Preparation method of universal affinity monolithic column

Abstract

The invention discloses a preparation method of a universal affinity monolithic column, and belongs to the technical field of adenovirus, and the preparation method comprises the following steps: S1, firstly establishing a nano antibody library to construct an immune library, immunizing by using alpaca, taking 100ml of venous blood, respectively screening out nano antibodies with high affinity to AAV8 and AAV9, then constructing strains, and expressing and purifying the obtained nano antibodies; s2, manufacturing the monolithic column with the volume of 1.25 ml; s3, activating the monolithic column supported in the step S2, and then coupling the activated monolithic column with the AAV8 nano antibody and the AAV9 nano antibody obtained in the step S1, so that the monolithic column is coupled with the nano antibodies; s4, obtaining the AAV virus; the AAV affinity filler can reduce operation of a TFF unit in AAV purification, time can be greatly shortened, the virus recovery rate can be improved, the monolithic column reduces the usage amount of the nano antibody to achieve the same binding load of macroporous microspheres, the monolithic column has high affinity to AAV8 and AAV9, and universality of an AAV chromatographic material is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of adenovirus, and in particular relates to a method for preparing a universal affinity monolithic column. Background Art

[0002] Adeno-associated virus is considered to be a highly promising gene therapy delivery vehicle with the advantages of safety, stability, wide host range, and long-term expression in host cells. At present, based on the existing preclinical research data, AAV vectors have successfully achieved safe, efficient, and long-term gene expression in human organs, and can achieve the purpose of long-term treatment. With the launch of AAV gene drugs, clinical research on AAV has gradually increased, and large-scale purification has become a problem that needs to be solved urgently. At present, the affinity resins used commercially for AAV purification include six adsorbents, such as POROS TM CaptureSelect TM AAVX, AAV8 (CSAL8) and AAV9 (CSAL9), AVBSepharoseHP, CaptoAVB and AVIPure, all rely on single-chain alpaca antibody fragments derived from animal immunization or biodisplay libraries. These ligands provide the required high binding strength and selectivity to separate AAV from current raw materials with low product titers and a large number of impurities. However, these ligands require the use of harsh aqueous buffers (pH ≤ 3) to release bound capsids and limit the reuse of the resins. At the same time, their limited biochemical stability leads to high costs. For this reason, we propose a method for preparing a universal affinity monolithic column. Summary of the invention

[0003] The present invention provides a method for preparing a universal affinity monolithic column to solve the problems raised in the above background technology.

[0004] To achieve the above object, the present invention provides the following technical solution: a method for preparing a universal affinity monolithic column, comprising the following steps:

[0005] S1. First, establish a nano-antibody library to construct an immune library. Use alpacas for immunization, collect 100 ml of venous blood, and screen out nano-antibodies with high affinity to AAV8 and AAV9, then construct strains, and express and purify the obtained nano-antibodies;

[0006] S2, making a 1.25 ml monolithic column;

[0007] S3, activating the monolith supported in step S2, and then coupling the activated monolith with the AAV8 nanobody and the AAV9 nanobody obtained in step S1, so that the monolith and the nanobody are coupled to each other;

[0008] S4. Acquire AAV virus;

[0009] S5. Combine the monolithic column and nano-antibody coupling material in step S3 with the AAV virus obtained in S4, so as to calculate the AAV binding amount and recovery rate.

[0010] Furthermore, the specific steps in step S1 are:

[0011] Nanoantibody library construction immune library: Alpacas were used for immunization 4 times, 100 ml of venous blood was collected, lymphocytes were separated and mRNA was extracted. After reverse transcription and nested PCR, the mRNA was cloned to obtain the plasmid library in phagemid, and then TG1 was transformed to amplify and purify the phage library.

[0012] Nanobody screening: Add the antigen to PBS, and add the antigen added with PBS to the immunotube, and incubate at 4°C; then add the appropriate amount of amplified phage to 1% BSA, incubate at room temperature for 2 hours, and add 1% BSA to the coated immunotube, and incubate at room temperature for 2 hours; wash the blocked immunotube with PBS containing pictures for 3 times, 5 minutes each time, add the blocked phage library to the blocked immunotube, and incubate at room temperature for 1 hour; the immunotube after incubation of antigen and phage is washed with 0.1 % Tween PBS for washing; add 0.1M Trimethymime to the immune tube, incubate at room temperature for 10 minutes, then add 1M Tris-HCL to neutralize Trimethymime, transfer the eluted phage to a new centrifuge tube, amplify and purify the phage library according to the eluted phage, and repeat the screening process twice after amplification; sequence the eluted phage to obtain a candidate nanoantibody DNA sequence library that binds to the antigen; identify the phage library obtained by the initial screening by ELISA, and obtain the nanoantibody by sequencing.

