Double-strand circular DNA vector

Abstract

The present invention addresses the problem of providing a new replication initiation region capable of increasing the yield of plasmid DNA.　Provided is a double-strand circular DNA vector having a replication initiation region in which at least the base corresponding to position 22 in the base sequence represented by SEQ ID NO: 1 is an adenine base.

Description

double-stranded circular DNA vector

[0001] The present invention relates to a replication initiation region, a double-stranded circular DNA vector, a transformed cell, a method for amplifying a double-stranded circular DNA vector, a method for expressing and producing a target nucleic acid or a target protein or a fragment thereof, and a method for increasing the replication efficiency of a double-stranded circular DNA vector.

[0002] Mass production of DNA for applications such as DNA vaccines and nucleic acid medicines is carried out by amplifying a plasmid DNA vector in a host cell such as Escherichia coli. Typical productivity of plasmid DNA in high-density culture of Escherichia coli is approximately several hundred mg to 1 g per liter of broth. Therefore, large-scale culture of tens to thousands of liters is required for mass production of plasmid DNA on a gram or kilogram scale.

[0003] In order to improve the productivity of plasmid DNA, various replication origins have long been developed in the art. A replication origin is a sequence region that is present on a double-stranded circular DNA vector, such as a plasmid DNA, and is capable of initiating the DNA replication initiation reaction. Various replication origins have been developed to date in order to increase the number of plasmid DNA copies maintained per cell.

[0004] For example, ColE1-type plasmid DNA is a typical example of a double-stranded circular DNA vector that replicates in Escherichia coli, and is a general term for plasmid DNA that has a replication origin derived from ColE1 plasmid DNA or a replication origin region including its surrounding sequences, or a sequence similar thereto. An example of ColE1-type plasmid DNA is pBR322 plasmid DNA, which is often used as a cloning vector in the field of genetic engineering.

[0005] In the replication origin of ColE1-type plasmid DNA, a single-stranded RNA called RNA II precursor is transcribed from a promoter sequence approximately 550 bases upstream of the replication origin. The RNA II precursor forms a DNA / RNA hybrid duplex with the DNA of the replication origin, which is then cleaved by RNase H at the replication origin to generate mature RNA II (primer RNA). Replication of the plasmid DNA is initiated by the function of the primer RNA.

[0006] Another example of ColE1-type plasmid DNA is pUC-type plasmid DNA, which is widely used in genetic engineering. The replication origin of pUC-type plasmid DNA is similar to that of ColE1-type plasmid DNA, but a mutation has been introduced into the base sequence encoding the RNA II precursor in the replication origin. Compared to ColE1-type plasmid DNA, pUC-type plasmid DNA maintains a higher number of plasmid DNA copies per cell (Non-Patent Document 1).

[0007] In order to further improve the efficiency of plasmid DNA production, a new replication origin that allows for a further increase in the copy number of plasmid DNA is needed.

[0008] Yanisch-Perron, C. , et al. , Gene, 1985;33(1):103-119.

[0009] An object of the present invention is to provide a new origin of replication that can increase the yield of plasmid DNA.

[0010] Previous studies on ColE1-type plasmid DNA have attempted to increase the number of copies of plasmid DNA maintained per cell by introducing mutations into the nucleotide sequence encoding the RNA II precursor, as in pUC-type plasmid DNA, or by introducing mutations into the -35 Box or -10 Box sequence in the promoter sequence controlling the transcription of the RNA II precursor (Rouches M.V., et al., Nat Commun., 2022; 13(1):3908.; Camps, M., Recent Pat DNA Gene Seq., 2010; 4(1):58-73.). However, no studies have investigated modifications to sequences other than the -35 Box and -10 Box in the promoter sequence controlling the transcription of the RNA II precursor.

[0011] In order to solve the above-mentioned problems, the present inventors introduced substitution mutations into the region between -35 Box and -10 Box in the replication origin region of pUC19 plasmid DNA, a pUC-type plasmid DNA, and evaluated the effect on the yield of plasmid DNA. As a result, they found that when the cytosine residue corresponding to position 22 from -35 Box to -10 Box in the region between -35 Box and -10 Box (the cytosine residue at position 22 in the base sequence shown in SEQ ID NO: 1) was substituted with an adenine base, the yield of plasmid DNA and the specific yield of plasmid DNA were significantly increased. The present invention is based on the above-mentioned findings and provides the following:

[0012] (1) A double-stranded circular DNA vector comprising a replication origin region, wherein in the replication origin region, at least the base corresponding to position 22 of the base sequence shown in SEQ ID NO: 1 below is an adenine base. (2) The double-stranded circular DNA vector according to (1), wherein, in the replication origin region, the base corresponding to position 23 of the base sequence is a cytosine base, a guanine base, or a thymine base. (3) The double-stranded circular DNA vector according to (1) or (2), wherein, in the replication origin region, the base corresponding to position 23 of the base sequence is a guanine base or a thymine base. (4) The double-stranded circular DNA vector according to any one of (1) to (3), wherein, in the replication origin region, the base corresponding to position 20 of the base sequence is a cytosine base or a guanine base. (5) The double-stranded circular DNA vector according to any one of (1) to (4), wherein, in the replication origin region, the base corresponding to position 21 of the base sequence is a guanine base. (6) The double-stranded circular DNA vector according to any one of (1) to (5), wherein, in the replication origin region, the base corresponding to position 20 of the base sequence is a cytosine base, the base corresponding to position 21 of the base sequence is a guanine base, the base corresponding to position 22 of the base sequence is an adenine base, and the base corresponding to position 23 of the base sequence is a guanine base. (7) The double-stranded circular DNA vector according to any one of (1) to (6), wherein, in the replication origin region, the sequence corresponding to the -35 box sequence consisting of positions 1 to 6 of the base sequence is identical to the -35 box sequence or consists of the -35 box sequence with one to four bases deleted, substituted, or added, and / or the sequence corresponding to the -10 box sequence consisting of positions 24 to 29 of the base sequence is identical to the -10 box sequence or consists of the -10 box sequence with one to four bases deleted, substituted, or added. (8) The double-stranded circular DNA vector according to any one of (1) to (7), comprising a nucleic acid sequence of interest and / or an antibiotic resistance gene that confers resistance to an antibiotic. (9) The double-stranded circular DNA vector according to (8), wherein the nucleic acid sequence of interest comprises a gene sequence of interest that encodes a protein of interest or a fragment thereof. (10) The double-stranded circular DNA vector according to any one of (1) to (9), which is a ColE1-type plasmid DNA. (11) The double-stranded circular DNA vector according to (10), wherein the ColE1-type plasmid DNA is a pUC-type plasmid DNA or a pET-type plasmid DNA.(12) The double-stranded circular DNA vector according to (10) or (11), wherein the replication origin region contains, except for the base corresponding to position 22 of the base sequence, (a) the base sequence shown in SEQ ID NO: 2 or 3, (b) a base sequence in which one or more bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 2 or 3, or (c) a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 2 or 3. (13) A transformed cell comprising the double-stranded circular DNA vector according to any of (1) to (12). (14) The transformed cell according to (13), which is a bacterium. (15) A method for amplifying a double-stranded circular DNA vector, comprising a culturing step of culturing the transformed cell according to (13) or (14). (16) A method for expressing a target nucleic acid or a target protein or a fragment thereof in a transformed cell, comprising: a culturing step of culturing a transformed cell containing the double-stranded circular DNA vector according to any one of (1) to (12), wherein the double-stranded circular DNA vector contains a nucleic acid sequence constituting the target nucleic acid, or a target gene sequence encoding the target protein or a fragment thereof in an expressible state. (17) The method according to (16), wherein the target protein is an enzyme, an antibody, or a functional peptide. (18) A method for producing a target nucleic acid, comprising: a culturing step of culturing a transformed cell containing the double-stranded circular DNA vector according to any one of (1) to (12), and a nucleic acid extraction step of extracting the target nucleic acid from the transformed cell after the culturing step, wherein the double-stranded circular DNA vector contains a nucleic acid sequence constituting the target nucleic acid. (19) A method for producing a target protein or a fragment thereof, comprising: a culturing step of culturing a transformed cell containing the double-stranded circular DNA vector according to any one of (1) to (12); and an isolation step of isolating the target protein or a fragment thereof from the transformed cell and / or culture supernatant after the culturing step, wherein the double-stranded circular DNA vector contains a target gene sequence encoding the target protein or a fragment thereof in an expressible state.(20) The method according to (19), comprising an expression induction step of inducing expression of the target protein or a fragment thereof after the culturing step and before the isolating step. (21) A method for increasing the replication efficiency of a double-stranded circular DNA vector, comprising a step of substituting an adenine base for the base corresponding to position 22 of the base sequence shown in SEQ ID NO: 1 in the replication origin region of the double-stranded circular DNA vector. (22) A method for increasing the replication efficiency of a double-stranded circular DNA vector other than pUC-type plasmid DNA, comprising: substituting a replication origin region of the double-stranded circular DNA vector other than pUC-type plasmid DNA with a replication origin region derived from pUC-type plasmid DNA, wherein in the replication origin region derived from pUC-type plasmid DNA, at least the base corresponding to position 22 of the base sequence shown in SEQ ID NO: 1 below is an adenine base. (23) The method according to (22), wherein the double-stranded circular DNA vector other than a pUC-type plasmid DNA is an R6K-type plasmid DNA. This specification incorporates the disclosure of Japanese Patent Application No. 2024-023712, from which the present application claims priority.

