Stable maintenance of poly(A) tails

Abstract

The present invention relates to a method for stably retaining a polyA tail.

Description

Technical Field

[0001] The present invention relates to a method for stably retaining a polyA tail.

Background Art

[0002] In recent years, mRNA has attracted attention as an active pharmaceutical ingredient (API) of pharmaceuticals. mRNA requires a component called a poly(A) tail, and the polyA tail plays an important role in the stability of RNA and the translation process. In the mRNA constituting the mRNA vaccine, the length of the polyA tail is recognized to be 120 as the standard size (Non-Patent Document 2). The mRNA transcribed in cells has a polyA signal, and undergoes a process in which a polyA tail is added after the transcription process. mRNA produced by in vitro transcription (IVT) also requires a polyA tail to enhance in vivo stability and translation efficiency.

[0003] The method of adding a polyA tail is divided into two types. One is a method of synthesizing a polyA tail using polyA polymerase after IVT, and the other is a method in which an adenine repeat sequence is included in the template used for IVT so that a polyA tail is generated by IVT.

[0004] In the former case, a separate process of synthesizing a polyA tail on mRNA by utilizing polyA polymerase is required, and the length of the generated polyA tail is not constant. However, in the latter case, no separate process other than the IVT process is required, and the length of the generated polyA tail becomes constant. Therefore, from the viewpoints of the efficiency of the process and the uniformity of the length of the polyA tail, it is preferable that the template includes a polyA tail sequence.

[0005] For this purpose, it is necessary to clone and retain a polyA tail sequence in plasmid DNA. However, when such a process is carried out by utilizing Escherichia coli in a general method, recombination and deletion occur, so it is difficult to secure plasmid DNA containing 100 or more adenine repeat sequences, which is the standard size for use in mRNA vaccines.

[0006] To solve the above problems, a method has been devised to improve stability by including a linker sequence between adenine repeat sequences (Non-Patent Document 3). However, since the polyA tail composed only of adenine repeat sequences is the original form existing in nature, the development of other methods for cloning and retaining in plasmids without including a linker sequence between polyA tails is still required.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0008] The object of the present invention is to provide a method for stably retaining a polyA tail.

Means for Solving the Problems

[0009] The present invention provides a method for stably retaining a polyA tail.

Effects of the Invention

[0010] By the method of the present invention, the polyA tail can be easily cloned, and the polyA sequence is stably retained.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Embodiments for Carrying Out the Invention

[0012] One aspect for realizing the present invention is a method for stably retaining a polyA tail in a plasmid. In one specific example, the method includes culturing a cell line transformed with a plasmid containing a sequence encoding a poly(A) tail under temperature conditions of 31°C or lower.

[0013] In any of the above-described specific examples, the culturing step includes (1) recovering the cell line under temperature conditions of 31°C or lower. In any of the above-described specific examples, after the recovery step of (1), the method includes (2) culturing the cell line on a solid medium under temperature conditions of 31°C or lower to form colonies.

[0014] In any of the above-described specific examples, the culturing further includes (3) culturing the colonies formed by the solid medium culturing of (2) in a liquid medium under temperature conditions of 31°C or lower.

[0015] In any of the above-described specific examples, the method includes (1) a step of recovering a cell line transformed with a plasmid containing a sequence encoding a poly(A) tail under temperature conditions of 31°C or lower, (2) a step of forming colonies by solid-culturing the recovered cell line under temperature conditions of 31°C or lower, and (3) a step of performing liquid culture under temperature conditions of 31°C or lower.

[0016] In any of the above-described specific examples, the step of culturing in the liquid medium includes performing subculture. In any of the above-described specific examples, the temperature conditions of steps (1) to (3) are 16°C to 31°C.

[0017] In any of the above-described specific examples, the temperature conditions of steps (1) to (3) are 19°C to 25°C. In any of the above-described specific examples, the cell line is Escherichia coli (E. coli).

[0018] In any of the above-described specific examples, the transformation includes introducing a plasmid containing a sequence encoding a polyA tail into a cell line using heat shock or electroporation.

[0019] In any of the above-described specific examples, the introduction is performed at a temperature of 37°C to 42°C. In any of the above-described specific examples, the method further includes a step of storing the cultured cell line.

