Method for preparing and purifying mRNA

Abstract

The invention belongs to the technical field of biology, and provides a novel method suitable for small-scale production in preclinical research or laboratory preparation and purification of mRNA with a complete structure. According to the method, mRNA is obtained through in-vitro transcription of biotinylated T7 RNA polymerase, and the mRNA with high purity and complete structure is finally obtained through a novel purification step of biotin / streptomycin affinity purification and capping. The purification loss rate of mRNA is reduced in the novel purification process, and the final yield of mRNA production is increased by 10% or above on the basis of a conventional purification method. Therefore, the method is a novel method for preparing and purifying mRNA with a complete structure, which is quicker and more convenient to operate and higher in production yield, is suitable for being widely applied and popularized in various preclinical researches of mRNA therapeutic agents and vaccines, and promotes the development of related nucleic acid drug industries.

Description

Technical Field

[0001] The invention belongs to the biological field, and in particular relates to a method for preparing and purifying mRNA. Background Art

[0002] The latest progress in mRNA technology and its delivery system has also ushered in a new era of medicine for mRNA nucleic acid therapy. Currently, mRNA technology has been applied to biomedical fields such as vaccines, cancer immunotherapy, genome editing and protein replacement, and is expanding to many other biomedical fields; mRNA nucleic acid therapy has also shown great potential in the treatment of a variety of diseases including infectious diseases, metabolic genetic diseases, cancer, cardiovascular and cerebrovascular diseases, etc.

[0003] mRNA is a single-stranded ribonucleic acid transcribed from a DNA chain, carrying the coding information for protein synthesis, and is further transcribed and processed into functional proteins. In eukaryotic cells, mature mRNA molecules include five basic elements: 5' cap structure, 5'-untranslated region (5'-UTR), open reading frame (ORF), 3'-untranslated region (3'-UTR) and 3' poly (A) tail. Keeping the mRNA structure intact is beneficial to the stability of mRNA and its ability to express proteins.

[0004] Numerous studies have shown that mRNA drugs have greater advantages than DNA and traditional protein drugs: mRNA can not only mediate better transfection efficiency and longer-lasting protein expression, but also initiate protein transient translation in the cytoplasm without inserting into the genome, so its risk of inserting into the genome is lower. Secondly, mRNA is easily synthesized through an in vitro transcription process, is relatively easy to produce, and can be quickly applied to various therapies. Obviously, these advantages of mRNA over DNA or protein / peptide enable mRNA-based technologies and products to quickly enter various branches of the biomedical field, benefiting all aspects of human life, especially the millions of patients with incurable diseases, which will also lead to a surge in the use and production of mRNA-based treatments. mRNA has become one of the most attractive areas for drug development.

[0005] In order to unleash the full potential of mRNA-based therapeutics and vaccines in various applications, both the scientific and industrial communities have conducted extensive research efforts to optimize the structure and preparation of mRNA, among which optimizing in vitro transcription and mRNA purification protocols will help overcome some of the development barriers of mRNA therapeutics and vaccines. To date, significant progress has been made in simplifying mRNA production. Synthetic mRNA for clinical use is obtained by in vitro transcription from DNA plasmids using bacteriophage T7 RNA polymerase (or using T3 and SP6 polymerases). It is co-transcriptionally capped with a cap structure analog (CleanCap, developed by TriLink BioTechnologies) and purified by high-performance liquid chromatography to remove double-stranded RNA (dsRNA) contaminants, reactants, and incomplete transcripts to meet the quality requirements of GMP production. Preclinical mRNA therapeutics and vaccines are also obtained by in vitro transcription based on bacteriophage T7 RNA polymerase (or using T3 and SP6 polymerases), and capping can be added by post-transcriptional reaction using vaccinia virus capping enzyme and 2′-O-methyltransferase.

[0006] mRNA therapeutics and vaccines used in preclinical studies are usually produced on a small scale or in the laboratory, and are also obtained by in vitro transcription from linear plasmid DNA or synthetic DNA prepared by PCR, and the poly (A) tail is usually encoded in the DNA template to eliminate reaction steps and reduce overall production time and material loss. Commonly used capping methods include enzymatic capping and co-transcriptional capping. Enzymatic capping is based on the capping principle in eukaryotic cells. After transcription is completed, cap0, cap1 or cap2 capping is performed by using vaccinia capping enzyme (VCE), GTP and S-adenosylmethionine (SAM). The capping efficiency is close to 100%, and all cap structures are added in the correct direction. Co-transcriptional capping refers to the addition of cap structure analogs during transcription for capping, which directly produces mRNA with cap0 structure. The standard cap structure analog [m7G (5') ppp (5') G] can be integrated in two directions [m7G (5') pppG-RNA] or [G (5') pppm7G-RNA], producing a mixture of two transcripts. If the cap structure analog is incorporated in the wrong direction ([G (5') pppm7G-RNA]), the mRNA cannot be effectively translated, resulting in low production of the target protein. To date, the structural optimization of cap analogs has also undergone three generations of changes. The second-generation cap analog ARCA solves the problem of reverse capping; the third-generation cap analog CleanCap can directly introduce Cap1, and the capping rate can reach more than 90%.

