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Anti-tumor protein and preparation method and application thereof

An anti-tumor and protein technology, applied in the field of anti-lymphoma protein and its preparation

Inactive Publication Date: 2010-06-09
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention isolates a class of protein with a molecular weight of about 26kD from the mushroom body, which has relatively specific anti-lymphoma activity, and no existing literature reports

Method used

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  • Anti-tumor protein and preparation method and application thereof
  • Anti-tumor protein and preparation method and application thereof
  • Anti-tumor protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Separation and purification of RE26 protein

[0064] (1) Obtaining of crude extract:

[0065] Take the fresh fruiting body of Rozites emodensis (Berk.) Moser that has been fully expanded, discard the stem, wash the canopy with distilled water, drain, and add 0.02 volume of pH 8.0 mol / l Tris-HCl buffer solution, homogenate at 4°C, adjust the pH of the solution to 8.0 with 1 mol / l NaOH, and gently stir overnight, after centrifugation, filter the supernatant to remove suspended matter to obtain RE26 crude extract.

[0066] (2) Purification of RE26: High-purity RE26 protein can be obtained by three ion exchange methods.

[0067] The first ion-exchange chromatography: use the Sepharose Q Fast Flow column (particle diameter 45-165 μ m) that effective column height is 10cm to carry out anion exchange, use the Tris-HCl buffer solution of pH 8.0, 0.02mol / l as mobile phase A, Using 1 mol / l NaCl, pH 8.0, 0.02 mol / l Tris-HCl buffer as the mobile phase B, the linear flow...

Embodiment 2

[0071] Partial physicochemical property research of embodiment two RE26

[0072] (1) SDS-PAGE electrophoresis analysis:

[0073] SDS-PAGE electrophoresis (gel concentration is 12%) is used to identify the purity of the RE26 obtained by the separation of Example 1, and determine the molecular weight. The experimental results are as attached Figure 4 As shown, it shows that the purity of RE26 sample is higher than 95%, and the molecular size of RE26 protein is about 26kD.

[0074] (2) Isoelectric focusing experiment:

[0075] The purified RE26 was desalted using a desalting column and replaced with a pure aqueous solution. Isoelectric focusing was performed on a Bio-Rad thin-layer isoelectric focusing instrument, using pH 3-10 ampholytes to establish a pH gradient. After focusing, compare the isoelectric point Marker to determine the isoelectric point of RE26, which is about 4.3, as shown in the attached Figure 5 shown. At the same time, the purified RE26 also showed a si...

Embodiment 3

[0081] Example 3 The minimum inhibitory concentration MIC of RE26 to various cells 50 Determination of

[0082] MIC of RE26 on various cells 50The value is obtained by measuring the cell viability with the MTT method using the drug after doubling dilution to act on the test cells for 48 hours. Tested cells include: Yac-1 (mouse T lymphoma cells), Raji (human Burkitt's lymphoma), HPC (human hepatocytes), Hepg2 (human liver cancer cells), El-4 (mouse lymphoma cells), MSC (mouse mesenchymal stem cells), ECV-304 (human umbilical vein endothelial cells), Vero-E6 (African green monkey kidney cells), primary spleen lymphocytes of mice, and red blood cells of rats.

[0083] Dissolve RE26 in 1640-calf serum medium to 1 mg / ml, and then use the same medium to double-dilute it 12 times in sequence for use.

[0084] Various cells were formulated into 5 × 10 4 Cell suspension at a concentration of 1 / ml was inoculated in a 96-well plate at 100 μl per well, and each type of cell was inocu...

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Abstract

The invention provides an anti-tumor protein RE26, which has a molecular weight of 26 kD and an isoelectric point of 4.3. The preparation method comprises the steps of: obtaining an RE25 crude extract through crude separation, and further separating and purifying through three times of ion-exchange column chromatography to obtain an RE26 pure protein which is for preparing medicines or reagents for treating, diagnosing and researching tumor especially lymphoma diseases. The anti-tumor proteins RE26 of the invention has the special activity of recognizing lymphoma cells and inducing the apoptosis of the lymphoma cells , thereby providing a new way for treating, diagnosing and researching tumor diseases and especially lymphoma diseases.

Description

technical field [0001] The invention relates to a protein and a preparation method thereof, in particular to an anti-lymphoma protein derived from fungi and a preparation method thereof. Background technique [0002] Malignant lymphoma is a kind of malignant tumor. It often occurs in young and middle-aged people, and has a great impact on the labor force. Therefore, it has attracted special attention from the medical field. The age of onset is more common among young and middle-aged people aged 20 to 40. The average death rate All were significantly younger than 45 years of age. [0003] Lymphoma is a malignant tumor originating in the lymphatic system, which can be divided into two categories: Hodgkin's disease and non-Hodgkin's lymphoma. Its occurrence is related to the malignant transformation of immune cells produced by the proliferation and differentiation of lymphocytes. Its etiology is not yet fully clear, and it may be related to some viral infections, such as Epste...

Claims

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Application Information

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IPC IPC(8): C07K14/375C07K1/18A61K38/16A61P35/00G01N33/68
Inventor 龚萌刘旭阳曹桂群程惊秋
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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