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Biosynthesis gene cluster of 2'-chloropentostatin and 2'-amino-2'-deoxyadenosine and application thereof

A technology of chlorpentastatin and deoxyadenosine, which is applied in the field of microbial genetic resources and genetic engineering, can solve the problems of little knowledge about the biosynthesis of purine nucleoside antibiotics

Active Publication Date: 2017-05-17
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although 2'-chloropentastatin and 2'-amino-2'-deoxyadenosine have been widely used clinically and have good research progress in chemical synthesis, in the past few decades, the Biosynthesis of two purine nucleoside antibiotics remains poorly understood

Method used

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  • Biosynthesis gene cluster of 2'-chloropentostatin and 2'-amino-2'-deoxyadenosine and application thereof
  • Biosynthesis gene cluster of 2'-chloropentostatin and 2'-amino-2'-deoxyadenosine and application thereof
  • Biosynthesis gene cluster of 2'-chloropentostatin and 2'-amino-2'-deoxyadenosine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] [Example 1] Extraction of 2'-chloropentastatin and 2'-amino-2'-deoxyadenosine producing bacteria Actinomyces madura ATCC39365 total DNA:

[0061] Take 30 μL of Actinomyces madurai ATCC 39365 spores into 50 mL of TSB medium, culture at 30°C, 200 rpm for 24-36 hours, until the medium becomes turbid. 50 mL of Actinomyces madurai ATCC 39365 bacterial liquid was centrifuged at 4,000 rpm at 4°C for 10 min to remove the supernatant and collect the bacterial cells. Dissolve the bacteria in 25mL of 10.3% sucrose solution, oscillate and wash the bacteria, centrifuge at 4,000rpm, 4°C, 10min to remove the supernatant; then dissolve the bacteria in 15mL of set buffer, shake and mix, 4,000rpm, 4 ℃, 10min centrifugation to remove the supernatant, and repeat twice; dissolve the bacteria in 10mL set buffer, shake and mix well, add 50μL lysozyme solution (100mg / mL) and place it in a 37℃ water bath for 30min; then add 280 μL of proteinase K solution (50 mg / mL), after mixing, add 600 μL o...

Embodiment 2

[0062] [Example 2] The establishment of a genomic library of Actinomyces madurai, a 2'-chloropentastatin and 2'-amino-2'-deoxyadenosine producing bacterium.

[0063] First, through a series of dilution experiments to determine the amount of Sau 3AI, prepare a total volume of 500 μL enzyme digestion system (5 μL 0.1u / μL Sau 3AI, 495 μL 10Xbuffer diluted DNA) solution 1, take 300 μl solution 1 and 100 μL DNA and mix well To form solution 2 (Sau 3AI final concentration 0.075u / 100μL), take 200μL solution 1 and 200μL DNA and mix to form solution 3 (Sau 3AI final concentration 0.05u / 100μL), take 200μL solution 2 and 200μL DNA and mix to form solution 4 ( Sau 3AI final concentration 0.0375u / 100 μL), take 200 μL solution 4 and 200 μl DNA and mix to form solution 5 (Sau 3AI final concentration 0.01875u / 100 μL). Mix the above solutions evenly and put them on ice, then put them in a water bath at 37°C for 1 hour, take them out and put them on ice immediately, and electrophoresis on 1% ag...

Embodiment 3

[0069] [Example 3] Fermentation conditions of 2'-chloropentastatin and 2'-amino-2'-deoxyadenosine producing bacteria Actinomyces madura ATCC39365, product high performance liquid phase (HPLC) detection conditions.

[0070] The spores of Actinomyces madurai ATCC 39365 were inoculated in the seed medium, cultured at 30°C, 220rmp for 48h, transferred to a fermentation shaker flask according to the inoculum size of 4%, and cultivated at 32°C, 220rmp for 6 days. Collect the fermented liquid, centrifuge the fermented liquid at 9,000rpm for 20min, take the supernatant and extract it with an equal volume of n-butanol repeatedly for 3 times. After high-speed centrifugation, the supernatant was taken, and passed through a 0.22 μm microporous membrane for detection and analysis by HPLC.

[0071] HPLC detection conditions: phase A is ultrapure water added with 0.15% trifluoroacetic acid (TFA), and phase B is methanol. The initial 95% phase A gradient eluted to 80% within 30min, and phase...

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Abstract

The invention relates to cloning, sequencing, analysis and functional research of a biosynthesis gene cluster of 2'-chloropentostatin and 2'-amino-2'-deoxyadenosine and application thereof. The 2'-chloropentostatin is a natural product produced by actinomadura madurae ATCC 39365 and is used for treating leukemia; the 2'-amino-2'-deoxyadenosine is a natural product of RNA (ribonucleic acid) viruses of anti-mycoplasma virus, measles virus and the like. The whole gene cluster contains 13 genes, wherein five genes are related with synthesis of the 2'-chloropentostatin; four genes are related with synthesis of the 2-amino-2'-deoxyadenosine; four genes are related with transfer and regulation of the 2'-chloropentostatin and the 2-amino-2'-deoxyadenosine. The biosynthesis of the 2'-chloropentostatin and the 2-amino-2'-deoxyadenosine is blocked or improved by the genetic manipulation on the biosynthesis gene. The gene and the protein thereof can be applied to the gene engineering, protein expression, enzyme catalytic reaction and the like of the compound, and be used for looking and finding compounds, genes and proteins for medicines, industry or agriculture.

Description

technical field [0001] The invention belongs to the field of microbial genetic resources and genetic engineering, and specifically relates to nucleoside antibiotics 2'-chloropentastatin (2'-chloropentostatin, 2'-Cl PTN) and 2'-amino-2'-deoxyadenosine (2'-amino-2'-deoxyadenosine, 2'-amino dA) biosynthetic gene cluster cloning, sequence analysis, gene function verification in vivo, in vitro biochemical research and its application. Background technique [0002] In 1979, American scientists isolated the purine nucleoside antibiotic 2'-amino-2'-deoxyadenosine for the first time from the fermentation broth of Actinomadura sp. ATCC 39365 (JAntibiot (Tokyo), 1979 , 32, 1367-1369; Arch Biochem Biophys, 1989, 270, 374-382). 2'-amino-2'-deoxyadenosine has a wide range of anti-RNA virus activity, and has been used in the treatment of RNA viruses such as mycoplasma virus and measles (JAntibiot (Tokyo), 1979, 32, 1367-1369; Agr Biol Chem Tokyo, 1985, 49, 2711-2717). Since 2'-amino-2'-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/54C12N15/53C12N15/52C12N15/55C12N15/61C12N9/14C12P19/40C12P19/38C12R1/03
CPCC07K14/195C12N9/0006C12N9/1048C12N9/1096C12N9/14C12N9/90C12N9/93C12N15/52C12P19/38C12P19/40C12Y101/01184C12Y306/01C12Y503/0102C12Y603/02006
Inventor 陈文青高耀杰邓子新徐顾丹巫攀
Owner WUHAN UNIV
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