Construct for producing recombinant minivector not including prescribed sequence

Abstract

The present invention provides a method for producing a recombinant minivector not including a prescribed sequence from a construct that includes the prescribed sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, the prescribed sequence being arranged so as to be sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence being a type IIS restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence being a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence. This method includes: 1) a step for cleaving the construct by using a first restriction enzyme and a second restriction enzyme to obtain a cleaved fragment containing the prescribed sequence and the first restriction enzyme recognition sequence and a cleaved fragment not containing the first restriction enzyme recognition sequence or the second restriction enzyme recognition sequence; and 2) a step for subjecting the two types of obtained cleaved fragments to a ligation reaction to obtain a reaction product containing two types of cyclic nucleic acids in which the two types of cleaved fragments are self-ligated.

Description

A construct for creating recombinant minivectors that do not contain a predetermined sequence.

[0001] The present invention broadly relates to constructs, etc., for producing recombinant minivectors that do not contain a predetermined sequence.

[0002] Plasmid DNA is produced in large quantities by growing E. coli transformed with plasmid DNA and then purifying the plasmid DNA from the grown E. coli. When plasmid DNA is produced in large quantities using E. coli in this way, the plasmid DNA contains selection markers and replication origins, but these regions are unnecessary when introducing the gene into mammalian cells.

[0003] As a technique for removing such unnecessary regions, a method is known in which plasmid DNA is amplified in E. coli and then miniaturized by inducing the expression of recombinase and the endonuclease I-SceI (Patent Document 1, Non-Patent Document 1). However, since the locations in which E. coli expressing recombinase and I-SceI can be used are limited, there is a need for a simpler method.

[0004] International Publication No. 2010 / 002470

[0005] Silva-Santos, R. , et al. , “Minicircle Biopharmaceuticals-An Overview of Purification Strategies.” Frontiers in Biotechnology, 2019.

[0006] The problem that this invention aims to solve is to provide a simple and readily usable method for producing recombinant minivectors that do not contain a predetermined sequence.

[0007] As a result of diligent research by the present inventors, it has been found that a recombinant minivector that does not contain the predetermined sequence can be produced from a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first and second restriction enzyme recognition sequences, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence.

[0008] In other words, the present invention encompasses the following inventions: [1] A construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is positioned so as to be sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence, for the production of a recombinant minivector that does not include the predetermined sequence. [2] The construct according to [1], wherein the predetermined sequence comprises a selection marker and an origin of replication. [3] The construct according to [1] or [2], wherein a portion of the other region of the two regions sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the region in which the predetermined sequence is not positioned, is included in the recombinant minivector, and the other region comprises a cleavage site by the first restriction enzyme and a target gene. [4] The construct according to any one of [1] to [3], wherein the region where a predetermined sequence is located among the two regions sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence does not contain a cleavage site by the second restriction enzyme. [5] The construct according to any one of [1] to [4], wherein the recombinant minivector has a superhelical structure. [6] The construct according to any one of [1] to [5], wherein the recombinant minivector is a recombinant minivector for transforming animal cells. [7] The construct according to any one of [1] to [6], wherein the recombinant minivector is introduced into cells with higher efficiency compared to the construct described above. [8] The construct according to any one of [1] to [7], wherein the recombinant minivector has a base length of 1,000 to 5,000. [9] The construct according to any one of [1] to [8], having a base length of 3,000 or more.

[10] A recombinant minivector that does not contain an origin of replication and contains a target gene.

[11] The recombinant minivector according to

[10] , having a base length of 1000 to 5000.

[12] A method for producing a recombinant minivector that does not contain a predetermined sequence from a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence, the method comprising: 1) cleaving the construct with a first restriction enzyme and a second restriction enzyme to obtain a cleavage fragment comprising the predetermined sequence and the first restriction enzyme recognition sequence, and a cleavage fragment that does not contain the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence; and 2) subjecting the two obtained cleavage fragments to a ligation reaction to obtain a reaction product comprising two self-ligated cyclic nucleic acids.

[13] The method according to

[13] , further comprising the step of cleaving the nucleic acid ligated by the two cleavage fragments, or the cyclic nucleic acid self-ligated by the cleavage fragment comprising the predetermined sequence and the first restriction enzyme recognition sequence, contained in the reaction product, with a first restriction enzyme.

[14] The method according to

[12] or

[13] , wherein each of the steps is carried out in a solution containing a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, a first restriction enzyme, a second restriction enzyme, and a ligase.

[15] The method according to any one of

[12] to

[14] , wherein the predetermined sequence comprises a selection marker and an origin of replication.

[16] The method according to any one of

[12] to

[15] , wherein the recombinant minivector is a self-ligated cyclic nucleic acid in which the cleavage fragments not comprising the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence are self-ligated.

[17] The method according to any one of

[12] to

[16] , wherein the recombinant minivector has a superhelical structure.

[18] The method according to any one of

[12] to

[17] , wherein the recombinant minivector is a recombinant minivector for transforming animal cells.

[19] The method according to any one of

[12] to

[18] , wherein the recombinant minivector is introduced into cells with higher efficiency compared to the construct.

[20] The method according to any one of

[12] to

[19] , further comprising the step of digesting a linear nucleic acid contained in the reaction product with an exonuclease.

[21] The method according to any one of

[12] to

[20] , wherein a cleavage fragment containing a predetermined sequence and a first restriction enzyme recognition sequence further comprises a third restriction enzyme recognition sequence different from the first and second restriction enzyme recognition sequences, and further comprising the step of cleaving a nucleic acid contained in the reaction product that has been ligated by the two types of cleavage fragments, or a circular nucleic acid that has been self-ligated by a cleavage fragment containing a predetermined sequence and a first restriction enzyme recognition sequence, with the third restriction enzyme.

[0009] According to the present invention, it is possible to provide a simple and readily usable method for producing recombinant minivectors that do not contain a predetermined sequence. Furthermore, according to the present invention, it is also possible to provide a construct for producing recombinant minivectors that do not contain a predetermined sequence.

[0010] Examples of the first restriction enzyme recognition sequence, the cleavage site by the first restriction enzyme, the target gene, the second restriction enzyme recognition sequence, the cleavage site by the second restriction enzyme, and a construct containing the predetermined sequence, the cleavage fragments obtained when the construct is cleaved with the first and second restriction enzymes, and the ligation reaction product obtained when the cleavage fragments are ligated are shown. A simplified plasmid DNA map is shown. Results of confirming restriction enzyme activity in T4 DNA ligase reaction buffer and results of confirming conjugation of linear DNA by T4 DNA ligase under reaction conditions of 37°C are shown. Results of confirming the reaction product of the co-existence reaction of restriction enzyme and T4 DNA ligase are shown. Results of confirming the reaction product of the co-existence reaction of restriction enzyme and T4 DNA ligase are shown using two types of restriction enzymes. Results of investigating the conditions for the co-existence reaction of restriction enzyme and T4 DNA ligase are shown. Results of confirming that the extra band in Figure 8 is the target reaction product are shown. This report presents the results of investigating the conditions for the coexistence reaction of restriction enzymes and T4 DNA ligase. It also presents the results of investigating the digestion of by-products by exonuclease III. Furthermore, it shows the results of investigating the purification and Xho I treatment of the target reaction product after the coexistence reaction of restriction enzymes and T4 DNA ligase. The sequence analysis results of the junction in the target reaction product obtained from the coexistence reaction of restriction enzymes and T4 DNA ligase are presented. The results of investigating the open-circularization of the purified target reaction product are presented. A comparison of the yields of the coexistence reaction of restriction enzymes and T4 DNA ligase with the yield of a reaction in which restriction enzyme treatment is followed by purification and then ligation is presented. The results of investigating the amount of ligase and reaction time in the reaction in which restriction enzyme treatment is followed by purification and then ligation are presented. Finally, the results of investigating the higher-order structural changes of plasmid DNA are presented. The superiority of MiniVector is evaluated. The results of evaluating the superiority of MiniVector are shown. The results of evaluating the superiority of MiniVector are shown. The results of evaluating the superiority of MiniVector are shown. The results of examining the amount of gene transfer by MiniVector are shown. The results of evaluating the superiority of MiniVector are shown.The results of evaluating the superiority of MiniVector are shown. The results of evaluating the superiority of MiniVector are shown. The results of evaluating the superiority of MiniVector are shown. The results of evaluating the superiority of MiniVector are shown. The results of evaluating the superiority of MiniVector are shown. The results of evaluating the superiority of MiniVector are shown. The results of evaluating the superiority of MiniVector are shown.

[0011] The following describes embodiments of the present invention (hereinafter referred to as "these embodiments"), but the scope of the present invention is not limited to these embodiments.

[0012] In a first embodiment, a method is provided for producing a recombinant minivector that does not contain a predetermined sequence from a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence, the method comprising: 1) cleaving the construct with the first restriction enzyme and the second restriction enzyme to obtain a cleavage fragment comprising the predetermined sequence and the first restriction enzyme recognition sequence, and a cleavage fragment comprising the cleavage fragment that does not contain the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence; and 2) subjecting the two obtained cleavage fragments to a ligation reaction to obtain a reaction product comprising two self-ligated cyclic nucleic acids of the two cleavage fragments.

