Preparation method of actinomycin D and application thereof

A technology of actinomycin and actinomycetes, applied in the field of preparation of actinomycin D, to achieve the effect of broad-spectrum and high-efficiency antagonistic activity

Active Publication Date: 2015-03-25
TARIM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently registered antibiotics for the prevention and treatment of Verticillium wilt have not been reported

Method used

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  • Preparation method of actinomycin D and application thereof
  • Preparation method of actinomycin D and application thereof
  • Preparation method of actinomycin D and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The source and isolation culture of embodiment one Streptomyces mutans TRM45540

[0022] From the 0-30cm soil samples in the ancient city of Loulan, Xinjiang (1400m above sea level, 89 ° 22' 22 " in east diameter, 40 ° 29' 55 " in north latitude), adopt the coating plate method, with 5%, 10%, 15%, 20 % and 25% sodium chloride in 8 kinds of media, 28 ℃ constant temperature culture, a total of 35 strains of actinomycetes of 7 genera were isolated, and finally separated and screened out The biocontrol bacteria TRM45540 with the best antibacterial effect.

Embodiment 2

[0023] The identification of embodiment two Streptomyces mutans TRM45540

[0024] In order to further confirm the taxonomic status of the biocontrol bacteria TRM45540, the present invention uses traditional classification methods and molecular classification methods to classify and identify the strain.

[0025] 1 Culture medium for morphology observation

[0026] Inorganic salt starch agar (ISP4) medium: starch 10g L -1 ,(NH 4 ) 2 SO 4 4g·L -1 ,MgSO 4 2g·L -1 ,K 2 HPO 4 2g·L -1 ,CaCO 3 1g·L -1 ,NaCl 40g·L -1 , agar 18g·L -1 , pH 7.2-7.4.

[0027] 2 primers

[0028] Synthesized by Aoke Biotechnology (Beijing) Co., Ltd. using actinomycetes universal primers. The sequences are: 27F (5'-AGAGTTTGATCCTGGCTC-3') and 1492R (5'-CGGCTACCTTGTTACGACTT-3')

[0029] 3 test method

[0030] 3.1 Morphological observation

[0031] Ordinary microscope observation: using the insert method, cultivate the biocontrol bacteria TRM45540 at 28°C. Use an optical microscope to observe...

Embodiment 3

[0052] Example 3 Utilize Streptomyces mutans TRM45540 to ferment and prepare Actinomycin D

[0053] Inoculate a small amount of mycelium in the ISP4 medium, cultivate at 28°C for 4 days, and obtain the seed liquid of Streptomyces vulbilis TRM45540, take 2mL (8% inoculum size) of the seed liquid and inoculate it in the corn flour fermentation medium for fermentation and cultivation. The fermentation conditions It is: rotation speed 180rpm, initial pH 7.2-7.4, liquid volume 250mL, fermentation temperature 28°C, fermentation 7d. Collect the fermentation broth, filter the fermentation broth with four layers of gauze, and load the sample to the macroporous resin chromatographic column by wet method (fill 2.0kg of D101 or AB-8 macroporous resin per 100L of fermentation filtrate); then use 2 times the column volume of water, Eluted with 10% ethanol solution, 30% ethanol solution, 50% ethanol solution, 70% ethanol solution, 95% ethanol solution; combined 70-95% ethanol eluate, concent...

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Abstract

The invention discloses a novel actinomycin strain which generates actinomycin D: Streptomycesmutabilis TRM45540, preserved in U. S. Department of Agriculture Agricultural Research Service (ABS) Culture Collection on 14th, April, 2014 with the preservation number of NRRL B-59117. Meanwhile, the invention further discloses a seed medium which efficiently produces actinomycin D by using the strain, a fermentation medium and a preparation process of extracting and separating actinomycin D from fermentation liquor. An antibacterial activity test shows that the strain and the actinomycin D generated by fermentation of the strain have broad-spectrum and efficient antagonistic activity on pathogenic bacteria such as cotton verticillium and fusarium wilts, walnut pythium ultimum, staphylococcus aureus, staphylococcus epidermidis, candida albicans and the like and have an important application value and a very good application and development prospect on safe production of crops such as cotton, walnuts and the like and in preventing and controlling diseases such as bovine mastitis, women's vaginitis and the like.

Description

technical field [0001] The invention relates to the fields of agriculture and medicine, in particular to a new application of actinomycin D, a new bacterial strain capable of producing actinomycin D and a preparation method for producing actinomycin D by using the bacterial strain. Background technique [0002] The two strains, Smelanochromogenes No.1779 and Streptomyces parvullus, can produce actinomycetes D. Their main mechanism of action is to hinder the function of RNA polymerase and inhibit the synthesis of RNA, especially mRNA, thereby hindering protein synthesis and inhibiting tumor growth. Combined use of radiation therapy can improve the sensitivity of tumors to radiation therapy. However, the effect of actinomycin D on plant pathogens of cotton Fusarium wilt, walnut pythium and the pathogenic bacteria of women's vaginitis, Candida albicans, has not been reported yet. [0003] Cotton fusarium wilt and cotton verticillium wilt are the most important diseases that har...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P21/02A01P3/00A61P31/10A61P31/04C12R1/465
CPCC12P21/02C12N1/205C12R2001/465
Inventor 张利莉万传星罗晓霞吕玲玲仼晓镤夏占峰
Owner TARIM UNIV
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