[0007] When in vitro transcription and enzymatic capping are used to produce structurally intact mRNA on a small scale in preclinical studies, the in vitro transcription reaction mixture contains residual NTPs, enzymes, incorrectly formed mRNA, and DNA plasmid templates. Several impurities need to be removed by a purification step before enzymatic capping reaction; after capping, another purification step is required to obtain mRNA with higher purity for subsequent preclinical studies. Therefore, in the process of small-scale production of structurally intact mRNA, two purification steps are required, which will inevitably lose a certain amount of mRNA and reduce the final yield of mRNA production. Based on the above mRNA production process, the factors affecting the final yield of small-scale production of mRNA are mainly the production volume of in vitro transcription and the purification recovery rate. Therefore, in order to increase the production volume of small-scale production and laboratory-prepared mRNA in preclinical studies, it is urgent to provide a novel mRNA preparation and purification method.

[0008] The purification steps for producing structurally intact mRNA on a small scale or laboratory scale involve removing the DNA transcription template by DNAse digestion, and removing other reactants and various enzymes after transcription and capping by two lithium chloride (LiCL) precipitations or column purification. Among them, the principle of column purification or magnetic bead purification is that RNA adsorption columns or RNA magnetic beads bind to mRNA, and then mRNA is eluted from the adsorption columns or magnetic beads by elution liquid. However, this process has the problem of mRNA loss caused by improper operation or insufficient elution, so the purification recovery rate of mRNA products still has a lot of room for improvement. In order to maximize the purification recovery rate in the mRNA preparation process, it is urgent to provide an mRNA preparation and purification method that is simpler and faster to operate, reduces the probability of operating errors, and has a higher purification recovery rate. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a method for preparing and purifying mRNA. The method of the present invention is simple and rapid to operate, has a low probability of operating errors, and has a high purification recovery rate.

[0010] The technical solution provided by the present invention is as follows:

[0011] A T7 RNA polymerase with an affinity tag added thereto, which is a protein described in any one of a1 to a5 below:

[0012] a1: a protein with biotin site-specifically labeled at the N-terminus of His-T7 RNA polymerase with an amino acid sequence as shown in SEQ ID NO.2;

[0013] a2: a protein having substantially the same enzyme activity and function obtained by labeling the His-T7 RNA polymerase shown in a1 with biotin at its C-terminus or / and N-terminus in addition to labeling the His-T7 RNA polymerase at its N-terminus as described above;

[0014] a3: The His-T7 RNA polymerase shown in a1 is labeled with biotin at the N-terminus or / and the C-terminus of its His tag in addition to the aforementioned labeling with biotin at the N-terminus to obtain a protein having substantially the same enzyme activity and function;

[0015] a4: a protein having substantially the same enzyme activity and function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in a1-a3;

[0016] a5: a protein that has more than 90% sequence homology with the proteins of a1-a3 and whose enzyme activity and function are basically equivalent to those of the proteins of a1-a2;

[0017] a6: A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of any of the amino acid sequences described in a1-6, wherein the tag does not affect the function of the T7 RNA polymerase mutant.

[0018] Furthermore, the method of labeling biotin with His-T7 RNA polymerase is chemical labeling or site-specific labeling using Avi-tag technology; preferably, biotin is site-specifically labeled using Avi-tag technology.

[0019] Furthermore, the method for site-specifically labeling biotin at the N-terminus of the His-T7 RNA polymerase comprises:

[0020] Constructing Avi-His-T7 RNA polymerase with an amino acid sequence as shown in SEQ ID NO.3;

[0021] Biotin ligase is used to activate biotin and specifically link biotin to the lysine of the Avi tag.

[0022] The present invention also provides a method for preparing and purifying mRNA, which adopts biotinylated T7 RNA polymerase for in vitro transcription and streptavidin affinity purification to obtain target mRNA.

[0023] Furthermore, the preparation method of the biotinylated T7 RNA polymerase includes: adding an Avi short peptide tag to the N-terminus of the His-T7 RNA polymerase, and then using a biotin ligase to perform Avi-tag site-specific biotinylation labeling on the N-terminus of the T7 RNA polymerase to obtain a biotinylated Avi-His-T7 RNA polymerase, wherein the sequence of the Avi short peptide tag is shown in SEQ ID NO.1, and the sequence of the Avi-His-T7 RNA polymerase is shown in SEQ ID NO.2.

[0024] Further, the reaction product transcribed by the biotinylated Avi-His-T7 RNA polymerase is mixed with the streptavidin agarose precipitate, the biotinylated Avi-His-T7 RNA polymerase is bound to the agarose, and then the agarose bound to the biotinylated Avi-His-T7 RNA polymerase is removed by centrifugal filtration through a microporous filter, and the reaction solution containing mRNA in the lower centrifuge tube is collected.

[0025] The present invention also provides an mRNA obtained by the above method.

[0026] The present invention also provides the use of the above mRNA or the above method in a capping reaction.

[0027] Furthermore, after capping, the reaction impurities are removed by magnetic bead purification or column purification.

[0028] The present invention also provides the application of the above method in preparing mRNA therapeutic agents and vaccines.

