Production method and application of plasmid for mRNA in-vitro transcription

Abstract

The invention belongs to the technical field of genetic engineering, and relates to a separated escherichia coli cell population containing exogenous plasmids. A method for preparing RCB, PCB, MCB, and WCB using the cell population; a production plasmid for producing mRNA using the WCB; and a method for producing mRNA using the WCB. Compared with traditional WCB, the yield and purity of plasmids produced by the method and the purity of mRNA transcribed in vitro are higher.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering. Background Art

[0002] The rapid development of IVT (in vitro transcription)-based synthesis platforms, nucleotide chemistry, delivery systems and other related technologies has enabled the rapid development of mRNA drugs for different diseases. Traditionally, mRNA is synthesized through IVT reactions, using T3, T7 or SP6 RNA polymerase to catalyze the formation of RNA in the 5'→3' direction from linearized plasmid DNA. The DNA template contains a promoter, a 5' untranslated region (5'UTR), an open reading frame (ORF), a 3' untranslated region (3'UTR) and a poly A tail.

[0003] Plasmid DNA can exist in three conformations: 1) supercoiled (sc), also commonly referred to as covalently closed circular DNA (ccc); 2) open circular (oc) DNA; and 3) linear DNA. In addition, plasmids usually appear in the form of polymers, which is caused by homologous recombination or continuous replication in the host bacteria.

[0004] In industry, it is necessary to continuously provide plasmid DNA with stable sequence for IVT mRNA production. Therefore, changes in strains and plasmids should be avoided. In addition, when the strain is contaminated, it is also necessary to ensure that there are backup strains to ensure subsequent plasmid production. Based on the above requirements, it is necessary to establish a strain cell bank (Cell Bank) to provide cells that are qualified, of the same quality, and can be continuously and stably propagated for the production of plasmid DNA.

[0005] In industrial production, cell banks are mainly divided into research cell banks (RCB), pre-master cell banks (PCB), master cell banks (MCB) and working cell banks (WCB). Among them, the construction method of RCB mainly includes: transforming the correctly sequenced plasmid into host cells (such as Escherichia coli cells), picking the monoclonal clones grown on the corresponding resistance plate after culturing under appropriate conditions, picking the monoclonal clones and inoculating the culture medium, and culturing under appropriate conditions until the bacterial solution becomes turbid. According to the experimental data such as the plasmid morphology extracted from the monoclonal bacterial solution (i.e., selecting the cell group that is basically a monomeric plasmid), the plasmid sequencing results, etc., the cell group that meets the requirements is initially screened. After that, the cell group after the above screening is further screened, mainly including continuous subculture of the cell group after the above screening according to the specified conditions, and the plasmid morphology extracted before and after the subculture, the plasmid yield, the polyA integrity (for example, the CE method test), the comprehensive mRNA purity, the cell expression and other aspects are examined, so as to screen out the RCB that meets the requirements. For details, please refer to: "Establishment of engineered bacteria for hepatitis B therapeutic DNA vaccine and preliminary optimization of fermentation medium", "Screening of highly stable host bacteria for hepatitis B therapeutic replicable DNA vaccine pSVK-HBVA and selection of basic culture medium", and "Establishment and quality research of bacterial strains for the production of recombinant trivalent human papillomavirus vaccine".

[0006] Use the screened RCB as a seed to build a PCB library: take a RCB and thaw it at room temperature, mix it and inoculate it into the culture medium according to a certain ratio, culture it under appropriate conditions until the bacterial liquid becomes turbid, then add an equal volume of glycerol solution to mix it and package it, first temporarily store it in an ultra-low temperature refrigerator overnight, and then transfer it to liquid nitrogen for long-term storage. Then prepare MCB and WCB: take a PCB / MCB glycerol bacteria and thaw it at room temperature, mix it and inoculate it into the culture medium according to a certain ratio, culture it under appropriate conditions until the bacterial liquid becomes turbid, then add an equal volume of glycerol solution to mix it and package it. MCB is stored in an ultra-low temperature refrigerator for 12 to 48 hours and then transferred to a liquid nitrogen tank for long-term storage; WCB is directly stored in an ultra-low temperature refrigerator for long-term storage.

[0007] The inventors found that in the process of constructing RCB, PCB, MCB and WCB, and using WCB to ferment and produce plasmids, the integrity of the poly A tail of the template plasmid was reduced due to multiple replications of the plasmid in the host bacteria. Specifically, when the plasmid produced by WCB is transcribed in vitro, mRNA containing poly A tails of different lengths will be produced, thereby reducing the uniformity or purity of the mRNA API. Therefore, it is urgent to establish a method for maintaining the stability of the plasmid sequence for mRNA transcription during cell passage.

[0008] In order to solve the above problems, the inventors found that after the exogenous mRNA production plasmid was transferred into the competent E. coli cells, the transformed E. coli could be divided into the following three categories according to the topological structure of the plasmid in the E. coli: 1) Class I clones, in which the supercoiled monomer of the plasmid was greater than 10%; 2) Class II clones, in which the supercoiled monomer of the plasmid was less than 10% and the supercoiled dimer of the plasmid was 10-100%; and Class III clones, in which the supercoiled monomer of the plasmid was less than 10% and the supercoiled dimer of the plasmid was less than 10%. The inventors found that after multiple passages, the plasmid topological structure of Class II clones remained basically unchanged, the plasmid yield was higher, and the poly A integrity in the plasmid was better than that of Class I and Class III clones. The purity and cell expression of the mRNA transcribed by the cells after multiple passages and linearization were also better than those of the other two clones. The above results show that the class II clones obtained by the method of the present invention have a higher plasmid yield after multiple passages, and can maintain the structural integrity of the plasmid poly A tail during multiple passages, and the mRNA purity and cell expression effect obtained by transcription using this linearized plasmid as a template are better. The present invention provides a new idea for the selection of plasmids in IVT reactions, and can be used to construct relevant cell banks (such as RCB, PCB, MCB and WCB) for commercial production of mRNA. Summary of the invention

[0009] In one aspect of the present invention, the present invention provides an isolated Escherichia coli cell population containing an exogenous plasmid, wherein the exogenous plasmid can be transcribed into mRNA in vitro, and the exogenous plasmid comprises a plasmid backbone, a promoter for regulating an exogenous coding gene (such as a T7 promoter, an SP6 promoter, a T3 promoter or a T5 promoter), a 5' untranslated region (5'UTR), an exogenous coding gene, a 3' untranslated region (3'UTR) and a polyA tail, wherein the topological structure of the exogenous plasmid in the Escherichia coli cell population accounts for: less than 10% (preferably less than 5%, more preferably less than 2%, most preferably less than 1%) of the plasmid supercoiled monomer, and 10-100% (preferably 30-100%, more preferably 40-100%, more preferably 50-100%, more preferably 60-80%, more preferably 70-80%, more preferably 70-90%) of the plasmid supercoiled dimer.

[0010] In one embodiment, the plasmid backbone is pVAX1 (as shown in SEQ ID NO: 1) or pDNA2.0 (ATUM).

[0011] In the present invention, the term "plasmid backbone" refers to a plasmid vector that does not contain a promoter that regulates an exogenous coding gene, a 5'UTR, an exogenous coding gene, a 3'UTR, and a polyA tail. The aforementioned promoter that does not regulate an exogenous coding gene, a 5'UTR, an exogenous coding gene, a 3'UTR, and a polyA tail, and a linearization restriction site can be inserted into the plasmid backbone by conventional means in the art to prepare the plasmid for in vitro transcription of mRNA of the present invention.

[0012] In the present invention, the promoter for regulating the exogenous coding gene may be a T7 promoter.

[0013] In one embodiment, the exogenous encoding gene encodes a peptide or protein of interest, preferably selected from the following group: cytokines (e.g., interleukins (preferably IL-12 or IL-2 or IL-7)); for example, erythropoietin; adhesion molecules, such as integrins; immunoglobulins; immunologically active compounds, such as antigens, such as tumor-associated antigens, pathogen-associated antigens (e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) viral antigens, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) human papillomavirus (HPV) antigens, monkeypox virus antigens, influenza virus (A, B or C) antigens, CMV antigens or RSV antigens), allergens or autoantigens; hormones, such as vasopressin, pancreatic insulin or growth hormone; growth factors, such as VEGFA; enzymes, such as herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatin or lactase; receptors, such as growth factor receptors; protease inhibitors, such as α1-antitrypsin; apoptosis regulators, such as BAX; transcription factors, such as FOXP3; tumor suppressor proteins, such as p53; structural proteins, such as surfactant proteins; reprogramming factors, such as OCT4, SOX2, c-MYC, KLF4, LIN28 or NANOG; genome engineering proteins, such as clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9); and blood proteins, such as fibrinogen.