[0013] The method of this embodiment allows for the production of recombinant minivectors that do not contain a predetermined sequence by removing that sequence from a construct. For example, the method of this embodiment is preferably used to produce vectors for introduction into animal cells. When a vector is amplified using E. coli, the vector needs to contain replication origins and selection markers for growing and selecting E. coli into which the vector has been introduced, but these replication origins and selection markers are regions that are unnecessary for introduction into animal cells. Using the method of this embodiment, after amplifying the construct using E. coli, a minivector can be obtained from which the regions necessary only for the amplification and selection of E. coli have been removed. Furthermore, compared to a method that uses a recombinase and the endonuclease I-SceI to obtain a vector from which a predetermined sequence has been removed, the method of this embodiment does not use reagents whose use is restricted, and therefore can be used more easily.

[0014] (Construct) As used herein, “construct” means a nucleic acid used to introduce a target gene into a host. A construct is a nucleic acid for introducing a target gene by recombination, and may be a nucleic acid that carries the target gene to the gene sequence to be recombined. A construct is not particularly limited as long as the target gene introduced by the construct can be stably expressed in the host. A construct may be a transformation vector or a recombinant vector.

[0015] The construct in this embodiment includes a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence. The construct in this embodiment may be one that has been amplified in E. coli.

[0016] In this specification, the "predetermined sequence" may be any sequence, but when removing sequences necessary for amplification in E. coli from the construct, the predetermined sequence may include, for example, selection markers such as antibiotic resistance genes, or origins of replication for the construct. Examples of antibiotic resistance genes that may be included in the predetermined sequence include ampicillin resistance genes, kanamycin resistance genes, and chloramphenicol resistance genes.

[0017] As used herein, "first restriction enzyme" refers to an IIS-type restriction enzyme. An IIS-type restriction enzyme generally refers to a restriction enzyme characterized by having a restriction enzyme recognition sequence and cleavage sites that are separated. Among IIS-type restriction enzymes, those having all cleavage sites on one side separated from the restriction enzyme recognition sequence are preferred. Examples of IIS-type restriction enzymes include BbsI, BbvI, BbvII, BcefI, BccI, BcgI, BciVI, BinI, BmrI, BpmI, BsaI, BseRI, BsgI, BsmAI, BsmBI, BspMI, BsrDI, BstF5I, BsmI, BsrI, BsmFI, BseMII, BspQI, BtgZI, AcuI, AlwI, Al Examples include wXI, Alw26I, EarI, Eco31I, Eco57I, Esp3I, Esp3I, FauI, FokiI, GsuI, HgaI, HinGUII, HphI, Ksp632I, MboII, MmeI, Mn1I, NgoVIII, PaqCI, PleI, PsrI, RleAI, SapI, SfaNI, TaqII, and Tth111II. Among these, BbsI is preferred.

[0018] In the construct of this embodiment, the predetermined sequence is positioned so as to be sandwiched between a first restriction enzyme recognition sequence and a second restriction enzyme recognition sequence. Of the two regions sandwiched between the first and second restriction enzyme recognition sequences, a portion of the other region not containing the predetermined sequence is included in the recombinant minivector, and this other region includes a cleavage site by the first restriction enzyme. The first restriction enzyme recognition sequence, the second restriction enzyme recognition sequence, and the predetermined sequence are positioned in the construct such that when the construct is cleaved with the first and second restriction enzymes, cleavage fragments containing the predetermined sequence and the first restriction enzyme recognition sequence, and cleavage fragments not containing the first and second restriction enzyme recognition sequences, are obtained. Specifically, in the construct, each sequence and cleavage site are located in the order of the first restriction enzyme recognition sequence, the cleavage site by the first restriction enzyme, the region included in the recombinant minivector, the second restriction enzyme recognition sequence, and the predetermined sequence. In this case, the cleavage site by the second restriction enzyme is located within the second restriction enzyme recognition sequence. Alternatively, in the construct, each sequence and each cleavage site are located in the following order: first restriction enzyme recognition sequence, first restriction enzyme cleavage site, region contained in the recombinant minivector, second restriction enzyme cleavage site, second restriction enzyme recognition sequence, and predetermined sequence. In this case, the second restriction enzyme cleavage site is located in the region between the first and second restriction enzyme recognition sequences that is not the region where the predetermined sequence is located.

[0019] As used herein, "second restriction enzyme" refers to a restriction enzyme having a restriction enzyme recognition sequence different from that of the first restriction enzyme, and is not particularly limited, but is preferably a restriction enzyme such that when a construct is cleaved with the first and second restriction enzymes, a cleavage fragment containing a predetermined sequence and the first restriction enzyme recognition sequence, and a cleavage fragment not containing the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, are obtained. Examples of the second restriction enzyme include NotI, BssHII, and MluI.

[0020] As used herein, "recombinant minivector" refers to a vector from which a specific sequence has been removed. Examples of recombinant minivectors include non-viral vectors such as transposon vectors and plasmid vectors, and viral vectors.

[0021] The region included in the recombinant minivector in this embodiment may be any sequence, but when introducing a target gene into cells using the recombinant minivector, the region included in the recombinant minivector may include, for example, the target gene, its promoter, a poly-A tail, etc. In this case, of the two regions sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the other region that does not contain the predetermined sequence includes the cleavage site by the first restriction enzyme, the target gene, its promoter, and a poly-A tail. Preferably, of the two regions sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the other region that does not contain the predetermined sequence preferably includes at least the cleavage site by the first restriction enzyme and the target gene. If the other region that does not contain the predetermined sequence contains the target gene, the target gene does not need to include a selection marker. Preferably, the selection marker that can be included in the recombinant minivector is different from the selection marker that can be included in the predetermined sequence. The selection markers included in the recombinant minivector may be antibiotic resistance genes or reporter genes, such as puromycin resistance genes, hygromycin resistance genes, geneticin resistance genes, blastosidine resistance genes, green fluorescent protein (GFP) expression genes, mCherry expression genes, nanoluciferase expression genes, β-glucuronidase expression genes, and LacZ genes. When the selection markers included in the recombinant minivector are antibiotic resistance genes, and the selection markers included in a predetermined sequence are also antibiotic resistance genes, it is preferable that these antibiotic resistance genes are resistance genes for different antibiotics.

[0022] To distinguish between a selection marker that may be included in a predetermined sequence and a selection marker that may be included in a recombinant minivector, these are referred to as selection marker I and selection marker II, respectively, in this specification. It is preferable that different selection markers are used for selection marker I and selection marker II. In one embodiment, this embodiment also provides a method for producing a recombinant minivector from a construct including selection marker I, a replication origin, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, which does not include selection marker I and a replication origin, and includes selection marker II, which is a different selection marker from selection marker I.

[0023] The length of the recombinant minivector varies depending on the length of the target gene contained in the recombinant minivector, but may be, for example, 1,000 to 5,000 base pairs long. The length of the construct varies depending on the length of the recombinant minivector and the predetermined sequence, but may be, for example, 3,000 base pairs or longer.

[0024] (Step of cleaving the construct with a first restriction enzyme and a second restriction enzyme) The method of this embodiment includes the step of 1) cleaving the construct with a first restriction enzyme and a second restriction enzyme to obtain a cleaved fragment containing a predetermined sequence and a first restriction enzyme recognition sequence, and a cleaved fragment not containing the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence (hereinafter also referred to as "step 1").

[0025] When using a construct in which each sequence and each cleavage site is located in the following order: first restriction enzyme recognition sequence, first restriction enzyme cleavage site, region contained in recombinant minivector, second restriction enzyme cleavage site, second restriction enzyme recognition sequence, and predetermined sequence, the cleavage fragment obtained by step 1) containing the predetermined sequence and the first restriction enzyme recognition sequence also contains the second restriction enzyme recognition sequence.

[0026] The reaction conditions, such as the reaction time and temperature for the first and second restriction enzymes and the construct, can be appropriately determined by a person skilled in the art based on the types of first and second restriction enzymes.

[0027] The cleavage of the construct with the first restriction enzyme and the cleavage of the construct with the second restriction enzyme may be carried out in separate reaction systems, but it is preferable that they be carried out in parallel. That is, it is preferable that step 1) be carried out in a solution in which the construct, the first restriction enzyme, and the second restriction enzyme are present together.

[0028] (Step of subjecting the two types of cleavage fragments to a ligation reaction) This embodiment includes the step of subjecting the two types of cleavage fragments obtained to a ligation reaction to obtain a reaction product containing two types of self-ligated cyclic nucleic acids (hereinafter also referred to as "step 2").

[0029] As used herein, "ligation" refers to a reaction in which nucleic acids are linked together by a phosphate diester bond using a ligase. The ligase used in the ligation reaction, the reaction time with the ligase, and the processing temperature, as well as other reaction conditions, can be appropriately determined by those skilled in the art. Commercially available ligation reagents may also be used. Self-ligation refers to a reaction in which both ends of a single linear nucleic acid are joined. When both ends of a linear nucleic acid are joined, it becomes a cyclic nucleic acid.

[0030] Step 2) yields a reaction product containing two types of cyclic nucleic acids, each obtained by self-ligating two types of cleavage fragments: one containing a predetermined sequence and a first restriction enzyme recognition sequence, and another containing a cleavage fragment that does not contain the first restriction enzyme recognition sequence or the second restriction enzyme recognition sequence. Of these two types of cyclic nucleic acids, the cyclic nucleic acid obtained by self-ligating the cleavage fragment that does not contain the first restriction enzyme recognition sequence or the second restriction enzyme recognition sequence is a recombinant minivector that does not contain the predetermined sequence.

