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Lentivirus production method

A production method and lentivirus technology, applied in biochemical equipment and methods, viruses/phages, microorganisms, etc., can solve the problems of unsuitability for large-scale production of lentiviruses, unstable temperature of ultracentrifuges, and low recovery rates. The effect of increasing the amount of poison, optimizing the packaging ratio, and simplifying the production process

Pending Publication Date: 2022-07-01
SHANGHAI GENECHEM
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Problems solved by technology

Existing purification processes such as ultra-centrifuge purification are time-consuming and labor-intensive, the recovery rate is not high, and the temperature of the ultracentrifuge is required to be stable at 4-6°C for a long time. In fact, the temperature of the ultracentrifuge is unstable. At the same time, the adapter of the ultracentrifuge Can only centrifuge 6 tubes (large tube) or 12 tubes (small tube), not suitable for large-scale production of lentivirus
After the virus is centrifuged, placing it at 4°C will not completely dissolve the virus particles in the resuspension, and it will also cause the loss of the virus.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Optimization of packaging conditions for transfection reagent Zymofect

[0071] The Zymofect 293 transfection reagent virus packaging process is as follows:

[0072] 1) Preparation before transfection: Add DMEM: 1 ml, H1 plasmid: 5.6 μg, H2 plasmid: 1.9 μg, shuttle plasmid (containing the target gene): 7.5 μg, Zymofect: 45 μl according to the amount of cells in each dish to calculate the required reagents volume of.

[0073] 2) Preparation of transfection system: Take a 50ml centrifuge tube, add the corresponding volumes of DMEM, H1 plasmid, H2 plasmid, and shuttle plasmid according to the amount calculated in step 1. After shaking and mixing, add the corresponding volume of Zymofect, and then centrifuge upside down. Tube 10-20 times, mix the transfection solution evenly, and place at room temperature for 5 min.

[0074] 3) Transfection: When the transfection solution is placed for 5 minutes, according to the amount of 1ml / dish, the transfection solution is ...

Embodiment 2 3

[0105] Example 2 Comparison of three packaging methods

[0106] 1. Comparison of stability and toxicity

[0107] The PEI packaging method is as follows:

[0108] 1) Preparation of transfection solution: Calculate the volume of reagents required to be added according to the amount of PBS: 1 ml, H1 plasmid: 5.6 μg, H2 plasmid: 1.9 μg, shuttle plasmid: 7.5 μg, and PEI reagent: 58 μl required for each dish of cells. Take out the corresponding number of cell culture dishes that have been cultured for about 48 hours in the cell incubator. The cells to be transfected with a cell confluence of 80%-85% are replaced with 5ml of DMEM medium with 2% FBS. After the medium change is completed, put the cells back 37°C incubator for use.

[0109] 2) Preparation of transfection solution: Take a 50ml centrifuge tube, add the corresponding volumes of PBS, H1 plasmid, H2 plasmid, and shuttle plasmid according to the amount calculated in step 1. After shaking and mixing, add the corresponding vo...

Embodiment 3

[0136] Embodiment 3 Purification mode condition exploration and optimization

[0137] 1. Pre-purification treatment

[0138] The collected lentiviral supernatant was passed through a 0.45 μm PES filter or 2000 rpm, and centrifuged at 4°C for 10 min before treatment.

[0139] The result is as Figure 15 and Figure 16 As shown, compared with the recovery rate of centrifugation and 0.45Hm filter, the recovery rate of 0.45μm filter is higher than that of centrifugation.

[0140] 2. Comparison of recovery rate between PEG concentration and purification method and ultra-ionization method

[0141] Virus concentration and purification by ultracentrifugation:

[0142] 1) Filtration: In the ultra-clean workbench, pass the collected lentivirus supernatant through a 0.45 μm PES filter membrane, and filter it into an ultra-centrifugation tube (Backman, Type 50.2 Ti or Type 45 Ti rotor with labeled lentivirus information) The supernatant volume in the supernatant tube should not be to...

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Abstract

The invention relates to the field of lentivirus production, in particular to a lentivirus production method which comprises the following steps: 1) mixing plasmids containing a lentivirus vector genome sequence with a polycation transfection reagent with a quaternary ammonium salt structure to form a transfection solution according to a mass-volume ratio of 1: 2.5-1: 3.5; (2) adding the transfection liquid into the cultured cells, and continuously culturing; and 3) collecting the cell culture supernatant, namely the culture solution containing the lentivirus. According to the lentivirus production method disclosed by the invention, the dosage of plasmids is reduced, the packaging proportion is optimized, the virus output is improved, and meanwhile, the material cost is reduced; the production process is simplified, and the step of changing the liquid 6 hours after transfection is reduced; the virus is concentrated by adopting a PEG8000 precipitation method, so that the labor cost is saved; the resuspended virus liquid can fully release viruses, so that the recovery rate of the viruses is improved. Purification efficiency is improved, machine loss is reduced, and virus quality is guaranteed.

Description

technical field [0001] The present invention relates to the field of lentivirus production, in particular to a lentivirus production method. Background technique [0002] The existing lentivirus packaging production process mostly uses calcium transfection method or PEI transfection method. The reagent needs to strictly adjust the pH value during the preparation process. The medium was changed 6 h after staining. Existing purification processes such as ultracentrifugation are time-consuming and labor-intensive, the recovery rate is not high, and the temperature of the ultracentrifuge is required to be stable at 4-6 ° C for a long time. Only 6 tubes (large tubes) or 12 tubes (small tubes) can be centrifuged, which is not suitable for large-scale production of lentivirus. If the virus is centrifuged and placed at 4°C, the virus particles cannot be completely dissolved in the resuspension, which will also cause the loss of the virus. Therefore, the risk of low toxicity cause...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00
CPCC12N7/00C12N2740/10051
Inventor 蔡丽娥王庆强王秀秀吴飞唐小丽韩芳婷
Owner SHANGHAI GENECHEM
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