Double-stranded circular DNA vector, preparation method of straight-chain covalently closed DNA, and fusion polypeptide containing front telomerase and endonuclease

Abstract

The present invention addresses the problem of providing a novel method for producing linear covalently closed DNA in a simple and efficient manner. The invention provides a double-stranded circular DNA (deoxyribonucleic acid) vector. The present invention relates to a vector comprising: a prolomerase gene sequence encoding a prolomerase or an active fragment thereof; an endonuclease gene sequence encoding an endonuclease or an active fragment thereof; and a pair of prolomerase recognition sequences for recognizing the prolomerase or the active fragment thereof and cleaving the vector. The double-stranded circular DNA vector comprises at least one endonuclease recognition sequence for recognizing the endonuclease or an active fragment thereof and cleaving the vector, and a target nucleic acid sequence. The foretelomerase gene sequence, the endonuclease gene sequence, and the endonuclease recognition sequence are arranged in the same region between the pair of foretelomerase recognition sequences, and the target nucleic acid sequence is arranged in another region between the pair of foretelomerase recognition sequences. And the foretelomerase gene sequence and the endonuclease gene sequence are configured under the control of a promoter capable of controlling expression.

Description

Technical Field

[0001] The present invention relates to a double-stranded circular DNA vector, a transformed cell, and a method for preparing a linear covalently closed DNA. Background Art

[0002] Double-stranded circular DNA vectors have been widely used as DNA vectors that can be easily handled. In addition to the target gene sequence, the double-stranded circular DNA vector also contains a replication origin sequence, an antibiotic resistance gene, and the like, which are sequences required for the maintenance and replication of the plasmid. When the double-stranded circular DNA vector is introduced into the tissues and cells of an animal, these sequences are also delivered together with the target gene sequence, but this is not desirable from the perspective of avoiding immunogenicity.

[0003] As a method for removing unnecessary sequences from a double-stranded circular DNA vector and obtaining only a target gene sequence, linear covalently closed DNA (LCC DNA) is used (Patent Documents 1 and 2).

[0004] Linear covalently closed DNA is a linear double-stranded DNA whose end portion is closed by a hairpin structure, and can be prepared by utilizing the activity of forming a hairpin structure at the cut end when the protelomerase cuts the double-stranded DNA. For example, a double-stranded circular DNA vector is prepared in which a target gene sequence is configured between a pair of protelomerase recognition sequences. Then, by allowing the protelomerase activity to act on the double-stranded circular DNA vector, a linear covalently closed DNA containing only the target gene sequence and whose end is closed by a hairpin structure can be prepared. The linear covalently closed DNA prepared in this way has tolerance to nuclease exonucleases, and is therefore stable, and due to its small size, it also has the advantage of high efficiency of introduction into cells. In addition, by configuring the protelomerase recognition sequence, the replication origin sequence, antibiotic resistance gene, etc. can be removed from the linear covalently closed DNA containing the target gene, so the risk of immunogenicity, etc. can also be reduced.

[0005] Patent document 1 discloses a method for preparing linear covalently closed DNA in vitro. The method described in patent document 1 is to synthesize double-stranded DNA molecules in vitro using DNA polymerase, and then treat them with front end polymerase to cut the double-stranded DNA molecules, thereby preparing a method for linear covalently closed DNA. However, the steps of making various enzymes react in vitro in this method are very complicated. In addition, in order to decompose and remove the unnecessary second linear covalently closed DNA from a vector sequence other than the target gene generated simultaneously with the first linear covalently closed DNA containing the target gene, various enzymes need to be separated, so the cost is very high.

[0006] Patent document 2 discloses a method for preparing linear covalently closed DNA in cells. The method described in patent document 2 is a method for preparing linear covalently closed DNA by amplifying double-stranded DNA molecules in cells, and then inducing the expression of front end telomerase in cells to cut the double-stranded DNA molecules. However, this method also generates unnecessary second linear covalently closed DNA from vector sequences other than the target gene together with the first linear covalently closed DNA containing the target gene. Therefore, it is still necessary to selectively decompose and remove the second linear covalently closed DNA in vitro after separating the first and second linear covalently closed DNAs from Escherichia coli.

[0007] Therefore, new methods for preparing linear covalently closed DNA in a simple and efficient manner are needed.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: U.S. Patent No. 9109250

[0011] Patent Document 2: International Publication No. 2022 / 209987 Summary of the invention

[0012] Problems to be solved by the invention

[0013] An object of the present invention is to provide a novel method for preparing linear covalently closed DNA simply and efficiently.

[0014] Solution to the problem

[0015] In the reaction in which a double-stranded circular DNA vector is cleaved by front end polymerase activity to generate linear covalently closed DNA, in addition to the first linear covalently closed DNA containing the target gene, linear covalently closed DNA derived from a vector sequence other than the target gene is also generated as an unnecessary second linear covalently closed DNA.

[0016] The present inventors have come up with the following method to solve the above-mentioned problems: by introducing all necessary elements into a double-stranded circular DNA vector when generating linear covalently closed DNA in cells, linear covalently closed DNA can be simply produced. Specifically, in order to induce the decomposition of the second linear covalently closed DNA that is not needed and generated at the same time as the first linear covalently closed DNA is generated by the double-stranded circular DNA vector in cells, a target gene located between the front end merase recognition sequences, and a fusion gene sequence encoding a fusion polypeptide containing the front end merase and the endonuclease located in other regions between the front end merase recognition sequences, and an endonuclease recognition sequence are arranged in a double-stranded circular DNA vector. The endonuclease and the endonuclease recognition sequence are introduced to induce the decomposition of the second linear covalently closed DNA that is not needed.

[0017] The inventors of the present invention introduced the above-mentioned double-stranded circular DNA vector into Escherichia coli as a host cell and induced the expression of a fusion polypeptide containing an endomerase and an endonuclease. As a result, it was found that by inducing the decomposition of the second linear covalently closed DNA, the first linear covalently closed DNA containing the target gene sequence can be produced simply and efficiently.

[0018] In the present invention, since all the elements required for producing linear covalently closed DNA in cells are introduced into one double-stranded circular DNA vector, there is an advantage that linear covalently closed DNA can be produced extremely simply. Based on the above findings, the present invention provides the following aspects.

[0019] (1) A double-stranded circular DNA vector comprising:

[0020] A fusion gene sequence encoding a fusion polypeptide comprising an anterior terminal merase or an active fragment thereof and an endonuclease or an active fragment thereof,

[0021] A pair of premerase recognition sequences for recognizing the premerase or its active fragment and cutting the vector,

[0022] at least one endonuclease recognition sequence for recognizing the endonuclease or its active fragment and cleaving the vector, and

[0023] The target gene sequence encoding the target protein or its fragment,

[0024] In the double-stranded circular DNA vector, the fusion gene sequence and the endonuclease recognition sequence are arranged in the same region between the pair of front end merase recognition sequences, and the target gene sequence is arranged in another region between the pair of front end merase recognition sequences, and

[0025] The above fusion gene sequence is placed under the control of a promoter capable of controlling expression.

[0026] (2) The double-stranded circular DNA vector according to (1), wherein in the fusion polypeptide, the front endomerase or the active fragment thereof is arranged on the N-terminal side of the endonuclease or the active fragment thereof.

[0027] (3) The double-stranded circular DNA vector according to (1) or (2), wherein the fusion polypeptide further comprises a linker sequence between the telomerase or an active fragment thereof and the endonuclease or an active fragment thereof.

[0028] (4) The double-stranded circular DNA vector according to any one of (1) to (3), further comprising a spacer sequence disposed between the endonuclease recognition sequence and the telomerase recognition sequence.

[0029] (5) The double-stranded circular DNA vector according to (4), wherein the spacer sequence is 200 bases or less in length.

[0030] (6) The double-stranded circular DNA vector according to any one of (1) to (3), wherein the endonuclease recognition sequence is arranged adjacent to one of the telomerase recognition sequences.

[0031] (7) The double-stranded circular DNA vector according to any one of (1) to (6), which is a plasmid.

[0032] (8) The double-stranded circular DNA vector according to any one of (1) to (7), wherein the anterior terminal telomerase is a TelN anterior terminal telomerase, a TelA anterior terminal telomerase or a TelK anterior terminal telomerase.

[0033] (9) The double-stranded circular DNA vector according to any one of (1) to (8), wherein the endonuclease is a homing endonuclease.

[0034] (10) A transformed cell comprising the double-stranded circular DNA vector according to any one of (1) to (9).

[0035] (11) A method for preparing a linear covalently closed DNA, the method comprising:

[0036] a step of culturing the transformed cells according to (10),

[0037] an expression induction step of inducing expression of the fusion polypeptide in the transformed cells after the culturing step, and

[0038] A DNA extraction step is a step of extracting DNA from the transformed cells after the above expression induction step.

[0039] (12) The method according to (11), further comprising: a separation step of separating the linear covalently closed DNA after the DNA extraction step.

[0040] (13) The method according to (11) or (12), further comprising: before the above-mentioned culturing step, an introduction step of introducing the double-stranded circular DNA vector described in any one of (1) to (9) into a host cell to obtain a transformed cell.

[0041] (14) The method according to any one of (11) to (13), wherein the host cell or transformed cell expresses an exonuclease.

[0042] (15) A fusion polypeptide or a nucleic acid encoding the same, wherein the fusion polypeptide comprises a front endomerase or an active fragment thereof at the N-terminal side and an endonuclease or an active fragment thereof at the C-terminal side.

[0043] In addition, the present inventors arranged a target gene located between the front end merase recognition sequences and a front end merase gene sequence, an endonuclease gene sequence, and an endonuclease recognition sequence located in another region between the front end merase recognition sequences in a double-stranded circular DNA vector in order to induce the degradation of an unwanted second linear covalently closed DNA, wherein the second linear covalently closed DNA is generated from the double-stranded circular DNA vector in the cell at the same time as the first linear covalently closed DNA is generated. The endonuclease and the endonuclease recognition sequence are introduced in order to induce the degradation of the unwanted second linear covalently closed DNA.

[0044] The present inventors introduced the double-stranded circular DNA vector into Escherichia coli as a host cell and induced the expression of front endomerase and endonuclease, and found that by inducing the degradation of the second linear covalently closed DNA, the first linear covalently closed DNA containing the target gene sequence can be simply and efficiently produced. Based on the above findings, the present invention provides the following aspects.

[0045] (1) A double-stranded circular DNA vector comprising:

[0046] A pre-telomerase gene sequence encoding a pre-telomerase or an active fragment thereof,

[0047] An endonuclease gene sequence encoding an endonuclease or an active fragment thereof,

[0048] A pair of premerase recognition sequences for recognizing the premerase or its active fragment and cutting the vector,

[0049] at least one endonuclease recognition sequence for recognizing the endonuclease or its active fragment and cleaving the vector, and

[0050] a target nucleic acid sequence (e.g., a target gene sequence encoding a target protein or a fragment thereof),

[0051] In the above-mentioned double-stranded circular DNA vector, the above-mentioned front-end merase gene sequence, the above-mentioned endonuclease gene sequence and the above-mentioned endonuclease recognition sequence are arranged in the same region between the above-mentioned pair of front-end merase recognition sequences, and the above-mentioned target nucleic acid sequence (for example, the above-mentioned target gene sequence) is arranged in another region between the above-mentioned pair of front-end merase recognition sequences, and

[0052] The anterior telomerase gene sequence and the endonuclease gene sequence are placed under the control of a promoter capable of controlling expression.

[0053] (2) The double-stranded circular DNA vector according to (1), which is a plasmid.

[0054] (3) The double-stranded circular DNA vector according to (1) or (2), wherein the aforementioned terminal endomerase is a TelN terminal endomerase, a TelA terminal endomerase, or a TelK terminal endomerase.

[0055] (4) The double-stranded circular DNA vector according to any one of (1) to (3), wherein the endonuclease is a homing endonuclease.

[0056] (5) The double-stranded circular DNA vector according to (4), wherein the homing endonuclease is I-SceI homing endonuclease, I-CeuI homing endonuclease or I-CreI homing endonuclease.

[0057] (6) A transformed cell comprising the double-stranded circular DNA vector according to any one of (1) to (5).

[0058] (7) The transformed cell according to (6), wherein the cell is a bacterial cell or a eukaryotic cell.

[0059] (8) The transformed cell according to (7), wherein the bacterium is Escherichia coli or Bacillus subtilis.

[0060] (9) The transformed cell according to any one of (6) to (8), which expresses an exonuclease.

[0061] (10) A method for preparing linear covalently closed DNA, the method comprising:

[0062] a step of culturing the transformed cell according to any one of (6) to (9),

[0063] an expression induction step of inducing the expression of the telomerase or an active fragment thereof and the endonuclease or an active fragment thereof in the transformed cells after the culturing step, and

[0064] A DNA extraction step is a step of extracting DNA from the transformed cells after the above expression induction step.

[0065] (11) The method according to (10), further comprising: a separation step of separating the linear covalently closed DNA after the DNA extraction step.

[0066] (12) The method according to (10) or (11), further comprising: before the above-mentioned culturing step, an introduction step of introducing the double-stranded circular DNA vector described in any one of (1) to (5) into a host cell to obtain a transformed cell.

[0067] (13) The method according to any one of (10) to (12), wherein the transformed cell expresses an exonuclease.

[0068] Furthermore, the present invention provides the following aspects.

[0069] (1) A double-stranded circular DNA vector comprising:

[0070] A pre-telomerase gene sequence encoding a pre-telomerase or an active fragment thereof,

[0071] An endonuclease gene sequence encoding an endonuclease or an active fragment thereof,

[0072] A pair of premerase recognition sequences for recognizing the premerase or its active fragment and cutting the vector,

[0073] at least one endonuclease recognition sequence for recognizing the endonuclease or its active fragment and cleaving the vector, and

[0074] a target nucleic acid sequence (e.g., a target gene sequence encoding a target protein or a fragment thereof),

[0075] In the above-mentioned double-stranded circular DNA vector, the above-mentioned front-end merase gene sequence, the above-mentioned endonuclease gene sequence and the above-mentioned endonuclease recognition sequence are arranged in the same region between the above-mentioned pair of front-end merase recognition sequences, and the above-mentioned target nucleic acid sequence (for example, the above-mentioned target gene sequence) is arranged in another region between the above-mentioned pair of front-end merase recognition sequences, and

[0076] The anterior telomerase gene sequence and the endonuclease gene sequence are placed under the control of a promoter capable of controlling expression.

