A method for purifying and preparing dsRNA standard

Abstract

The present invention discloses a method for purifying and preparing a dsRNA standard. The dsRNA standard is prepared using a two-step chromatography method. During the ion exchange chromatography purification step, the present invention employs the addition of basic amino acids to reduce the interaction between long-chain dsRNA and the chromatography filler, increase the saline elution strength, and improve the recovery rate, thereby resolving the problem of low purification recovery rates in the preparation of dsRNA standards. The method provided by the present invention is highly efficient and low-cost. Using a linearized plasmid as a vector for in vitro transcription templates, large-scale in vitro transcription can be achieved, yielding gram-level dsRNA products in a single batch, meeting the standard product requirements for registration and production quality testing in the field of nucleic acid pesticides.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid preparation and purification, and in particular relates to a method for purifying and preparing a dsRNA standard. Background Art

[0002] Nucleic acid pesticides developed based on RNAi technology are a new type of biopesticide that has recently entered the agrochemical market. They have been reported to be effective in controlling a variety of pests and diseases, including plant pests, viruses, nematodes, and pathogenic fungi. Double-stranded RNA (dsRNA) is the primary active ingredient in nucleic acid pesticides. During both the production of parent drugs and formulation development, high-purity dsRNA standards are required for quantitative analysis and quality control of the main component.

[0003] Currently, dsRNA standards are not commercially available, requiring companies to independently produce them. Most companies opt for in vitro transcription kits, which are expensive and produce products of insufficient purity, making them difficult to meet standards for use as standards. dsRNA is also a byproduct of mRNA production, necessitating the preparation of standards for quantification. CN116926149A discloses the synthesis of single-stranded RNA (ssRNA) by in vitro transcription. The in vitro transcribed DNA template in the reaction product is then degraded to single nucleotides; dsRNA is then annealed, and the ssRNA in the annealed product is degraded using S1 nuclease. The reaction product is then purified to yield dsRNA. This method utilizes PCR and gel extraction to obtain the transcription template, followed by the use of a transcription kit to prepare dsRNA. However, due to the high cost and small production yield of the kit (approximately 80–100 ng), it is not suitable for the preparation of dsRNA standards in the nucleic acid pesticide field.

[0004] dsRNA shares similar properties with mRNA, making it suitable for purification and preparation using chromatography. Currently, the nucleic acid pharmaceutical industry primarily uses affinity chromatography to purify mRNA components from in vitro transcription systems. This method relies primarily on the polyadenylic acid tail structure within the mRNA molecule, which dsRNA lacks, leading to the need for improved chromatography purification processes. The applicant has recently developed ion exchange chromatography methods (CN113717984A and CN116144646A) based on the molecular properties of dsRNA, which effectively isolate and purify dsRNA. However, with the increasing demand for dsRNA research and use, these methods are no longer able to meet the purity requirements for dsRNA standards. Therefore, a cost-effective and efficient method for the large-scale preparation of high-purity dsRNA standards remains to be developed. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the present invention provides a method for preparing dsRNA standards. Using commercially available common materials, dsRNA is prepared by in vitro transcription and then purified through a two-step chromatography process followed by ultrafiltration to produce gram-level, high-purity dsRNA standards. To improve the recovery rate of ion exchange chromatography, the present invention employs the addition of basic amino acids to reduce the electrostatic interaction between larger RNA molecules and the column packing. Furthermore, the type and concentration of chaotropic salts in the buffer are optimized to enhance dsRNA recovery.

[0006] In one aspect, the present invention provides a method for preparing a dsRNA standard, the method comprising the processes of preparing an in vitro transcription template, preparing a crude dsRNA solution, preparing a pre-purified dsRNA solution, and preparing a refined dsRNA solution; the pre-purified dsRNA solution preparation process comprises ion exchange chromatography, the ion exchange chromatography comprising the steps of column equilibration, loading, and elution; the ion exchange chromatography is anion exchange chromatography; the loading step comprises diluting the dsRNA solution with a loading buffer and then loading the solution onto an ion exchange chromatography column, the loading buffer comprising one or more amino acids selected from lysine, arginine, and histidine.

[0007] In one or more embodiments, the loading buffer comprises 300-400 mM of one or more amino acids selected from lysine, arginine, and histidine.

[0008] Preferably, the loading buffer comprises 0-150 mM Tris-HCl, 100-500 mM NaCl, 300-400 mM of one or more amino acids selected from lysine, arginine, and histidine, and has a pH of 7.0-8.5. More preferably, the loading buffer comprises 10-30 mM Tris-HCl, 300-400 mM NaCl, 300-400 mM of one or more amino acids selected from lysine, arginine, and histidine, and has a pH of 7.0-8.0. Further preferably, the loading buffer comprises 15-25 mM Tris-HCl, 340-360 mM NaCl, 340-360 mM of one or more amino acids selected from lysine, arginine, and histidine, and has a pH of 7.4-7.6.

