Immobilized enzyme for catalyzing RNA ligation reaction and preparation method and application thereof

Abstract

The present invention provides an immobilized enzyme for catalyzing RNA ligation reaction, and a preparation method and application thereof. The immobilized enzyme comprises an immobilized carrier and an enzyme fixed on the immobilized carrier, the immobilized carrier is an affinity carrier, and the enzyme is an enzyme catalyzing RNA ligation reaction. The present invention can solve the problem that free enzymes capable of catalyzing RNA ligation reaction in the prior art are not suitable for industrial production, and is applicable to the field of immobilized enzyme preparation.

Description

Technical Field

[0001] The invention relates to the field of immobilized enzyme preparation, and in particular to an immobilized enzyme for catalyzing RNA ligation reaction, and a preparation method and application thereof. Background Art

[0002] Small nucleic acid drugs refer to nucleotide sequences of no more than 30 base pairs. Their mechanism of action is to act on mRNA through specific nucleotide sequences, thereby inhibiting the expression of target proteins and achieving the effect of treating diseases. Small nucleic acid drugs include various types of small molecule nucleic acids such as RNA small interfering drugs (siRNA), microRNA (miRNA), mRNA, and RNA aptamers. Nucleic acid ligases can be divided into DNA ligases and RNA ligases. Existing studies have found that RNA ligases are involved in the repair, splicing, and editing of damaged RNA molecules or in changing the primary structure of damaged RNA molecules. Therefore, the use of RNA ligases to catalyze the synthesis of small nucleic acid drugs in vitro is of great clinical significance.

[0003] Enzyme immobilization technology has been widely used throughout the world. Compared with free enzymes, immobilized enzymes not only maintain their high efficiency, specificity and mild enzyme catalytic reaction characteristics, but also overcome the shortcomings of free enzymes, showing a series of advantages such as high storage stability, easy separation and recovery, repeated use, continuous and controllable operation, and simple process. Therefore, if the enzyme that catalyzes the RNA ligation reaction can be immobilized, the enzyme can be reused, and after immobilization, it will also go through steps such as cleaning to fix the target protein on the carrier, which is equivalent to purifying the enzyme without nuclease residues. The application of immobilized enzymes in the continuous synthesis of RNA will reduce the steps of separating the reaction product from the enzyme, reduce production costs, and improve production efficiency and capacity.

[0004] At present, no relevant reports on this type of immobilized enzyme have been found. If an immobilized enzyme with high enzyme activity recovery and good repeated use stability for catalyzing RNA ligation reactions can be developed, it will not only save the purification step and remove nucleases, but also greatly save costs and improve production efficiency. Summary of the invention

[0005] The main purpose of the present invention is to provide an immobilized enzyme for catalyzing RNA ligation reaction and a preparation method and application thereof, so as to solve the problem that the free enzyme capable of catalyzing RNA ligation reaction in the prior art is not suitable for industrial production.

[0006] In order to achieve the above-mentioned object, according to the first aspect of the present invention, an immobilized enzyme for catalyzing RNA ligation reaction is provided, wherein the immobilized enzyme comprises an immobilized carrier and an enzyme immobilized on the immobilized carrier, wherein the immobilized carrier is an affinity carrier, and the enzyme is an enzyme catalyzing RNA ligation reaction; the ligand of the immobilized carrier is one or more of nitrilotriacetic acid, iminodiacetic acid, glutathione, Halo Tag, dextrin or Strep-Tactin.

[0007] Furthermore, the matrix of the immobilized carrier is one or more of styrene, polystyrene, acrylic acid, polyacrylic acid, methacrylate, butyl methacrylate, silica, dextran and agarose; preferably, the matrix of the immobilized carrier is agarose.

[0008] Furthermore, the linker arm of the immobilization carrier is C6~C14.

[0009] Further, the immobilized carrier comprises one or more of the affinity carriers EziG Amber, EziG Coral, EziG Opal, IB-HIS-1, IB-HIS-2, IMAC Chromstar FF, Ni Chromstar FF, Ni-IDA Purose 6 FF, Ni-NTAPurose 6 FF, AEC-9001, AEC-9002, AEC-9007, AEC-9008, AEC-9050, AEC-9051, AEC-9052, AEC-9060, AEC-9061, AEC-9062, AEC-9063, Glutathione Beads 4FF, Halo LinkTM Resin, MBPseq Dextrin Agarose Resin 6FF or Streptactin Agarose Resin 4FF; preferably, the immobilized carrier comprises the affinity carrier Ni Chromstar FF, Ni-IDA Purose 6 One or more of FF, Ni-NTAPurose 6 FF, AEC-9002, AEC-9007, AEC-9008, AEC-9060 or AEC-9063; more preferably, the immobilized carrier comprises affinity carrier AEC-9007 or AEC-9008.

[0010] Furthermore, the enzyme used to catalyze the RNA ligation reaction is any one or more of RNA ligase families 1, 2 and 3.

[0011] Furthermore, the enzyme used to catalyze the RNA ligation reaction is derived from a pure enzyme solution or a crude enzyme solution.

[0012] In order to achieve the above object, according to the second aspect of the present invention, a method for preparing the enzyme for catalyzing RNA ligation reaction is provided, the preparation method comprising: mixing an immobilized carrier with the enzyme catalyzing RNA ligation reaction for immobilization and incubation to obtain an immobilized enzyme.

[0013] Furthermore, the preparation method includes: S1, performing a first cleaning on the immobilized carrier; S2, mixing the cleaned immobilized carrier with an enzyme that catalyzes RNA ligation reaction for immobilization incubation; S3, performing a second cleaning on the incubated immobilized enzyme to obtain an immobilized enzyme for catalyzing RNA ligation reaction.

[0014] Further, S1 comprises: performing a first wash twice on the immobilized carrier using a first buffer, the first buffer comprising 0.05-0.2M PB, 0.2-1M NaCl, 20-100mM imidazole, pH 7.0-8.0; preferably, the first buffer comprises 0.1M PB, 0.5M NaCl, 50mM imidazole, pH 7.0-8.0.

[0015] Furthermore, the immobilization incubation in S2 includes: mixing the cleaned immobilized carrier with the enzyme that catalyzes the RNA ligation reaction, incubating with stirring at 20°C for 4h~48h and then filtering; preferably, the enzyme that catalyzes the RNA ligation reaction includes an enzyme solution, and the volume ratio of the enzyme solution to the cleaned immobilized carrier is 2-6:1, preferably 4:1; more preferably, the enzyme solution includes a crude enzyme solution or a pure enzyme solution; further preferably, the protein concentration in the crude enzyme solution is 20~50mg / mL, and the protein concentration in the pure enzyme solution is 1~5mg / mL; preferably, the stirring time is 4h~16h.

