Method for promoting recombinant virus vector to be secreted out of cells

A technology of recombinant virus vectors and exocrine, applied in the field of technology to promote the harvest of virus vectors, can solve the problems of increasing the complexity and cost of the process, and achieve the effect of high reference value and simple process

Pending Publication Date: 2022-04-26
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are also some viruses, such as adenovirus, herpes simplex virus, pox virus and other viral vectors, which mainly exist in the cells when harvested, so the cells need to be lysed, such as detergent treatment, freeze-thawing or ultrasonic lysing cells to release viruses, but These methods for lysing cells have the following disadvantages, 1) the step of increasing the complexity of the process requires expensive professional equipment; 2) the introduction of harmful chemicals such as detergents; 3) impurities such as a large amount of host DNA and proteins will be released after cell lysis, Brings great challenges to virus purification

Method used

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  • Method for promoting recombinant virus vector to be secreted out of cells
  • Method for promoting recombinant virus vector to be secreted out of cells

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Amplification and Harvest of Human Type 5 Adenovirus Vector

[0026] 1) Cell recovery: Take a HEK293 suspension cell from liquid nitrogen, transfer it to a water bath in liquid nitrogen or ice bath, and quickly melt it in a water bath at 37.0°C±1.0°C (120s±30s), aseptically transfer it to a cell containing Dyanmis medium. In a 15ml centrifuge tube, centrifuge at 526×g for 5 minutes at 25°C, and discard the supernatant. The cell pellet was resuspended in 10ml medium and transferred aseptically to a 125ml shake flask, and the volume was made up to 30ml with Dyanmis medium, and the sampling count was 0.5×10 6 cells / ml, cultivated in a shaker, 37.0°C, 5.0% CO 2 cultivated under conditions.

[0027] 2) Cell subculture: After the cells were cultured for 72 hours, the samples were taken and counted, which was 4.5×10 6 cells / ml. According to the passage cell density (0.5×10 6 cells / ml), dilute 30ml of cell culture medium to 270ml with Dyanmis medium; 37.0°C, 5.0...

Embodiment 2

[0034] Example 2: Amplification and Harvesting of Herpes Simplex Virus Type I Vectors

[0035]1) Cell recovery: Take a Vero cell working bank and place the cells in liquid nitrogen, transfer them to a water bath, and quickly thaw them in a water bath at 37°C±1°C, and the time is controlled for 120s±30s. The cells were transferred to a 15ml centrifuge tube containing 9ml of four kinds of DMEM medium (containing 10% FBS) pre-warmed at 37°C±1°C. The diluted cell suspension was centrifuged at room temperature (125×g) for 7-9 minutes. The supernatant was discarded, and the cell pellets were resuspended in 10 ml of 37°C±1°C preheated MEM medium (containing 10% FBS). After mixing, take samples and count them, transfer them to T25 flasks, put 4 T25 flasks in total at 37°C±1°C, 5±0.5% CO 2 cultured in an incubator.

[0036] 2) Cell subculture: recover and culture for 72-96 hours, and the confluence rate is greater than 95%. Discard the supernatant, add 10ml / T25 DPBS to rinse twice,...

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Abstract

The invention relates to preparation of a virus vector, and provides a method for promoting secretion of a recombinant virus vector to the outside of a cell. The technical scheme is as follows: in the process of cell culture or/and virus inoculation, a polysaccharide chemical substance containing sulfo groups is added into a culture medium to promote a virus vector to be secreted out of cells. The method specifically comprises the following steps: (1) cell resuscitation; (2) cell passage; (3) virus inoculation and culture; and (4) harvesting viruses. According to the method, a polysaccharide chemical substance containing a sulfo group is added in the virus amplification process, and the virus is promoted to be secreted from inside to outside of cells, so that in the virus harvesting process, cells do not need to be split, a culture supernatant is directly harvested to obtain the virus, and the harvesting process of a virus vector mainly located in the cells is simplified. The method provided by the invention is simple in process, harmful chemical substances such as a detergent are not introduced, the virus can be harvested without a complicated cell lysis step, and the method is economical and environment-friendly.

Description

technical field [0001] The invention relates to the preparation of viral vectors, in particular to a process for promoting the harvesting of viral vectors. Background technique [0002] Gene therapy refers to a therapy that introduces exogenous normal genes into target cells to correct or replace disease-causing genes. Through this therapy, the target gene is either integrated with the host cell chromosome or not located outside the chromosome but can be expressed in the cell for the purpose of treating diseases. Although viral or non-viral vectors can be used as gene therapy vectors, viral vectors are the mainstream of gene therapy due to their excellent ability to infect cells and deliver genes. [0003] The preparation of viral vectors is a very complicated task, and the optimization of the virus production process can improve the efficiency of virus production. The production of viral vectors includes: cell culture and expansion, virus inoculation, virus amplification,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/02C12N5/071
CPCC12N7/00C12N5/0686C12N2501/90C12N2710/10051C12N2710/10052C12N2710/16651C12N2710/16652
Inventor 兰永发潘讴东于春淼高晗韦厚良由庆睿
Owner OBIO TECH SHANGHAI CORP LTD
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