[0014] Therefore, in one embodiment, the exogenous plasmid backbone in the isolated Escherichia coli cell population containing an exogenous plasmid of the present invention, and the RCB, PCB, MCB and WCB derived from the cell population can be pVAX1 (SEQ ID NO: 1) or pDNA2.0 (ATUM company); the 5'UTR sequence in the exogenous plasmid can be as shown in SEQ ID NO: 2 or 5; the 3'UTR sequence in the exogenous plasmid can be as shown in SEQ ID NO: 3 or 6; or the encoding gene can be a hIL12 encoding gene (for example, as shown in SEQ ID NO: 4) or an HPV E6-E7 encoding gene (for example, as shown in SEQ ID NO: 7); the polyA tail contains at least 30 consecutive adenine deoxynucleotides, and the total length of the polyA tail is between 50 and 160 nucleotides (for example, the total length of the polyA tail is 120 nucleotides or 124 nucleotides, preferably the polyA tail is 120A or 124A).

[0015] In one embodiment, the plasmid supercoiled monomers in the Escherichia coli cell population of the present invention are less than 5%, more preferably less than 2%, and most preferably less than 1%, and the plasmid supercoiled dimers are 30-100%, more preferably 40-100%, more preferably 50-100%, more preferably 60-100%, more preferably 70-100%, and most preferably 80-100%.

[0016] In one embodiment, the plasmid supercoiled monomers in the Escherichia coli cell population of the present invention are less than 10% (preferably less than 5%, more preferably less than 2%, and most preferably less than 1%), and the plasmid supercoiled dimers are 10-100% (preferably 30-100%, more preferably 40-100%, more preferably 50-100%, more preferably 60-80%, more preferably 70-80%, and more preferably 70-90%).

[0017] In the present invention, the proportion of plasmid supercoiled monomers, plasmid supercoiled dimers, plasmid supercoiled trimers or plasmid supercoiled tetramers in the E. coli cell population refers to the proportion of the aforementioned monomers, dimers, trimers or tetramers in the aforementioned four topological structures. Therefore, the proportion of plasmid supercoiled monomers and the proportion of plasmid supercoiled dimers are calculated as follows:

[0018]

[0019]

[0020] Therefore, "the percentage of plasmid supercoiled monomers in the E. coli cell population of the present invention is less than 10% (preferably less than 5%, more preferably less than 2%, and most preferably less than 1%), and the percentage of plasmid supercoiled dimers is 10-100% (preferably 30-100%, more preferably 40-100%, more preferably 50-100%, more preferably 60-80%, more preferably 70-80%, and more preferably 70-90%)" means that "the percentage of the topological structure of the exogenous plasmid in the E. coli cell population is: the percentage of plasmid supercoiled monomers in the plasmid supercoiled monomers, the percentage of plasmid supercoiled dimers in the plasmid supercoiled monomers, and the percentage of plasmid supercoiled dimers in the plasmid supercoiled dimers in the plasmid supercoiled monomers. The proportion of supercoiled dimers, plasmid supercoiled trimers and plasmid supercoiled tetramers is less than 10% (preferably less than 5%, more preferably less than 2%, and most preferably less than 1%), and the proportion of plasmid supercoiled dimers in plasmid supercoiled monomers, plasmid supercoiled dimers, plasmid supercoiled trimers and plasmid supercoiled tetramers is 10-100% (preferably 30-100%, more preferably 40-100%, more preferably 50-100%, more preferably 60-80%, more preferably 70-80%, and more preferably 70-90%)."

[0021] Methods for measuring supercoiled monomers, supercoiled dimers, supercoiled trimers and supercoiled tetramers in Escherichia coli cells are known in the art. For example, the grayscale analysis method of Image Lab can be used for measurement. The specific method is as follows: extract plasmids from the cell population, identify the plasmid topology by 1% agarose gel electrophoresis, obtain gel imaging images using a gel imaging system (Tanon-1600B), and then use Image Lab software to analyze the plasmid topology: (1) remove the highlighted saturated pigments in the image transformation option, adjust the positions of the lanes and bands, and select the bands to be analyzed to the correct position; (2) adjust the background subtraction to the highest value in the background adjustment option; (3) in the display gel option, select the display band properties, and apply the band percentage to all lanes; (4) select the detection band in the band option, and adjust the custom sensitivity to 100%; at this time, the selected band will display the grayscale percentage. (5) Select the lane to be analyzed in the lane contour option, adjust the band brightness area range, and obtain the final grayscale analysis data.

[0022] In one embodiment, the E. coli cell is selected from Stbl3, DH5α, Top10, JM108, JM109, EPI300 or Stbl4.

[0023] In one aspect of the present invention, the present invention provides a method for preparing a research cell bank (RCB), comprising: mixing the Escherichia coli cell population of the present invention with a cryoprotectant (e.g., 30-70% glycerol (v / v), (preferably 40%-50% (v / v) glycerol) (such as an equal volume with the cryoprotectant) and then packaging (preferably 1 mL / tube) to prepare the RCB; preferably, the prepared RCB is stored in an ultra-low temperature refrigerator (≤-70°C) or liquid nitrogen.

[0024] In one aspect of the present invention, the present invention provides a method for preparing a primary cell bank (PCB), comprising: thawing the RCB described in the present invention at room temperature, and inoculating (such as at about 1% (v / v)) into (such as about 25 mL) an antibiotic resistance culture medium (such as LB antibiotic resistance culture medium (such as LB Kan)), culturing in a shaker at about 37° C. and about 220 rpm for about 12 hours, adding a cryoprotectant (such as 30-70% glycerol (v / v), preferably 40-50% glycerol (v / v)) and then mixing, and storing after aliquoting (preferably first storing in an ultra-low temperature refrigerator (≤-70° C.) overnight, and then transferring to liquid nitrogen for long-term storage), thereby obtaining a primary cell bank (PCB).

[0025] In one aspect of the present invention, the present invention provides a method for preparing a master cell bank (MCB), which comprises: thawing the PCB described in the present invention, mixing, inoculating (such as at about 1% (v / v)) into (such as about 125 mL) an antibiotic resistance culture medium (such as LB antibiotic resistance culture medium (such as LB Kan)), placing in a constant temperature culture shaker at about 37° C., about 220 rpm, and culturing for about 12 hours, adding a cryoprotectant (such as 30-70% glycerol (v / v), preferably 40-50% glycerol (v / v)) and then mixing, and storing after aliquoting (preferably first storing in an ultra-low temperature refrigerator (≤-70° C.) overnight, and then transferring to liquid nitrogen for long-term storage), thereby obtaining a master cell bank (MCB).

[0026] In one aspect of the present invention, the present invention provides a method for preparing a working cell bank (WCB), comprising: thawing the MCB of the present invention, mixing, inoculating (e.g., at about 1% (v / v)) into (e.g., about 125 mL) an antibiotic resistance medium (e.g., LB antibiotic resistance medium (e.g., LB Kan)), placing in a constant temperature culture shaker at about 37° C., about 220 rpm, and culturing for about 12 hours, adding a cryoprotectant (e.g., 30-70% glycerol (v / v), preferably 40-50% glycerol (v / v)), mixing, and directly storing in an ultra-low temperature refrigerator (≤-70° C.) for a long time after packaging, thereby obtaining a working cell bank (WCB).

[0027] In one aspect of the present invention, the present invention provides a research cell bank (RCB) prepared by the method of the present invention.

[0028] In one aspect of the present invention, the present invention provides a primary cell bank (PCB) prepared by the method of the present invention.

[0029] In one aspect of the present invention, the present invention provides a master cell bank (MCB) prepared by the method of the present invention.

[0030] In one aspect of the present invention, the present invention provides a working cell bank (WCB) prepared by the method of the present invention.