[0029] Beneficial Effects

[0030] The invention provides an mRNA preparation and purification method which is simpler and faster to operate, reduces the probability of operation errors and has a higher purification recovery rate. The preparation method comprises in vitro transcription based on biotinylated His-T7 RNA polymerase, biotin / streptavidin affinity purification to remove biotinylated proteins in the transcription product, enzymatic capping, and conventional RNA purification steps to prepare and purify mRNA with a complete structure.

[0031] The present invention uses biotinylated T7 RNA polymerase to transcribe in vitro to obtain mRNA, and caps after a novel purification step-biotin / streptavidin affinity purification, and finally obtains mRNA with high purity and complete structure. In the present invention, the purification step of removing biotinylated protease by streptavidin agarose (streptavidin agarose) only takes 3 to 5 minutes to complete, and the operation is fast and simple. It not only eliminates the influence of protease on subsequent capping reaction as much as possible, and ensures 100% capping of subsequent mRNA, but also because agarose does not adsorb or low-adsorbs mRNA during the purification process, compared with conventional RNA adsorption columns or RNA magnetic beads purification, the novel purification process in the present invention reduces the mRNA loss rate, so that the final yield of mRNA production is increased by more than 10% on the basis of conventional purification methods. Compared with conventional methods, the purification operation is faster and simpler, and the mRNA loss rate in the purification process is reduced. Therefore, the present invention is a new method for preparing and purifying structurally intact mRNA with faster and simpler operation and higher production yield. It is suitable for wide application and promotion in various preclinical studies of mRNA therapeutics and vaccines, and promotes the development of related nucleic acid drug industries.

[0032] The present invention is based on the in vitro transcription method of synthesizing mRNA by T7 RNA polymerase, and biotinylates T7 RNA polymerase. The N-terminus of T7 RNA polymerase is subjected to Avi-tag site-specific biotinylation labeling by biotin ligase (BirA), and then the biotinylated T7 RNA polymerase is used to perform in vitro transcription on a DNA transcription template containing a T7 promoter site and a poly (A) tail to obtain the target mRNA. Secondly, based on the principle that streptavidin-agarose has extremely high affinity for biotinylated T7 RNA polymerase, and has extremely low or even no adsorption to ordinary nucleic acids, we mix the transcription product with streptavidin-agarose precipitation, and the biotinylated T7 RNA polymerase will quickly bind to the agarose, and then the agarose bound to the T7 RNA polymerase is removed by centrifugation through a microporous filter, and the reaction solution containing mRNA in the lower centrifuge tube is collected. Because most of the protein, T7 RNA polymerase, has been removed from the collected reaction solution, the impact on the subsequent capping reaction is greatly reduced. Therefore, we directly use the collected reaction solution for the subsequent capping reaction, and the capping rate is 100%; after the capping is completed, most of the reaction impurities are removed by the second step of magnetic bead purification, and finally a high-purity mRNA with complete structure is obtained, which can be used for preclinical research. Compared with the preparation process of performing two RNA adsorption columns or RNA magnetic bead purification, since the first step of purification of the present invention uses streptavidin agarose to bind and remove biotinylated proteins without involving the column binding and column elution process of mRNA, the purification loss of mRNA in the transcription product is reduced, so the final yield of mRNA prepared and purified using the present invention is increased by more than 10% on the basis of RNA magnetic bead purification, and the purification process of streptavidin agarose is simpler and faster than the column purification and magnetic bead purification, and it can be completed in only 3 to 5 minutes. Therefore, our invention is a new method that is faster and simpler to operate, has a higher purification yield, and is suitable for various small-scale production or laboratory preparation and purification to obtain structurally complete mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Conventional and novel flow charts for preparing and purifying structurally intact mRNA for laboratory-scale production.

[0034] Figure 2 The figure is a flow chart of the purification of mRNA obtained by in vitro transcription using Biotin-His-T7 RNA polymerase via streptavidin agarose affinity.

[0035] Figure 3 Schematic diagram of the fusion protein structure of Avi-tag and His-T7 RNA polymerase.

[0036] Figure 4Diagram of the mechanism of site-directed biotinylation of Avi-His-T7 RNA polymerase by biotin ligase (BirA).

[0037] Figure 5 The figure shows the preparation and plasmid verification results of Avi-His-T7 RNA polymerase plasmid. A shows that the His-T7 RNA polymerase plasmid was digested with EcoRⅠ and BsaBⅠ restriction enzymes and then purified and recovered by gel purification; b shows that a DNA insert containing a partial fragment of Avi-His-T7 was synthesized, and then digested with EcoRⅠ restriction enzyme and purified and recovered; c shows the PCR test result of Avi-His-T7 RNA polymerase bacterial solution; d shows the single digestion test result of EcoRⅠ and BsaBⅠ of Avi-His-T7 RNA polymerase plasmid.

[0038] Figure 6 This is an 8% SDS-PAGE protein gel image of the induced expression of Avi-His-T7 RNA polymerase in BL21 Escherichia coli competent cells and elution from a nickel column.

[0039] Figure 7 This is the 8% SDS-PAGE protein gel image of the preparation of Biotin-His-T7 RNA polymerase and elution from the nickel column.

[0040] Figure 8 8% SDS-PAGE protein gel showing the biotinylation efficiency of Avi-His-T7 RNA polymerase.