[0009] Preferably, the arginine is L-arginine.

[0010] In one or more embodiments, the elution step comprises flushing the chromatography column with elution buffer A and collecting the eluate; the elution buffer A comprises chloride salt or perchlorate.

[0011] In one or more embodiments, the elution buffer A comprises 0.1-1.2 M sodium chloride or 0.1-1.2 M sodium perchlorate.

[0012] Preferably, the elution buffer A comprises 300-700 mM sodium chloride or 300-700 mM sodium perchlorate.

[0013] Preferably, the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaCl, and a pH value of 7.0-8.5.

[0014] More preferably, step S3 adopts linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 300-700 mM NaCl, and a pH value of 7.0-8.5. More preferably, step S3 adopts linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 400-600 mM NaCl, and a pH value of 7.0-8.0. Further preferably, step S3 adopts linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 400-600 mM NaCl, and a pH value of 7.4-7.6.

[0015] More preferably, step S3 adopts gradient elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaCl, and a pH value of 7.0-8.5. Further preferably, step S3 adopts gradient elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.0 M NaCl, and a pH value of 7.0-8.0, wherein the concentration of NaCl changes gradually as the elution proceeds; further preferably, step S3 adopts gradient elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.0 M NaCl, and a pH value of 7.4-7.6, wherein the concentration of NaCl increases gradually from 0.3 M to 1.0 M as the elution proceeds.

[0016] Preferably, the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaClO4, and a pH value of 7.0-8.5.

[0017] More preferably, step S3 adopts linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 300-700 mM NaClO4, and a pH value of 7.0-8.5. More preferably, step S3 adopts linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 400-600 mM NaClO4, and a pH value of 7.0-8.0. Further preferably, step S3 adopts linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 400-600 mM NaClO4, and a pH value of 7.4-7.6.

[0018] More preferably, step S3 adopts gradient elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaClO4, and a pH value of 7.0-8.5. Further preferably, step S3 adopts gradient elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.0 M NaClO4, and a pH value of 7.0-8.0, wherein the concentration of NaClO4 changes gradually as the elution proceeds; further preferably, step S3 adopts gradient elution, and the elution buffer A comprises 0-150 mMTris-HCl, 0.3-1.0 M NaClO4, and a pH value of 7.4-7.6, wherein the concentration of NaClO4 increases gradually from 0.3 M to 1.0 M as the elution proceeds.

[0019] In one or more embodiments, the process for preparing the crude dsRNA solution comprises adding DNase digestion, adding protease digestion and membrane filtration to the in vitro transcription reaction solution.

[0020] In one or more embodiments, the method further comprises a freeze-drying process.

[0021] Preferably, the method for preparing the dsRNA standard includes an in vitro transcription template preparation process, and the in vitro transcription template preparation process includes the following steps:

[0022] S1: DNA template fragment amplification;

[0023] S2: enzyme digestion of amplified products;

[0024] S3: The enzyme digestion products were connected to obtain the recombinant plasmid;

[0025] S4: Verification of transformed host bacteria;

[0026] S5: recombinant plasmid extraction;

[0027] S6: Enzyme digestion of recombinant plasmid.

[0028] More preferably, in the in vitro transcription template preparation process, in step S1, the two ends of the amplified DNA template fragment respectively include a restriction endonuclease site and a promoter.

[0029] More preferably, in the in vitro transcription template preparation process, in step S6, a sufficient amount of DNA fragment including a bidirectional promoter and an RNA expression gene is obtained after enzyme digestion to serve as an in vitro transcription template.

[0030] Preferably, the method for preparing the dsRNA standard includes a process for preparing a crude dsRNA solution, and the process for preparing the crude dsRNA solution includes the following steps:

[0031] S1: in vitro transcription;

[0032] S2: DNase digestion;

[0033] S3: protease digestion;

[0034] S4: Membrane filtration.

[0035] More preferably, during the preparation of the crude dsRNA solution, step S1 comprises preparing an in vitro transcription reaction system using the reaction solution of the recombinant plasmid digestion to carry out the reaction.

[0036] More preferably, in the process of preparing the crude dsRNA solution, in step S2, the DNase is DNase I.

[0037] More preferably, in the process of preparing the crude dsRNA solution, in step S3, the protease is proteinase K.