[0020] In any of the above-described specific examples, the step of storing includes freezing and / or drying the cell line. In any of the above-described specific examples, the culturing includes a step of culturing and activating the stored strain.

[0021] In any of the above-described specific examples, the method is characterized in that the stability of the polyA tail in the plasmid is improved as compared with a method including a step of culturing a cell line transformed with a plasmid at a temperature of 37°C or higher.

[0022] In any of the above-described specific examples, the polyA tail contains 20 to 400 adenines (A). Another aspect of realizing the present invention is a method for mass-producing a plasmid for mRNA production.

[0023] In one specific example, the method includes a step of culturing a cell line transformed with a plasmid containing a sequence encoding an mRNA containing a poly(A) tail under a temperature condition of 31°C or lower.

[0024] In any of the above-described specific examples, the mRNA is a constituent component of an immunogenic composition and / or a therapeutic agent. Yet another aspect of realizing the present invention is a method for producing an mRNA containing a polyA tail.

[0025] In one specific example, the method includes culturing a cell line transformed with a plasmid containing a sequence encoding an mRNA containing a poly(A) tail under temperature conditions of 31°C or lower.

[0026] In any of the above-described specific examples, the method includes recovering the plasmid from the cell line and performing transcription in vitro. In any of the above-described specific examples, the mRNA is a component of an immunogenic composition and / or a therapeutic agent.

[0027] Hereinafter, these will be specifically described. Note that each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. That is, any combination of various elements disclosed in the present invention is included in the present invention. Also, the present invention is not limited to the following specific description.

[0028] Moreover, those having ordinary knowledge in the art will be able to recognize and confirm many equivalents of the specific aspects of the present invention described in the present invention using only ordinary experiments. Furthermore, it is intended that such equivalents are also included in the present invention.

[0029] Furthermore, many papers and patent documents are referenced throughout this specification, and their citations are indicated. The entire disclosure content of the cited papers and patent documents is incorporated herein by reference, whereby the level of the technical field to which the present invention belongs and the content of the present invention are more clearly explained.

[0030] One aspect of the present invention is a method for stably retaining a poly(A) tail in a plasmid. The method includes culturing a cell line transformed with a plasmid containing a sequence encoding a poly(A) tail under temperature conditions of 31°C or lower.

[0031] As used herein, the "poly A tail" refers to a continuous or discontinuous sequence of adenylate residues typically located at the 3'-end of an RNA molecule. The poly A tail may follow the 3'-end of the mRNA, for example, after the 3'-UTR. Such a poly A tail may be composed of, or include, about 20 or more, 25 or more, 40 or more, 60 or more, 80 or more, or 100 or more adenyl (A) nucleotides.

[0032] As an example, the poly A tail may contain 20 to 400 adenines (A). For example, it may contain 20 to 300, 40 to 200, 50 to 190, 60 to 180, 70 to 170, 60 to 160, 70 to 150, 80 to 140 adenines. For example, it contains about 120 adenines, but is not limited thereto.

[0033] As an example, based on the number of nucleotides of the poly A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% are A nucleotides, while the remaining nucleotides may be nucleotides other than A nucleotides, such as U, T, or C. For example, the poly A tail may include a modification that delays the degradation of the mRNA.

[0034] In the present invention, a plasmid containing a poly A tail may include a promoter that initiates transcription, any operator sequence for regulating the transcription, a sequence encoding the target mRNA, and a sequence for regulating the termination of transcription and translation. As an example, the plasmid may include a restriction enzyme binding site that is easy to utilize for cloning.

[0035] As an example, the plasmid may include a selection marker for confirming whether it has been transformed. Markers that confer selectable phenotypes such as drug resistance, auxotrophy, and resistance to cytotoxic agents are used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit different phenotypes, so transformed cells can be selected. However, it is not limited to these.

[0036] The plasmid of the present invention can be prepared by introducing a sequence encoding a polyA tail into the plasmid. For example, a sequence encoding a polyA tail can be amplified by PCR, treated with a restriction enzyme, and then ligated to a plasmid treated with the restriction enzyme to prepare a plasmid containing a sequence encoding a poly(A) tail. For example, a polynucleotide sequence encoding mRNA may be ligated upstream of the sequence encoding the polyA tail. For example, the plasmid may be used as a template for transcribing the target mRNA, which includes a polynucleotide sequence encoding mRNA upstream of the sequence encoding the polyA tail.