[0031] The construct is designed and the first and second restriction enzymes are selected so that cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence, as well as cleavage fragments not containing the first and second restriction enzyme recognition sequences, can self-ligate. For example, if cleavage by the first restriction enzyme produces sticky ends, the construct is designed and the first and second restriction enzymes are selected so that cleavage by the second restriction enzyme produces the same sticky ends as cleavage by the first restriction enzyme. Specifically, if cleavage of the construct by the first restriction enzyme produces 5'-GCCC-3' and 5'-GGGC-3' sticky ends, the construct is designed and the first and second restriction enzymes are selected so that cleavage of the construct by the second restriction enzyme also produces the same 5'-GCCC-3' and 5'-GGGC-3' sticky ends. If cleavage by the first and second restriction enzymes produces blunt ends, the above-described construct design and restriction enzyme selection are not necessary.

[0032] Step 2) may be performed after step 1), but it is more preferable that steps 1) and 2) be performed in parallel. That is, it is preferable that steps 1) and 2) be performed in a solution in which a construct containing a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, the first restriction enzyme, the second restriction enzyme, and a ligase are present together.

[0033] The reaction products in step 2) may include, in addition to self-ligated cyclic nucleic acids obtained from cleavage fragments that do not contain the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, and self-ligated cyclic nucleic acids obtained from cleavage fragments that contain the predetermined sequence and the first restriction enzyme recognition sequence, nucleic acids obtained from the ligation of two types of cleavage fragments (cyclic nucleic acids obtained from the ligation of two types of cleavage fragments or linear nucleic acids obtained from the ligation of two types of cleavage fragments), linear nucleic acids obtained from the ligation of either one of the cleavage fragments, and two types of cleavage fragments that were not ligated. When steps 1) and 2) are carried out in parallel, among the nucleic acids contained in the reaction products obtained in step 2), nucleic acids derived from cleavage fragments containing the predetermined sequence and the first restriction enzyme recognition sequence, such as self-ligated cyclic nucleic acids obtained from cleavage fragments containing the predetermined sequence and the first restriction enzyme recognition sequence, nucleic acids obtained from the ligation of two types of cleavage fragments, and linear nucleic acids obtained from the ligation of cleavage fragments containing the predetermined sequence and the first restriction enzyme recognition sequence, are cleaved by the first restriction enzyme present in the same solution. When using a construct in which each sequence and each cleavage site is located in the following order: first restriction enzyme recognition sequence, cleavage site by the first restriction enzyme, region contained in the recombinant minivector, cleavage site by the second restriction enzyme, second restriction enzyme recognition sequence, and predetermined sequence, the cleavage fragment containing the predetermined sequence and the first restriction enzyme recognition sequence also contains the second restriction enzyme recognition sequence. Therefore, nucleic acids derived from the cleavage fragment containing the predetermined sequence and the first restriction enzyme recognition sequence may also be cleaved by the second restriction enzyme. A recombinant minivector, a self-ligated cyclic nucleic acid, is not cleaved by the first and second restriction enzymes. The method of this embodiment may further include a step of cleaving a nucleic acid ligated by two types of cleavage fragments contained in the reaction product, or a self-ligated cyclic nucleic acid containing a predetermined sequence and the first restriction enzyme recognition sequence, with the first restriction enzyme. This step is preferably achieved by carrying out steps 1) and 2) in parallel, but may also be achieved by carrying out steps 1) and 2) separately and then reacting with the first restriction enzyme.

[0034] Figure 1 shows an example of a construct containing a first restriction enzyme recognition sequence, a cleavage site by the first restriction enzyme, a target gene, a second restriction enzyme recognition sequence, a cleavage site by the second restriction enzyme, and a predetermined sequence; cleavage fragments obtained when the construct is cleaved with the first and second restriction enzymes; and ligation reaction products obtained when the cleavage fragments are ligated.

[0035] When steps 1) and 2) are carried out in parallel, that is, when steps 1) and 2) are carried out in a solution in which a construct containing a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, a first restriction enzyme, a second restriction enzyme, and a ligase are present, for example, after steps <1> and <2> below, steps <3> and <4> below are repeated and the reaction proceeds. <1> The construct is cleaved by the first and second restriction enzymes, yielding two types of cleavage fragments: one containing the predetermined sequence and the first restriction enzyme recognition sequence, and another not containing the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence. <2> The two types of cleavage fragments react with the ligase to obtain a ligation reaction product. <3> Among the reaction products, nucleic acids containing the first restriction enzyme recognition sequence (such as cyclic nucleic acids in which cleavage fragments containing a predetermined sequence and the first restriction enzyme recognition sequence are self-ligated, cyclic nucleic acids in which two types of cleavage fragments are ligated, linear nucleic acids in which two types of cleavage fragments are ligated, and linear nucleic acids in which cleavage fragments containing a predetermined sequence and the first restriction enzyme recognition sequence are ligated to each other) are cleaved by the first restriction enzyme coexisting in the same solution. <4> The two types of cleavage fragments produced by the cleavage of the reaction product by the first restriction enzyme react with ligase to obtain a ligation reaction product.

[0036] Among the reaction products, the recombinant minivector, which is a cyclic nucleic acid that has self-ligated from a cleavage fragment that does not contain the first and second restriction enzyme recognition sequences, is not cleaved by the first and second restriction enzymes. Therefore, by performing steps 1) and 2) in parallel, recombinant minivectors can be accumulated.

[0037] When steps 1) and 2) are carried out in parallel, the ratio of the concentrations of the first and second restriction enzymes to the ligase can be appropriately determined by a person skilled in the art depending on the concentration of the construct, etc. For example, the ratio of the concentration of the first or second restriction enzyme to the concentration of the ligase may be 1:0.1 to 10. When steps 1) and 2) are carried out in parallel, the reaction time can be appropriately determined by a person skilled in the art depending on the concentrations of the first and second restriction enzymes, the ligase, and the construct, etc. For example, it may be 2 hours or more.

[0038] (Step of cleavage with a third restriction enzyme) The construct may also be designed such that the cleavage fragment containing the predetermined sequence and the first restriction enzyme recognition sequence further contains a third restriction enzyme recognition sequence different from the first and second restriction enzyme recognition sequences. This makes it possible to cleave the nucleic acid derived from the cleavage fragment containing the predetermined sequence and the first restriction enzyme recognition sequence, which is contained in the reaction product, with the third restriction enzyme after steps 1) and 2). If the third restriction enzyme is reacted after steps 1) and 2) are carried out in parallel, the nucleic acid product derived from the cleavage fragment containing the predetermined sequence and the first restriction enzyme recognition sequence, which was not completely cleaved by the first restriction enzyme, is cleaved by the third restriction enzyme. The first restriction enzyme may also be reacted together with the third restriction enzyme. The method of this embodiment may further include a step of cleaving the nucleic acid ligated from the two types of cleavage fragments contained in the reaction product, or a cyclic nucleic acid in which the cleavage fragment containing the predetermined sequence and the first restriction enzyme recognition sequence is self-ligated, with the third restriction enzyme. Furthermore, the method of this embodiment may further include the step of cleaving a nucleic acid ligated by two types of cleavage fragments contained in the reaction product, or a cyclic nucleic acid in which cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence are self-ligated, with a first restriction enzyme and a third restriction enzyme.

[0039] (Step of digesting with exonuclease) After the steps 1) and 2), linear nucleic acids contained in the reaction product may be degraded by the exonuclease by reacting the reaction product with the exonuclease. Examples of the linear nucleic acid contained in the reaction product include a linear nucleic acid formed by ligation of two types of cleaved fragments, a linear nucleic acid formed by ligation of cleaved fragments of either one of the two types, and two types of unligated cleaved fragments. The method of the present embodiment may further comprise a step of digesting linear nucleic acids contained in the reaction product with the exonuclease.

[0040] After steps 1) and 2), both the step of cleaving with a third restriction enzyme and the step of digesting with an exonuclease may be performed. In this case, the step of cleaving with the third restriction enzyme and the step of digesting with the exonuclease may be performed separately from each other, or may be performed in parallel. When the step of digesting with the exonuclease is performed after the step of cleaving with the third restriction enzyme, or when both steps are performed in parallel, linear cleaved fragments generated by cleavage with the third restriction enzyme are digested with the exonuclease. Therefore, nucleic acids other than the target recombinant minivector can be digested to a greater extent than when the step of cleaving with the third restriction enzyme is performed after the step of digesting with the exonuclease.