[0031] In one aspect of the present invention, the present invention provides a method for producing a plasmid, wherein the plasmid can transcribe an mRNA containing a polyA tail in vitro, the method comprising: (1) inoculating the WCB of the present invention into a fermentation medium, culturing on a shaking table at about 37° C. and about 220 rpm for about 12 hours to obtain a seed solution, and then fermenting and culturing in a fermentation tank (e.g., about 10 L) according to OD 600 =0.1, calculate the inoculum size, inoculate the seed solution into (e.g., about 4 L) fermentation medium, and culture at about 37° C. and about 220 rpm for about 22-26 h, (2) separate the cultured E. coli cells, and (3) extract the plasmid from the separated E. coli cells.

[0032] In one aspect of the present invention, the present invention provides a method for in vitro transcription to produce mRNA containing a polyA tail, the method comprising: (1) inoculating the WCB of the present invention into a fermentation medium, culturing at about 37° C. and about 220 rpm on a shaking table for about 12 h to obtain a seed solution, and then using a fermentation tank (e.g., about 10 L) for fermentation culturing, and culturing the mRNA according to OD 600 =0.1, inoculating the seed solution into (e.g., about 4 L) fermentation medium, culturing at about 37° C. and about 220 rpm for about 22-26 h, (2) isolating the cultured E. coli cells, (3) extracting plasmids from the isolated E. coli cells, and (4) transcribing the extracted plasmids into mRNA in vitro.

[0033] In one aspect of the present invention, the present invention also provides a method for improving the purity of mRNA produced by in vitro transcription, wherein the mRNA comprises a polyA tail, the method comprising:

[0034] (1) preparing a working cell bank (WCB) using the isolated Escherichia coli cell population containing an exogenous plasmid of the present invention;

[0035] wherein optionally, the E. coli cell population comprises an exogenous plasmid, the exogenous plasmid can be transcribed into mRNA in vitro, the exogenous plasmid comprises a plasmid backbone, a promoter for regulating an exogenous coding gene (such as a T7 promoter, an SP6 promoter, a T3 promoter or a T5 promoter), a 5' untranslated region (5'UTR), an exogenous coding gene, a 3' untranslated region (3'UTR) and a polyA tail,

[0036] Optionally, the topological structure of the exogenous plasmid in the E. coli cell population accounts for less than 10% (preferably less than 5%, more preferably less than 2%, most preferably less than 1%) of the plasmid supercoiled monomer, and 10-100% (preferably 30-100%, more preferably 40-100%, more preferably 50-100%, more preferably 60-80%, more preferably 70-80%, more preferably 70-90%) of the plasmid supercoiled dimer;

[0037] Wherein, optionally, the E. coli cell population is selected from Stbl3, DH5α, Top10, JM108, JM109, EPI300 or Stbl4 cells;

[0038] Wherein, optionally, the plasmid backbone of the exogenous plasmid is pVAX1 or pDNA2.0;

[0039] Wherein, optionally, the sequence of the 5'UTR is as shown in SEQ ID NO: 2 or 5, the sequence of the 3'UTR is as shown in SEQ ID NO: 3 or 6, or the coding gene is human IL12 (for example, as shown in SEQ ID NO: 4) or HPV E6-E7 protein coding gene (for example, as shown in SEQ ID NO: 7);

[0040] wherein, optionally, the polyA tail contains at least 30 consecutive adenine deoxynucleotides, and the total length of the polyA tail is between 50 and 160 nucleotides (for example, the total length of the polyA tail is 120 nucleotides or 124 nucleotides, preferably the polyA tail is 120A or 124A);

[0041] Wherein, preferably, the ratio of the supercoiled monomer, supercoiled dimer, supercoiled trimer and supercoiled tetramer in the E. coli cell population is measured by Image Lab grayscale analysis;

[0042] (2) Thawing the WCB at room temperature, inoculating (e.g., at 1% (v / v)) into (e.g., about 100 mL) of fermentation medium, and culturing at about 37° C. and 220 rpm on a shaking table for about 12 h to obtain a seed solution, and then fermenting the solution in a fermenter (e.g., about 10 L). 600= 0.1 Calculate the inoculation amount, inoculate the seed solution into (e.g., about 4 L) fermentation medium, and culture at about 37° C. and about 220 rpm for about 22-26 h;

[0043] (3) isolating the cultured Escherichia coli cells;

[0044] (4) extracting plasmids from the isolated E. coli cells, and

[0045] (5) transcribing the extracted plasmid into mRNA in vitro;

[0046] The purity of the mRNA transcribed according to step (5) is higher than that of the mRNA transcribed under the same conditions (i.e., steps (2) to (5)) from WCB prepared from type I cell clones (i.e., E. coli cell populations containing more than 10% of exogenous plasmid supercoiled monomers) and / or type III cell clones (i.e., E. coli cell populations containing less than 10% of exogenous plasmid supercoiled monomers and supercoiled dimers).

[0047] Therefore, in the "method for improving the purity of mRNA produced by in vitro transcription" described in the present invention, the method for preparing WCB from class I cell clones, class III cell clones, or from the E. coli cell population (class II cell clone) of the present invention is the same, and all include:

[0048] (a) mixing the Escherichia coli cell population with a cryoprotectant (e.g., 30-70% glycerol (v / v), preferably 40%-50% (v / v) glycerol) (e.g., equal volumes) and packaging the mixture (preferably 1 mL / tube) to prepare a research cell bank (RCB);

[0049] (b) thawing the RCB described in step (a) at room temperature, and inoculating (e.g., at about 1% (v / v)) into (e.g., about 25 mL) an antibiotic resistance medium (e.g., LB antibiotic resistance medium (e.g., LB Kan)), culturing in a shaker at about 37° C. and about 220 rpm for about 12 h, adding a cryoprotectant (e.g., 30-70% glycerol (v / v), preferably 40-50% glycerol (v / v)), mixing, and storing after aliquoting (preferably first storing in an ultra-low temperature refrigerator (≤-70° C.) overnight, and then transferring to liquid nitrogen for long-term storage), thereby obtaining a primary cell bank (PCB);

[0050] (c) thawing the PCB, mixing it, and inoculating (e.g., at about 1% (v / v)) it into (e.g., about 125 mL) an antibiotic resistance medium (e.g., LB antibiotic resistance medium (e.g., LB Kan)), placing it in a constant temperature culture shaker at about 37° C. and about 220 rpm for about 12 h, adding a cryoprotectant (e.g., 30-70% glycerol (v / v), preferably 40-50% glycerol (v / v)), mixing it, and storing it after aliquoting (preferably first storing it in an ultra-low temperature refrigerator (≤-70° C.) overnight, and then transferring it to liquid nitrogen for long-term storage), thereby obtaining a master cell bank (MCB);

[0051] (d) thawing the MCB, mixing it, and inoculating (e.g., at about 1% (v / v)) it into (e.g., about 125 mL) an antibiotic resistance medium (e.g., LB antibiotic resistance medium (e.g., LB Kan)), placing it in a constant temperature culture shaker at about 37° C., about 220 rpm, and culturing it for about 12 h, adding a cryoprotectant (e.g., 30-70% glycerol (v / v), preferably 40-50% glycerol (v / v)), mixing it, and storing it directly in an ultra-low temperature refrigerator (≤-70° C.) for a long time after packaging, thereby obtaining a working cell bank (WCB).

