Purification and preparation method of dsRNA standard substance

Abstract

The invention discloses a purification and preparation method of a dsRNA standard substance. According to the present invention, the dsRNA standard product is prepared by using the two-step chromatography method, and in the ion exchange chromatography purification step, the alkaline amino acid adding method is adopted, such that the interaction force between the long-chain dsRNA and the chromatography filler is reduced, the saline solution elution strength is increased, the recovery rate is improved, and the problem of low purification recovery rate during the dsRNA standard product preparation is solved. The method provided by the invention has the characteristics of high efficiency and low cost, a linearized plasmid is adopted as a carrier of an in-vitro transcription template, large-scale in-vitro transcription can be realized, gram-level dsRNA products can be obtained in one batch, and the standard product requirements of registration and production quality detection in the field of nucleic acid pesticides can be met.

Description

Technical Field

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

[0002] Nucleic acid pesticides developed based on RNAi technology are new biological pesticides in the agrochemical market in recent years. It has been reported that they can effectively control various pests and diseases such as plant pests, viruses, nematodes, and pathogenic fungi. Double-stranded RNA (dsRNA) is the main active ingredient of nucleic acid pesticides. In the production of its technical material and the development of formulations, it is necessary to prepare high-purity dsRNA standard products for the quantitative analysis and quality detection of the main component.

[0003] Currently, dsRNA standard products are not commercially available and need to be prepared independently by enterprises. Most enterprises choose to use in vitro transcription kits, which are expensive and the purity of the products obtained is not high enough to meet the usage standards of standard products. dsRNA is also a by-product in the process of preparing mRNA. As a by-product, a standard product needs to be prepared for quantification. CN116926149A discloses the use of in vitro transcription to synthesize single-stranded RNA (ssRNA), and then degrade the in vitro transcribed DNA template in the reaction product into individual nucleotides; then anneal to form dsRNA, and then use S1 nuclease to degrade the ssRNA in the annealed product, and purify the reaction product to obtain dsRNA. This method uses PCR and gel recovery to obtain the transcription template, and then uses a transcription kit to prepare dsRNA. However, due to the high price of the kit and the small preparation amount (about 80 - 100 ng), it is not suitable for the preparation of dsRNA standard products in the field of nucleic acid pesticides.

[0004] dsRNA has similar properties to mRNA, so chromatography methods can be used for purification and preparation. Currently, the nucleic acid medicine industry mostly uses affinity chromatography to purify the mRNA component in the in vitro transcription system, which mainly relies on the design of the polyadenylate tail structure in mRNA molecules. However, dsRNA does not have this structure, so its chromatographic purification process still needs to be improved. The applicant has recently developed an ion exchange chromatography method (CN113717984A and CN116144646A) based on the molecular characteristics of dsRNA, which can better achieve the separation and purification of dsRNA. However, with the increasing research and use requirements for dsRNA, the aforementioned methods are difficult to meet the purity requirements for dsRNA standard products. Therefore, there is still a need to develop an economical and efficient method for large-scale preparation of high-purity dsRNA standard products. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a method for preparing a dsRNA standard product. Using commonly available materials on the market, dsRNA is prepared by in vitro transcription, and then purified by two-step chromatography and ultrafiltration buffer exchange to prepare a gram-level high-purity dsRNA standard product. In order to improve the recovery rate of ion exchange chromatography, the present invention weakens the electrostatic interaction between RNA molecules with larger molecular weights and the column packing by adding basic amino acids, and optimizes the type and concentration of chaotropic salts in the buffer to improve the recovery rate of dsRNA.

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

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

[0008] Preferably, the loading buffer includes 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 the pH value is 7.0-8.5. More preferably, the loading buffer includes 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 the pH value is 7.0-8.0. Further preferably, the loading buffer includes 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 the pH value is 7.4-7.6.

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

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

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

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

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

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

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

[0016] Preferably, the elution buffer A comprises 0 to 150 mM Tris-HCl, 0.3 to 1.2 M NaClO4, with a pH value of 7.0 to 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, with 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, with 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, with 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, with 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, with a pH value of 7.0 - 8.0, wherein the concentration of NaClO4 changes in a gradient manner 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 NaClO4, with a pH value of 7.4 - 7.6, wherein the concentration of NaClO4 increases from 0.3 M to 1.0 M in a gradient manner as the elution proceeds.

