In-vitro transcription buffer system, in-vitro transcription solution containing in-vitro transcription buffer system and in-vitro transcription method using in-vitro transcription buffer system

Abstract

The invention provides an in-vitro transcription buffer system, an in-vitro transcription solution containing the in-vitro transcription buffer system and an in-vitro transcription method using the in-vitro transcription buffer system. The in-vitro transcription buffer system comprises the following components with the following concentrations: 200-600mM of Tris-HCl, 10-20mM of Tris-HCl The concentration of the divalent metal ions is 165 to 300 mM; 20 to 50 mM of spermidine; and Triton X-100 with the concentration of 0.01%-0.05%. When the in-vitro transcription buffer system and the in-vitro transcription solution containing the in-vitro transcription buffer system provided by the invention are used for in-vitro transcription, the use amount of hat analogues can be greatly reduced while the high capping rate is ensured.

Description

[0001] This application claims the priority of Chinese patent application with application date of 2023 / 6 / 21, application number of 2023107430122, and invention name of "In vitro transcription method". This application cites the full text of the above Chinese patent application. Technical Field

[0002] The present invention relates to the field of biotechnology, in particular to an in vitro transcription buffer system, an in vitro transcription solution containing the same and an in vitro transcription method using the same. Background Art

[0003] With the outbreak and spread of the new coronavirus, mRNA vaccines with short R&D cycles and high biosafety have been favored by major research institutions and pharmaceutical companies. mRNA vaccines are to introduce mRNA containing encoded antigenic proteins into the human body and directly translate them to generate corresponding antigenic proteins, thereby inducing the body to produce specific immune responses and achieve the effect of preventive immunity. The production process of mRNA vaccines mainly includes the preparation of plasmid stock solution, the preparation and purification of mRNA stock solution, and the production of mRNA vaccine preparations. Among them, the mRNA stock solution preparation process directly affects the quality and production cost of mRNA vaccines, so it is very important to optimize the production process of mRNA stock solution. The production process of mRNA stock solution begins with the mass production of amplified plasmid DNA (pDNA), using linearized plasmids or plasmid PCR products as templates, using RNA polymerases such as T7, T3 or Sp6 phage polymerases to transcribe mRNA, and after DNase I enzyme degradation of the template and purification, the stability of mRNA is enhanced by adding a cap structure at the 5′ end and a ployA structure at the 3′ end.

[0004] So far, there are two main large-scale capping methods, namely two-step enzymatic capping and one-step co-transcriptional capping. Enzymatic capping is the most traditional capping method, which can achieve almost 100% capping rate, but the capping enzyme is relatively expensive, the enzymatic cost required is high, and the batch production cost is high. In addition, this method also introduces additional proteins and S-adenosylmethionine (SAM), which requires multiple purifications and a cumbersome process. Although the one-step co-transcriptional capping has the advantage of simple process, the capping efficiency is low.

[0005] Therefore, we must improve the existing in vitro transcription (IVT) mRNA production scheme to improve the capping efficiency while reducing the production cost of mRNA. Summary of the invention

[0006] Among the existing in vitro transcription capping methods, the two-step method has the problems of expensive capping enzymes, high enzymatic costs and complicated process flow, and the one-step method has the problem of low capping efficiency. To solve the above problems, the present invention provides an in vitro transcription buffer system, an in vitro transcription solution containing the same and an in vitro transcription method using the same.

[0007] The present invention solves the above technical problems by the following technical means:

[0008] The first aspect of the present invention provides an in vitro transcription buffer system, wherein the in vitro transcription buffer system comprises components of the following concentrations:

[0009]

[0010] In some embodiments of the present invention, the in vitro transcription buffer system comprises components at the following concentrations:

[0011]

[0012] In some optional technical solutions of the present invention, the transcription buffer system further comprises 50-100 mM dithiothreitol; and / or, in the transcription buffer, the divalent metal ion is Mg 2+ .

