A kind of separation and purification method of bleomycin derivatives

A technology of bleomycin family and purification method, which is applied in the fields of peptide preparation methods, chemical instruments and methods, organic chemistry, etc., and can solve problems such as inability to meet clinical research industrial production, complex molecular structure, and lack of activity

Active Publication Date: 2017-10-20
CHANGSHA CHARISM BIOSCIENCES CO LTD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the molecular structure of bleomycin compounds is relatively complex, the process of obtaining related derivatives through traditional chemical total synthesis methods is cumbersome and inefficient, which cannot meet the needs of clinical research and the application of industrial production. The bleomycin derivatives also did not show better activity or lower toxicity

Method used

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  • A kind of separation and purification method of bleomycin derivatives
  • A kind of separation and purification method of bleomycin derivatives
  • A kind of separation and purification method of bleomycin derivatives

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Experimental program
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Embodiment 1

[0056] Both Streptomyces chrysogenum SB9001 and the genetically engineered mutant strain obtained in the experiment were cultured on ISP4 agar solid medium at 30°C to obtain spores. Streptomyces verticillium ATCC15003 was grown in TSBY liquid medium containing 0.5% glycine at 28°C and 250 rpm to obtain genomic DNA. Escherichia coli was grown in LB liquid medium at 37°C and 250rpm for daily plasmid cloning and establishment of BAC library.

[0057] 1) Construction of Streptomyces chrysanthemum SB9025 mutant strain

[0058]A 10kb DNA fragment containing the complete zbmVIII gene was isolated from the cosmid containing a part of the zobermycin biosynthetic gene cluster zbm, and cloned into the Litmus 28 plasmid through BamHI and BglII restriction sites superior. The resulting plasmid was digested with SbfI to remove the insert fragment with a size of about 3.7 kb, and then the plasmid was self-ligated to complete the knockout of the zbmVIII gene. The modified DNA insert was di...

Embodiment 2

[0065] Separation, purification and detection of novel bleomycin analogs BLM S (Tianci 102, compound 1) and 6'-dehydroxy-BLM S (Tianci 103, compound 2)

[0066] The supernatant solution obtained after the fermentation broth was centrifuged was detected and analyzed by high-performance liquid phase (HPLC). The mobile phase A was 99.9% deionized water and 0.1% acetic acid, and the mobile phase B was 99.9% methanol and 0.1% acetic acid. The flow rate was 0.8mL / min, the UV detector wavelength is 300nm. The linear gradient analysis program was: 0-30 minutes, 100%A / 0%B to 0%A / 100%B. The rest of the supernatant was adjusted to pH 7.0 with 1N hydrochloric acid and loaded into Amberlite IRC50 (NH 4 + type) resin column, washed with 10 times column volume of deionized water, and then eluted with 2 liters of 20% ammonium acetate solution. The resulting eluate was mixed with Diaion HP-20 resin and gently shaken at room temperature for 45 minutes for adsorption, then the HP-20 resin w...

Embodiment 3

[0080] BLM S (Tianci 102, compound 1) and 6'-dehydroxy-BLM S (Tianci 103, compound 2) DNA cleavage activity test

[0081] in Fe 2+ In the presence of conditions, respectively for BLM S, 6'-dehydroxy-BLM S and bleomycin A 2 (BLM A 2 ) to test and compare their biological activity. Based on analysis of plasmid relaxation activity of supercoiled plasmid DNA pBluescript II SK(+), bleomycin family compound-mediated single-strand cleavage first converts supercoiled plasmid DNA (form A) into open circular plasmid DNA (form B) , followed by double-strand cleavage to convert it to linear plasmid DNA (form C). The total reaction volume of the DNA cleavage activity test experiment was 10 μl, which contained 25 mM Tris-HCl buffer (pH 7.5), about 0.65 μg of pBluescript SKII(+) plasmid DNA, 5 μM Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O (in 1mM H 2 SO 4 freshly prepared in solution) and a certain concentration of active compounds to be tested (BLM S, 6'-dehydroxy-BLM S and BLM A 2 ). After ...

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Abstract

The invention relates to the technical field of biomedicine, in particular to a method for isolating and purifying the bleomycin derivative 6'-dehydroxy-BLM S from the fermentation broth of Streptomyces chrysanthemum recombinant engineered bacteria SB9026 (preservation number CCTCC M2011292). The method includes steps such as precipitation separation pretreatment, D‑113 and Diaion HP‑20 resin adsorption elution, Silica Flash column equal gradient elution, copper removal and purification. The method simplifies and reduces separation and purification steps, reduces production cost and increases recovery rate. The general chemical structure formula of the bleomycin family derivatives of the present invention is as shown in the description.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a method for efficiently separating and purifying a novel active bleomycin derivative 6'-dehydroxyl-BLMS from the fermentation liquid of Streptomyces chrysogenum recombinant engineered bacteria SB9026. Background technique [0002] Tumor is one of the serious diseases that endanger human health. At present, there are about 14 million malignant tumor patients in the world. In my country, the annual incidence of cancer is about 1.8 million to 2 million, and the death rate is 1.4 million to 1.5 million. For every 5 deaths among Chinese residents, 1 person died of cancer. The World Health Organization predicts that by 2020, the number of new cancer patients will reach 15 million each year. Cancer has become the number one killer of human beings in the new century and has become the largest public health problem in the world. [0003] Bleomycin (abbreviated as BLM) is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K9/00C07K1/36C07K1/18
Inventor 段燕文沈奔朱湘成杨虎
Owner CHANGSHA CHARISM BIOSCIENCES CO LTD
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