[0038] Preferably, the preparation method of the dsRNA standard comprises a process for preparing a dsRNA pre-purified solution, wherein the process comprises exchanging the membrane solution of the crude dsRNA solution with the membrane solution, and then purifying the solution by ion exchange chromatography, wherein the ion exchange chromatography comprises the following steps:

[0039] S1: column equilibrium;

[0040] S2: sample loading;

[0041] S3: Elution.

[0042] More preferably, during the preparation of the dsRNA primary pure solution, the liquid used for membrane coating exchange is equilibrium buffer A.

[0043] More preferably, the ion exchange chromatography further comprises the following steps:

[0044] S4: column regeneration;

[0045] S5: Column cleaning.

[0046] Preferably, the preparation method of the dsRNA standard comprises a process for preparing a dsRNA pure solution, wherein the preparation process of the dsRNA pure solution comprises the steps of performing a first membrane exchange, hydrophobic chromatography purification, and then a second membrane exchange.

[0047] Preferably, the hydrophobic chromatography comprises the following steps:

[0048] S1: column equilibrium;

[0049] S2: sample loading;

[0050] S3: Elution.

[0051] More preferably, the hydrophobic chromatography further comprises the following steps:

[0052] S4: column regeneration;

[0053] S5: Column cleaning.

[0054] More preferably, during the preparation of the dsRNA pure solution, the liquid used in the first membrane exchange is the equilibrium buffer solution B, and the liquid used in the second membrane exchange is pure water.

[0055] In another aspect, the present invention provides a method for purifying dsRNA, comprising ion exchange chromatography, the ion exchange chromatography comprising the steps of column equilibration, loading, and elution; the ion exchange chromatography is anion exchange chromatography; the loading step comprises diluting the dsRNA solution with a loading buffer and then loading the solution onto the ion exchange chromatography column, wherein the loading buffer comprises one or more amino acids selected from lysine, arginine, and histidine.

[0056] In one or more embodiments, the loading buffer comprises 300-400 mM of one or more amino acids selected from lysine, arginine, and histidine.

[0057] Preferably, the loading buffer comprises 0-150 mM Tris-HCl, 100-500 mM NaCl, 300-400 mM of one or more amino acids selected from lysine, arginine, and histidine, and has a pH of 7.0-8.5. More preferably, the loading buffer comprises 10-30 mM Tris-HCl, 300-400 mM NaCl, 300-400 mM of one or more amino acids selected from lysine, arginine, and histidine, and has a pH of 7.0-8.0. Further preferably, the loading buffer comprises 15-25 mM Tris-HCl, 340-360 mM NaCl, 340-360 mM of one or more amino acids selected from lysine, arginine, and histidine, and has a pH of 7.4-7.6.

[0058] Preferably, the arginine is L-arginine.

[0059] In one or more embodiments, the elution step comprises flushing the chromatography column with elution buffer A and collecting the eluate; the elution buffer A comprises chloride salt or perchlorate.

[0060] In one or more embodiments, the elution buffer A comprises 0.1-1.2 M sodium chloride or 0.1-1.2 M sodium perchlorate.

[0061] Preferably, the elution buffer A comprises 300-700 mM sodium chloride or 300-700 mM sodium perchlorate.

[0062] Preferably, the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaCl, and a pH value of 7.0-8.5.

[0063] More preferably, step S3 adopts linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 300-700 mM NaCl, and a pH value of 7.0-8.5. More preferably, step S3 adopts linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 400-600 mM NaCl, and a pH value of 7.0-8.0. Further preferably, step S3 adopts linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 400-600 mM NaCl, and a pH value of 7.4-7.6.

[0064] More preferably, step S3 adopts gradient elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaCl, and a pH value of 7.0-8.5. Further preferably, step S3 adopts gradient elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.0 M NaCl, and a pH value of 7.0-8.0, wherein the concentration of NaCl changes gradually as the elution proceeds; further preferably, step S3 adopts gradient elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.0 M NaCl, and a pH value of 7.4-7.6, wherein the concentration of NaCl increases gradually from 0.3 M to 1.0 M as the elution proceeds.

[0065] Preferably, the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaClO4, and a pH value of 7.0-8.5.

[0066] More preferably, step S3 adopts linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 300-700 mM NaClO4, and a pH value of 7.0-8.5. More preferably, step S3 adopts linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 400-600 mM NaClO4, and a pH value of 7.0-8.0. Further preferably, step S3 adopts linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 400-600 mM NaClO4, and a pH value of 7.4-7.6.

