Marine bacillus polypeptide and preparation and application thereof

A marine bacillus, fermentation culture technology, applied to marine bacillus polypeptides and the fields of preparation and application thereof, can solve the problems of low content, difficult drug sources, difficult large-scale preparation and the like

Inactive Publication Date: 2015-05-13
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the vast majority of anti-tumor lead compounds have low content in the body, and it is difficult to prepare the

Method used

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  • Marine bacillus polypeptide and preparation and application thereof
  • Marine bacillus polypeptide and preparation and application thereof
  • Marine bacillus polypeptide and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The fermentation culture of embodiment 1 marine bacillus N11-8

[0056] The marine bacillus N11-8 (the Latin name is Bacillus sp.N11-8, was preserved in the China Center for Type Culture Collection on November 15, 2012, the preservation address: Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2012459 .) connected in the test tube containing 3mL seed culture solution, seed culture solution: beef extract 3g / L, peptone 10g / L, NaCl 5g / L, the above is the final concentration of each component, pH7.5. Place in a 30°C, 200rmp constant temperature shaking incubator and cultivate overnight; make a gradient dilution coating plate, solid medium: beef extract 3g / L, peptone 10g / L, NaCl 5g / L, agar 2g / L, the above are the components final concentration, pH 7.5. Place the coated plate in a constant temperature incubator at 30°C and incubate it upside down for 18-24 hours; pick a single colony and inoculate it in a 3mL test tube seed culture solution, and cul...

Embodiment 2

[0057] The preparation method of embodiment 2 marine bacillus polypeptide

[0058] (1) ultrafiltration

[0059] 60L of the collected fermentation supernatant was concentrated by ultrafiltration. Then use ultrafiltration membranes with a molecular weight of 3kDa and 30kDa to obtain different components with a molecular weight of less than 3kDa, 3-30kDa and greater than 30kDa, and detect the anti-tumor activity of each component. The results show that the components with a molecular weight of 3-30kDa and greater than 30kDa All have anti-tumor activity, and the anti-tumor activity of the 3-30kDa component is the best.

[0060] (2) Ammonium sulfate precipitation

[0061]Slowly add ammonium sulfate powder to the 3-30kDa component, stir gently to make the concentration reach 50% (mass percentage concentration), control the temperature at 4°C during the period, and let it stand in the refrigerator for 2h after it is completely dissolved; at 10000rpm, 30min Centrifuge under the con...

Embodiment 3

[0068] Molecular weight and N-terminal amino acid sequence of embodiment 3PBN11-8

[0069] (1) Determination of metabolites and PBN11-8 concentration

[0070] The concentration of the polypeptide was determined by the BCA method (quinic acid), using the BCA Protein Assay Kit, and operating according to the instructions.

[0071] (2) Electrophoretic pure detection of sample purity and molecular weight

[0072] Using the Laemmli method, SDS-PAGE discontinuous gel electrophoresis, the concentration of the stacking gel is 5%, and the concentration of the separating gel is 12%. The fermentation concentrate, the ammonium sulfate precipitation is less than 45%, the breakthrough peak Q2 and PBN11-8 are subjected to SDS- PAGE gel electrophoresis, such as image 3 , lane 1 in the illustration is Marker, lane 2 is fermentation concentrate, lane 3 is the sample with less than 45% ammonium sulfate precipitation, lane 4 is the breakthrough peak Q2 sample and lane 5 is PBN11-8; as shown by...

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Abstract

The invention discloses a marine bacillus polypeptide and a preparation and an application thereof and belongs to the technical field of marine microorganism medicines. The marine bacillus polypeptide is from metabolites of antarctic marine bacillus N11-8 subjected to fermentation cultivation; 15 amino acid sequences at N-ends of the active polypeptide are ASTGSQKVTVYAVAD; the active polypeptide has relatively high cytotoxicity to a plurality of tumor cells such as human hepatoma carcinoma cell BEL-7402, human ovarian carcinoma cells NIH:OVCAR-3, human renal clear cell carcinoma cells 786-0 and human large cell lung cancer cells NCI-H460, and has relatively good research and application value. The polypeptide can be applied to medicines for preventing and/or treating the cancers.

Description

technical field [0001] The invention belongs to the technical field of marine microbial medicine, and in particular relates to a marine bacillus polypeptide and its preparation and application. Background technique [0002] Malignant tumor is a major disease that seriously endangers human health and is one of the main causes of population death in my country. In 2005, about 7.5 million people died of malignant tumors in the world, and about 1.3 million people died of cancer in my country every year, and the incidence rate is increasing year by year. Upward trend. Drug therapy is an important part of comprehensive therapy for malignant tumors. Classical cytotoxic antineoplastic drugs are the main means of chemotherapy at present, and will still occupy a dominant position for a long time in the future. Therefore, looking for anti-tumor active ingredients with new structure types or new mechanisms of action from natural organisms is a new trend in current anti-tumor drug resear...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12P21/02C07K1/36C07K1/34C07K1/30C07K1/18C07K1/16A61K38/10A61P35/00C12R1/07
Inventor 郑兰红杨康利孙谧吕梅康道乐郑媛
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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