[0041] Fig. 9 1% agarose gel image of in vitro transcription test based on Biotin-His-T7 RNA polymerase (left) and 20% urea PAGE gel image of mRNA integrity obtained by ribozyme-based method (right).

[0042] Fig.10 This is an 8% SDS-PAGE protein gel image of Biotin-His-T7 RNA polymerase binding to different amounts of streptavidin agarose and different binding time tests.

[0043] Fig.11 8% SDS-PAGE protein gel for testing the binding time of Biotin-His-T7 RNA polymerase to streptavidin agarose.

[0044] Fig.12 The RBD mRNA obtained by in vitro transcription of Biotin-His-T7 RNA polymerase was purified by streptavidin agarose, capped, purified by RNA magnetic beads, and then digested with ribozymes to detect the capping efficiency on 20% urea PAGE gel.

[0045] Fig.13 The transcription product of Biotin-His-T7 RNA polymerase was purified by streptavidin agarose, filtered by a microporous filter, capped, purified by RNA magnetic beads, and then the capping efficiency was detected by ribozyme digestion on a 20% urea PAGE gel. DETAILED DESCRIPTION

[0046] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0047] Example 1: Laboratory-scale preparation of intact mRNA

[0048] Typically, small-scale production and laboratory-scale preparation and purification of structurally intact mRNA require in vitro transcription with T7 RNA polymerase, column purification or RNA magnetic bead purification to remove the reaction solution, and then capping. After capping, column purification or RNA magnetic bead purification is performed to remove the reaction solution to obtain mRNA with high purity and intact structure for various preclinical studies.

[0049] In order to improve the production yield of mRNA in small-scale production or laboratory-scale preparation, the present invention provides a novel preparation and purification method: first, based on the in vitro transcription method of synthesizing mRNA vaccine by T7 RNA polymerase, His-T7 RNA polymerase is subjected to site-specific biotinylation treatment, and the N-terminus of His-T7 RNA polymerase is subjected to Avi-tag site-specific biotinylation labeling by biotin ligase (BirA), and then the biotinylated T7 RNA polymerase is used to perform in vitro transcription of a DNA transcription template containing a T7 promoter site and a poly (A) tail to obtain the target mRNA. The specific steps are as follows: Figure 1 shown.

[0050] Secondly, based on the principle that streptavidin-agarose has a very high affinity for biotinylated T7 RNA polymerase, but has very low or even no adsorption to ordinary nucleic acids, we mixed the transcription product with the streptavidin-agarose precipitate, and the biotinylated T7 RNA polymerase will quickly bind to the agarose. Then, the agarose bound to the T7 RNA polymerase was removed by centrifugation through a microporous filter, and the reaction solution containing mRNA in the lower centrifuge tube was collected. Because most of the protein, T7 RNA polymerase, has been removed from the collected reaction solution, the impact on the subsequent capping reaction is greatly reduced. Therefore, we directly used the collected reaction solution for the subsequent capping reaction, and the capping rate was tested to be 100%. After the capping is completed, most of the reaction impurities are removed by the second step of magnetic bead purification, and finally a high-purity, structurally complete mRNA is obtained, which can be used for preclinical research. Compared with the preparation process of performing RNA adsorption column or RNA magnetic bead purification twice, since the first step of purification of the present invention uses streptavidin agarose binding to remove biotinylated proteins without involving column binding and column elution processes of mRNA, the purification loss of mRNA in the transcription product is reduced, and the yield of structurally intact mRNA finally obtained is increased by more than 10% based on RNA magnetic bead purification; and the purification process of streptavidin agarose is simpler and faster, and purification can be completed in only 3 to 5 minutes. Therefore, our invention is a new method for preparing and purifying structurally intact mRNA vaccines in preclinical studies that is faster and simpler to operate, has a higher recovery rate, and is suitable for.

[0051] Example 2: mRNA obtained by in vitro transcription using Biotin-His-T7 RNA polymerase and purified by streptavidin agarose affinity

[0052] Based on the principle that streptavidin agarose has a very high affinity for biotinylated proteases, but has very low or even no adsorption to mRNA in the transcription product, the reaction product transcribed by Biotin-His-T7 RNA polymerase is mixed with streptavidin agarose precipitation, and the biotinylated T7 RNA polymerase in the reaction product is quickly bound to the agarose. The agarose bound to the protease is filtered out by centrifugation through a microporous filter, and the reaction solution containing mRNA in the lower centrifuge tube is collected. The specific steps are as follows:

[0053] Pipette the commercial streptavidin agarose suspension, centrifuge, discard the supernatant as much as possible, and keep the agarose precipitate; rinse the millipore filter with DEPC water. Add the transcription product to the agarose precipitate, aspirate with the pipette tip 5 times, let stand at room temperature for 1 minute, add all the transcription product and streptavidin agarose suspension to the millipore filter, and immediately centrifuge at 12000rpm for 10 seconds. At this time, the agarose precipitate is located in the upper filter and the reaction liquid is at the bottom of the centrifuge tube; aspirate the agarose precipitate in the filter with DEPC water of the same volume as the transcription product, and then immediately centrifuge at 12000rpm for 10 seconds. The washing solution is collected at the bottom of the tube and mixed with the original reaction solution; repeat the above agarose washing operation 2 times, and mix the 3 washing solutions with the original reaction solution and collect them together. The entire purification process can be completed within 3 to 5 minutes. The specific process is as follows: Figure 2 shown.