[0013] In some preferred technical solutions of the present invention, Mg 2+ It is added in the form of magnesium acetate, magnesium chloride or magnesium sulfate; the concentration of dithiothreitol is 50 mM or 100 mM; the concentration of Tris-HCl is 400 mM; and the concentration of Triton X-100 is 0.02%.

[0014] In some more preferred technical solutions of the present invention, the components of the transcription buffer are selected from any one of the following groups:

[0015]

[0016]

[0017] The second aspect of the present invention provides an in vitro transcription solution, which includes a first solution and a second solution; optionally, it also includes a transcription template, preferably a linearized plasmid transcription template; the first solution contains an in vitro transcription buffer system, a cap analog, guanine triphosphate, adenosine triphosphate, cytosine triphosphate, pseudouridine triphosphate and RNA polymerase; the second solution contains guanine triphosphate and pseudouridine triphosphate.

[0018] In some preferred technical embodiments of the present invention, the RNA polymerase is T7 RNA polymerase.

[0019] In some preferred technical solutions of the present invention, the in vitro buffer system is as described in the first aspect of the present invention.

[0020] In some more preferred technical solutions of the present invention, in the first solution, the initial concentration of the in vitro transcription buffer system is 10×;

[0021] The concentration of the cap analog is 1-8 mM;

[0022] The concentration of guanosine triphosphate is 0.25mM-1.5mM;

[0023] The concentration of adenosine triphosphate is 9mM-11mM;

[0024] The concentration of cytosine triphosphate is 9mM-11mM;

[0025] The concentration of pseudouridine triphosphate is 0.25mM-1.5mM;

[0026] The concentration of the linearized plasmid transcription template is 5mM-20mM;

[0027] The concentration of the RNA polymerase is 5mM-20mM;

[0028] And / or, in the second solution, the concentration of guanosine triphosphate is 8.5 mM-9.75 mM; the concentration of pseudouridine triphosphate is 8.5 mM-9.75 mM.

[0029] In some specific technical solutions of the present invention, in the first solution, the concentration of the cap analog is 1 mM, 3 mM, 6 mM or 8 mM;

[0030] The concentration of guanosine triphosphate was 0.25 mM, 0.5 mM, 1 mM or 1.5 mM;

[0031] The concentration of adenosine triphosphate was 10 mM;

[0032] The concentration of cytidine triphosphate was 10 mM;

[0033] The concentration of pseudouridine triphosphate was 0.25 mM, 0.5 mM, 1 mM, or 1.5 mM;

[0034] The concentration of the linearized plasmid transcription template is 10 mM;

[0035] The concentration of the RNA polymerase is 10 mM;

[0036] In the second solution, the concentration of guanosine triphosphate is 8.5 mM, 9 mM, 9.5 mM or 9.75 mM; the concentration of pseudouridine triphosphate is 8.5 mM, 9 mM, 9.5 mM or 9.75 mM.

[0037] In some embodiments of the present invention, in the in vitro transcription solution, the total guanosine triphosphate concentration of the first solution and the second solution is 8-12 mM; the total pseudouridine triphosphate concentration is 8-12 mM.

[0038] In some specific embodiments of the present invention, in the first solution, the total guanosine triphosphate concentration of the first solution and the second solution is 10 mM; the total pseudouridine triphosphate concentration is 10 mM.

[0039] The concentrations of the cap analog, guanosine triphosphate, adenosine triphosphate, cytosine triphosphate, pseudouridine triphosphate, linearized plasmid transcription template, and RNA polymerase are all working concentrations.

[0040] The third aspect of the present invention provides an in vitro transcription solution comprising a first solution and a second solution, wherein the second solution is used to be added to the first solution after the first solution has reacted for 30 minutes; the first solution comprises an in vitro transcription buffer, a cap analog, guanine triphosphate, adenosine triphosphate, cytosine triphosphate, pseudouridine triphosphate, a linearized plasmid transcription template, T7 RNA polymerase, and the balance is ultrapure water; the second solution consists of guanine triphosphate and pseudouridine triphosphate.

[0041] Optionally, the initial concentrations of the cap analog, guanosine triphosphate, adenosine triphosphate, cytosine triphosphate, and pseudouridine triphosphate in the in vitro transcription solution provided by the present invention are each 100 millimoles per liter (mM).

