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Application of ciona intestinalis linnaeus peptides

A technology of ascidian and B liquid, which is applied in the preparation method of peptide, medical preparations containing active ingredients, peptides, etc., to achieve the effects of novel structure, low drug resistance and prolonging 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

A 13-amino acid cyclic peptide found in two species of ascidian Didemnum cuculiferum and Polysyncranton lithostrotum significantly inhibited microtubule polymerization and prolonged the survival of p388 lymphocytic leukemia in mice; but isolated from Ciona intestinalis Anti-tumor protein has not been reported

Method used

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  • Application of ciona intestinalis linnaeus peptides
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  • Application of ciona intestinalis linnaeus peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation method of Ciona polypeptide:

[0028] 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 the supernatant, add the volume of the supernatant to the supernatant Three times of acetone with a volume percentage of 70% was left standing overnight at 4°C; after standing overnight, it was centrifuged at 1000 rpm for 20 minutes to collect the precipitate. After the precipitate was redissolved, it was filtered through a 5KDa membrane for ultrafiltration, and the part larger than 5KDa was retained for use.

[0029] 2) DEAE Sepharose TM Fast Flow Anion Exchange Column Chromatography:

[0030] 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 th...

Embodiment 2

[0039] Embodiment 2: the determination of antitumor spectrum

[0040] 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.

[0041] 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).

[0042] Instruments: ultra-low temperature refrigerator (Nature, USA), cell culture incubat...

Embodiment 3

[0048] Example 3: Observation of apoptosis by scanning electron microscope

[0049] Use a six-well plate to make cell slides → incubate at 37°C for 24 hours → add Ciona protein extract with a final concentration of 100ug / ml prepared in the above example → incubate at 37°C for 20 hours → wash with PBS → fix with 2.5% glutaraldehyde → 30 %, 50%, 70%, 80%, 90%, 100% ethanol gradient dehydration→observe under scanning electron microscope.

[0050] Scanning electron microscope to observe the external morphology of apoptotic cells: Observe BEL-7402-treated cells under a scanning electron microscope. Compared with normal cells, the microvilli on the surface of the ascidian polypeptide-treated cells prepared in the above examples are reduced or disappeared, and the cells become round and shrunk. Honeycomb-shaped cavities appear on the surface, and apoptotic bodies can be found in a few cells (see Figure 3).

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Abstract

The invention belongs to the fields of biochemistry and biomedicines, in particular to application of ciona intestinalis linnaeus peptides in preparation of antineoplastic medicaments. In vitro methods such as MTT assay, scanning electron microscopy, nucleus staining, mitochondrial membrane potential changing and the like prove that the ciona intestinalis linnaeus peptides have a significant antineoplastic effect and can induce apoptosis of tumor cells. The induction mechanism of the ciona intestinalis linnaeus peptides is based on apoptosis, which indicates novel functions and means of cionaintestinalis linnaeus peptides in prevention and treatment of tumors.

Description

technical field [0001] The invention belongs to the field of biochemistry and biomedicine, and specifically relates to the application of Ciona polypeptide. Background technique [0002] Malignant tumors are the number one killer of human health, and their morbidity and mortality rates rank first among all diseases. The research and development of anticancer drugs has become a major topic of new drug research in this century. The isolation and purification of antitumor active substances from the ocean has become one of the hot spots in the research of anticancer drugs. In the process of screening anti-tumor drugs, we found that a polypeptide (PI5931) isolated and purified from Ciona has a strong inhibitory effect on tumor cell growth, and has great potential for developing into new anti-cancer drugs. [0003] Sea squirts (Ascidian) belong to the phylum Chordates, the subphylum Urochordata, and the class Ascidiacea, and there are about 1,600 species in the world. Many cycl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17C07K1/36A61P35/00
Inventor 林秀坤程林友刘明吴宁
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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