[0052] In one aspect of the present invention, the present invention also provides a method for plasmid yield, the method comprising:

[0053] (1) preparing a working cell bank (WCB) using the isolated Escherichia coli cell population containing an exogenous plasmid of the present invention;

[0054] wherein optionally, the E. coli cell population comprises an exogenous plasmid, the exogenous plasmid can be transcribed into mRNA in vitro, the exogenous plasmid comprises a plasmid backbone, a promoter for regulating an exogenous coding gene (such as a T7 promoter, an SP6 promoter, a T3 promoter or a T5 promoter), a 5' untranslated region (5'UTR), an exogenous coding gene, a 3' untranslated region (3'UTR) and a polyA tail,

[0055] Optionally, the topological structure of the exogenous plasmid in the E. coli cell population accounts for less than 10% (preferably less than 5%, more preferably less than 2%, most preferably less than 1%) of the plasmid supercoiled monomer, and 10-100% (preferably 30-100%, more preferably 40-100%, more preferably 50-100%, more preferably 60-80%, more preferably 70-80%, more preferably 70-90%) of the plasmid supercoiled dimer;

[0056] Wherein, optionally, the E. coli cell population is selected from Stbl3, DH5α, Top10, JM108, JM109, EPI300 or Stbl4 cells;

[0057] Wherein, optionally, the plasmid backbone of the exogenous plasmid is pVAX1 or pDNA2.0;

[0058] Wherein, optionally, the sequence of the 5'UTR is as shown in SEQ ID NO: 2 or 5, the sequence of the 3'UTR is as shown in SEQ ID NO: 3 or 6, or the coding gene is human IL12 (for example, as shown in SEQ ID NO: 4) or HPV E6-E7 protein coding gene (for example, as shown in SEQ ID NO: 7);

[0059] wherein, optionally, the polyA tail contains at least 30 consecutive adenine deoxynucleotides, and the total length of the polyA tail is between 50 and 160 nucleotides (for example, the total length of the polyA tail is 120 nucleotides or 124 nucleotides, preferably the polyA tail is 120A or 124A);

[0060] Wherein, preferably, the ratio of the supercoiled monomer, supercoiled dimer, supercoiled trimer and supercoiled tetramer in the E. coli cell population is measured by Image Lab grayscale analysis;

[0061] (2) Thawing the WCB at room temperature, inoculating (e.g., at 1% (v / v)) into (e.g., about 100 mL) of fermentation medium, and culturing at about 37° C. and 220 rpm on a shaking table for about 12 h to obtain a seed solution, and then fermenting the solution in a fermenter (e.g., about 10 L). 600 = 0.1 Calculate the inoculation amount, inoculate the seed solution into (e.g., about 4 L) fermentation medium, and culture at about 37° C. and about 220 rpm for about 22-26 h;

[0062] (3) isolating the cultured E. coli cells; and

[0063] (4) extracting plasmids from the isolated Escherichia coli cells,

[0064] The amount of the plasmid extracted according to step (4) is higher than that of the plasmid extracted under the same conditions (i.e., steps (2) to (4)) from WCB prepared from type I cell clones (i.e., E. coli cell populations containing more than 10% of supercoiled monomers of exogenous plasmids) and / or type III cell clones (i.e., E. coli cell populations containing less than 10% of supercoiled monomers and supercoiled dimers of exogenous plasmids).

[0065] Therefore, in the "method for improving the purity of mRNA produced by in vitro transcription" described in the present invention, the method for preparing WCB from class I cell clones, class III cell clones, or from the E. coli cell population (class II cell clone) of the present invention is the same, and all include:

[0066] (a) mixing the Escherichia coli cell population with a cryoprotectant (e.g., 30-70% glycerol (v / v), preferably 40%-50% (v / v) glycerol) (e.g., equal volumes) and packaging the mixture (preferably 1 mL / tube) to prepare a research cell bank (RCB);

[0067] (b) thawing the RCB described in step (a) at room temperature, and inoculating (e.g., at about 1% (v / v)) into (e.g., about 25 mL) an antibiotic resistance medium (e.g., LB antibiotic resistance medium (e.g., LB Kan)), culturing in a shaker at about 37° C. and about 220 rpm for about 12 h, adding a cryoprotectant (e.g., 30-70% glycerol (v / v), preferably 40-50% glycerol (v / v)), mixing, and storing after aliquoting (preferably first storing in an ultra-low temperature refrigerator (≤-70° C.) overnight, and then transferring to liquid nitrogen for long-term storage), thereby obtaining a primary cell bank (PCB);

[0068] (c) thawing the PCB, mixing it, and inoculating (e.g., at about 1% (v / v)) it into (e.g., about 125 mL) an antibiotic resistance medium (e.g., LB antibiotic resistance medium (e.g., LB Kan)), placing it in a constant temperature culture shaker at about 37° C. and about 220 rpm for about 12 h, adding a cryoprotectant (e.g., 30-70% glycerol (v / v), preferably 40-50% glycerol (v / v)), mixing it, and storing it after aliquoting (preferably first storing it in an ultra-low temperature refrigerator (≤-70° C.) overnight, and then transferring it to liquid nitrogen for long-term storage), thereby obtaining a master cell bank (MCB);

[0069] (d) thawing the MCB, mixing it, and inoculating (e.g., at about 1% (v / v)) it into (e.g., about 125 mL) an antibiotic resistance medium (e.g., LB antibiotic resistance medium (e.g., LB Kan)), placing it in a constant temperature culture shaker at about 37° C., about 220 rpm, and culturing it for about 12 h, adding a cryoprotectant (e.g., 30-70% glycerol (v / v), preferably 40-50% glycerol (v / v)), mixing it, and storing it directly in an ultra-low temperature refrigerator (≤-70° C.) for a long time after packaging, thereby obtaining a working cell bank (WCB). BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG1 shows a map of the plasmid used in the present invention, wherein Figure 1A This is the pVAX1 plasmid map; Figure 1B This is the plasmid map of pVAX1-IL12-BspQI; Figure 1C is the pVAX1-IL12-BsaI plasmid map; and Figure 1D The plasmid map is pVAX1-IL12-no poly A.

[0071] Figure 2 shows the topological structure of the 0th generation plasmid. Figure 2A The 0th generation pVAX1-IL12-BspQI plasmid, wherein lanes 1-3 are Class I clones; lanes 4-6 are Class II clones; and lanes 7-9 are Class III clones; Figure 2B It is the 0th generation pVAX1-IL12-BsaI plasmid, among which lanes 10-12 are type I clones; lanes 13-15 are type II clones; and lanes 16-18 are type III clones.

[0072] FIG3 shows the plasmid yield results of different types of clones.

[0073] Figure 4 shows the 7th generation plasmid topology. Figure 4A The topological structure of the 7th generation pVAX1-IL12-BspQI plasmid, where lanes 1-3 are class I clones; lanes 4-6 are class II clones; and lanes 7-9 are class III clones; Figure 4B This is the topological structure of the 7th generation pVAX1-IL12-BsaI plasmid, where lanes 10-12 are type I clones; lanes 13-15 are type II clones; and lanes 16-18 are type III clones.

[0074] FIG. 5 shows the results of plasmid poly A integrity detection. Figure 5A The results of capillary electrophoresis (CE) detection of the integrity of the poly A tail of the plasmid are shown in Figure 2. The % in the brackets indicates the percentage of plasmids with complete poly A tails. A plasmid without a poly A tail was used as a control. Figure 5B The CE statistical results of the integrity of the poly A tail of different types of cloned plasmids.

[0075] FIG. 6 shows the results of HPLC-IPRP detection of mRNA purity. Fig. 6A The purity of mRNA transcribed from the three types of cloned plasmids at generation 0; Figure 6B The purity of mRNA transcribed from the 7th generation Class III clone plasmid; Figure 6C These are the statistical results of the mRNA purity obtained by transcribing the 0th and 7th generation plasmids.

[0076] FIG. 7 shows the results of ELISA Kit detection of cell IL-12 expression. Fig. 7A The expression results of three types of cloned IL-12 from pVAX1-IL12-BspQI plasmid; Figure 7B The expression results of three types of cloned IL-12 in pVAX1-IL12-BsaI plasmid.

[0077] FIG8 shows a map of the plasmid used in the present invention, wherein Fig. 8Ais the plasmid map of pDNA2.0-HPV16-E6_E7-120A-BspQI; Figure 8B This is the plasmid map of pDNA2.0-HPV16-E6_E7-nopolyA-BspQI.

[0078] FIG. 9 shows the results of plasmid polyA integrity detection. Fig. 9A The results of capillary electrophoresis (CE) detection of the integrity of the plasmid polyA tail. The percentage in brackets indicates the percentage of plasmids with complete polyA tails. Meanwhile, a plasmid without a polyA tail was used as a control. Fig. 9B The CE statistical results of the integrity of the polyA tail of different types of cloned plasmids.

[0079] FIG. 10 shows the results of HPLC-IPRP detection of mRNA purity. Fig. 10A is the purity of mRNA transcribed from two types of cloned plasmids at generation 0; Fig. 10B This is the purity of mRNA transcribed from two types of cloned plasmids at the 7th generation; Fig. 10C These are the statistical results of the mRNA purity obtained by transcribing the 0th and 7th generation plasmids.