[0077] (2) The double-stranded circular DNA vector according to (1), wherein the pre-telomerase gene sequence and the pre-endonuclease gene sequence encode a fusion polypeptide comprising the pre-telomerase or an active fragment thereof and the pre-endonuclease or an active fragment thereof.

[0078] (3) The double-stranded circular DNA vector according to (2), wherein in the fusion polypeptide, the front endomerase or the active fragment thereof is arranged on the N-terminal side of the endonuclease or the active fragment thereof.

[0079] (4) The double-stranded circular DNA vector according to (2) or (3), wherein the fusion polypeptide further comprises a linker sequence between the telomerase or an active fragment thereof and the endonuclease or an active fragment thereof.

[0080] (5) The double-stranded circular DNA vector according to any one of (1) to (4), further comprising a spacer sequence disposed between the endonuclease recognition sequence and the telomerase recognition sequence.

[0081] (6) The double-stranded circular DNA vector according to (5), wherein the spacer sequence is 200 bases or less in length.

[0082] (7) The double-stranded circular DNA vector according to any one of (1) to (6), wherein the endonuclease recognition sequence is arranged adjacent to one of the telomerase recognition sequences.

[0083] (8) The double-stranded circular DNA vector according to any one of (1) to (7), which is a plasmid.

[0084] (9) The double-stranded circular DNA vector according to any one of (1) to (8), wherein the anterior terminal endomerase is a TelN anterior terminal endomerase, a TelA anterior terminal endomerase or a TelK anterior terminal endomerase.

[0085] (10) The double-stranded circular DNA vector according to any one of (1) to (9), wherein the endonuclease is a homing endonuclease.

[0086] (11) The double-stranded circular DNA vector according to (10), wherein the homing endonuclease is I-SceI homing endonuclease, I-CeuI homing endonuclease or I-CreI homing endonuclease.

[0087] (12) A transformed cell comprising the double-stranded circular DNA vector according to any one of (1) to (11).

[0088] (13) The transformed cell according to (12), which is a bacterial cell or a eukaryotic cell.

[0089] (14) The transformed cell according to (13), wherein the bacterium is Escherichia coli or Bacillus subtilis.

[0090] (15) The transformed cell according to any one of (12) to (14), which expresses a nuclease.

[0091] (16) A method for preparing linear covalently closed DNA, the method comprising:

[0092] a step of culturing the transformed cell according to any one of (12) to (15),

[0093] an expression induction step of inducing the expression of the telomerase or an active fragment thereof and the endonuclease or an active fragment thereof in the transformed cells after the culturing step, and

[0094] A DNA extraction step is a step of extracting DNA from the transformed cells after the above expression induction step.

[0095] (17) The method according to (16), further comprising: a separation step of separating the linear covalently closed DNA after the DNA extraction step.

[0096] (18) The method according to (16) or (17), further comprising: before the culturing step, a step of introducing the double-stranded circular DNA vector according to claim 1 into a host cell to obtain a transformed cell.

[0097] (19) The method according to any one of (16) to (18), wherein the transformed cell expresses an exonuclease.

[0098] (20) A fusion polypeptide or a nucleic acid encoding the same, wherein the fusion polypeptide comprises a front endomerase or an active fragment thereof at the N-terminal side and an endonuclease or an active fragment thereof at the C-terminal side.

[0099] This specification includes the disclosure of Japanese Patent Application No. 2022-144282 and Japanese Patent Application No. 2022-144285 on which the priority of this application is based.

[0100] Effects of the Invention

[0101] According to the present invention, a new method for preparing linear covalently closed DNA simply and efficiently can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 This is a diagram showing a control vector for preparing linear covalently closed DNA.

[0103] Figure 2It is a diagram showing the isolated expression vector for the preparation of linear covalently closed DNA.

[0104] Figure 3 The diagram shows a fusion polypeptide and DNA in which I-SceI homing endonuclease is fused to the C-terminal side of TelN frontomerase via a linker sequence.

[0105] Figure 4 It is a diagram showing a fusion expression vector for preparing linear covalently closed DNA.

[0106] Figure 5 It is a diagram showing the electrophoresis results of linear covalently closed DNA. The linear covalently closed DNA containing the nucleic acid encoding the packaging protein is represented as "target linear covalently closed DNA", and the linear covalently closed DNA containing the nucleic acid encoding the TelN front endomerase and the I-SceI homing endonuclease is represented as "unnecessary linear covalently closed DNA". "Con" represents the control vector, and "M" represents the DNA marker (New England Biolabs, N3200).

[0107] Figure 6 It is a figure showing the results of studying the expression induction conditions. In the figure, "Method A" represents a method of shaking culture at 37°C for 6 hours, and "Method B" represents a method of shaking culture at 30°C for 4 hours and then shaking culture at 37°C for 2 hours. pH represents the pH of the Plusgrow II medium added during expression induction. "M" represents a DNA marker (New England Biolabs, N3200).

[0108] Figure 7 This is a diagram showing a control vector (T6) among the two vectors for preparing linear covalently closed DNA prepared in Comparative Example 1.

[0109] Figure 8 This is a diagram showing the I-SceI vector among the two vectors for preparing linear covalently closed DNA prepared in Comparative Example 1.

[0110] Fig. 9 : is a diagram showing the electrophoresis results of the linear covalently closed DNA in Comparative Example 2. The linear covalently closed DNA containing the nucleic acid encoding the packaging protein is represented as "target linear covalently closed DNA", and the other linear covalently closed DNA is represented as "unnecessary linear covalently closed DNA". In the figure, the band with high mobility corresponds to the residual I-SceI vector. "M" represents a DNA marker (manufactured by New England Biolabs, N3200). DETAILED DESCRIPTION

[0111] 1. Double-stranded circular DNA vector

[0112] 1-1. Overview

[0113] The first embodiment of the present invention is a double-stranded circular DNA vector. The double-stranded circular DNA vector of this embodiment contains a fusion gene sequence encoding a fusion polypeptide containing a front-end merase or its active fragment and an endonuclease or its active fragment, and an endonuclease recognition sequence in the same region between a pair of front-end merase recognition sequences, and contains a target nucleic acid sequence in another region between the pair of front-end merase recognition sequences.

[0114] According to the double-stranded circular DNA vector of this embodiment, a linear covalently closed DNA containing a target nucleic acid sequence can be prepared simply and efficiently.

[0115] 1-2. Definition of terms

[0116] The following terms commonly used in this specification are defined.

[0117] In this specification, "linear covalently closed DNA (LCCDNA)" refers to a linear double-stranded DNA whose ends are closed by a hairpin structure. At each end of the linear covalently closed DNA, the 5' end and the 3' end of the two DNA chains are connected to form a hairpin structure. The end portion of the linear covalently closed DNA protected by the hairpin structure is resistant to degradation by exonucleases.

[0118] In the present specification, "telomerase" refers to a polypeptide having the following activity (hereinafter referred to as "telomerase activity"), which is to recognize a specific sequence in a double-stranded DNA molecule, cut the double-stranded DNA molecule at a position within or near the recognition sequence, and connect the 5' end and the 3' end of the cut end to form a covalently closed end. In the present specification, the type of telomerase is not particularly limited as long as it has the above-mentioned activity. For example, the telomerase derived from Agrobacterium fabrum (TelA telomerase, ACCESSION No. AAK88254), the telomerase derived from Halomonas virus HAP1 (ACCESSION No. ABY90402), the telomerase derived from Vibrio virus VP882 (ACCESSION No. ABM73418), the telomerase derived from Klebsiella phage phiKO2 (TelK telomerase, ACCESSION No. AAR83042), the telomerase derived from Rhizobium pusense (ACCESSION No. QKJ91773), the telomerase derived from Feldmanniaspecies virus (ACCESSION No. ACH46812), the telomerase derived from Vibrio phage vB_VpaM_MAR (ACCESSION No. AFV81380), the telomerase derived from Yersinia phage PY54-derived telomerase (Tel telomerase, ACCESSION No. CAD91792), Escherichia virus N15-derived telomerase (TelN telomerase, ACCESSION No. AAB81106), or mutants of any of the telomerases described above. In the present specification, telomerase is not included in the endonuclease described below.

[0119] In the present specification, the "active fragment" of the front-end telomerase is a polypeptide fragment that contains a part of the front-end telomerase and retains the activity of the front-end telomerase, for example, retains 1% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more of the activity of the full-length protein. The length of the amino acids of the polypeptide constituting the active fragment is not particularly limited. For example, it can be a continuous region of at least 100, 150, 200, 250, 300, 350 or 400 amino acids that contains the catalytic domain in the front-end telomerase.

[0120] In the present specification, a "front-end telomerase gene" is a gene encoding a front-end telomerase or an active fragment thereof. Examples of front-end telomerase genes include genes encoding any of the above-mentioned front-end telomerases or active fragments thereof. Examples include, for example, a front-end telomerase gene encoding a TelN front-end telomerase formed by the amino acid sequence shown in SEQ ID NO: 24, a TelN front-end telomerase gene formed by the base sequence shown in SEQ ID NO: 2, and the like.

[0121] In this specification, "telomerase recognition sequence" refers to a sequence recognized by telomerase. Telomerase recognizes the telomerase recognition sequence, cuts the double-stranded DNA molecule at a position within or near the telomerase recognition sequence, and rejoins the cut ends. It is known that the telomerase recognition sequence is a palindromic sequence or a palindrome-like sequence.

[0122] In this specification, "endonuclease" refers to an enzyme having the activity of cutting phosphodiester bonds inside a nucleotide chain. Among endonucleases, endonucleases that recognize a specific sequence and cut double-stranded DNA molecules within or near the sequence are particularly referred to as restriction enzymes. In addition to restriction enzymes of natural origin, restriction enzymes can also include artificial restriction enzymes such as TALEN and zinc finger nucleases (ZFN), genome editing enzymes such as Cas9 proteins, or mutants of any of the above endonucleases. It should be noted that the endonuclease can be any one of an enzyme having the activity of cutting two DNA chains or an enzyme (Nickase) that cuts only one DNA chain.

[0123] Among restriction enzymes, those found in inteins, introns, etc. are specifically called "homing endonucleases." In general, homing endonucleases are known to recognize long recognition sequences of 12 to 40 bp, and it is also known that the recognition sequence is not a palindromic sequence. In this specification, the type of homing endonuclease is not particularly limited. Examples include: I-SceI (ACCESSION No. NP009324), I-CeuI (ACCESSION No. CAA78934), I-CreI (ACCESSION No.CAA26008), I-AniI, I-ChuI, I-DmoI, I-CsmI, PI-SceI, PI-TIiI, PI-MtuI, I-SceII, I-SceIII, HO, PI-Civ I, PI-CtrI, PI-AaeI, PI-BsuI, PI-DhaI, PI-DraI, PI-MavI, PI-MchI, PI-MfuI, PI-MflI, PI-MgaI, PI-MgoI, PI -MinI, PI-MkaI, PI-MmaI, PI-MshI, PI-MsmI, I-MsoI, PI-MthI, PI-MtuI, PI-MxeI, PI-NpuI, PI-PfuI, PI-RmaI, PI-SpbI, PI-SspI, PI-FacI, PI-MjaI, PI-PhoI, PI-TagI, PI-ThyI, PI-TkoI, PI-TspI, or mutants of any of the above homing nucleases, etc. Among them, preferred are I-SceI, I-ChuI, I-DmoI, I-CreI, I-CsmI, PI-PfuI, PI-SceI, PI-TliI, I-MsoI, PI-MtuI, I-CeuI, I-SceII, I-SceIII, HO, or mutants of any of the above homing endonucleases, etc.; more preferred are I-SceI, I-ChuI, I-DmoI, I-CreI, I-CsmI, PI-SceI, PI-PfuI, PI-TliI, I-MsoI, PI-MtuI, I-CeuI, or mutants of any of the above homing endonucleases, etc.; further preferred are I-SceI, I-CeuI, I-CreI, or mutants of any of the above homing endonucleases, etc.

[0124] In this specification, the "active fragment" of endonuclease is a polypeptide fragment that contains a part of the region of endonuclease and the fragment keeps endonuclease activity, for example, keeps more than 1%, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98% or equivalent of the activity of the full-length protein. The amino acid length of the polypeptide constituting this active fragment is not particularly limited. For example, it can be a continuous region of at least 100, 150, 200, 250, 300, 350 or 400 amino acids that contain a catalytic domain in the endonuclease.

[0125] In this specification, "endonuclease gene" is a gene encoding an endonuclease or its active fragment. As an example of an endonuclease gene, a gene encoding any of the above-mentioned endonucleases or its active fragment can be cited. For example, a gene encoding an I-SceI homing endonuclease formed by the amino acid sequence shown in sequence number 25, for example, an I-SceI homing endonuclease gene encoding a base sequence shown in sequence number 4, etc. can be cited.

[0126] In the present specification, "endonuclease recognition sequence" refers to a sequence recognized by an endonuclease. An endonuclease recognizes the endonuclease recognition sequence and cuts a double-stranded DNA molecule at a position within or near the endonuclease recognition sequence.

[0127] In the present invention, "double-stranded circular DNA vector" refers to a circular double-stranded DNA molecule that can be maintained and / or replicated in a cell. The double-stranded circular DNA vector may contain sequences required for maintenance and replication in the cell, such as genes encoding replication origins and / or antibiotic resistance proteins. The double-stranded circular DNA vector may be, for example, a plasmid vector or a Bacmid vector. In addition, the vector may be a shuttle vector that can replicate between bacteria such as Escherichia coli and mammalian cells.

[0128] 1-3. Composition

[0129] The double-stranded circular DNA vector of the present embodiment comprises a fusion gene sequence, a pair of front-end merase recognition sequences, at least one endonuclease recognition sequence, and a target nucleic acid sequence. In the double-stranded circular DNA vector of the present embodiment, the fusion gene sequence and the endonuclease recognition sequence are arranged in the same region between the pair of front-end merase recognition sequences, and the target nucleic acid sequence is arranged in another region between the pair of front-end merase recognition sequences. In addition, the fusion gene sequence is arranged under the control of a promoter that can control expression.

