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Daunorubicin hydrochloride bacterium and yield increasing method thereof

A technology for daunorubicin hydrochloride and strains, applied to daunorubicin hydrochloride strains and high-yield fields, can solve problems such as gaps, difficulty in breeding strains producing antitumor antibiotics, and the like

Inactive Publication Date: 2014-09-24
JIANGSU HECHANG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Due to the considerable difficulty in the strain breeding of anti-tumor antibiotic-producing bacteria, through the above work, although the fermentation yields of daunorubicin of different strains have been improved to varying degrees, compared with the fermentation levels of other types of antibiotics, there is still a considerable gap

Method used

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  • Daunorubicin hydrochloride bacterium and yield increasing method thereof
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Experimental program
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Embodiment 1

[0067] Step Q1: the preparation of protoplasts comprises the following sub-steps:

[0068] Q11: Take the cultured mycelia (ATCC31276), centrifuge at 8000 rpm for 5 minutes to obtain a precipitate, add normal saline to wash, discard the clarified part after centrifuging again, and disperse the mycelium by vortexing;

[0069] Q12: Add 1ml of physiological saline to the suspended mycelia treated in step Q11 to suspend the mycelium, then add 0.2ml of lysozyme solution, blow and inhale with a pipette 5 times and mix well, then place in a 30° water bath Carry out enzymatic hydrolysis in the medium, every 10 minutes, use the pipette to inhale 3 times, stop the enzymatic hydrolysis after 90 minutes;

[0070] Q13: Centrifuge the treatment solution after the above step Q12 at a rate of 2000 rpm for 5 minutes, make the remaining mycelium fragments sink to the bottom of the tube, take the upper clarified part, which is the protoplast suspension, and transfer it to another tube. Centrifug...

Embodiment 2

[0073] Step Q2: regeneration of protoplasts, including the following sub-steps:

[0074] Q21: Take 0.5ml of the protoplast suspension obtained in step Q14, mix it with 25ml of soft agar, and pour it into the medium on the upper layer of the R2YE plate;

[0075] Q22: Take another R2YE plate, cover the R2YE plate in step Q21, and culture the double-layer medium plate upside down at 28°.

[0076] Among them, the components of the medium are: sucrose, glucose, yeast extract, magnesium chloride, hydrolyzed casein, potassium sulfate, TES, trace elements, the content of each component in the medium is: sucrose 103g / L; glucose 10g / L; Yeast extract 5g / L; Magnesium chloride 10g / L; Hydrolyzed casein 0.1g / L; Potassium sulfate 0.25g / L; TES6.0g / L; 2 B 4 o 7 10H 2 O: 10mg / 100ml, ZnCl 2 : 40mg / 100ml, (NH 4 )6MO 7 o 24 4H 2 O: 10mg / 100ml, FeCl 3 ·6H 2 O: 200mg / 100ml, MnCl 2 4H 2 O: 10mg / 100ml, CuCl 2 2H 2 O: 10mg / 100ml.

Embodiment 3

[0078] Step Q3: UV mutagenesis of protoplasts, including the following sub-steps:

[0079] Q31: Perform ultraviolet mutagenesis on the protoplast suspension prepared and diluted according to step Q14:

[0080] Q311: The intensity of ultraviolet irradiation is 15W, 254nm, the distance of the light source remains unchanged at 30cm, and the one-time continuous ultraviolet irradiation time is 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, and 6 minutes;

[0081] Q312: Keep the ultraviolet irradiation intensity at 15W, 254nm, and set the irradiation distance to 10cm, 20cm, 30cm, 40cm, 50cm respectively, and the irradiation time for each distance is 3 minutes;

[0082] Q32: After completion, follow the steps of Q2, under the culture environment of 28°, and culture in the dark for 7 days.

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Abstract

The invention provides a daunorubicin hydrochloride bacterium and a yield increasing method thereof. The method comprises the following steps: Q1, preparing protoplast; Q2, regenerating the protoplast; Q3, performing ultraviolet mutagenesis of the protoplast; Q4, performing ion implantation mutagenization treatment of the prepared protoplast suspension according to the following steps; Q5, performing genome shuffling treatment by using bacterial colonies respectively treated in Q3 step and cultured in Q4 step as parents with different genetic backgrounds; Q6, culturing heterozygous regenerated offspring mycelia; Q7, preparing heterozygous offspring protoplast and performing protoplast fusion; Q8, performing multi-time fusion and regeneration of offspring protoplast, that is, performing multi-time repeated fusion and of offspring according to Q1-Q7, and screening to obtain a mutant strain with greatly increased daunorubicin yield.

Description

technical field [0001] The invention relates to the technical field of bioengineering and pharmacy, in particular to a strain of daunorubicin hydrochloride and a high-yield method thereof. Background technique [0002] Anthracyclines (Anthracyclines) are an important class of anti-tumor drugs, they are all composed of anthracyclone chromophores linked with one or several different sugars through glycosidic bonds. Most anthracycline antibiotics have strong bone marrow suppression and cardiotoxicity and cannot be used clinically. Only daunorubicin-doxorubicin-type anthracycline antibiotics have become the most clinically applicable antitumor drugs, including daunorubicin-doxorubicin. Erythromycin, Adriamycin, Epirubicin, Carmine etc. Daunorubicin is mainly used clinically to treat acute leukemia. For acute lymphocytic leukemia and acute myelogenous leukemia, daunorubicin can be used as the drug of choice. Daunorubicin can also treat lymphosarcoma and neuroblastoma , Rhabdomy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/01C12N13/00C12N15/03C12R1/465C12R1/545
Inventor 余宏徐超波陈欢斌王波姚定余
Owner JIANGSU HECHANG BIOLOGICAL TECH
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