[0080] Fig.11 The results of protein expression detected by flow cytometry are shown. The mean fluorescence intensity of protein expression of two clones of passage 0 and passage 7 was detected 24h and 48h after transfection. DETAILED DESCRIPTION

[0081] Example 1 Plasmid Construction

[0082] Experimental purpose: To construct pVAX1-IL12-BspQI and pVAX1-IL12-BsaI plasmids for the preparation of RCB.

[0083] Experimental methods: Human interleukin 12 (IL-12) (SEQ ID NO: 4), 5'UTR (SEQ ID NO: 2), 3'UTR (SEQ ID NO: 3) and polyA tail (124A) sequences were designed and synthesized under the control of T7 phage promoter, and the aforementioned sequence elements were inserted into pVAX1 plasmid vector (Thermofisher, SEQ ID NO: 1) ( Figure 1A ), thereby obtaining plasmid pVAX1-IL12-BspQI ( Figure 1B ) and pVAX1-IL12-BsaI( Figure 1C ), which are 3996 bp and 3995 bp in length, respectively. In addition, a pVAX1-IL12-no poly A plasmid was constructed. This plasmid is a pVAX1-IL12-BspQI plasmid lacking a poly A tail sequence ( Figure 1D), with a length of 3872bp. The constructed plasmid was transformed into Escherichia coli Stbl3, plated, and cultured overnight to obtain a single colony. The single colony was inoculated into LB liquid culture medium, and the plasmid was extracted after overnight culture. All plasmids were used after Sanger sequencing to verify the accuracy of the sequence.

[0084] Example 2 Preparation of RCB

[0085] Experimental purpose: To prepare Stbl3 E. coli cells containing the plasmid described in Example 1, and to screen out three types of clones containing different plasmid topological structures for subsequent passage evaluation.

[0086] Experimental method: Take out the Stbl3 competent cells, place them on ice to thaw, adjust the concentration of the correctly sequenced pVAX1-IL12-BspQI and pVAX1-IL12-BsaI plasmids to 1-100 ng / μL, take 1-2μl and add them to the competent cells, mix gently, incubate on ice for 10-30 minutes, heat shock at 42.0℃ for 90 seconds, place on ice for 2 minutes, add 1mL LB medium, 37℃, 220rpm to recover for about 60 minutes, spread the recovered bacterial solution on the LB Kan plate in gradients, and invert and culture at 37℃ overnight to prepare for the selection of single colonies. Pick 20 single colonies from each competent cell transformation plate and inoculate them into 5mL LB Kan medium respectively. The culture container is a 10mL centrifuge tube, which is cultured in a shaker at 37℃, 220rpm until the bacterial solution becomes turbid. After the bacterial solution was mixed with 40% glycerol (v / v) in equal volumes, it was dispensed at 1 mL / tube to prepare RCB. Eight tubes of each RCB were prepared and stored in an ultra-low temperature refrigerator (≤-70°C). Thus, a total of 40 single colony RCBs (i.e., the 0th generation) were preserved. 100 μL of the excess glycerol bacteria were inoculated into 10 mL of LB Kan medium in a 100 mL conical flask. Incubate overnight in a shaker at 37°C, 220 rpm (about 12 hours), and record the OD value of the bacterial solution at the end of the culture. 600 According to the results of plasmid sequencing (polyA tail length is the length of the exogenous plasmid polyA tail length before transformation ± 5A), 1% agarose gel electrophoresis and Image Lab image analysis, 18 RCBs were selected from the selected competent cells for subsequent experiments.

[0087] Experimental results: According to the grayscale analysis results of Image Lab, 9 RCBs (a total of 18) were obtained after transformation with the two plasmids, and according to the polymer state of the supercoiled plasmids in the cells, the above 18 RCBs were divided into three types of clones (Class I, Class II and Class III). The experimental results are shown in Figure 2 and Table 1.

[0088] As shown in Table 1, Class I clones contain more than 10% of plasmid supercoiled monomers; Class II clones contain less than 10% of plasmid supercoiled monomers, and are mainly diploid supercoiled plasmids. Most plasmids in Class III clones are triploid or tetraploid supercoiled plasmids, and the content of supercoiled monomers and supercoiled dimer plasmids is less than 10%.

[0089] Table 1 Results of topological structure analysis of the 0th generation plasmid

[0090]

[0091] Example 3 RCB passage stability screening

[0092] Purpose: To subculture the three types of clones (18 RCBs) obtained from the initial screening of RCBs, and to screen out the RCBs with the best subculture stability for PCB library construction based on the analysis results of plasmid yield, plasmid electrophoresis, poly A integrity (CE method), purity of transcribed mRNA (IPRP-HPLC method) and cell expression (ELISAKit).

[0093] Experimental methods:

[0094] 3.1 Strain subculture

[0095] After thawing and mixing the 18 RCBs obtained by screening at room temperature, 50 μL was taken and inoculated into 5 mL LB Kan medium (inoculation ratio 1% (v / v)). The culture container was a 10 mL centrifuge tube, which was cultured in a shaker at 37°C and 220 rpm for about 12 hours. After the culture was completed, it was recorded as the first generation. 50 μL of the first generation bacterial solution was inoculated into 5 mL LB Kan medium (inoculation ratio 1% (v / v)). The culture container was a 10 mL centrifuge tube, which was cultured in a shaker at 37°C and 220 rpm for about 12 hours. After the culture was completed, it was recorded as the second generation, and the third generation was transferred in sequence until the end of the seventh generation. After passage to the seventh generation, the seventh generation plasmid was extracted, the yield of the 0th and seventh generation plasmids was compared, and the changes in the plasmid topological structure were observed by 1% agarose gel electrophoresis (results shown in Figure 4) and Image Lab (results shown in Table 2).

[0096] 3.2 Plasmid yield detection

[0097] Take 100 μL of the 0th or 7th generation glycerol bacteria and inoculate it into 10 mL of LB Kan medium in a 100 mL conical flask. Incubate overnight (about 12 hours) at 37°C and 220 rpm in a shaker and record the OD value of the bacterial solution at the end of the culture. 600The plasmids were extracted using UE Plasmid Miniprep Kit (US Everbright) and the plasmid yields of each clone were compared.

[0098] 3.3 Poly A integrity detection

[0099] The pVAX1-IL12-BspQI plasmid was double-digested with MfeI and BspQI (New England Biolabs) to obtain the poly A tail sequence. The integrity of the poly A tail sequence of each type of clone after digestion was analyzed using PA 800Plus Pharmaceutical Analysis System (SCIEX).

[0100] 3.4 mRNA purity detection

[0101] The pVAX1-IL12-BspQI plasmid was linearized by single restriction digestion with BspQI, and the pVAX1-IL12-BsaI plasmid was linearized by single restriction digestion with BsaI, and the linearized plasmid was used as the DNA template for the IVT reaction. The IVT process was performed using HiScribe TM T7 mRNA Kit (New England Biolabs) and CleanCap Reagent AG (TriLink BioTechnologies) were used for the reaction, and BeaverBeads Oligo(dT) beads (BEAVER) were used to purify and recover mRNA. The concentration of mRNA was detected at 260 nm using Nanodrop One spectrophotometer (Thermo Fisher Scientific), and the purity of mRNA was finally detected using IPRP-HPLC (Thermo Fisher Scientific).

[0102] 3.5 Cell expression detection

[0103] Hela cells were seeded into 24-well cell culture plates at 1x10 per well. 5 cells, and the culture medium was Hyclone (cytiva). After 24 h of culture, mRNA was transfected using Lipofectamine MessengerMAX Reagent (Thermo Fisher Scientific). After 48 h of transfection, the cell culture medium was replaced, and the supernatant was collected at 24 h and 72 h, and the concentration of IL-12 in the supernatant was detected using Human IL-12p70 Valukine ELISA Kit (Novus Biologicals).

[0104] Experimental results:

[0105] 3.1 Plasmid yield

[0106] Plasmids were extracted after overnight culture of the 0th and 7th generation bacterial suspensions. The plasmid yields are shown in Figure 3A Before and after subculture, the plasmid yield of type II clones was higher than that of the other two types of clones.