[0130] In this specification, "fusion gene sequence" refers to a gene sequence encoding a fusion polypeptide comprising a front telomerase or an active fragment thereof and an endonuclease or an active fragment thereof. The fusion gene sequence can be codon-optimized according to the codon usage frequency in the cell into which the double-stranded circular DNA vector is to be introduced.

[0131] In the present specification, "a fusion polypeptide comprising a front end merase or an active fragment thereof and an endonuclease or an active fragment thereof" may be a fusion polypeptide in which the front end merase or an active fragment thereof is arranged at the N-terminal side of the endonuclease or an active fragment thereof, or a fusion polypeptide in which the front end merase or an active fragment thereof is arranged at the C-terminal side of the endonuclease or an active fragment thereof. Preferably, the front end merase or an active fragment thereof is arranged at the N-terminal side of the endonuclease or an active fragment thereof.

[0132] The type of the front-end telomerase or its active fragment contained in the fusion polypeptide is not particularly limited as long as it has the front-end telomerase activity, as long as it does not recognize sequences other than a pair of front-end telomerase recognition sequences in the double-stranded circular DNA vector, especially the target nucleic acid sequence. In addition, there may be no front-end telomerase recognition sequence in the genome sequence of the cell to be introduced into the double-stranded circular DNA vector. For example, the length of the front-end telomerase recognition sequence recognized by the front-end telomerase or its active fragment contained in the fusion polypeptide is not limited, and can be 5 bp or more, 6 bp or more, 7 bp or more, 8 bp or more, 9 bp or more, 10 bp or more, 15 bp or more, 20 bp or more, 25 bp or more, 30 bp or more, 35 bp or more, 40 bp or more, 45 bp or more, 50 bp or more, 55 bp or more, 60 bp or more, 65 bp or more, 70 bp or more, 80 bp or more or 90 bp or more. As particularly preferred examples of the above-mentioned front-end telomerases, TelN front-end telomerase, TelA front-end telomerase, or TelK front-end telomerase, or mutants thereof can be cited. As mutants, there are no particular restrictions and can be appropriately selected according to the purpose. For example, the homology with the original amino acid sequence may be 80% or more, 90% or more, 95% or more, or 99% or more.

[0133] The endonuclease or its active fragment included in the fusion polypeptide can be, as long as it has endonuclease activity, not particularly limited, as long as it does not recognize sequences other than the endonuclease recognition sequence in the double-stranded circular DNA vector, particularly the target nucleic acid sequence. In addition, there can be no endonuclease recognition sequence in the genomic sequence of the cell to be introduced into the double-stranded circular DNA vector. The endonuclease or its active fragment can be a restriction enzyme, or can be a nickase (Nickase). For example, the length of the endonuclease recognition sequence identified by the endonuclease or its active fragment included in the fusion polypeptide is not limited, and can be more than 4bp, more than 5bp, more than 6bp, more than 7bp, more than 8bp, more than 9bp, more than 10bp, more than 15bp, more than 20bp, more than 25bp, more than 30bp, more than 35bp, more than 40bp, more than 45bp, more than 50bp, more than 60bp, more than 70bp, more than 80bp, more than 90bp or more than 100bp.

[0134] In one embodiment of the present invention, the endonuclease can be a homing endonuclease. As the homing endonuclease, there is no limitation, and examples thereof include I-SceI, I-CeuI, I-CreI, I-AniI, I-ChuI, I-DmoI, I-CsmI, PI-SceI, PI-TIiI, PI-MtuI, I-CeuI, I-SceII, I-SceIII, HO, PI-CivI, PI-CtrI, PI-AaeI, PI-BsuI, PI-DhaI, PI-DraI, PI-MavI, PI-MchI, PI-MfuI, PI-Mfl I, PI-Mgal, PI-MgoI, PI-MinI, PI-MkaI, PI-MkaI, PI-MmaI, PI-MshI, PI-MsmI, I-MsoI, PI-MthI, PI-MtuI, PI-MxeI, PI-NpuI, PI-PfuI, PI-RmaI, PI-SpbI, PI-SspI, PI-FacI, PI-MjaI, PI-PhoI, PI-TagI, PI-ThyI, PI-TkoI, PI-TspI, or mutants thereof. As mutants, there are no particular restrictions and can be appropriately selected according to purpose. For example, the homology with the original amino acid sequence can be more than 80%, more than 90%, more than 95% or more than 99%.

[0135] In the double-stranded circular DNA vector of the present invention, the fusion gene sequence is placed under the control of a promoter capable of controlling expression.

[0136] In this specification, "a promoter capable of controlling expression" refers to a promoter capable of inducing the expression of a target gene, etc., in a cell at any time, and is a gene expression regulatory region capable of controlling the expression of a gene, etc., arranged downstream (3' end side) in a cell into which a double-stranded circular DNA vector has been introduced. A promoter capable of controlling expression can be induced, for example, by the presence or absence of a specific factor, temperature, and other conditions. The type of promoter capable of controlling expression is not limited and can be appropriately selected according to the purpose. Examples thereof include heat-inducible promoters (lambda PR, lambda PL, etc.), arabinose-inducible promoters (araBAD promoter, etc.), IPTG-inducible promoters (LAC promoter, LACUV5 promoter, TAC promoter, Trc promoter, LPP promoter, etc.), T7 promoter, low temperature-inducible promoters (cspA promoter, etc.), Trp promoter, rhamnose-inducible promoters (rhaT promoter, etc.), proU promoter, prpB promoter, phoA promoter, recA promoter, tetA promoter, cadA promoter, or mutants thereof, preferably IPTG-inducible promoters and arabinose-inducible promoters.

[0137] In the present specification, “arranged under the control of a promoter capable of controlling expression” means that a gene to be expressed or the like is arranged in a promoter downstream region under the control of a promoter capable of controlling expression.

[0138] The above-mentioned fusion polypeptide may include a linker sequence between the front end telomerase or its active fragment and the endonuclease or its active fragment. In this specification, "linker sequence" refers to a peptide that can be inserted between the front end telomerase or its active fragment and the endonuclease or its active fragment in the fusion polypeptide in order to enable the fused parts to perform the target functions. The length of the linker sequence is not limited, and is, for example, 1 amino acid length or more, 2 amino acids length or more, 3 amino acids length or more, 4 amino acids length or more, 5 amino acids length or more, 6 amino acids length or more, 7 amino acids length or more, 8 amino acids length or more, 9 amino acids length or more, 10 amino acids length or more, 11 amino acids length or more, 12 amino acids length or more, 13 amino acids length or more, 14 amino acids length or more, 15 amino acids length or more, 20 amino acids length or more, 25 amino acids length or more, 30 amino acids length or more, 35 amino acids length or more, 40 amino acids length or more, 45 amino acids length or more, 50 amino acids length or more, 60 amino acids length or more, 70 amino acids length or more, 80 amino acids length or more, 90 amino acids length or more, 100 amino acids length or more, 110 amino acids length or more, 120 amino acids length or more, 150 amino acids length or more, 200 amino acids length or more, 30 The length of the polypeptide chain may be 100 amino acids or more, 400 amino acids or more, or 500 amino acids or more, and / or 5000 amino acids or less, 4000 amino acids or less, 3000 amino acids or less, 2000 amino acids or less, 1000 amino acids or less, 900 amino acids or less, 800 amino acids or less, 700 amino acids or less, or 600 amino acids or less. Examples include 1 to 500 amino acids in length, 2 to 450 amino acids in length, The length of the linker portion is 3 to 400 amino acids, 3 to 350 amino acids, 3 to 300 amino acids, 3 to 250 amino acids, 3 to 200 amino acids, 3 to 150 amino acids, 4 to 100 amino acids, 5 to 50 amino acids, 6 to 40 amino acids, 7 to 30 amino acids, 8 to 20 amino acids, 9 to 15 amino acids, 10 to 14 amino acids, 11 to 13 amino acids, or 12 amino acids. In many cases, peptides containing a large number of amino acids with smaller side chains, such as serine and glycine, are used in the linker sequence.

[0139] The pair of antenomerase recognition sequences in the double-stranded circular DNA vector of the present embodiment are antenomerase recognition sequences recognized by antenomerase or an active fragment thereof contained in the fusion polypeptide. Specific examples of antenomerase recognition sequence include a base sequence recognized by antenomerase of TelA (SEQ ID NO: 26), a base sequence recognized by antenomerase derived from Halomonas virus HAP1 (SEQ ID NO: 27), a base sequence recognized by antenomerase derived from Vibrio virus VP882 (SEQ ID NO: 28), a base sequence recognized by antenomerase of TelK (SEQ ID NO: 29), a base sequence recognized by antenomerase derived from Rhizobiumpusense, a base sequence recognized by antenomerase derived from Feldmannia species virus, a base sequence recognized by antenomerase derived from Vibrio phage vB_VpaM_MAR, a base sequence recognized by antenomerase of Tel (SEQ ID NO: 30), and a base sequence recognized by antenomerase of TelN (telRL sequence) (SEQ ID NO: 3).

[0140] The endonuclease recognition sequence in the double-stranded circular DNA vector of the present embodiment is an endonuclease recognition sequence recognized by the endonuclease or its active fragment contained in the fusion polypeptide. As the endonuclease recognition sequence, it can be enumerated: restriction enzyme recognition sequence, TALEN recognition sequence, ZFN recognition sequence, CRISPR / Cas9 recognition sequence, etc. In the case where the endonuclease is a homing endonuclease, as an example of a homing endonuclease recognition sequence, it can be enumerated: I-SceI recognition sequence (sequence number 31), I-CeuI recognition sequence (sequence number 32) and I-CreI recognition sequence (sequence number 33), etc.

[0141] The number of endonuclease recognition sequences in the double-stranded circular DNA vector of the present invention may be 1 or more. For example, the double-stranded circular DNA vector of the present invention may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more endonuclease recognition sequences. However, the region containing the target nucleic acid sequence between a pair of front endomerase recognition sequences does not contain an endonuclease recognition sequence.

[0142] In this specification, the "target nucleic acid sequence" is not particularly limited as long as it is a sequence containing more than two nucleotides. More specifically, the target nucleic acid sequence is not limited to a sequence encoding a protein or a fragment thereof or an RNA, and may be, for example, a sequence that can function as a nucleic acid medicine, or a sequence that can be used to introduce a target nucleic acid sequence. As a specific example of a nucleic acid sequence, there is no limitation, and examples include: a gene sequence encoding a protein or a fragment thereof; a nucleic acid sequence encoding a non-coding RNA such as siRNA, shRNA, miRNA, lncRNA, or ribozyme; or nucleic acid medicines such as antisense nucleic acids, aptamers, or decoy nucleic acids. In addition, as examples of sequences that can be used to introduce a target nucleic acid sequence, examples include: sequences that can be recognized / cut off by nucleases such as restriction enzymes and genome editing enzymes, and multiple cloning sites containing sequences that can be recognized / cut off by more than two nucleases.

[0143] In this specification, "target gene sequence" refers to a gene sequence encoding a target protein or a fragment thereof or a target RNA molecule. There is no particular limitation on the target protein or its fragment, and it can be appropriately selected according to the purpose. Examples include polypeptides constituting viruses, polypeptides produced by animals, plants, fungi, algae, bacteria, viruses, etc., and their fragments. They can be used as cell / gene therapy drugs, vaccines, disease treatment drugs, etc. There is no particular limitation on the polypeptides produced by animals, plants, fungi, algae, bacteria, viruses, etc., and they can be appropriately selected according to the purpose. Examples thereof include enzymes such as phytase, amylase, glucosidase, cellulase, lipase, protease, glutaminase, peptidase, oxidase, lactase, xylanase, trypsin, pectinase, isomerase, antibody-binding proteins such as protein A, protein G, and protein L, human antibodies, humanized antibodies, chimeric antibodies, llama antibodies, alpaca antibodies, single-chain antibodies, heavy-chain antibodies, multivalent antibodies, Fab, F(ab'), F(ab')2, Fc, Fc fusion proteins, bispecific antibodies, heavy chains (H chains), light chains (L chains), single-chain Fv (scFv), sc(Fv)2, disulfide-bonded Fv (sdFv), Diabody, and antibody-like molecular target peptides. Antibodies or antibody fragments such as (microantibodies), antibodies such as other functional proteins, complexes of compounds other than antibody fragments and antibody fragments (antibody-like molecules), serum albumins such as human serum albumin, epithelial growth factors such as human epidermal growth factor, insulin, growth hormone, erythropoietin, interferon, coagulation factor VIII, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), thrombopoietin, IL-1, IL-6, tissue plasminogen activator (TPA), urokinase, leptin, stem cell growth factor (SCF), silk protein, fluorescent protein, hepatitis B virus surface antigen, hirudin, etc.

[0144] The target nucleic acid sequence such as the target gene sequence may further include a promoter in addition to the target nucleic acid sequence (e.g., a sequence encoding a target protein or a fragment thereof). In addition, it may further include constituent elements such as introns, enhancers, terminators, and / or poly A signals as needed.

[0145] In the double-stranded circular DNA vector of the present embodiment, the fusion gene sequence and the endonuclease recognition sequence are arranged in the same region between a pair of front-end merase recognition sequences, and the target nucleic acid sequence is arranged in another region between a pair of front-end merase recognition sequences. The endonuclease recognition sequence can be arranged adjacent to one of the front-end merase recognition sequences, or a spacer sequence can be arranged between the endonuclease recognition sequence and the front-end merase recognition sequence.