[0013] Strain construction: The sequences obtained by screening in the above steps are outsourced for plasmid sequencing, and the plasmid is transferred into E. coli for culture to obtain the strain;

[0014] Nanobody expression and purification: Use TB medium, inoculate at 5% inoculation volume, culture at 37°C for 3-5h, add inducer for overnight induction, centrifuge the induced liquid to obtain wet bacteria containing nanoantibodies, use a high-pressure crusher to break the cells, and take the supernatant after centrifugation at 4°C and 10,000rpm; filter with 0.1 flat ultrafiltration membrane, and then pass through PrePack Finedex 75pg16 / 600 chromatography column to separate and purify AAV nanoantibodies.

[0015] Furthermore, the specific steps in step S2 are:

[0016] Preparation of liquid: prepare glycidyl methacrylate, ethylene glycol dimethacrylate, cyclohexanol, dodecanol and sodium persulfate, and mix the above raw materials according to 20:20:50:9:1, dissolve them by ultrasonic oscillation, take 6 ml, pour them into the mold, then put the mold filled with the mixed agent into an 80°C water bath for reaction for 24 hours to form it, and demold it after forming;

[0017] Monolithic column shaping: The above monolithic column was trimmed into a 1.25 ml columnar monolithic column with a bottom diameter of 20 mm and a height of 4 mm using a special tool, placed in a specific assembly, and the pore-forming agent was washed with alcohol;

[0018] Test: Test pressure, flow rate and integrity.

[0019] Furthermore, the specific steps in step S3 are:

[0020] Activation of the whole column: dissolve 1g of ethylenediamine in 10g of water. After dissolution, immerse 1.25ml of the column in the ethylenediamine solution, heat the liquid to 70°C, and keep the temperature at 70°C for 30h.

[0021] Coupling: Take 0.02 g SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and dissolve it in pH 7.0 MES buffer. Circulate the monolithic column prepared in step S2 at 1 ml / min for 2 h until it is empty.

[0022] Dissolve 2 mg of AAV8 nanobody in 5 ml of pH 7.0 MES and circulate at a flow rate of 1 ml / min for 2 h. Test whether the nanobody is completely attached to the column and use deionized water to circulate and clean the buffer.

[0023] Furthermore, the specific steps in step S4 are:

[0024] Culture: HEK293T cells were cultured for 12-24 h in Hyclone high-glucose DMEM and then changed to serum-free medium before transfection.

[0025] Transfect cells: slowly drop the prepared transfection system into the culture dish, shake the dish slowly to mix, and culture in an incubator; change to serum-free culture medium 24 hours after transfection.

[0026] Virus collection: Virus collection The virus can be collected 48 to 72 hours after transfection. The virus-producing cells and the culture medium are collected into a centrifuge tube, and the cell pellet is obtained by centrifugation. The cell pellet is lysed to collect the virus.

[0027] Furthermore, the specific steps in step S4 are:

[0028] Column: 1.25 ml monolithic column prepared in step S2;

[0029] Flow rate: 2.5 ml / min (CV);

[0030] Loading conditions: 10 mM Tris + 300 mM NaCl pH 8.0;

[0031] Equilibration buffer: 10 mM Tris + 300 mM NaCl pH 8.0;

[0032] Elution buffer: 100 mM HAc + 500 mM NaCl pH 3.0.

[0033] The beneficial effects of the present invention are:

[0034] 1. AAV affinity fillers can reduce TFF unit operations in AAV purification, which can significantly shorten the time and improve the virus recovery rate.

[0035] 2. The monolithic column reduces the amount of nanoantibodies used to achieve the same binding capacity as macroporous microspheres.

[0036] 3. It has high affinity for both AAV8 and AAV9, achieving the versatility of AAV chromatography materials. DETAILED DESCRIPTION

[0037] In order to further understand the content, features and effects of the present invention, the following embodiments are listed and described in detail as follows.