[0067] More preferably, step S3 adopts gradient elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaClO4, and a pH value of 7.0-8.5. Further preferably, step S3 adopts gradient elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.0 M NaClO4, and a pH value of 7.0-8.0, wherein the concentration of NaClO4 changes gradually as the elution proceeds; further preferably, step S3 adopts gradient elution, and the elution buffer A comprises 0-150 mMTris-HCl, 0.3-1.0 M NaClO4, and a pH value of 7.4-7.6, wherein the concentration of NaClO4 increases gradually from 0.3 M to 1.0 M as the elution proceeds.

[0068] Compared with the prior art, the dsRNA preparation and purification method provided by the present invention has the following beneficial effects:

[0069] (1) The method for preparing dsRNA standards provided by the present invention is characterized by high efficiency and low cost. One batch can produce 1-2 g of high-purity standards, which can meet the standard product requirements for registration and production quality testing in the field of nucleic acid pesticides.

[0070] (2) Specifically, in terms of dsRNA synthesis, compared with the method of obtaining by PCR amplification, the use of plasmid as a vector for in vitro transcription template and then obtained by enzyme digestion can achieve large-scale in vitro transcription and obtain gram-level dsRNA products.

[0071] (3) Specifically, in terms of dsRNA purification, the present invention utilizes a two-step chromatography method to prepare a dsRNA standard product, which has a purity greater than 99% as determined by molecular sieve liquid chromatography. This invention avoids the use of nuclease S1 during the preparation process, significantly reducing preparation costs. It also avoids the use of organic solvents such as phenol and chloroform, achieving green production.

[0072] (4) Specifically, in the ion exchange chromatography purification step, the present invention adopts a method of adding basic amino acids to reduce the interaction between long-chain dsRNA and the chromatography filler, increase the salt solution elution strength, and improve the recovery rate. This solves the problem of low purification recovery rate in standard preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 is a map of an exemplary recombinant plasmid.

[0074] Figure 2 The figure below shows the agarose gel electrophoresis of the EcoRI digested sample. “1” represents the marker, and “2” and “3” represent the parallel samples.

[0075] Figure 3 This is a chromatogram of dsRNA purified by ion exchange chromatography using conventional methods.

[0076] Figure 4 This is a chromatogram of dsRNA purified by ion exchange chromatography using the optimized method.

[0077] Figure 5 This is a liquid chromatography detection chart of dsRNA in the eluted sample E2 of the optimized method.

[0078] Figure 6 It is the recovery rate of dsRNA purified by ion exchange chromatography using loading buffer with different arginine concentrations.

[0079] Figure 7 is the recovery rate of dsRNA purified by ion exchange chromatography using elution buffers with different chaotropic salts.

[0080] Figure 8This is the agarose gel electrophoresis image of the dsRNA standard solution.

[0081] Figure 9 This is a liquid chromatography detection chart of dsRNA standard solution. DETAILED DESCRIPTION

[0082] In the present invention, during the in vitro transcription process for synthesizing dsRNA, the pH of the in vitro transcription reaction system is maintained at 7.2 to 7.8, and the reaction time is 10 to 16 hours, until the reaction solution changes from clear and transparent to a certain degree of turbidity. After the in vitro transcription is completed, DNase I (10 to 20 U / μL) is added to the reaction system and digested at 37°C for 1 to 2 hours to ensure complete digestion of the DNA template. 50 mg of proteinase K is then added and digested at 57°C for 1 to 2 hours to remove protein impurities in the reaction system. Finally, the digestion solution is filtered through a 3 μm microporous filter membrane to obtain a crude dsRNA solution, which is then further purified.

[0083] Generally speaking, purifying low-molecular-weight RNA by ion exchange chromatography can achieve a high recovery rate. However, the dsRNA used in the field of nucleic acid pesticides can be up to several hundred bp in length and contain double strands in the molecule, so it carries a much greater negative charge than low-molecular-weight RNA, resulting in excessive binding to conventional strong anionic fillers and a low recovery rate in the eluted sample obtained under conventional conditions. If a weak anionic filler is used, the separation degree between dsRNA and impurities is low, and the ideal purification effect cannot be achieved. The present invention purifies the in vitro transcription product through a two-step chromatography method, wherein ion exchange chromatography can remove impurities such as small RNA, proteinase K, and NTP to obtain a dsRNA pre-purified solution with a purity greater than 95%; hydrophobic chromatography can further remove unannealed ssRNA and mRNA fragments during the transcription process; after the eluate from the hydrophobic chromatography is concentrated and replaced with pure water, a dsRNA pure solution with a purity greater than 99% is obtained, which can be directly diluted for use as a standard or frozen for storage.

[0084] In the present invention, the anion medium used in ion exchange chromatography is preferably a strong anion medium. More preferably, the strong anion medium has a ligand that is a quaternary ammonium group, and the ligand is a high-flow agarose rigid microsphere or a stabilized regenerated cellulose membrane.