[0054] Example 3: Site-directed biotinylation of His-T7 RNA polymerase based on Avi-tag technology

[0055] like Figure 3 As shown in the figure, a short peptide tag consisting of 15 amino acid residues, the Avi tag, is added to the N-terminus of the His-T7 RNA polymerase. Then, biotin ligase (BirA) can be used to efficiently activate biotin (D-Biotin) in the presence of ATP to form biotinyl-5' adenylate. Biotinyl-5' adenylate specifically connects biotin to the lysine residue (K) of the Avi tag, thereby achieving fast and efficient site-specific biotin labeling of our His-T7 RNA polymerase ( Figure 4 ), the specific steps of preparing biotinylated Avi-His-T7 RNA polymerase are shown in the following Examples 4 to 6. Since biotinylation occurs on the lysine residue of the Avi tag, it is a site-specific tag and thus does not affect the activity of the protease.

[0056] Example 4: Preparation of Avi-His-T7 RNA polymerase plasmid

[0057] The His-T7 RNA polymerase plasmid was digested with EcoRⅠ and BsaBI restriction enzymes and then purified by gel purification. Figure 5 As shown in a;

[0058] A DNA insert containing a partial fragment of Avi-His-T7 was synthesized, and then digested with EcoRⅠ restriction endonuclease and purified and recovered, such as Figure 5 As shown in b;

[0059] The DNA insert and the double-digested purified T7 RNA polymerase vector were ligated with T4 DNA ligase and transformed into DH5α competent cells. The competent cells were plated on ampicillin-resistant plates and cultured overnight at 37°C in the culture phase.

[0060] Pick three single clones (marked as ①, ②, ③) and shake the bacteria for PCR detection. The PCR procedure is as follows:

[0061] Table 1 Avi-His-T7 RNA polymerase bacterial liquid PCR system

[0062]

[0063] Table 2 Avi-His-T7 RNA polymerase bacterial liquid PCR program

[0064]

[0065] After PCR, take 5 μL of the product, add 6× DNA loading buffer, run on 1% agarose gel, and analyze by imaging. Figure 5 As shown in c, monoclonal clone No. ① showed an obvious positive band.

[0066] The PCR-positive single clone No. ① was shaken to extract the plasmid (plasmid ①), and the plasmid was further verified by single restriction endonuclease digestion with EcoRⅠ and BsaBI restriction endonucleases (such as Figure 5 d), the enzyme digestion system is as follows:

[0067] Table 3 Single restriction enzyme digestion system of Avi-His-T7 RNA polymerase plasmid with EcoRI and BsaBI restriction enzymes

[0068]

[0069] After enzyme digestion, run 1% agarose gel and analyze the image. Figure 5 As shown in Figure d, the full length of Avi-His-T7 RNA plasmid is 7330 bp, and plasmid ① can obtain a band of 7330 bp after single restriction digestion with EcoR Ⅰ and BsaBI Ⅰ, respectively. This verification shows that plasmid ① is constructed with the correct Avi-His-T7 RNA polymerase.

[0070] Example 5: Expression and purification of Avi-His-T7 RNA polymerase

[0071] ⑴ Expression and purification of Avi-His-T7 RNA polymerase. The plasmid was transformed into BL21 competent cells, and a single clone was picked for expression test: one thousandth of the bacterial solution was inoculated into a new culture medium containing ampicillin resistance and shaken at 37°C for 3 hours, and one thousandth of 1M IPTG was added to induce expression at 37°C for 3 hours. The bacterial solution was centrifuged at 4000rpm for 20 minutes to collect the bacteria, lysis buffer was added to resuspend the bacteria, the bacteria were broken with an ultrasonic device, and the protein solution was collected by centrifugation at 16000rpm for 30 minutes; the protein solution was hung on a Ni column and the protein was eluted. The results are as follows Figure 6 shown.

[0072] ⑵ Desalting of Avi-His-T7 RNA polymerase. Since Avi-His-T7 RNA polymerase is present in a high concentration salt solution after being eluted from the nickel column, in order to carry out the biochemical reaction, the protein is desalted and the protein buffer is replaced with 1× PBS.

[0073] Example 6: Biotinylation with Avi-His-T7 RNA polymerase and detection of biotinylation efficiency

[0074] Avi-His-T7 RNA polymerase was biotinylated in the presence of 2× BirA (purchased from Bio-Tech, P0630M) at a final concentration for 1 h. The Biotin-His-T7 RNA polymerase obtained after the reaction was re-loaded with a nickel column, eluted, and desalted ( Figure 7 ).

[0075] The biotinylation efficiency was tested by affinity binding with streptavidin agarose. After affinity binding of Biotin-His-T7 RNA polymerase with streptavidin agarose, the mixture of protein and agarose was centrifuged for solid-liquid separation. After the liquid was collected, it was heated and denatured for Western blot detection to determine whether the remaining protein in the liquid was biotinylated Biotin-His-T7 RNA polymerase or unbiotinylated Avi-His-T7 RNA polymerase. The results are shown in Figure 2. Figure 8 As shown in the Coomassie brilliant blue gel image, after affinity binding with streptavidin agarose, most of the protease was captured by streptavidin agarose, indicating that Avi-His-T7 RNA polymerase has been biotinylated in large quantities and can be used for subsequent experiments and uses.