[0016] Further, S3 includes: performing a second washing 3 to 4 times on the incubated immobilized enzyme using a second buffer, wherein the second buffer includes 0.05-0.2M PB, 10-50mM imidazole, pH 7.0-8.0; preferably, the second buffer includes 0.1M PB, 30mM imidazole, pH 7.0-8.0.

[0017] In order to achieve the above-mentioned purpose, according to the third aspect of the present invention, there is provided an immobilized enzyme for catalyzing RNA ligation reaction, or an immobilized enzyme prepared by the preparation method of the immobilized enzyme for catalyzing RNA ligation reaction, and its use in synthesizing RNA.

[0018] Furthermore, the application includes: placing an immobilized enzyme for catalyzing RNA ligation reaction in a continuous flow device to achieve continuous synthesis of RNA.

[0019] By applying the technical solution of the present invention and using the above-mentioned immobilized enzyme as an affinity carrier, the enzyme used to catalyze RNA ligation reaction can be reused, thereby achieving efficient enzyme immobilization, improving enzyme utilization and stability, and reducing the pressure of enzyme purification before the reaction, including but not limited to application in continuous RNA synthesis, reducing the steps of separating reaction products from enzymes, reducing production costs, and improving production efficiency and capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings in the specification, which constitute a part of this application, are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 The UPLC result diagram according to Example 8 of the present invention is shown.

[0022] Figure 2 A schematic diagram of a device for single-feed continuous reaction according to Example 13 of the present invention is shown.

[0023] Figure 3 A schematic diagram of the apparatus for continuous reaction of split feed according to Example 13 of the present invention is shown.

[0024] Among them, the above-mentioned drawings include the following figure marks: A: substrate system; B: peristaltic pump; C: packed bed reactor; D: post-reaction system; A1: substrate 1 system; A2: substrate 2 system; A3: substrate 3 system; A4: substrate 4 system; B1: substrate 1 peristaltic pump; B2: substrate 2 peristaltic pump; B3: substrate 3 peristaltic pump; B4: substrate 4 peristaltic pump; C1: packed bed reactor; D1: post-reaction system; E: mixer. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0026] At present, in the prior art, the synthesis of in vitro small molecule RNA is mainly carried out by in vitro transcription and solid phase synthesis. However, the existing solid phase synthesis method is mainly achieved by primer synthesis, and the synthesis scale is limited and difficult to be scaled up to production scale. And limited by the characteristics of solid phase synthesis, the increase of chain length will lead to a decrease in purity and yield, and there will be many problems such as reduced coupling efficiency, decreased physical stability of the carrier, and increased branched impurities. At present, the enzyme that catalyzes RNA ligation reaction is used to catalyze and synthesize RNA in vitro. The enzyme needs to be purified or secondary purified to remove nuclease before it can be used, otherwise the oligonucleotide chain substrate is easily degraded, it is difficult to react, and impurities such as endotoxins are easily introduced. The purification process involved requires the use of affinity resins, purification instruments, and manual operations, etc., and this enzyme is unstable and easy to inactivate. The purified enzyme is used to catalyze and synthesize RNA, which can only be used once and cannot be reused. After the addition of various factors, the cost of synthesizing RNA in vitro is high. In this application, the inventor attempts to develop an immobilized enzyme for catalyzing RNA ligation reaction, and thus proposes a series of protection schemes of this application.

[0027] In a first typical embodiment of the present application, an immobilized enzyme for catalyzing RNA ligation reaction is provided, wherein the immobilized enzyme comprises an immobilized carrier and an enzyme fixed on the immobilized carrier, the immobilized carrier is an affinity carrier, and the enzyme is an enzyme catalyzing RNA ligation reaction; the ligand of the immobilized carrier is one or more of nitrilotriacetic acid, iminodiacetic acid, glutathione, Halo Tag, dextrin or Strep-Tactin.

[0028] In the present application, the inventor unexpectedly discovered that the use of an affinity carrier containing the above-mentioned ligand as an immobilized carrier of an enzyme for catalyzing RNA ligation reaction can form a stable bond with little effect on enzyme activity. This bond is not only conducive to the purification of the ligase, but also enables the ligase to be reused, thereby improving its economy and environmental protection in the process of catalyzing the synthesis of non-natural RNA.

[0029] Halo Tag ligand is a chloroalkane-PEG compound, which is itself a genetically modified substrate of bacterial dehalogenase (DhaA from Rhodococcus sp.) and can effectively covalently bind to dehalogenase under physiological conditions. Strep-Tactin ligand is a specially modified streptavidin derivative that can efficiently and specifically bind to the short peptide tag Strep-tag.

[0030] Nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA) can react with Ni 2+The metal ions such as glutathione, Halo Tag, dextrin or Strep-Tactin form a stable chelate structure, which has a good recognition effect on proteins containing His tags. Affinity carriers modified with ligands such as glutathione, Halo Tag, dextrin or Strep-Tactin also have specific recognition capabilities for proteins containing specific tags. When these ligands are covalently bound to carrier materials such as resins and agarose, the enzymes that catalyze RNA ligation reactions are better fixed through recognition and binding of protein sequences, and immobilized enzymes with better performance for catalyzing RNA ligation reactions are obtained.

[0031] Using a carrier with the above-mentioned ligand as an enzyme immobilization carrier can immobilize the enzyme that catalyzes RNA ligation reaction with better performance. Compared with other ligands, such as primary amine, secondary amine, epoxy, cyano, etc., it can avoid modification of amino acid residues near the active center, and can directionally fix the enzyme to the carrier, eliminate the influence of foreign enzymes, retain high enzyme activity, and at the same time improve the stability of the immobilized enzyme to achieve enzyme reuse.

[0032] In a preferred embodiment, the matrix of the immobilization carrier is one or more of styrene, polystyrene, acrylic acid, polyacrylic acid, methacrylate, butyl methacrylate, silica, dextran and agarose; preferably, the matrix of the immobilization carrier is agarose.

[0033] In a preferred embodiment, the linker arm of the immobilization carrier is C6-C14.