[0037] A cell line transformed with the plasmid containing the sequence encoding the poly(A) tail of the present invention may be prepared by introducing the plasmid containing the sequence encoding the polyA tail into the cell line.

[0038] In the present invention, introducing the plasmid containing the sequence encoding the polyA tail into the cell line may be said to transform the cell line with the plasmid. In the present invention, the "introduction" or "transformation" of a plasmid means transmitting the plasmid to a cell line. Such introduction can be easily carried out by ordinary methods in the art. In order to introduce a plasmid into a cell line, the cells may be processed so as to be permeable to DNA molecules. Cells that have undergone such a process are called competent cells.

[0039] As an example, the introduction may be carried out using heat shock or electroporation. The transmission of a plasmid using heat shock is carried out by mixing DNA and cells and instantaneously applying heat. For example, the introduction by heat shock is carried out under temperature conditions of about 37°C or higher, specifically under temperature conditions of about 37°C to 42°C. The step of cooling the cells on ice may be included before and / or after applying the heat shock.

[0040] Electroporation is a method of transmitting a plasmid using electricity. When a short and high-voltage electrical pulse stimulation is applied to change the membrane potential of the cell membrane, small pores of nanometer size are generated on the surface of the cell membrane, and the permeability of DNA becomes higher. For example, the introduction by electroporation is carried out under temperature conditions of about 37°C or higher, specifically under temperature conditions of about 37°C to 42°C. The step of cooling the cells on ice may be included before and / or after applying the electrical stimulation.

[0041] As other examples, the CaCl2 precipitation method, the Hanahan method in which the efficiency is improved by using a reducing agent DMSO (dimethyl sulfoxide) in the CaCl2 method, the calcium phosphate precipitation method, the protoplast fusion method, the stirring method using silicon carbide fibers, the transformation method using PEG, dextran sulfate, lipofectamine, and the dry / suppression medium transformation method, etc. are used. However, it is not limited thereto.

[0042] In the case of mRNA for therapeutic purposes such as mRNA vaccines, a poly-A tail is necessary for the stability and translation efficiency of the mRNA. The applicant of the present application newly provides a method for culturing Escherichia coli cloned by a method capable of stably cloning, mass-producing, and preserving the sequence encoding the poly-A tail present in the plasmid without disappearance.

[0043] In the present invention, the step of culturing the cell line into which the plasmid containing the sequence encoding the poly-A tail is introduced under a temperature condition of 31°C or lower may include one or more of (i) a step of recovering the cell line, (ii) a step of culturing the cell line on a solid medium, and (iii) a step of culturing the cell line in a liquid medium.

[0044] The step (i) of recovering the cell line is a step of culturing the cell line in a competent state after transformation in a medium to recover its physiological function. For example, the medium in the recovery step may be a non-selective medium.

[0045] For example, the culture temperature in the recovery step may be about 31°C or lower, such as about 16°C to 31°C, about 16°C to 30°C, about 16°C to 29°C, about 16°C to 28°C, about 16°C to 27°C, about 16°C to 26°C, about 16°C to 25°C, about 17°C to 25°C, about 18°C to 25°C, or about 19°C to 25°C.

[0046] For example, the culture time in the recovery step may be about 5 minutes to 2 hours. By the step (ii) of culturing the cell line on a solid medium, the transformed cell line can be cultured to form colonies.

[0047] "Colony" means a cluster of cell lines into which the target plasmid is introduced. "Solid medium" is also referred to as "solid culture medium". In solid medium culture, it is possible to confirm whether the strain has grown and whether the plasmid has been introduced, and a selective medium may be used to confirm whether the plasmid has been introduced. For example, the selective medium may contain an antibiotic.

[0048] For example, the temperature of the step of culturing on the solid medium may be about 31°C or lower, for example, about 16°C - 31°C, about 16°C - 30°C, about 16°C - 29°C, about 16°C - 28°C, about 16°C - 27°C, about 16°C - 26°C, about 16°C - 25°C, about 17°C - 25°C, about 18°C - 25°C, or about 19°C - 25°C.

