A method for purifying dsRNA

Abstract

The application provides a method for purifying dsRNA, wherein after fermentation of Escherichia coli, the bacterial bodies are broken and centrifuged to obtain a liquid phase; the liquid phase is filtered through a hollow fiber column; the obtained filtrate is adjusted in conductivity and pH through a buffer to obtain a sample for loading; and finally, the sample for loading is subjected to gradient elution through column chromatography to obtain the dsRNA. The purity of the dsRNA obtained through the purification method is above 90%, the yield reaches above 85% due to the simple steps; the purification method does not use organic solvents and expensive protein purification platforms, is green and environmentally friendly, and is economical and energy-saving; the method can be easily scaled up in proportion, from 1L to 100L, and the effect is better after scaling up.

Description

Technical Field

[0001] This invention belongs to the field of biopesticide technology, and in particular relates to a method for purifying dsRNA. Background Technology

[0002] RNA interference (RNA silencing), also known as gene silencing, works by using specially designed dsRNA in insects. The Dicer enzyme then cleaves it into siRNA, which consists of approximately 20 base pairs. The antisense strand of the siRNA binds to a RISC protein, which specifically recognizes and cleaves the mRNA, rendering it unable to translate proteins. The cleaved fragments then enter the next cycle, leading to the death of the insect.

[0003] RNA interference technology has been effectively applied in the fields of insecticides and viruses both domestically and internationally. For example, Renaissance Biosciences used dsRNA to kill potato beetles with a kill rate as high as 98.3%; GreenLight Biosciences achieved excellent results using dsRNA to kill Varroa mites. This demonstrates that RNA interference technology has a very promising future in plant protection, animal health, and human health.

[0004] Currently, the main methods for synthesizing dsRNA are in vitro transcription and microbial fermentation. In vitro transcription requires a large amount of expensive enzymes, resulting in huge costs, and subsequent purification is also expensive, making it unsuitable for large-scale scale-up. Microbial fermentation is currently the most feasible method to reduce the cost of dsRNA, but traditional fermentation methods involve cumbersome extraction steps and are expensive. For example, in the phenol-chloroform method for purifying RNA described in CN114891782 A, cells are broken down, extracted with organic solvents such as chloroform reagent, then precipitated, washed, and dried before finally dissolving. This method requires a large amount of organic solvents, which is detrimental to human health and the environment, making large-scale scale-up impossible. CN113136383A discloses a method suitable for large-scale dsRNA extraction, including cell pretreatment, buffer preparation and addition, two-step homogenization, tangential flow concentration, packing material pretreatment, and two-step combined elution. The extracted dsRNA can achieve a purity of over 90% and a yield of over 80% after low-temperature drying. However, this method is cumbersome, requires the use of organic solvents such as alcohol, does not involve purification elution, and has not yet been implemented for large-scale scale-up. CN114921457A discloses a method for extracting dsRNA, which mainly includes steps such as processing the crude extract, positive pressure-bell jar double filtration, and alcohol precipitation. The purity of dsRNA prepared by this method is above 97%. However, this method mainly emphasizes the effectiveness of double filtration and still requires alcohol elution, without involving purification elution. Large-scale scale-up has not yet been implemented. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a green, environmentally friendly, economical, energy-saving, high-purity, high-yield, and scalable dsRNA purification method.

[0006] This invention discloses a method for purifying dsRNA, comprising the following steps:

[0007] (1) Sample pretreatment: Add buffer 1 to the fermented Escherichia coli cells to adjust the pH to 5-9, stir to disperse and suspend the cells, use a high pressure homogenizer to break the dispersed cells, and then collect the liquid phase after high-speed centrifugation.

[0008] (2) Sample reprocessing: Add salt to the liquid phase, stir to dissolve, and place at a low temperature of 0-4℃ until the layers are separated; the supernatant is poured out and filtered through a hollow fiber column to obtain filtrate; use buffer 2 to adjust the conductivity of the filtrate to 48-55 S / m, and use buffer 3 to adjust the pH of the filtrate to 5-9, thus obtaining the sample to be loaded;

[0009] (3) Obtain dsRNA, load the sample using a peristaltic pump until the packing material in the column is saturated, and perform three gradient elutions using buffer 4 with increasing concentration. Collect the sample after the third elution.

