Regulation method of production of taxane by long-period culture of taxus chinensis cells

A taxus cell and taxane technology, which is applied in the field of plant cell culture and production, can solve problems such as the decline in the productivity of plant cells, and achieve the effects of improving long-term stability and a stable production system

Inactive Publication Date: 2017-05-10
TIANJIN ACELBIO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The two-stage regulation process can only solve the problem of production capacity in the early and middle stages of long-term subculture of plant cells. Considering that the plant cells cultivated on a large scale are all high-generation and located in the late stage of long-term subculture, we need a kind of subculture and Regulation method to cope with the problem of decreased production capacity of long-term subcultured plant cells

Method used

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  • Regulation method of production of taxane by long-period culture of taxus chinensis cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The first unregulated subculture method of the new line of yew plant cells in the initial stage of shake flask:

[0022] Taxus cells Southern Taxus solid O38-2-2 line is converted to suspension culture of the 25th generation shake flask line suspension culture, the seed line is numbered 81#, use a 500mL shake flask, and the liquid volume is 100mL of fresh B5 Culture medium, the initial concentration of sucrose is 15g / L, 6-BA is 1mg / L, 2,4-D is 0.5mg / L, the inoculum is 10g of fresh cells on a 100-mesh sieve, protected from light, shake culture , 2cm pitch, 100rpm. After 14 days of culture, subculture was carried out, and the doubling ratio of fresh and living cells was 2.68.

Embodiment 2

[0024] The second method of unregulated subculture of yew plant cells in the mid-shake flask stage:

[0025] The yew cells of the southern yew solid O38-2-2 line were transformed into the suspension culture of the shake flask line of the 48th generation, and the seed line numbered 81# was used in the culture method of Example 1. After 14 days of culture, subculture was carried out, and the doubling ratio of fresh and living cells increased to 2.92.

Embodiment 3

[0027] The weakly regulated subculture method of yew plant cells in the long-term stage after shaking flasks three:

[0028] The yew cells of the southern yew solid O38-2-2 line were transformed into the suspension culture of the 85th generation shake flask line, and the seed line of the 81# line was numbered. Use a shaker flask with a capacity of 500mL, the liquid volume is 100mL of fresh B5 medium, the initial concentration of sucrose is 20g / L, the 6-BA content is 1.5 mg / L, and the inoculum is 16g of fresh cells on a 100-mesh sieve. Protect from light, shake culture, 2cm pitch, 100rpm. On the 2nd to 7th day of subculture, an aqueous solution of silver nitrate with a concentration of 10 mMol / L was added. After 14 days of culture, subculture was carried out, and the doubling ratio of fresh and living cells was 2.95.

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Abstract

The invention discloses a regulation method of production of taxane by long-period culture of taxus chinensis cells. The regulation method comprises the following steps: after carrying out suspension culture on taxus chinensis cell liquid for 50 generations, adopting a silver nitrate water solution added with a wide weak regulation agent at a first phase; sub-culturing by adopting a weak regulation manner with the concentration of 1mM to 40mM; at a second phase, producing paclitaxel and the taxane through a moderate intensity regulation manner by adopting a strong regulation agent, wherein at the second phase, the strong regulation agent is a coronatine water solution, the utilization concentration is 10nM to 2000nM, the number of days for adding coronatine is 1 day to 8 days; or the strong regulation agent is a jasmonic acid type ethanol solution, the utilization concentration is 10nM to 200nM, the number of days for adding jasmonic acid is 1 day to 8 days. At a subculture phase, the weak regulation agent also is added; at a production phase, a moderate intensity regulation agent is added; in a long-period culture process, the culture of plant cells reaches harmonious join of the two phases; finally, high-yield and stable-yield paclitaxel and the taxane midbody 10DBA and BAT III are obtained.

Description

technical field [0001] The invention belongs to plant cell culture production technology in biopharmaceuticals, in particular to a subculture and control method for long-term culture and stable taxane production of yew cells. Background technique [0002] The artificial planting of yew has a long period and low yield; in terms of yew plant cell culture, due to the toxicity of secondary metabolites to itself, the cell line is unstable in the long-term subculture process, and the output of secondary metabolites is getting lower and lower. Large-scale commercial production faces great difficulties. The current traditional process for the production of taxanes by yew plant cell culture is a two-stage method. The first stage is the relatively rapid scale-up of cells in the growth medium without regulators, and the main purpose is to obtain a large amount of usable plant cell mass biomass. The second stage is to take a part of the plant cell mass and put it into the production m...

Claims

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Application Information

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IPC IPC(8): C12P15/00C12P17/02C12N5/04
CPCC12N5/04C12N2500/05C12N2500/30C12P15/00C12P17/02
Inventor 丁靖志刘晓月其他发明人请求不公开姓名
Owner TIANJIN ACELBIO BIOTECH
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