[0038] Embodiment 1:

[0039] A method for preparing a universal affinity monolithic column comprises the following steps:

[0040] S1. First, establish a nano-antibody library to construct an immune library. Use alpacas for immunization, collect 100 ml of venous blood, and screen out nano-antibodies with high affinity to AAV8 and AAV9, then construct strains, and express and purify the obtained nano-antibodies;

[0041] Nanoantibody library construction immune library: Alpacas were used for immunization 4 times, 100 ml of venous blood was collected, lymphocytes were separated and mRNA was extracted, and the mRNA was cloned after reverse transcription and nested PCR, so as to clone the plasmid library in phagemid, and then TG1 was transformed, and the phage library was amplified and purified;

[0042] Nanobody screening: Add the antigen to PBS, and add the antigen added with PBS to the immunotube, and incubate at 4°C; then add the appropriate amount of amplified phage to 1% BSA, incubate at room temperature for 2 hours, and add 1% BSA to the coated immunotube, and incubate at room temperature for 2 hours; wash the blocked immunotube with PBS containing pictures for 3 times, 5 minutes each time, add the blocked phage library to the blocked immunotube, and incubate at room temperature for 1 hour; the immunotube after incubation of antigen and phage is washed with 0.1 % Tween PBS for washing; add 0.1M Trimethymime to the immune tube, incubate at room temperature for 10 minutes, then add 1M Tris-HCL to neutralize Trimethymime, transfer the eluted phage to a new centrifuge tube, amplify and purify the phage library according to the eluted phage, and repeat the screening process twice after amplification; sequence the eluted phage to obtain a candidate nano-antibody DNA sequence library that binds to the antigen; identify the phage library obtained by the initial screening by ELISA, and obtain the nano-antibody by sequencing;

[0043] Strain construction: The sequences obtained by screening in the above steps are outsourced for plasmid sequencing, and the plasmid is transferred into E. coli for culture to obtain the strain;

[0044] Nanobody expression and purification: Use TB medium, inoculate at 5% inoculation volume, culture at 37°C for 3-5h, add inducer for overnight induction, centrifuge the induction liquid to obtain wet bacteria containing nanoantibodies, use a high-pressure crusher to crush the cells, and take the supernatant after centrifugation at 10000rpm at a temperature of 4°C; filter with a 0.1 flat ultrafiltration membrane, and then pass through a PrePack Finedex 75pg16 / 600 chromatography column to separate and purify the AAV nanoantibody;

[0045] The same method was used for AAV2, AAV5, AAV8, and AAV9 to screen antigens AAV2, AAV5, AAV8, and AAV9;

[0046] S2, making a 1.25 ml monolithic column;

[0047] Preparation of liquid: prepare glycidyl methacrylate, ethylene glycol dimethacrylate, cyclohexanol, dodecanol and sodium persulfate, and mix the above raw materials according to 20:20:50:9:1, dissolve them by ultrasonic oscillation, take 6 ml, pour them into the mold, then put the mold filled with the mixed agent into an 80°C water bath for reaction for 24 hours to form it, and demold it after forming;

[0048] Monolithic column shaping: The above monolithic column was trimmed into a 1.25 ml columnar monolithic column with a bottom diameter of 20 mm and a height of 4 mm using a special tool, placed in a specific assembly, and the pore-forming agent was washed with alcohol;

[0049] Side view: test pressure, flow rate and integrity;

[0050] S3, activating the monolith supported in step S2, and then coupling the activated monolith with the AAV8 nanobody and the AAV9 nanobody obtained in step S1, so that the monolith and the nanobody are coupled to each other;

[0051] Activation of the whole column: dissolve 1g of ethylenediamine in 10g of water. After dissolution, immerse 1.25ml of the column in the ethylenediamine solution, heat the liquid to 70°C, and keep the temperature at 70°C for 30h.

[0052] Coupling: Take 0.02 g SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and dissolve it in pH 7.0 MES buffer. Circulate the monolithic column prepared in step S2 at 1 ml / min for 2 h until it is empty.

[0053] Dissolve 2 mg of AAV8 nanobody in 5 ml of pH 7.0 MES and circulate at a flow rate of 1 ml / min for 2 h. Test that the nanobody is completely attached to the column and wash the buffer with deionized water.