[0010] The buffer solution 1 is prepared from ammonium chloride solution and potassium chloride solution;

[0011] The buffer solution 2 is prepared from potassium chloride solution, potassium dihydrogen phosphate solution and tris(hydroxymethyl)aminomethane solution;

[0012] The buffer solution 3 is prepared from potassium chloride solution, potassium dihydrogen phosphate solution and tris(hydroxymethyl)aminomethane solution;

[0013] The buffer solution 4 is prepared from potassium chloride solution and tris(hydroxymethyl)aminomethane solution.

[0014] According to the present invention, the buffer solution 1 in step (1) is prepared by mixing 600mM ammonium chloride and 450mM potassium chloride in a volume ratio of 1:6 to 6:1.

[0015] According to the present invention, the pressure of the high-pressure homogenizer in step (1) is 5000-15000 psi, and the rotation speed of the centrifuge is 5000-10000 rpm.

[0016] According to the present invention, the salt in step (2) is selected from one or more of sodium sulfate, ammonium sulfide or ammonium chloride.

[0017] According to the present invention, the buffer solution 2 in step (2) is prepared by mixing 350mM potassium chloride, 110mM potassium dihydrogen phosphate and 10mM tris(hydroxymethyl)aminomethane in a volume ratio of 10:5:3 to 30:2:1.

[0018] According to the present invention, the filter membrane pore size of the hollow fiber column is 0.45-5 μm.

[0019] According to the present invention, the buffer solution 3 in step (2) is prepared by mixing 300mM potassium chloride, 50mM potassium dihydrogen phosphate and 10mM tris(hydroxymethyl)aminomethane in a volume ratio of 1:5:3 to 10:2:1.

[0020] According to the present invention, the filler in step (3) is surface-modified silica.

[0021] According to the present invention, the packing is composed of three different particle sizes, packing 1, packing 2 and packing 3, in a volume ratio of 1:3:5 to 1:1:1, wherein:

[0022] The particle size of filler 1 is 5-25 μm, the particle size of filler 2 is 25-55 μm, and the particle size of filler 3 is 55-95 μm.

[0023] According to the present invention, the buffer solution 4 used for gradient elution in step (3) is prepared according to the following three concentration gradients:

[0024] Buffer 4-1: Prepared by mixing 350 mM potassium chloride and 20 mM tris(hydroxymethyl)aminomethane in a volume ratio of 1:3 to 10:1;

[0025] Buffer 4-2: Prepared from 375 mM potassium chloride and 25 mM tris(hydroxymethyl)aminomethane in a volume ratio of 1:3 to 10:1;

[0026] Buffer 4-3: Prepared by mixing 375mM potassium chloride and 40mM tris(hydroxymethyl)aminomethane in a volume ratio of 1:3 to 10:1.

[0027] The method for purifying dsRNA of the present invention has the following beneficial effects:

[0028] The purification method does not use organic solvents or expensive protein purification platforms, making it green, environmentally friendly, economical, and energy-saving. It eliminates the need to purchase ethanol and treat ethanol waste, saving over a million yuan annually. The purified dsRNA has a purity of over 90%, and due to the simple steps, the yield reaches over 85%. It can be easily scaled up proportionally from 1L to 100L, and the effect is even better after scaling up. Attached Figure Description

[0029] Figure 1This is a chromatogram of dsRNA before purification in Example 1;

[0030] Figure 2 The chromatogram of dsRNA purified in Example 1;

[0031] Figure 3 This is a chromatogram of dsRNA before purification in Example 2;

[0032] Figure 4 The chromatogram of dsRNA purified in Example 2;

[0033] Figure 5 This is a chromatogram of dsRNA before purification in Example 3;

[0034] Figure 6 The chromatogram of dsRNA purified in Example 3;

[0035] Figure 7 This is a chromatogram of dsRNA before purification in Example 4;

[0036] Figure 8 The chromatogram of dsRNA purified in Example 4;

[0037] Figure 9 The chromatogram of dsRNA before purification in Comparative Example 1 is shown.

[0038] Figure 10 The chromatogram of dsRNA purified in Comparative Example 1 is shown.

[0039] Figure 11 The chromatogram of dsRNA before purification in Comparative Example 2 is shown.

[0040] Figure 12 The chromatogram is of the dsRNA purified in Comparative Example 2. Detailed Implementation

[0041] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. It should be understood that the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of the present invention.