[0085] In the present invention, the specific method of ion exchange chromatography includes:

[0086] S1: Column equilibration: Use 3 to 5 column volumes (CV) of equilibration buffer A to equilibrate the column packing until the baseline is flat;

[0087] S2: Sample loading: The in vitro transcription system mixture is replaced with loading buffer through the membrane, diluted to an appropriate concentration, and then loaded onto the ion exchange chromatography column; after the sample loading is completed, the chromatography column is rinsed with 3-5CV of equilibration buffer A until the UV absorbance at 255-265 nm drops to near the baseline level; optionally, the chromatography column is rinsed with 3-5CV of impurity removal buffer and the impurity removal peak is collected;

[0088] S3: Elution: Use 3-5CV of elution buffer A for linear elution or gradient elution, and collect the eluate until the absorbance at 255-265 nm drops to near the baseline level, then stop collecting; perform liquid chromatography on the eluate, and collect samples with a purity greater than 95% for storage or standby use as the dsRNA primary pure solution.

[0089] Preferably, the ion exchange chromatography further comprises:

[0090] S4: Column regeneration: flush the column with 3-5 CV of washing buffer;

[0091] S5: Column cleaning: Rinse the chromatography column with 5 to 10 CV of pure water.

[0092] Preferably, the equilibration buffer A comprises 0-150 mM Tris-HCl, 300-500 mM NaCl, and a pH value of 7.0-8.5.

[0093] Preferably, the loading buffer comprises 0-150 mM Tris-HCl, 100-500 mM NaCl, 300-400 mM basic amino acid, and a pH of 7.0-8.5; wherein the basic amino acid is lysine, arginine, or histidine. More preferably, the basic amino acid is L-lysine, L-arginine, or L-histidine.

[0094] Preferably, the impurity removal buffer comprises 0-150 mM Tris-HCl, 400-500 mM NaCl, and a pH value of 7.0-8.5.

[0095] Preferably, the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaCl, and a pH value of 7.0-8.5. More preferably, step S3 employs linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 300-700 mM NaCl, and a pH value of 7.0-8.5. More preferably, step S3 employs gradient elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaCl, and a pH value of 7.0-8.5.

[0096] Preferably, the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaClO4, and a pH value of 7.0-8.5. More preferably, step S3 employs linear elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 300-700 mM NaClO4, and a pH value of 7.0-8.5. More preferably, step S3 employs gradient elution, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaClO4, and a pH value of 7.0-8.5.

[0097] In the present invention, preferably, the ligand of the hydrophobic chromatography medium is an aliphatic butyl group, and the ligand is a rigid microsphere of agarose.

[0098] In the present invention, the specific method of hydrophobic chromatography includes:

[0099] S1: Column equilibration: Use 3 to 5 CV of equilibration buffer B to equilibrate the column packing until the baseline is level;

[0100] S2: Sample loading: Load the concentrated dsRNA solution onto the hydrophobic chromatography column. After loading, rinse the column with 5-10 CV of equilibration buffer B until the UV absorbance at 255-265 nm drops to near the baseline level.

[0101] S3: Elution: Perform linear elution with 3-5CV of elution buffer B, collect all the eluate, and stop collecting when the absorbance at 255-265 nm drops to near the baseline level; perform liquid chromatography on the eluate, and collect samples with a purity greater than 99% for storage or standby use.

[0102] Preferably, the equilibration buffer B comprises 0-150 mM Tris-HCl, 1.5-2.0 M ammonium sulfate, and a pH value of 7.0-8.5.

[0103] Preferably, the elution buffer B comprises 0-150 mM Tris-HCl with a pH value of 7.0-8.5.

[0104] In the present invention, the purity of dsRNA in the eluate corresponding to the elution peak is determined by liquid chromatography. After hydrophobic chromatography, samples with a purity greater than 99% are collected. The collected fractions from the elution peak are concentrated using a membrane cassette or hollow fiber column with a pore size of 30-200 kD, and the buffer is replaced with pure water to obtain a pure dsRNA solution. The pure dsRNA solution is freeze-dried to obtain a dsRNA standard powder, which is stored at -20°C.

[0105] Example 1: Preparation of in vitro transcription template

[0106] In this example, EcoRI restriction sites were added to both ends of a DNA template fragment carrying a bidirectional T7 promoter, and then cloned into a replicative plasmid vector of E. coli to obtain a recombinant plasmid. The E. coli host can be JM109, DH5α, HT115, etc., and the replicative plasmid vector can be a pMD series, pUC series, pGEM series, etc. The pMD series, pUC series, or pGEM series plasmid vectors described in the present invention are commercially available. An exemplary recombinant plasmid map is shown in FIG. Figure 1 shown.