[0041] In one embodiment, the method of the present embodiment comprises the steps of: 1) cleaving a construct with a first restriction enzyme and a second restriction enzyme to obtain a cleaved fragment comprising a predetermined sequence and a first restriction enzyme recognition sequence, and a cleaved fragment that does not comprise the first restriction enzyme recognition sequence or the second restriction enzyme recognition sequence; 2) subjecting the two obtained cleaved fragments to a ligation reaction to obtain a reaction product comprising two types of circular nucleic acids formed by self-ligation of the two types of cleaved fragments; 3) cleaving, with a third restriction enzyme, a nucleic acid formed by ligation of the two types of cleaved fragments, or a circular nucleic acid formed by self-ligation of a cleaved fragment comprising the predetermined sequence and the first restriction enzyme recognition sequence, contained in the reaction product (hereinafter also referred to as "step 3)"); and 4) digesting linear nucleic acids contained in the reaction product with an exonuclease (hereinafter also referred to as "step 4)"). Steps 1) and 2) are preferably carried out in a solution in which the construct comprising the predetermined sequence, the first restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence, the first restriction enzyme, the second restriction enzyme, and a ligase coexist, and steps 3) and 4) are preferably carried out in a solution in which the ligation reaction product, the third restriction enzyme, and the exonuclease coexist. A larger amount of recombinant minivector can be produced when steps 1) and 2) are carried out in a solution in which the construct comprising the predetermined sequence, the first restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence, the first restriction enzyme, the second restriction enzyme, and a ligase coexist, compared to when steps 1) and 2) are carried out separately.

[0042] When steps 3) and / or 4) are carried out after steps 1) and 2), the method may further comprise a step of purifying the recombinant minivector from the reaction solution after steps 3) and / or 4). Reagents used for purification and each reaction condition can be appropriately determined by those skilled in the art, and commercially available purification kits may also be used.

[0043] Whether a recombinant minivector has been successfully produced by the method of the present embodiment can be confirmed using a conventionally known method, for example, by electrophoresis, sequence analysis, or the like.

[0044] The recombinant minivectors produced by the method of this embodiment preferably have a superhelical structure.

[0045] The recombinant minivectors obtained by the method of this embodiment are not particularly limited in their applications and can be used in the same applications as general vectors.

[0046] The cells into which the target gene is introduced by the recombinant minivector are not particularly limited and may be prokaryotic or eukaryotic cells, adherent or suspension cells, or primary cells. Examples include bacterial cells, fungal cells, plant cells, animal cells, and insect cells, but animal cells are preferred, and mammalian cells are more preferred. Examples of mammalian cells include hematopoietic cells, cancer cells, and ovarian cells. Specifically, examples include Jurkat cells, Ramos cells, Razi cells, THP-1 cells, Namalwa cells, HL60 cells, U266B1 cells, T cells, B cells, NK cells, dendritic cells, macrophages, monocytes, and CHO cells.

[0047] The gene introduced into cells by a recombinant minivector, i.e., the target gene in this embodiment, is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. For example, the target gene may be a gene for expressing a target protein, a reporter gene, an internal standard gene, a gene for gene therapy, hairpin RNA (shRNA), guide RNA (gRNA), etc.

[0048] The method of introducing genes using recombinant minivectors is not particularly limited and includes, for example, electroporation, heat shock, lipofection, calcium phosphate, and methods using dextran derivatives such as DEAE-dextran.

[0049] When a recombinant minivector produced by the method of this embodiment is used to introduce a gene into cells, the gene transfer efficiency, the amount of gene introduced, and / or cell viability may be improved compared to using the construct before the predetermined sequence is removed by the method of this embodiment. The recombinant minivector produced by the method of this embodiment may have a superhelical structure, which may further improve the gene transfer efficiency, the amount of gene introduced per cell, and / or cell viability.

[0050] This embodiment also provides recombinant minivectors manufactured by the method of this embodiment.

[0051] Embodiments of methods for producing recombinant minivectors that do not contain a predetermined sequence also apply to constructs for producing recombinant minivectors that do not contain a predetermined sequence, recombinant minivectors, and kits for producing recombinant minivectors, as described below.

[0052] This embodiment also provides a construct for producing a recombinant minivector that does not include a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first and second restriction enzyme recognition sequences, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence. As one embodiment, this embodiment also provides a construct for producing a recombinant minivector that does not include a selection marker I and an origin of replication, but includes a target gene, and includes a selection marker I, an origin of replication, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence. Here, the target gene may include a selection marker II, which is a selection marker different from selection marker I.

[0053] This embodiment also provides a recombinant minivector that does not contain a replication origin and contains the target gene. This recombinant minivector may be manufactured by the method of this embodiment.

[0054] This embodiment also provides: 1) a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first and second restriction enzyme recognition sequences, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence; 2) a first restriction enzyme; and 3) a kit for producing a recombinant minivector comprising the second restriction enzyme.

[0055] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0056] <Restriction Enzyme Reaction> The samples were treated at 37°C for 1 hour with a 1×rCutSmart Buffer (New England Biolabs, Cat: B6004S) reaction solution containing one or more restriction enzymes: 10 ng / μL template plasmid DNA, 2 ng / μL purified nucleic acid, or 20 U / μg DNA. The restriction enzymes used were MuI-HF (Cat: R3198L), BssHII (Cat: R0199L), BbsI-HF (Cat: R3539L), NotI-HF (Cat: R3189L), and XhoI (Cat: R0146L), all products of New England Biolabs. Prior to the ligase co-reaction, the components necessary for each ligase reaction were added to confirm that they would not affect the restriction enzyme reaction.

[0057] <Ligases> When using DNA ligases derived from various thermophilic bacteria, use 1 mM dithiothreitol (DTT, Fujifilm Wako, Cat: 042-29222), 1 mM β-nicotinamide adenine dinucleotide (NAD, Nacalai Tesque, Cat: 24338-57), 0.1% Triton® X-100 (Fujifilm Wako, Cat: 160-24751) in 1×rCutSmart Buffer or Ideal Buffer (20 mM). The samples were treated at 37°C for 1 hour in Tris-HCl (Nacalai Tesque, Cat: 35434-05, pH 8.5), 150 mM potassium chloride (Nacalai Tesque, Cat: 28538-75), 10 mM magnesium chloride (Nacalai Tesque, Cat: 20937-72), 10 mM DTT, 1 mM NAD, and 0.1% Triton® X-100. When using T4 DNA Ligase, the samples were treated at 16°C or 37°C for 1 hour in either 1X T4 DNA Ligase Buffer (the same enzyme buffer) containing 8750 U / μg DNA T4 DNA Ligase (TaKaRa, Cat:2011A) and 2 ng / μL of template DNA, or in 1× CutSmart Buffer containing 1 mM DTT, 1 mM NAD, and 0.1 mM adenosine 5'-triphosphate (ATP, Sigma-Aldrich, Cat:A7699-1G).

[0058] <Restriction enzyme and ligase co-reaction> The samples were treated at 37°C for 1 to 16 hours with a reaction mixture of 1 mM DTT containing 10 ng / μL template plasmid DNA, 1–1750 U / μL T4 DNA Ligase, and 2.5–20 U / μg DNA restriction enzymes (combination of MuI-HF and BssHII, or combination of BbsI-HF and NotI-HF), and 0.1 mM ATP in 1×rCutSmart Buffer (New England Biolabs, Cat: B6004S).

[0059] <Exonuclease III Reaction> 2 ng / μL nucleic acid, 5000 U / μg DNA exonuclease III (RaKaRa, Cat: 2170A) was treated in a 1× Exonuclease III Buffer (the buffer provided with the enzyme) reaction solution at 37°C for 1 hour, followed by inactivation at 65°C for 5 minutes. To carry out co-reaction with restriction enzymes, 1 mM DTT-containing 1X rCutSmart Buffer was used instead of the attached buffer. The restriction enzymes used in the co-reaction were BssHII, PstI-HF (New England Biolabs, Cat: R3140L), HindIII-HF (New England Biolabs, Cat: R3104L), XhoI, PvuI-HF (New England Biolabs, Cat: R3150L), and EcoRI-HF (Cat: R3101L). Enzyme concentrations of 10 U or 20 U / μg DNA were added, and the mixture was treated at 37°C for 1 hour, followed by inactivation at 65°C for 10 minutes.

[0060] <Suspension Cell Line Culture> Jurkat beta-del (JCRB Cell Bank, Cat: JCRB0147, hereinafter referred to as Jurkat), Raji (JCRB Cell Bank, Cat: JCRB1647), and Ramos (JCRB Cell Bank, Cat: JCRB9119) were cultured in RPMI-1640 (Fujifilm Wako Pure Chemical Industries, Cat: 189-02025) (hereinafter referred to as complete medium) containing 10% (v / v) inactivated (56°C, 30 minutes) fetal bovine serum (Nichirei Bioscience, Cat: 175012-500ML) at 37°C and 5% CO2. ２ CO under certain conditions ２ The cells were cultured in an incubator.

[0061] <Primary T cell culture> Frozen PBMCs were seeded at a rate of 200,000 / well in 24-well plates pre-coated with 0.5 μg / mL anti-CD3 antibody (BioLegend, Cat: BL317302). The culture volume was 1.0 mL / well, and the culture medium used was 0.5 μg / mL anti-CD28 antibody (BioLegend, Cat: BL302902), 40 U / mL IL-2 (Peprotech, Cat: AF-200-02), and AIM-V (gibco, Cat: 12055-091) containing 5% inactivated human type AB serum (GeminiBio, Cat: 100-512). Three days after the start of culture, 1 mL / well of AIM-V containing 80 U / mL IL-2 and 5% inactivated human type AB serum is added, and the culture is continued for a total of 5 days, with subculturing repeated as needed.