[0013] According to the present invention, a new origin of replication is provided that can increase the yield of plasmid DNA.

[0014]

[0023] Figure 2 is a diagram showing the overall structure of pUC19 plasmid DNA and the sequence portion containing the -35 Box and -10 Box in its replication origin region. In the diagram, "bp" indicates a base pair. Figure 2 is a diagram showing the base sequence targeted for substitution mutagenesis between the -35 Box and the -10 Box in the replication origin region of pUC19 plasmid DNA. Figure 2A shows the base targeted for substitution mutagenesis in Example 1 (a cytosine base (C base) at position 22). Figure 2B shows the base sequence targeted for substitution mutagenesis in Example 2 (a CGCG base sequence consisting of a cytosine base at position 20, a guanine base at position 21, a cytosine base at position 22, and a guanine base at position 23).

[0015] 1. Double-stranded circular DNA vector 1-1. Overview A first aspect of the present invention is a double-stranded circular DNA vector. In the replication origin region of the double-stranded circular DNA vector of this aspect, the base corresponding to position 22 in the base sequence shown in SEQ ID NO: 1 is an adenine base. When the double-stranded circular DNA vector of this aspect is introduced into a host cell such as Escherichia coli, a large number of plasmid DNA copies are retained per cell, and the yield of plasmid DNA that can be produced using the host cell is high.

[0016] 1-2. Definition of Terms The following terms frequently used in this specification are defined below. In the present invention, a "double-stranded circular DNA vector" refers to a circular double-stranded DNA molecule that can be maintained and / or replicated in a cell. As used herein, a double-stranded circular DNA vector essentially includes a replication origin, and may optionally include a sequence other than the replication origin that is necessary for the intracellular maintenance of the double-stranded circular DNA vector, such as a gene encoding an antibiotic resistance protein. The double-stranded circular DNA vector may be, for example, a plasmid DNA vector, a phagemid DNA vector, a cosmid DNA vector, or a bacmid DNA vector. The double-stranded circular DNA vector may also be a shuttle DNA vector that can replicate between bacteria such as E. coli and mammalian cells.

[0017] As used herein, the term "origin of replication (ori)" refers to the position where DNA replication starts. The origin of replication is also called the replication origin.

[0018] As used herein, the term "replication origin region" refers to a sequence region capable of initiating DNA replication initiation, such as a sequence region that enables DNA replication in a double-stranded circular DNA vector such as a plasmid DNA.

[0019] Various base sequences are known as replication origin regions for double-stranded circular DNA vectors that can replicate in E. coli. Plasmid DNA vectors that can replicate in E. coli are classified into ColE1-type plasmid DNA (e.g., pUC-type plasmid DNA and pET-type plasmid DNA), p15A-type plasmid DNA (e.g., pACYC-type plasmid DNA), pSC101-type plasmid DNA (e.g., pSC101-type plasmid DNA), R6K-type plasmid DNA, etc., based on the replication origin, the base sequence of the replication origin region, and / or the replication mechanism.

[0020] As used herein, "ColE1-type plasmid DNA" is a general term for plasmid DNA having a replication origin derived from ColE1 plasmid DNA or a replication origin having a sequence similar thereto. Specific examples of ColE1-type plasmid DNA include ColE1 plasmid DNA, pBR322 plasmid DNA, and the pUC-type and pET-type plasmid DNAs described below. The copy number per cell of ColE1 plasmid DNA and pBR322 plasmid DNA is known to be approximately 15 to 20.

[0021] In ColE1-type plasmid DNA replication, a sequence from a promoter approximately 550 bases upstream of the replication origin to approximately 150 bases downstream of the replication origin is transcribed to generate a single-stranded RNA called an RNA II precursor. The sequence at the 3' end of the RNA II precursor forms a DNA / RNA hybrid duplex with the DNA of the replication initiation region, which is cleaved by RNase H at the replication origin to generate mature RNA II (primer RNA) from the RNA II precursor. Using mature RNA II as a primer, synthesis of the leading strand of the plasmid DNA is initiated from the replication origin. In ColE1-type plasmid DNA, the initiation site of leading strand synthesis in the above-mentioned replication mechanism corresponds to the replication origin.

[0022] In the replication origin region of ColE1-type plasmid DNA, a single-stranded RNA called RNAI is transcribed in the opposite direction to RNAII, starting approximately 455 bases upstream of the replication origin. RNAI is a 108-base antisense RNA of RNAII, and its sequence is completely complementary to the 5'-terminal sequence of the RNAII precursor. RNAI forms an RNA / RNA hybrid with the RNAII precursor, thereby suppressing the production of mature RNAII and functioning as a negative regulator of plasmid DNA replication (leading strand synthesis initiation).

[0023] Furthermore, in the replication origin region of ColE1-type plasmid DNA, such as ColE1 plasmid DNA and pBR322 plasmid DNA, there is a sequence encoding a protein called Rom, approximately 400 bases downstream from the replication origin. The Rom protein forms a homodimer and binds to RNAI and RNAII to form a stable RNAI / RNAII precursor hybrid, and is known to function as a factor that enhances the inhibitory effect of RNAI on plasmid DNA replication.

[0024] As used herein, "pUC-type plasmid DNA" refers to a plasmid DNA vector in which the replication origin of ColE1-type plasmid DNA lacks the sequence encoding the Rom protein and has a replication origin modified to have a single point mutation (a mutation from cytosine (C) to thymine (T)) one base upstream of the RNAI transcription start site in the RNAII coding region (referred to as a "pUC-type replication origin" herein). pUC19 plasmid DNA, which corresponds to pUC-type plasmid DNA, is known as a multicopy plasmid DNA, with a copy number of several hundred or more per cell.