[0076] Example 7: In vitro transcription test and ribozyme digestion test of Biotin-His-T7 RNA polymerase

[0077] The prepared Biotin-His-T7 RNA polymerase was used to in vitro transcribe the SARS-COV-2 RBD mRNA containing a T7 promoter site and a poly (A) tail, and the system was as follows:

[0078] Table 4 In vitro transcription system of Biotin-His-T7 RNA polymerase

[0079]

[0080] Place in a 37℃ water bath for 3 hours. After the reaction, take 4μL of the transcription product and run it on 1% agarose gel for imaging analysis. Fig. 9 (Left) As shown, Biotin-His-T7 RNA polymerase can perform normal in vitro transcription reaction, indicating that biotinylation does not affect the protease activity.

[0081] Both sets of transcription products were subjected to one-step RNA magnetic bead purification (magnetic bead purification), with the following specific steps: add 1.8 times the volume of RNA magnetic beads to the transcription products, mix by pipetting, and let stand at room temperature for 5 minutes; place on a magnetic stand for 5 minutes, carefully aspirate and discard the liquid with a gun tip to avoid aspirating RNA magnetic beads and causing mRNA loss; carefully add 200 μL 80% ethanol to wash the RNA magnetic beads, carefully aspirate and discard the ethanol, repeat once, and dry at room temperature for 3 minutes; remove the EP tube from the magnetic stand, add DEPC water to resuspend the RNA magnetic beads, and let stand at room temperature for 5 minutes; place on a magnetic stand, let stand for 3 minutes, aspirate the transparent liquid, avoid aspirating RNA magnetic beads, and measure the concentration with Nano drop. The mRNA obtained by in vitro transcription based on Biotin-His-T7 RNA polymerase was further tested for quality using a cleavage-type ribozyme targeting the 5'-UTR sequence of mRNA, and the degradation of mRNA during in vitro transcription by Biotin-His-T7 RNA polymerase was observed by urea PAGE gel image. RBD mRNA (10 μg) and ribozyme (60 pmol) were mixed in a flask containing a final concentration of 10 mM MgCl. 2 The reaction was carried out in a 37°C water bath in a saline solution for 2-3 hours. After the reaction was completed, the loading buffer was added and the 5'-enzyme-cleaved short fragments were separated by 20% urea PAGE gel. The 20% urea PAGE gel was stained for nucleic acid and imaged for analysis. The results are shown in Fig. 9 As shown in (right), the 5'-UTR of mRNA cleaved by ribozyme has clear enzyme cleavage fragments on the gel image, indicating the integrity of the 5'-end structure of mRNA obtained by in vitro transcription by Biotin-His-T7 RNA polymerase.

[0082] Example 9: Test of the Binding Time of Biotin-His-T7 RNA Polymerase and Streptavidin Agarose

[0083] To determine the time limit of affinity binding of Biotin-His-T7 RNA polymerase during streptavidin agarose purification, we tested the experimental conditions of incubating with equal amounts of protease and different amounts of streptavidin agarose for the same time and incubating with equal amounts of protease and different amounts of streptavidin agarose for different times. The specific operation is as follows:

[0084] Take the streptavidin agarose suspension and centrifuge to remove the supernatant, add an equal volume of 1× PBS to wash once, centrifuge to remove the supernatant, and keep the agarose precipitate for use. Add different amounts of streptavidin agarose to equal amounts of Biotin-His-T7 RNA polymerase, incubate for the same time, and use protein gel to detect the amount of remaining protein; add equal amounts of streptavidin agarose to equal amounts of Biotin-His-T7 RNA polymerase, incubate for different times, and use protein gel to detect the amount of remaining protein. The results are as follows Fig.10 As shown, 1 μL Biotin-His-T7 RNA polymerase (2 mg / mL) needs to be combined with 20 μL streptavidin agarose (300 μg / μL) and the incubation time is 2 min. Fig.11 As shown, 1 μL Biotin-His-T7 RNA polymerase (2 mg / mL) and 20 μL streptavidin agarose (300 μg / μL) can be combined by incubation for 1 min.

[0085] Example 10: Capping test of mRNA obtained by in vitro transcription with Biotin-His-T7 RNA polymerase after purification with streptavidin agarose

[0086] ① In vitro transcription. Use the prepared Biotin-His-T7 RNA polymerase to in vitro transcribe the SARS-COV-2 RBD mRNA containing the T7 promoter site and the poly (A) tail. The system is as follows:

[0087] Table 5 Biotin-His-T7 RNA polymerase in vitro transcription system

[0088]

[0089] Place in a 37°C water bath and react for 3 hours.