[0049] By the step of culturing the (iii) cell line in a liquid medium, the cell line can be cultured in large quantities. The step of culturing in the liquid medium may be to culture a single colony obtained in the solid medium culture step of (ii).

[0050] For example, the temperature of the step of culturing in the liquid medium may be about 31°C or lower, for example, about 16°C - 31°C, about 16°C - 30°C, about 16°C - 29°C, about 16°C - 28°C, about 16°C - 27°C, about 16°C - 26°C, about 16°C - 25°C, about 17°C - 25°C, about 18°C - 25°C, or about 19°C - 25°C.

[0051] For example, the step of culturing in the liquid medium may include a subculture step. Subculture means transplanting a part of the cells in the original culture into a new medium and culturing them newly. By subculture, the density of the cells can be adjusted appropriately. For example, the subculture is performed 2 times, 3 times, or more, but is not limited thereto, and those skilled in the art can adjust it appropriately.

[0052] The method of the present invention may further include a step of storing the cultured cell line. The step of preservation includes freezing and / or drying the cell line. As the cryopreservative, substances known in the art such as glycerol and dimethylsulfoxide can be used.

[0053] In any of the above-described specific examples, the culturing includes the step of culturing and activating the stored strain. The stored strain may be in a frozen state or a dried state. The culturing in the step of activation may be solid culturing or liquid culturing.

[0054] According to the method of the present invention, in the steps of preserving and activating the cultured cell line, the polyA tail in the plasmid is stably retained. The medium used in the culturing step of the present invention may contain nutrients necessary for culturing the cell line. For example, a normal medium containing a carbon source, a nitrogen source, a phosphorus source, inorganic compounds, amino acids and / or vitamins is used. Also, in the culturing step, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in a suitable manner.

[0055] The cell line of the present invention is not particularly limited, but may be Escherichia coli (E. coli). Escherichia coli is a host cell widely used for large-scale production and cloning of DNA, and the cell line of the present invention includes not only wild-type Escherichia coli but also mutant Escherichia coli engineered to have characteristics advantageous for cloning. For example, a strain lacking RecA is used. However, it is not limited thereto.

[0056] The polyA tail contained in the plasmid becomes shorter due to recombination or deletion in the cell line. However, when the cells are cultured by the method of the present invention, the length of the polyA tail contained in the plasmid can be stably retained.

[0057] For example, compared with a method that includes culturing a cell line transfected with a plasmid at a temperature of 37°C or higher, the method is characterized in that the stability of the polyA tail in the plasmid is improved. The culturing may include one or more of the steps of (i) recovering the cell line, (ii) culturing the cell line on a solid medium, and (iii) culturing the cell line in a liquid medium.

[0058] Another aspect of the present invention is a method for mass-producing a plasmid for mRNA production. The method includes culturing a cell line transformed with a plasmid containing a sequence encoding mRNA containing a poly(A) tail under temperature conditions of 31°C or lower.

[0059] The poly(A) tail, plasmid, cell line, culturing, and temperature conditions are as described above. For example, the produced plasmid may be used as a template for synthesizing mRNA in vitro. For example, the method may further include the step of recovering the plasmid from the cells. However, it is not limited thereto.

[0060] For example, the mRNA containing a poly(A) tail synthesized using the plasmid as a template may be used as a constituent component of an immunogenic composition. For example, the mRNA containing a poly(A) tail synthesized using the plasmid as a template may be used as a constituent component of a therapeutic agent.

[0061] Still another aspect of the present invention is a method for mRNA production. The method includes culturing a cell line transformed with a plasmid containing a sequence encoding mRNA containing a poly(A) tail under temperature conditions of 31°C or lower, and performing transcription using the plasmid of the cell line as a template.

[0062] The poly(A) tail, the mRNA, plasmid, cell line, culture, and temperature conditions containing the same are as described above. For example, after the culturing step, the plasmid may be recovered from the cell line.

[0063] For example, the transcription step may be performed in vitro. For example, the production method may further include a step of formulating the produced mRNA. When formulating, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be used. For example, the production method may include a step of preparing the mRNA in a form contained in lipid nanoparticles. However, it is not limited thereto.