[0025] In ColE1-type plasmid DNA, the replication origin region corresponds to the promoter that controls the transcription of the RNAII precursor (hereinafter referred to as the "RNAII promoter") and the sequence region that includes the region encoding the RNAII precursor. Examples include the region in the RNAII promoter that includes from -35 Box to the sequence encoding the RNAII precursor or the transcription termination sequence of the RNAII precursor, and in some cases the region that includes the sequence encoding the Rom protein. Specific examples of replication origin regions in ColE1-type plasmid DNA include the replication origin region of ColE1 plasmid DNA consisting of the nucleotide sequence shown in SEQ ID NO:2 and the replication origin region of pUC19 plasmid DNA consisting of the nucleotide sequence shown in SEQ ID NO:3.

[0026] As used herein, the term "-35 Box" refers to a consensus sequence consisting of the nucleotide sequence TTGAGA (SEQ ID NO: 4), or a nucleotide sequence similar thereto, which is found approximately 35 nucleotides upstream from the transcription start site in the promoter sequence of E. coli. Unless otherwise specified, the term "-35 Box" refers to the -35 Box in the RNAII promoter. In a previous report (Rouches M.V., et al., Nat Commun., 2022; 13(1):3908.), it was found that mutations were introduced into the -35 Box sequence in the RNAII promoter, and that the function as a replication origin region was maintained even when mutations were introduced into four nucleotides of the six-nucleotide consensus sequence (TTGAGA; SEQ ID NO: 4).

[0027] As used herein, the term "-10 Box" refers to a consensus sequence consisting of the base sequence TAATCT (SEQ ID NO: 5), or a base sequence similar thereto, found approximately 10 bases upstream from the transcription start site in the promoter sequence of E. coli. Unless otherwise specified, the term "-10 Box" refers to the -10 Box in the RNAII promoter. In a previous report (Rouches M.V., et al., Nat Commun., 2022; 13(1):3908.), it was found that mutations were introduced into the -10 Box sequence in the RNAII promoter, and that the function as a replication initiation region was maintained even when mutations were introduced into four bases of the six-base consensus sequence (TAATCT; SEQ ID NO: 5).

[0028] In this specification, the term "plurality" refers to an integer of 2 or more, for example, an integer of 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.

[0029] As used herein, the "identity (base identity)" of a base sequence refers to the percentage (%) of the number of matching bases in the total number of bases when two base sequences to be compared are aligned by inserting appropriate gaps into one or both of them as necessary to maximize the number of matching bases. Identity can be determined using methods well known to those skilled in the art, sequence analysis software, etc. Examples include the blastn program of the BLAST algorithm and the fasta program of the FASTA algorithm.

[0030] 1-3. Configuration The double-stranded circular DNA vector of the present invention contains a replication origin region. In the replication origin region of the double-stranded circular DNA vector of the present invention (hereinafter referred to as the "replication origin region of the present invention"), at least the base corresponding to position 22 of the base sequence shown in SEQ ID NO: 1 below is an adenine base.

[0031] As used herein, "a base corresponding to position 22 in the base sequence shown in SEQ ID NO: 1" refers to a base in a replication origin region that corresponds to the cytosine base (C base) at position 22 in the base sequence shown in SEQ ID NO: 1 when comparing an arbitrary replication origin region with the base sequence shown in SEQ ID NO: 1. For example, when two base sequences are aligned by inserting appropriate gaps into one or both as necessary to maximize the number of matching bases, the base corresponds to the cytosine base (C base) at position 22 in the base sequence shown in SEQ ID NO: 1.

[0032] In the replication origin region of the double-stranded circular DNA vector of the present invention, the base corresponding to position 22 in the base sequence shown in SEQ ID NO: 1 is an adenine base (A base), and the bases corresponding to positions 20, 21, and 23 in the base sequence shown in SEQ ID NO: 1 may each independently be an adenine base (A base), a guanine base (G base), a thymine base (T base), or a cytosine base (C base).

[0033] For example, in the replication origin region of the present invention, the base corresponding to position 23 of the base sequence shown in SEQ ID NO: 1 is a cytosine base (C base), a guanine base (G base), or a thymine base (T base). Preferably, the base corresponding to position 23 of the base sequence shown in SEQ ID NO: 1 is a guanine base (G base) or a thymine base (T base).

[0034] In one embodiment, in the replication origin region of the present invention, the base corresponding to position 20 of the base sequence shown in SEQ ID NO: 1 is a cytosine base (C base) or a guanine base (G base).

[0035] In one embodiment, in the replication origin region of the present invention, the sequence corresponding to positions 20 to 23 of the base sequence shown in SEQ ID NO: 1 is SNAK. In the sequence, "S" represents either a cytosine base (C base) or a guanine base (G base); "N" represents either an adenine base (A base), a guanine base (G base), a thymine base (T base), or a cytosine base (C base); and "K" represents either a guanine base (G base) or a thymine base (T base).

[0036] In a further embodiment, in the replication origin region of the present invention, the sequence corresponding to positions 20 to 23 of the base sequence shown in SEQ ID NO: 1 is NGAN, where "N" represents any of adenine (A base), guanine (G base), thymine (T base), and cytosine (C base).

[0037] In a further embodiment, in the replication origin region of the present invention, the sequence corresponding to positions 20 to 23 of the base sequence shown in SEQ ID NO: 1 is NGAB. In the sequence, "N" represents any of adenine base (A base), guanine base (G base), thymine base (T base), and cytosine base (C base), and "B" represents any of cytosine base (C base), guanine base (G base), and thymine base (T base).

[0038] In a further embodiment, in the replication origin region of the present invention, the sequence corresponding to positions 20 to 23 of the base sequence shown in SEQ ID NO: 1 is NGAK. In the sequence, "N" represents any of the bases adenine (A base), guanine (G base), thymine (T base), and cytosine (C base), and "K" represents any of the bases guanine (G base) and thymine (T base).

[0039] In a further embodiment, in the replication origin region of the present invention, the sequence corresponding to positions 20 to 23 of the base sequence shown in SEQ ID NO: 1 is SGAK. In the sequence, "S" represents either a cytosine base (C base) or a guanine base (G base), and "K" represents either a guanine base (G base) or a thymine base (T base).

[0040] In a further embodiment, in the replication origin region of the present invention, the sequence corresponding to positions 20 to 23 of the nucleotide sequence shown in SEQ ID NO: 1 is GGAT.

[0041] In a further embodiment, in the replication origin region of the present invention, the sequence corresponding to positions 20 to 23 of the nucleotide sequence shown in SEQ ID NO: 1 is CGAG.

[0042] In one embodiment, in the replication origin region of the present invention, the sequences corresponding to positions 7 to 19 and positions 30 to 36 of the base sequence shown in SEQ ID NO: 1 are identical to the sequences consisting of positions 7 to 19 and positions 30 to 36 of the base sequence shown in SEQ ID NO: 1.

[0043] In the replication origin region of the present invention, there are no particular limitations on bases other than the base corresponding to position 22 in the base sequence shown in SEQ ID NO: 1. For example, the replication origin region of the present invention is a replication origin region derived from any plasmid DNA vector that can replicate in E. coli (e.g., ColE1-type plasmid DNA, p15A-type plasmid DNA, pSC101-type plasmid DNA, or R6K-type plasmid DNA), and comprises or consists of a replication origin region in which the base corresponding to position 22 in the base sequence shown in SEQ ID NO: 1 is an adenine base. More specifically, examples of such origins include a replication origin region in which the base corresponding to position 22 in the base sequence shown in SEQ ID NO: 1 is an adenine base in the replication origin region of any of ColE1-type plasmid DNA (e.g., pUC-type plasmid DNA or pET-type plasmid DNA), p15A-type plasmid DNA (e.g., pACYC-type plasmid DNA), pSC101-type plasmid DNA (e.g., pSC101-type plasmid DNA), or R6K-type plasmid DNA; a replication origin region in which the base corresponding to position 22 in the base sequence shown in SEQ ID NO: 1 is an adenine base in a base sequence in which one or more bases have been deleted, substituted, or added in any of the above-mentioned replication origin regions; and a replication origin region in which the base corresponding to position 22 in the base sequence shown in SEQ ID NO: 1 is an adenine base in a base sequence that has 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 99.5% or more identity to any of the above-mentioned replication origin regions.