[0090] ②Purification. Divide 100μL of mRNA transcribed in vitro by Biotin-His-T7 RNA polymerase into two parts: take 45μL of the transcription product for RNA magnetic bead purification and measure the concentration with Nano drop. Take 55μL of the transcription product for streptavidin agarose purification in the other part. The specific purification steps are as follows: aspirate 100μL of streptavidin agarose suspension (300μg / μL), centrifuge, discard the supernatant as much as possible, and leave the agarose precipitate; add 55μL of the transcription product to the precipitate, aspirate 5 times with the pipette tip, and let it stand at room temperature for 1 minute; immediately centrifuge at 12000rpm for 10 seconds, and carefully aspirate the supernatant and transfer it to a new EP tube. The above two mRNAs transcribed in vitro by Biotin-His-T7 RNA polymerase were capped. The capping reaction is as follows:

[0091] Table 6 Capping system for mRNA purified by streptavidin agarose

[0092]

[0093] The capping reaction was incubated in a 37°C water bath for 30 minutes. After the reaction, RNA magnetic beads were used for purification and the concentration was measured by Nano drop.

[0094] ③ Use ribozymes to detect capping reactions. Use ribozymes to digest the 5'-UTR of capped and uncapped mRNAs to generate detectable 5'-digested short fragments with cap structures (length (n+1) nt) and 5'-digested short fragments without cap structures (length n nt) to indicate whether the capping reaction is complete. The digestion reaction is as follows: RBD mRNA (10 μg) and ribozyme (60 pmol) are mixed in a solution containing a final concentration of 10 mM MgCl 2 The reaction was carried out in a 37°C water bath in a saline solution for 2-3 hours. After the reaction, the loading buffer was added and the 5'-enzyme-cleaved short fragments were separated by 20% urea PAGE gel. The 20% urea PAGE gel was stained for nucleic acid and imaged for analysis.

[0095] The results are as follows Fig.12 As shown, the mRNA obtained by this synthesis and purification method based on Biotin-His-T7 RNA polymerase in vitro transcription and streptavidin agarose purification can be completely capped by enzymatic capping reaction. This shows that the mRNA synthesis and purification method we invented is feasible, and the streptavidin agarose purification process can be completed in only 3 to 5 minutes, with fast purification speed and simple operation.

[0096] Example 11: Final yield test of Biotin-His-T7 RNA polymerase transcription product after streptavidin affinity purification, capping, and RNA magnetic bead purification

[0097] ① In vitro transcription. The reaction system based on Biotin-His-T7 RNA polymerase in vitro transcription is as follows:

[0098] Table 7 In vitro transcription system based on Biotin-His-T7 RNA polymerase

[0099]

[0100] ② Purification based on streptavidin agarose (filtered using a microporous filter). The above transcription product was divided into two equal parts: one part was used for two RNA magnetic bead purifications, and the concentration was measured using Nano drop. The other part was used for two purifications of streptavidin agarose + RNA magnetic beads, and the concentration was measured using Nano drop. Specific streptavidin agarose purification steps: aspirate 160 μL of diluted commercial streptavidin agarose suspension (300 μg / μL), centrifuge, discard the supernatant as much as possible, and leave the agarose precipitate; rinse the microporous filter once with DEPC water. Add 200 μL of transcription product to the agarose precipitate, aspirate with a pipette tip 5 times, let stand at room temperature for 1 minute, add all the suspension of transcription product and streptavidin agarose to the microporous filter, and immediately centrifuge at 12000rpm for 10 seconds. At this time, the agarose precipitate is located in the upper filter and the reaction solution is located at the bottom of the centrifuge tube; immediately draw 200 μL of DEPC water to rinse the agarose precipitate in the filter, and then centrifuge at 12000rpm for 10 seconds, collect the washing solution at the bottom of the tube and mix it with the original reaction solution; repeat the above agarose washing operation 2 times, and mix the 3 washing solutions with the original reaction solution and collect them together. Purification can be completed in 3-5 minutes. Then perform a second RNA magnetic bead purification. Column purification (RNA magnetic beads + RNA magnetic beads) group and (streptavidin agarose + RNA magnetic beads) purification group were carried out at the same time, and 3 independent comparative experiments were carried out, and the experimental data were statistically analyzed. The final recovery of the two groups, column purification (RNA magnetic beads + RNA magnetic beads) group and (streptavidin agarose + RNA magnetic beads) purification group, was compared with the recovery of the column purification (RNA magnetic beads + RNA magnetic beads) group after the first RNA magnetic bead purification to calculate the final yield of the two groups. The results are shown in Table 8. The final yield of mRNA in the column purification (RNA magnetic beads + RNA magnetic beads) group was 81% ± 2% (n = 3), while the final yield of mRNA in the (streptavidin agarose + RNA magnetic beads) purification group was 93% ± 2% (n = 3), which was increased by more than 10%. This shows that the method we invented based on the preparation of Biotin-His-T7 RNA polymerase and the purification of streptavidin agarose can significantly improve the final yield of mRNA.

[0101] Table 8 Final yield of Biotin-His-T7 RNA polymerase transcription products after streptavidin affinity purification, microporous filter filtration, and RNA magnetic bead purification (n=3)

[0102]

[0103] ③ mRNA capping reaction test based on streptavidin agarose purification (filtered using a microporous filter). Pipette 200 μL of the transcription product for streptavidin agarose purification (filtered using a microporous filter) in step ② above. The amount of mRNA in the reaction solution obtained at this time can be detected using the Qubit system. The purified reaction solution is directly subjected to the capping reaction. The reaction system is as follows:

[0104] Table 9 mRNA capping reaction system based on streptavidin agarose purification (using microporous filter filtration)

[0105]

[0106] The capping reaction was incubated in a 37°C water bath for 30 minutes. After the reaction, RNA was purified using RNA magnetic beads and the concentration was measured using Nanodrop.