[0146] In the present embodiment, "spacer sequence" refers to the following sequence, which can be inserted between the endonuclease recognition sequence and the front end merase recognition sequence in a manner that the front end merase or its active fragment contained in the fusion polypeptide can recognize the front end merase recognition sequence, and the endonuclease or its active fragment contained in the fusion polypeptide can recognize the endonuclease recognition sequence. The length of the spacer sequence is not limited as long as it is more than 1 base, for example, more than 1 base, more than 2 bases, more than 3 bases, more than 4 bases, more than 5 bases, more than 6 bases, more than 7 bases, more than 8 bases, more than 9 bases, more than 10 bases, more than 11 bases, more than 12 bases, more than 13 bases, more than 14 bases, more than 15 bases, more than 20 bases, more than 25 bases. , 30 bases or more, 35 bases or more, 40 bases or more, 45 bases or more, 50 bases or more, 55 bases or more, 60 bases or more, 65 bases or more, 70 bases or more, 75 bases or more, 80 bases or more, 85 bases or more, 90 bases or more, 95 bases or more, 100 bases or more, 120 bases or more, 150 bases or more, or 200 bases or more, and / or or less than 1000 bases in length, less than 900 bases in length, less than 800 bases in length, less than 700 bases in length, less than 600 bases in length, less than 500 bases in length, less than 400 bases in length, less than 300 bases in length, or less than 250 bases in length, and examples thereof include 1 to 1000 bases in length, 1 to 500 bases in length, 1 to 400 bases in length, 1 to 300 bases in length, 1~250 bases in length, 1~200 bases in length, 1~150 bases in length, 1~100 bases in length, 1~70 bases in length, 1~50 bases in length, 1~40 bases in length, 1~30 bases in length, 1~20 bases in length, 1~10 bases in length, 2~10 bases in length, 3~10 bases in length, 4~9 bases in length, 5~9 bases in length, or 6~8 bases in length.

[0147] In one embodiment, the length of the spacer sequence can be determined based on the length of the linker sequence.

[0148] The double-stranded circular DNA vector of this mode may also include other sequences in addition to the fusion gene sequence, a pair of front endomerase recognition sequences, at least one endonuclease recognition sequence, and the target nucleic acid sequence. As other sequences, there is no particular limitation and they can be appropriately selected according to the purpose. For example, the sequences required for the maintenance and replication of the vector in the cell, the necessary elements of the general plasmid vectors such as the genes encoding the replication origin and / or the antibiotic resistance protein, the sequences encoding the activator protein or the repressor protein such as the LacI gene and the AraC gene, the cloning site, the overlapping regions for utilizing the In-Fusion cloning system of Clontech, the Gibson Assembly system of New England Biolabs, etc. It should be noted that as examples of genes encoding antibiotic resistance proteins, the beta-lactamase gene (sometimes recorded as "ampR gene") that imparts ampicillin resistance, the aminoglycoside 3'-phosphotransferase gene (sometimes recorded as "kanR gene") that imparts kanamycin resistance, the tetracycline efflux transporter gene that imparts tetracycline resistance, the CAT (chloramphenicol acetyltransferase) gene that imparts chloramphenicol resistance, etc.

[0149] In this embodiment, other sequences can be arranged in the same region as the fusion gene sequence and the endonuclease recognition sequence between a pair of front-end merase recognition sequences, or in the same region as the target nucleic acid sequence between a pair of front-end merase recognition sequences, and more preferably arranged in the same region as the fusion gene sequence and the endonuclease recognition sequence between a pair of front-end merase recognition sequences. For example, all elements other than the target nucleic acid sequence can also be arranged in the same region as the fusion gene sequence and the endonuclease recognition sequence.

[0150] The preparation method of the double-stranded circular DNA vector of this mode is not particularly limited and can be appropriately selected according to the purpose. For example, methods such as total synthesis, PCR, and the use of the In-Fusion cloning system of Clontech, the Gibson Assembly system of New England Biolabs, etc. can be cited. For example, it can also be prepared based on commercially available or known vectors, and it can also be prepared based on pUC vectors, pET vectors, pGEM vectors, etc. In particular, when designed based on a plasmid having ori of the pUC system represented by a pUC19 vector, it is preferred from the perspective of the high efficiency of DNA replication. The double-stranded circular DNA vector of the present invention can also be prepared by adding a fusion gene sequence, a pair of front endomerase recognition sequences, at least one endonuclease recognition sequence, and a target nucleic acid sequence to these commercially available vectors.

[0151] 1-4. Effect

[0152] When the expression of the fusion polypeptide is induced by the double-stranded circular DNA vector of the present embodiment, the front end of the fusion polypeptide is recognized by the front end of the fusion polypeptide or its active fragment, and the cut ends are recombined, thereby generating the first linear covalently closed DNA containing the target nucleic acid sequence and the second linear covalently closed DNA containing the fusion gene sequence and the endonuclease recognition sequence. The second linear covalently closed DNA is recognized by the endonuclease or its active fragment contained in the fusion polypeptide and the endonuclease recognition sequence and cut. As a result, the second linear covalently closed DNA is decomposed by the endogenous exonuclease activity, so that the first linear covalently closed DNA can be efficiently obtained.

[0153] The double-stranded circular DNA vector of the present invention contains all the elements required for producing linear covalently closed DNA in cells in one DNA vector, and thus can easily and efficiently produce linear covalently closed DNA containing a target nucleic acid sequence.

[0154] According to the present invention, a fusion polypeptide comprising an endomerase or an active fragment thereof at the N-terminal side and an endonuclease or an active fragment thereof at the C-terminal side can also be provided. In addition, a nucleic acid encoding the fusion polypeptide can also be provided.

[0155] 2. Transformed cells containing double-stranded circular DNA vectors

[0156] 2-1. Overview

[0157] The second aspect of the present invention is a transformed cell. The transformed cell of this aspect comprises the double-stranded circular DNA vector described in the first aspect.

[0158] 2-2. Composition

[0159] The transformed cell of this embodiment contains a double-stranded circular DNA vector as an essential component.

[0160] In this specification, the type of transformed cells is not limited. The transformed cells can be cells that can introduce double-stranded circular DNA vectors, can maintain and / or can replicate double-stranded circular DNA vectors. Examples include: archaea, bacteria, or eukaryotic cells. Bacteria can be Escherichia coli, lactic acid bacteria, or Bacillus subtilis. Examples of eukaryotic cells include: fungal cells (e.g., yeast cells), algae cells, plant cells, protozoan cells, insect cells, nematode cells, fish cells, bird cells (e.g., chicken cells), and mammalian cells (e.g., mouse cells, chimpanzee cells, and human cells). Among them, cells that can replicate double-stranded circular DNA vectors are preferred.

[0161] Examples of Escherichia coli include those belonging to the genus Escherichia, and more specifically, Escherichia coli (for example, Escherichia coli K-12 strain, Escherichia coli B strain) and the like.

[0162] In addition, commercially available E. coli strains and E. coli strains from biological resource centers can also be used, such as JM109 strain, DH5 strain, DH5α strain, DH10B strain, NEB10β strain, HST08 strain, HST16CR strain, HB101 strain, W3110 strain, MG1655 strain, BL21 strain, BL21 DE3 strain, etc. These strains can be obtained from New England Biolabs, Takara Bio, Thermo Fisher Scientific, Toyobo Co., Ltd., ATCC (American Type Culture Collection), NBRP (National Bio Resource Project), etc.

[0163] In the present invention, induced strains derived from the above-mentioned Escherichia coli strains can also be used. Examples thereof include JW3973 strain (available from NBRP) for methionine auxotrophy, JW2806 strain (available from NBRP) for leucine auxotrophy, JW3582 strain (available from NBRP) for cysteine ​​auxotrophy, and ME5305 strain (available from NBRP) for thiamine and histidine auxotrophy.

[0164] In one embodiment, the transformed cell of this mode expresses an exonuclease. Here, the exonuclease is not particularly limited as long as it can decompose the DNA cut by the endonuclease or its active fragment contained in the fusion polypeptide from its cut end. The exonuclease can be, for example, an endogenous exonuclease.

[0165] 2-3. Effect

[0166] When the expression of the fusion polypeptide is induced by the double-stranded circular DNA vector in the transformed cells of the present embodiment, a linear covalently closed DNA containing the target nucleic acid sequence can be efficiently produced.

[0167] After the second linear covalently closed DNA including the fusion gene sequence and the endonuclease recognition sequence is cut by the endonuclease, it is rapidly degraded due to the endogenous exonuclease activity.

[0168] 3. Preparation of linear covalently closed DNA

[0169] 3-1. Overview

[0170] The third aspect of the present invention is a method for preparing linear covalently closed DNA. The preparation method of this aspect includes a culture step, an expression induction step, and a DNA extraction step as essential steps, and includes a separation step and / or an introduction step as an optional step. According to the preparation method of this aspect, linear covalently closed DNA can be prepared simply and efficiently.

[0171] 3-2. Methods

[0172] Hereinafter, each step in the method for producing linear covalently closed DNA according to the present embodiment will be described in detail.

[0173] (Introduction process)

[0174] The "introduction step" refers to a step of introducing the double-stranded circular DNA vector of the first embodiment into host cells to obtain transformed cells.

[0175] In this step, the method for double-stranded circular DNA vector to be imported into host cell is not particularly limited. For example, Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York and other records of gene introduction methods known in the art (transformation methods) can be used. Specifically, heat shock method, liposome transfection, electroporation, microinjection, calcium phosphate method, DEAE dextran method, based on the introduction of cationic lipids, based on the introduction of cationic polymers (such as polyethyleneimine (PEI)), based on the introduction of nanoparticles, based on the introduction of viruses, particle impact, etc.

[0176] The host cell into which the double-stranded circular DNA vector is introduced in this step can be appropriately selected in a medium containing antibiotics based on the gene encoding the antibiotic resistance protein in the double-stranded circular DNA vector.

[0177] (Cultivation process)

[0178] The "culturing step" refers to a step of culturing transformed cells. The purpose of this step is to increase the copy number of the double-stranded circular DNA vector in the cells before entering the expression induction step described below by proliferating the transformed cells before the expression induction step described below.

[0179] The method for culturing transformed cells in this step is not particularly limited and can be appropriately selected according to the type of transformed cells. For example, a culture method known in the art as recorded in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc. can be used.

[0180] The culture medium used to culture the transformed cells in this step can be any of a liquid culture medium or a solid culture medium. As a culture medium, it can be a culture medium containing, for example, one or more of the following components: protease degradation products such as peptone, tryptone, biological extracts such as potato glucose, yeast extract, amino acids such as glutamic acid or their salts, glucose, glycerol, sucrose and other sugars, sodium chloride, magnesium chloride, potassium dihydrogen phosphate and other inorganic salts. As specific culture medium and composition, LB culture medium (tryptone, yeast extract, sodium chloride), YPG culture medium (yeast extract, peptone, glucose), PD culture medium (potato glucose), TB culture medium (tryptone, yeast extract, dipotassium hydrogen phosphate, potassium dihydrogen phosphate) and the like can be cited.

[0181] When the double-stranded circular DNA vector contains a gene encoding an antibiotic resistance protein, this step can be performed in the presence of an antibiotic. Examples of antibiotics include ampicillin, carbenicillin, kanamycin, tetracycline, and chloramphenicol.

[0182] The culture conditions in this step can be appropriately selected depending on the type of transformed cells. For example, the culture in this step can be performed at 20 to 42°C, 25 to 40°C, 30 to 38°C, or 35 to 37°C.

[0183] The culturing time in this step is not limited as long as it is a time that can obtain a sufficient amount of double-stranded circular DNA vector, for example, it can be 1 hour or more, 2 hours or more, 4 hours or more, 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, or 1 week or more.

[0184] In addition, the pH of the culture medium used in this step is not particularly limited as long as it can generate transformed cells. For example, it can be pH3 or above, pH4 or above, pH5 or above, pH6 or above, pH7 or above, pH7.5 or above, pH8 or above, pH8.5 or above, pH9 or above, or pH9.5 or above, and / or pH11 or below, pH10.5 or below, pH10 or below, pH9.5 or below, pH9 or below, pH8.5 or below, pH8 or below, or pH7.5 or below, for example, pH3 to 11, pH4 to 10, pH5 to 9, or pH7 to 9, or pH7.5 to pH10.5, pH8 to pH10, or pH8.5 to pH9.5.

[0185] (Expression induction step)

[0186] In the present embodiment, the "expression induction step" refers to a step of inducing expression of the fusion polypeptide in the transformed cells after the culturing step.

[0187] The method for inducing expression of the fusion polypeptide in this step is not particularly limited and can be appropriately selected depending on the type of promoter that can control expression.

[0188] In one embodiment, in this step, an expression inducer may be added to the culture medium after the culture step. For example, in the case of an arabinose-inducible promoter, an IPTG-inducible promoter, or a rhamnose-inducible promoter, a method of adding arabinose, IPTG, or rhamnose as an expression inducer to the culture medium containing the transformed cells after the culture step can be cited.

[0189] In this step, the time for culturing the transformed cells in the presence of the expression inducer is not particularly limited as long as it is a time that can sufficiently induce the expression of the fusion polypeptide. For example, it can be 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 6 hours or more, 12 hours or more, or 24 hours or more.

[0190] The pH of the medium used in this step can be any pH as long as it is within the range in which the fusion polypeptide can function in the cell, that is, within the range in which both the anterior endomerase and the posterior endonuclease can function, and can be, for example, pH 3 to 11, pH 4 to 10, pH 5 to 9, or pH 7 to 9. For example, a pH range between the optimal pH of the anterior endomerase and the optimal pH of the posterior endonuclease can also be used.

[0191] In this step, the temperature at which the transformed cells are cultured in the presence of the expression inducer may be, for example, 20°C or higher, 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, 36°C or higher, or 37°C or higher, and / or 42°C or lower, 40°C or lower, 39°C or lower, 38°C or lower, 37°C or lower, 36°C or lower, 35°C or lower, 34°C or lower, 33°C or lower, 32°C or lower, or 31°C or lower, and may be, for example, 30-38°C, 32-38°C, 33-38°C, 34-37°C, 35-37°C, or 36-37°C, or 25-35°C, 26-34°C, 27-33°C, 28-32°C, or 29-31°C. For example, a temperature range between the optimum temperature of an endomerase and the optimum temperature of an endonuclease may also be used.

[0192] In a further embodiment, in this process, the transformed cells can be cultured under multiple temperature conditions in the presence of an expression inducer. As multiple temperature conditions, the optimum temperature range of the enzyme activity of the front end merase and the endonuclease can be exemplified. The optimum temperature of the enzyme activity of the front end merase can be, for example, 26-37°C, 27-35°C, 28-33°C, or 29-31°C (for example, 30°C). The optimum temperature of the enzyme activity of the homing endonuclease can be, for example, 30-42°C, 32-40°C, 34-39°C, or 36-38°C (for example, 37°C). The culture time under each temperature condition in this embodiment can be more than 10 minutes, more than 20 minutes, more than 30 minutes, more than 1 hour, more than 2 hours, more than 3 hours, more than 4 hours, more than 5 hours, more than 6 hours, more than 8 hours, more than 10 hours, more than 12 hours, or more than 24 hours. The culture of this process can be carried out by any combination of the above-mentioned temperature and culture time. For example, the culture may be carried out at 28 to 32° C. for 2 hours or more (eg, 4 hours or more), and then at 35 to 39° C. for 2 hours or more (eg, 4 hours or more).