[0054] S4. Acquire AAV virus;

[0055] Culture: HEK293T cells were cultured for 12 h in Hyclone high-glucose DMEM and then changed to serum-free medium before transfection;

[0056] Transfect cells: slowly drop the prepared transfection system into the culture dish, shake the dish slowly to mix, and place it in the incubator for culture; change to serum-free culture medium 24 hours after transfection;

[0057] Virus collection: Virus collection The virus can be collected 48 hours after transfection. The virus-producing cells and the culture medium are collected into a centrifuge tube, centrifuged to obtain the cell pellet, and the cell pellet is lysed to collect the virus;

[0058] S5, combining the monolithic column and the nano-antibody coupling material in step S3 with the AAV virus obtained in S4, thereby calculating the AAV binding amount and the recovery rate;

[0059] Column: 1.25 ml monolithic column prepared in step S2;

[0060] Flow rate: 2.5 ml / min (CV);

[0061] Loading conditions: 10 mM Tris + 300 mM NaCl pH 8.0;

[0062] Equilibration buffer: 10 mM Tris + 300 mM NaCl pH 8.0;

[0063] Elution buffer: 100 mM HAc + 500 mM NaCl pH 3.0.

[0064] Embodiment 2:

[0065] S1. First, establish a nano-antibody library to construct an immune library. Use alpacas for immunization, collect 100 ml of venous blood, and screen out nano-antibodies with high affinity to AAV8 and AAV9, then construct strains, and express and purify the obtained nano-antibodies;

[0066] Nanoantibody library construction immune library: Alpacas were used for immunization 4 times, 100 ml of venous blood was collected, lymphocytes were separated and mRNA was extracted, and the mRNA was cloned after reverse transcription and nested PCR, so as to clone the plasmid library in phagemid, and then TG1 was transformed, and the phage library was amplified and purified;

[0067] Nanobody screening: Add the antigen to PBS, and add the antigen added with PBS to the immunotube, and incubate at 4°C; then add the appropriate amount of amplified phage to 1% BSA, incubate at room temperature for 2 hours, and add 1% BSA to the coated immunotube, and incubate at room temperature for 2 hours; wash the blocked immunotube with PBS containing pictures for 3 times, 5 minutes each time, add the blocked phage library to the blocked immunotube, and incubate at room temperature for 1 hour; the immunotube after incubation of antigen and phage is washed with 0.1 % Tween PBS for washing; add 0.1M Trimethymime to the immune tube, incubate at room temperature for 10 minutes, then add 1M Tris-HCL to neutralize Trimethymime, transfer the eluted phage to a new centrifuge tube, amplify and purify the phage library according to the eluted phage, and repeat the screening process twice after amplification; sequence the eluted phage to obtain a candidate nano-antibody DNA sequence library that binds to the antigen; identify the phage library obtained by the initial screening by ELISA, and obtain the nano-antibody by sequencing;

[0068] Strain construction: The sequences obtained by screening in the above steps are outsourced for plasmid sequencing, and the plasmid is transferred into E. coli for culture to obtain the strain;

[0069] Nanobody expression and purification: Use TB medium, inoculate at 5% inoculation volume, culture at 37°C for 3-5h, add inducer for overnight induction, centrifuge the induction liquid to obtain wet bacteria containing nanoantibodies, use a high-pressure crusher to crush the cells, and take the supernatant after centrifugation at 10000rpm at a temperature of 4°C; filter with a 0.1 flat ultrafiltration membrane, and then pass through a PrePackFinedex75pg16 / 600 chromatography column to separate and purify the AAV nanoantibody;

[0070] The same method was used for AAV2, AAV5, AAV8, and AAV9 to screen antigens AAV2, AAV5, AAV8, and AAV9;

[0071] S2, making a 1.25 ml monolithic column;

[0072] Preparation of liquid: prepare glycidyl methacrylate, ethylene glycol dimethacrylate, cyclohexanol, dodecanol and sodium persulfate, and mix the above raw materials according to 20:20:50:9:1, dissolve them by ultrasonic oscillation, take 6 ml, pour them into the mold, then put the mold filled with the mixed agent into an 80°C water bath for reaction for 24 hours to form it, and demold it after forming;

[0073] Monolithic column shaping: The above monolithic column was trimmed into a 1.0 ml columnar monolithic column with a bottom diameter of 20 mm and a height of 4 mm using a special tool, placed in a specific assembly, and the pore-forming agent was washed with alcohol;

[0074] Side view: test pressure, flow rate and integrity;

[0075] S3, activating the monolith supported in step S2, and then coupling the activated monolith with the AAV8 nanobody and the AAV9 nanobody obtained in step S1, so that the monolith and the nanobody are coupled to each other;

[0076] Activation of the whole column: dissolve 1g of ethylenediamine in 10g of water. After dissolution, immerse 1.25ml of the column in the ethylenediamine solution, heat the liquid to 70°C, and keep the temperature at 70°C for 30h.