[0062] <Gene transfer by DEAE / Dextran method> Cells in the logarithmic growth phase are harvested, washed with D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Cat: 049-29793), 2 million cells are separated into 1.5 mL microcentrifuge tubes, and after centrifugation (400x g, 5 min, room temperature), they are pelletized. 1.25 μg of GFP expression vector (if different doses are tested, the results should be noted) is added to STBS buffer (25 mM Tris (Nacalai Tesque, Cat: 35434-21)-HCl (Nacalai Tesque, Cat: 18321-05), pH 7.5, 137 mM NaCl (Nacalai Tesque, Cat: 31333-45), 5 mM KCl (Nacalai Tesque, Cat: 28538-75), 0.6 mM Na ２ HPO ４ (Nacalai Tesque, Cat: 31738-55), 0.7 mM CaCl ２ (Fujifilm Wako Pure Chemical Industries, Cat: 3819735), 0.5 mM MgCl ２(Nacalai Tesque, Cat: 20937-72) was diluted in 62.5 μL (Tube 1). Average molecular weight 500 kDa DEAE / Dextran (Fujifilm Wako Pure Chemical Industries, Cat: 591-03161) was diluted in STBS buffer to 250 μg / mL (Tube 2). Equal volumes of the solution in Tube 2 were mixed with Tube 1, and the cell pellet was suspended in the mixture (DEAE / Dextran-DNA complex). The suspended cell solution was treated at 29°C for 30 minutes, then 1 mL of ice-cold STBS buffer was added and the mixture was rapidly centrifuged. The resulting cell pellet was washed once with ice-cold STBS and once with RPMI-1640 medium, suspended in 2 mL of complete medium, and the entire amount was seeded into a 6-well multi-well plate. ２ The samples were incubated in an incubator for 48 hours. The above are the basic conditions, but any conditions that were changed during the study are described in each test example and example.

[0063] <Gene transfer using Lipofectamine 3000> Cells in the logarithmic growth phase were harvested, and 0.5 mL of cell suspension prepared in complete medium at a concentration of 200,000 cells / mL was seeded into 24-well multi-well plates. 0.125–0.5 μg of EGFP expression vector (promoter: PGK1) was diluted in 50 μL of OptiMEM (Thermo Fisher Scientific, Cat: 31985-062) (Tube 1). On the other hand, 3 μL of Lipofectamine 3000 (Thermo Fisher Scientific, Cat: L3000-01), 0.5–4 μL of P3000 reagent (included), and 50 μL of OptiMEM were mixed in a separate tube (Tube 2). The entire volume of liquid was transferred from tube 1 to tube 2, mixed thoroughly, and then allowed to stand at room temperature for 15 minutes. The mixture was then added dropwise to the wells on which the cells had been seeded, and CO2 was added. ２ The cells were incubated in an incubator for 48 hours.

[0064] <Gene Transfer Using Neon Electroporation System> For gene transfer, a Neon NxT Electroporation System (Thermo Fisher Scientific) and Neon NxT Buffer Kit, 10 μL (Thermo Fisher Scientific, Cat: N1096) or Neon NxT Buffer Kit, 100 μL (Thermo Fisher Scientific, Cat: N10096) were used. Jurkat cells in the logarithmic growth phase were collected, and a cell suspension at 2×10^7 cells / mL was prepared in Buffer R containing plasmid DNA. 10 μL of the cell suspension was subjected to electroporation (Voltage: 1,325, Width: 10, Pulse: 3), and the cells were immediately transferred to a 24-well multi-plate containing 1 mL of pre-warmed complete medium per well. Then, CO ２ The cells were cultured in an incubator for 48 hours. The basic amount of nucleic acid used for gene transfer was 500 ng / reaction for the original vector and 100 ng / reaction for MiniVector. Raji cells in the logarithmic growth phase were collected, and a cell suspension at 7×10^6 cells / mL was prepared in Buffer R containing plasmid DNA. 100 μL of the cell suspension was subjected to electroporation (Voltage: 1,300, Width: 30, Pulse: 1), and the cells were immediately transferred to a 6-well multi-plate containing 2 mL of pre-warmed complete medium per well. Then, CO ２ The cells were cultured in an incubator for 48 hours. The basic amount of nucleic acid used for gene transfer was 5000 ng / reaction for the original vector and 250 ng / reaction for MiniVector. Ramos cells in the logarithmic growth phase were collected, and a cell suspension at 7.6×10^6 cells / mL was prepared in Buffer R containing plasmid DNA. 100 μL of the cell suspension was subjected to electroporation (Voltage: 1,550, Width: 20, Pulse: 1), and the cells were immediately transferred to a 6-well multi-plate containing 2 mL of pre-warmed complete medium per well. Then, CO ２The cells were cultured in an incubator for 48 hours. The basic amounts of nucleic acid used for gene transfer were 10 μg / reaction for the original vector and 250 ng / reaction for MiniVector.

[0065] <Evaluation of GFP-transformed cells> After 48 hours following gene transfer, viability was calculated using a trypan blue (Gibco, Cat: 15250-061) efflux test. Cells were collected and the GFP transfer rate was evaluated using a flow cytometer (KSRFortessa X-20, Beckton Dickenson or CytoFLEX LX 4, Beckman Coulter).

[0066] <Evaluation of NanoLuciferase-introduced cells> 48 hours after gene transfer, viability was calculated using a trypan blue (Gibco, Cat: 15250-061) efflux test. Cells were collected, and 5,000 viable cells / 100 μL were transferred to a 96-well Whitewell plate. Using a Nano-Glo Luciferase Assay (Promega, Cat: N1120), luminescence due to NanoLuciferase was measured using a plate reader according to the instructions provided.

[0067] <Evaluation of CAR-transformed cells> 48 hours after gene transfer, viability was calculated by trypan blue (Gibco, Cat: 15250-061) excretion test. Cells were harvested, and 500,000 viable cells were reacted with a biotin-labeled anti-ScFv antibody cocktail (GeneScript, Cat: A02283-200), and then stained with PE-labeled streptavidin (BioLegend, Cat: 405245). PE-positive CAR-expressing cells were evaluated using a flow cytometer (CytoFLEX LX 4, Beckman Coulter).

[0068] [Test Example 1] Confirmation of restriction enzyme activity in T4 DNA ligase reaction buffer and confirmation of conjugation of linear DNA by T4 DNA ligase under 37°C reaction conditions Plasmid DNA shown in the simplified plasmid DNA map in Figure 2 was cut with MuI, BssHII, or both restriction enzymes. In addition to the basic reaction buffer, 1X rCutSmart Buffer, 0.1 mM ATP or an additional 1 mM DTT was added (ALL). It was confirmed that the plasmid DNA was cut and became linear after a reaction at 37°C for 1 hour, without any effect from the additives on either restriction enzyme. Each restriction enzyme product (MuI / BssHII) processed under conditions without the above additives was purified using the QIA quick PCR Purification Kit. The purified product was reacted in 1X T4 DNA Ligase Buffer at 16°C or 37°C, as shown in Figure 3. In addition to T4 DNA Ligase Buffer (T4), rCutSmart Buffer conditions (CB) with the aforementioned ATP and DTT added were used as the reaction buffer. Since MuI produces a 5' overhang (5'-CGCG-3') and BssHII also produces the same overhang, conjugation is possible even between cut surfaces derived from different restriction enzymes. Under both conditions, the linear DNA disappeared and a shift towards the high-molecular-weight side was observed, suggesting that a conjugation reaction occurred.

[0069] [Test Example 2] Co-existence reaction of restriction enzyme and T4 DNA ligase. The restriction enzyme reaction was performed under the same conditions as ALL in Test Example 4. During the restriction enzyme reaction, 1 / 5 the amount of T4 DNA ligase (1750 U / μg DNA) as in Test Example 4 was co-existed ("T4 co-existence," hereinafter referred to as the co-existence reaction). In Figure 4, "T4 separate treatment" is under the same conditions as in Test Example 4 (37°C, CB) (hereinafter referred to as the continuous reaction), but as with Test Example 4, no linear nucleic acids were detected, so it was considered that the ligase activity was sufficient even when the amount of enzyme was reduced to 1 / 5. A band pattern different from both before restriction enzyme treatment (Non-cut) and under the T4 separate treatment conditions was detected by the co-existence reaction. It was decided to confirm that the product obtained by the co-existence reaction was joined and cyclic.

[0070] [Test Example 3] Confirmation of Cyclization of Coexisting Reaction Products A continuous reaction (RE treatment followed by T4 treatment) or a coexisting reaction (T4 coexisting RE treatment) was performed under the same conditions as in Test Example 5. Each reaction product was purified and treated with exonuclease III or MuI / BssHII (Figure 5). Almost no unconjugated linear nucleic acids were observed in the continuous reaction product, and its resistance to exonuclease III indicated cyclization. Furthermore, it was re-cleaved by restriction enzyme treatment, and the two target fragments were detected. The coexisting reaction product showed a supershift towards the high molecular weight side under unpurified conditions, but the band was detected at the same position as the continuous reaction product after purification. The presence of some unconjugated linear nucleic acids was also confirmed, and since only this band was digested by exonuclease III, the main band is a cyclized product due to self-ligation. Similar to the continuous reaction product, restriction enzyme treatment produced two fragments. The results suggest that the coexistence reaction leads to antagonism between restriction enzyme and ligase reactions, resulting in the formation of a cyclic product and some kind of higher-order structure. We decided to investigate whether self-ligation products of different restriction enzyme fragments, which are the target product, are produced by coexisting two types of restriction enzymes that produce two nucleic acid fragments with T4 DNA Ligase.