[0064] For example, the produced mRNA containing the poly(A) tail may be used as a constituent component of an immunogenic composition. For example, the produced mRNA containing the poly(A) tail may be used as a constituent component of a therapeutic agent.

Examples

[0065] Hereinafter, the present invention will be described in more detail with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present invention, and the present invention is not limited to these examples and experimental examples.

[0066] Comparative Example 1. Cloning under normal conditions using a general strain In order to obtain plasmid DNA inserted with the Poly(A) sequence, the commonly used DH5α strain and method were used.

[0067] Preparation of Poly(A) fragment and conjugate To prepare the Poly(A) fragment to be inserted into the plasmid, a DNA oligo (custom-made by Cosmo Genetech) consisting of the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 was mixed at a molar ratio of 1:1, annealed by lowering the temperature by 1 degree per minute from 95 degrees, and the gap site was filled with Klenow large fragment (NEB) to prepare a double-stranded polyA DNA fragment. Then, it was cleaved with restriction enzymes SacII and HindIII (NEB), and then inserted into a plasmid (pSKBS01, SEQ ID NO: 3) cleaved with the same restriction enzymes using T4 DNA ligase to prepare a conjugate.

[0068] Transformation (DH5α) The prepared conjugate was transformed into Escherichia coli DH5α (Enzynomics, CP010). The conjugate was placed in the transformation Escherichia coli on ice and allowed to stand for 30 minutes, then heat-shocked at 42 degrees Celsius for 30 seconds. Then, it was cooled on ice for 2 minutes, then SOC medium was added and allowed to recover at 37 degrees Celsius for 1 hour, and then the Escherichia coli was smeared on an antibiotic selection solid medium. The medium smeared with Escherichia coli was cultured at 37 degrees Celsius to generate colonies.

[0069] Confirmation of Poly(A) sequence insertion For the colonies formed on the selection medium after transformation, colony PCR was used to determine the possibility of insertion of the poly(A) fragment. Then, plasmids were isolated from the colonies where the possibility of fragment insertion was confirmed, and then the inserted sequences were confirmed by nucleotide sequence analysis.

[0070] For colony PCR, M13-forward and M13-reverse primers (SEQ ID NOs: 4, 5) were used. When poly(A) was inserted, a PCR product with a size of 550 bp was generated, and when the plasmid without insertion was used, a product with a size of 446 bp was generated. PCR was performed on 40 colonies, and the PCR products were confirmed by agarose gel. As a result of colony PCR, 19 colonies clearly showed 446 bp, indicating that poly(A) was not inserted. The remaining 21 colonies showed a multi-band pattern, but it seemed that a PCR product with a size of 550 bp was included (Figure 1). Those 21 colonies with confirmed fragment insertion possibility were cultured in liquid at 37 °C, and then the plasmid was isolated and subjected to nucleotide sequence analysis by Sanger sequencing.

[0071] However, in all colonies analyzed for the nucleotide sequence, it was confirmed that the poly(A) sequence was not inserted. This result supports that all poly(A) tails disappear in normal condition cloning.

[0072] Comparative Example 2. Cloning using a strain specialized for cloning unstable fragments To obtain plasmid DNA inserted with the poly(A) sequence, the Stbl3 strain, which is one of the strains specialized for cloning unstable fragments containing repetitive sequences, was used. The culture temperature during transformation was set at 37 °C as in Comparative Example 1 described above.

[0073] Transformation (Stbl3) The conjugants prepared in Comparative Example 1 were transformed into Escherichia coli Stbl3 (Invitrogen, C737303). The conjugants were placed into the transformation Escherichia coli on ice and allowed to stand for 30 minutes, and then heat shock was applied at 42 °C for 4 seconds. Then, it was cooled on ice for 2 minutes, and then SOC medium was added and allowed to recover at 37 °C for 1 hour, and then Escherichia coli was smeared on an antibiotic selection solid medium. The medium smeared with Escherichia coli was cultured at 37 °C to generate colonies.

