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Ciona intestinalis polypeptide and preparation method thereof

A Ciona and B solution technology, applied in the field of Ciona polypeptide and its preparation, to achieve the effects of novel structure, low drug resistance, and prolonged shelf life

Active Publication Date: 2011-09-07
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been no reports on the separation and purification of polypeptides using Ciona as raw materials for the prevention and treatment of tumor diseases

Method used

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  • Ciona intestinalis polypeptide and preparation method thereof
  • Ciona intestinalis polypeptide and preparation method thereof
  • Ciona intestinalis polypeptide and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] The preparation method of embodiment 1 Ciona polypeptide:

[0024] 1) Preparation of crude extract by acetone precipitation: take fresh ascidian, wash, crush, disperse and homogenize with a homogenizer, heat at 70°C for 30 minutes, and then centrifuge at 10,000 rpm for 20 minutes to obtain supernatant, and add supernatant to the supernatant Three times the volume of acetone to obtain a 70% acetone solution by volume. Stand overnight at 4°C; centrifuge at 10,000 rpm for 20 minutes after standing overnight, take the precipitate, redissolve the precipitate and pass it through a 5KDa filter membrane for ultrafiltration interception, and keep the part larger than 5KDa for use.

[0025] 2) DEAE Sepharose TM Fast Flow Anion Exchange Column Chromatography:

[0026] First, put the crude extract into a dialysis bag with a molecular cut-off of 3.5kD, perform preliminary dialysis with pure water, and then fully dialyze it against the initial buffer solution; take out the dialyzed...

Embodiment 2

[0035] The mensuration of embodiment 2 antitumor spectrum:

[0036]Cells: human cervical cancer epithelial cells (Hela), human liver cancer cells (BEL-7402), human colon cancer cells (HCT-116), human breast cancer cells (MCF-7), human lung cancer cells (A549), human glia Glioma cells (U8T). The above cells were provided by the Marine Drug Laboratory of the Institute of Oceanography, Chinese Academy of Sciences.

[0037] Drugs and reagents: Ciona polypeptide: provided by the Marine Drug Laboratory of the Institute of Oceanography, Chinese Academy of Sciences; 5-fluorouracil (5-FU), a product of Jiangsu Nantong Jingjing Pharmaceutical Co., Ltd., batch number 100402; MTT (SIGMA, USA); DMSO (SIGMA, USA); trypsin (SIGMA, USA); RPMI1640 (GIBCO, Invitragen Co., USA); DMEM (GIBCO, Invitragen Co., USA); premium fetal bovine serum (GIBCO, Invitragen Co., USA); Fetal Bovine Serum (Hyclone).

[0038] Instruments: ultra-low temperature refrigerator (Nature, USA), cell culture incubator ...

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Abstract

The invention belongs to the filed of pharmaceutical sciences and particularly relates to ciona intestinalis polypeptide and a preparation method thereof. The ciona intestinalis polypeptide has the N- terminal sequence of YQPDKFMLRGELRNN, the molecular weight of 5931Da and the isoelectric point of 7.25. The preparation method of the ciona intestinalis polypeptide comprises the following steps: precipitating fresh ciona intestinalis by using 10-90% acetone, dialyzing and desalting active components, and purifying by adopting ion-exchange chromatography, gel filtration chromatography and HPLC (high-performance liquid chromatography) to obtain the ciona intestinalis polypeptide which has the molecular weight of 5931Da. The in vivo and in vitro activity experiments verify and confirm that theapplication of the ciona intestinalis polypeptide to malignant tumor prevention and treatment.

Description

technical field [0001] The invention relates to the field of pharmaceutical sciences, in particular to a Ciona polypeptide and its preparation. Background technique [0002] Marine drugs are currently a hot spot in drug research, among which the research results of anti-tumor drugs are the most eye-catching. Scientists from various countries have isolated and purified a variety of anti-tumor compounds with novel structures and specific activities from marine animals and plants. However, the content of active substances in marine animals and plants is low, and the separation and purification are difficult, which is a major problem in the separation and purification of marine anti-tumor active substances. To separate and obtain a highly active compound requires a large amount of raw materials, a reasonable test route and advanced separation and purification equipment. So far, there have been no reports on the separation and purification of polypeptides using Ciona as raw mate...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/36C07K1/34C07K1/30C07K1/16A61P35/00
Inventor 林秀坤程林友魏建腾郑兰红王翠翠
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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