[0071] [Test Example 4] Confirmation of Cyclization of Coexisting Reaction Products (Using Two Restriction Enzymes) A ​​continuous reaction (MluI / BssHII, purified → T4lig) or a coexisting reaction (MluI / BssHII + T4lig) was performed under the same conditions as in Test Example 5 (Figure 6). However, the restriction enzyme concentration was 10 U / μg DNA, and the T4 DNA Ligase was 875 U / μg DNA. Each reaction product, either unpurified or purified, was treated in Exonuclease III Buffer (EB) or DTT-containing rCutSmart Buffer (CB) with either Exonuclease III alone or in the coexistence of MuI / BssHII (MB). It was confirmed that Exonuclease III had the same activity in CB as in EB. Similar to Test Example 4, the original vector band was not detected by enzymatic treatment alone, suggesting that digestion was sufficient with half the amount of restriction enzyme. Furthermore, similar to Test Example 5, no linear nucleic acids were detected in the continuous reaction, suggesting that ligase activity was sufficient even when the enzyme amount was reduced by half. Treatment of the purified product after MuI / BssHII with exonuclease III resulted in the complete disappearance of two bands (fragment 1 and fragment 2), indicating that the amount of nuclease in the reaction solution was sufficient to digest the linear double-stranded DNA, and no differences between buffers were observed. Treatment of the continuous reaction product with exonuclease III resulted in the detection of three bands. However, the uppermost band partially disappeared after treatment with MB-coexisting exonuclease III, suggesting that the uppermost band contained fragments 1 and 2 joined in the forward direction to regenerate the original vector, and fragments 1 and 2 joined in the reverse direction, appearing to be the same size as the original vector but no longer being re-cleaved by the restriction enzyme. These were all unintended by-products. The two lower bands are thought to be the products of self-ligation of fragment 1 and fragment 2, respectively, and are the products we are trying to obtain. On the other hand, many of the coexisting reaction products were products that were re-cleaved by restriction enzymes (purified, ExoIII(-), CB / MB), and since most of the bands disappeared with ExoIII alone, it was thought that the product contained many linear fragments or cyclized products formed by joining these restriction enzyme fragments.However, the number of cyclized by-products, fragments 1 and 2 ligated in either the forward or reverse direction, was smaller compared to the continuous reaction. We believed that by optimizing the reaction conditions, we could selectively obtain the target product, and therefore decided to proceed with further investigations.

[0072] [Test Example 5] Investigation of Coexistence Reaction Conditions (Restriction Enzyme Amount, T4 DNA Ligase Amount) A coexistence reaction was performed using the same buffer and reaction conditions as in Test Example 7. The amounts of restriction enzyme and T4 DNA Ligase used are shown in Figure 7. After the coexistence reaction, exonuclease III was added to digest linear nucleic acids. Compared to the conditions in Test Example 7 (restriction enzyme: 10 U / μg DNA, T4 DNA Ligase: 875 U / μg DNA), reducing the amount of T4 Ligase significantly increased the amount of target product detected after exonuclease III treatment (the multiple bands detected on the lower side). Almost the same results were obtained in the concentration range of 1 to 25 U / μg DNA for T4 DNA Ligase. On the other hand, there was no significant change in the amount of target product when the restriction enzyme amount was halved, but it decreased dramatically when it was halved again. Interestingly, when the amount of T4 DNA ligase was reduced, multiple extra bands were detected below the two main bands that had been detected under high-concentration T4 DNA ligase conditions. In the next investigation, we decided to confirm that these extra bands were the target product.

[0073] [Test Example 6] Confirmation that the extra band is the target product Eight samples from the samples prepared in Test Example 8 were reacted with exonuclease III in the presence of either PstI or HindIII (Figure 8). The lower series of multiple bands detected during the reaction with exonuclease III alone disappeared in the presence of PstI, suggesting that both are self-ligating products of 3282 bp fragments and take on different higher-order structures. All bands disappeared in the presence of HindIII. The restriction enzyme concentration range was narrowed to 2.5–10 U / μg DNA, and the T4 DNA Ligase concentration range to 50 U / μg DNA or less, and the reaction time was investigated.

[0074] [Test Example 7] Investigation of coexistence reaction conditions (restriction enzyme amount, T4 DNA ligase amount, reaction time) A coexistence reaction was carried out in the same manner as in Test Example 8. The restriction enzyme and T4 DNA ligase amounts used are shown in Figure 9. After the coexistence reaction, linear nucleic acid digestion treatment was performed with exonuclease III. It was observed that increasing the reaction time tended to increase both the amount of the target product and the number of multiple bands. Since there was almost no difference between the conditions at the enzyme concentrations investigated this time, the enzyme concentration was set to 5 U / μg DNA in all cases, and further investigation of the conditions was carried out.

[0075] [Example 11] Investigation of coexistence reaction conditions using a new plasmid DNA (reaction time) As shown in the results of the investigations up to Test Example 10, by mixing restriction enzymes and T4 DNA Ligase in an appropriate ratio and causing competition between cleavage and conjugation reactions, we were able to obtain self-ligation products of the target nucleic acid fragment. To obtain the target product even more efficiently, we constructed the plasmid DNA shown in Figure 10. This plasmid DNA has recognition sequences for BbsI and NotI on both sides of the target gene product (the 3252 bp fragment shown in Figure 10). BbsI is an IIS-type restriction enzyme and cleaves sites far from the recognition site, but this plasmid DNA was designed so that digestion with BbsI produces sticky ends identical to those of NotI. By coexisting with ligase, a reaction occurs in which the BbsI or NotI cleavage sites are rejoined, and a reaction occurs in which the two fragments resulting from the digestion of both restriction enzymes self-ligate. The self-ligation product of the 2008 bp fragment has an intact BbsI recognition site and is therefore re-cleaved by BbsI. On the other hand, the self-ligation product of the 3252 bp fragment (target product) lacks a BbsI recognition site, and the NotI recognition site also disappears, so it is not digested by either coexisting restriction enzyme. We hypothesized that we could accumulate the self-ligation product of the target 3252 bp fragment by utilizing this property of plasmid DNA. When we reacted this plasmid with restriction enzymes at concentrations of 5 U / μg DNA and T4 DNA Ligase, we detected multiple bands at the bottom in a time-dependent manner, reproducing the results of Test Example 10. Furthermore, the bands observed above 7000 kDa derived from the original vector were clearly reduced compared to the results of Test Example 10, suggesting that the target self-ligation product was accumulated as expected. The bands that disappeared after exonuclease III treatment are the remaining linear nucleic acids. The bands above 7000 bp that disappeared due to the presence of XhoI are thought to be the original plasmid DNA, and the bands around 3000-5000 bp are thought to be the target product (self-ligation product of the 3252 bp fragment). The remaining bands detected around 1500-2000 bp at this time are the self-ligation product of the 2008 bp fragment (by-product).Comparing the amounts of the target product and by-products when digested by exonuclease III alone, it was found that the amount of by-products was smaller than the target product, especially at reaction times of 2-6 hours. As expected, this suggests that the by-products underwent re-digestion by BbsI, existed partially in a linear form, and were digested by exonuclease III. Increasing the coexistence reaction time clearly reduced the amount of bands derived from the original plasmid DNA, while increasing the amounts of both the target product and by-products. Furthermore, the ratio of multiple bands tended to converge to a certain ratio. Based on these results, a reaction time of 2 hours or more was considered sufficient, but in future studies, the coexistence reaction time will be increased to 16 hours or more.

[0076] [Example 12] Examination of by-product digestion by exonuclease III In Example 11, most of the by-products remained as cyclized products without being re-cleaved by BbsI after a reaction time of 12 hours. Therefore, it was considered that the activity of BbsI was being lost, and in order to completely digest products other than the target product with exonuclease III, BbsI or PvuI (10 U / μg DNA) was added during the exonuclease III digestion following the coexistence reaction. As shown in Figure 11, most of the by-products were digested, but some were detected. Therefore, the amount of PvuI, which was digested more efficiently, was doubled and digested, and then purified using QIAprep Spin Miniprep Kit (QIAGEN, Cat: 27106) or QIAquick PCR Purification Kit (QIAGEN, Cat: 28106).

[0077] [Example 13] Purification of the target product after coexistence reaction and cleavage with structural restriction enzymes was examined using the same method as in Example 12, but the additional PvuI concentration during exonuclease III treatment was set to 20 U / μg DNA. After exonuclease III treatment, purification was performed using two different purification kits, and the QIAprep Spin Miniprep Kit yielded a higher yield than the QIAquick PCR Purification Kit. Multiple bands were still detected after purification, suggesting that the higher-order structure was preserved. These multiple bands converged into a single linear nucleic acid of 3252 bp by XhoI treatment (Figure 12). From these results, it was considered that the multiple bands obtained by the coexistence reaction are the same structure with multiple higher-order structures.

[0078] [Example 14] Sequence analysis of the junction of the target product after coexistence reaction The target product purified in Example 13 was sequenced using Eurofins Genomics. It was confirmed that the NotI and BbsI sections of the original vector were junctioned (Figure 13).