[0193] In another embodiment, if the promoter that can control expression is a heat-inducible promoter or a low-temperature-inducible promoter, heat shock or low-temperature shock can also be performed. The conditions of heat shock or low-temperature shock are not particularly limited as long as the temperature and time can sufficiently induce the expression of the fusion polypeptide. For example, in the case of heat shock, it can be carried out under the following conditions: 36°C to 45°C, 37°C to 44°C, 38°C to 43°C, 39°C to 42°C or 40°C to 41°C, and 10 seconds or more, 20 seconds or more, 30 seconds or more, 40 seconds or more, 50 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, or 1 hour or more, and / or 24 hours or less, 12 hours or less, 6 hours or less, 3 hours or less, or 2 hours or less, for example, 10 seconds to 24 hours, 20 seconds to 12 hours, 25 seconds to 6 hours, 30 seconds to 3 hours, 40 seconds to 2 hours, 50 seconds to 1 hour, 1 minute to 30 minutes, or 2 minutes to 5 minutes.

[0194] Through this process, the fusion polypeptide is expressed in the transformed cells, generating a first linear covalently closed DNA comprising the target nucleic acid sequence and a second linear covalently closed DNA comprising the fusion gene sequence and the endonuclease recognition sequence. The second linear covalently closed DNA is cut and decomposed by endogenous exonuclease activity.

[0195] (DNA extraction process)

[0196] The "DNA extraction step" refers to a step of extracting DNA from transformed cells after the expression induction step.

[0197] The method for extracting DNA in this step can use the common method known in the art. For example, it can be prepared according to the method described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc. In addition, it can also be prepared using a commercially available DNA extraction kit.

[0198] Through this step, the first linear covalently closed DNA containing the target nucleic acid sequence can be extracted.

[0199] (Separation process)

[0200] The "separation step" refers to a step of separating the linear covalently closed DNA after the DNA extraction step. The purpose of this step is to remove DNA components other than the linear covalently closed DNA containing the target nucleic acid sequence extracted in the DNA extraction step, such as genomic DNA fragments, DNA fragments left over from cleavage, and unnecessary linear covalently closed DNA containing the endonuclease recognition sequence.

[0201] The method for separating the linear covalently closed DNA in this step is not particularly limited and can be appropriately selected, for example, a method for purification using a commercially available kit, gel purification, a method for separation using cation chromatography, anion chromatography, size exclusion chromatography, a filter, or an ultrafiltration membrane, etc.

[0202] 3-3. Effect

[0203] According to the method for preparing linear covalently closed DNA of this embodiment, linear covalently closed DNA can be prepared simply and efficiently. According to the preparation method of the present invention, a large amount of linear covalently closed DNA can be prepared with a small number of steps.

[0204] 4. Double-stranded circular DNA vector

[0205] 4-1. Overview

[0206] The fourth aspect of the present invention is a double-stranded circular DNA vector. The double-stranded circular DNA vector of this aspect contains a pre-telomerase gene sequence, an endonuclease gene sequence, and an endonuclease recognition sequence in the same region between a pair of pre-telomerase recognition sequences, and contains a target nucleic acid sequence in another region between the pair of pre-telomerase recognition sequences.

[0207] According to the double-stranded circular DNA vector of this embodiment, a linear covalently closed DNA containing a target nucleic acid sequence can be prepared simply and efficiently.

[0208] 4-2. Composition

[0209] The double-stranded circular DNA vector of the present embodiment comprises a front-end merase gene sequence, an endonuclease gene sequence, a pair of front-end merase recognition sequences, at least one endonuclease recognition sequence, and a target nucleic acid sequence. In the double-stranded circular DNA vector of the present embodiment, the front-end merase gene sequence, the endonuclease gene sequence, and the endonuclease recognition sequence are arranged in the same region between the pair of front-end merase recognition sequences, and the target nucleic acid sequence is arranged in another region between the pair of front-end merase recognition sequences. In addition, the front-end merase gene sequence and the endonuclease gene sequence are arranged under the control of a promoter that can control expression.

[0210] In the present specification, "anterior telomerase gene sequence" refers to a gene sequence encoding anterior telomerase or an active fragment thereof. The anterior telomerase gene sequence may be codon-optimized according to the codon usage frequency in the cell into which the double-stranded circular DNA vector is to be introduced.

[0211] The type of the pre-telomerase or its active fragment encoded by the pre-telomerase gene sequence is not particularly limited as long as it has pre-telomerase activity, as long as it does not recognize sequences other than a pair of pre-telomerase recognition sequences in the double-stranded circular DNA vector, especially the target nucleic acid sequence. In addition, there may be no pre-telomerase recognition sequence in the genome sequence of the cell to be introduced into the double-stranded circular DNA vector. For example, the length of the pre-telomerase recognition sequence recognized by the pre-telomerase or its active fragment is not limited, and can be 5 bp or more, 6 bp or more, 7 bp or more, 8 bp or more, 9 bp or more, 10 bp or more, 15 bp or more, 20 bp or more, 25 bp or more, 30 bp or more, 35 bp or more, 40 bp or more, 45 bp or more, 50 bp or more, 55 bp or more, 60 bp or more, 65 bp or more, 70 bp or more, 80 bp or more, or 90 bp or more. Among the above-mentioned pre-telomerases, TelN pre-telomerase, TelA pre-telomerase or TelK pre-telomerase, or mutants thereof can be cited as particularly preferred examples. There are no particular limitations on the mutants, and they may be appropriately selected depending on the intended purpose. For example, the homology to the original amino acid sequence may be 80% or more, 90% or more, 95% or more, or 99% or more.

[0212] In the present specification, "endonuclease gene sequence" refers to a gene sequence encoding an endonuclease or an active fragment thereof. The endonuclease gene sequence may be codon-optimized according to the codon usage frequency in the cell into which the double-stranded circular DNA vector is to be introduced.

[0213] As long as the endonuclease or its active fragment encoded by the endonuclease gene sequence has endonuclease activity, it is not particularly limited, as long as the sequence outside the endonuclease recognition sequence in the double-stranded circular DNA vector is not recognized, particularly the target nucleic acid sequence. In addition, there can be no endonuclease recognition sequence in the genome sequence of the cell to be introduced into the double-stranded circular DNA vector. Endonuclease or its active fragment can be a restriction enzyme, or it can also be a nickase (Nickase). For example, the length of the endonuclease recognition sequence identified by the endonuclease or its active fragment is not limited, and can be more than 4bp, more than 5bp, more than 6bp, more than 7bp, more than 8bp, more than 9bp, more than 10bp, more than 15bp, more than 20bp, more than 25bp, more than 30bp, more than 35bp, more than 40bp, more than 45bp, more than 50bp, more than 60bp, more than 70bp, more than 80bp, more than 90bp or more than 100bp.

[0214] In the double-stranded circular DNA vector of the present invention, the front endomerase gene sequence and the endonuclease gene sequence are placed under the control of a promoter capable of controlling expression.

[0215] In the present embodiment, for example, the pre-telomerase gene sequence and the endonuclease gene sequence can be placed under the control of promoters capable of controlling expression separately, or can be placed under the control of the same promoter capable of controlling expression. In the case where the pre-telomerase gene sequence and the endonuclease gene sequence are placed under the control of the same promoter capable of controlling expression, the pre-telomerase gene sequence and the endonuclease gene sequence can be placed in the same operon, or a sequence encoding a self-cleaving peptide, an IRES sequence, etc. can be placed between the pre-telomerase gene sequence and the endonuclease gene sequence.

[0216] The pair of pre-telomerase recognition sequences in the double-stranded circular DNA vector of the present embodiment are pre-telomerase recognition sequences recognized by the pre-telomerase encoded by the pre-telomerase gene sequence or an active fragment thereof. Specific examples of the telomerase recognition sequence include: a base sequence recognized by the TelA telomerase (SEQ ID NO: 26), a base sequence recognized by the telomerase derived from Halomonas virus HAP1 (SEQ ID NO: 27), a base sequence recognized by the telomerase derived from Vibrio virus VP882 (SEQ ID NO: 28), a base sequence recognized by the TelK telomerase (SEQ ID NO: 29), a base sequence recognized by the telomerase derived from Rhizobium pusense, a base sequence recognized by the telomerase derived from Feldmannia species virus, a base sequence recognized by the telomerase derived from Vibrio phage vB_VpaM_MAR, a base sequence recognized by the Tel telomerase (SEQ ID NO: 30), and a base sequence recognized by the TelN telomerase (telRL sequence) (SEQ ID NO: 3).

[0217] The endonuclease recognition sequence in the double-stranded circular DNA vector of this embodiment is an endonuclease recognition sequence recognized by an endonuclease encoded by an endonuclease gene sequence or its active fragment. As an endonuclease recognition sequence, it can be enumerated: restriction enzyme recognition sequence, TALEN recognition sequence, ZFN recognition sequence, CRISPR / Cas9 recognition sequence, etc. In the case where the endonuclease is a homing endonuclease, as an example of a homing endonuclease recognition sequence, it can be enumerated: I-SceI recognition sequence (sequence number 31), I-CeuI recognition sequence (sequence number 32) and I-CreI recognition sequence (sequence number 33), etc.

[0218] In the double-stranded circular DNA vector of the present embodiment, the pre-telomerase gene sequence, the endonuclease gene sequence and the endonuclease recognition sequence are arranged in the same region between a pair of pre-telomerase recognition sequences, and the target nucleic acid sequence is arranged in another region between a pair of pre-telomerase recognition sequences. The endonuclease recognition sequence can be arranged adjacent to one of the pre-telomerase recognition sequences, or a spacer sequence can be arranged between the endonuclease recognition sequence and the pre-telomerase recognition sequence.

[0219] In the present specification, "spacer sequence" refers to the following sequence, which can be inserted between the endonuclease recognition sequence and the front end merase recognition sequence in a manner that the front end merase or its active fragment can recognize the front end merase recognition sequence, and the endonuclease or its active fragment can recognize the endonuclease recognition sequence. The length of the spacer sequence is not limited as long as it is more than 1 base, for example, more than 1 base, more than 2 bases, more than 3 bases, more than 4 bases, more than 5 bases, more than 6 bases, more than 7 bases, more than 8 bases, more than 9 bases, more than 10 bases, more than 11 bases, more than 12 bases, more than 13 bases, more than 14 bases, more than 15 bases, more than 20 bases, more than 25 bases. , 30 bases or more, 35 bases or more, 40 bases or more, 45 bases or more, 50 bases or more, 55 bases or more, 60 bases or more, 65 bases or more, 70 bases or more, 75 bases or more, 80 bases or more, 85 bases or more, 90 bases or more, 95 bases or more, 100 bases or more, 120 bases or more, 150 bases or more, or 200 bases or more, and / or or less than 1000 bases in length, less than 900 bases in length, less than 800 bases in length, less than 700 bases in length, less than 600 bases in length, less than 500 bases in length, less than 400 bases in length, less than 300 bases in length, or less than 250 bases in length, and examples thereof include 1 to 1000 bases in length, 1 to 500 bases in length, 1 to 400 bases in length, 1 to 300 bases in length, 1~250 bases in length, 1~200 bases in length, 1~150 bases in length, 1~100 bases in length, 1~70 bases in length, 1~50 bases in length, 1~40 bases in length, 1~30 bases in length, 1~20 bases in length, 1~10 bases in length, 2~10 bases in length, 3~10 bases in length, 4~9 bases in length, 5~9 bases in length, or 6~8 bases in length.

[0220] The double-stranded circular DNA vector of this mode may also contain other sequences in addition to the front end merase gene sequence, the endonuclease gene sequence, a pair of front end merase recognition sequences, at least one endonuclease recognition sequence, and the target nucleic acid sequence. As other sequences, there are no particular restrictions and they can be appropriately selected according to the purpose. For example, sequences required for the maintenance and replication of the vector in the cell, such as genes encoding replication origins and / or antibiotic resistance proteins, necessary elements for general plasmid vectors, sequences encoding activator proteins or repressor proteins such as LacI genes and AraC genes, cloning sites, overlapping regions for utilizing the In-Fusion cloning system of Clontech, the Gibson Assembly system of New England Biolabs, etc. It should be noted that as examples of genes encoding antibiotic resistance proteins, the beta-lactamase gene (ampR gene) that confers ampicillin resistance, the aminoglycoside 3'-phosphotransferase gene (kanR gene) that confers kanamycin resistance, the tetracycline efflux transporter gene that confers tetracycline resistance, the CAT (chloramphenicol acetyltransferase) gene that confers chloramphenicol resistance, etc.

[0221] In this embodiment, other sequences can be arranged in the same region as the pre-terminus myramase gene sequence, the endonuclease gene sequence and the endonuclease recognition sequence between a pair of pre-terminus myramase recognition sequences, or in the same region as the target nucleic acid sequence between a pair of pre-terminus myramase recognition sequences, and more preferably arranged in the same region as the pre-terminus myramase gene sequence, the endonuclease gene sequence and the endonuclease recognition sequence between a pair of pre-terminus myramase recognition sequences. For example, all elements other than the target nucleic acid sequence can also be arranged in the same region as the pre-terminus myramase gene sequence, the endonuclease gene sequence and the endonuclease recognition sequence.

[0222] The preparation method of the double-stranded circular DNA vector of this mode is not particularly limited and can be appropriately selected according to the purpose. For example, methods such as total synthesis, PCR, and the use of Clontech's In-Fusion cloning system, New England Biolabs' Gibson Assembly system, etc. can be cited. For example, it can also be prepared based on commercially available or known vectors, and can also be prepared based on pUC vectors, pET vectors, pGEM vectors, etc. In particular, when designed based on a plasmid having ori of the pUC system represented by a pUC19 vector, it is preferred from the perspective of high DNA replication efficiency. The double-stranded circular DNA vector of the present invention can also be prepared by adding a front end polymerase gene sequence, an endonuclease gene sequence, a pair of front end polymerase recognition sequences, at least one endonuclease recognition sequence, and a target nucleic acid sequence to these commercially available vectors.