[0019] In one or more embodiments, during the preparation of the dsRNA crude solution, steps of adding DNase digestion, protease digestion and membrane filtration to the in vitro transcription reaction solution are included.

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

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

[0022] S1: Amplification of the DNA template fragment;

[0023] S2: Digestion of the amplification product with an enzyme;

[0024] S3: Ligation of the digestion product to obtain a recombinant plasmid;

[0025] S4: Verification by transforming the host bacteria;

[0026] S5: Extraction of the recombinant plasmid;

[0027] S6: Digestion of the recombinant plasmid with an enzyme.

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

[0029] More preferably, during the preparation of the in vitro transcription template, in step S6, after enzymatic digestion, a sufficient amount of DNA fragments including a bidirectional promoter and an RNA expression gene are obtained as the in vitro transcription template.

[0030] Preferably, the method for preparing the dsRNA standard product 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 process for preparing the crude dsRNA solution, step S1 includes taking the reaction solution of recombinant plasmid digestion to prepare an in vitro transcription reaction system for reaction.

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

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

[0038] Preferably, the method for preparing the dsRNA standard product includes a process for preparing a preliminary purified dsRNA solution, and the process for preparing the preliminary purified dsRNA solution includes subjecting the crude dsRNA solution to membrane buffer exchange and then purifying it by ion exchange chromatography. Among them, the ion exchange chromatography includes the following steps:

[0039] S1: Column equilibration;

[0040] S2: Sample loading;

[0041] S3: Elution.

[0042] More preferably, during the process for preparing the preliminary purified dsRNA solution, the liquid used for membrane buffer exchange is equilibration buffer A.

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

[0044] S4: Column regeneration;

[0045] S5: Column cleaning.

[0046] Preferably, the method for preparing the dsRNA standard includes the process of preparing a dsRNA purified solution, and the process of preparing the dsRNA purified solution includes the steps of performing a first membrane buffer exchange on the dsRNA preliminarily purified solution, hydrophobic chromatography purification, and then performing a second membrane buffer exchange.

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

[0048] S1: Column equilibration;

[0049] S2: Sample loading;

[0050] S3: Elution.

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

[0052] S4: Column regeneration;

[0053] S5: Column cleaning.

[0054] More preferably, in the process of preparing the dsRNA purified solution, the liquid used for the first membrane buffer exchange is equilibration buffer B, and the liquid used for the second membrane buffer exchange is pure water.

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

[0056] In one or more embodiments, the sample loading buffer includes 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 value 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 value 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 value of 7.4 - 7.6.

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

[0059] In one or more embodiments, the elution step comprises rinsing the chromatography column with elution buffer A and collecting the eluate; the elution buffer A comprises a chloride salt or a 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 has a pH value of 7.0 - 8.5.

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

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

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

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

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

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

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

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

[0071] (3)Specifically, in terms of dsRNA purification, the dsRNA standard prepared by the two-step chromatography method of the present invention has a purity greater than 99% detected by molecular sieve liquid chromatography. In the preparation process of the present invention, the use of nuclease S1 is avoided, which can greatly reduce the preparation cost. At the same time, the use of organic solvents such as phenol and chloroform is also avoided, realizing green production.

[0072] (4)Specifically, in the ion exchange chromatography purification step, the method of adding basic amino acids is adopted in the present invention, which reduces the interaction force between long-chain dsRNA and chromatography packing material, increases the elution strength of salt solution, and improves the recovery rate. It solves the problem of low purification recovery rate in the preparation of standard. Description of the Drawings

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

[0074] Figure 2 is the agarose gel electrophoresis diagram of EcoRI digestion sample. Among them, "1" represents marker, and "2" and "3" are parallel samples.

[0075] Figure 3 is the chromatogram of ion exchange chromatography purification of dsRNA by conventional method.