[0079] [Example 15] Investigation of open-circularization of the target purified product after coexistence reaction Two lots of the target product purified using the method of Example 13 were treated with a restriction enzyme (Nb.BtsI) that cleaves only one strand of the recognition site. As a result, an open-circular band (white arrowhead in Figure 14) was detected as the main band. A small amount of linear bands in which both strands were cleaved were also detected, as shown by the black arrowhead. From these results, it was considered that the multiple bands detected by NC represent multiple superhelical structures. Hereafter, this target product will be referred to as MiniVector.

[0080] [Example 16] Comparison of Yields of Coexistence Reaction and Normal Reaction In previous studies, byproducts have been digested by coexisting PvuI and exonuclease III after the coexistence reaction. To minivectorize various gene products, vectors were created in which BamHI, HindIII, and ExoRI recognition sequences were inserted as restriction enzyme sites to be coexisted during byproduct digestion (see Figure 15, simplified map). It was confirmed in advance that changing the coexisting restriction enzyme during digestion does not affect the yield (data omitted). Normally, when obtaining self-ligation products of restriction enzyme fragments, nucleic acid fragments are purified after restriction enzyme treatment and then ligated (see Figure 15, right side of the flow chart). In this study, we confirmed whether the coexistence reaction is superior in terms of yield. First, a T4 DNA Ligase (5 U / μg DNA, 16 hr) reaction was attempted on the restriction enzyme-treated purified product under the same conditions as the coexistence reaction. However, the target 2230 bp fragment was fragmented into two by XhoI digestion after the reaction, suggesting that no conjugation occurred at all (Figure 16, left four lanes). Therefore, when the amount of T4 DNA Ligase was increased by more than 200 times, the original fragment was no longer detectable even after a 30-minute reaction, and the 2230 bp linear target fragment was detected after XhoI treatment, suggesting that a sufficient conjugation reaction occurred and the fragment was cyclic (Figure 16, right four lanes). In a yield comparison test with the coexistence reaction under these conditions, the yield of MiniVector was 55% in the coexistence reaction, while the yield was less than 2% in the method on the right side of the flow chart in Figure 15.

[0081] [Example 17] Structural changes of plasmid DNA by ligation reaction of restriction enzyme fragments In the previous study, it was found that MiniVectors could be produced even if the restriction enzyme treatment step and the ligation step were separated, although the yield decreased. Therefore, the same treatment was applied to plasmid DNA purified from E. coli to confirm that a change in higher-order structure occurred. The plasmid DNA used in the previous study was digested with NotI alone, subjected to heat inactivation, and then ligated with T4 ligase (Figure 17, flow chart). As a result of the study, MiniVector-like multiple bands were detected, which were not seen in the electrophoresis image of the original plasmid DNA, as shown in lanes 3-4 of the right panel of Figure 17.

[0082] [Example 18] Evaluation of the superiority of MiniVector (Jurkat cells, Nano Luciferase, DEAE / Dextran method) Purified Nano Luciferase-expressing MiniVector (MiniVector) after coexistence reaction and its original vector (Nluc MV-1, -2) were introduced into Jurkat cells using the DEAE / Dextran method. Two days after introduction, the cells were lysed, and the Luciferase activity in the lysate was compared (Figure 18). Compared with the use of the original vector, approximately six times higher Luciferase activity was detected when MiniVector was used, suggesting that the gene transfer efficiency and the amount introduced per cell increased due to the use of MiniVector. The Nluc MV vector used in this study had a total length of 5159 bp, which was reduced to 3160 bp after MiniVectorization. When the same amount of nucleic acid was introduced, the amount of MiniVector increased by approximately 1.6 times. Therefore, we decided to investigate the Luciferase activity dependent on the amount of nucleic acid introduced by the MiniVector.

[0083] [Example 19] Dose-dependent analysis of MiniVector (Jurkat cells, Nano Luciferase, DEAE / Dextran method) Purified Nano Luciferase-expressing MiniVector (MiniVector) after coexistence reaction and its original vector were introduced into Jurkat cells using the DEAE / Dextran method. Two days after introduction, the cells were lysed, and the Luciferase activity in the lysate was compared (Figure 19). However, the amount of introduced nucleic acid of MiniVector was also evaluated at four levels of reduction from 1.25 μg with a common ratio of 2. When using normal pDNA, the introduction efficiency showed a gradual bell-shaped curve with a peak at 1.25 μg in the range of introduced nucleic acid amounts from 0.5 to 2.0 μg (data omitted). Surprisingly, it was found that the introduction rate of MiniVector plateaued even at approximately 0.3 μg, which is below the equivalent molar amount of the original vector (0.78 μg). Unless otherwise noted, the amount of MiniVector introduced was assumed to be 0.3 μg.

[0084] [Example 20] Evaluation of the superiority of MiniVector (Jurkat cells, GFP, DEAE / Dextran method) Purified GFP-expressing MiniVector (mvEGFP) after coexistence reaction, its original vector (EGFP / pMV), and a GFP-expressing vector (EGFP / pRP) as a control were introduced into Jurkat cells using the DEAE / Dextran method, and the percentage of GFP-positive cells and the average fluorescence intensity of GFP-positive cells were compared two days after introduction (Figure 20; the promoters and sizes of each vector are shown in the table on the right). Compared to when both EGFP expression vectors were used, the gene transfer efficiency was more than 1.5 times higher when MiniVector was used, and the MFI, an indicator of the amount of expression per cell, was more than 6 times higher. From these results, it was suggested that MiniVector is useful not only for increasing the transfer efficiency but also for increasing the amount of target gene expression per cell.

[0085] [Example 21] Evaluation of the superiority of MiniVector (Jurkat cells, GFP, DEAE / Dextran method) Using three lots of MiniVector (mvEGGFP) prepared on a separate test day, independent tests were conducted with the original vector (EGGFP / pMV) in n=2 and n=3 trials, following the example of Test Example 9 (Figure 21). The results showed that using MiniVector compared to the original vector resulted in a statistically significant improvement in transduction efficiency of approximately 1.6 times and an increase in transgene expression per cell of approximately 4 times.

[0086] [Example 22] Dose-dependent analysis of MiniVector (Jurkat cells, GFP, DEAE / Dextran method) Purified EGFP-expressing MiniVector after coexistence reaction was reduced in three steps from 600 ng using a common ratio of 2. Gene transfer was performed on Jurkat cells using the DEAE / Dextran method, and the percentage of GFP-positive cells and the average fluorescence intensity of GFP-positive cells were analyzed two days after transfer. The results showed that the gene transfer efficiency (Figure 22, line graph) plateaued at 150 ng, but the expression level per cell (Figure 22, bar graph) increased in a nucleic acid-dependent manner and did not reach a plateau within the scope of this study. At least in terms of gene transfer efficiency, 150 ng / reaction was found to be sufficient, and the required amount was found to be less than 1 / 8 of the original vector. However, based on the results of the studies up to Example 10, the amount of nucleic acid used when introducing MiniVector was set to be approximately 1 / 4 of the amount of the original vector. [Example 23] Evaluation of the superiority of MiniVector (Jurkat cells, GFP, Lipofection method) To evaluate the usefulness of MiniVector in other gene transfer methods, gene transfer into Jurkat cells using Lipofectamine 3000 was attempted. The amount of nucleic acid introduced was 500 ng for the original vector according to the package insert, and 1 / 4 of that amount, 125 ng, for MiniVector. The results showed that Jurkat cells had very low transfection efficiency using the lipofection method, with the original vector transfection efficiency being only a few percent. However, applying MiniVector improved this efficiency by approximately three times, and the MFI, an indicator of the amount of transfection per cell, also improved by approximately three times (Figure 23). Since it was thought that MiniVectorization would also improve other gene transfection methods, we decided to investigate this using the electroporation method.

[0087] [Example 24] Evaluation of the superiority of MiniVector (Jurkat cells, GFP, Electroporation method) To evaluate the usefulness of MiniVector in electroporation, gene introduction into Jurkat cells was attempted using the Neon NxT Electroporation system (Figure 24). The amount of introduced nucleic acid was set according to the package insert, with the original vector being 100 ng and 30 ng based on 500 ng, and MiniVector being 100 ng and 30 ng. Under the condition of introducing 500 ng of original vector, the introduction efficiency was high at over 90%, but the survival rate was low at approximately 60%. When the amount of introduced nucleic acid was reduced to 30 ng, the survival rate recovered to over 90%, but the introduction efficiency fell below 90%. On the other hand, even with a nucleic acid dose of 100 ng, MiniVector achieved a survival rate of over 90% and nearly 100% delivery efficiency. Cell-level expression levels followed the same trend as GFP positivity, with the 100 ng MiniVector condition showing the highest level. Even when the amount of nucleic acid dose was reduced to 30 ng, the GFP positivity was almost equivalent to that of the 500 ng original vector delivery condition. These results demonstrate that MiniVector is an effective tool for improving cell survival and delivery efficiency even in electroporation.

[0088] [Example 25] Evaluation of the superiority of MiniVector (Raj cells, GFP, Electrification method) Next, in order to evaluate the superiority of MiniVector over suspension cell lines other than Jurkat, we attempted to introduce MiniVector using Neon NxT Electrification with Burkitt lymphoma cell lines, Raj cells and Ramos cells. For Raj cells, the amount of nucleic acid introduced was 250 ng and 100 ng based on the original vector of 5000 ng as per the package insert, and 250 ng and 100 ng of MiniVector were used (Figure 25). Under the condition of introducing 5000 ng of original vector, the introduction efficiency was high at over 80%, but the survival rate was less than 20%. Reducing the amount of introduced nucleic acid to 250 ng restored the survival rate to over 90%, but the translocation efficiency fell below 80%. On the other hand, with MiniVector, the survival rate remained above 90% even with an introduced nucleic acid amount of 250 ng, and the translocation efficiency was almost 100%. Even when the introduced nucleic acid amount was reduced to 100 ng, the GFP positivity rate was equivalent to or better than that under the conditions of introducing 5000 ng of the original vector.