[0107] The recombinant plasmid is then transformed into E. coli and the positive transformant strains are fermented to achieve large-scale plasmid amplification. After plasmid extraction, the amplified plasmid is digested with EcoRI to obtain a sufficient amount of DNA fragment containing the bidirectional T7 promoter and the RNA expression gene, which serves as a template for in vitro transcription.

[0108] Taking the preparation of cact::snap dsRNA in patent CN116042620A as an example, first prepare its in vitro transcription template:

[0109] S1: Using the plasmid vector pT7B-cact::snap as a template and cact::snap-EcoRI-F and cact::snap-EcoRI-R as primers (primer sequences are shown in Table 1 below), a DNA template fragment with EcoRI restriction site sequences and T7 promoter sequences at both ends was amplified;

[0110] S2: Use EcoRI enzyme to digest the DNA template fragment and plasmid vector pUC19 respectively;

[0111] S3: Use DNA ligase to connect the DNA template fragment and the enzyme digestion product of plasmid vector pUC19 to obtain the recombinant plasmid pUC19-EcoRI-cact::snap;

[0112] S4: The recombinant plasmid was transformed into Escherichia coli DH5α, and the positive transformant strain was fermented and cultured to extract the recombinant plasmid; 360 mg of the recombinant plasmid was taken and 1.2 L of enzyme digestion reaction system was prepared according to Table 2. The enzyme digestion reaction was carried out in a 5 L bioreactor with the reaction temperature set at 37°C and the stirring speed at 150 rpm for 2 h; after the reaction, two parallel samples of the reaction solution were taken for agarose gel electrophoresis detection. The results are as follows Figure 2 As shown in the gel imaging diagram, two clear bands of different sizes can be seen at the corresponding positions of the two samples, confirming that the template required for the in vitro transcription reaction was prepared.

[0113] Table 1: Primers for in vitro transcription template amplification

[0114]

[0115] Table 2: Enzyme digestion reaction system

[0116]

[0117] Example 2: In vitro transcription synthesis of dsRNA

[0118] The enzyme digestion reaction solution containing the in vitro transcription template in Example 1 was taken to prepare the in vitro transcription reaction system shown in Table 3, which was thoroughly mixed. The temperature was controlled at 37°C, and a 10 M NaOH base bottle was added. The pH value was controlled at 7.5, and the stirring speed was 150 rpm. The in vitro transcription reaction was carried out for 16 h.

[0119] Table 3: In vitro transcription reaction system

[0120]

[0121] After in vitro transcription, take the in vitro transcription reaction solution, add 20 ml of DNaseI (15 U / μL), digest at 37°C for 2 h, then add 50 mg of proteinase K, digest at 57°C for 1 h, and finally use a bell filter to filter with a 3 μm pore size filter membrane to obtain a crude dsRNA solution.

[0122] Example 3: Purification of dsRNA by ion exchange chromatography

[0123] 3.1 Purification by conventional methods

[0124] The crude dsRNA solution obtained in Example 2 was replaced with equilibration buffer A (20 mM Tris-HCl, 350 mM NaCl, pH 7.5) using a 100 kD membrane packing. Ion exchange chromatography was then performed using a chromatography column containing a strong anion filler, and the purified dsRNA was collected. The dsRNA had a UV absorption peak at 260 nm. Preferably, the strong anion filler was Q Bestarose FF chromatography filler (purchased from Boglon Biotechnology Co., Ltd., Catalog No. AI0024). The specific steps of the ion exchange chromatography were as follows:

[0125] S1: Column equilibration: Wash the column with 5 column volumes (CV) of equilibration buffer A (20 mM Tris-HCl, 350 mM NaCl, pH 7.5) and adjust the UV absorbance of the effluent to zero.

[0126] S2: Sample loading: Take 1.2 g of crude dsRNA solution, dilute it to 1 L with loading buffer (20 mM Tris-HCl, 350 mM NaCl, pH 7.5), and then load it onto the chromatography column. After all samples are loaded, continue to rinse the chromatography column with 3CV of equilibration buffer A. Stop rinsing after the UV absorbance basically reaches the baseline, and collect the eluted sample (E1) with a UV absorbance value greater than 50 mAU at 260 nm.

[0127] S3: Elution: Wash the column with 3-5 CV of elution buffer A (20 mM Tris-HCl, 1 M NaCl, pH 7.5) in a linear elution mode, and collect the eluted sample (E2) with a UV absorbance value greater than 50 mAU at 260 nm.

