.g/ml BSA, and the pH is 7.5-8.0; for example, the restriction endonuclease buffer contained 50mM potassium acetate, 20mM Tris acetate, 10mM magnesium acetate and 100. Mu.g/ml BSA, pH7.9.

6. A kit comprising the cleavage system according to any one of claims 1 to 5.

7. Use of the cleavage system according to any one of claims 1 to 5 or the kit according to claim 6 for the preparation of a linear covalently closed DNA microcarrier.

8. A method for preparing a linear covalently closed DNA microcarrier, comprising the steps of: in vitro digestion of target DNA comprising at least 1 recognition site for a prokaryotic telomerase using the digestion system of any one of claims 1 to 5 or the kit of claim 6;

preferably, the mass ratio of the target DNA to the prokaryotic telomerase is 3 (0.25-3.75), e.g., 3 (0.5-1).

9. The method of claim 8, wherein the target DNA comprises a sequence of interest and a redundant sequence; both ends of the target sequence comprise recognition sites of the prokaryotic telomerase;

when the target DNA is a linear plasmid, the recognition sites of the prokaryotic telomerase at the two ends of the linear plasmid and the recognition sites of the prokaryotic telomerase at the two ends of the target sequence are the recognition sites of different types of prokaryotic telomerase; the backbone of the linear plasmid, e.g., pJAZZ-OK plasmid;

10. The method of claim 9, wherein the target DNA is a circular plasmid comprising at least 1 restriction endonuclease site and wherein the sequence of interest does not comprise the restriction endonuclease site, and wherein the method further comprises linearizing the circular plasmid with a restriction endonuclease prior to the in vitro cleavage;

preferably, the time for linearizing the restriction endonuclease is 4 to 16 hours; and/or, the temperature at which the restriction endonuclease linearizes is 37 ℃.