[0034] The relationship between the matrix and the linker of the carrier is mainly reflected in how they work together to achieve enzyme immobilization, thereby improving the stability and reusability of the enzyme during the synthesis of non-natural RNA. The carrier matrix is ​​usually a solid material with a high surface area and good mechanical stability, which provides a physical platform for the enzyme to attach. The linker is a molecule that acts as a bridge between the carrier matrix and the enzyme molecule, and its length affects the spatial arrangement and activity of the enzyme molecule.

[0035] The length of the connecting arm of the immobilization carrier used in the present application is preferably C6-C14, which indicates that the connecting arm is a molecule composed of 6 to 14 carbon atoms. The connecting arm within the above length range can better fix the enzyme on the carrier, without affecting the catalytic efficiency, and can ensure the stable connection between the enzyme and the carrier.

[0036] In a preferred embodiment, the immobilized carrier affinity carrier is one or more of EziG Amber, EziG Coral, EziGOpal, IB-HIS-1, IB-HIS-2, IMAC Chromstar FF, Ni Chromstar FF, Ni-IDA Purose 6 FF, Ni-NTA Purose 6 FF, AEC-9001, AEC-9002, AEC-9007, AEC-9008, AEC-9050, AEC-9051, AEC-9052, AEC-9060, AEC-9061, AEC-9062, AEC-9063, Glutathione Beads 4FF, Halo LinkTM Resin, MBPseq Dextrin Agarose Resin 6FF or Streptactin Agarose Resin 4FF.

[0037] Preferably, the immobilized carrier comprises one or more of the affinity carriers Ni Chromstar FF, Ni-IDA Purose 6 FF, Ni-NTA Purose 6 FF, AEC-9002, AEC-9007, AEC-9008, AEC-9060 or AEC-9063; more preferably, the immobilized carrier comprises the nickel affinity carrier AEC-9007 or AEC-9008.

[0038] Affinity carriers applicable to the present application are shown in Tables 1 and 2. The carriers in Table 2 are all commercially available carriers.

[0039] Table 1

[0040] .

[0041] Table 2

[0042] .

[0043] The above affinity carriers can be used to prepare immobilized enzymes for catalyzing RNA ligation reactions. Due to differences in carrier structure and other factors among different types of carriers, the activity, stability and reusability of the immobilized enzymes also vary. Among the enzymes used to catalyze RNA ligation reactions, the immobilized enzymes prepared using AEC-9007 or AEC-9008 resins have the best activity and stability.

[0044] In a preferred embodiment, the enzyme catalyzing RNA ligation is any one or more of RNA ligase families 1, 2 and 3.

[0045] In a preferred embodiment, the enzyme is derived from pure enzyme solution or crude enzyme solution.

[0046] In a second typical embodiment of the present application, a method for preparing the above-mentioned immobilized enzyme for catalyzing RNA ligation reaction is provided, and the preparation method comprises: mixing an immobilized carrier with an enzyme catalyzing RNA ligation reaction for immobilization and incubation to obtain an immobilized enzyme.

[0047] In a preferred embodiment, the preparation method includes: S1, performing a first cleaning on the immobilized carrier; S2, mixing the cleaned immobilized carrier with an enzyme that catalyzes RNA ligation reaction for immobilization incubation; S3, performing a second cleaning on the incubated immobilized enzyme to obtain the immobilized enzyme.

[0048] In the above preparation method, an optimized process is proposed for the preparation of immobilized enzymes that catalyze RNA ligation reactions, aiming to improve the enzyme's immobilization efficiency, purity, and activity retention, thereby ensuring its performance in non-natural RNA synthesis. The following is a detailed analysis of the principles and effects of each step:

[0049] In the first cleaning step, since the immobilized carrier may be adsorbed with residual chemicals, microorganisms or their metabolites during storage or transportation, these impurities may not only affect the adsorption efficiency of the enzyme, but also introduce nonspecific reactions or contamination in subsequent catalytic reactions. Therefore, the first cleaning step can effectively remove these impurities by using a buffer solution to create a clean environment for enzyme fixation. Through the first cleaning, the surface of the carrier is cleaned, the nonspecific adsorption that may be encountered in the subsequent immobilization process is reduced, and the purity and specific adsorption rate of the ligase are improved. This step is crucial for the activity and stability of the enzyme in the later stage, and helps to improve the efficiency and yield of RNA synthesis.

[0050] In the second cleaning step, after incubation, the surface of the immobilized enzyme may still have unbound enzyme molecules or chemical components in the washing solution, and these residues may affect the catalytic activity of the enzyme or the stability of the reaction system. Therefore, the second cleaning step is intended to completely remove these residues and ensure the purity of the immobilized enzyme. The above-mentioned second cleaning step further improves the purity of the immobilized enzyme and removes residues that may inhibit enzyme activity or cause non-specific reactions. By using a buffer containing an appropriate concentration of imidazole for cleaning, enzyme desorption caused by excessive cleaning can be avoided, while ensuring the purity of the reaction system, which is conducive to improving the efficiency of RNA synthesis and the purity of the product.

[0051] The above-mentioned method for preparing immobilized RNA ligase can effectively avoid the complex steps and cost issues in the enzyme purification process, and at the same time, through optimized immobilization and cleaning steps, ensure the stability and activity of the enzyme on the carrier, as well as the purity of the reaction system. This method not only simplifies the enzyme preparation process, but also significantly improves the efficiency and yield of RNA synthesis, making large-scale production possible, and is more in line with the requirements of green chemistry and environmental protection.

[0052] In a preferred embodiment, S1 includes: performing two first washes on the immobilized carrier using a first buffer, the first buffer including 0.05-0.2M PB, 0.2~1M NaCl, 20~100mM imidazole, pH 7.0~8.0; preferably, the first buffer includes 0.1M PB, 0.5M NaCl, 50mM imidazole, pH 7.0~8.0.

[0053] In a preferred embodiment, the immobilization incubation in S2 includes: mixing the cleaned immobilized carrier with the enzyme that catalyzes the RNA ligation reaction, stirring and incubating at 20°C for 4h~48h and then filtering; preferably, the enzyme that catalyzes the RNA ligation reaction includes an enzyme solution, and the volume ratio of the enzyme solution to the cleaned immobilized carrier is 2-6:1, preferably 4:1; more preferably, the enzyme solution includes a crude enzyme solution or a pure enzyme solution; further preferably, the protein concentration in the crude enzyme solution is 20~50mg / mL, and the protein concentration in the pure enzyme solution is 1~5mg / mL; preferably, the stirring time is 4h~16h.

