AMP-18 antigen polypeptide and application in preparing medicament for treating tumor

A technology of AMP-18 and antigenic peptides, applied in the field of bioengineering, can solve the problem of uncontrollable tumor spread throughout the body, and achieve a good effect of tumor suppression

Inactive Publication Date: 2010-06-16
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

From a medical point of view, there is no completely effective method for the treatment of tumors, especially for pa

Method used

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  • AMP-18 antigen polypeptide and application in preparing medicament for treating tumor
  • AMP-18 antigen polypeptide and application in preparing medicament for treating tumor
  • AMP-18 antigen polypeptide and application in preparing medicament for treating tumor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of AMP-18 Antigen Polypeptide

[0036] 1. Protein extraction

[0037] The tissue frozen in liquid nitrogen was ground by a metal mortar, precipitated with 10% TCA / acetone for 2 hours, centrifuged at 35,000 g for 15 minutes at 4°C, and the supernatant was discarded. The precipitate was washed three times with pre-cooled acetone at -20°C and dried in vacuo, and washed with lysis buffer (8M urea, 4% CHAPS, 0.5% Ampholyte (3-10), 20mM Tris pH8.5, when used, add 2mM EDTA, 1mM PMSF, 10mM DTT, Protease Inhibitor Cocktail) was redissolved, sonicated for 5 minutes, centrifuged at 40,000 g for 30 minutes at 15°C, and the supernatant was taken as the protein used in the experiment.

[0038] 2. Protein separation

[0039] After the protein was quantified by the Bradford method, it was separated by two-dimensional electrophoresis. The strip is 18cm, pH3-10, and the focusing process is set as follows: 0V*4h, 50V*8h, 500V*1h, 1000V*1h, 8000V*1h, 8000V, 60,000V...

Embodiment 2

[0050] Example 2 Determination of AMP-18 as a secreted protein

[0051] 1. Identify AMP-18 as a secreted protein

[0052] We constructed pEGFP-AMP-18 eukaryotic expression plasmid (pEGFP expression plasmid is a commercial plasmid), and transfected it and empty vector into gastric cancer cell line, BGC-823. After 24 hours of transfection, the cells and culture medium were collected, and the protein expression of AMP-18 was detected by immunoblotting, as shown in image 3 As shown, the results show that the expression of AMP-18 can be detected in both cells and medium. This means that the AMP-18 protein is secreted outside the cell.

[0053] In general, classic autocrine proteins are mediated by signal peptides, enter the endoplasmic reticulum, and are further processed through the Golgi apparatus to become proteins with specific structures and functions, which are further secreted outside the cell. Therefore, the co-localization of secreted proteins with the Golgi apparatus ...

Embodiment 3

[0058] Example 3 Effect of AMP-18 on tumor cell proliferation

[0059] 1. In vitro experiments, the effect of AMP-18 on the proliferation of tumor cells

[0060] At present, the methods used to detect cell proliferation mainly include MTT assay, Brdu incorporation assay, cell colony formation assay, nude mouse tumorigenicity assay, and the like. Currently, we mainly apply MTT such as Figure 6 As shown in A, Brdu is as Figure 6 As shown in B, the colony generation experiment, such as Figure 6 As shown in C, the effect of AMP-18 on the proliferation of the gastric cancer cell line BGC-823 was examined. Such as Figure 6 A, AMP-18 was incubated with BGC-823 for 72 hours, and AMP-18 could significantly inhibit the proliferation of BGC-823 from 48 hours. Figure 6 B shows that AMP-18 can significantly inhibit the proliferation of BGC-823 after incubation of AMP-18 and BGC-823 for 48 hours. Figure 6 C shows that after the cells were treated with AMP-18, they basically coul...

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Abstract

The invention provides a tumor marker AMP-18 antigen polypeptide which has an amino acid sequence shown as SEQ ID No.1 in a sequence table. The invention also discloses a method for cloning and expressing the protein by a gene and carrying out gastric cancer biological behavior judgment and an animal test, wherein two different test technologies of immunoblot and confocal are applied to obtain the same conclusion that the AMP-18 can be combined with a gastric mucosa epithelial cell and is the autocrine protein of the cell. Because the AMP-18 has favorable tumor inhibiting effect, the AMP-18 antigen polypeptide can be used for the application in preparing a medicament for treating a tumor.

Description

technical field [0001] The invention relates to an AMP-18 antigen polypeptide, more specifically a tumor marker AMP-18 antigen polypeptide, and the application of autoantibodies in the preparation of drugs for treating tumors, belonging to the field of bioengineering. Background technique [0002] Gastric cancer is one of the most common tumors that seriously endanger the health of the population. Its morbidity and mortality rate are only listed after lung cancer, ranking second in the cause of cancer death. At present, the main methods of treating tumors in the world are surgery, radiotherapy, chemotherapy and biological therapy. From a medical point of view, there is no completely effective method for the treatment of tumors, especially for patients with metastatic tumors. Traditional treatment methods cannot control the spread of tumors throughout the body. Studies have confirmed that tumor diameter is closely related to the 5-year survival rate after surgery. If the tu...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12A61K38/17A61P35/00
Inventor 邢蕊张军康滨刘斯奇吕有勇
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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