[0042] The samples used for purifying dsRNA in the following examples are fermented Escherichia coli cells that can express dsRNA, such as the E. coli genetically engineered strain E. coli DH 5α / pT7B-TMV-EcoRI-EcoRI-cp that expresses tobacco mosaic virus resistance dsRNA disclosed in CN113717984A, E. coli expressing diamondback moth dsRNA, and E. coli expressing potato beetle dsRNA, etc.

[0043] The buffers used in the following examples of dsRNA purification include Buffer 1, Buffer 2, Buffer 3, Buffer 4-1, Buffer 4-2, and Buffer 4-3, with the specific composition and proportions as follows:

[0044] Buffer 1: prepared from ammonium chloride solution and potassium chloride solution; preferably, prepared from 600mM ammonium chloride and 450mM potassium chloride in a volume ratio of 1:6 to 6:1.

[0045] Buffer 2: prepared from potassium chloride solution, potassium dihydrogen phosphate solution and tris(hydroxymethyl)aminomethane solution; preferably, prepared from 350 mM potassium chloride, 110 mM potassium dihydrogen phosphate and 10 mM tris(hydroxymethyl)aminomethane in a volume ratio of 10:5:3 to 30:2:1.

[0046] Buffer 3: prepared from potassium chloride solution, potassium dihydrogen phosphate solution and tris(hydroxymethyl)aminomethane solution; preferably, prepared from 300 mM potassium chloride, 50 mM potassium dihydrogen phosphate and 10 mM tris(hydroxymethyl)aminomethane in a volume ratio of 1:5:3 to 10:2:1.

[0047] Buffer 4: Prepared from potassium chloride solution and tris(hydroxymethyl)aminomethane solution, preferably prepared in the following three concentration gradients:

[0048] Buffer 4-1: Prepared by mixing 350 mM potassium chloride and 20 mM tris(hydroxymethyl)aminomethane in a volume ratio of 1:3 to 10:1;

[0049] Buffer 4-2: Prepared from 375 mM potassium chloride and 25 mM tris(hydroxymethyl)aminomethane in a volume ratio of 1:3 to 10:1;

[0050] Buffer 4-3: Prepared by mixing 375mM potassium chloride and 40mM tris(hydroxymethyl)aminomethane in a volume ratio of 1:3 to 10:1.

[0051] Example 1: Preparation of 1L of high-purity dsRNA

[0052] 1.1 Sample Pretreatment

[0053] Add buffer 1 to the fermented E. coli cells to adjust the pH of the cell disruption solution to 5, and maintain mechanical stirring for 20 minutes to ensure that the cells are fully dispersed and suspended.

[0054] The dispersed bacterial cells were broken up using a high-pressure homogenizer at 5000 psi, and then centrifuged at 5000 rpm to collect the liquid phase.

[0055] After pretreatment, the purity of dsRNA was detected, and the results are as follows: Figure 1 As shown, the retention time of dsRNA was 10.027 min, the purity of dsRNA was 15%, and the concentration was 6000 ng / μL.

[0056] 1.2 Sample Reprocessing

[0057] Add 150 mL of 680 mM sodium sulfate to the liquid phase obtained in step 1.1, stir until dissolved, and let stand at a low temperature of 4°C for 24 h until the upper and lower layers separate.

[0058] Pour off the supernatant and filter it using a hollow fiber column (model YWD15) with a pore size of 2μm. Adjust the conductivity of the filtrate to 50S / m using buffer 2 and adjust the pH of the filtrate to 5 using buffer 3. The sample is then loaded and the sample processing is complete.

[0059] 1.3 Obtaining dsRNA

[0060] A 1L atmospheric pressure column (model SG14) with a diameter-to-height ratio of 1:4 was selected. Surface-modified silica was chosen as the packing material, with a particle size ratio of 25:55:95 mm in a volume ratio of 1:2:3. The packing material was mixed and packed into the column (1L). The sample obtained in step 1.2 was directly loaded using a peristaltic pump (model BT300) at 80% of its full speed. The sample flow was connected to a separate detector. Based on detector data adjustment, the absorbance of dsRNA at 260 nm (UV260) was positively correlated with the concentration of dsRNA. Loading continued until the column was saturated, reaching the maximum absorbance of 1150 nm at UV260.