[0054] In a preferred embodiment, S3 includes: performing a second wash 3 to 4 times on the immobilized enzyme after incubation using a second buffer, wherein the second buffer includes 0.05-0.2M PB, 10-50mM imidazole, pH 7.0-8.0; preferably, the second buffer includes 0.1M PB, 30mM imidazole, pH 7.0-8.0. Preferably, the salt ion concentration in the second buffer is less than the salt ion concentration in the first buffer, and the above-mentioned salt ions include but are not limited to salt ions in sodium chloride and phosphate.

[0055] In the preparation method of the above-mentioned immobilized enzyme, the incubated enzyme solution can be a pure enzyme solution or a crude enzyme solution. This method is a method for rapidly preparing a high-purity immobilized enzyme that catalyzes RNA ligation reactions. It is quick and easy, and can be prepared using a crude enzyme solution, which greatly simplifies the preparation process of the immobilized enzyme and greatly saves costs. At the same time, the purified enzyme can be used to prepare the immobilized enzyme, and the enzyme can be easily recovered for multiple uses, thereby improving the utilization rate of the enzyme. By cleaning with salts, impurities such as host proteins, RNA or DNA that are free or bound to the immobilized carrier can be removed to obtain a pollution-free immobilized enzyme. By using this kind of pollution-free immobilized enzyme catalyzed reaction, the impurity content in the reaction product is low, which can meet the requirements of subsequent production with higher quality requirements such as injection drug production, reduce additional purification steps, thereby saving costs and yields, and is conducive to industrialized amplification production.

[0056] In a third typical embodiment of the present application, there is provided an enzyme that catalyzes RNA ligation reaction, or an immobilized enzyme that catalyzes RNA ligation reaction prepared by the above-mentioned method for preparing an immobilized enzyme, and its use in synthesizing RNA.

[0057] In a preferred embodiment, the application includes: placing an immobilized enzyme for catalyzing RNA ligation reaction in a continuous flow device to achieve continuous synthesis of RNA.

[0058] In a preferred embodiment, the continuous synthesis of RNA includes synthesizing RNA using a single-channel continuous reaction or a multi-channel continuous reaction.

[0059] In a preferred embodiment, the continuous flow device comprises a pump, a reaction system and a reaction column.

[0060] In a preferred embodiment, the continuous flow device comprises a pump, a reaction system, a mixer and a reaction column.

[0061] In a preferred embodiment, the pump comprises one or more of a micro peristaltic pump, a high performance liquid phase pump or a syringe pump.

[0062] In a preferred embodiment, the reaction system comprises 1, 2, 3 or 4 RNA substrates.

[0063] In a preferred embodiment, the reaction column is filled with the above-mentioned immobilized enzyme for catalyzing RNA ligation reaction.

[0064] In a preferred embodiment, the mixer comprises a dynamic mixer or a static mixer; preferably, the static mixer comprises a static baffle mixer, a perforated plate mixer or a three-way mixer.

[0065] In a preferred embodiment, the synthetic RNA product includes natural RNA and non-natural RNA, preferably, the non-natural RNA includes: ribonucleotides having one or more of ribose 2' position modification, ribose backbone modification or base modification; preferably, the ribose 2' position modification includes 2'-methoxy modification, 2'-fluorine modification, 2'-hydrogen modification, 2'-methoxyethyl modification, 2'-FANA modification, locked nucleic acid modification or hexitol nucleic acid modification.

[0066] Among them, the locked nucleic acid is modified to ; Hexitol nucleic acid modified to ; 2' methoxyethyl modified to ; 2' methoxy modified to ; 2' fluorine modified to ; 2'-FANA is ; In the above structures, Base represents a base.

[0067] Non-natural RNA is an important tool in the research of biomedicine and life sciences. Nowadays, most non-natural RNA is synthesized by solid-phase phosphoramidite chemistry using automated equipment. The specific process consists of four cycles: deprotection, coupling, oxidation and capping. However, the solid-phase synthesis method has serious drawbacks and can only be used for the synthesis of relatively short RNA fragments. As the chain length increases, the yield drops sharply. In addition, the yield of solid-phase synthesis is low, and the current price of RNA fragment synthesis is still high, and large-scale production cannot be achieved. At the same time, due to the excessive number of toxic chemical reagents in the process, the treatment of the three wastes generated brings great treatment pressure. The use of enzymes that catalyze RNA ligation reactions to connect and synthesize long-chain RNA products requires the preparation of purified enzymes, but the purification of enzymes is very time-consuming and labor-intensive, and requires a lot of material and labor costs. At the same time, the purified liquid enzyme cannot be recovered from the reaction system, which has a certain impact on the post-treatment enzyme removal process. Therefore, it is very necessary to develop a more environmentally friendly and efficient method for synthesizing non-natural RNA with immobilized enzymes.

[0068] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0069] Example 1 Construction and expression of RNA ligase

[0070] This example uses the enzyme used in Chinese patent CN117070512B as an example. The RNA ligase protein is codon-optimized for host bacteria recombinantly expressed in Escherichia coli. The optimized codons are synthesized to obtain the target gene, and the target gene is constructed into the pET28a expression vector with NcoI and XhoI as restriction sites, with a His tag at the N-terminus. The recombinant plasmid with the ligase gene is transformed into the cloning strain DH5α, and cultured overnight on a medium containing LB+1.5% agar containing 50μg / mL of kanamycin to obtain a monoclonal clone. The sequence of the monoclonal clone is confirmed by sequencing.

[0071] Single clones were picked into liquid LB medium containing 50µg / mL kanamycin, cultured at 37°C for 16h, centrifuged, and the supernatant was discarded to obtain bacterial sludge. The obtained bacterial sludge was used to extract plasmids using a plasmid extraction kit, and the obtained plasmids were transformed into BL21 (DE3) expression strains. The single clones were cultured overnight on LB + 1.5% agar medium containing 50µg / mL kanamycin to obtain single clones. Single clones were picked into 5mL LB medium containing 50µg / mL kanamycin, cultured at 37°C for 16h, and seed solution was obtained. The seed solution was inoculated into 500mL LB medium (containing 50µg / mL kanamycin) at a 0.1% inoculum, and cultured at 37°C until OD 600 =0.6, 0.1 M IPTG was added for induction, the culture temperature was adjusted to 20°C, and the induction was carried out at 220 rpm for 16 h to obtain the induced expression of the ligase Escherichia coli bacterial solution.