[0076] Figure 4 is the chromatogram of ion exchange chromatography purification of dsRNA by optimized method.

[0077] Figure 5 is the liquid chromatography detection diagram of dsRNA in the elution sample E2 by optimized method.

[0078] Figure 6 is the recovery rate of dsRNA purified by ion exchange chromatography using loading buffers 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 8It is the agarose gel electrophoresis pattern of the dsRNA standard solution.

[0081] Figure 9 It is the liquid chromatography detection pattern of the dsRNA standard solution. Specific implementation manners

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

[0083] Generally speaking, purifying low-molecular-weight RNA by ion exchange chromatography can achieve a relatively high recovery rate. However, the dsRNA used in the field of nucleic acid pesticides can reach several hundred base pairs in length and contains double strands in the molecule, so the negative charge carried is much greater than that of low-molecular-weight RNA, resulting in too strong a binding force with conventional strong anion fillers and a low recovery rate in the eluted samples obtained under conventional conditions; while if using weak anion fillers, it will lead to a low separation degree between dsRNA and impurities and cannot achieve an ideal purification effect. The present invention purifies the in vitro transcription product by a two-step chromatography method. Among them, ion exchange chromatography can remove impurities such as small RNA, proteinase K, and NTP to obtain a preliminarily purified dsRNA solution with a purity greater than 95%; hydrophobic chromatography can further remove unannealed ssRNA and mRNA fragments during the transcription process; after concentrating and changing the buffer of the eluate of hydrophobic chromatography with pure water, a highly purified dsRNA solution with a purity greater than 99% is obtained, which can be directly diluted for use as a standard product or stored frozen.

[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 of quaternary ammonium group and a ligand of high-flow agarose rigid microspheres or stabilized regenerated cellulose membrane.

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

[0086] S1: Column equilibration: Equilibrate the column packing with 3 - 5 column volumes (CV) of equilibration buffer A until the baseline is washed flat;

[0087] S2: Loading: Replace the in vitro transcription system mixture with a loading buffer through a membrane package, dilute it to an appropriate concentration, and then load it onto an ion exchange chromatography column; after the loading is completed, wash the chromatography column with 3 - 5 column volumes (CV) of equilibration buffer A until the ultraviolet absorption value at 255 - 265 nm drops to near the baseline level; optionally, wash the chromatography column with 3 - 5 CV of impurity removal buffer and collect the impurity removal peak.

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

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

[0090] S4: Column regeneration: Wash the chromatography column with 3 - 5 CV of cleaning buffer.

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

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

[0093] Preferably, the loading buffer includes 0 - 150 mM Tris-HCl, 100 - 500 mM NaCl, 300 - 400 mM basic amino acid, and has a pH value of 7.0 - 8.5; where 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 includes 0 - 150 mM Tris-HCl, 400 - 500 mM NaCl, and has a pH value of 7.0 - 8.5.

[0095] Preferably, the elution buffer A includes 0 - 150 mM Tris-HCl, 0.3 - 1.2 M NaCl, and has a pH value of 7.0 - 8.5. More preferably, in step S3, linear elution is used, and the elution buffer A includes 0 - 150 mM Tris-HCl, 300 - 700 mM NaCl, and has a pH value of 7.0 - 8.5. More preferably, in step S3, gradient elution is used, and the elution buffer A includes 0 - 150 mM Tris-HCl, 0.3 - 1.2 M NaCl, and has 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, with a pH value of 7.0-8.5. More preferably, in step S3, linear elution is adopted, and the elution buffer A comprises 0-150 mM Tris-HCl, 300-700 mM NaClO4, with a pH value of 7.0-8.5. Even more preferably, in step S3, gradient elution is adopted, and the elution buffer A comprises 0-150 mM Tris-HCl, 0.3-1.2 M NaClO4, with a pH value of 7.0-8.5.

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

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

[0099] S1: Column equilibration: Equilibrate the column packing with 3-5 CV of equilibration buffer B until the baseline is leveled.

[0100] S2: Sample loading: Load the concentrated solution of the primary purified dsRNA solution onto the hydrophobic chromatography column; after the sample loading is completed, rinse the chromatography column with 5-10 CV of equilibration buffer B until the UV absorption value at 255-265 nm drops to near the baseline level.