[0042] Optionally, based on 1000 parts by volume of the total volume of the first solution, the content of the cap analog is 10 to 20 parts by volume.

[0043] Optionally, based on the total volume of the first solution being 1000 parts by volume, the sum of the volumes of guanosine triphosphate in the first solution and the guanosine triphosphate in the second solution is 80 to 120 parts by volume.

[0044] Optionally, based on 1000 parts by volume of the total volume of the first solution, the sum of the volumes of the pseudouridine triphosphate in the first solution and the pseudouridine triphosphate in the second solution is 80 to 120 parts by volume.

[0045] Optionally, based on the total volume of the first solution being 1000 parts by volume, the volume of the second solution is 160 to 240 parts by volume.

[0046] A fourth aspect of the present invention provides an in vitro transcription method, which uses the in vitro transcription solution described in any of the above technical solutions to perform a capping reaction.

[0047] In some embodiments of the present invention, the second solution is used to be added to the first solution after the first solution has reacted.

[0048] In some preferred embodiments of the present invention, the reaction time is 5-60 minutes.

[0049] In some more preferred embodiments of the present invention, the in vitro transcription buffer system is the in vitro transcription buffer system as described in the first aspect of the present invention.

[0050] In some technical schemes of the present invention, in the capping reaction, the first solution is first reacted at 34-39° C. for 0.5-1 hour, and then the second solution is added to the first solution, and the reaction is continued for 1.5-2 hours;

[0051] Optionally, in the capping reaction of the in vitro transcription method, the first solution is first reacted at 37° C. for 0.5 hour, and then the second solution is added to the first solution and the reaction is continued for 1.5 to 2 hours.

[0052] In a specific embodiment of the present invention, in the capping reaction of the in vitro transcription method, the first solution is first reacted at 37° C. for 0.5 hour, and then the second solution is added to the first solution and the reaction is continued for 1.5 hours.

[0053] The fifth aspect of the present invention provides a kit for in vitro transcription of mRNA, the kit comprising the transcription buffer system as described in the first aspect of the present invention or the in vitro transcription solution as described in the second aspect or the third aspect of the present invention.

[0054] The sixth aspect of the present invention provides use of the in vitro transcription buffer system as described in the first aspect of the present invention or the in vitro transcription solution as described in the second aspect or the third aspect of the present invention in in vitro transcription.

[0055] The positive and progressive effects of the present invention are:

[0056] The in vitro transcription buffer system, in vitro transcription solution and in vitro transcription method using the same provided by the present invention can significantly reduce the amount of cap analogs used while ensuring a high capping rate. DETAILED DESCRIPTION

[0057] Unless otherwise specified, the terms of the present invention are defined as follows.

[0058] In certain aspects, "polynucleotide" includes mRNA. In one aspect, the mRNA is a synthetic mRNA. In some aspects, the synthetic mRNA includes at least one non-natural base. In some aspects, all core bases of a certain class are replaced with non-natural bases (for example, all uridines in the polynucleotides disclosed herein can be replaced with non-natural bases, for example, 5-methoxyuridine). In some aspects, polynucleotides (for example, synthetic RNA or synthetic DNA) only include natural bases, i.e., A (adenosine), G (guanosine), C (cytidine) and T (thymidine) in the case of synthetic DNA, or A, C, G and U (uridine) in the case of synthetic RNA.

[0059] Those skilled in the art will appreciate that the T in the codon map disclosed herein is present in DNA, and T will be replaced by U in the corresponding RNA. For example, the codon nucleotide sequence disclosed herein in the form of DNA, such as a vector or in vitro translation (IVT) template, will be transcribed as U in its corresponding transcribed mRNA. In this regard, codon-optimized DNA sequences (including T) and their corresponding mRNA sequences (including U) are both considered to be codon-optimized nucleotide sequences. In addition, equivalent codon maps can also be generated by replacing one or more bases with non-natural bases. Thus, for example, TTC codons (DNA maps) correspond to UUC codons (RNA maps), and UUC codons correspond to ΨΨC codons (RNA maps, wherein U is replaced by pseudouridine).

