Rapamycin biosynthesis gene from actinoplanes, and separation method and application thereof

A technology of actinomycetes mobilis and rapamycin, which is applied in the field of genetic engineering and can solve problems such as unreported research

Inactive Publication Date: 2010-11-17
SHANGHAI INST OF PHARMA IND CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the rapamycin biosynthetic gene cluster of another strain of rapamycin high-yielding Actinomycetes mobilis discovered in 1995.

Method used

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  • Rapamycin biosynthesis gene from actinoplanes, and separation method and application thereof
  • Rapamycin biosynthesis gene from actinoplanes, and separation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Extraction of total DNA from rapamycin producing bacteria Actinomycetes mobilis

[0043] Source of bacteria

[0044]Actinomycetes mobilis is consistent with the literature source: Nishida, H., T. Sakakibara, et al. (1995). "Generation of novel rapamycin structures by microbial manipulations." J Antibiot (Tokyo) 48 (7): 657 -66.

[0045] Inoculate a small amount of actinomycete mycelium into a test tube containing 5ml YEME medium, 30°C, shake culture for about 72 hours, inoculate 5% to 50ml YEME medium, 30°C, shake culture for 24 hours, centrifuge at 3500rpm The cell phone mycelium was washed twice with lysate and stored at -20°C for the extraction of genomic DNA. Add 10ml of lysate (containing lysozyme 5mg / ml) to the mycelium of the mobile phone, vortex until uniform, bathe in 37°C water bath for 60 minutes, add 0.1ml proteinase K (4mg / ml), 1ml 10% SDS, mix well Quickly put it into a 55°C water bath for 60 minutes until the solution is clarified. Cool on ice, add 2....

Embodiment 2

[0047] Construction of Genomic Library of Rapamycin-producing Bacteria

[0048] Firstly, the total DNA of Actinomycetes mobilis obtained in Example 1 was mechanically disrupted to obtain a DNA fragment with a size of 20-40 kb, which was recovered. Escherichia coli EPI100 was streaked on a blank LB plate and cultivated overnight at 37°C. Pick EPI100 monoclonal and inoculate in 3mlLB (containing 10mM MgSO 4 ) in liquid medium, shake the bacteria at 37°C until OD 600 =0.8-1.0 and set aside at 4°C. The total DNA size of 20-30kb fragments were ligated to the fosmid vector overnight. Take out the packaging mixture (containing 50ul of packaged protein) from the -80°C refrigerator and place it in a dry ice bucket. Melt the packaged material immediately. Take out 25ul of the packaged protein and put it in a 1.5ml centrifuge tube and add 6ul of the ligated product just after it starts to melt. , and mix well in a 30°C water bath for 90 minutes. The remaining 25ul of packaged protei...

Embodiment 3

[0050] Nucleic acid molecular hybridization

[0051] 1. DIG DNA labeling: Dilute the DNA to be labeled with sterile water to a total volume of 15ul, heat and denature in boiling water for 10 minutes, and immediately cool in ice. Then add 2ul of primer mixture, 2ul of dNTP mixture, and 1ul of enzyme, mix well, and put in a 37°C water bath for about 16 hours. Add 0.8ul 0.8M EDTA (pH8.0) to terminate the reaction, add 2.5ul 4M LiCl and mix well, then add 75ul pre-cooled absolute ethanol to precipitate the labeled DNA, and place it at -80°C for 40 minutes. The DNA was collected by centrifugation at 12000 rpm for 20 minutes at 4°C, washed with pre-cooled 70% ethanol, and redissolved in 50ul TE (pH8.0) after vacuum drying.

[0052] 2. Quality detection after DIG DNA probe labeling: Dilute the labeled DNA probe to the following six gradients, 1, 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 . Dilute labeled control DNA to concentrations of 1 ug / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, 100 pg / ml,...

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Abstract

The invention discloses a Rapamycin biosynthesis gene from antinoplanes, and a separation method and application thereof. The gene is a nucleotide sequence shown by SEQ ID NO:1.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a rapamycin biosynthetic gene from Actinomycetes mobilis and its separation method and application. Background technique [0002] The development of immunosuppressants has greatly improved the success rate of organ transplantation. In the 1960s, Murray et al. applied azathioprine (Aza) to organ transplantation and achieved a series of successes in kidney transplantation. For this reason, azathioprine and prednisone (Prednisone, PRD) became the first generation of immunosuppressants. Due to disadvantages such as poor selectivity and high toxicity, the success rate of organ transplantation is affected. Cyclosporin A (Cyclosporin, CSA), an immunosuppressant derived from microorganisms, was discovered in 1976 and successfully applied to organ transplantation in 1978, opening up a new era of organ transplantation and greatly reducing the toxicity of immunosuppressants. side effect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/63C12N1/00C12N9/00C12Q1/68C12N15/10C12P17/18C12R1/045
Inventor 胡海峰郭铭朱宝泉
Owner SHANGHAI INST OF PHARMA IND CO LTD
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