A method for inducing the expression of S. cellulosus to prepare epothilone

A technology of Cystobacter cellulis and inducing fibers, which is applied in the field of microbial fermentation of anti-tumor drugs, can solve problems such as low expression efficiency of epothilone, and achieve the effects of shortening fermentation culture time, improving fermentation efficiency, and simple analysis and elution method.

Active Publication Date: 2014-10-29
广州市微生物研究所集团股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the deficiency of low epothilone expression efficiency in the prior art, and provide a method for inducing the expression of S. cellulosus to prepare epothilone

Method used

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  • A method for inducing the expression of S. cellulosus to prepare epothilone

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Embodiment 1

[0056] S1. Preparation of fermented strains: add the freeze-dried strains of S. cellulosus strain ATCC25532 into a test tube containing 3 mL of normal saline, shake evenly, and add 1 mL of the solution to a petri dish containing solid medium for coating uniform. Solid medium formula (all components are percentages by weight): 2% paper pulp, 2% potato starch, 1% peptone, 2% corn steep liquor, 0.2% dipotassium hydrogen phosphate, 2% agar powder, pH7.0, cultured on the basis of After sterilizing at 121°C for 20 minutes, add 20 mg of kanamycin B per liter of medium on the ultra-clean workbench, mix well, and pour it into a petri dish. Placed in a 28°C incubator for 48 hours. Scrape and inoculate evenly growing orange colony moss into a 500mL Erlenmeyer flask filled with 100mL of liquid medium, and place it in a constant temperature air bath shaker with a temperature of 28°C and a rotation speed of 220rpm for 72 hours. When the biomass reaches 10 9 CFU / mL is used as the fermenta...

Embodiment 2

[0063] S1. Preparation of fermented strains: add the freeze-dried strains of S. cellulosus strain ATCC25532 into a test tube containing 3 mL of normal saline, shake evenly, and add 1 mL of the solution to a petri dish containing solid medium for coating uniform. Solid medium formula (each component is weight percent): 1% paper pulp, 1% potato starch, 1% peptone, 1% corn steep liquor, 0.1% dipotassium hydrogen phosphate, 1.5% agar powder, pH7.0, cultured based on After sterilizing at 121°C for 20 minutes, add 15 mg of kanamycin B per liter of culture medium on the ultra-clean workbench, mix well, and pour it into a petri dish. Placed in a 25°C incubator for 40 hours. Scraped and inoculated the evenly growing and orange colony moss into a 500mL Erlenmeyer flask filled with 100mL of liquid medium, and placed it in a constant temperature air bath shaker with a temperature of 25°C and a rotation speed of 240rpm for 60 hours. When the biomass reached 10 9 CFU / mL is used as the fe...

Embodiment 3

[0070] S1. Preparation of fermented strains: add the freeze-dried strains of S. cellulosus strain ATCC25532 into a test tube containing 3 mL of normal saline, shake evenly, and add 1 mL of the solution to a petri dish containing solid medium for coating uniform. Solid medium formula (each component is weight percent): 5% paper pulp, 2% potato starch, 5% peptone, 2% corn steep liquor, 0.2% dipotassium hydrogen phosphate, 2% agar powder, pH7.0, cultured on the basis of After sterilizing at 121°C for 20 minutes, add 30 mg of kanamycin B per liter of culture medium on the ultra-clean workbench, mix well, and pour it into a petri dish. Placed in a 30°C incubator for 50 hours. Scraped and inoculated the evenly growing and orange colony moss into a 500mL Erlenmeyer flask filled with 100mL liquid medium, and placed it in a constant temperature air bath shaker with a temperature of 28°C and a rotation speed of 260rpm for 80 hours. When the biomass reached 10 9 CFU / mL is used as the ...

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Abstract

The invention relates to the field of microbial fermentation of antineoplastic drugs, and discloses a method for preparing epothilone by inducing sorangium cellulosum expression. The method comprises the following preparing steps of: S1, culturing a sorangium cellulosum strain until the biomass of the sorangium cellulosum is 0.8-1.2*109 cfu / mL, and using the sorangium cellulosum strain as a fermentation strain for standby application; S2, adding the fermentation strain in the step S1 into a fermentation culture medium for cultivation, and stopping fermentative cultivation when the biomass of the sorangium cellulosum in the fermentation liquid is greater than or equal to 1010 cfu / mL so as to obtain a fermentation culture; S3, adding an inductive composition in the fermentation culture in step S2 for the induced fermentation cultivation so as to obtain an induced fermentation culture; and S4, separating epothilone in the induced fermentation culture. As the multi-factor induced sorangium cellulosum high-efficiency expression epothilone is adopted, the unit expression amount of the epothilone is improved obviously.

Description

technical field [0001] The invention relates to the field of microbial fermentation of antineoplastic drugs, and more specifically relates to a method for inducing expression of Sonocystis cellulosus to prepare epothilone. Background technique [0002] Epothilone (epothilone) is a class of macrolide compounds, epothilone can be isolated from the fermentation broth of S. cellulosus strain of Myxobacteria suborder, and its main components are Epothilone A and B. It has the same mechanism of action as paclitaxel to inhibit the growth of tumor cells, and exhibits strong anticancer activity against multidrug-resistant tumor cells and paclitaxel-resistant tumor cells. In addition to its advantages in activity, epothilone has a simpler chemical structure and better water solubility than paclitaxel, and can be produced by fermentation. The molecular structure of epothilone is as follows: [0003] [0004] Epothilones A (R = H) and B (R = CH 3 ) [0005] It was first repor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/18C12R1/01
Inventor 杜少平夏枫耿石笛戴南艺黄魁英赵培静秦鹏蓝碧锋张竞立陈勉华
Owner 广州市微生物研究所集团股份有限公司
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