[0060] The term "nucleotide sequence encoding" refers to a nucleic acid (e.g., mRNA or DNA molecule) coding sequence encoding a polypeptide. The coding sequence may further include start and stop signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may further include a sequence encoding a signal peptide.

[0061] "PolyA" or "poly (A)" is a region of an mRNA that is located downstream of the 3'UTR, for example, directly downstream (i.e., 3'), and contains multiple consecutive adenosine monophosphates. A polyA tail can contain 10 to 300 adenosine monophosphates. For example, a polyA tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a poly (A) tail contains 50 to 250 adenosine monophosphates. In relevant biological contexts (e.g., in cells, in vivo), the function of the poly(A) tail is to protect the mRNA from enzymatic degradation, such as in the cytoplasm, and to aid in transcription termination, mRNA export from the nucleus, and translation.

[0062] As used herein, the term "synthesize" refers to artificial production, preparation and / or manufacture. The synthesis of polynucleotides or other molecules can be chemical or enzymatic. As used herein, "expression" of a nucleic acid sequence refers to the translation of a polynucleotide (such as mRNA) into a polypeptide or protein and / or post-translational modification of a polypeptide or protein. Methods of transfection include, but are not limited to, chemical methods, physical treatments, and cationic lipids or mixtures.

[0063] The term "transcription" refers to the process of producing mRNA (e.g., mRNA sequence or template) from DNA (e.g., DNA template or sequence). "Transfection" refers to the introduction of a polynucleotide (e.g., exogenous nucleic acid) into a cell, wherein the polypeptide encoded by the polynucleotide is expressed (e.g., mRNA) or the polypeptide modulates a cellular function (e.g., siRNA, miRNA). For example, transfection can occur in vitro, ex vivo, or in vivo.

[0064] As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting the survival, growth, and / or spread of a tumor. Subjects who do not show signs of a disease, disorder, and / or condition and / or subjects who show only early signs of a disease, disorder, and / or condition may be treated to reduce the risk of developing pathology associated with the disease, disorder, and / or condition.

[0065] The term "therapeutic agent" or "prophylactic agent" refers to any agent that has a therapeutic, diagnostic and / or prophylactic effect and / or causes a desired biological and / or pharmacological effect when administered to a subject. Therapeutic agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins and nucleic acids.

[0066] In certain embodiments, the treatment and / or preventive measure is an mRNA. The mRNA can encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0067] As used herein, "and / or" should be considered to specifically disclose each of the two specific features or components, whether or not there is another. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" or "one or more" used in phrases such as "A, B and / or C" or "one or more of A, B, and C" is intended to include the following: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0068] It should also be noted that the term "comprising" is intended to be open and allowable, but not require the inclusion of additional elements or steps. When the term "comprising" is used, the terms "consisting essentially of" and "consisting of" are also included. When a composition is described as having, including, or comprising a particular ingredient, it is contemplated that the composition also consists essentially of or consists of the described ingredient. Similarly, when a method or process is described as having, including, or comprising a particular step, the method or process also consists essentially of or consists of the described steps. In addition, it should be understood that the order of the steps or the order in which certain actions are performed is unimportant as long as the invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0069] When using a 1190 μL system, the actual concentration of each component should be divided by 1.19 or approximately divided by 1.2 to convert it to the concentration of a 1000 μL system. Similarly, when using a 119, 118, 117, 119.5 μL system, the actual concentration of each component should be divided by 1.19, 1.18, 1.17, 1.195 or approximately divided by 1.2 to convert it to the concentration of a 100 μL system.