[0072] The bacterial solution was centrifuged at 8000rpm, and the supernatant was discarded to obtain the induced expression of E. coli bacterial sludge. The obtained ligase E. coli bacterial sludge was resuspended in 100mM Tris-CL buffer to prepare a 10% bacterial concentration. Ultrasonic disruption was performed at a power of 20%, ultrasonic for 2s, and stopped for 6s. The enzyme solution after ultrasonication was centrifuged at 4°C and 12000rpm for 30min. The supernatant was the RNA ligase crude enzyme solution. 10mM imidazole was added to the crude enzyme solution, and the enzyme solution obtained after filtration with a 0.22µm filter membrane was purified with a nickel column. After desalting and changing the solution, it was purified again with a strong anion column. The obtained enzyme solution was stored in 30% glycerol after desalting and changing the solution, and the RNA ligase pure enzyme solution was obtained.

[0073] It should be noted that in subsequent experiments, the His tag at the N-terminus of the RNA ligase used for immobilization with a glutathione ligand affinity carrier was replaced with a GST tag; the His tag at the N-terminus of the RNA ligase used for immobilization with a Halo Tag ligand affinity carrier was replaced with a Halo Tag tag; the His tag at the N-terminus of the RNA ligase used for immobilization with a dextrin ligand affinity carrier was replaced with an MBP tag; the His tag at the N-terminus of the RNA ligase used for immobilization with a Strep-Tactin ligand affinity carrier was replaced with a Strep-II Tag tag.

[0074] Example 2 Preparation of immobilized RNA ligase

[0075] a. Wash the vector 1-2 times with buffer 0.1M PB 7.0-8.0 (containing 0.2-1M NaCl + 20-100mM imidazole); b. Mix the vector with 4 times (4V) of the vector volume of pure RNA ligase or crude enzyme solution, stir at 20°C for 4h-48h, filter, and wash 3-4 times with buffer 0.1M PB 7.0-8.0 (containing 0.5M NaCl + 50mM imidazole) to obtain the immobilized RNA ligase on the affinity vector.

[0076] The method for preparing the immobilized RNA ligase is not limited to a specific RNA ligase. The immobilized RNA ligase described in the present application can be prepared using the method for RNA ligases with different sources and sequences.

[0077] Example 3 Synthesis of natural tRNA using immobilized RNA ligase prepared from pure enzyme

[0078] Substrate 1 and substrate 2 were added to a clean reagent bottle (substrate 1 and substrate 2 are shown in Table 3), and the reaction system was prepared so that the final volume was 100µL, the concentration of substrate 1 and substrate 2 were both 500µM, the concentration of ATP was 1mM, the concentration of Tris-HCl (pH 7.5 @25℃) was 50mM, the concentration of MgCl2 was 10 mM, the concentration of DTT was 1 mM, and 30mg of the immobilized ligase prepared from the pure RNA ligase solution prepared in Example 2 was added. The reaction system was reacted at 16℃ for 16h. The obtained reaction system was centrifuged at 3000rpm for 2min, the immobilized ligase solid was removed, and the supernatant was detected by SDS-PAGE and MS. The results showed that the immobilized ligase could catalyze the synthesis of the final product, and the results of UPLC showed that the substrate had been basically reacted and converted completely. The molecular weight was 23560.3 identified by mass spectrometry, and the theoretical molecular weight was 23564±5, which was the target product. It shows that the method of catalyzing the synthesis of natural tRNA by immobilized ligase is successful.

[0079] Table 3

[0080] .

[0081] P represents the phosphate group on the 5th carbon atom of the 5'-terminal nucleotide.

[0082] The vectors used and the specific results are shown in Table 4:

[0083] Table 4

[0084] .

[0085] Example 4 Synthesis of non-natural tRNA using immobilized RNA ligase prepared from crude enzyme

[0086] The reaction system is as shown in Example 3 (substrate 3 and substrate 4 are shown in Table 5), wherein the difference is that 30 mg of immobilized ligase prepared from crude RNA ligase solution prepared in Example 2 is added. The results show that the immobilized ligase can catalyze the synthesis of the final product, and the results of UPLC show that the substrate has been basically reacted and converted completely. The molecular weight identified by mass spectrometry is 24601.6, and the theoretical molecular weight is 24606.2±5, which is the target product. This shows that the method of catalyzing the synthesis of non-natural tRNA by immobilized ligase is successful.

[0087] Table 5

[0088] .

[0089] The m before A, C, G or U represents the 2' methoxy modification of the ribonucleotide, and P represents the phosphate group on the 5th C atom of the 5' terminal nucleotide.

[0090] Substrates 3 and 4 have 2' methoxy modifications on all ribonucleotides.

[0091] The vectors used and the specific results are shown in Table 6:

[0092] Table 6

[0093] .

[0094] Example 5: Using immobilized RNA ligase prepared from crude enzyme to catalyze the synthesis of double-stranded ligation of non-natural RNA

[0095] Substrate 5 to substrate 8 were mixed in equal molar proportions (substrate 5 to substrate 8 are shown in Table 7), 10 μL of 1 mM ATP was added, 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, 1 mM DTT) was added, water was added so that the substrate concentration was 50 μM, 30 mg of the immobilized ligase prepared in Example 2 was added, and the reaction was carried out at 25° C. for 3 h. The obtained reaction system was centrifuged at 3000 rpm for 2 min, the immobilized ligase solid was removed to obtain the supernatant, and the supernatant was analyzed and detected by denaturing polyacrylamide gel electrophoresis. As a result, new bands were generated and the substrate bands disappeared. The sample was subjected to UPLC detection, and the results showed that a target peak with increased molecular weight was obtained, which was judged to be the product peak, and there was basically no substrate remaining. After MS identification, 4613.12 and 6633.07 were detected. The theoretical molecular weight of the positive chain was 4609.91±5 and the molecular weight of the antisense chain was 6634.99±6, indicating that the product catalyzed by the immobilized ligase was generated to generate a double-stranded RNA after connection.

[0096] Table 7

[0097] .

[0098] The m after A, C, G or U represents a 2' methoxy modification of the ribonucleotide, and the f represents a 2' fluorine modification of the ribonucleotide.

[0099] Substrate 5 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 5 and 6, and 2' fluorine modifications on ribonucleotides at positions 3 and 4.

[0100] Substrate 6 has 2' methoxy modifications on ribonucleotides at positions 3, 4, 7 and 8, and 2' fluorine modifications on ribonucleotides at positions 1, 2, 5 and 6.

