Bleomycin derivative separating and purifying method

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 no activity, low toxicity, and low quality

Active Publication Date: 2014-08-27
CHANGSHA CHARISM BIOSCIENCES CO LTD +2
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  • Summary
  • 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

Method used

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  • Bleomycin derivative separating and purifying method
  • Bleomycin derivative separating and purifying method
  • Bleomycin derivative separating and purifying method

Examples

Experimental program
Comparison scheme
Effect test

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 250rpm to obtain genomic DNA. E. coli was grown in LB liquid medium at 37°C and 250rpm for routine plasmid cloning, BAC library establishment.

[0057] 1) Construction of Streptomyces aeruginosa 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 the 10kb DNA fragment was cloned into the Litmus28 plasmid through the BamHI and BglII restriction sites . The obtained plasmid was digested with SbfI to remove the insert fragment of about 3.7 kb size, and then the plasmid was self-ligated to complete the knockout of the zbmVIII gene. The modified DNA insert was digest...

Embodiment 2

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

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

Embodiment 3

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

[0081] in Fe 2+ In the presence of BLM S, 6'-dehydroxy-BLM S and bleomycin A, respectively 2 (BLM A 2 ) were tested to compare the biological activity. Based on the analysis of the plasmid relaxation activity of the supercoiled plasmid DNA pBluescript II SK(+), single-strand cleavage mediated by bleomycin family compounds firstly transformed the supercoiled plasmid DNA (form A) into an open-circular plasmid DNA (form B). , followed by double-strand cleavage into 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 pBluescriptSK II(+) plasmid DNA, 5 μM Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O (at 1 mM H 2 SO 4 freshly prepared in solution) and concentrations of the active compounds to be tested (BLM S, 6'-dehydroxy-BLM S, and BLM A 2 ). The s...

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Abstract

The invention relates to the technical field of biomedicines, and concretely relates to a method for separating and purifying bleomycin (BLM) derivatives 6'-dehydroxy-BLMs from a recombinant engineering bacterium Streptomyces flavovirens SB9026 (with the preservation number of CCTCC M2011292) fermentation liquid. The method comprises the steps of deposition separation pretreatment, D-113 and Diaion HP-20 resin adsorption elution, Silica Flash column isogradient elution, copper removing refining and the like. The method has the advantages of simplification and reduction of the separating and purifying steps, production cost reduction and recovery rate increase. The chemical structure of the bleomycin derivatives is represented by a general formula shown in the specification.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for efficiently separating and purifying a novel active bleomycin family derivative 6'-dehydroxy-BLMS from the fermentation broth of Streptomyces chrysogenum recombinant engineering 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. The number of cancer cases in my country is about 1.8 million to 2 million each year, and 1.4 million to 1.5 million die. For every 5 people who die in our country, 1 person will die 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 the largest public health problem in the world. [0003] Bleomycin (BLM for short) is a class of glyc...

Claims

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Application Information

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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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