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A kind of cultivation method of defective adenovirus adc68-gp

A culture method, adc68-gp technology, applied in the field of virus culture, can solve the problems of difficult control of serum quality, unfavorable purification, low toxin production, etc., and achieve the effect of increasing virus amplification multiple, facilitating process amplification, and increasing virus production

Active Publication Date: 2020-11-10
长睿生物技术(成都)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The existing methods for culturing defective adenovirus AdC68-GP have problems such as difficult process amplification, use of serum in the cultivation process, which is not conducive to later purification, and the quality of serum is not easy to control, resulting in difficult control of product quality; MOI is generally controlled during inoculation Lesions can only be produced at 0.1-10, the amount of poison received is large, and the toxin production is not high

Method used

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  • A kind of cultivation method of defective adenovirus adc68-gp
  • A kind of cultivation method of defective adenovirus adc68-gp

Examples

Experimental program
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Effect test

Embodiment 1

[0029] The method for culturing the defective adenovirus AdC68-GP provided in this embodiment comprises the following steps:

[0030] 1 Cell Recovery:

[0031] Take out a human embryonic kidney 293 suspension cell (purchased from Thermofisher) from the cell bank, transfer it to a constant temperature water bath at 37°C for quick thawing (about 2 minutes), and after complete thawing, 293 suspension cells (1×10 7 cells, 1ml) transferred to 29ml EX-CELL TM 293 serum-free medium (purchased from sigma) in a 125ml shake flask, mix well and take samples for counting, place at 37°C, 8% CO 2 And the suspension culture under the condition of rotating speed 130rpm.

[0032] 2 Subculture and inoculation:

[0033] Cultivate for 2 to 3 days, and culture in suspension until the cell density is equal to 1.1×10 6 cells / ml, and the cell viability rate is 90%, for subculture and inoculation culture, the subculture density is 3×10 5 cells / ml; Simultaneously inoculate defective adenovirus AdC...

Embodiment 2

[0048] The method for culturing the defective adenovirus AdC68-GP provided in this embodiment comprises the following steps:

[0049] 1 Cell Recovery:

[0050] Take out a human embryonic kidney 293 suspension cell from the cell bank, transfer it to a 37°C constant temperature water bath for quick thawing (about 2 minutes), and transfer the 293 suspension cell to the 29ml EX-CELL after complete thawing TM 293 serum-free medium in a 125ml shake flask, mix well and take samples for counting, place at 36°C, 7% CO 2 , 150rpm shaker culture.

[0051] 2 Subculture and inoculation:

[0052] Cultivate for 2-3 days, and culture in suspension until the cell density is equal to 1.4×10 6 cells / ml, and the cell viability rate is 98%, for subculture and inoculation culture, the subculture density is 3.5×10 5 cells / ml; Simultaneously inoculate defective adenovirus AdC68-GP at a ratio of MOI value 0.0008;

[0053] After passage, place at 38°C, 8% CO 2 , 120rpm shaker culture. Harvest th...

Embodiment 3

[0056] The method for culturing the defective adenovirus AdC68-GP provided in this embodiment comprises the following steps:

[0057] 1 Cell Recovery:

[0058] Take out a human embryonic kidney 293 suspension cell from the cell bank, transfer it to a 37°C constant temperature water bath for quick thawing (about 2 minutes), and transfer the 293 suspension cell to the 29ml EX-CELL after complete thawing TM 293 serum-free medium in a 125ml shake flask, mix well and take samples for counting, place at 38°C, 9% CO 2 , 110rpm shaker culture.

[0059] 2 Subculture and inoculation:

[0060] Cultivate for 2-3 days, and culture in suspension until the cell density is equal to 1.8×10 6 cells / ml, and the cell viability rate is 94%, for subculture and inoculation culture, the subculture density is 4×10 5 cells / ml; Simultaneously inoculate defective adenovirus AdC68-GP at a ratio of MOI of 0.0014;

[0061] After passage, place at 37°C, 8% CO 2 , 130rpm shaker culture. Harvest the vir...

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Abstract

The invention discloses a method for cultivating defective adenovirus AdC68-GP, and relates to the technical field of virus cultivation. It includes: the cell recovery step of inoculating packaging cells into serum-free medium for suspension culture, and according to the cell density value, that is, the cell density is 1‑2×10 6 The cells / ml and cell survival rate are equal to or greater than 90% of the subculture inoculation culture step. This culture method uses serum-free medium to carry out suspension culture of packaging cells. On the one hand, it is beneficial to process amplification. For adenovirus AdC68‑GP, lowering the MOI value and controlling the MOI in the range of 0.0001 to 0.01 can increase the virus amplification factor and increase the virus yield.

Description

technical field [0001] The invention relates to the technical field of virus culture, in particular to a method for culturing defective adenovirus AdC68-GP. Background technique [0002] Defective adenovirus AdC68-GP is an adenovirus that can effectively induce neutralizing antibodies against rabies virus and produce strong resistance to virus infection after modification based on chimpanzee adenovirus AdC68. [0003] The existing methods for culturing defective adenovirus AdC68-GP have problems such as difficult process amplification, use of serum in the cultivation process, which is not conducive to later purification, and the quality of serum is not easy to control, resulting in difficult control of product quality; MOI is generally controlled during inoculation Lesions can only be produced at 0.1-10, the amount of poison received is large, and the toxin production is not high. Contents of the invention [0004] The object of the present invention is to provide a metho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12R1/93
CPCC12N7/00C12N2710/10352
Inventor 冉鸿宇王鑫杰邓飞贾芳苗张仁怀
Owner 长睿生物技术(成都)有限公司
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