[0044] Furthermore, the base sequence TTGAGA (SEQ ID NO: 4) consisting of positions 1 to 6 of the base sequence shown in SEQ ID NO: 1 is a consensus sequence called the -35 box. In the replication origin region of the present invention, the sequence corresponding to positions 1 to 6 of the base sequence shown in SEQ ID NO: 1 may be identical to this -35 box sequence (i.e., the base sequence TTGAGA; SEQ ID NO: 4), or may consist of a sequence in which one or more, for example, 1 to 4, 1 to 3, or 1 or 2 bases have been deleted, substituted, or added in this -35 box sequence (i.e., the base sequence TTGAGA; SEQ ID NO: 4). Furthermore, the base sequence TAATCT (SEQ ID NO: 5) consisting of positions 24 to 29 of the base sequence shown in SEQ ID NO: 1 is a consensus sequence called -10 box. In the replication origin region of the present invention, the sequence corresponding to positions 24 to 29 of the base sequence shown in SEQ ID NO: 1 may be identical to this -10 box sequence (i.e., the base sequence TAATCT; SEQ ID NO: 5), or may consist of a sequence in which one or more, for example, 1 to 4, 1 to 3, or 1 or 2 bases have been deleted, substituted, or added in this -10 box sequence (i.e., the base sequence TAATCT; SEQ ID NO: 5).

[0045] In a further embodiment, the replication origin region of the present invention comprises or consists of, excluding the base corresponding to position 22 of the base sequence shown in SEQ ID NO: 1, (a) the base sequence shown in SEQ ID NO: 2 or 3, (b) a base sequence in which one or more (e.g., 1 to 4, 1 to 3, or 1 or 2) bases have been deleted, substituted, or added in the base sequence shown in SEQ ID NO: 2 or 3, or (c) a base sequence having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 99.5% or more identity to the base sequence shown in SEQ ID NO: 2 or 3.

[0046] The double-stranded circular DNA vector of the present invention can contain any base sequence other than the replication origin region in addition to any of the replication origin regions described above. The sequence other than the replication origin region is not particularly limited and can be selected appropriately depending on the purpose. Examples include sequences necessary for the maintenance and replication of DNA vectors within cells, such as elements contained in common plasmid DNA vectors, such as genes encoding antibiotic resistance proteins, sequences encoding activator or repressor proteins, such as LacI and AraC genes, cloning sites, and overlap regions for use with Clontech's In-Fusion cloning system or New England Biolabs' Gibson Assembly system. Furthermore, the double-stranded circular DNA vector of the present invention may contain a protelomerase gene and / or endonuclease gene, a protelomerase recognition sequence and / or an endonuclease recognition sequence that can be used to produce linear covalently closed DNA.

[0047] In one embodiment, the double-stranded circular DNA vector of the present invention contains a nucleic acid sequence of interest. As used herein, the term "nucleic acid sequence of interest" is not particularly limited as long as it contains two or more nucleotides. More specifically, the nucleic acid sequence of interest is not limited to a sequence encoding a protein or a fragment thereof, or RNA. It may be, for example, a sequence that can function as a nucleic acid drug, or a sequence that can be used to introduce a nucleic acid sequence of interest. Specific examples of nucleic acid sequences include, but are not limited to, gene sequences encoding proteins or fragments thereof (e.g., mRNA or cDNA); nucleic acid sequences encoding non-coding RNA such as siRNA, shRNA, miRNA, lncRNA, tRNA, or ribozymes; or nucleic acid drugs such as antisense nucleic acids, aptamers, or bait nucleic acids. Examples of sequences that can be used to introduce a nucleic acid sequence of interest include sequences that can be recognized / cleaved by nucleases such as restriction enzymes or genome editing enzymes, and multicloning sites containing sequences that can be recognized / cleaved by two or more nucleases.

[0048] As used herein, the term "gene sequence of interest" refers to a gene sequence that encodes a protein of interest or a fragment thereof, or an RNA molecule of interest. The protein of interest or a fragment thereof is not particularly limited and can be selected appropriately depending on the purpose. Examples include polypeptides that constitute viruses, polypeptides produced by animals, plants, fungi, algae, bacteria, viruses, etc., and fragments thereof. These can be used as cell / gene therapy drugs, vaccines, disease therapy drugs, etc. The polypeptides produced by animals, plants, fungi, algae, bacteria, viruses, etc. are not particularly limited and can be selected appropriately depending on the purpose. For example, any enzyme such as phytase, amylase, glucosidase, cellulase, lipase, protease, glutaminase, peptidase, oxidase, lactase, xylanase, trypsin, pectinase, isomerase, etc., antibody-binding proteins such as protein A, protein G, protein L, etc., 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 protein, bispecific antibody, heavy chain (H chain), light chain (L chain), single chain Fv (scFv), sc(Fv) ２ Examples of the antibody fragment include antibodies or antibody fragments such as disulfide-linked Fv (sdFv), diabodies, and antibody-like molecule targeting peptides (microantibodies); conjugates of antibody fragments with compounds other than antibodies or antibody fragments, such as other functional proteins (antibody-like molecules); serum albumins such as human serum albumin; epidermal growth factors such as human epidermal growth factor; insulin; growth hormone; erythropoietin; interferon; blood 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), fibroin, fluorescent proteins, hepatitis B virus surface antigen, and hirudin.

[0049] A nucleic acid sequence of interest, such as a gene sequence of interest, can include a promoter in addition to the nucleic acid sequence of interest (e.g., a sequence encoding a protein of interest or a fragment thereof), and may also include further components such as introns, enhancers, terminators, and / or polyA signals, as needed.

[0050] In one embodiment, the double-stranded circular DNA vector of the present invention comprises an antibiotic resistance gene that confers resistance to an antibiotic. Examples of genes encoding antibiotic resistance proteins include a β-lactamase gene (sometimes referred to as an "ampR gene") that confers resistance to ampicillin, an aminoglycoside 3' phosphotransferase gene (sometimes referred to as an "kanR gene") that confers resistance to kanamycin, a tetracycline efflux transporter gene that confers resistance to tetracycline, and a CAT (chloramphenicol acetyltransferase) gene that confers resistance to chloramphenicol.

[0051] In one embodiment, the double-stranded circular DNA vector of the present invention comprises an auxotrophy-complementing gene capable of complementing the growth of an auxotrophic strain. The auxotrophic strain herein is not limited to any particular strain, as long as it does not grow or grows at a low rate in a medium that does not sufficiently contain a particular medium component. Specific examples of auxotrophy include amino acid auxotrophy, vitamin auxotrophy, and nucleic acid auxotrophy. Examples of amino acid auxotrophy include methionine auxotrophy, leucine auxotrophy, cysteine ​​auxotrophy, histidine auxotrophy, and arginine auxotrophy. Examples of vitamin auxotrophy include biotin auxotrophy and thiamine auxotrophy. Examples of nucleic acid auxotrophy include uracil auxotrophy, adenine auxotrophy, and thymine auxotrophy.