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

[0102] Preferably, the equilibration buffer B comprises 0-150 mM Tris-HCl, 1.5-2.0 M ammonium sulfate, with 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 detected by liquid chromatography. After hydrophobic chromatography, collect the samples with a detected purity greater than 99%, and concentrate and change the buffer of the collected components of the elution peak using a membrane package or hollow fiber column with a pore size of 30-200 kD, and replace the buffer with pure water to obtain a purified dsRNA solution. Perform freeze-drying treatment on the purified dsRNA solution to obtain a dry powder of the dsRNA standard product, and store it at -20°C.

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

[0106] In this embodiment, EcoRI restriction enzyme sites are added to both ends of the DNA template fragment with a bidirectional T7 promoter, and then it is cloned into a replicative plasmid vector of Escherichia coli to obtain a recombinant plasmid. Among them, the Escherichia coli host can be JM109, DH5α, HT115, etc., and the replicative plasmid vector can be 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. The map of an exemplary recombinant plasmid is as Figure 1 shown.

[0107] Furthermore, the recombinant plasmid is transformed into Escherichia coli, and the positive transformant strain is fermented and cultured to achieve a large amount of plasmid amplification. After extracting the plasmid, the amplified plasmid is digested with EcoRI enzyme to obtain a sufficient amount of DNA fragments including the bidirectional T7 promoter and the RNA expression gene as an in vitro transcription template.

[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 (the primer sequences are shown in Table 1 below) to amplify the DNA template fragment with EcoRI restriction enzyme site sequences and T7 promoter sequences at both ends;

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

[0111] S3: Using DNA ligase to ligate the digestion products of the DNA template fragment and the plasmid vector pUC19 respectively to obtain the recombinant plasmid pUC19-EcoRI-cact::snap;

[0112] S4: Transform this recombinant plasmid into Escherichia coli DH5α, ferment and culture the positive transformant strain, and extract the recombinant plasmid; take 360 mg of the recombinant plasmid, prepare a 1.2 L digestion reaction system according to Table 2, use a 5 L bioreactor to carry out the digestion reaction, set the reaction temperature at 37 °C, the stirring speed at 150 rpm, and carry out the digestion reaction for 2 h; after the reaction is completed, take two parallel samples of the reaction solution for agarose gel electrophoresis detection, and the results are as Figure 2 shown. In the gel imaging diagram, two clear bands of large and small sizes can be seen at the corresponding positions of the two samples, which can confirm that the template required for the in vitro transcription reaction is prepared.

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

[0114]

[0115] Table 2: Restriction Enzyme Reaction System

[0116]

[0117] Example 2: In Vitro Transcription to Synthesize dsRNA

[0118] Take the restriction enzyme reaction solution containing the in vitro transcription template in Example 1, prepare the in vitro transcription reaction system as shown in Table 3, mix well, control the temperature at 37°C, connect a 10 M NaOH alkali bottle, control the pH value at 7.5, and stir at a speed of 150 rpm for 16 h for in vitro transcription reaction.

[0119] Table 3: In Vitro Transcription Reaction System

[0120]

[0121] After the in vitro transcription is completed, 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 filter through a bell filter with a 3 μm pore size membrane to obtain a crude dsRNA solution.

[0122] Example 3: Purification of dsRNA by Ion Exchange Chromatography

[0123] 3.1 Purification by Conventional Method

[0124] Exchange the buffer of the crude dsRNA solution obtained in Example 2 with a 100 kD membrane package to the equilibration buffer A (20 mM Tris-HCl, 350 mM NaCl, pH 7.5), and then perform ion exchange chromatography using a chromatography column containing a strong anion packing material, and collect the purified dsRNA; among them, dsRNA has an ultraviolet absorption peak at 260 nm. Preferably, the strong anion packing material is Q Bestarose FF chromatography packing material (purchased from Shanghai Bogolong Biotechnology Co., Ltd., product number AI0024). The specific steps of ion exchange chromatography are as follows:

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

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

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

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

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

[0130] The chromatogram during the ion exchange chromatography process is as Figure 3 shown. 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 - E4 in sequence. Analyze Figure 3 using the SDL chromatography system (purchased from Suzhou Sepure Instruments Co., Ltd.) to obtain the results shown in Table 4. Combining Figure 3 and Table 4, it can be seen that the peak area ratio of the eluted sample E2 in the linear elution is only 14.73%, while the peak area ratio of the eluted sample E3 in the column regeneration is as high as 58.50%, indicating that a large amount of dsRNA is eluted in the column regeneration step, and the dsRNA collected in this step has been denatured due to the treatment with the alkali solution in the column cleaning buffer, ultimately resulting in a low purification recovery rate (35.68%).

[0131] Table 4: Chromatographic analysis results of purifying dsRNA by ion exchange chromatography with the conventional method

[0132]

[0133] 3.2 Purification by the optimized method

[0134] The dsRNA crude solution obtained in Example 2 was subjected to buffer exchange using a 100 kD membrane package and replaced with equilibration buffer A (20 mM Tris-HCl, 350 mM NaCl, pH 7.5). Then, the sample was subjected to ion exchange chromatography with reference to the method of Example 3.1. Among them, the equilibration buffer A used in the S2 loading step contained an appropriate concentration of arginine to reduce the electrostatic interaction between dsRNA molecules and the strong anion packing material, thereby increasing the dsRNA elution rate in the S3 linear elution step. The loading buffer included 20 mM Tris-HCl, 350 mM NaCl, 300 mM L-arginine, pH 7.5.

[0135] The chromatogram during the ion exchange chromatography is as Figure 4 shown, and no obvious ultraviolet absorption peak of E3 was observed in the figure. Analyzed using the SDL chromatography system Figure 4 to obtain the results shown in Table 5. Combining Figure 4 with Table 5, it can be seen that the peak area of the eluted sample E2 in the linear elution using the optimized method reached 80.34%, significantly increasing the dsRNA elution rate compared to the conventional method; while the peak area of the eluted sample E3 for column regeneration was almost 0.

[0136] The eluted sample E2 was detected by liquid chromatography using a SEC1000 chromatographic column to obtain the Figure 5 liquid chromatogram shown. The sample purity of dsRNA reached 95%, indicating that a dsRNA crude purification solution with relatively high purity was obtained and could be used for further purification.

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

[0138]

[0139] 3.3 Arginine concentration optimization

[0140] The dsRNA crude solution obtained in Example 2 was subjected to buffer exchange using a 100 kD membrane package and replaced with equilibration buffer A (20 mM Tris-HCl, 350 mM NaCl, pH 7.5). Then, the sample was subjected to ion exchange chromatography with reference to the method of Example 3.2. Among them, the equilibration buffer A used in the S2 loading step contained 100, 200, 300, 400, 500, 600, 700, 800, or 900 mM of arginine. The loading buffer included 20 mM Tris-HCl, 350 mM NaCl, 100 - 900 mM L-arginine, pH 7.5.

[0141] The chromatogram of the ion exchange chromatography process using a loading buffer containing different concentrations of arginine was analyzed by an SDL chromatography system. The peak area percentage of the eluted sample E2 with linear elution was used as the recovery rate of dsRNA, and the results shown in Figure 6 were obtained. As the addition amount of arginine increased, the recovery rate first increased and then decreased. When the addition amount was 300 - 400 mM, the recovery rate could reach a peak value of over 80%.

[0142] 3.4 Optimization of the elution buffer

[0143] The dsRNA crude solution obtained in Example 2 was exchanged using a 100 kD membrane package to the equilibration buffer A (20 mM Tris-HCl, 350 mM NaCl, pH 7.5), and then the sample was subjected to ion exchange chromatography with reference to the method of Example 3.2. Among them, in the S3 elution step, the chromatography column was rinsed 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 an elution buffer containing different chaotropic salts was analyzed by an SDL chromatography system. The peak area percentage of the eluted sample E2 with linear elution was used as the recovery rate of dsRNA, and the results shown in Figure 7 were obtained. Using NaCl and NaClO4 as chaotropic salts could achieve a better elution effect, and the recovery rate using NaClO4 was 10.57% higher than that using NaCl.