[0101] Substrate 7 has 2' methoxy modifications on ribonucleotides at positions 2, 3, 6, 7, 10 and 11, and 2' fluorine modifications on ribonucleotides at positions 1, 4, 5, 8 and 9.

[0102] Substrate 8 has 2' methoxy modifications on ribonucleotides at positions 1, 4, 5, 8 and 9, and 2' fluorine modifications on ribonucleotides at positions 2, 3, 6 and 7.

[0103] The vectors used and the specific results are shown in Table 8:

[0104] Table 8

[0105] .

[0106] Example 6 Using immobilized ligase prepared from crude enzyme to catalyze nick ligation of non-natural RNA

[0107] Substrates 9 to 12 were mixed in equal molar proportions (substrates 9 to 12 are shown in Table 9), added to the reaction bottle, 10 μL of 1 mM ATP, 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, 1 mM DTT), 50 mg of the immobilized ligase prepared in Example 2, and water was added to make the substrate concentration 400 μM. The reaction was carried out at 25°C for 16 hours. The obtained reaction system was centrifuged at 3000 rpm for 2 minutes, and the immobilized ligase solid was removed to obtain the supernatant. The supernatant was analyzed and detected by denaturing polyacrylamide gel electrophoresis. The catalytic systems of immobilized enzymes using different types of carriers all generated product bands with very high brightness. The samples were subjected to UPLC detection, and the results showed that the target peak with increased molecular weight was obtained, which was judged to be the product peak. After MS identification, the results were consistent with the standard product, and the generation of the product was confirmed, indicating that the immobilized ligase can synthesize non-natural RNA products using the gapped double-stranded non-natural RNA as a substrate.

[0108] Table 9

[0109] .

[0110] The m after A, C, G or U represents a 2' methoxy modification of the ribonucleotide, and the f represents a 2' fluorine modification of the ribonucleotide.

[0111] Substrate 9 has 2' methoxy modifications on the 1st and 3rd ribonucleotides and 2' fluorine modifications on the 2nd and 4th ribonucleotides.

[0112] The substrate 10 has 2' methoxy modifications on the 1st, 3rd and 5th ribonucleotides, and 2' fluorine modifications on the 2nd and 4th ribonucleotides.

[0113] The substrate 11 has 2' methoxy modifications on the 2nd, 4th and 6th ribonucleotides, and 2' fluorine modifications on the 1st, 3rd and 5th ribonucleotides.

[0114] Substrate 12 has 2' methoxy modifications on ribonucleotides at positions 1, 3, 5, 7, 9, 11, 13 and 15, and 2' fluorine modifications on ribonucleotides at positions 2, 4, 6, 8, 10, 12 and 14.

[0115] Example 7: Ligation of double-stranded RNA containing thiolation modification catalyzed by immobilized RNA ligase

[0116] Substrates 13 to 16 were mixed in an equal molar ratio of 50 μM (substrates 13 to 16 are shown in Table 10), added to the reaction bottle, and the ligation reaction system was prepared to be 50 mM Tris-HCl (pH7.5), 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 20 mg of the immobilized RNA ligation crude enzyme prepared in Example 2, and water was added to make the substrate concentration 50 μM, and the reaction was carried out at 16°C for 16 hours. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5 minutes. The samples were tested by UPLC, and the results showed that the target peak with increased molecular weight was obtained in the catalytic system of the immobilized enzyme using different types of carriers, which was judged to be the product peak. After MS identification, the results were consistent with the standard, and the generation of the product was confirmed, indicating that the immobilized ligase can synthesize non-natural RNA products using the gapped double-stranded non-natural RNA as a substrate.

[0117] Table 10

[0118] .

[0119] The m after A, C, G or U represents a 2' methoxy modification of the ribonucleotide, f represents a 2' fluorine modification of the ribonucleotide, and s represents a thio modification of the 5' phosphate of the ribonucleotide.

[0120] The substrate 13 has 2' methoxy modification on the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th and 12th ribonucleotides, 2' fluorine modification on the 7th, 9th, 10th and 11th ribonucleotides, and 5' phosphate thio modification on the 2nd and 3rd ribonucleotides.

[0121] The substrate 14 has 2' methoxy modification on the 1st to 9th ribonucleotides, and 5' phosphate thio modification on the 1st, 8th and 9th ribonucleotides.

[0122] The substrate 15 has 2' methoxy modification on the 1st, 3rd, 4th, 5th, 6th, 7th and 8th ribonucleotides, 2' fluorine modification on the 2nd ribonucleotide, and 5' phosphate thio modification on the 1st, 8th and 9th ribonucleotides.

[0123] Substrate 16 has 2' methoxy modification on ribonucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11 and 12, 2' fluorine modification on ribonucleotides at positions 2, 6 and 14, and 5' phosphate thio modification on ribonucleotides at positions 2 and 3.

[0124] Example 8 Using immobilized ligase to catalyze double-stranded ligation of non-natural RNA

[0125] Mix substrates 17 to 20 in equal molar proportions (substrates 17 to 20 are shown in Table 11), add to the reaction bottle, add 10 μL of 1 mM ATP, add 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, 1 mM DTT), add 50 mg of immobilized RNA ligase prepared in Example 2, add water so that the substrate concentration is 800 μM, react at 25°C for 16 hours, centrifuge the obtained reaction system at 3000 rpm for 2 minutes, remove the immobilized ligase solid to obtain the supernatant, and analyze and detect by denaturing polyacrylamide gel electrophoresis, resulting in the generation of a very bright product band. The sample was subjected to UPLC detection, and it was found that in the catalytic system of the immobilized enzyme using different types of carriers, the target peak with increased molecular weight was obtained, which was judged to be the product peak. The UPLC results are as follows Figure 1 The results of MS identification were consistent with those of the standard product, confirming the generation of the product, indicating that the immobilized ligase can synthesize double-stranded non-natural RNA.

[0126] Table 11

[0127] .

[0128] Wherein, m after A, C, G or U represents 2' methoxy modification of the ribonucleotide, f represents 2' fluorine modification of the ribonucleotide, s represents thio modification of 5' phosphate of the ribonucleotide; dT represents thymine.

[0129] Substrate 17 has 2' methoxy modification on ribonucleotides at positions 1, 2, 3, 4, 5, 6, 8, 10, 12, 13 and 14, 2' fluorine modification on ribonucleotides at positions 7 and 9, 5' phosphate thio modification on ribonucleotides at positions 2 and 3, and deoxyribonucleotide thymine at position 11.