[0223] In one embodiment of this embodiment, the front end merase gene sequence and the endonuclease gene sequence encode a fusion polypeptide comprising a front end merase or an active fragment thereof and an endonuclease or an active fragment thereof. This embodiment corresponds to the first embodiment described above, and the gene sequence encoding the fusion polypeptide in this embodiment corresponds to the fusion gene sequence in the first embodiment.

[0224] 4-3. Effect

[0225] When the expression of a nuclease or its active fragment and a front-end mitogenase or its active fragment is induced by the double-stranded circular DNA vector of the present method, the front-end mitogenase recognition sequence is recognized by the front-end mitogenase or its active fragment, and the cut ends are recombined, thereby generating a first linear covalently closed DNA comprising a target nucleic acid sequence, and a second linear covalently closed DNA comprising a front-end mitogenase gene sequence, an endonuclease gene sequence, and an endonuclease recognition sequence. The second linear covalently closed DNA recognizes the endonuclease recognition sequence and is cut by the endonuclease or its active fragment. As a result, the second linear covalently closed DNA is decomposed by endogenous exonuclease activity, so that the first linear covalently closed DNA can be efficiently obtained.

[0226] 5. Transformed cells containing double-stranded circular DNA vectors

[0227] 5-1. Summary

[0228] The fifth aspect of the present invention is a transformed cell. The transformed cell of this aspect comprises the double-stranded circular DNA vector described in the fourth aspect.

[0229] 5-2. Composition

[0230] The transformed cell of this embodiment contains a double-stranded circular DNA vector as an essential component.

[0231] In one embodiment, the transformed cell of this mode expresses exonuclease. Here, exonuclease is not particularly limited as long as it can decompose from the end of DNA cut by the endonuclease encoded by the endonuclease gene sequence or its active fragment. Exonuclease can be, for example, endogenous exonuclease.

[0232] 5-3. Effect

[0233] When the expression of an endonuclease or an active fragment thereof and an endomerase or an active fragment thereof is induced by a double-stranded circular DNA vector in the transformed cells of the present embodiment, a linear covalently closed DNA containing a target nucleic acid sequence can be efficiently produced.

[0234] After the second linear covalently closed DNA including the pre-telomerase gene sequence, the endonuclease gene sequence and the endonuclease recognition sequence is cut by the endonuclease, it is rapidly degraded due to the endogenous exonuclease activity.

[0235] 6. Preparation of linear covalently closed DNA

[0236] 6-1. Overview

[0237] The sixth aspect of the present invention is a method for preparing linear covalently closed DNA. The preparation method of this aspect includes a culture step, an expression induction step, and a DNA extraction step as essential steps, and includes a separation step and / or an introduction step as an optional step. According to the preparation method of this aspect, linear covalently closed DNA can be prepared simply and efficiently.

[0238] 6-2. Methods

[0239] Hereinafter, each step in the method for producing linear covalently closed DNA according to the present embodiment will be described in detail.

[0240] (Introduction process)

[0241] The "introduction step" is a step of introducing the double-stranded circular DNA vector of the first embodiment into a host cell to obtain a transformed cell. The configuration of this step is the same as that of the third embodiment, and therefore its description is omitted here.

[0242] (Cultivation process)

[0243] The "culturing step" refers to the step of culturing transformed cells. The purpose of this step is to increase the copy number of the double-stranded circular DNA vector in the cell before entering the expression induction step described later by proliferating the transformed cells before entering the expression induction step described later. The structure of this step is in accordance with the description of the third embodiment, so the description is omitted here.

[0244] (Expression induction step)

[0245] In the present embodiment, the "expression induction step" refers to a step of inducing the expression of an endonuclease or an active fragment thereof and an endomerase or an active fragment thereof in the transformed cells after the culturing step.

[0246] In this step, the method for inducing the expression of the endonuclease or its active fragment and the endomerase or its active fragment is not particularly limited and can be appropriately selected according to the type of promoter that can control the expression.

[0247] In one embodiment, in this step, an expression inducer may be added to the culture medium after the culture step. For example, in the case of an arabinose-inducible promoter, an IPTG-inducible promoter, or a rhamnose-inducible promoter, a method of adding arabinose, IPTG, or rhamnose as an expression inducer to the culture medium containing the transformed cells after the culture step can be cited.

[0248] In this step, the time for culturing the transformed cells in the presence of the expression inducer is not particularly limited as long as it is a time that can sufficiently induce the expression of the endonuclease or its active fragment and the anterior terminal telomerase or its active fragment. For example, it can be 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 6 hours or more, 12 hours or more, or 24 hours or more.

[0249] The pH of the medium used in this step can be any pH within the range where the endonuclease or its active fragment and / or the anterior telomerase or its active fragment can function in the cell, for example, pH 3 to 11, pH 4 to 10, pH 5 to 9, or pH 7 to 9. For example, a pH range between the optimal pH of the anterior telomerase and the optimal pH of the endonuclease can also be used.

[0250] The temperature at which the transformed cells are cultured in the presence of the expression inducer in this step may be, for example, 20° C. or higher, 25° C. or higher, 28° C. or higher, 30° C. or higher, or 31° C. or higher, and / or 42° C. or lower, 40° C. or lower, 39° C. or lower, 38° C. or lower, 37° C. or lower, 36° C. or lower, 35° C. or lower, 34° C. or lower, 33° C. or lower, 32° C. or lower, or 31° C. or lower, and may be, for example, 26 to 38° C., 27 to 37° C., 28 to 36° C., 29 to 35° C., 30 to 34° C., 30 to 33° C., 30 to 32° C., or 30 to 31° C. For example, a temperature range between the optimum temperature of an endomerase and the optimum temperature of an endonuclease may also be used.

[0251] In a further embodiment, in this step, the transformed cells can be cultured under multiple temperature conditions in the presence of an expression inducer. Multiple temperature conditions can be selected from the optimum temperature ranges for the enzyme activity of the front end merase and the endonuclease. The optimum temperature for the enzyme activity of the front end merase can be, for example, 26-37°C, 27-35°C, 28-33°C, or 29-31°C (e.g., 30°C). The optimum temperature for the enzyme activity of the homing endonuclease can be, for example, 30-42°C, 32-40°C, 34-39°C, or 36-38°C (e.g., 37°C). The culture time under each temperature condition in this embodiment can be more than 10 minutes, more than 20 minutes, more than 30 minutes, more than 1 hour, more than 2 hours, more than 3 hours, more than 6 hours, more than 12 hours, or more than 24 hours.

[0252] In another embodiment, if the promoter capable of controlling expression is a heat-inducible promoter or a low-temperature-inducible promoter, heat shock or low-temperature shock may also be performed. The conditions for heat shock or low-temperature shock are not particularly limited as long as the temperature and time can sufficiently induce the expression of the endonuclease or its active fragment and the front end merase or its active fragment. For example, in the case of heat shock, it can be carried out under the following conditions: 36°C to 45°C, 37°C to 44°C, 38°C to 43°C, 39°C to 42°C or 40°C to 41°C, and 10 seconds or more, 20 seconds or more, 30 seconds or more, 40 seconds or more, 50 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, or 1 hour or more, and / or 24 hours or less, 12 hours or less, 6 hours or less, 3 hours or less, or 2 hours or less, for example, 10 seconds to 24 hours, 20 seconds to 12 hours, 25 seconds to 6 hours, 30 seconds to 3 hours, 40 seconds to 2 hours, 50 seconds to 1 hour, 1 minute to 30 minutes, or 2 minutes to 5 minutes.

[0253] Through this process, a nuclease or an active fragment thereof and a front end polymerase or an active fragment thereof are expressed in the transformed cells, thereby generating a first linear covalently closed DNA comprising a target nucleic acid sequence and a second linear covalently closed DNA comprising a front end polymerase gene sequence, an endonuclease gene sequence and an endonuclease recognition sequence. The second linear covalently closed DNA is cut and decomposed by endogenous exonuclease activity.

[0254] (DNA extraction process)

[0255] The "DNA extraction step" is a step of extracting DNA from the transformed cells after the expression induction step. The configuration of this step is the same as that of the third embodiment, and therefore its description is omitted here.

[0256] (Separation process)

[0257] The "separation step" refers to a step of separating the linear covalently closed DNA after the DNA extraction step. The purpose of this step is to remove DNA components other than the linear covalently closed DNA containing the target nucleic acid sequence extracted in the DNA extraction step, such as genomic DNA fragments, DNA fragments left over from cutting, and unnecessary linear covalently closed DNA containing the endonuclease recognition sequence. The composition of this step is in accordance with the description of the third embodiment, so the description is omitted here.

[0258] 6-3. Effect

[0259] According to the method for preparing linear covalently closed DNA of this embodiment, linear covalently closed DNA can be prepared simply and efficiently. According to the method for preparing linear covalently closed DNA of this embodiment, a large amount of linear covalently closed DNA can be produced with a small number of steps.

[0260] Example

[0261] Hereinafter, the present invention will be specifically described by way of examples. It should be noted that these examples are used to illustrate the present invention and do not limit the scope of the present invention.

[0262] <Example 1: Construction of a linear covalently closed DNA preparation vector>

[0263] (Purpose)

[0264] Preparation of linear covalently closed DNA vectors.

[0265] (Methods and Results)

[0266] In the following examples, nucleic acid manipulations based on recombinant DNA technology were performed with reference to the following two documents: Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989), and Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience).

[0267] In addition, Prime STAR MAX DNA Polymerase (manufactured by Takara Bio) and the like were used for PCR in the following Examples, and the reaction conditions were in accordance with the method described in the attached manual.

[0268] (1) Preparation of nucleic acid fragments

[0269] The nucleic acid fragment used for constructing a linear covalently closed DNA production vector was prepared as follows.

[0270] (a) Nucleic acid encoding fusion polypeptide (SEQ ID NO: 1)

[0271] A nucleic acid encoding a fusion polypeptide (sequence number 1) was prepared by PCR using primer 1 (forward, sequence number 14) and primer 4 (reverse, sequence number 17), wherein the fusion polypeptide is formed by connecting, in sequence from the N-terminal side, the TelN front endomerase (the front endomerase derived from Escherichiavirus N15), the linker sequence (the amino acid sequence encoded by the base sequence shown in sequence number 34), and the I-SceI homing endonuclease (the homing endonuclease derived from Saccharomyces cerevisiae).

[0272] (b) Nucleic acid encoding TelN telomerase (SEQ ID NO: 2)

[0273] Using the above-mentioned nucleic acid encoding the fusion polypeptide (SEQ ID NO: 1) as a template, a nucleic acid encoding TelN terminal telomerase (SEQ ID NO: 2) was prepared by PCR using primer 1 (forward, SEQ ID NO: 14) and primer 2 (reverse, SEQ ID NO: 15).

[0274] (c) A pair of telomerase recognition sequences (telRL sequence, SEQ ID NO: 3)

[0275] A nucleic acid having a pair of telomerase recognition sequences (telRL sequence, SEQ ID NO: 3) was prepared by total synthesis.

[0276] (d) Nucleic acid encoding I-SceI homing endonuclease (SEQ ID NO: 4)

[0277] Using the above-mentioned nucleic acid encoding the fusion polypeptide (sequence number 1) as a template, a nucleic acid (sequence number 4) encoding the I-SceI homing nuclease from Saccharomyces cerevisiae was prepared by PCR using primer 3 (forward, sequence number 16) and primer 4 (reverse, sequence number 17).

[0278] (e) Sequence containing telRL sequence and I-SceI recognition sequence (SEQ ID NOs: 5 to 9)

[0279] Sequences (SEQ ID NOs. 5 to 9) comprising a telRL sequence and an I-SceI recognition sequence were prepared by total synthesis. Sequence No. 5 is a sequence in which the telRL sequence and the I-SceI recognition sequence are adjacent to each other without a spacer sequence. Sequence Nos. 6, 7, 8, and 9 respectively comprise T0 (no spacer), T2, T4, T6, and T8 as spacer sequences between the telRL sequence and the I-SceI recognition sequence.

[0280] (f) AraC gene sequence under promoter control (SEQ ID NO: 11)

[0281] The AraC gene sequence (SEQ ID NO: 11) under the control of the promoter was prepared by PCR using primer 5 (forward, SEQ ID NO: 18) and primer 6 (reverse, SEQ ID NO: 19).

[0282] (g) Arabinose-inducible promoter sequence (SEQ ID NO: 12)

[0283] The arabinose-inducible promoter sequence (SEQ ID NO: 12) was prepared by total synthesis.

[0284] (h) Arabinose-inducible promoter sequence containing control sequences (SEQ ID NO: 13)

[0285] An arabinose-inducible promoter sequence (SEQ ID NO: 13) including a control sequence was prepared by PCR using primer 7 (forward, SEQ ID NO: 20) and primer 8 (reverse, SEQ ID NO: 21).

[0286] (2) Preparation of control vector

[0287] As a vector for preparing linear covalently closed DNA, a vector expressing TelN-terminal endomerase under the control of an arabinose-inducible promoter (hereinafter referred to as a "control vector") was prepared. Figure 1 ).

[0288] The control vector was prepared as follows: First, the telRL sequence of (c) was inserted into the EcoRV site and SnaBI site of the pRC2-mi342 vector (manufactured by Takara Bio). Next, (b), (f) and (g) were inserted into the SmaI site of the vector.

[0289] In the control vector, the Rep gene (SEQ ID NO: 22) and the Cap gene (SEQ ID NO: 23) are arranged between a pair of telomerase recognition sequences (telRL sequences) ( Figure 1 ). It should be noted that the Rep gene and the Cap gene encode packaging proteins of adeno-associated virus, respectively.

[0290] (3) Preparation of isolated expression vector

[0291] As a linear covalently closed DNA vector, a vector containing the TelN frontomerase gene and the I-SceI homing endonuclease gene (hereinafter referred to as "isolated expression vector") was prepared ( Figure 2 ).