[0089] [Example 26] Evaluation of the superiority of MiniVector (Ramos cells, GFP, Electrification method) For Ramos cells, the amount of introduced nucleic acid was set to 250 ng and 100 ng for the original vector, based on 10 μg as per the package insert, and 250 ng and 100 ng for MiniVector (Figure 26). Under the condition of introducing 10 μg of original vector, the introduction efficiency was high at over 50%, but the survival rate was approximately 20%. When the amount of introduced nucleic acid was reduced to 250 ng, the survival rate recovered to about 80%, similar to the decoy gene introduction sample, but the introduction efficiency fell below 10%. On the other hand, even with an introduced nucleic acid amount of 250 ng, the survival rate of MiniVector was as high as that of the control sample, and the introduction efficiency was also over 40%. These results demonstrate the usefulness of MiniVector in electroporation for other suspension cell lines as well.

[0090] [Example 27] Evaluation of MiniVector's Superiority (Raj / Ramos Cells, GFP, DEAE / Dextran Method) Next, the superiority of MiniVector (MV) over Raji cells and Ramos cells using the DEAE / Dextran method was evaluated (Figure 27). The gene transfer efficiency using the original vector was approximately 5% for both cell types, but by using MiniVector, it improved to approximately 14% for Raji cells and approximately 10% for Ramos cells. The expression level per cell also increased by approximately 25% in Raji cells and approximately doubled in Ramos cells. On the other hand, no change was observed in viability. From these results, the usefulness of MiniVector in the DEAE / Dextran method was demonstrated for other suspension cell lines as well.

[0091] [Example 28] Evaluation of the superiority of MiniVector (Primary T cells, GFP, DEAE / Dextran method) Next, we evaluated the superiority of MiniVector (MV) over primary T cells using the DEAE / Dextran method. The gene transfer efficiency using the original vector was 2.44% (Figure 28, Δ value in the second quadrant), but using MiniVector improved it to 16.5%, an improvement of approximately 10 times. The expression level per cell also clearly improved, and the survival rate improved from approximately 50% when using the original vector to over 70% when using MiniVector (data omitted). From these results, the usefulness of MiniVector over primary T cells in the DEAE / Dextran method was demonstrated.

[0092] [Example 29] Production of CAR-T cells using MiniVector (Primary T cells, CAR, DEAE / Dextran method) We decided to confirm that other genes could also be introduced into primary T cells using MiniVector. We attempted to express CD19 CAR (chimeric antigen receptor), which is attracting attention as a new treatment method for diffuse large B-cell lymphoma and other diseases. As shown in Figure 29, the Δ value in the second quadrant indicating CAR-positive cells was approximately 30% compared to non-genetically modified cells. This suggests that CAR-T cells can be prepared by chemical methods by applying MiniVector.

[0093] [Example 30] Establishment of stable expression cells using the Tol2 Transposon System (Jurkat cells, NanoLuciferase, DEAE / Dextran method) Conventionally, viral vector methods such as lentiviruses and retroviruses are used to obtain suspension cell lines that stably express the target gene. Viral vector production is technically difficult, time-consuming, and labor-intensive, and requires equipment that meets biosafety requirements for use. There are also methods to insert the target gene into genomic DNA using transposon systems such as PiggyBac, SleepingBeauty, and Tol2, but when suspension cells are the target, sufficient introduction efficiency cannot be obtained with chemical methods, and electroporation is used. Electroporation is not versatile because it requires specialized equipment and instruments. We evaluated whether stable expression cells could be efficiently obtained by the transposon method by utilizing the characteristics of MiniVector, which showed high gene introduction efficiency even with chemical gene introduction methods. Both the Puromycin-selection marker-equipped Nano Luciferase-expressing Tol2 vector and the transposase (TPase) vector were mini-vectorized. This reduced the vector size from 5180 bp to 3160 bp for the former and from 6042 bp to 4025 bp for the latter. The Tol2 original vector and the transposase original vector, or the Tol2 MiniVector and TPase MiniVector, were introduced into Jurkat cells in various ratios using the DEAE / Dextran method. Luciferase activity was measured the day after introduction, Puromycin selection was performed for 3 days, and then the Luciferase activity of the proliferating cells was evaluated over the following 2 weeks. When the original vector was introduced, stable Jurkat cells were obtained in only one case each under two of the three conditions. In contrast, when MiniVector was used, stable expression cells were obtained in both cases under all conditions (Table 1). This suggests that applying MiniVector to the transposon method allows for stable expression of Jurkat cells even using chemical methods.

[0094]

[0095] [Example 31] Evaluation of the superiority of MiniVector-like plasmid DNA (Jurkat cells, GFP, DEAE / Dextran method) We evaluated the superiority of the plasmid DNA that underwent MiniVector-like structural changes, prepared in Example 17, compared to the original plasmid DNA in terms of gene transfer efficiency using the DEAE / Dextran method. As shown in Figure 30, by giving it a MiniVector-like structure, the gene transfer efficiency was significantly improved compared to the original pDNA.

Claims

1. A construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence, for the purpose of producing a recombinant minivector that does not contain the predetermined sequence.

2. The construct according to claim 1, wherein the predetermined sequence includes a selection marker and a replication origin.

3. The construct according to claim 1 or 2, wherein a portion of the other region of the two regions sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the region not containing the predetermined sequence, is included in the recombinant minivector, and the other region contains the cleavage site by the first restriction enzyme and the target gene.

4. The construct according to claim 3, wherein, of the two regions sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the region in which a predetermined sequence is located does not include a cleavage site by the second restriction enzyme.

5. The construct according to claim 1 or 2, wherein the recombinant minivector has a superhelical structure.

6. The construct according to claim 1 or 2, wherein the recombinant minivector is a recombinant minivector for transforming animal cells.

7. The construct according to claim 1 or 2, wherein the recombinant minivector is introduced into cells with higher efficiency compared to the construct.

8. The construct according to claim 1 or 2, wherein the recombinant minivector has a base length of 1,000 to 5,000.

9. The construct according to claim 1 or 2, having a base length of 3000 or more.

10. A recombinant minivector containing the target gene but without an origin of replication.

11. The recombinant minivector according to claim 10, having a base length of 1,000 to 5,000.

12. A method for producing a recombinant minivector that does not contain a predetermined sequence from a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, wherein the predetermined sequence is arranged so as to be sandwiched between the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence, the first restriction enzyme recognition sequence is an IIS-type restriction enzyme recognition sequence, and the second restriction enzyme recognition sequence is a restriction enzyme recognition sequence different from the first restriction enzyme recognition sequence, the method comprising: 1) cleaving the construct with the first restriction enzyme and the second restriction enzyme to obtain a cleavage fragment comprising the predetermined sequence and the first restriction enzyme recognition sequence, and a cleavage fragment comprising the cleavage fragments that do not contain the first restriction enzyme recognition sequence and the second restriction enzyme recognition sequence; and 2) subjecting the two obtained cleavage fragments to a ligation reaction to obtain a reaction product comprising two self-ligated cyclic nucleic acids.

13. The method according to claim 12, further comprising the step of cleaving a nucleic acid ligated by the two types of cleavage fragments contained in the reaction product, or a cyclic nucleic acid self-ligated by cleavage fragments containing a predetermined sequence and a first restriction enzyme recognition sequence, with a first restriction enzyme.

14. The method according to claim 12 or 13, wherein each of the steps is carried out in a solution containing a construct comprising a predetermined sequence, a first restriction enzyme recognition sequence, and a second restriction enzyme recognition sequence, a first restriction enzyme, a second restriction enzyme, and a ligase.

15. The method according to claim 12 or 13, wherein the predetermined sequence includes a selection marker and a replication origin.

16. The method according to claim 12 or 13, wherein the recombinant minivector is a self-ligated cyclic nucleic acid in which the cleavage fragments not containing a first restriction enzyme recognition sequence and a second restriction enzyme recognition sequence are self-ligated.

17. The method according to claim 12 or 13, wherein the recombinant minivector has a superhelical structure.

18. The method according to claim 12 or 13, wherein the recombinant minivector is a recombinant minivector for transforming animal cells.

19. The method according to claim 12 or 13, wherein the recombinant minivector is introduced into cells with higher efficiency compared to the construct.

20. The method according to claim 12 or 13, further comprising the step of digesting the linear nucleic acid contained in the reaction product with an exonuclease.

21. The method according to claim 12 or 13, wherein a cleavage fragment containing a predetermined sequence and a first restriction enzyme recognition sequence further contains a third restriction enzyme recognition sequence different from the first and second restriction enzyme recognition sequences, and further comprises the step of cleaving a nucleic acid ligated by the two types of cleavage fragments, or a cyclic nucleic acid self-ligated by a cleavage fragment containing a predetermined sequence and a first restriction enzyme recognition sequence, contained in the reaction product, with the third restriction enzyme.

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