[0052] In one embodiment, the double-stranded circular DNA vector of the present invention includes a nucleic acid sequence encoding a non-translated or non-coding RNA as a selection marker (hereinafter referred to as an "RNA selection marker"). The non-translated or non-coding RNA transcribed from the nucleic acid sequence regulates the expression of a target gene from the chromosome, thereby enabling the selection of transformed cells. Specific examples of RNA selection markers include antisense RNA, siRNA, suppressor tRNA, RNA-OUT (WO 2008 / 153733), RNA II precursor, mature RNA II, and RNA I (antisense RNA of RNA II).

[0053] The size of the double-stranded circular DNA vector of the present invention is not limited as long as it can be maintained and / or replicated in a host cell, and may be, for example, 1,000 base pairs or more, 2,000 base pairs or more, 5,000 base pairs or more, 10,000 base pairs or more, 12,000 base pairs or more, 15,000 base pairs or more, or 20,000 base pairs or more, and / or 30,000 base pairs or less, 25,000 base pairs or less, 20,000 base pairs or less, 15,000 base pairs or less, 12,000 base pairs or less, or 10,000 base pairs or less. Exemplary ranges include 5,000 to 30,000 base pairs, 10,000 to 25,000 base pairs, 12,000 to 20,000 base pairs, or 15,000 to 18,000 base pairs.

[0054] The present invention also provides a nucleic acid fragment obtainable by cleaving the double-stranded circular DNA vector of the present invention with an endonuclease such as an exonuclease or a restriction enzyme, etc. Also provided is a nucleic acid obtainable by amplifying the double-stranded circular DNA vector of the present invention as a template by a nucleic acid amplification method such as PCR, which comprises the replication origin region of the present invention.

[0055] The present invention also provides a transformation composition. The transformation composition of the present invention contains any of the above-described double-stranded circular DNA vectors of the present invention as an essential component, and may contain a solvent and / or additives as optional components. The solvent may be, for example, water, an aqueous solution, or an organic solvent. The aqueous solution may be, for example, physiological saline, phosphate buffer, sodium acetate buffer, Tris buffer, etc. These are preferably sterilized. Examples of additives include chelating agents, pH adjusters, suspending agents, surfactants, stabilizers, excipients, preservatives, diluents, isotonicity agents, buffers, solubilizers, etc. Chelating agents such as EDTA and EGTA are particularly preferred because they can inactivate trace amounts of nucleases when they are present.

[0056] The present invention also provides a transformation kit, which contains any of the above-described double-stranded circular DNA vectors of the present invention as an essential component, and can optionally contain other reagents required for transformation, such as buffers, transformation reagents, antibiotics used to select transformed cells, and / or instructions describing the transformation method.

[0057] 1-4. Effects The double-stranded circular DNA vector of the present invention has extremely high replication efficiency in host cells. As a result, a large number of plasmid DNA copies are retained per cell, and the yield of plasmid DNA is also high. For example, compared to a plasmid DNA vector containing a replication origin in which the base corresponding to position 22 of the base sequence shown in SEQ ID NO: 1 is a base other than adenine, such as cytosine, guanine, or thymine, the replication efficiency of the double-stranded circular DNA vector of the present invention is high, and a large number of plasmid DNA copies are retained per cell.

[0058] 2. Transformed Cells 2-1. Overview A second aspect of the present invention is a transformed cell. The transformed cell of this aspect contains the double-stranded circular DNA vector of the first aspect and is capable of efficiently producing the double-stranded circular DNA vector.

[0059] 2-2. Configuration The transformed cell of this embodiment contains the double-stranded circular DNA vector of the first embodiment as an essential component. The type of double-stranded circular DNA vector of the first embodiment contained in the transformed cell of this embodiment is not limited, and may be one type or two or more types. Furthermore, the transformed cell of this embodiment may additionally contain a double-stranded circular DNA vector other than the double-stranded circular DNA vector of the first embodiment.

[0060] In this embodiment, the type of transformed cell is not limited. The transformed cell may be any cell into which the double-stranded circular DNA vector of the first embodiment can be introduced and which can maintain and / or replicate the double-stranded circular DNA vector of the first embodiment. Examples of transformed cells include archaea, bacteria, and eukaryotic cells. Bacteria may be Escherichia, lactic acid bacteria, Bacillus, 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, avian cells (e.g., chicken cells), and mammalian cells (e.g., mouse cells, chimpanzee cells, Chinese hamster cells, and human cells).

[0061] Examples of Escherichia include Escherichia coli, more specifically Escherichia coli K-12 strain and Escherichia coli B strain.

[0062] As for E. coli, commercially available E. coli strains or E. coli strains from the Biological Resource Center can be used, and strains such as JM109, DH5, DH5α, DH10B, NEB10β, HST08, HST16CR, HB101, W3110, MG1655, BL21, and BL21 DE3 can also be used. These strains can be obtained from New England Biolabs, Takara Bio, Thermo Fisher Scientific, Toyobo, ATCC (American Type Culture Collection), NBRP (National Bio Resource Project), etc.

[0063] In the present invention, strains derived from the above-mentioned E. coli strains can also be used, such as the methionine-requiring JW3973 strain (available from NBRP), the leucine-requiring JW2806 strain (available from NBRP), the cysteine-requiring JW3582 strain (available from NBRP), and the thiamine- and histidine-requiring ME5305 strain (available from NBRP).

[0064] 3. Amplification Method, Expression Method, and Production Method 3-1. Overview A third aspect of the present invention is a method for amplifying a double-stranded circular DNA vector (hereinafter simply referred to as the "amplification method"), a method for expressing a target nucleic acid or a target protein or a fragment thereof in a transformed cell (hereinafter simply referred to as the "expression method"), a method for producing a target nucleic acid (hereinafter simply referred to as the "nucleic acid production method"), and a method for producing a target protein or a fragment thereof (hereinafter simply referred to as the "protein production method") (hereinafter in this aspect, the amplification method, expression method, nucleic acid production method, and protein production method of the present invention are collectively referred to as the "method of this aspect").

[0065] 3-2. Methods The amplification method and expression method of the present invention include a culture step as an essential step and an introduction step as an optional step. Furthermore, the nucleic acid production method of the present invention includes a culture step and a nucleic acid extraction step as essential steps and an introduction step as an optional step. The protein production method of the present invention includes a culture step and an isolation step as essential steps and an introduction step and / or an expression induction step as optional steps. Each step in the method of this embodiment will be specifically described below.

[0066] (Introduction step) In the method of this embodiment, 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 containing the double-stranded circular DNA vector of the first embodiment.

[0067] The double-stranded circular DNA vector used in the expression method of the present invention contains a nucleic acid sequence constituting a nucleic acid of interest, or contains a gene sequence of interest encoding the protein of interest or a fragment thereof in an expressible state. The protein of interest may be an enzyme, an antibody, or a functional peptide. As used herein, "expressible state" refers to the gene to be expressed being placed under the control of a promoter and downstream of the promoter.

[0068] In one embodiment, this step involves introducing the transformation composition according to the first aspect into a host cell.

[0069] In one embodiment, this step involves introducing the double-stranded circular DNA vector of the first aspect into a host cell using the transformation kit described in the first aspect.

[0070] In this step, the method for introducing the double-stranded circular DNA vector of the first embodiment into a host cell is not particularly limited. For example, a gene transfer method (transformation method) known in the art, such as that described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, may be used. Specific examples include the heat shock method, lipofection, electroporation, microinjection, calcium phosphate method, DEAE-dextran method, introduction using a cationic lipid, introduction using a cationic polymer (e.g., polyethyleneimine (PEI)), introduction using nanoparticles, introduction using a virus, and particle bombardment.