[0070] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0071] Example 1 Comparison of the effects of in vitro transcription buffer and traditional buffer

[0072] Reagents

[0073] Ultrapure water;

[0074] In vitro transcription buffer 1, 10×, the components of which include, for example, Tris·HCl (400 mM, pH=8.0), 165 mM magnesium acetate, 100 mM dithiothreitol (DTT), 20 mM spermidine, and 0.02% (vol / vol) Triton X-100;

[0075] cap analog Cap1 (APE×BIO, catalog number B8176);

[0076] Adenosine triphosphate, ATP (Thermo Fisher, catalog number R0481 or equivalent);

[0077] Cytosine triphosphate, CTP (Thermo Fisher, catalog number R0481 or equivalent);

[0078] Guanosine triphosphate, GTP (Thermo Fisher, catalog number R0481 or equivalent);

[0079] Pseudouridine triphosphate, pseudo UTP (APE×BIO, catalog number B7972);

[0080] T7 RNA polymerase (APE×BIO, catalog number K1083R);

[0081] Based on the standard of generating approximately 5 mg of mRNA per 1 mL reaction, in other embodiments of the present invention, the reaction can be scaled up or down according to these ratios.

[0082] equipment

[0083] 37°C water bath.

[0084] Nuclease-free microcentrifuge tubes (VWR, catalog number 87003-294 or similar).

[0085] Benchtop shaker.

[0086] Microcentrifuge.

[0087] Micropipette.

[0088] Chill enzyme cooler or ice bucket.

[0089] Parafilm (sealing film).

[0090] Prepare reagents: Clean the work area before starting a reaction to avoid common lab contaminants. Treat surfaces and pipettes with a 10% bleach solution to ensure there is no nucleic acid cross-contamination. Allow the bleach solution to remain on the surfaces for 10 minutes. Wipe all bleach-treated surfaces and pipettes with RNase-free water and then wipe with 70% ethanol. Change gloves frequently to ensure there is no RNase contamination from other areas of the lab.

[0091] The water bath was kept at a constant temperature of 37°C.

[0092] Thaw NTPs, IVT (in vitro transcription) buffer, and linearized plasmid transcription template (hereinafter referred to as template) at room temperature. Vortex NTPs and 10× IVT buffer to mix well. Check the in vitro transcription buffer for precipitation. If precipitation is observed, heat the buffer to 37°C for 2 minutes. Vortex again to mix and check for precipitation. Repeat the heating and mixing steps until no precipitation is observed.

[0093] Prepare the reaction: Mix the components in an RNase-free tube. Example 1 and Comparative Examples 1 to 4 provided by the present invention are for a 1 mL reaction, as shown in Table 1. After adding 10× in vitro transcription buffer, mix the solution with an oscillator to ensure that the reaction is evenly mixed before adding other reagents. Centrifuge the tube slightly to collect the liquid on the lid. Mix the linearized plasmid transcription template by pipetting up and down or gently inverting, and then briefly centrifuge in a centrifuge of appropriate size. Longer templates (>8kb) can be added to a final concentration of 100μg / mL to achieve a molar concentration similar to that of shorter templates. When on the countertop, the enzyme should be kept cool, and an ice bucket or enzyme cooler can be used. Gently shake the enzyme or gently invert to mix. Due to the high viscosity of the enzyme, operate slowly when pipetting. Mix by gently pipetting until uniform. Close the tube with a sealing film. Avoid vigorous stirring or bubbles. In this step, whether in the embodiment or the comparative example, only the first solution is added, and the second solution is not added. Different from the comparative examples, the amount of the cap analogue used in the examples of the present invention is much lower than that used in the traditional reaction system (as shown in comparative examples 1 to 4).

[0094] Table 1

[0095]

[0096]

[0097] Capping reaction: Incubate the reaction in a 37°C water bath or heat block. Use sealing film to prevent sample evaporation and possible water bath contamination. For transcripts less than 4 kb in length, react for 2 to 3 hours. For transcripts greater than 4 kb in length, react for 2 hours to minimize heat exposure that may cause RNA degradation. The total reaction time for the embodiments of the present invention and the comparative examples is 2 hours. Unlike the comparative examples, the embodiments of the present invention add the second solution after 30 minutes of reaction and continue the reaction for another 90 minutes.