[0107] 3.2 Changes in plasmid topology

[0108] The topological structure of the plasmid at the 7th generation is shown in Figure 4. As can be seen from Figures 2 and 4, Tables 1 and 2, when the subculture reached the 7th generation, the ratio of supercoiled monomers and dimers of Class I clones decreased, but they were still Class I clones; the ratio of supercoiled monomers of Class II clones increased, and some clones turned into Class I clones; the ratio of supercoiled monomers and dimers of Class III clones increased, and all of them turned into Class I clones, that is, the ratio of supercoiled monomers and dimers increased.

[0109] Table 2 Results of topological structure analysis of the 7th generation plasmid

[0110]

[0111] 3.3 Poly A integrity detection

[0112] The results of the integrity test of poly A of pVAX1-IL12-BspQI plasmid at generation 0 and generation 7 are shown in Figure 5. The integrity of the poly A tail of the 0th generation plasmid of the three types of clones was above 80%, and after passage, the integrity of the poly A tail of the 7th generation plasmid decreased. However, the integrity of the poly A tail of the plasmid of type II clones was least affected by passage, indicating that the structural stability of the plasmid of type II clones was the best, and the integrity of the poly A tail was higher than that of the other two types of clones.

[0113] 3.4 mRNA purity detection

[0114] The results of the purity test of the mRNA transcribed in vitro from the two plasmids are shown in Figure 6 and Table 3. The purity of the mRNA was analyzed by IPRP-HPLC and can be divided into three main peaks: no poly A peak, incomplete poly A peak, and complete poly A peak. At the 0th generation, the average purity of the mRNA transcribed from the three types of cloned plasmids was about 80%; at the 7th generation, the purity of all mRNAs decreased, among which the purity of the mRNA transcribed from the type II clone decreased the least, while the mRNA transcribed from the type III clone with incomplete poly A accounted for the largest proportion.

[0115] Table 3 Statistical results of mRNA purity obtained by transcription of different types of cloned plasmids

[0116]

[0117] 3.5 Cell expression detection

[0118] The results of cell expression detection are shown in Figure 7. After the 7th generation, the expression levels of mRNA IL-12 transcribed from the three types of cloned plasmids all decreased; and due to the transient expression of mRNA, the expression levels of IL-12 all decreased 72 hours after transfection. At the 0th generation, the expression levels of type II and type III cloned plasmids were higher; and at the 7th generation, the expression level of type II cloned plasmid was higher than that of the other two types of clones.

[0119] It can be seen from the above examples that plasmids replicate and amplify in cells. As the number of cell division and amplification increases, the ratio of plasmid supercoiled monomers to polymers will change (that is, the type of clones described in the present invention changes). This change will not only affect the integrity of the plasmid polyA tail, but also affect the quality and cell expression of mRNA obtained by IVT. The inventors of the present invention found that the plasmids in Class II clones had the highest plasmid yield after passage, and the integrity of the poly A tail was higher than that of the other two types of clones; the purity and cell expression of the transcribed mRNA were also better than those of the other two types of clones. Based on the above experiments, Class II clones with the best passage stability were selected as research cell banks (RCBs) for subsequent PCB, MCB and WCB library construction.

[0120] Example 4 Construction and propagation of pDNA2.0-HPV16-E6_E7-120A-BspQI plasmid

[0121] Purpose of the experiment: Use new vectors and genes to construct the pDNA2.0-HPV16-E6_E7-120A-BspQI plasmid, pass it through the same method, observe the changes between the supercoiled monomers and dimers of the plasmid, and the quality and cellular expression differences of the mRNA prepared thereby.

[0122] Experimental methods:

[0123] 4.1 Plasmid construction

[0124] HPV 16 antigens E6 and E7 (SEQ ID NO: 7), 5'UTR (SEQ ID NO: 5), 3'UTR (SEQ ID NO: 6) and polyA tail sequences were designed and synthesized under the control of T7 phage promoter, and the aforementioned sequence elements were inserted into pDNA2.0 plasmid vector (ATUM), thereby obtaining pDNA2.0-HPV16-E6_E7-120A-BspQI plasmid ( Fig. 8A), which is 3819 bp in length. In addition, a pDNA2.0-HPV16-E6_E7-nopolyA-BspQI plasmid was constructed. The plasmid lacks the poly A tail sequence ( Figure 8B ), with a length of 3699bp. The constructed plasmid was transformed into Escherichia coli Stbl3, plated, and cultured overnight to obtain a single colony. The single colony was inoculated into LB liquid medium, and the plasmid was extracted after overnight culture. All plasmids were used after Sanger sequencing to verify the accuracy of the sequence.

[0125] 4.2 Plasmid yield detection

[0126] Take 100 μL of the 0th or 7th generation glycerol bacteria and inoculate it into 10 mL of LB Kan medium in a 100 mL conical flask. Incubate overnight (about 12 hours) at 37°C and 220 rpm in a shaker and record the OD value of the bacterial solution at the end of the culture. 600 The plasmids were extracted using UE Plasmid Miniprep Kit (US Everbright) and the plasmid yields of each clone were compared.

[0127] 4.3 Preparation of RCB

[0128] Take out the Stbl3 competent cells, place them on ice to thaw, adjust the concentration of the correctly sequenced pDNA2.0-HPV16-E6_E7-120A-BspQI plasmid to 1-100 ng / μL, take 1-2μl and add it to the competent cells, mix gently, incubate on ice for 10-30 minutes, heat shock at 42.0℃ for 90 seconds, place on ice for 2 minutes, add 1mL LB medium, 37℃, 220rpm to recover for about 60 minutes, spread the recovered bacterial solution on the LB Kan plate in gradients, and invert and culture at 37℃ overnight to prepare for the selection of single colonies. Pick 20 single colonies from the competent cell transformation plate and inoculate them into 5mL LB Kan medium. The culture container is a 10mL centrifuge tube, which is cultured in a shaker at 37℃, 220rpm until the bacterial solution becomes turbid. After mixing equal volumes of bacterial solution and 40% glycerol (v / v), 1 mL / tube was dispensed to prepare RCB. 8 tubes of each RCB were prepared and stored in an ultra-low temperature refrigerator (≤-70°C). Thus, a total of 20 single colony RCBs (i.e., generation 0) were preserved. 100 μL of the excess glycerol bacteria were inoculated into 10 mL of LBKan medium in a 100 mL conical flask. Incubate overnight in a shaker at 37°C, 220 rpm (about 12 hours), and record the OD of the bacterial solution at the end of the culture. 600According to the plasmid Sanger sequencing results (polyA tail length is the exogenous plasmid polyA tail length before transformation ± 5A), 1% agarose gel electrophoresis results and Image Lab image analysis results, 4 RCBs were selected in the selected competent cells for subsequent experiments.

[0129] 4.4 Subculture of strains

[0130] After thawing and mixing the four RCBs obtained by screening at room temperature, 50 μL of each was inoculated into 5 mL LB Kan medium (inoculation ratio 1% (v / v)). The culture container was a 10 mL centrifuge tube, which was cultured in a shaker at 37°C, 220 rpm for about 12 hours. After the culture was completed, it was recorded as the first generation. 50 μL of the first generation bacterial solution was inoculated into 5 mL LB Kan medium (inoculation ratio 1% (v / v)). The culture container was a 10 mL centrifuge tube, which was cultured in a shaker at 37°C, 220 rpm for about 12 hours. After the culture was completed, it was recorded as the second generation, and the third generation was transferred in sequence until the seventh generation. After subculture to the seventh generation, the seventh generation plasmid was extracted, and the changes in the plasmid topological structure were observed by 1% agarose gel electrophoresis and Image Lab.

[0131] 4.5 PolyA integrity detection

[0132] The pDNA2.0-HPV16-E6_E7-120A-BspQI plasmid was double-digested with PmLI and BspQI (Thermofisher) to obtain the poly A tail sequence. The integrity of the poly A tail sequence of each type of clone after digestion was analyzed using PA 800Plus Pharmaceutical Analysis System (SCIEX).

[0133] 4.6 mRNA purity detection

[0134] The pDNA2.0-HPV16-E6_E7-120A-BspQI plasmid was linearized by single restriction digestion with BspQI, and the linearized plasmid was used as the DNA template for the IVT reaction. The IVT process was performed using HiScribe TMT7 mRNA Kit (New England Biolabs) and CleanCap Reagent AG (TriLink BioTechnologies) were used for reaction, and BeaverBeadsOligo(dT)beads (BEAVER) were used to purify and recover mRNA. Nanodrop One spectrophotometer (ThermoFisher Scientific) was used to detect the concentration of mRNA at 260 nm, and finally IPRP-HPLC (Thermo Fisher Scientific) was used to detect the purity of mRNA.