[0071] In this step, host cells into which the double-stranded circular DNA vector of the first aspect has been introduced can be appropriately selected in a medium containing an antibiotic, based on the gene encoding an antibiotic resistance protein in the double-stranded circular DNA vector of the first aspect. Furthermore, host cells into which the double-stranded circular DNA vector of the first aspect has been introduced can be appropriately selected in a medium that does not sufficiently contain a medium component corresponding to the auxotrophy-complementing gene in the double-stranded circular DNA vector of the first aspect. Furthermore, host cells into which the double-stranded circular DNA vector of the first aspect has been introduced can be appropriately selected in this step based on the RNA selection marker in the double-stranded circular DNA vector of the first aspect.

[0072] (Culturing step) In the method of this embodiment, the "culturing step" refers to a step of culturing transformed cells into which the double-stranded circular DNA vector of the first embodiment has been introduced. In the method of this embodiment, the purpose of this step is to increase the yield of the double-stranded circular DNA vector by growing the transformed cells. In addition, in the expression method of the present invention, the purpose is to increase the copy number of the double-stranded circular DNA vector and to express a nucleic acid or protein of interest, or a fragment thereof, in the transformed cells.

[0073] The method for culturing the transformed cells in this step is not particularly limited and can be appropriately selected depending on the type of transformed cells. For example, a culture method known in the art, such as that described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc., may be used.

[0074] The medium for culturing the transformed cells in this step may be either a liquid medium or a solid medium. The medium may contain one or more components selected from the group consisting of enzymatic hydrolysates of proteins such as peptone and tryptone, biological extracts such as potato dextrose and yeast extract, amino acids such as glutamic acid or salts thereof, sugars such as glucose, glycerol, and sucrose, and inorganic salts such as sodium chloride, magnesium chloride, and potassium dihydrogen phosphate. Specific media and compositions include LB medium (tryptone, yeast extract, sodium chloride), YPG medium (yeast extract, peptone, glucose), PD medium (potato dextrose), and TB medium (tryptone, yeast extract, dipotassium hydrogen phosphate, potassium dihydrogen phosphate).

[0075] When the double-stranded circular DNA vector of the first embodiment contains a gene encoding an antibiotic resistance protein, this step can be carried out in the presence of an antibiotic corresponding to the gene, such as ampicillin, carbenicillin, kanamycin, tetracycline, or chloramphenicol.

[0076] The culture conditions for this step can be appropriately selected depending on the type of transformed cells. For example, the culture can be performed at 20 to 42°C, 25 to 40°C, 30 to 38°C, or 35 to 37°C.

[0077] The culture time in this step is not limited as long as a sufficient amount of double-stranded circular DNA vector can be obtained, and may be, for example, 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.

[0078] The pH of the medium used in this step is not particularly limited as long as it allows the transformed cells to grow. For example, the medium may have a pH of 3 or higher, 4 or higher, 5 or higher, 6 or higher, 7 or higher, 7.5 or higher, 8 or higher, 8.5 or higher, 9 or higher, or 9.5 or higher, and / or a pH of 11 or lower, 10.5 or lower, 10 or lower, 9.5 or lower, 9 or lower, 8.5 or lower, 8 or lower, or 7.5 or lower, e.g., a pH of 3 to 11, 4 to 10, 5 to 9, or 7 to 9, or a pH of 7.5 to 10.5, 8 to 10, or 8.5 to 9.5.

[0079] (Nucleic Acid Extraction Step) In the nucleic acid production method of the present invention, the "nucleic acid extraction step" is a step of extracting the target nucleic acid from the transformed cells after the culture step.

[0080] In this step, nucleic acids such as DNA may be extracted using conventional methods known in the art. For example, they may 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. Alternatively, they may be prepared using a commercially available DNA extraction kit. An example of a commercially available DNA extraction kit is the QIA prep Spin Miniprep Kit (manufactured by QIAGEN).

[0081] (Expression Induction Step) In the protein production method of the present invention, the "expression induction step" is a step of inducing the expression of a target protein or a fragment thereof in the transformed cells after the culture step.

[0082] In this step, the method for inducing the expression of the target protein or a fragment thereof is not particularly limited and can be appropriately selected depending on the type of promoter capable of regulating expression.

[0083] In one embodiment, in this step, an expression inducer is added to the medium after the culturing step. When an arabinose-inducible promoter, an IPTG-inducible promoter, or a rhamnose-inducible promoter is used as the promoter for driving the expression of a gene encoding a target protein or a fragment thereof, an example of a method is to add arabinose, IPTG, or rhamnose, respectively, as an expression inducer to the medium containing the transformed cells after the culturing step.

[0084] 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 sufficiently induces expression of the target protein or a fragment thereof, and may be, for example, 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.

[0085] The temperature at which the transformed cells are cultured in the presence of an 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, 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.

[0086] In another embodiment, if the expression-controllable promoter is a heat-inducible promoter or a cold-inducible promoter, a heat shock or cold shock may be applied. The conditions for the heat shock or cold shock are not particularly limited, as long as the temperature and time are such that the expression of the target protein or a fragment thereof is sufficiently induced. For example, the heat shock may be performed at 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 for 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, e.g., 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. Furthermore, for example, in the case of cold shock, temperature conditions of 4°C to 35°C, 10°C to 30°C, 11°C to 25°C, 12°C to 20°C, or 13°C to 17°C and treatment times of 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 can be used.

[0087] This step allows the target protein or a fragment thereof to be expressed in the transformed cells.

[0088] (Isolation Step) In the protein production method of the present invention, the "isolation step" is a step of isolating the target protein or a fragment thereof from the transformed cells and / or culture supernatant after the culture step.

[0089] The method for isolating the target protein or a fragment thereof in this step is not particularly limited and can be selected appropriately, for example, purification using a commercially available kit, gel extraction, cation chromatography, anion chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, or separation using a filter or ultrafiltration membrane.

[0090] 3-3. Effects The amplification method of the present invention enables the mass amplification of double-stranded circular DNA vectors. Furthermore, the nucleic acid production method and protein production method of the present invention enable the mass production of a target nucleic acid and a target protein or fragments thereof, respectively, based on the efficient amplification of double-stranded circular DNA vectors in transformed cells.

[0091] When the double-stranded circular DNA vector used in the expression method of the present invention contains a target gene sequence encoding an enzyme or an enzyme fragment in an expressible state, the enzyme or enzyme fragment that has been mass-expressed in the bacterial cells can be reacted with a substrate that is a low-molecular-weight compound in the bacterial cells and / or in the culture medium to produce an additional low-molecular-weight compound as a reaction product.

[0092] 4. Method for Increasing Replication Efficiency 4-1. Overview A fourth aspect of the present invention is a method for increasing the replication efficiency of a double-stranded circular DNA vector (hereinafter referred to as the "method for increasing replication efficiency"). The method of this aspect can increase the replication efficiency of both pUC-type plasmid DNA and double-stranded circular DNA vectors other than pUC-type plasmid DNA.

[0093] The method of this embodiment includes a substitution step as an essential step. The substitution step has different configurations depending on whether the target is an arbitrary double-stranded circular DNA vector or a double-stranded circular DNA vector other than pUC-type plasmid DNA. Therefore, each case will be described separately below.

[0094] When any double-stranded circular DNA vector is the target, the substitution step involves substituting an adenine base for the base corresponding to position 22 of the base sequence shown in SEQ ID NO: 1 below in the replication origin region of the double-stranded circular DNA vector. In this case, the type of double-stranded circular DNA vector to be used in this step is not limited, and may be, for example, ColE1 type plasmid DNA such as pUC type plasmid DNA or pET type plasmid DNA.

