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Method for preparing high-titer lentiviral vector by conventional centrifugation

A lentiviral vector and lentivirus technology, which is applied in the field of preparing high-titer lentiviral vectors, can solve the problems of increasing the cost of lentiviral vector equipment, expensive ultracentrifuges, etc., and achieve simple operation, increased yield and low preparation cost. Effect

Inactive Publication Date: 2020-12-08
南京艾德免疫治疗研究院有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, most of the methods for concentrating lentiviral vectors require ultra-high-speed centrifugation (centrifugal force above 70,000 g), and ultra-high-speed centrifuges are very expensive, thus greatly increasing the equipment cost of lentiviral vector preparation

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  • Method for preparing high-titer lentiviral vector by conventional centrifugation
  • Method for preparing high-titer lentiviral vector by conventional centrifugation
  • Method for preparing high-titer lentiviral vector by conventional centrifugation

Examples

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Effect test

Embodiment 1

[0020] Example 1: A method for preparing high-titer lentivirus products by conventional centrifugation, with a centrifugal force of 25,000 g, centrifuged for 10 hours, and concentrated 100 times. The steps are as follows:

[0021] (1) Preparation of lentivirus concentrate: take Tris-HCl solution, NaCl solution and EDTA solution with a pH of 7.4, and mix them according to the volume ratio of Tris-HCl:NaCl:EDTA=100:200:1 to obtain lentivirus concentrate, In the lentivirus concentrate, the concentration of Tris-HCl is 50mmol / L, the concentration of NaCl is 100mmol / L, and the concentration of EDTA is 0.5mmol / L;

[0022] (2) Prepare the virus extract: dissolve the sucrose solid with the lentivirus concentrate to obtain the virus extract, the mass volume concentration of sucrose in the virus extract is 0.1g / mL;

[0023] (3) Centrifugation: Add 10ml of virus extract to a centrifuge tube, then slowly add 40ml of lentivirus supernatant to the virus extract, put the centrifuge tube cont...

Embodiment 2

[0025] Example 2: A method for preparing high-titer lentivirus products by ultrahigh-speed centrifugation, with a centrifugal force of 70,000 g, centrifuged for 2 hours, and concentrated 100 times. The steps are as follows:

[0026] (1) Centrifugation: Take 40ml lentiviral supernatant exactly the same as in Example 1, add it to a centrifuge tube and put it into a centrifuge, select the operation mode with the slowest ascent and descent speed of the centrifuge to centrifuge the mixed solution, Centrifugal force 70000g, centrifuge at 20°C for 2 hours;

[0027] (4) Preparation of high-titer lentiviral vector: Remove the supernatant of the solution in the centrifuge tube after centrifugation, and then add 400ul lentivirus lysate (10mM Tris-HCl (pH8.0), 2mM MgCl2, 5 % sucrose) to dissolve the lentiviral pellet, and store in -80°C.

Embodiment 3

[0028] Embodiment 3: Titer determination

[0029] 1) Two 6-well plates were inoculated with 293T cells. 5×10 cells per well 5 For each, the volume of the medium added is 2ml, the growth rate of different types of cells is different, and the cell fusion rate during virus infection is 40%-60%;

[0030] 2) Prepare 6 sterile EP tubes, add 900ul of fresh complete medium (high sugar DMEM+10% FBS) to each tube; 24 hours after inoculating the cells, count the cells in two wells to determine the time of infection The actual number of cells, denoted as N;

[0031] 3) Take 100ul of the virus stock solution under different centrifugal force conditions (25000g and 70000g) and add it to the first tube, mix gently, then take 100ul and add it to the second tube, mix it and add it to the third tube middle;

[0032] 4) Add 100 ul of lentivirus at different dilutions to each well, remove the culture supernatant after 24 hours of infection, replace with 2 ml of complete medium (high sugar DME...

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Abstract

The invention provides a method for preparing a high-titer lentiviral vector by conventional centrifugation. The method comprises the following steps of mixing a Tris HCl solution, a NaCl solution andan EDTA solution to obtain a lentiviral concentrated solution; dissolving cane sugar in the lentivirus concentrated solution to obtain a lentivirus extracting solution; putting a mixed solution of the lentivirus extracting solution and lentivirus supernatant into a centrifugal machine, and performing centrifugation for 10 h at the centrifugal force of 10000 g to 25000 g and the temperature of 4 DEG C; and discarding supernatant after centrifugation, and adding a lentivirus dissolving solution to obtain the high-titer lentiviral vector. The method has the advantages that the operation is simple; an ultra-high-speed centrifugal machine is not needed; the high-titer lentiviral vector can be prepared by a conventional centrifugal machine; the preparation cost is low; and the method can be widely applied to most laboratories.

Description

technical field [0001] The invention relates to a method for preparing a high-titer lentiviral vector, in particular to a method for preparing a high-titer lentiviral vector without ultra-high-speed centrifugation and using conventional centrifugal force. Background technique [0002] The research on lentiviral vectors is developing rapidly, and the research is also very in-depth. The vector can effectively integrate the foreign gene into the host chromosome, so as to achieve persistent expression. In terms of infection ability, it can effectively infect various types of cells such as neuron cells, liver cells, cardiomyocytes, tumor cells, endothelial cells, stem cells, etc., so as to achieve good gene therapy effects. Extensive clinical research has been carried out abroad. For example, the use of lentiviral vectors to prepare CAR-T for tumor immunotherapy has broad application prospects. [0003] At present, most methods for concentrating lentiviral vectors require ultra...

Claims

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Application Information

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IPC IPC(8): C12N15/867
CPCC12N15/86C12N2740/15043C12N2740/15051
Inventor 王恩秀张海汪晨
Owner 南京艾德免疫治疗研究院有限公司
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