[0077] Coupling: Take 0.02 g SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and dissolve it in pH 7.0 MES buffer. Circulate the monolithic column prepared in step S2 at 1 ml / min for 2 h until it is empty.

[0078] Dissolve 2 mg of AAV8 nanobody in 5 ml of pH 7.0 MES and circulate at a flow rate of 1 ml / min for 2 h. Test that the nanobody is completely attached to the column and wash the buffer with deionized water.

[0079] S4. Acquire AAV virus;

[0080] Culture: HEK293T cells were cultured for 24 h in Hyclone high-glucose DMEM as the culture medium, and then changed to serum-free medium before transfection;

[0081] Transfect cells: slowly drop the prepared transfection system into the culture dish, shake the dish slowly to mix, and place it in the incubator for culture; change to serum-free culture medium 24 hours after transfection;

[0082] Virus collection: Virus collection The virus can be collected 72 hours after transfection. The virus-producing cells and the culture medium are collected into a centrifuge tube, centrifuged to obtain the cell pellet, and the cell pellet is lysed to collect the virus;

[0083] S5, combining the monolithic column and the nano-antibody coupling material in step S3 with the AAV virus obtained in S4, thereby calculating the AAV binding amount and the recovery rate;

[0084] Column: 1.25 ml monolithic column prepared in step S2;

[0085] Flow rate: 2.5 ml / min (CV);

[0086] Loading conditions: 10 mM Tris + 300 mM NaCl pH 8.0;

[0087] Equilibration buffer: 10 mM Tris + 300 mM NaCl pH 8.0;

[0088] Elution buffer: 100 mM HAc + 500 mM NaCl pH 3.0.

[0089] Table 1 is the comparison data between step S5 and macroporous microspheres in Example 1-2

[0090] Dwell time(S) Yield (%) Binding capacity (E12vg / mL) 60 80 22 30 85 52

[0091] Example AAV affinity fillers can reduce TFF unit operations in AAV purification, significantly shorten the time and improve virus recovery rate.

[0092] In this technical solution:

[0093] AAV affinity fillers can reduce TFF unit operations in AAV purification, significantly shorten the time and improve virus recovery rates. The monolithic column reduces the amount of nanoantibodies used to achieve the same binding capacity as large-pore microspheres. It has high affinity for both AAV8 and AAV9, achieving the versatility of AAV chromatography materials and being compatible with AAV2, AAV5, AAV8 and AAV9.

[0094] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a universal affinity monolithic column, characterized in that: The following steps are involved: S1. First, establish a nano-antibody library to construct an immune library. Use alpacas for immunization, collect 100 ml of venous blood, and screen out nano-antibodies with high affinity to AAV8 and AAV9, then construct strains, and express and purify the obtained nano-antibodies; S2, making a 1.25 ml monolithic column; S3, activating the monolith supported in step S2, and then coupling the activated monolith with the AAV8 nanobody and the AAV9 nanobody obtained in step S1, so that the monolith and the nanobody are coupled to each other; S4. Acquire AAV virus; S5. Combine the monolithic column and nano-antibody coupling material in step S3 with the AAV virus obtained in S4, so as to calculate the AAV binding amount and recovery rate.