[0098] After the capping reaction, the linearized plasmid transcription template was digested by DNaseI, and 50 μL of each sample was taken for sample detection. The mRNA yield and capping rate are shown in Table 2. It can be seen that the solution provided by the present invention still maintains a high capping rate when the amount of Cap1 capping analog is the lowest.

[0099] Table 2

[0100]

[0101] Example 2 Reaction Step Adjustment

[0102] Using in vitro transcription buffer 1, add all substances in a stepwise or one-step manner according to the reaction system shown in Table 3.

[0103] Table 3

[0104]

[0105]

[0106] Experimental steps of the step-by-step method:

[0107] 1. In the embodiment of this experiment, the second solution was added after the reaction lasted for 30 minutes, and the reaction was continued for another 90 minutes.

[0108] 2. After the capping reaction is completed, the linearized plasmid transcription template is digested with DNaseI, purified, and the mRNA yield is calculated.

[0109] One-step experimental steps: direct 37°C water bath for 2 h. The experimental results are shown in Table 4.

[0110] Table 4

[0111] Grouping mRNA yield mg / mL Step-by-step approach 5.4 One-step method 1.9

[0112] Example 3 In vitro transcription buffer formulation and reaction method

[0113] The in vitro transcription buffer formula is shown in Tables 5 to 7, the reaction system is the system of the step-by-step method in Table 3, and the experimental steps are the steps of the step-by-step method in Example 2.

[0114] Table 5

[0115]

[0116] Table 6

[0117]

[0118] Table 7

[0119]

[0120] The experimental results are shown in Table 8.

[0121] Table 8

[0122] Grouping mRNA yield mg / mL In vitro transcription buffer 1 5.4 In vitro transcription buffer 2 4.5 In vitro transcription buffer 3 3.5 In vitro transcription buffer 4 2.1 In vitro transcription buffer 5 0.7 In vitro transcription buffer 6 5.4 In vitro transcription buffer 7 0.7 In vitro transcription buffer 8 1.0 In vitro transcription buffer 9 5.7 In vitro transcription buffer 12 5.7 In vitro transcription buffer 10 5.6 In vitro transcription buffer 11 5.1

[0123] Example 4 Reaction system adjustment

[0124] In vitro transcription buffer 1 was used, the reaction system was as shown in Table 9, and the experimental steps were the same as the step-by-step method in Example 2.

[0125] Table 9

[0126]

[0127]

[0128] The results are shown in Table 10.

[0129] Table 10

[0130]

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of implementation of the present invention. Therefore, any changes or modifications made according to the claims and description of the present invention should fall within the scope of the patent of the present invention.

Claims

1. An in vitro transcription buffer system, characterized in that: The in vitro transcription buffer system comprises the following components at the following concentrations: Optionally, the in vitro transcription buffer system further comprises 50-100 mM dithiothreitol; and / or, in the transcription buffer system, the divalent metal ion is Mg 2+ ; Preferably, Mg 2+ It is added in the form of magnesium acetate, magnesium chloride or magnesium sulfate; the concentration of dithiothreitol is 50 mM or 100 mM; the concentration of Tris-HCl is 400 mM; and the concentration of Triton X-100 is 0.02%.

2. An in vitro transcription solution, characterized in that: The in vitro transcription solution comprises a first solution and a second solution; optionally, further comprises a transcription template, preferably a linearized plasmid transcription template; the first solution comprises an in vitro transcription buffer system, a cap analog, guanine triphosphate, adenosine triphosphate, cytidine triphosphate, pseudouridine triphosphate and RNA polymerase; the second solution comprises guanine triphosphate and pseudouridine triphosphate; Preferably, the RNA polymerase is T7 RNA polymerase; and / or, the in vitro buffer system is as described in claim 1; More preferably, in the first solution, the initial concentration of the in vitro transcription buffer system is 10×; The concentration of the cap analog is 1-8 mM; The concentration of guanosine triphosphate is 0.25mM-1.5mM; The concentration of adenosine triphosphate is 9mM-11mM; The concentration of cytosine triphosphate is 9mM-11mM; The concentration of pseudouridine triphosphate is 0.25mM-1.5mM; The concentration of the linearized plasmid transcription template is 5mM-20mM; The concentration of the RNA polymerase is 5mM-20mM; And / or, in the second solution, the concentration of guanosine triphosphate is 8.5 mM-9.75 mM; the concentration of pseudouridine triphosphate is 8.5 mM-9.75 mM.