[0128] S4: Column regeneration: Wash the column with 5CV of column wash buffer (20 mM Tris-HCl, 1 M NaCl, 0.5 M NaOH, pH 7.5) and collect the eluted sample (E3) with a UV absorbance greater than 50 mAU at 260 nm.

[0129] S5: Column cleaning: Rinse the column with 5-10 CV of pure water and collect the eluted sample (E4) with a UV absorbance greater than 50 mAU at 260 nm.

[0130] The chromatogram during ion exchange chromatography is as follows Figure 3 As shown in the figure, the left vertical axis is used to represent the UV absorption value of the sample, and the right vertical axis is used to represent the conductivity of the sample. Four obvious UV absorption peaks can be observed in the figure, corresponding to the four samples E1 to E4. Figure 3 The results are shown in Table 4. Figure 3 As can be seen from Table 4, the peak area of ​​sample E2 eluted by linear elution accounts for only 14.73%, while the peak area of ​​sample E3 eluted by column regeneration accounts for as high as 58.50%, indicating that a large amount of dsRNA was eluted in the column regeneration step. In addition, the dsRNA collected in this step has been denatured due to the alkaline solution treatment in the column washing buffer, which ultimately leads to a low purification recovery rate (35.68%).

[0131] Table 4: Chromatographic analysis results of dsRNA purified by ion exchange chromatography using conventional methods

[0132]

[0133] 3.2 Optimization of purification methods

[0134] The crude dsRNA solution obtained in Example 2 was replaced with equilibration buffer A (20 mM Tris-HCl, 350 mM NaCl, pH 7.5) using a 100 kD membrane cassette. Ion exchange chromatography was then performed using the method described in Example 3.1. The equilibration buffer A used in step S2 contained an appropriate concentration of arginine to reduce electrostatic interactions between dsRNA molecules and the strongly anionic filler, thereby increasing the dsRNA elution rate in step S3. The loading buffer consisted of 20 mM Tris-HCl, 350 mM NaCl, and 300 mM L-arginine, pH 7.5.

[0135] The chromatogram during ion exchange chromatography is as follows Figure 4 As shown in the figure, no obvious UV absorption peak of E3 was observed. Figure 4 The results are shown in Table 5. Figure 4 As shown in Table 5, the peak area of ​​the eluted sample E2 using the optimized linear elution method reached 80.34%, which significantly improved the elution rate of dsRNA compared with the conventional method; while the peak area of ​​the eluted sample E3 using the column regeneration method was almost 0.

[0136] The eluted sample E2 was subjected to liquid chromatography using a SEC1000 column, and the following Figure 5 As shown in the liquid chromatogram, the purity of the dsRNA sample reached 95%, indicating that a high-purity dsRNA primary solution was obtained and can be used for further purification.

[0137] Table 5: Chromatographic analysis results of dsRNA purified by ion exchange chromatography using the optimized method

[0138]

[0139] 3.3 Optimization of arginine concentration

[0140] The crude dsRNA solution obtained in Example 2 was replaced with equilibration buffer A (20 mM Tris-HCl, 350 mM NaCl, pH 7.5) using a 100 kD membrane cassette. Ion exchange chromatography was then performed on the sample using the method of Example 3.2. The equilibration buffer A used in step S2 contained arginine at a concentration of 100, 200, 300, 400, 500, 600, 700, 800, or 900 mM. The loading buffer comprised 20 mM Tris-HCl, 350 mM NaCl, 100-900 mM L-arginine, pH 7.5.

[0141] The chromatograms of the ion exchange chromatography process using the loading buffer containing different concentrations of arginine were analyzed using the SDL chromatography system, and the peak area ratio of the eluted sample E2 in the linear elution was used as the recovery rate of dsRNA, and the following results were obtained: Figure 6 The results show that as the amount of arginine added increases, the recovery rate first increases and then decreases. When the addition amount is 300-400 mM, the recovery rate can reach a peak of more than 80%.

[0142] 3.4 Optimization of elution buffer

[0143] The crude dsRNA solution obtained in Example 2 was replaced with equilibration buffer A (20 mM Tris-HCl, 350 mM NaCl, pH 7.5) using a 100 kD membrane package. The sample was then subjected to ion exchange chromatography according to the method of Example 3.2. In the S3 elution step, the chromatography column was flushed with elution buffer A (20 mM Tris-HCl, pH 7.5) containing 1 M Na2HPO4, 1 M sodium citrate, 1 M NaCl, or 1 M NaClO4 as a chaotropic salt.

