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Polyene macrolides compound, preparation and application thereof

A kind of macrolide and compound technology, applied in polyene macrolide compound and its preparation and application fields

Inactive Publication Date: 2010-05-19
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, except for Paenibacillus polymyxa, new compounds produced by other Paenibacillus species with clear structures have rarely been reported, so Paenibacillus species, especially the new species resources, may be new secondary metabolites an important source of

Method used

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  • Polyene macrolides compound, preparation and application thereof
  • Polyene macrolides compound, preparation and application thereof
  • Polyene macrolides compound, preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Isolation of Paenibacillus F6-B70

[0061] 1. Collect soil samples at an altitude of 400-1500 meters, and use the dilution coating method to isolate bacterial strains. The specific steps are: weigh 5g of soil samples and add them to a 250ml Erlenmeyer flask filled with 45ml of sterile water, and mix well. Use a pipette to take 1ml and add it to a test tube filled with 9ml sterile water, mix well, and record it as 10 -1 ; take 10 -1 Add 1ml of the solution into a test tube filled with 9ml of sterile water, mix well, and record it as 10 -2 ; Repeatedly diluted in turn until 10 -8 . Take 0.1ml of each dilution gradient and spread evenly on the nutrient agar plate.

[0062] 2. Place the plate at 30°C for 3 days;

[0063] 3. Pick a single colony, dilute it in the same medium and spread it on a plate for culture at 30°C. After repeated separation and purification for three times, check under the microscope and confirm that it is a pure species, pick a purebred...

Embodiment 2

[0065] Embodiment 2: the extraction of Paenibacillus F6-B70 genome

[0066] Referring to the "Guidelines for Molecular Biology Experiments", the details are as follows: the Paenibacillus F6-B70 bacteria obtained in Example 1 were cultured to the late logarithmic growth stage, and 1.5 ml was taken and centrifuged for 2 minutes. Add 567ul of TE buffer, 30ul of 10% SDS and 3ul of 20mg / ml proteinase K to the precipitate, mix well, and incubate at 37°C for 1h. Add 100ul 5mol / L NaCl, 80ul CTAB / NaCl solution (10%CTAB, 0.7M NaCl; preparation method: dissolve 4.1gNaCL in 80ml water, slowly add 10gCTAB, heat to dissolve), mix well, and incubate at 65°C for 1h. Add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1, v / v / v), mix well, centrifuge for 5 min, transfer the supernatant to a new tube, and repeat this step once. Add 0.6 volume of isopropanol, mix gently until the DNA precipitates, transfer the precipitate to 1 ml of 70% ethanol with a sealed Pasteur tube and wash. C...

Embodiment 3

[0067] Embodiment 3: PCR amplification and sequencing of Paenibacillus F6-B70 strain 16S rDNA

[0068]The primers for PCR amplification of 16S rDNA fragments use E8F: 5-AGAGTTTGATCCTGGCTCAG-3 and E1541R: 5-AAGGAGGTGATCCAGCCGCA-3, which can amplify fragments with a length of about 1500bp; the template is the Paenibacillus F6-B70 genome obtained according to Example 2 . Through the optimization of PCR reaction conditions, the optimal PCR conditions and procedures for amplifying 16S rDNA were finally determined: PCR reaction system 20 μl, the specific composition is as follows:

[0069] dNTP (10mM) 2μl

[0070] E8F (20μM) 0.5μl

[0071] E1541R (20μM) 0.5μl

[0072] rTaq (takara) 1.25U

[0073] Template 10ng

[0074] wxya 2 O 12μl

[0075] 10×buffer (for TaKaRa-Taq PCR) 2μl

[0076] The reaction program is: 95°C for 5 min; 95°C for 1 min, 58°C for 1 min, 72°C for 2 min, 30 cycles; 72°C for 12 min.

[0077] The PCR amplification product was purified by electrophoresis firs...

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Abstract

The invention provides a new compound having medicinal value, a preparation method thereof and a new bacterial strain which can generate the compound. The compound with formula (I) is chosen and prepared by rapidly choosing bacillus from soil sample with specific primer, using 16Ss rRNA gene to preliminarily identify and separate bacterial strain, amplifying, sequencing and comparing partial sequence of NRPSs / PKSs gene cluster after choosing potential new resources, analyzing the diversity of metabolite thereof, acquiring bacterial strain F6-B70 which contains new NRPSs / PKSs gene cluster and has new potential after choosing a large amount, fermenting in a fermentation medium, extracting active substance from bacterial cell and fermenting liquid, and then separating and purifying by using multiple purification techniques. The compound is a polyene macrolides compound which has an obvious function of restraining drug-resisting staphylococcus and an activity of resisting tumor.

Description

(1) Technical field [0001] The invention relates to a polyene macrolide compound and its preparation and application. (2) Background technology [0002] Clinically, the increasingly serious problem of drug resistance of pathogenic microorganisms and the emergence of new diseases such as AIDS, SARS, and avian flu have made research and development of new drugs more urgent and important. Since the discovery of penicillin by Fleming, microbial secondary metabolites have gradually been recognized as a classic source of therapeutic drugs with novel chemical backbones. In the 1940s and 1960s, a variety of important antibiotics were successively discovered from actinomycetes, especially streptomyces, making actinomycetes a particularly concerned group of microorganisms for screening new antibiotics in the following decades. Most of the currently marketed antibiotics are secondary metabolites and their derivatives produced by actinomycetes. With the large-scale screening of strain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D313/00C12P17/08A61K31/365A61P31/04A61P35/00C12R1/01
Inventor 吴雪昌钱朝东李欧方海环周建英白骅杜加秋林建秋
Owner ZHEJIANG UNIV
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