[0074] Confirmation of Poly(A) Sequence Insertion Colony PCR was performed in the same manner as in Comparative Example 1. PCR was carried out on 25 colonies, and the PCR products were confirmed by agarose gel. Seventeen colonies clearly showed 446 bp, indicating that no poly(A) was inserted. The remaining eight colonies showed a multi-band pattern, and it seemed that a PCR product with a size of 550 bp was included (Figure 2). Those eight colonies in which fragment insertion was confirmed were cultured in liquid at 37°C, and then the plasmids were isolated and subjected to nucleotide sequence analysis by Sanger sequencing.

[0075] However, it was confirmed that no poly(A) sequence was inserted in all the colonies analyzed for the nucleotide sequence. This result supports the fact that the poly(A) tail is completely lost even in special strains that can stably clone unstable fragments, and it is unstable for producing stable mRNA vaccines.

Example

[0076] Cloning Using Low-Temperature Conditions From the above comparative examples, it was confirmed that in the normal method of introducing the poly(A) sequence into Escherichia coli and then culturing it at 37°C, there is a problem that the poly(A) sequence is not correctly inserted or stably retained.

[0077] Therefore, a new method for stably retaining the poly(A) tail was established. For this purpose, the culture temperature of Escherichia coli during transformation was lowered below the normal 37°C. Transformation (DH5α, Stbl3) The conjugates prepared in Comparative Example 1 were transformed into Escherichia coli DH5α (Enzynomics, CP010) and Stbl3 (Invitrogen, C737303), respectively. Other conditions except for the Escherichia coli culture temperature were the same as those in Comparative Examples 1 and 2. Escherichia coli was heat-shocked and recovered at 25°C, and then Escherichia coli was smeared on an antibiotic selection solid medium and cultured at 25°C to generate colonies.

[0078] Confirmation of Poly(A) Sequence Insertion Colony PCR was performed in the same manner as in Comparative Example 1. PCR was performed on 10 colonies per strain, and the PCR products were confirmed using an agarose gel.

[0079] In DH5α, 5 colonies and in Stbl3, 7 colonies clearly showed 446 bp, indicating that poly(A) was not inserted. In the remaining colonies, unlike Comparative Examples 1 and 2, no multi-band pattern was shown, and a PCR product with a size of 550 bp was clearly observed (Figure 3). Those colonies in which the possibility of fragment insertion was confirmed were cultured in liquid at 25°C, and then the plasmids were isolated and subjected to nucleotide sequence analysis by Sanger sequencing.

[0080] As a result of the experiment, it was confirmed that the poly(A) sequence was inserted in all the colonies whose nucleotide sequences were analyzed. Therefore, it was confirmed that lower temperature conditions than 37°C are advantageous for stably cloning the poly(A) tail in all of the general strains and the strains specialized for unstable fragment cloning.

Example

[0081] Exploration of Temperature Conditions: Transformation and Sequence Confirmation Using General Strains To explore the temperature range in which the poly(A) tail can be stably retained, plasmid DNA containing the poly(A) tail was transformed under various temperature conditions and cultured in liquid, and their nucleotide sequences were analyzed to confirm that the poly(A) tail was retained.

[0082] Transformation (DH5α) Using the method described above (Comparative Example 1), a plasmid DNA with a poly(A) tail length of 124 was prepared, and the plasmid DNA was transformed into Escherichia coli DH5α (Enzynomics, CP010). The plasmid DNA was introduced into Escherichia coli and allowed to stand on ice for 30 minutes, followed by a heat shock at 42°C for 40 seconds. Then, it was cooled on ice for 2 minutes, and then SOC medium was added and cultured at 37°C, 34°C, 32°C, 31°C, 28°C, 25°C, 22°C, and 19°C for 1 hour each to recover. Subsequently, Escherichia coli was spread on an antibiotic selection solid medium. The medium on which Escherichia coli was spread was cultured at the same temperature as the temperature at which Escherichia coli was recovered so that colonies were formed.

[0083] Confirmation of the length of the Poly(A) sequence To isolate the plasmid for nucleotide sequence analysis, 24 or 25 colonies formed on the solid medium were selected and cultured in liquid at the same temperature as the temperature at which the solid medium was cultured. After liquid culture, the plasmid was isolated, and the length and nucleotide sequence of the poly(A) sequence were analyzed by Sanger sequencing. The Atail-seq-R (SEQ ID NO: 6) primer was used for nucleotide sequence analysis.