[0130] The ribonucleotides at positions 1 to 7 of substrate 18 all have 2' methoxy modifications.

[0131] Substrate 19 has 2' methoxy modification on ribonucleotides at positions 2, 4, 6, 7, 8, 9 and 10, 2' fluorine modification on ribonucleotides at positions 1, 3 and 5, and 5' phosphate thio modification on ribonucleotides at positions 9 and 10.

[0132] The substrate 20 has 2' methoxy modification on the 1st, 3rd, 7th, 9th, 11th and 13th ribonucleotides, 2' fluorine modification on the 2nd, 4th, 5th, 6th, 8th, 10th and 12th ribonucleotides, and 5' phosphate thio modification on the 2nd and 3rd ribonucleotides.

[0133] The structure of L96 is:

[0134] .

[0135] Example 9

[0136] The preparation method of the immobilized ligase is the same as that in Example 2, and the carrier AEC-9008 of Asymchem is selected. The difference is that the carrier is washed with solutions of different concentrations, and the first washing is performed to prepare the affinity resin immobilized ligase. The method of catalyzing the synthesis of natural tRNA ligation using this immobilized ligase is the same as that in Example 3. The conversion rate is detected, and the results are shown in Table 12.

[0137] Table 12

[0138] .

[0139] Example 10

[0140] The preparation method of the immobilized ligase is the same as that in Example 2, and the carrier AEC-9008 of Asymchem is selected. The difference is that the crude RNA ligase solution with different protein concentrations is fixed to prepare the affinity resin immobilized ligase. The method of catalyzing the synthesis of natural tRNA ligation using this immobilized ligase is the same as that in Example 3. The conversion rate is detected, and the results are shown in Table 13.

[0141] Table 13

[0142] .

[0143] Embodiment 11

[0144] The preparation method of the immobilized ligase is the same as that in Example 2, and the carrier AEC-9008 of Asymchem is selected. The difference is that the immobilized enzyme is washed with different concentrations of imidazole, and a second washing is performed to prepare the affinity resin immobilized ligase. The method of catalyzing the synthesis of natural tRNA ligation using this immobilized ligase is the same as that in Example 3. The conversion rate is detected, and the results are shown in Table 14.

[0145] Table 14

[0146] .

[0147] Example 12

[0148] In a clean reaction bottle, 0.5 g of each of substrates 17 to 20 in Example 8 was added, reaction buffer was added to a final concentration of Tris-Cl 50 mM, MgCl2 10 mM, DTT 1 mM, ATP was added to a final concentration of 1 mM, and the immobilized enzyme prepared in Example 2 (using Asymchem's carrier AEC-9008) was added to make its protein amount 0.4 mg / mL and the final substrate concentration 800 µM. The system was mixed and shaken on a shaker. After reacting at 16°C for 16 h, it was heated at 85°C for 15 min to inactivate the protein, centrifuged at 12000 rpm for 10 min, and the supernatant was taken for UPLC detection.

[0149] In another clean reaction bottle, 0.5 g of each of substrates 17 to 20 in Example 8 was added, reaction buffer was added to a final concentration of Tris-Cl 50 mM, MgCl2 10 mM, DTT 1 mM, ATP was added to a final concentration of 1 mM, pure enzyme was added to make its protein amount 0.4 mg / mL, the final substrate concentration was 800 µM, the system was mixed and shaken on a shaker, reacted at 16 ° C for 16 h, heated at 85 ° C for 15 min to inactivate the protein, centrifuged at 12000 rpm for 10 min, and the supernatant was taken for UPLC detection.

[0150] After UPLC detection, both groups of reactions were able to achieve complete reaction, and the reaction system was subjected to ion column purification to purify the product, and the final product was obtained after ultrafiltration concentration and freeze drying. The purity, water content and endotoxin content of the product were tested, and the results are shown in Table 15 below.

[0151] Table 15

[0152] .

[0153] The results showed that the immobilized RNA ligase had higher activity, resulting in higher conversion rate. At the same time, the product prepared after post-treatment had better purity and higher yield. Since the product prepared by the immobilized ligase had higher purity, although the reaction was carried out in a non-clean laboratory, endotoxins still remained. The product was endotoxin-removed in a clean area, and the level of injection-grade drugs could reach 0.25EU / mg. However, the product prepared by the liquid pure enzyme had too high initial endotoxin content, and it was difficult to control the endotoxin to the standard range of injection-grade drugs in the clean area.

[0154] Example 13 Continuous catalytic ligation of double-stranded non-natural RNA using immobilized ligase

[0155] Single-strand feed continuous reaction: Substrates 17 to 20 in Example 8 were mixed in equal molar proportions (substrates 17 to 20 are shown in Table 11), added to a reaction bottle, 1 mM ATP, 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, 1 mM DTT), and water were added to make the substrate concentration 600 μM. This was the reaction system. The affinity carrier-immobilized ligase prepared in Example 2 (Asymchem's carrier AEC-9008 was selected) was added to a 5 mL reaction column, about 3.5 g of the immobilized enzyme was filled, and the reaction system was continuously passed through the reaction column (reaction device as shown in Figure 11) at 25°C by the action of a peristaltic pump. Figure 2 As shown in the figure), the retention time was set to 5-20 minutes, and samples were taken for detection every 30 minutes. The samples were tested by UPLC, and the results showed that the product conversion rate reached more than 92%, and it could run continuously for more than 100 hours without decreasing the conversion rate.

[0156] Feed continuous reaction in fractions: Prepare substrates 17-20 at a concentration of 600 µM, add 1 mM ATP, 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, 1 mM DTT), and then obtain 4 substrate systems. Add the affinity carrier-immobilized ligase prepared in step 2 into a 5 mL reaction column, filling about 3.5 g of immobilized enzyme. Connect the 4 substrate systems to a peristaltic pump respectively, and pass the 4 reaction systems through the reaction column (reaction device as shown in the figure) at 25 °C. Figure 3 As shown in the figure), the retention time is set to 5-20 minutes, and samples are taken for detection every 30 minutes. When a certain substrate is detected to have a large amount of residue, the flow rate of this substrate is immediately reduced to maximize the utilization of the substrate. The sample was tested by UPLC, and the results showed that the product conversion rate reached more than 94%, and it can run continuously for more than 100 hours without a decrease in conversion rate.

[0157] The above two continuous flow reaction modes can achieve a production capacity of >1 kg of product per liter of column reactor per day. Compared with the batch reaction of immobilized enzymes, the conversion rate is basically the same, but the production capacity is increased by about 20 times, and the steps of separating the enzyme from the product are reduced.

