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Biosynthetic gene cluster of pentostatin and arabinofuranosyladenine and application of biosynthetic gene cluster

A kind of arabinoside biological and pentostatin technology, applied in the field of microbial gene resources and genetic engineering, can solve the problem of little understanding of biosynthesis and so on

Active Publication Date: 2017-05-24
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although adenosine vidarabine and pentostatin have been widely used clinically and good research progress has been made in chemical synthesis, the biosynthesis of these two purine nucleoside antibiotics remains unclear in the past few decades. little is known

Method used

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  • Biosynthetic gene cluster of pentostatin and arabinofuranosyladenine and application of biosynthetic gene cluster
  • Biosynthetic gene cluster of pentostatin and arabinofuranosyladenine and application of biosynthetic gene cluster
  • Biosynthetic gene cluster of pentostatin and arabinofuranosyladenine and application of biosynthetic gene cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] [Example 1] Extraction of total DNA of pentostatin and vidarabine-producing bacteria Streptomyces NRRL 3238 total DNA:

[0060] Take 30 μL of Streptomyces NRRL 3238 spores into 50 mL of TSBY medium, culture at 30°C and 200 rpm for 24-36 hours until the medium becomes turbid. 50 mL of Streptomyces NRRL 3238 bacteria solution was centrifuged at 4000 rpm at 4°C for 10 min to remove the supernatant and collect the bacteria. Dissolve the bacteria in 25mL of 10.3% sucrose solution, oscillate and wash the bacteria, centrifuge at 4000rmp, 4°C, 10min to remove the supernatant; then dissolve the bacteria in 15mL set buffer, shake and mix, 4000rmp, 4°C, Centrifuge for 10 minutes to remove the supernatant, and repeat twice; dissolve the bacteria in 10 mL of set buffer, shake and mix well, add 50 μL of lysozyme solution (100 mg / mL) and place it in a 37°C water bath for 30 minutes; then add 280 μL of protease K solution (50mg / mL), after mixing, add 600μL of 10% SDS, mix it upside do...

Embodiment 2

[0061][Example 2] Establishment of Genomic Library of Streptomyces NRRL 3238 Genomic Library of Pentostatin and Vidarabine Producing Bacteria

[0062] First, determine the amount of Sau 3AI through a series of dilution experiments, 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 mix 100 μL DNA 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% agarose gel with a length of ...

Embodiment 3

[0068] [Example 3] Fermentation conditions of pentostatin and vidarabine producing bacteria Streptomyces NRRL 3238, product high performance liquid phase (HPLC) detection conditions

[0069] The spores of Streptomyces NRRL 3238 were inoculated into the seed medium, cultured at 28°C, 220rmp for 48h, transferred to a fermentation shaker flask at 4% inoculum size, and cultured at 28°C, 220rmp for 6 days. Collect the fermentation broth, centrifuge the fermentation broth at 12000rmp for 20min, take the supernatant and pass it through a 0.22μm microporous membrane for detection and analysis by HPLC and LC-MS.

[0070] 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 A was converted to 10% at 31min and maintained at this concentration for 45min, and phase A was converted to 95% at 46min and maintained for 65min. The flow rate is 0.5mL / min,...

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Abstract

The invention relates to cloning, sequencing, analysis and functional study of a biosynthetic gene cluster of a natural product pentostatin which is produced from streptomyces antibioticus and is used for treating lymphocytic leukemia and a natural product arabinofuranosyladenine for resisting Koi herpes virus and varicella-zoster virus and application of the biosynthetic gene cluster. Biosynthetic genes of the two compounds are included in the same gene cluster and are independent from each other. The whole gene cluster comprises ten genes, namely three genes related to pentostatin synthesis, five genes related to arabinofuranosyladenine synthesis and two genes related to transportation regulation of the pentostatin and arabinofuranosyladenine. According to genetic manipulations of the previous biosynthetic genes, biosynthesis of the pentostatin or arabinofuranosyladenine can be blocked or improved. The genes provided by the invention and proteins thereof can be used for genetic engineering, protein expression, enzymic catalytic reaction and the like of the compounds, and can be further used for searching and discovering compounds or genes and proteins which can be used 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 the cloning, sequence analysis, and gene cluster biosynthesis of nucleoside antibiotics Pentostatin (PTN) and arabinofuranosyladenine (Ara-A). In vivo functional verification, in vitro biochemical studies and their applications. Background technique [0002] In 1967, scientists isolated the purine nucleoside antibiotic vidarabine for the first time from the fermentation broth of Streptomyces NRRL 3238 (S.antibioticus NRRL 3238) (Biochemistry, 1972, 11, 911-916). Adenosine vidarabine has a wide range of anti-DNA virus activity, and has been used in the treatment of herpes simplex encephalitis, neonatal herpes, herpes zoster and chronic myelogenous leukemia (Biochemistry, 1972, 11, 911-916; Trends Pharmacol Sci, 2010, 31, 255-265). Since adenosine vidarabine is easily transformed into arabinofuransylhypoxanthine (Ara-I) by adenosine de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/52C12N15/53C12N15/54C12N15/55C12N15/76C12N9/04C12P19/38C12P19/40C12R1/48
Inventor 陈文青巫攀邓子新万丹徐顾丹
Owner WUHAN UNIV
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