[0145] Example 4: Hydrophobic chromatography purification of dsRNA

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

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

[0148] S2: Sample loading: Load the concentrated solution of 0.5 L (about 3 g) of the primary purified dsRNA solution onto a hydrophobic chromatography column. After all the samples are loaded, continue to rinse the chromatography column with 5 - 10 CV of equilibration buffer B until the UV absorption basically reaches the baseline and then stop.

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

[0150] Example 5: Preparation of dsRNA standard

[0151] Concentrate and exchange the eluate obtained in Example 4 using a membrane package with a pore size of 100 kD, and replace it with pure water to obtain a purified dsRNA solution. Perform freeze-drying on the purified dsRNA solution to obtain a dry powder of the dsRNA standard, and store it at -20°C.

[0152] Take 10 mg of the dry powder of the standard and dissolve it in 100 mL of nuclease-free water to fully dissolve it to prepare a standard solution, and perform agarose gel electrophoresis detection and liquid chromatography SEC1000 chromatographic column detection on the standard solution respectively. The results of agarose gel electrophoresis are as Figure 8 shown. The electrophoresis band is single without other impurity bands, indicating that the dsRNA in the dry powder of the standard has a high purity; the results of liquid chromatography are as Figure 9 shown. It can be seen that the purity of the collected sample dsRNA reaches more than 99%, which can meet the usage requirements of the dsRNA standard for nucleic acid pesticides.

Claims

1. A method for preparing a dsRNA standard product, characterized in that, The method includes the processes of in vitro transcription template preparation, crude dsRNA solution preparation, primary purified dsRNA solution preparation, and highly purified dsRNA solution preparation; In the process of preparing the crude dsRNA solution, it includes the steps of adding DNase digestion, protease digestion, and membrane filtration to the in vitro transcription reaction solution; The process of preparing the primary purified dsRNA solution includes ion exchange chromatography, and the ion exchange chromatography includes the steps of column equilibration, sample loading, and elution; the ion exchange chromatography is anion exchange chromatography; the sample loading step includes diluting the dsRNA solution with a sample loading buffer and then loading it onto an ion exchange chromatography column, and the sample loading buffer includes one or more amino acids selected from lysine, arginine, and histidine; In the process of preparing the highly purified dsRNA solution, it includes the steps of performing the first membrane buffer exchange on the primary purified dsRNA solution, hydrophobic chromatography purification, and then performing the second membrane buffer exchange.

2. The method according to claim 1, characterized in that The sample loading buffer includes 300 - 400 mM of one or more amino acids selected from lysine, arginine, and histidine.

3. The method according to claim 1, wherein The elution step includes rinsing the chromatography column with elution buffer A and collecting the eluate; the elution buffer A includes chloride salt or perchlorate.

4. The method according to claim 3, characterized in that, The elution buffer A includes 0.1 - 1.2 M sodium chloride or 0.1 - 1.2 M sodium perchlorate.

5. The method according to any one of claims 1 to 4, characterized in that, The method also includes the process of freeze-drying.

6. A method for purifying dsRNA, characterized in that, The method includes ion exchange chromatography, and the ion exchange chromatography includes the steps of column equilibration, sample loading, and elution; the ion exchange chromatography is anion exchange chromatography; the sample loading step includes diluting the dsRNA solution with a sample loading buffer and then loading it onto an ion exchange chromatography column, and the sample loading buffer includes one or more amino acids selected from lysine, arginine, and histidine.

7. The method according to claim 6, wherein The sample loading buffer includes 300 - 400 mM of one or more amino acids selected from lysine, arginine, and histidine.

8. The method according to claim 6, wherein The elution step includes rinsing the chromatography column with elution buffer A and collecting the eluate; the elution buffer A includes chloride salt or perchlorate.

9. The method according to claim 8, characterized in that The elution buffer A includes 0.1 - 1.2 M sodium chloride or 0.1 - 1.2 M sodium perchlorate.

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