Application of ligand antrum mucosal protein-18(AMP-18) of laminin receptor in treatment of tumor

A technology of receptors and tumors, applied in the field of bioengineering, can solve problems such as the inability to maintain cell functions, and achieve a good effect of tumor suppression

Inactive Publication Date: 2012-12-19
BEIJING PROTEIN INNOVATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in gastric cancer tissues, because cancer cells do not express or lowly express AMP-18, the microenvironment they live in lacks sufficient

Method used

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  • Application of ligand antrum mucosal protein-18(AMP-18) of laminin receptor in treatment of tumor
  • Application of ligand antrum mucosal protein-18(AMP-18) of laminin receptor in treatment of tumor
  • Application of ligand antrum mucosal protein-18(AMP-18) of laminin receptor in treatment of tumor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of AMP-18 recombinant protein

[0034] Using normal gastric tissue cDNA as a template, design specific primers according to the gene sequence of AMP-18 mature peptide (SEQ ID No.2), and introduce BamHI and SalI restriction sites at the 5' ends of the upstream and downstream primers respectively: upstream primer: 5'- TGAAGGATCCAACTATAATATCAACGTC-3'; downstream primer: 5'-AAATGTCGACGTTTCTCCACCGTGTCTCC-3'. The gene encoding the mature peptide sequence of AMP-18 was amplified by PCR, and then double-digested with BamHI and SalI after recovery from the gel. The enzyme-digested fragments were recovered by double gel and ligated with the BamHI / SalI double-digested vector pQE30a overnight at 16°C to chemically transform the competent cell JM109. Pick a single clone to extract the plasmid for double enzyme digestion verification, and sequence the positive clone to verify that the sequence is correct. Inoculate and culture the positive bacteria with correc...

Embodiment 2

[0036] Example 2 Autocrine and membrane localization of AMP-18

[0037] 1. Western blot detection of AMP-18 expression in cells and culture medium

[0038] The eukaryotic expression plasmid EGFP-AMP-18 cloned on the carrier pEGFP-N1 (Invitrogen Company) and its empty vector EV were transfected into BGC-823 cells by the method of Lipofectamin 2000 (Invitrogen Company), at 37°C, 5% CO 2 Cells and medium were collected after culturing for 24 hrs. The culture medium was centrifuged and concentrated 50 times by ultrafiltration tube (Millipore Company). The signal of AMP-18 protein was detected by immunoblotting.

[0039] The result is as figure 2 As shown in A, the AMP-18 signal can be detected in both the EGFP-AMP-18 transfected cells and the medium, while the AMP-18 signal cannot be detected in the EV transfected cells and the medium. This shows that AMP-18 protein can be secreted by cells.

[0040] 2. Localization of exogenous AMP-18 by immunofluorescence experiment

[004...

Embodiment 3

[0043] Example 3 LR is the receptor of AMP-18

[0044] 1. Co-immunoprecipitation method to verify the interaction between AMP-18 and LR

[0045] BGC-823 cells were treated with the recombinant AMP-18 prepared in Example 1 or the control solution for 5 minutes, and then the cell membrane proteins were extracted. Harvest about 5 x 10 7 Cells were washed twice with PBS and then incubated with 0.1XPBS at 4°C for 5min. Cells were collected and frozen and thawed five times in liquid nitrogen and 37°C water bath, 5 min each time. Centrifuge the cells at 35,000g at 4°C for 30min, take the supernatant and centrifuge at 150,000g at 4°C for 2hrs, and the pellet is the cell membrane fraction. and 1% protease inhibitor cocktail), lyse in ice bath for 30 min, centrifuge at 12,000 g at 4°C for 30 min, and transfer the supernatant to a new eppendorf tube. After quantification, take 3 mg of protein and dilute to 1 ml with lysate, add 40 μl of Protein A Sepharose column material that has be...

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Abstract

The invention provides a ligand antrum mucosal protein-18(AMP-18) of a laminin receptor (LR), which has an amino acid sequence as shown in SEQ ID No.1 in a sequence table. The ligand antrum mucosal protein-18(AMP-18) can bind with LR on the surface of gastric epithelial cells to inhibit the growth of stomach cancer cells and can be used to prepare drugs for treating tumor.

Description

technical field [0001] The invention relates to a ligand of laminin receptor, more specifically the application of a recombinant protein containing the ligand gastric antrum mucosal protein-18 in the preparation of drugs for treating tumors, and belongs to the field of bioengineering. Background technique [0002] Gastric cancer is the most common malignant tumor of the digestive tract, which seriously endangers the health of the population. Its morbidity and mortality rate are only listed after lung cancer, ranking second in the cause of cancer death. The formation of gastric cancer is a multi-step complex process, which has an inseparable relationship with the biological characteristics of tumor cells, the immune status of the body, and the tumor microenvironment, and secreted proteins play a vital role in it. [0003] Currently, surgical resection is the main treatment for gastric cancer. For patients with unresectable advanced disease, chemotherapy is the main treatmen...

Claims

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Application Information

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IPC IPC(8): C07K14/47A61K38/17A61P35/00
Inventor 不公告发明人
Owner BEIJING PROTEIN INNOVATION
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