[0158] The specific results are shown in Table 16:

[0159] Table 16

[0160] .

[0161] Embodiment 14

[0162] The preparation method of the immobilized ligase is the same as that of Example 2, except that the linker arms of the selected carriers are different, and the carriers AEC-9002, AEC-9008, AEC-9050, AEC-9051, and AEC-9052 of Asymchem are selected to prepare affinity resin immobilized ligases respectively. The method of using such immobilized ligase to catalyze the synthesis of double-stranded ligation of non-natural RNA is the same as that of Example 8. The conversion rate is detected, and the results are shown in Table 17.

[0163] Table 17

[0164] .

[0165] Embodiment 15

[0166] The preparation method of the immobilized ligase is the same as that of Example 2, except that the chelated metal ions of the selected carriers are different, and the carriers AEC-9008, AEC-9060, AEC-9061, AEC-9062, and AEC-9063 of Asymchem are selected to prepare affinity resin immobilized ligases respectively. The method of using such immobilized ligase to catalyze the synthesis of double-stranded ligation of non-natural RNA is the same as that of Example 8. The conversion rate is detected, and the results are shown in Table 18.

[0167] Table 18

[0168] .

[0169] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the above immobilized RNA ligase is prepared by using the above preparation method with an immobilized enzyme carrier, and the RNA ligase activity can be improved and reused by using the above RNA ligase, thereby reducing the purification pressure of the RNA ligase before the reaction, reducing the steps of separating the reaction product from the enzyme, reducing the production cost, improving the production efficiency and capacity, and the endotoxin content in the prepared product is low, and a reaction product with an impurity content that meets the requirements and can be directly used to prepare injectable drugs can be prepared, thereby reducing the steps and costs required for product post-processing.

[0170] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An immobilized enzyme for catalyzing RNA ligation reaction, characterized in that: The immobilized enzyme comprises an immobilized carrier and an enzyme immobilized on the immobilized carrier; The immobilized carrier is an affinity carrier; The enzyme is an enzyme that catalyzes RNA ligation reactions; The ligand of the immobilized carrier is nitrilotriacetic acid or iminodiacetic acid; The immobilized carrier is one or more of affinity carriers AEC-9007, AEC-9008, Ni Chromstar FF, Ni-IDA Purose 6FF, Ni-NTA Purose 6 FF, AEC-9002, AEC-9060 or AEC-9063; The AEC-9002 is a matrix of agarose, a linker of C6, a ligand of IDA, and a metal ion of Ni. 2+ , affinity carrier with a particle size of 40 μm; The AEC-9007 is agarose matrix, the linker is C14, the ligand is IDA, and the metal ion is Ni 2+ , affinity carrier with a particle size of 90 μm; The AEC-9008 has a matrix of agarose, a linker of C14, a ligand of IDA, and a metal ion of Ni 2+ , affinity carrier with a particle size of 40 μm; The AEC-9060 is agarose as the matrix, C14 as the linker, IDA as the ligand, and Cu as the metal ion. 2+ , affinity carrier with a particle size of 40 μm; The AEC-9063 is agarose as the matrix, C14 as the linker, IDA as the ligand, and Fe as the metal ion. 2+ , affinity carrier with a particle size of 40μm.

2. The immobilized enzyme for catalyzing RNA ligation reaction according to claim 1, characterized in that: The matrix of the immobilization carrier is one or more of styrene, polystyrene, acrylic acid, polyacrylic acid, methacrylate, butyl methacrylate, silica, dextran or agarose.

3. The immobilized enzyme for catalyzing RNA ligation reaction according to claim 1, characterized in that: The connecting arm of the immobilization carrier is C6-C14.

4. The immobilized enzyme for catalyzing RNA ligation reaction according to claim 1, characterized in that: The enzyme includes any one or more of RNA ligase families 1, 2 and 3.

5. A method for preparing an immobilized enzyme for catalyzing RNA ligation reaction according to any one of claims 1 to 4, characterized in that: The preparation method comprises: The immobilized carrier is mixed with the enzyme catalyzing the RNA ligation reaction for immobilization and incubation to obtain the immobilized enzyme.

6. The preparation method according to claim 5, characterized in that: The preparation method comprises: S1, performing a first cleaning on the immobilized carrier; S2, mixing the cleaned immobilized carrier with the enzyme catalyzing the RNA ligation reaction for immobilization and incubation; S3, performing a second cleaning on the incubated immobilized enzyme to obtain the immobilized enzyme for catalyzing RNA ligation reaction.

7. The preparation method according to claim 6, characterized in that: The S1 comprises: performing the first washing twice on the immobilized carrier using a first buffer solution, wherein the first buffer solution comprises 0.05-0.2M PB, 0.2-1M NaCl, 20-100mM imidazole, and pH 7.0-8.

0.

8. The preparation method according to claim 6, characterized in that: The immobilization incubation in S2 comprises: The immobilized carrier after cleaning is mixed with the enzyme catalyzing the RNA ligation reaction, incubated with stirring at 20° C. for 4 h to 48 h, and then filtered.

9. The preparation method according to claim 6, characterized in that: The enzyme includes enzyme solution, and the volume ratio of the enzyme solution to the immobilized carrier after washing is 2-6:

1.

10. The preparation method according to claim 9, characterized in that: The enzyme solution includes crude enzyme solution or pure enzyme solution.

11. The preparation method according to claim 10, characterized in that: The protein concentration in the crude enzyme solution is 20-50 mg / mL, and the protein concentration in the pure enzyme solution is 1-5 mg / mL.

12. The preparation method according to claim 8, characterized in that: The stirring time is 4h~16h.

13. The preparation method according to claim 7, characterized in that: The S3 comprises: performing the second washing 3 to 4 times on the immobilized enzyme after the incubation using a second buffer, wherein the second buffer comprises 0.05-0.2 M PB, 10 to 50 mM imidazole, and pH 7.0 to 8.

0.

14. Use of the immobilized enzyme for catalyzing RNA ligation reaction according to any one of claims 1 to 4, or the immobilized enzyme for catalyzing RNA ligation reaction prepared by the preparation method according to any one of claims 6 to 13, in synthesizing RNA.

15. The use according to claim 14, characterized in that: The application comprises: arranging the immobilized enzyme for catalyzing RNA ligation reaction in a continuous flow device to realize continuous synthesis of RNA.

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