2. The method for preparing a universal affinity monolithic column according to claim 1, characterized in that: The specific steps in step S1 are: Nanoantibody library construction immune library: Alpacas were used for immunization 4 times, 100 ml of venous blood was collected, lymphocytes were separated and mRNA was extracted. After reverse transcription and nested PCR, the mRNA was cloned to obtain the plasmid library in phagemid, and then TG1 was transformed to amplify and purify the phage library. Nanobody screening: Add the antigen to PBS, and add the antigen added with PBS to the immune tube, and incubate at 2℃~4℃; then add the appropriate amount of amplified phage to 1% BSA, incubate at room temperature for 2h, and add 1% BSA to the coated immune tube, and incubate at room temperature for 2h; wash the blocked immune tube with PBS containing pictures for 3 times, 5min each time, add the blocked phage library to the blocked immune tube, and incubate at room temperature for 1h; the immune tube after incubation of antigen and phage is washed with PBS containing 0 .1% Tween PBS was used for washing; 0.1M Trimethymime was added to the immune tube and incubated at room temperature for 10 minutes, followed by adding 1M Tris-HCL to neutralize Trimethymime, and the eluted phage was transferred to a new centrifuge tube, and the eluted phage was amplified and purified according to the phage library, and the screening process was repeated twice after amplification; the eluted phage was sequenced to obtain a candidate nano-antibody DNA sequence library that binds to the antigen; the phage library obtained from the initial screening was identified by ELISA and sequenced to obtain the nano-antibody. Strain construction: The sequences obtained by screening in the above steps are outsourced for plasmid sequencing, and the plasmid is transferred into E. coli for culture to obtain the strain; Nanobody expression and purification: Use TB medium, inoculate at 5% inoculation volume, culture at 37°C for 3-5h, add inducer for overnight induction, centrifuge the induced liquid to obtain wet bacteria containing nanoantibodies, use a high-pressure crusher to break the cells, and take the supernatant after centrifugation at 4°C and 10,000rpm; filter with 0.1 flat ultrafiltration membrane, and then pass through PrePack Finedex 75pg 16 / 600 chromatography column to separate and purify AAV nanoantibodies.

3. The method for preparing a universal affinity monolithic column according to claim 1, characterized in that: The specific steps in step S2 are: Preparation of liquid: prepare glycidyl methacrylate, ethylene glycol dimethacrylate, cyclohexanol, dodecanol and sodium persulfate, and mix the above raw materials according to 20:20:50:9:1, dissolve them by ultrasonic oscillation, take 6 ml, pour them into the mold, then put the mold filled with the mixed agent into an 80°C water bath for reaction for 24 hours to form it, and demold it after forming; Monolithic column shaping: The above monolithic column was trimmed into a 1.25 ml columnar monolithic column with a bottom diameter of 20 mm and a height of 4 mm using a special tool, placed in a specific assembly, and the pore-forming agent was washed with alcohol; Test: Test pressure, flow rate and integrity.

4. The method for preparing a universal affinity monolithic column according to claim 1, characterized in that: The specific steps in step S3 are: Activation of the whole column: dissolve 1g of ethylenediamine in 10g of water. After dissolution, immerse 1.25ml of the column in the ethylenediamine solution, heat the liquid to 70°C, and keep the temperature at 70°C for 30h. Coupling: 0.02 g SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) was dissolved in pH 7.0 MES buffer, and the monolithic column prepared in step S2 was circulated at 1 ml / min for 2 h until it was empty; Dissolve 2 mg of AAV8 nanobody in 5 ml of pH 7.0 MES and circulate at a flow rate of 1 ml / min for 2 h to test whether the nanobody is completely attached to the column. Use deionized water to circulate and clean the buffer.

5. The method for preparing a universal affinity monolithic column according to claim 1, characterized in that: The specific steps in step S4 are: Culture: HEK293T cells were cultured for 12-24 h in Hyclone high-glucose DMEM and then changed to serum-free medium before transfection. Transfect cells: slowly drop the prepared transfection system into the culture dish, shake the dish slowly to mix, and culture in an incubator; change to serum-free culture medium 24 hours after transfection. Virus collection: Virus collection The virus can be collected 48 to 72 hours after transfection. The virus-producing cells and the culture medium are collected into a centrifuge tube, centrifuged to obtain the cell pellet, and the cell pellet is lysed to collect the virus.

6. The method for preparing a universal affinity monolithic column according to claim 1, characterized in that: The specific steps in step S4 are: Column: 1.25 ml monolithic column prepared in step S2; Flow rate: 2.5 ml / min (CV); Loading conditions: 10 mM Tris + 300 mM NaCl pH 8.0; Equilibration buffer: 10 mM Tris + 300 mM NaCl pH 8.0; Elution buffer: 100 mM HAc + 500 mM NaCl pH 3.0.