[0095] When a double-stranded circular DNA vector other than pUC-type plasmid DNA is used as the target, the substitution step involves substituting the replication origin of the double-stranded circular DNA vector other than pUC-type plasmid DNA with the replication origin derived from pUC-type plasmid DNA, in which at least the base corresponding to position 22 of the base sequence shown in SEQ ID NO: 1 below is an adenine base. In this case, the type of double-stranded circular DNA vector targeted in this step is not limited as long as it is other than pUC-type plasmid DNA, and may be, for example, R6K-type plasmid DNA.

[0096] 4-3. Effects The replication efficiency enhancing method of the present invention can increase the replication efficiency in host cells for pUC-type plasmid DNA or double-stranded circular DNA vectors other than pUC-type plasmid DNA. The replication efficiency enhancing method of the present invention can increase the plasmid DNA yield by improving the number of plasmid DNA copies retained per cell for any plasmid DNA. The replication efficiency enhancing method of the present invention can increase the plasmid DNA yield, for example, compared to the double-stranded circular DNA vector before the substitution step.

[0097] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0098] Example 1: Introduction of a mutation at position 22 in a pUC-type replication origin region (Purpose) In ColE1-type plasmid DNA, attempts have been made to increase the number of plasmid DNA copies maintained per cell by introducing mutations into the nucleotide sequence encoding the RNAII precursor or by introducing mutations into the -35 Box or -10 Box sequence in the promoter sequence controlling the transcription of the RNAII precursor (Rouches M.V., et al., Nat Commun., 2022;13(1):3908.). On the other hand, no studies have been conducted to date on sequences other than the -35 Box and -10 Box in the promoter sequence controlling the transcription of the RNAII precursor.

[0099] In this example, the region between −35 Box and −10 Box in the promoter sequence that controls the transcription of RNA II precursor was targeted for mutagenesis, and the cytosine base (C base) corresponding to position 22 of the base sequence shown in SEQ ID NO: 1 was substituted with other bases (guanine base (G base), thymine base (T base), or adenine base (A base)), and the effect of this substitution on the yield of plasmid DNA was evaluated.

[0100] (Methods and Results) (1) Mutation Introduction into the Replication Origin pUC19 plasmid DNA (total length 2686 bp) is a plasmid DNA vector for cloning in Escherichia coli, containing the pUC ori (SEQ ID NO: 3) of the pUC-type replication origin and an ampicillin resistance gene (Figure 1). pUC19 plasmid DNA was purchased from New England Biolabs, and mutant plasmid DNAs were prepared by substituting the cytosine base corresponding to position 22 of the nucleotide sequence shown in SEQ ID NO: 1 below (hereinafter referred to as the "22C base") with a guanine base, a thymine base, or an adenine base (hereinafter referred to as the "C22G mutant," "C22T mutant," and "C22A mutant," respectively) in the replication origin (Figure 2A).

[0101]

[0102] Specifically, a nucleic acid fragment was prepared by PCR using pUC19 plasmid DNA as a template and primer 1 (SEQ ID NO: 7) and primer 2 (SEQ ID NO: 8). In the sequence shown in SEQ ID NO: 7, "d" represents any of the following bases: guanine base (G base), thymine base (T base), or adenine base (A base). Next, the overlapping regions at the ends of the obtained nucleic acid fragments were used to circularize them using the Gibson Assembly system from New England Biolabs, to prepare the C22G mutant, C22T mutant, and C22A mutant as the desired mutant plasmid DNA.

[0103] (2) Transformation E. coli was transformed with pUC19 plasmid DNA containing 22C bases, as well as the plasmid DNAs of the C22G mutant, C22T mutant, and C22A mutant. Specifically, 25 μL of competent cell solution of E. coli DH10B strain was mixed with a solution containing each plasmid DNA and allowed to stand on ice for 30 minutes. After standing for 30 minutes, the mixture was heat-treated at 42°C for 45 seconds and then allowed to stand on ice for 2 minutes. Next, 225 μL of SOC medium was added and spread on LB agar medium (1% tryptone, 0.5% dry yeast extract, 1% sodium chloride, 0.005% carbenicillin disodium (Nacalai Tesque)). Strains that grew after static culture at 37°C for 1 day were selected, and E. coli containing each plasmid DNA was obtained.

[0104] (3) Cultivation and Plasmid DNA Extraction The transformed E. coli was inoculated into 2 mL of Plusgrow II medium (4% Plusgrow II (Nacalai Tesque), 0.005% carbenicillin disodium), and cultured with shaking at 37°C for 16 hours. Then, 500 μL of the culture medium was centrifuged to recover bacterial cells. Plasmid DNA was extracted from the recovered bacterial cells using a QIA prep Spin Miniprep Kit (QIAGEN).

[0105] (4) Evaluation of Plasmid DNA Yield The plasmid DNA yield and specific plasmid DNA yield obtained from 2 mL of culture solution were calculated based on measurements using Nanodrop (Thermo Fisher Scientific). The results are shown in columns 4 and 5 of Table 1 below.

[0106]

[0107] In the replication origin region of pUC19 plasmid DNA, the C22G mutant or C22T mutant, in which the 22C base in the sequence region between -35 Box and -10 Box was substituted with a guanine base or a thymine base, decreased the plasmid DNA yield and specific plasmid DNA yield. In contrast, the C22A mutant, in which the 22C base was substituted with an adenine base, significantly increased the plasmid DNA yield and specific plasmid DNA yield.

[0108] Example 2: Introduction of mutations at positions 20 to 23 in the pUC-type replication origin region (Objective) In the replication origin region of pUC19 plasmid DNA, the 22C base was substituted with an adenine base, and the two bases located on the 5' side and one base located on the 3' side were further substituted with various bases to evaluate the effect on the yield of plasmid DNA.

[0109] (Methods and Results) (1) Introduction of mutations into the replication origin region Mutant plasmid DNA was prepared by substituting the sequence corresponding to positions 20 to 23 of the nucleotide sequence shown in SEQ ID NO: 1 below (hereinafter referred to as the "CGCG sequence") with various sequences containing an adenine base corresponding to position 22 in the replication origin region of pUC19 plasmid DNA (Figure 2B).

[0110]

[0111] Specifically, in order to replace the 22C base in the replication origin region of pUC19 plasmid DNA with an adenine base and further replace the two bases located on the 5' side and the two bases located on the 3' side with various bases, nucleic acid fragments were prepared by PCR using pUC19 plasmid DNA as a template and primer 3 (SEQ ID NO: 9) and primer 2 (SEQ ID NO: 8). In the sequence shown in SEQ ID NO: 9, "n" represents any of the following bases: adenine base (A base), guanine base (G base), thymine base (T base), or cytosine base (C base). Next, the overlapping regions at the ends of the obtained nucleic acid fragments were circularized using the Gibson Assembly system from New England Biolabs, to prepare a mutant plasmid DNA library containing various base combinations at the position corresponding to "n" in the sequence shown in SEQ ID NO: 9 (hereinafter referred to as the "first-generation mutant plasmid DNA library").