[0292] The method for preparing the separation expression vector is as follows. First, the telRL sequence of (c) above is inserted into the SnaBI site of the pRC2-mi342 vector (manufactured by Takara Bio). Next, (b), (d), (f), (g) and (h) above are inserted into the SmaI site of the vector. By inserting the five sequences (sequence numbers 5 to 9) containing the telRL sequence and the I-SceI recognition sequence of (e) above into the EcoRV site of the obtained vector, separation expression vectors (T0), separation expression vectors (T2), separation expression vectors (T4), separation expression vectors (T6) and separation expression vectors (T8) were prepared ( Figure 2 ).

[0293] In the separate expression vector, the I-SceI homing endonuclease and the TelN front-end telomerase are designed to be separately expressed under the control of an arabinose-inducible promoter. In addition, the Rep gene and the Cap gene ( Figure 2 ).

[0294] <Example 2: Construction of fusion expression vector>

[0295] (Purpose)

[0296] As a vector for preparing linear covalently closed DNA, a vector containing a gene encoding a fusion polypeptide of TelN terminal endomerase and I-SceI homing endonuclease (hereinafter referred to as a “fusion expression vector”) was prepared.

[0297] (Methods and Results)

[0298] (1) Result prediction

[0299] The three-dimensional structure of TelN-terminal oligomerase was predicted by the following method.

[0300] As enzymes that catalyze DNA cleavage / rebinding reactions similar to TelN front-end micromerase, the crystal structures of TelK front-end micromerase (sequence homology ~83%) and TelA front-end micromerase (sequence homology ~8%) have been partially resolved (PDBID: 2V6E, 4DWP, 4E0G, etc.), but the three-dimensional structures of the entire length including the C-terminal side have not been resolved. In order to efficiently perform DNA cleavage / rebinding reactions and decomposition of unnecessary linear covalently closed DNA simultaneously using a fusion polypeptide of TelN front-end micromerase and I-SceI homing endonuclease, the configuration of I-SceI homing endonuclease and TelN front-end micromerase in the fusion polypeptide (fusion on the N-terminal side or the C-terminal side), and the relative positions relative to the I-SceI homing endonuclease recognition sequence and the TelN front-end micromerase recognition sequence (telRL sequence) are important. Therefore, the full-length three-dimensional structure of the TelN frontomerase was predicted using the high-precision protein three-dimensional structure prediction software ColabFold (Mirdita M, et al., Nature Methods(2022) doi:10.1038 / s41592-022-01488-1).

[0301] The predicted structure of TelN-terminal telomerase shows that the N-terminal side of TelN-terminal telomerase is located near the center of the DNA recognition sequence (telRL sequence), while the C-terminal side (positions 606 to 641) is folded and located away from the DNA recognition sequence, and can take various relative configurations with respect to DNA. This is because the loop region (positions 535 to 583) predicted between the N-terminal side and the C-terminal side is flexible, so even when the N-terminal side is bound to DNA, the C-terminal side bound via the loop region can take various configurations ( Figure 3 ). Therefore, it can be expected that for the fusion polypeptide (PH type fusion polypeptide of Example 5) in which the N-terminal side of the I-SceI nuclease is fused to the C-terminal side of the TelN front telomerase, the activities of the two polypeptides can function more efficiently compared with the fusion polypeptide (HP type fusion polypeptide of Example 5) configured in the opposite manner. The configuration of the PH type fusion polypeptide and the DNA recognition sequence is shown in Figure 3 .

[0302] (2) Preparation of fusion expression vector

[0303] The method for preparing a fusion expression vector is as follows. First, the telRL sequence of (c) in Example 1 is inserted into the SnaBI site of the pRC2-mi342 vector (manufactured by Takara Bio). Next, (a), (f) and (g) in Example 1 are inserted into the SmaI site of the vector. By inserting the five sequences (sequence numbers 5 to 9) containing the telRL sequence and the I-SceI recognition sequence of (e) in Example 1 into the EcoRV site of the obtained vector, fusion expression vectors (T0), (T2), (T4), (T6) and (T8) were prepared ( Figure 4 ).

[0304] In the fusion expression vector, the fusion polypeptide of I-SceI homing endonuclease and TelN front-end telomerase is designed to be expressed under the control of an arabinose-inducible promoter. In addition, the Rep gene and the Cap gene ( Figure 4 ).

[0305] <Example 3: Production of linear covalently closed DNA>

[0306] (Purpose)

[0307] Escherichia coli was transformed with the vector for preparing linear covalently closed DNA prepared in Example 1 to produce linear covalently closed DNA. The production amount of linear covalently closed DNA was evaluated.

[0308] (Methods and Results)

[0309] (1) Conversion

[0310] Escherichia coli was transformed using the control vector prepared in Example 1, the isolated expression vector (T0), the isolated expression vector (T2), the isolated expression vector (T4), the isolated expression vector (T6), the isolated expression vector (T8), the fusion expression vector (T0), the fusion expression vector (T2), the fusion expression vector (T4), the fusion expression vector (T6) and the fusion expression vector (T8).

[0311] 25 μL of competent cell solution of Escherichia coli DH10B strain was mixed with a solution containing 100 pg of each vector and allowed to stand on ice for 30 minutes. After standing for 30 minutes, heat treatment was performed at 42°C for 45 seconds and allowed to stand on ice for 2 minutes. Next, 225 μL of SOC medium was added, and Escherichia coli was spread on LB agar medium (1% tryptone, 0.5% dry yeast extract, 1% sodium chloride, 0.005% disodium carbenicillin (manufactured by Nacalai Tesque)), and strains that grew during static culture at 37°C for 1 day were selected to obtain Escherichia coli with the vector introduced.

[0312] (2) Cultivation

[0313] The obtained Escherichia coli was inoculated into 2 mL of Plusgrow II medium (4% Plusgrow II (manufactured by NacalaiTesque Co., Ltd.), 0.005% carbenicillin disodium), and cultured at 37°C for 6 hours with shaking to obtain a pre-culture solution. 200 μL of the pre-culture solution was subcultured in 100 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium) in a flask, and cultured at 37°C for 16 hours with shaking.

[0314] (3) Expression induction

[0315] To the obtained culture solution, 100 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium) at pH 7.0 and 2 mL of arabinose solution (10% arabinose) were added, and the culture was shaken at 30° C. or 37° C. for 6 hours. Then, the bacterial cells were recovered by centrifugation.

[0316] (4) DNA extraction

[0317] DNA was extracted from 1 mL of the recovered culture solution using QIAprep Spin Miniprep Kit (manufactured by QIAGEN).

[0318] (5) Evaluation of total DNA yield

[0319] The total DNA yield from 200 mL of the culture solution was calculated based on measurement using Nanodrop (manufactured by Thermo Fisher Scientific), and the results are shown in Table 1 below.

[0320] [Table 1]

[0321]

[0322] (6) Evaluation of linear covalently closed DNA

[0323] The linear covalently closed DNA was evaluated by electrophoresis. Specifically, the DNA solution obtained in (4) above was adjusted to 2 to 10 ng / μL and analyzed using the DNA / RNA analysis microchip electrophoresis device MultiNA (Shimadzu Corporation).

[0324] The electrophoresis results obtained by MultiNA are shown in Figure 5 .exist Figure 5 In the figure, the linear covalently closed DNA containing the nucleic acid encoding the packaging protein is indicated as "target linear covalently closed DNA", and the linear covalently closed DNA derived from the vector sequence other than the nucleic acid encoding the packaging protein is indicated as "unnecessary linear covalently closed DNA". In the figure, the left side shows the sample cultured with shaking at the expression induction culture temperature of 30°C for 6 hours, and the right side shows the sample cultured with shaking at the expression induction culture temperature of 37°C for 6 hours.

[0325] Furthermore, the residual rate of the unwanted linear covalently closed DNA is calculated based on the molar concentrations of the target linear covalently closed DNA and the unwanted linear covalently closed DNA. The calculation formula for calculating the residual rate is shown below.

[0326] Residual rate (%) = (the number of moles of unwanted linear covalently closed DNA) / (the number of moles of target linear covalently closed DNA) × 100

[0327] The calculation results of the residual ratio under each condition are shown in Table 2 below.

[0328] [Table 2]

[0329]

[0330] according to Figure 5 As shown in Table 2, in the case of the control vector, the residual rate of unnecessary linear covalently closed DNA was 115.8% (30°C) and 90.7% (37°C). On the other hand, in the case of the isolated expression vector and the fusion expression vector, the residual rate of unnecessary linear covalently closed DNA was greatly reduced. This result shows that in both the isolated expression vector and the fusion expression vector, the homing endonuclease recognition sequence is cut by the I-SceI homing nuclease and decomposed from its cut end by the endogenous exonuclease in the Escherichia coli.

[0331] <Example 4: Study of expression induction conditions>

[0332] (Purpose)

[0333] The culture conditions for producing linear covalently closed DNA were investigated. Specifically, the pH and temperature conditions for culturing transformed Escherichia coli were investigated.

[0334] (Methods and Results)

[0335] (1) Production of linear covalently closed DNA

[0336] Escherichia coli was transformed by the same method as in "(1) Transformation" of Example 3. The obtained Escherichia coli was inoculated into 2 mL of Plusgrow II medium (4% Plusgrow II (manufactured by Nacalai Tesque Co., Ltd.), 0.005% carbenicillin disodium), and cultured at 37°C for 6 hours with shaking to obtain a pre-culture solution. 100 μL of the pre-culture solution was subcultured in 1.4 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium) in a test tube, and cultured at 37°C with shaking for 16 hours.

[0337] Next, for expression induction, 1.5 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium) pre-adjusted to pH 7.0, pH 7.5, pH 8.0, and pH 9.0 using 1 M NaOH solution and 30 μL of arabinose solution (10% arabinose) were added, shake-cultured for 6 hours, and then the cells were recovered by centrifugation. The 6-hour shaking culture was performed by the following method: (Method A) shaking culture at 37°C for 6 hours; or (Method B) shaking culture at 30°C for 4 hours, followed by shaking culture at 37°C for 2 hours.

[0338] DNA extraction was performed in the same manner as in "(4) DNA extraction" of Example 2.

[0339] (2) Evaluation of total DNA yield

[0340] Furthermore, the total DNA yield was evaluated by the same method as in "(5) Evaluation of total DNA yield" of Example 2. The results are shown in Table 3 below.

[0341] [Table 3]

[0342]

[0343] (3) Evaluation of linear covalently closed DNA

[0344] The linear covalently closed DNA was analyzed by the same method as in "(6) Evaluation of linear covalently closed DNA" of Example 2, and the results are shown in FIG. Figure 6Furthermore, the residual rate of unnecessary linear covalently closed DNA was calculated, and the results are shown in Table 4 below.

[0345] [Table 4]

[0346]

[0347] exist Figure 6 In the results of Table 4, the residual rate of unnecessary linear covalently closed DNA in the Escherichia coli cells introduced with the isolated expression vector or the fusion expression vector was greatly reduced compared with the Escherichia coli cells introduced with the control vector. This result shows that in the isolated expression vector and the fusion expression vector, the homing endonuclease recognition sequence is cut by the I-SceI homing nuclease and decomposed from the cut end by the endogenous exonuclease in the Escherichia coli cells.

[0348] according to Figure 6 As shown in the results of Table 4, the culture temperature in the expression induction step is not particularly limited as long as it is within the range where the front endomerase and homing endonuclease can function in the cell. In the case of the condition of shaking culture at 37°C for 6 hours after adding the arabinose solution (method A), the residual rate of unnecessary linear covalently closed DNA in the separated expression vector is lower than that in the fusion expression vector. On the other hand, in the case of the condition of shaking culture at 30°C for 4 hours after adding the arabinose solution and then shaking culture at 37°C for 2 hours (method B), the residual rate of unnecessary linear covalently closed DNA in the fusion expression vector is lower than that in the separated expression vector.

[0349] This result indicates that since the optimum temperature for the enzyme activity of the TelN front endomerase is 30°C and the optimum temperature for the enzyme activity of the I-SceI homing endonuclease is 37°C, by setting the temperature after adding the arabinose solution to 30°C, the production efficiency of linear covalently closed DNA is increased by the activity of the TelN front endomerase, and then by switching to 37°C, the I-SceI homing endonuclease activity is increased, and the decomposition efficiency of unnecessary linear covalently closed DNA is increased.

[0350] In addition, similarly, according to Figure 6As shown in the results of Table 4, the pH in the expression induction step is not particularly limited as long as it is within the range where the telomerase and homing endonuclease can function in the cell. In the case of method A, for both the isolated expression vector and the fusion expression vector, when the medium added in the expression induction step is at pH 8.0, the residual rate of the unnecessary linear covalently closed DNA is lower than that at pH 7.0. In addition, in the case of method B, for both the isolated expression vector and the fusion expression vector, as the pH of the medium added in the expression induction step is increased from 7.0 to 9.0, the residual rate of the unnecessary linear covalently closed DNA is lower.

[0351] This result indicates that since the optimum pH for the enzymatic activity of the TelN front endomerase is 8.8 and the optimum temperature for the enzymatic activity of the I-SceI homing endonuclease is pH 7.9, by increasing the pH of the culture medium added in the expression induction step from 7.0 to 9.0, the production efficiency of linear covalently closed DNA is increased by the TelN front endomerase activity, and further, the I-SceI homing endonuclease activity is increased, thereby increasing the decomposition efficiency of unnecessary linear covalently closed DNA.

[0352] <Example 5: Study of fusion polypeptide>

[0353] (Purpose)

[0354] A fusion polypeptide comprising a TelN front endomerase at the N-terminal side and an I-SceI homing endonuclease at the C-terminal side (hereinafter referred to as a "PH-type fusion polypeptide"), and a fusion polypeptide comprising an I-SceI homing endonuclease at the N-terminal side and a TelN front endomerase at the C-terminal side (hereinafter referred to as a "HP-type fusion polypeptide") were prepared, and the production efficiency of linear covalently closed DNA was compared.

[0355] (Methods and Results)

[0356] (1) Preparation of PH and HP fusion expression vectors

[0357] The "fusion expression vector (T0)" prepared in Example 2 was used as a linear covalently closed DNA preparation vector (hereinafter referred to as "PH-type fusion expression vector") containing a gene encoding a PH-type fusion polypeptide, which contains, from the N-terminal side, a TelN front endomerase, a linker sequence (an amino acid sequence encoded by the base sequence shown in SEQ ID NO: 34), and an I-SceI homing endonuclease. In the PH-type fusion expression vector, it is designed in such a way that the PH-type fusion polypeptide is expressed under the control of an arabinose-inducible promoter.