[0084] As a result of nucleotide sequence analysis, it was confirmed that the poly(A) sequence was not retained at 37°C, and the stability of the poly(A) length was significantly improved when cultured at 31°C or lower. Therefore, it was confirmed that the stability of the poly(A) sequence improves as the Escherichia coli culture temperature decreases.

[0085] During nucleotide sequence analysis by Sanger sequencing, when analyzing a template in which one sequence is repeated long, due to the polymerase slip phenomenon, a nucleotide may be deleted or added by one in the analysis result (Non-Patent Document 4). Therefore, the ratio of A124±1 with an error range within 1 in the length of the inserted poly(A) sequence was used as an index. The results are shown in Table 1 and Figure 4.

[0086]

Table 1-1

[0087]

Table 1-2

[0088] As shown in Table 1 and Figure 4, when a cell line transformed with a plasmid containing a sequence encoding a poly(A) tail was cultured under temperature conditions of 31°C or lower, the ratio of A124±1 was 21.7 to 55.0%, indicating that the poly(A) tail was stably retained in the plasmid. However, since almost no growth of Escherichia coli occurs under temperature conditions below 16°C (Non-Patent Document 1), temperature conditions below 16°C cannot be applied to the mass vaccine production process. The inability to apply temperature conditions below 16°C to the process will be confirmed again in Example 5 described below.

Example

[0089] Stability during subculture: Transformation and subculture using a strain specialized for retaining unstable fragments To confirm whether the poly(A) sequence is normally retained during continuous subculture under low-temperature conditions, transformation and subculture were performed at 37°C and 25°C using the NEB stable strain, which is one of the strains specialized for retaining unstable fragments.

[0090] Transformation (NEB stable) The plasmid DNA used in Example 2 was transformed into Escherichia coli NEB stable (NEB, C3040H). The plasmid DNA was placed into the Escherichia coli for transformation on ice and left standing for 30 minutes, and then heat shock was applied at 42°C for 40 seconds. Then, it was cooled on ice for 5 minutes, and then NEB 10-beta / Stable Outgrowth medium was added and allowed to recover at 37°C or 25°C for 1 hour, and then the Escherichia coli was spread on an antibiotic selection solid medium. The medium on which the Escherichia coli was spread was cultured at the same temperature as the temperature at which the Escherichia coli was recovered so that colonies were formed.

[0091] Confirmation of the length of the poly(A) sequence after transformation Twenty-five colonies generated after transformation at each temperature were selected and cultured in liquid at the same temperature as the solid culture temperature. The plasmid was isolated and the length of the poly(A) sequence was analyzed by Sanger sequencing. The 3UTR-Xba1-F (SEQ ID NO: 7) primer was used for the nucleotide sequence analysis. It was confirmed that the proportion of colonies in which the length of the poly(A) sequence was maintained at 120 or more was relatively high during transformation at 37°C compared to the results obtained in the commonly used DH5α in Example 2 described above. These results are considered to have occurred by using a strain specialized for retaining unstable fragments.

[0092] However, as can be confirmed from the ratio of A124±1, the colonies that were retained close to the 124 poly(A)s originally possessed by the plasmid used for transformation were very low at 37°C compared to 25°C. Thus, it was reconfirmed that low temperature is advantageous for maintaining the length of the poly(A) sequence regardless of the strain.

[0093]

Table 2

[0094] Subculture Six colonies were selected at 37°C and nine colonies were selected at 25°C from the colonies in which the length of the poly(A) sequence was confirmed by the nucleotide sequence analysis after transformation described above. The culture solutions of the selected colonies were inoculated into a new liquid medium at a volume of 1 / 1000, and then subcultured at the same temperature. Subculture was carried out twice at 37°C and three times at 25°C.

[0095] Confirmation of the length of the poly(A) sequence by subculture The plasmid was isolated from some of the culture solutions during subculture, and the length of the poly(A) sequence was analyzed by Sanger sequencing. The 3UTR-Xba1-F (SEQ ID NO: 7) primer was used for the nucleotide sequence analysis.

[0096] As a result of subculture at 37°C, it was confirmed that the length of the poly(A) sequence was not maintained in most colonies (Table 3). In contrast, when subculture was performed at 25°C, it was confirmed that the length of the poly(A) sequence was maintained in all colonies (Table 4).