[0061] First, wash the column with buffer 4-1 until the UV260 drops below 50. Then, further elute with buffer 4-2, during which the UV260 first increases and then drops below 50. Finally, elute with buffer 4-3 and collect the sample. This sample contains dsRNA with the strongest adsorption capacity. The sample purity was determined using liquid chromatography, and the results are as follows. Figure 2 As shown.

[0062] Figure 2The results showed that the dsRNA retention time was 10.027 min, the dsRNA purity was 90.3%, the concentration detected by NanoDrop was 5130 ng / μL, and the yield was 86.2%.

[0063] Example 2: Preparation of 100L of high-purity dsRNA

[0064] 2.1 Sample Pretreatment

[0065] Add buffer 1 to the fermented E. coli cells to adjust the pH of the cell disruption solution to 5, and maintain mechanical stirring for 20 minutes to ensure that the cells are fully dispersed and suspended.

[0066] The dispersed bacterial cells were broken up using a high-pressure homogenizer at 5000 psi, and then centrifuged at 5000 rpm to collect the liquid phase.

[0067] After pretreatment, the purity of dsRNA was detected, and the results are as follows: Figure 3 As shown, the retention time of dsRNA was 9.984 min, the purity of dsRNA was 17.8%, and the concentration was 5500 ng / μL.

[0068] 2.2 Sample Reprocessing

[0069] Add 15 L of 680 mM sodium sulfate to the liquid phase obtained in step 2.1, stir until dissolved, and let stand at a low temperature of 4°C for 24 h until the upper and lower layers separate.

[0070] Pour off the supernatant and filter it using a hollow fiber column (model YWD15) with a pore size of 2μm. Adjust the conductivity of the filtrate to 50S / m using buffer 2 and adjust the pH of the filtrate to 5 using buffer 3. The sample is then loaded and the sample processing is complete.

[0071] 2.3 Acquisition of dsRNA

[0072] A 100L atmospheric pressure column (model SG14) with a diameter-to-height ratio of 1:4 was selected. Surface-modified silica was used as the packing material, with a particle size ratio of 25:55:95 mm in a volume ratio of 1:2:3. The mixed packing material was packed into the 100L column. The sample obtained in step 2.2 was directly loaded using a peristaltic pump (model BT300) at 80% of its full speed. The sample flow was connected to a separate detector. Based on detector data adjustment, the absorbance of dsRNA at 260 nm (UV260) was positively correlated with the concentration of dsRNA. Loading continued until the column was saturated, and the UV260 absorbance reached its maximum value of 1150 nm.

[0073] First, wash the column with buffer 4-1 until the UV260 drops below 50. Then, further elute with buffer 4-2, during which the UV260 first increases and then drops below 50. Finally, elute with buffer 4-3 and collect the sample. This sample contains dsRNA with the strongest adsorption capacity. The sample purity was determined using liquid chromatography, and the results are as follows. Figure 4 As shown.

[0074] Figure 4 The results showed that the dsRNA retention time was 9.984 min, the dsRNA purity was 91.5%, the concentration detected by NanoDrop was 5080 ng / μL, and the yield was 87.2%.

[0075] Example 3: Preparation of 100L of high-purity dsRNA

[0076] 3.1 Sample Pretreatment

[0077] Add buffer 1 to the fermented E. coli cells to adjust the pH of the cell disruption solution to 7, and maintain mechanical stirring for 20 minutes to ensure that the cells are fully dispersed and suspended.

[0078] The dispersed bacterial cells were broken up using a high-pressure homogenizer at 10,000 psi, and then centrifuged at 8,000 rpm to collect the liquid phase.

[0079] After pretreatment, the purity of dsRNA was detected, and the results are as follows: Figure 5 As shown, the retention time of dsRNA was 10.176 min, the purity of dsRNA was 15.5%, and the concentration was 5700 ng / μL.

[0080] 3.2 Sample Reprocessing

[0081] Add 14 L of 480 mM ammonium chloride to the liquid phase obtained in step 3.1, stir until dissolved, and let stand at 0°C for 24 h until the upper and lower layers separate.

[0082] Pour off the supernatant and filter it using a hollow fiber column (model YWD15) with a pore size of 0.45 μm. Adjust the conductivity of the filtrate to 48 S / m using buffer 2 and adjust the pH of the filtrate to 7 using buffer 3. The sample is then obtained and the sample processing is complete.