[0358] A linear covalently closed DNA preparation vector (hereinafter referred to as "HP-type fusion expression vector") containing a gene encoding an HP-type fusion polypeptide was prepared by exchanging a region encoding a TelN front endomerase with a region encoding an I-SceI homing endonuclease in the above-mentioned PH-type fusion expression vector, wherein the HP-type fusion polypeptide contains, in order from the N-terminal side, an I-SceI homing endonuclease, a linker sequence (an amino acid sequence encoded by a base sequence shown in SEQ ID NO: 34), and a TelN front endomerase. In the HP-type fusion expression vector, it is designed so that the HP-type fusion polypeptide is expressed under the control of an arabinose-inducible promoter.

[0359] (2) Evaluation of the production of linear covalently closed DNA

[0360] Using the PH type fusion expression vector or the HP type fusion expression vector, transformation, culture, expression induction, DNA extraction, evaluation of total DNA yield, and evaluation of linear covalently closed DNA were performed in the same manner as in Example 3. In this example, shaking culture was performed in 200 mL of culture solution at 30°C in the presence of arabinose for 6 hours.

[0361] The total DNA yield obtained from 200 mL of the culture solution was calculated based on the measurement using Nanodrop, and the results are shown in Table 5 below.

[0362] [Table 5]

[0363]

[0364] Next, the residual rate of unnecessary linear covalently closed DNA was evaluated by electrophoresis. The results are shown in Table 6 below.

[0365] [Table 6]

[0366]

[0367] The results shown in Tables 5 and 6 indicate that, compared with the HP-type fusion expression vector, the PH-type fusion expression vector has a higher total DNA yield and a lower residual rate of unwanted linear covalently closed DNA.

[0368] This result shows that by arranging the TelN front endomerase on the N-terminal side of the I-SceI homing endonuclease as expected from the structural prediction in Example 2, both the front endomerase activity and the homing endonuclease activity of the fusion polypeptide can function efficiently.

[0369] <Comparative Example 1: Construction of two vectors for preparing linear covalently closed DNA>

[0370] (Purpose)

[0371] As two types of vectors for preparing linear covalently closed DNA, a control vector (T6) and an I-SceI vector were prepared.

[0372] (Methods and Results)

[0373] (1) Preparation of nucleic acid fragments

[0374] As a nucleic acid fragment used for constructing the vector, a nucleic acid encoding an aminoglycoside 3'-phosphotransferase conferring kanamycin resistance (SEQ ID NO: 35) was prepared by total synthesis.

[0375] (2) Preparation of control vector (T6)

[0376] A vector containing T6 as a spacer sequence between the telRL sequence and the I-SceI recognition sequence (hereinafter referred to as "control vector (T6)") was prepared to express TelN frontomerase ( Figure 7 ).

[0377] The preparation method of the control vector (T6) is as follows. First, the isolated expression vector (T6) prepared in Example 1 was used as a template, and a nucleic acid fragment was prepared by PCR using primer 9 (forward, sequence number 36) and primer 10 (reverse, sequence number 37). Then, the overlapping region at the end of the obtained nucleic acid fragment was circularized using the Gibson Assembly system of New England Biolabs to prepare the control vector (T6) ( Figure 7 ). The control vector (T6) is a vector obtained by removing the I-SceI homing endonuclease gene designed to be expressed under the control of an arabinose-inducible promoter from the isolated expression vector (T6). In the control vector (T6), the Rep gene and the Cap gene are arranged between a pair of front endomerase recognition sequences (telRL sequences) ( Figure 7 ).

[0378] (3) Preparation of I-SceI vector

[0379] A vector (hereinafter referred to as "I-SceI vector") containing a nucleic acid encoding an aminoglycoside 3'-phosphotransferase conferring kanamycin resistance (SEQ ID NO: 35) was prepared, and the vector expressed an I-SceI homing endonuclease ( Figure 8 ).

[0380] The preparation method of I-SceI vector is as follows. First, the nucleic acid encoding the β-lactamase that confers ampicillin resistance contained in the separation expression vector (T6) prepared in Example 1 is replaced with a nucleic acid encoding an aminoglycoside 3'-phosphotransferase that confers kanamycin resistance (sequence number 35). Using the replaced vector as a template, a nucleic acid fragment was prepared by PCR using primer 7 (forward, sequence number 20) and primer 11 (reverse, sequence number 38). Then, the overlapping region at the end of the obtained nucleic acid fragment was circularized using the Gibson Assembly system of New England Biolabs to prepare the I-SceI vector ( Figure 8 The I-SceI vector does not contain the telomerase recognition sequence (telRL sequence) but contains the I-SceI recognition sequence ( Figure 8 ).

[0381] <Comparative Example 2: Production of linear covalently closed DNA>

[0382] (Purpose)

[0383] Escherichia coli was transformed with the two vectors prepared in Comparative Example 1 to produce linear covalently closed DNA. The production amount of linear covalently closed DNA was evaluated.

[0384] (Methods and Results)

[0385] (1) Conversion

[0386] Escherichia coli was transformed with the control vector (T6) and the I-SceI vector prepared in Comparative Example 1. In addition, as a control group, Escherichia coli transformed with only the control vector (T6) was also prepared.

[0387] 25 μL of competent cell solution of Escherichia coli DH10B strain was mixed with the solution containing each vector and allowed to stand on ice for 30 minutes. After standing for 30 minutes, heat treatment was performed at 42°C for 45 seconds and allowed to stand on ice for 2 minutes. Next, 225 μL of SOC medium was added. In the case where only the control vector (T6) was introduced, Escherichia coli was spread on LB agar medium (1% tryptone, 0.5% dry yeast extract, 1% sodium chloride, 0.005% disodium carbenicillin (manufactured by Nacalai Tesque)). In the case where the control vector (T6) and I-SceI vector were introduced, Escherichia coli was spread on LB agar medium (1% tryptone, 0.5% dry yeast extract, 1% sodium chloride, 0.005% disodium carbenicillin (manufactured by Nacalai Tesque), 0.005% kanamycin sulfate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)). A strain that grew during static culture at 37°C for 1 day was selected to obtain Escherichia coli into which the vector was introduced.

[0388] (2) Cultivation

[0389] The Escherichia coli introduced with the isolated expression vector (T6) obtained in Example 3 was inoculated into 2 mL of PlusgrowII medium (4% Plusgrow II (manufactured by Nacalai Tesque Co., Ltd.), 0.005% carbenicillin disodium), and cultured at 37°C with shaking for 6 hours to obtain a pre-culture solution. 200 μL of the pre-culture solution was subcultured in 100 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium) in a flask, and cultured with shaking at 37°C for 16 hours.

[0390] Escherichia coli into which only the control vector (T6) obtained above was introduced was inoculated into 2 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium), and cultured at 37°C with shaking for 6 hours to obtain a pre-culture solution. 200 μL of the pre-culture solution was subcultured in 100 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium) in a flask, and cultured at 37°C with shaking for 16 hours.

[0391] The Escherichia coli into which the control vector (T6) and the I-SceI vector obtained above were introduced was inoculated into 2 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium, 0.005% kanamycin sulfate), and the medium was shaken and cultured at 37°C for 6 hours to obtain a pre-culture solution. 200 μL of the pre-culture solution was subcultured in 100 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium, 0.005% kanamycin sulfate) in a flask, and the medium was shaken and cultured at 37°C for 16 hours.

[0392] (3) Expression induction

[0393] 100 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium) and 2 mL of arabinose solution (10% arabinose) at pH 7.0 were added to the culture solution obtained from Escherichia coli into which the isolated expression vector (T6) was introduced, and the culture was shaken at 30° C. or 37° C. for 6 hours. Then, the bacterial cells were recovered by centrifugation.

[0394] 100 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium) and 2 mL of arabinose solution (10% arabinose) at pH 7.0 were added to the culture solution obtained from Escherichia coli into which only the control vector (T6) was introduced, and the culture was shaken at 30°C or 37°C for 6 hours. Then, the bacterial cells were recovered by centrifugation.

[0395] 100 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium, 0.005% kanamycin sulfate) and 2 mL of arabinose solution (10% arabinose) at pH 7.0 were added to the culture solution obtained from Escherichia coli into which the control vector (T6) and the I-SceI vector were introduced, and the culture was shaken at 30°C or 37°C for 6 hours. Then, the bacterial cells were recovered by centrifugation.

[0396] (4) DNA extraction

[0397] DNA was extracted from 1 mL of the recovered culture solution using QIAprep Spin Miniprep Kit (manufactured by QIAGEN).

[0398] (5) Evaluation of total DNA yield

[0399] The total DNA yield from 200 mL of the culture solution was calculated based on measurement using Nanodrop (manufactured by Thermo Fisher Scientific), and the results are shown in Table 7 below.

[0400] [Table 7]

[0401]

[0402] (6) Evaluation of linear covalently closed DNA

[0403] The linear covalently closed DNA was evaluated by electrophoresis. Specifically, the DNA solution obtained in (4) above was adjusted to 2 to 10 ng / μL and analyzed using the DNA / RNA analysis microchip electrophoresis device MultiNA (Shimadzu Corporation).

[0404] The electrophoresis results obtained using MultiNA are shown in Fig. 9 .exist Fig. 9 In the figure, the linear covalently closed DNA containing the nucleic acid encoding the packaging protein is indicated as "target linear covalently closed DNA", and the linear covalently closed DNA derived from the vector sequence other than the nucleic acid encoding the packaging protein is indicated as "unnecessary linear covalently closed DNA". In the figure, the left side shows the sample cultured with shaking at the expression induction culture temperature of 30°C for 6 hours, and the right side shows the sample cultured with shaking at the expression induction culture temperature of 37°C for 6 hours.

[0405] Fig. 9 The results shown indicate that in E. coli introduced with the control vector (T6) and the I-SceI vector, a portion of the I-SceI vector remained, and thus the amount of DNA other than the target linear covalently closed DNA increased.

[0406] All publications, patents and patent applications cited in this specification are incorporated herein by direct reference.

Claims

1. A double-stranded circular DNA vector comprising: A pre-telomerase gene sequence encoding a pre-telomerase or an active fragment thereof, An endonuclease gene sequence encoding an endonuclease or an active fragment thereof, A pair of telomerase recognition sequences for recognizing the telomerase or its active fragment and cutting the vector, at least one endonuclease recognition sequence for recognizing the endonuclease or its active fragment and cleaving the vector, and Target nucleic acid sequence, In the above-mentioned double-stranded circular DNA vector, the above-mentioned front-end merase gene sequence, the above-mentioned endonuclease gene sequence and the above-mentioned endonuclease recognition sequence are arranged in the same region between the above-mentioned pair of front-end merase recognition sequences, and the above-mentioned target nucleic acid sequence is arranged in another region between the above-mentioned pair of front-end merase recognition sequences, and The anterior telomerase gene sequence and the endonuclease gene sequence are placed under the control of a promoter capable of controlling expression.

2. The double-stranded circular DNA vector according to claim 1, wherein The anterior terminal telomerase gene sequence and the anterior endonuclease gene sequence encode a fusion polypeptide comprising the anterior terminal telomerase or an active fragment thereof, and the anterior endonuclease or an active fragment thereof.

3. The double-stranded circular DNA vector according to claim 2, wherein In the above-mentioned fusion polypeptide, the above-mentioned premerase or an active fragment thereof is arranged on the N-terminal side of the above-mentioned endonuclease or an active fragment thereof.

4. The double-stranded circular DNA vector according to claim 2, wherein The fusion polypeptide further comprises a linker sequence between the telomerase or an active fragment thereof and the endonuclease or an active fragment thereof. 5 . The double-stranded circular DNA vector according to claim 1 , further comprising a spacer sequence disposed between the endonuclease recognition sequence and the front endomerase recognition sequence.

6. The double-stranded circular DNA vector according to claim 5, wherein The above-mentioned spacer sequence is less than 200 bases in length.

7. The double-stranded circular DNA vector according to claim 1 or 2, wherein The endonuclease recognition sequence is arranged adjacent to one of the telomerase recognition sequences. The double-stranded circular DNA vector according to claim 1 , which is a plasmid.

9. The double-stranded circular DNA vector according to claim 1, wherein The above-mentioned terminal endomerase is TelN terminal endomerase, TelA terminal endomerase or TelK terminal endomerase.

10. The double-stranded circular DNA vector according to claim 1, wherein The above endonuclease is a homing endonuclease.

11. The double-stranded circular DNA vector according to claim 10, wherein The above-mentioned homing endonuclease is I-SceI homing endonuclease, I-CeuI homing endonuclease or I-CreI homing endonuclease.

12. The double-stranded circular DNA vector according to claim 1, wherein The target nucleic acid sequence comprises a target gene sequence encoding a target protein or a fragment thereof. 13 . A transformed cell comprising the double-stranded circular DNA vector according to claim 1 .

14. The transformed cell according to claim 13, wherein The cell is a bacterial cell or a eukaryotic cell.

15. The transformed cell according to claim 14, wherein The bacteria are Escherichia coli or Bacillus subtilis. The transformed cell according to claim 13 , which expresses an exonuclease.

17. A method for preparing linear covalently closed DNA, the method comprising: a step of culturing the transformed cell according to claim 13, an expression induction step of inducing the expression of the telomerase or an active fragment thereof and the endonuclease or an active fragment thereof in the transformed cells after the culturing step, and A DNA extraction step is a step of extracting DNA from the transformed cells after the above expression induction step.

18. The method according to claim 17, further comprising: After the DNA extraction step, a separation step of isolating the linear covalently closed DNA is performed.

19. The method according to claim 17, further comprising: A step of introducing the double-stranded circular DNA vector according to claim 1 into host cells to obtain transformed cells before the culturing step.

20. The method according to claim 17, wherein: The transformed cells express exonuclease.

21. A fusion polypeptide or a nucleic acid encoding the same, wherein the fusion polypeptide comprises a frontomerase or an active fragment thereof at the N-terminal side and an endonuclease or an active fragment thereof at the C-terminal side.

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