[0144] The chromatogram of the ion exchange chromatography process using elution buffers containing different chaotropic salts was analyzed using the SDL chromatography system, and the peak area ratio of the eluted sample E2 in the linear elution was used as the recovery rate of dsRNA, and the following was obtained: Figure 7 The results shown in the figure show that using NaCl and NaClO4 as chaotropic salts can achieve better elution effects, among which the recovery rate using NaClO4 is 10.57% higher than that using NaCl.

[0145] Example 4: Purification of dsRNA by hydrophobic chromatography

[0146] The purified dsRNA solution obtained in step 3.2 was exchanged with equilibration buffer B (20 mM Tris-HCl, 1.5 M ammonium sulfate, pH 7.2) using a 100 kDa membrane cassette. The solution was concentrated to 0.5 L and then subjected to hydrophobic chromatography. Preferably, the medium used in the hydrophobic chromatography process is UniHR Butyl-30L (available from Nanovitamin Technology Co., Ltd., 06132-030100). The specific steps of the hydrophobic chromatography are as follows:

[0147] S1: Column equilibration: flush the column with 5 CV of equilibration buffer B (20 mM Tris-HCl, 1.5 M ammonium sulfate, pH 7.2) and adjust the UV absorbance of the effluent to zero.

[0148] S2: Sample loading: Load 0.5 L (about 3 g) of concentrated dsRNA primary pure solution onto the hydrophobic chromatography column. After all samples have been loaded, continue to flush the chromatography column with 5-10CV of equilibration buffer B until the UV absorbance reaches the baseline.

[0149] S3: Elution: Use 5CV of elution buffer B (20 mM Tris-HCl, pH 7.2) to wash the column in a linear elution manner and collect all the eluate; stop collecting when the UV absorbance at 260 nm drops to near the baseline level.

[0150] Example 5: Preparation of dsRNA Standards

[0151] The eluate obtained in Example 4 was concentrated using a 100 kD pore size membrane cassette and replaced with pure water to obtain a dsRNA pure solution. The dsRNA pure solution was freeze-dried to obtain a dsRNA standard powder, which was stored at -20°C.

[0152] Dissolve 10 mg of the standard powder in 100 mL of nuclease-free water to prepare a standard solution. The standard solution was tested by agarose gel electrophoresis and liquid chromatography SEC1000 column. Figure 8 As shown in the figure, the electrophoresis band is single and there are no other impurities, indicating that the dsRNA in the standard powder is of high purity; the liquid chromatography results are as follows Figure 9 As shown, the purity of the collected sample dsRNA reached more than 99%, which can meet the use requirements of nucleic acid pesticide dsRNA standards.

Claims

1. A method for preparing a dsRNA standard, characterized in that: The method includes the processes of preparing an in vitro transcription template, preparing a crude dsRNA solution, preparing a primary pure dsRNA solution and preparing a finely purified dsRNA solution; The process for preparing the crude dsRNA solution includes adding DNase digestion, adding protease digestion and membrane filtration to the in vitro transcription reaction solution; The dsRNA pre-purified solution preparation process includes ion exchange chromatography, which includes column equilibration, loading and elution steps; the ion exchange chromatography is anion exchange chromatography; the loading step includes diluting the dsRNA solution with a loading buffer and then loading it onto the ion exchange chromatography column, wherein the loading buffer includes 300-400 mM arginine; The preparation process of the dsRNA pure solution includes the steps of performing a first membrane coating solution exchange on the dsRNA primary pure solution, performing hydrophobic chromatography purification, and then performing a second membrane coating solution exchange on the dsRNA primary pure solution.

2. The method according to claim 1, wherein The elution step comprises flushing the chromatography column with an elution buffer A and collecting the eluate; the elution buffer A comprises chloride ion salt or perchlorate.

3. The method according to claim 2, wherein The elution buffer A includes 0.1-1.2 M sodium chloride or 0.1-1.2 M sodium perchlorate.

4. The method according to any one of claims 1 to 3, wherein The method also includes a freeze-drying process.

5. A method for purifying dsRNA, characterized in that: The method comprises ion exchange chromatography, which comprises the steps of column equilibration, loading and elution; the ion exchange chromatography is anion exchange chromatography; the loading step comprises diluting a dsRNA solution with a loading buffer and then loading the solution onto an ion exchange chromatography column, wherein the loading buffer comprises 300-400 mM arginine.

6. The method according to claim 5, wherein The elution step comprises flushing the chromatography column with an elution buffer A and collecting the eluate; the elution buffer A comprises chloride ion salt or perchlorate.

7. The method according to claim 6, wherein The elution buffer A includes 0.1-1.2 M sodium chloride or 0.1-1.2 M sodium perchlorate.

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