[0112] (2) Transformation E. coli was transformed with the first-generation mutant plasmid DNA library. Specifically, 25 μL of competent cell solution of E. coli DH10B strain was mixed with a solution containing the first-generation mutant plasmid DNA library and allowed to stand on ice for 30 minutes. After standing for 30 minutes, the mixture was heat-treated at 42 ° C for 45 seconds and then allowed to stand on ice for 2 minutes. Next, 225 μL of SOC medium was added, and inoculated into 100 mL of Plusgrow II medium (4% Plusgrow II, 0.005% carbenicillin disodium), which was then cultured with shaking at 37 ° C for 16 hours. The culture medium was then centrifuged to recover the bacterial cells. DNA was extracted from the recovered bacterial cells using a QIA prep Spin Miniprep Kit (manufactured by QIAGEN) to obtain a second-generation mutant plasmid DNA library. Next, the second-generation mutant plasmid DNA library was used to transform E. coli DH10B strain, followed by culturing, and DNA was extracted again to obtain a third-generation mutant plasmid DNA library. Next, the third-generation mutant plasmid DNA library was used to transform E. coli DH10B strain, followed by culturing, and DNA was extracted again to obtain a fourth-generation mutant plasmid DNA library. This resulted in a fourth-generation mutant plasmid DNA library enriched with mutant plasmid DNA and having a high plasmid DNA yield.

[0113] (3) Nucleotide Sequence Analysis The fourth-generation mutant plasmid DNA library was randomly cloned, and each clone was subjected to sequence analysis. As a result, in all mutant plasmid DNAs subjected to sequence analysis, the base corresponding to position 24 of the base sequence shown in SEQ ID NO: 1 was a thymine base (T base). Furthermore, the base corresponding to position 23 of the base sequence shown in SEQ ID NO: 1 was a cytosine base (C base), a guanine base (G base), or a thymine base (T base). Details of the sequences corresponding to positions 20 to 23 of the base sequence shown in SEQ ID NO: 1 of the mutant plasmid DNAs subjected to sequence analysis are shown in the second column of Table 2.

[0114] (4) Cultivation, Plasmid DNA Extraction, and Evaluation of Plasmid DNA Yield In the same manner as in Example 1, Escherichia coli was transformed with each of the mutant plasmid DNAs cloned in (3) above, and the transformed Escherichia coli was cultured, plasmid DNA was extracted, and the plasmid DNA yield was evaluated.

[0115] The yield of plasmid DNA and the specific yield of plasmid DNA obtained from 2 mL of culture medium were calculated based on measurements using Nanodrop (Thermo Fisher Scientific), and the results are shown in columns 4 and 5 of Table 2 below.

[0116]

[0117] It has been revealed that a GGAT mutant in which the CGCG sequence (the base sequence corresponding to positions 20 to 23 of the base sequence shown in SEQ ID NO: 1) in the sequence region between -35 Box and -10 Box in the replication origin region of pUC19 plasmid DNA is replaced with a GGAT sequence significantly increases the plasmid DNA yield and the specific plasmid DNA yield. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A double-stranded circular DNA vector comprising a replication origin region, wherein in the replication origin region, at least the base corresponding to position 22 of the base sequence shown in SEQ ID NO: 1 below is an adenine base.

2. The double-stranded circular DNA vector according to claim 1, wherein in the replication origin region, the base corresponding to position 23 of the base sequence is a cytosine base, a guanine base, or a thymine base.

3. The double-stranded circular DNA vector according to claim 2, wherein in the replication origin region, the base corresponding to position 23 of the base sequence is a guanine base or a thymine base.

4. The double-stranded circular DNA vector according to claim 3, wherein in the replication origin region, the base corresponding to position 20 of the base sequence is a cytosine base or a guanine base.

5. A double-stranded circular DNA vector according to any one of claims 1 to 4, wherein in the replication origin region, the base corresponding to position 21 of the base sequence is a guanine base.

6. The double-stranded circular DNA vector according to claim 5, wherein, in the replication origin region, the base corresponding to position 20 of the base sequence is a cytosine base, the base corresponding to position 21 of the base sequence is a guanine base, the base corresponding to position 22 of the base sequence is an adenine base, and the base corresponding to position 23 of the base sequence is a guanine base.

7. The double-stranded circular DNA vector according to claim 1, wherein in the replication origin region, the sequence corresponding to the -35 box sequence consisting of positions 1 to 6 of the base sequence is identical to the -35 box sequence or consists of the -35 box sequence with one to four bases deleted, substituted or added, and / or the sequence corresponding to the -10 box sequence consisting of positions 24 to 29 of the base sequence is identical to the -10 box sequence or consists of the -10 box sequence with one to four bases deleted, substituted or added.

8. The double-stranded circular DNA vector of claim 1, comprising a nucleic acid sequence of interest and / or an antibiotic resistance gene that confers resistance to an antibiotic.

9. The double-stranded circular DNA vector of claim 8, wherein the nucleic acid sequence of interest comprises a gene sequence of interest encoding a protein of interest or a fragment thereof.

10. The double-stranded circular DNA vector of claim 1, which is a ColE1-type plasmid DNA.

11. The double-stranded circular DNA vector according to claim 10, wherein the ColE1 type plasmid DNA is a pUC type plasmid DNA or a pET type plasmid DNA.

12. The double-stranded circular DNA vector described in claim 10, wherein the replication initiation region comprises, except for the base corresponding to position 22 of the base sequence, (a) the base sequence shown in SEQ ID NO: 2 or 3, (b) a base sequence in which one or more bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 2 or 3, or (c) a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 2 or 3.

13. A transformed cell comprising the double-stranded circular DNA vector of claim 1.

14. The transformed cell of claim 13, which is a bacterium.

15. A method for amplifying a double-stranded circular DNA vector, comprising a culturing step of culturing the transformed cell according to claim 13 or 14.

16. A method for expressing a target nucleic acid or a target protein or a fragment thereof in a transformed cell, comprising a culture step of culturing a transformed cell containing the double-stranded circular DNA vector described in claim 1, wherein the double-stranded circular DNA vector contains a nucleic acid sequence constituting the target nucleic acid, or contains a target gene sequence encoding the target protein or a fragment thereof in an expressible state.

17. The method of claim 16, wherein the protein of interest is an enzyme, an antibody, or a functional peptide.

18. A method for producing a target nucleic acid, comprising: a culturing step of culturing transformed cells containing the double-stranded circular DNA vector described in claim 1; and a nucleic acid extraction step of extracting the target nucleic acid from the transformed cells after the culturing step, wherein the double-stranded circular DNA vector contains a nucleic acid sequence that constitutes the target nucleic acid.

19. A method for producing a target protein or a fragment thereof, comprising: a culturing step of culturing transformed cells containing the double-stranded circular DNA vector described in claim 1; and an isolation step of isolating the target protein or a fragment thereof from the transformed cells and / or culture supernatant after the culturing step, wherein the double-stranded circular DNA vector contains a target gene sequence encoding the target protein or a fragment thereof in an expressible state.

20. The method according to claim 19, further comprising, after the culturing step and before the isolating step, an expression inducing step of inducing expression of the target protein or a fragment thereof.

21. A method for increasing the replication efficiency of a double-stranded circular DNA vector, comprising the step of substituting an adenine base for the base corresponding to position 22 of the base sequence shown in SEQ ID NO: 1 below in the replication origin region of the double-stranded circular DNA vector.

22. A method for increasing the replication efficiency of a double-stranded circular DNA vector other than pUC-type plasmid DNA, comprising the step of substituting a replication origin region of the double-stranded circular DNA vector other than pUC-type plasmid DNA with a replication origin region derived from pUC-type plasmid DNA, wherein in the replication origin region derived from pUC-type plasmid DNA, at least the base corresponding to position 22 of the base sequence shown in SEQ ID NO: 1 below is an adenine base.

23. The method of claim 22, wherein the double-stranded circular DNA vector other than pUC-type plasmid DNA is R6K-type plasmid DNA.

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