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A clam and buffer technology, which is applied in the field of clam polypeptide and its preparation, can solve the problems of prolonged survival time, low treatment efficiency, and drug resistance of tumor cells, and achieve the effects of small molecular weight, no immunogenicity, and good application prospects
Active Publication Date: 2011-08-24
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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At present, tumor chemotherapy technology has been improved, and the survival time of tumor patients has been significantly prolonged. In particular, breakthroughs have been made in the treatment of leukemia and malignant lymphoma. The treatment of tumors has not yet achieved satisfactory results, and there are widespread problems such as low treatment efficiency, poor selectivity, large toxic and side effects, and easy drug resistance of tumor cells.
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Embodiment 1
[0027] Preparation of Anti-tumor Polypeptide in Clam Body Fluid
[0028] (1) Take fresh clams, open their shells to collect their body fluids, and centrifuge at 10,000×g for 20 minutes to remove sediment. According to the ammonium sulfate precipitant dosage table, slowly add finely ground ammonium sulfate to the supernatant to 30% saturation, let stand for 12 hours at 4°C, centrifuge at 10000×g for 20 minutes, collect the precipitate and supernatant, and go up again Add ammonium sulfate to the supernatant to 70% saturation, let stand for 12 hours at 4°C, centrifuge at 10000×g for 20 minutes, collect the precipitate and supernatant, add ammonium sulfate to the supernatant again to 95% saturation according to the above method, and let it stand at 4°C. Leave it for 12 hours, centrifuge at 10000×g for 20 minutes, and collect the precipitate.
[0029] (2) Dissolve the precipitate where the active component is located with pure water, microfiltration to remove insolubles, and then pass ...
Embodiment 2
[0035] Example 2: Clam polypeptide inhibits tumor cell activity in vitro
[0036] Anti-tumor activity test:
[0037] Human lung cancer cells (A549), human liver cancer cells (BEL-7402), human cervical cancer epithelial cells (Hela), human colon cancer cells (HCT-116), human breast cancer cells (MCF-15), human pancreatic cancer cells ( ASPC-1) can be used to evaluate the activity of anti-tumor drugs, with simple operation and good reproducibility. Experimental method (MTT method):
[0038] Tetrazolium salt (MTT) is a dye that can accept hydrogen atoms. The NADH-related dehydrogenase in the mitochondria of living cells can convert yellow MTT into insoluble blue-purple formazan (formazan), but dead cells have no such function. The amount of Formazan crystals produced is proportional to the number of living cells. The crystallized DMSO solution has a maximum absorption peak at 570 nm. Therefore, the effect of the drug on cell proliferation can be evaluated by detecting the amount of f...
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Abstract
The invention belongs to the field of biological pharmacy, particularly relating to a clam polypeptide and a preparation method thereof. The N-tail end sequence of the clam polypeptide is IDEIQNTGGGTNFR, and the molecular weight of the clam polypeptide is 15KDa. The preparation method comprises the following steps: carrying out fractional precipitation, ultrafiltration, ion-exchange column chromatography, hydrophobic chromatography and opposite phase chromatography on a clam liquid by ammonium sulphate to obtain the polypeptide of which the molecular weight is 15KDa. The invention also relates to an application of the polypeptide in preventing and/ or treating malignant tumors. The preparation method is characterized in that the polypeptide is extracted from the clam and has higher inhibitory activity on various tumor cells. The polypeptide is continuously optimized into a polypeptide substance of which the molecular weight is 15KDa. The antitumor activity of the clam polypeptide is detected with an MTT (3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) colorimetric method, and the compound can obviously inhibit the growth of various tumor cells but does not have an inhibition action on normal cells. The characteristic that the polypeptide can selectively inhibit the proliferation of tumor cells enables the polypeptide to have the potential to become a new anti-cancer drug.
Description
Technical field [0001] The invention belongs to the field of biopharmaceuticals, and specifically relates to a clam polypeptide and a preparation method thereof. Background technique [0002] The diversity of marine life, the uniqueness of the marine environment, and the novel structure of marine biological active substances make the ocean a treasure house of innovative medicines and functional / health foods. Since the 1970s, tens of thousands of new active substances have been isolated from marine organisms, including peptides, proteins, polysaccharides, alkaloids, terpenes, macrocyclic polyesters and many other types. Among them, peptides are the largest group of compounds. It has been proven that a variety of marine peptides have physiological activities such as anti-tumor, anti-AIDS, anti-fungal, anti-virus and immune regulation. [0003] Malignant tumors are a kind of diseases that seriously threaten human health and life. At present, cancer chemotherapy technology has impro...
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