[0097] [Table 3]

[0098] [Table 4]

[0099] Therefore, it was confirmed that culturing at a temperature of 31°C or lower during subculture is a condition suitable for maintaining the poly(A) sequence of the plasmid. [Examples]

[0100] Confirmation of stability after preparation of cell line stocks To confirm the ease of storage of the cell line prepared by transformation, the transformed cell line in which the length of A was confirmed to be 124 at 25°C in Example 3 described above was made into the form of a glycerol stock and stored in a -70°C freezer. The stock stored in the frozen state was taken out, liquid cultured at 16°C, 25°C, and 30°C in each test tube, and then each plasmid was separated and subjected to sequence analysis. Since the length of A analyzed from the plasmid separated from the cultured Escherichia coli was all 124, it was confirmed that the length of A was completely maintained during culturing after stock preparation.

[0101] [Table 5] [Examples]

[0102] Confirmation of culture curves at each temperature The following experiments were conducted to confirm that culturing under conditions of less than 16 degrees is not appropriate for process application.

[0103] The transformed cell lines DH5α and NEB stable, in which the length of A was confirmed to be 124 at 25 degrees in Examples 2 and 3, were each made into the form of a glycerol stock and stored in a freezer at -70°C. To confirm the culture curves at each temperature, the stock was taken out and cultured at 200 rpm for 48 hours under the conditions of 13 degrees, 16 degrees, 19 degrees, 22 degrees, and 25 degrees, while measuring OD 600 at predetermined times. As a result of the experiment, it was confirmed that there was almost no cell growth at 13 degrees, which is less than 16 degrees (Figure 5).

[0104] From the above description, those skilled in the technical field to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea and essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be construed as including all changes and modifications derived from the meaning and scope of the claims and their equivalent concepts, rather than the description.

[0105] (Sequence Listing)

[0106] [Chemical formula]

[0107] [Chemical formula]

[0108] [Chemical formula] [Chemical formula]

[0109] [Chemistry]

[0110] [Chemistry]

[0111] [Chemistry]

[0112] [Chemistry]

Claims

1. A method for stably retaining a poly(A) tail in a plasmid, comprising culturing a cell line transformed with a plasmid containing a sequence encoding a poly(A) tail under temperature conditions of 19°C to 25°C. A method for stably retaining a poly(A) tail in a plasmid.

2. The method according to claim 1, wherein the culturing step comprises (1) recovering the cell line under temperature conditions of 19°C to 25°C.

3. The method according to claim 2, further comprising, after the recovery step of (1), (2) culturing the cell line on a solid medium under temperature conditions of 19°C to 25°C to form colonies.

4. The method according to claim 3, wherein the culturing further comprises (3) culturing the colonies formed by the solid medium culture of (2) in a liquid medium under temperature conditions of 19°C to 25°C.

5. The method according to claim 4, wherein the step of culturing the colonies in a liquid medium comprises performing subculture.

6. The method according to claim 1, wherein the cell line is Escherichia coli (E. coli).

7. The method according to claim 1, wherein the transformation comprises introducing a plasmid containing a sequence encoding a poly(A) tail into the cell line using heat shock or electroporation.

8. The method according to claim 7, wherein the introduction is performed at a temperature of 37°C to 42°C.

9. The method according to claim 1, wherein the stability of the poly(A) tail in the plasmid is improved compared to a method comprising culturing a cell line transformed with a plasmid containing a sequence encoding a poly(A) tail under temperature conditions of 37°C or higher.

10. The method according to claim 1, wherein the poly(A) tail contains 20 to 400 adenines (A).

11. A method for mass-producing a plasmid for mRNA production, comprising culturing a cell line transformed with a plasmid containing a sequence encoding an mRNA containing a poly(A) tail under temperature conditions of 19°C to 25°C. A method for mass-producing a plasmid for mRNA production.

12. Culturing a cell line transformed with a plasmid containing a sequence encoding an mRNA containing a poly(A) tail under temperature conditions of 19°C to 25°C, and A step of performing transcription using the plasmid of the cell line as a template, and A method for producing mRNA containing a poly-A tail.

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