[0107] The capped and purified mRNA was subjected to a ribozyme-based capping reaction test. The specific steps were as follows: RBD mRNA (10 μg) and ribozyme (60 pmol) were mixed in a solution containing a final concentration of 10 mM MgCl 2 The reaction was carried out in a 37°C water bath in a saline solution for 2-3 hours. After the reaction, the loading buffer was added and the 5'-enzyme-cleaved short fragments were separated by 20% urea PAGE gel. The 20% urea PAGE gel was stained for nucleic acid and imaged for analysis.

[0108] The results are as follows Fig.13 As shown, the capping efficiency of the mRNA prepared and purified by the method invented by us is 100%. This shows that the method invented by us based on in vitro transcription preparation by biotinylated His-T7 RNA polymerase combined with streptavidin agarose affinity purification can not only prepare mRNA quickly, simply and with higher purification recovery rate, but also the structure of the prepared mRNA is complete and the quality is guaranteed. It is a new method suitable for preparing mRNA in preclinical research.

[0109] Amino acid sequence of Avi-tag (SEQ ID NO.1)

[0110] GLNDIFEAQKIEWHE;

[0111] Amino acid sequence of Avi-His-T7 RNA polymerase SEQ ID NO.2

[0112] HHHHHHGSMNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWRRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA*

[0113] Amino acid sequence of Avi-His-T7 RNA polymerase SEQ ID NO.3

[0114] *。

Claims

1. A T7 RNA polymerase with an affinity tag, characterized in that: It is a protein described in any one of the following a1-a5: a1: a protein with biotin site-specifically labeled at the N-terminus of His-T7 RNA polymerase with an amino acid sequence as shown in SEQ ID NO.2; a2: a protein having substantially the same enzyme activity and function obtained by labeling the His-T7 RNA polymerase shown in a1 with biotin at its C-terminus or / and N-terminus in addition to labeling the His-T7 RNA polymerase at its N-terminus as described above; a3: The His-T7 RNA polymerase shown in a1 is labeled with biotin at the N-terminus or / and the C-terminus of its His tag in addition to the aforementioned labeling with biotin at the N-terminus to obtain a protein having substantially the same enzyme activity and function; a4: a protein having substantially the same enzyme activity and function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in a1-a3; a5: a protein that has more than 90% sequence homology with the proteins of a1-a3 and whose enzyme activity and function are basically equivalent to those of the proteins of a1-a2; a6: A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of any of the amino acid sequences described in a1-6, wherein the tag does not affect the function of the T7 RNA polymerase mutant.

2. The T7 RNA polymerase with an affinity tag according to claim 1, characterized in that: The method of labeling His-T7 RNA polymerase with biotin is chemical labeling or site-specific labeling using Avi-tag technology.

3. The T7 RNA polymerase with an affinity tag according to claim 1, characterized in that: The method for site-specifically labeling biotin at the N-terminus of the His-T7 RNA polymerase comprises: b1: constructing Avi-His-T7 RNA polymerase with an amino acid sequence as shown in SEQ ID NO.3; b2: Biotin ligase is used to activate biotin and specifically link biotin to the lysine of the Avi tag.

4. A method for preparing and purifying mRNA, characterized in that: The target mRNA was obtained by in vitro transcription using biotinylated T7 RNA polymerase and affinity purification using streptavidin.

5. The method for preparing and purifying mRNA according to claim 4, characterized in that The preparation method of the biotinylated T7 RNA polymerase comprises: adding an Avi short peptide tag to the N-terminus of the His-T7 RNA polymerase, and then using a biotin ligase to perform Avi-tag site-specific biotinylation labeling on the N-terminus of the T7 RNA polymerase to obtain a biotinylated Avi-His-T7 RNA polymerase, wherein the sequence of the Avi short peptide tag is shown in SEQ ID NO.1, and the sequence of the Avi-His-T7 RNA polymerase is shown in SEQ ID NO.

2.

6. The method for preparing and purifying mRNA according to claim 4, characterized in that: The reaction product transcribed by the biotinylated Avi-His-T7 RNA polymerase is mixed with the streptavidin agarose precipitate, the biotinylated Avi-His-T7 RNA polymerase is bound to the agarose, and then the agarose bound to the biotinylated Avi-His-T7 RNA polymerase is filtered out, and the reaction solution containing mRNA in the lower centrifuge tube is collected.

7. An mRNA, characterized in that The method according to any one of claims 4 to 6 is used to obtain the 8. Use of the mRNA according to claim 7 or the method according to any one of claims 4 to 6 in a capping reaction.

9. The use according to claim 8, characterized in that: After capping, the reaction impurities are removed by magnetic bead purification or column purification.

10. Use of the method according to any one of claims 4 to 6 in the preparation of mRNA therapeutic agents and vaccines.

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