3. The in vitro transcription solution according to claim 2, characterized in that In the in vitro transcription solution, the total guanosine triphosphate concentration of the first solution and the second solution is 8-12 mM; the total pseudouridine triphosphate concentration is 8-12 mM.

4. An in vitro transcription method, characterized in that The in vitro transcription method uses an in vitro transcription solution to perform a capping reaction, wherein the in vitro transcription solution includes a first solution and a second solution, wherein the second solution is added to the first solution after the first solution reacts; wherein The first solution comprises an in vitro transcription buffer system, a cap analog, guanosine triphosphate, adenosine triphosphate, cytosine triphosphate, pseudouridine triphosphate, a linearized plasmid transcription template, and T7 RNA polymerase, and the balance is ultrapure water; The second solution consists of guanosine triphosphate and pseudouridine triphosphate; Preferably, the reaction time is 5-60 minutes; More preferably, the in vitro transcription buffer system is the in vitro transcription buffer system as claimed in claim 1.

5. An in vitro transcription method, characterized in that: The in vitro transcription method uses an in vitro transcription solution to perform a capping reaction, wherein the in vitro transcription solution includes a first solution and a second solution, wherein the second solution is added to the first solution after the first solution has reacted for 30 minutes; wherein The first solution comprises an in vitro transcription buffer, a cap analog, guanosine triphosphate, adenosine triphosphate, cytosine triphosphate, pseudouridine triphosphate, a linearized plasmid transcription template, and T7 RNA polymerase, and the balance is ultrapure water; The second solution consists of guanosine triphosphate and pseudouridine triphosphate.

6. The in vitro transcription method according to claim 4 or 5, characterized in that The initial concentration of the cap analog, guanine nucleoside triphosphate, adenosine nucleoside triphosphate, cytosine nucleoside triphosphate, and pseudouridine nucleoside triphosphate in the in vitro transcription solution is 100 mmol / L.

7. The in vitro transcription method according to claim 4 or 5, characterized in that: Based on 1000 parts by volume of the total volume of the first solution, the content of the cap analog is 10-20 parts by volume.

8. The in vitro transcription method according to claim 4 or 5, characterized in that: Based on 1000 parts by volume of the total volume of the first solution, the sum of the volumes of guanosine triphosphate in the first solution and the guanosine triphosphate in the second solution is 80-120 parts by volume.

9. The in vitro transcription method according to claim 4 or 5, characterized in that: Based on 1000 parts by volume of the total volume of the first solution, the sum of the volumes of the pseudouridine triphosphate in the first solution and the pseudouridine triphosphate in the second solution is 80-120 parts by volume.

10. The in vitro transcription method according to claim 4 or 5, characterized in that: Based on the total volume of the first solution being 1000 parts by volume, the volume of the second solution is 160-240 parts by volume; preferably, based on the total volume of the first solution being 1000 parts by volume, the total volume of the second solution is 170-195 parts by volume.

11. The in vitro transcription method according to any one of claims 4 to 10, characterized in that: In the capping reaction, the first solution is first reacted at 34-39° C. for 5-60 minutes, and then the second solution is added to the first solution, and the reaction is continued for 1.5-2 hours; Preferably, in the capping reaction, the first solution is first reacted at 37° C. for 0.5 hour, and then the second solution is added to the first solution, and the reaction is continued for 1.5-2 hours, for example, 1.5 hours.

12. A kit for in vitro transcription of mRNA, characterized in that: The kit comprises the transcription buffer system as claimed in claim 1 or the in vitro transcription solution as claimed in claim 2 or 3.

13. Use of the in vitro transcription buffer system according to claim 1 or the in vitro transcription solution according to claim 2 or 3 in in vitro transcription.

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