Coprinus comatus laccase with activity of inhibiting tumor cell proliferation and preparation method thereof

A technology of Coprinus comatus and laccase, which is applied in the direction of antineoplastic drugs, medical preparations containing active ingredients, antiviral agents, etc., can solve the problems of toxic side effects and prominent drug resistance, and achieve HIV reverse transcriptase The effect of activity inhibition

Inactive Publication Date: 2013-01-23
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Mitsuya et al. first discovered that deoxythymidine AZT has a significant effect of inhibiting HIV, and then found that deoxynucleosides, acyclic nucleosides, and nucleoside analogs can effectively inhibit the replication of HIV, but the resistance of these inhibitors Drug problem is becoming more and more prominent, and has serious toxic side effects

Method used

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  • Coprinus comatus laccase with activity of inhibiting tumor cell proliferation and preparation method thereof
  • Coprinus comatus laccase with activity of inhibiting tumor cell proliferation and preparation method thereof
  • Coprinus comatus laccase with activity of inhibiting tumor cell proliferation and preparation method thereof

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Experimental program
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Embodiment 1

[0032] Embodiment 1, separation, purification and identification of Coprinus comatus laccase

[0033]The raw material of the present invention is to be isolated from the fruiting body of Coprinus comatus (Coprinus comatus) (China General Microorganisms Preservation Management Center, numbering CGMCC 5.615), utilizes PD medium (potato 200g, glucose 20g, add water to 1000ml), Obtain mycelium by fermentation under the following conditions: inoculate the Coprinus comatus species into a comprehensive PDA (200g potato, 20g glucose, 20g agar, add water to 1000ml) plate for activation, and cultivate at a constant temperature of 25°C until the mycelium is overgrown After plating, it was inoculated into PD medium, and after 7 days of shaking flask culture at 25°C and 160rpm, it was transferred to a liquid shake flask and cultured for 7 days to collect mycelium and store it in a -20°C refrigerator for use.

[0034] 1. Extraction and coarse separation

[0035] Suspend Coprinus comatus my...

Embodiment 2

[0057] Embodiment 2, the functional detection of Coprinus comatus laccase

[0058] 1. Functional detection of tumor cell proliferation inhibition in vitro

[0059] 1) Accurately weigh the pure laccase product obtained in Example 1, and prepare different doses of solutions (see Table 1) with serum-free culture medium to detect the activity of inhibiting tumor cell proliferation in vitro;

[0060] 2) Select human liver cancer cell line HepG2 (ATCC, HB-8065) ​​and human breast cancer cell line MCF-7 (ATCC, HTB-22) as detection objects, and culture HepG2 and MCF-7 until the cells enter the logarithmic growth phase , the cells were arranged at a density of 8×10 3 Inoculate into a 96-well plate at a ratio of cells / well, and culture for 6 hours with serum-containing culture medium to make all the cells adhere to the wall;

[0061] 3) Remove the serum culture medium, wash the cells with PBS (Beijing Dingguo Changsheng Biotechnology Co., Ltd., BF-0013), replace with serum-free culture ...

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Abstract

The invention discloses coprinus comatus laccase with activity of inhibiting tumor cell proliferation and a preparation method thereof. The coprinus comatus laccase provided in the invention is prepared by the following steps: 1) breaking up cells; 2) collecting a crude extract of proteins; 3) separating and purifying laccase from the crude extract of proteins collected in the step 2), wherein the obtained laccase is an extract of coprinus comatus, the molecular weight of the laccase is 64KD, and N terminal sequence of the laccase is described by sequence 1 in a sequence table. The coprinus comatus extract obtained through the preparation method in the invention has an obvious inhabiting effect on cancer cell proliferation and HIV reverse transcriptase activity and has an important value in the field of research on novel anti-cancer and anti-AIDS medicines.

Description

technical field [0001] The invention relates to a Coprinus comatus laccase with anti-tumor cell proliferation activity and a preparation method thereof, belonging to the field of biotechnology. Background technique [0002] Coprinus comatus, also known as Coprinus comatus, belongs to Basidiomycota, Phytomycetes, Agaricales, Coprinus family, Coprinus genus. Coprinus cuspidatum is a kind of edible fungus with crisp and smooth texture and delicious taste. It is traditionally eaten in my country and European countries. Coprinus comatus has excellent characteristics such as high protein and low fat, and has the function of homologous food and medicine. According to analysis, every 100g of dry product contains 25.4g of crude protein, 3.3g of fat, 58.8g of total sugar, 7.3g of fiber, and 12.5g of ash, and contains 20 kinds of amino acids, of which all 8 kinds of amino acids necessary for human body are available. It also contains elements such as potassium, sodium, calcium, magne...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02A61K38/44A61P35/00A61P31/18
Inventor 赵爽王贺祥刘宇王守现许峰
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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