[0083] 3.3 Acquisition of dsRNA

[0084] A 100L atmospheric pressure column (model SG14) with a diameter-to-height ratio of 1:2 was selected. Surface-modified silica was used as the packing material, with a particle size ratio of 5:25:55 of 1:1:1. The mixed packing material was packed into the 100L column. The sample obtained in step 3.2 was directly loaded using a peristaltic pump (model BT300) at 60% of its full speed. The sample flow was connected to a separate detector. Through detector data adjustment, the absorbance of dsRNA at 260nm (UV260) was positively correlated with the concentration of dsRNA. The loading continued until the column was saturated, and the UV260 absorbance reached its maximum value of 1150 nm.

[0085] First, wash the column with buffer 4-1 until the UV260 drops below 50. Then, further elute with buffer 4-2, during which the UV260 first increases and then drops below 50. Finally, elute with buffer 4-3 and collect the sample. This sample contains dsRNA with the strongest adsorption capacity. The sample purity was determined using liquid chromatography, and the results are as follows. Figure 6 As shown.

[0086] Figure 6 The results showed that the dsRNA retention time was 10.165 min, the dsRNA purity was 92.1%, the concentration detected by NanoDrop was 5260 ng / μL, and the yield was 85.9%.

[0087] Example 4: Preparation of 100L of high-purity dsRNA

[0088] 4.1 Sample Pretreatment

[0089] Add buffer 1 to the fermented E. coli cells to adjust the pH of the cell disruption solution to 9, and maintain mechanical stirring for 30 minutes to ensure that the cells are fully dispersed and suspended.

[0090] The dispersed bacterial cells were broken up using a high-pressure homogenizer at 15,000 psi, and then centrifuged at 10,000 rpm to collect the liquid phase.

[0091] After pretreatment, the purity of dsRNA was detected, and the results are as follows: Figure 7 As shown, the retention time of dsRNA was 9.920 min, the purity of dsRNA was 14.5%, and the concentration was 5680 ng / μL.

[0092] 4.2 Sample Reprocessing

[0093] Add 14 L of 480 mM ammonium sulfate to the liquid phase obtained in step 4.1, stir until dissolved, and let stand at a low temperature of 10°C for 24 h until the upper and lower layers separate.

[0094] Pour off the supernatant and filter it using a hollow fiber column (model YWD15) with a pore size of 5μm. Adjust the conductivity of the filtrate to 55S / m using buffer 2 and adjust the pH of the filtrate to 9 using buffer 3. The sample is then obtained and the sample processing is complete.

[0095] 4.3 Obtaining dsRNA

[0096] A 100L atmospheric pressure column (model SG14) with a diameter-to-height ratio of 1:10 was selected. Surface-modified silica was chosen as the packing material, with a particle size ratio of 15:35:75 mm in volume of 1:3:5. The mixed packing material was packed into the 100L column. The sample obtained in step 4.2 was directly loaded onto the column using a peristaltic pump (model BT300) at 90% of its full speed. The sample flow was connected to a separate detector. Based on detector data adjustment, the absorbance of dsRNA at 260 nm (UV260) was positively correlated with the concentration of dsRNA. Loading continued until the column was saturated, reaching the maximum absorbance of 1150 nm at UV260.

[0097] First, wash the column with buffer 4-1 until the UV260 drops below 50. Then, further elute with buffer 4-2, during which the UV260 first increases and then drops below 50. Finally, elute with buffer 4-3 and collect the sample. This sample contains dsRNA with the strongest adsorption capacity. The sample purity was determined using liquid chromatography, and the results are as follows. Figure 8 As shown.

[0098] Figure 8 The results showed that the dsRNA retention time was 9.909 min, the dsRNA purity was 91.3%, the concentration detected by NanoDrop was 5250 ng / μL, and the yield was 86.7%.

[0099] Using embodiments outside the preferred process range of the present invention as comparative examples, the experimental results are as follows:

[0100] Comparative Example 1: Preparation of 100L of high-purity dsRNA

[0101] The pore size of the filter membrane of the hollow fiber column in Example 2 was changed to 0.22 μm, and the rest of the operation was the same as in Example 2.

[0102] After pretreatment, the purity of dsRNA was detected, and the results are as follows: Figure 9 As shown, the retention time of dsRNA was 9.877 min, the purity of dsRNA was 13.8%, and the concentration was 5540 ng / μL.

[0103] After sample reprocessing and dsRNA acquisition, the sample purity test results are as follows: Figure 10 As shown.