[0135] 4.7 Cell expression detection

[0136] Hela cells were seeded into 24-well cell culture plates at 1x10 per well. 5 The culture medium was Hyclone (cytiva). After 24 h of culture, mRNA was transfected using Lipofectamine MessengerMAX Reagent (Thermo Fisher Scientific). The cells were collected at 24 h and 48 h, and the average fluorescence signal of protein expression was detected using a flow cytometer (Agilent NovoCyteQuanteon).

[0137] Experimental Results

[0138] 4.1 Plasmid yield

[0139] Plasmids were extracted after overnight culture of the 0th and 7th generation bacterial suspensions. The plasmid yields are shown in Figure 3B Before and after subculture, the plasmid yield of class II clones was higher than that of class I clones.

[0140] 4.2 Changes in plasmid topology

[0141] The ratio of supercoiled monomers to polymers of the 0th and 7th generation plasmids is shown in Tables 4 and 5. As can be seen from the table, when the 7th generation was subcultured, the types of clones of type I and type II did not change, only the ratio of supercoiled monomers to polymers changed.

[0142] Table 4 Results of topological structure analysis of the 0th generation plasmid

[0143]

[0144] Table 5 Results of topological structure analysis of the 7th generation plasmid

[0145]

[0146] 4.3 PolyA integrity detection

[0147] The results of the plasmid poly A integrity test are shown in Figure 9. The integrity of the poly A tail of the two types of cloned plasmids was above 80%. After subculture, the integrity of the poly A tail of the plasmid in the 7th generation decreased. However, the poly A integrity of the plasmid of type II clones was less affected by subculture, indicating that the structural stability of the plasmid of type II clones was better, and the integrity of the poly A tail was higher than that of type I clones.

[0148] 4.4 mRNA purity detection

[0149] The results of mRNA purity test are shown in Figure 10 and Table 6. The mRNA purity was analyzed by IPRP-HPLC and can be divided into three main peaks: no poly A peak, incomplete poly A peak, and complete poly A peak. At the 0th generation, the average purity of mRNA transcribed from the two types of clone plasmids was above 90%; after passage to the 7th generation, the purity of all mRNAs decreased, while the purity of mRNA transcribed from type II clones decreased less.

[0150] Table 6 Statistical results of mRNA purity obtained by transcription of different types of cloned plasmids

[0151]

[0152] 4.5 Cell expression detection

[0153] The results of cell expression detection are shown in Fig.11 After 7 generations, the expression levels of target proteins transcribed from the mRNA of the two types of cloned plasmids decreased; and due to the transient expression of mRNA, the expression levels of target proteins decreased 24 hours after transfection, but there was no significant difference in the expression levels of target proteins between the two types of clones.

Claims

1. An isolated Escherichia coli cell population containing an exogenous plasmid, wherein the exogenous plasmid can be transcribed into mRNA in vitro, wherein the exogenous plasmid comprises a plasmid backbone, a promoter (preferably a T7 promoter, an SP6 promoter, a T3 promoter or a T5 promoter), a 5' untranslated region (5'UTR), an exogenous coding gene, a 3' untranslated region (3'UTR) and a polyA tail, wherein the topological structure of the exogenous plasmid in the Escherichia coli cell population is as follows: the plasmid supercoil monomer is 1 / 4 of the plasmid supercoil monomer, the plasmid supercoil is 2 / 4 of the plasmid supercoil monomer, The proportion of plasmid supercoiled dimers in the sum of plasmid supercoiled monomers, plasmid supercoiled dimers, plasmid supercoiled trimers and plasmid supercoiled tetramers is less than 10% (preferably less than 5%, more preferably less than 2%, most preferably less than 1%), and the proportion of plasmid supercoiled dimers in the sum of plasmid supercoiled monomers, plasmid supercoiled dimers, plasmid supercoiled trimers and plasmid supercoiled tetramers is 10-100% (preferably 30-100%, more preferably 40-100%, more preferably 50-100%, more preferably 60-80%, more preferably 70-80%, more preferably 70-90%), in, Optionally, the E. coli cell population is selected from Stbl3, DH5α, Top10, JM108, JM109, EPI300 or Stbl4, Wherein, optionally, the plasmid backbone of the exogenous plasmid is pVAX1 or pDNA2.0; Wherein, optionally, the sequence of the 5'UTR is as shown in SEQ ID NO: 2 or 5, the sequence of the 3'UTR is as shown in SEQ ID NO: 3 or 6, or the coding gene is human IL-12 (preferably as shown in SEQ ID NO: 4) or HPV E6-E7 protein coding gene (preferably as shown in SEQ ID NO: 7); Wherein, optionally, the polyA tail contains at least 30 consecutive adenine deoxynucleotides, and the total length of the polyA tail is between 50 and 160 nucleotides (preferably the total length of the polyA tail is 120 nucleotides or 124 nucleotides, more preferably the polyA tail is 120A or 124A); Wherein, preferably, the ratio of the supercoiled monomer, supercoiled dimer, supercoiled trimer and supercoiled tetramer in the Escherichia coli cell population is measured by Image Lab grayscale analysis.

2. A method for preparing a research cell bank (RCB), comprising: The Escherichia coli cell population according to claim 1 is mixed with a cryoprotectant (preferably 30-70% glycerol (v / v), more preferably 40%-50% (v / v) glycerol) and then packaged (preferably 1 mL / vial) to prepare the RCB; preferably, The E. coli cell population is mixed with the cryoprotectant in equal volumes, and / or The prepared RCB is stored in an ultra-low temperature refrigerator (≤-70° C.) or liquid nitrogen.

3. A method for preparing a primary cell bank (PCB), comprising: The RCB of claim 2 is thawed, inoculated into an antibiotic resistance medium (preferably LB antibiotic resistance medium, more preferably LB Kan), cultured in a shaker at about 37° C. and about 220 rpm for about 12 h, mixed after adding a cryoprotectant (preferably 30-70% glycerol (v / v), more preferably 40-50% glycerol (v / v)), and stored after aliquoting to obtain a primary cell bank (PCB); preferably, The RCB was inoculated into the antibiotic resistance medium at 1% (v / v), The volume of the antibiotic resistance medium is about 25 mL, and / or The storage method after the subpackaging is to first store it in an ultra-low temperature refrigerator (≤-70°C) overnight, and then transfer it to liquid nitrogen for long-term storage.

4. A method for preparing a master cell bank (MCB), comprising: The PCB according to claim 3 is thawed, mixed, inoculated into an antibiotic resistance medium (preferably LB antibiotic resistance medium, more preferably LB Kan), placed in a constant temperature culture shaker at about 37° C., about 220 rpm, and cultured for about 12 hours, and a cryoprotectant (preferably 30-70% glycerol (v / v), more preferably 40-50% glycerol (v / v)) is added and mixed, and stored after aliquoting, thereby obtaining a master cell bank (MCB); preferably, The PCB was inoculated into the antibiotic resistance medium at 1% (v / v), The volume of the antibiotic resistance medium is about 125 mL, and / or The storage method after the subpackaging is to first store it in an ultra-low temperature refrigerator (≤-70°C) overnight, and then transfer it to liquid nitrogen for long-term storage.

5. A method for preparing a working cell bank (WCB), comprising: The MCB of claim 4 is thawed, mixed, inoculated into an antibiotic resistance medium (preferably LB antibiotic resistance medium, more preferably LB Kan), placed in a constant temperature culture shaker at about 37° C., about 220 rpm, and cultured for about 12 hours, and a cryoprotectant (preferably 30-70% glycerol (v / v), more preferably 40-50% glycerol (v / v)) is added, mixed, and stored after aliquoting to obtain a working cell bank (WCB); preferably, The MCB was inoculated into the antibiotic resistance medium at 1% (v / v), The volume of the antibiotic resistance medium is about 125 mL, and / or The storage method after the subpackaging is to directly store it in an ultra-low temperature refrigerator (≤-70° C.) for a long time.