[0104] Figure 10 The results showed that the dsRNA retention time was 9.931 min, the dsRNA purity was 42.3%, the concentration detected by NanoDrop was 3750 ng / μL, and the yield was 71.2%.

[0105] Comparative Example 2: Preparation of 100L of high-purity dsRNA

[0106] In Example 2, the particle size of the column chromatography packing was changed to a volume ratio of 35:60:95 particles of 1:2:3. The rest of the operation was the same as in Example 2.

[0107] After pretreatment, the purity of dsRNA was detected, and the results are as follows: Figure 11 As shown, the retention time of dsRNA was 9.909 min, the purity of dsRNA was 12.3%, and the concentration was 5100 ng / μL.

[0108] After sample reprocessing and dsRNA acquisition, the sample purity test results are as follows: Figure 12 As shown.

[0109] Figure 12 The results showed that the dsRNA retention time was 9.835 min, the dsRNA purity was 32.3%, the concentration detected by NanoDrop was 4130 ng / μL, and the yield was 68.2%.

[0110] The purity and yield of dsRNA purified in Examples 1-4 and Comparative Examples 1-2 were compared, and the results are shown in Table 1:

[0111] Table 1. Purity and yield of purified dsRNA in the examples and comparative examples.

[0112]

[0113] As can be seen from the comparison results in Table 1, the overall purity and yield of the example are significantly higher than those of the comparative example. This shows that the pore size of the hollow fiber column filter membrane and the particle size of the packing material during column chromatography have a significant impact on the purification effect. The process conditions selected by the inventors in this technical solution can significantly improve the purity and yield of dsRNA purification.

Claims

1. A method for purifying dsRNA, characterized by, The method comprises the following steps: (1) sample pretreatment, adding buffer 1 prepared by 600 mM of ammonium chloride and 450 mM of potassium chloride in a volume ratio of 1:6-6:1 to adjust the pH of the bacteria body after fermentation of Escherichia coli to 5-9, stirring to disperse and suspend the bacteria body, crushing the dispersed bacteria body using a high-pressure homogenizer, and collecting the liquid phase after high-speed centrifugation of the centrifuge; (2) sample reprocessing, adding salt to the liquid phase, stirring to dissolve, and placing at low temperature 0-4℃ until stratification; The supernatant is filtered through a hollow fiber column with a filter membrane pore size of 0.45-5 μm to obtain a filtrate; buffer 2 prepared by 350 mM of potassium chloride, 110 mM of potassium dihydrogen phosphate and 10 mM of tris-hydroxymethyl aminomethane in a volume ratio of 10:5:3-30:2:1 is used to adjust the conductivity of the filtrate to 48-55 S / m, and buffer 3 prepared by 300 mM of potassium chloride, 50 mM of potassium dihydrogen phosphate and 10 mM of tris-hydroxymethyl aminomethane in a volume ratio of 1:5:3-10:2:1 is used to adjust the pH of the filtrate to 5-9, to obtain a sample for loading; the salt in step (2) is selected from one or more of sodium sulfate, ammonium sulfide or ammonium chloride; (3) obtaining dsRNA, the sample for loading is loaded through a peristaltic pump, and the packing material in column chromatography is saturated with adsorption, three gradient elutions are carried out using buffer 4 with increasing concentration, and the sample is collected in the third elution; wherein buffer 4 is prepared in the following three concentration gradients: buffer 4-1: prepared by 350 mM of potassium chloride and 20 mM of tris-hydroxymethyl aminomethane in a volume ratio of 1:3-10:1; buffer 4-2: prepared by 375 mM of potassium chloride and 25 mM of tris-hydroxymethyl aminomethane in a volume ratio of 1:3-10:1; buffer 4-3: prepared by 375 mM of potassium chloride and 40 mM of tris-hydroxymethyl aminomethane in a volume ratio of 1:3-10:1; The packing material is surface-modified silica, which is composed of packing material 1 with a particle size of 5-25 μm, packing material 2 with a particle size of 25-55 μm and packing material 3 with a particle size of 55-95 μm in a volume ratio of 1:3:5-1:1:

1.

2. The method of purifying dsRNA according to claim 1, wherein, The pressure of the high-pressure homogenizer in step (1) is 5000-15000 psi, and the speed of the centrifuge is 5000-10000 rpm.

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