6. A research cell bank (RCB) prepared by the method of claim 2, a primary cell bank (PCB) prepared by the method of claim 3, a master cell bank (MCB) prepared by the method of claim 4, or a working cell bank (WCB) prepared by the method of claim 5.

7. A method for producing a plasmid capable of transcribing an mRNA containing a polyA tail in vitro, the method comprising: (1) The WCB of claim 6 is inoculated into a fermentation medium, and cultured in a shaking incubator at about 37° C. and about 220 rpm for about 12 h to obtain a seed solution, and then fermented in a fermenter (preferably with a volume of about 10 L) to obtain a seed solution. 600 = 0.1 Calculate the inoculation amount, inoculate the seed liquid into the fermentation medium (preferably about 4 L in volume), and culture at about 37°C and about 220 rpm for about 22-26 hours. (2) isolating the cultured E. coli cells, and (3) Extracting plasmids from the isolated Escherichia coli cells.

8. A method for producing mRNA containing a polyA tail by in vitro transcription, the method comprising: (1) The WCB of claim 6 is inoculated into a fermentation medium, and cultured in a shaking incubator at about 37° C. and about 220 rpm for about 12 h to obtain a seed solution, and then fermented in a fermenter (preferably with a volume of about 10 L) to obtain a seed solution. 600 = 0.1 Calculate the inoculation amount, inoculate the seed liquid into the fermentation medium (preferably about 4 L in volume), and culture at about 37°C and about 220 rpm for about 22-26 hours. (2) Isolating the cultured Escherichia coli cells, (3) extracting plasmids from the isolated E. coli cells, and (4) transcribing the extracted plasmid into mRNA in vitro.

9. A method for improving the purity of mRNA produced by in vitro transcription, wherein the mRNA contains a polyA tail, the method comprising: (1) Preparation of working cell bank (WCB): (a) mixing the Escherichia coli cell population with a cryoprotectant (preferably 30-70% glycerol (v / v), more preferably 40%-50% (v / v) glycerol) (e.g., equal volumes) and packaging the mixture (preferably 1 mL / tube) to prepare a research cell bank (RCB); The E. coli cell population comprises an exogenous plasmid, which can be transcribed into mRNA in vitro, and the exogenous plasmid comprises a plasmid backbone, a promoter (preferably a T7 promoter, an SP6 promoter, a T3 promoter or a T5 promoter), a 5' untranslated region (5'UTR), an exogenous coding gene, a 3' untranslated region (3'UTR) and a polyA tail, wherein the topological structure ratio of the exogenous plasmid in the E. coli cell population is as follows: the ratio of the plasmid supercoiled monomer to the sum of the plasmid supercoiled monomer, the plasmid supercoiled dimer, the plasmid supercoiled trimer and the plasmid supercoiled tetramer is less than 10% (preferably less than 5%, more preferably less than 2%, most preferably less than 1%), and the ratio of the plasmid supercoiled dimer to the sum of the plasmid supercoiled monomer, the plasmid supercoiled dimer, the plasmid supercoiled trimer and the plasmid supercoiled tetramer is 10-100% (preferably 30-100%, more preferably 40-100%, more preferably 50-100%, more preferably 60-80%, more preferably 70-90%); Wherein, optionally, the E. coli cell population is selected from Stbl3, DH5α, Top10, JM108, JM109, EPI300 or Stbl4; Wherein, optionally, the plasmid backbone of the exogenous plasmid is pVAX1 or pDNA2.0; Wherein, optionally, the sequence of the 5'UTR is as shown in SEQ ID NO: 2 or 5, the sequence of the 3'UTR is as shown in SEQ ID NO: 3 or 6, or the coding gene is human IL12 (preferably as shown in SEQ ID NO: 4) or HPV E6-E7 protein coding gene (preferably as shown in SEQ ID NO: 7); Wherein, optionally, the polyA tail contains at least 30 consecutive adenine deoxynucleotides, and the total length of the polyA tail is between 50 and 160 nucleotides (preferably the total length of the polyA tail is 120 nucleotides or 124 nucleotides, more preferably the polyA tail is 120A or 124A); Wherein, preferably, the ratio of the supercoiled monomer, supercoiled dimer, supercoiled trimer and supercoiled tetramer in the Escherichia coli cell population is measured by Image Lab grayscale analysis; (b) preparing primary cell bank (PCB); (c) preparing a master cell bank (MCB); (d) preparing a working cell bank (WCB); (2) fermenting and producing the exogenous plasmid; (3) isolating the cultured Escherichia coli cells; (4) extracting plasmids from the isolated E. coli cells, and (5) Transcribe the extracted plasmid into mRNA in vitro.

10. A method for increasing the yield of a plasmid, wherein the plasmid is capable of transcribing an mRNA containing a polyA tail in vitro, the method comprising: (1) Preparation of working cell bank (WCB): (a) mixing the Escherichia coli cell population with a cryoprotectant (preferably 30-70% glycerol (v / v), more preferably 40%-50% (v / v) glycerol) (e.g., equal volumes) and packaging the mixture (preferably 1 mL / tube) to prepare a research cell bank (RCB); The E. coli cell population comprises an exogenous plasmid, which can be transcribed into mRNA in vitro, and the exogenous plasmid comprises a plasmid backbone, a promoter (preferably a T7 promoter, an SP6 promoter, a T3 promoter or a T5 promoter), a 5' untranslated region (5'UTR), an exogenous coding gene, a 3' untranslated region (3'UTR) and a polyA tail, The proportion of the exogenous plasmid topological structure in the Escherichia coli cell population is as follows: the proportion of plasmid supercoiled monomers in the sum of plasmid supercoiled monomers, plasmid supercoiled dimers, plasmid supercoiled trimers and plasmid supercoiled tetramers is less than 10% (preferably less than 5%, more preferably less than 2%, most preferably less than 1%), and the proportion of plasmid supercoiled dimers in the sum of plasmid supercoiled monomers, plasmid supercoiled dimers, plasmid supercoiled trimers and plasmid supercoiled tetramers is 10-100% (preferably 30-100%, more preferably 40-100%, more preferably 50-100%, more preferably 60-80%, more preferably 70-90%); Wherein, optionally, the E. coli cell population is selected from Stbl3, DH5α, Top10, JM108, JM109, EPI300 or Stbl4; Wherein, optionally, the plasmid backbone of the exogenous plasmid is pVAX1 or pDNA2.0; Wherein, optionally, the sequence of the 5'UTR is as shown in SEQ ID NO: 2 or 5, and the 3' The UTR sequence is as shown in SEQ ID NO: 3 or 6, or the coding gene is human IL12 (preferably as shown in SEQ ID NO: 4) or HPV E6-E7 protein coding gene (preferably as shown in SEQ ID NO: 7); Wherein, optionally, the polyA tail contains at least 30 consecutive adenine deoxynucleotides, and the total length of the polyA tail is between 50 and 160 nucleotides (preferably the total length of the polyA tail is 120 nucleotides or 124 nucleotides, more preferably the polyA tail is 120A or 124A); Wherein, preferably, the ratio of the supercoiled monomer, supercoiled dimer, supercoiled trimer and supercoiled tetramer in the Escherichia coli cell population is measured by Image Lab grayscale analysis; (b) preparing primary cell bank (PCB); (c) preparing a master cell bank (MCB); (d) preparing a working cell bank (WCB); (2) fermenting and producing the exogenous plasmid; (3) isolating the cultured E. coli cells; and (4) Extracting plasmids from the isolated Escherichia coli cells.

11. The method of claim 10 or claim 11, wherein The method for preparing PCB in step (b) is as shown in claim 3; The method for preparing MCB in step (c) is as shown in claim 4; The method for preparing WCB in step (d) is as shown in claim 5, and / or The method for producing the exogenous plasmid by fermentation in step (2) comprises: After the WCB is thawed at room temperature, it is inoculated (e.g., at about 1% (v / v)) into (e.g., about 100 mL) of fermentation medium, and cultured at about 37° C. and about 220 rpm on a shaking table for about 12 h to obtain a seed solution, and then fermented in a fermenter (preferably with a volume of about 10 L) to obtain a seed solution. 600 =0.1 to calculate the inoculation amount, inoculate the seed liquid into the fermentation medium (preferably with a volume of about 4 L), and culture at about 37°C and about 220 rpm for about 22-26 h.

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