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A tumor necrosis factor-related apoptosis ligand fusion protein and its preparation method and application

A tumor necrosis factor and fusion protein technology, applied in the field of tumor necrosis factor-related apoptosis ligand fusion protein, can solve the problems of TRAIL instability, increased sensitivity, short half-life, etc., achieve good pharmacokinetic effect and prolong half-life Effect

Active Publication Date: 2018-05-25
JIANGSU TARGET BIOMEDICINE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, chemotherapy and radiotherapy combined with TRAIL is a commonly used method to increase the sensitivity of tumor cells to TRAIL, but radiotherapy and chemotherapy also have certain effects on normal cells
In addition, TRAIL is unstable, easy to denature, and has a short half-life, which have an impact on its application

Method used

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  • A tumor necrosis factor-related apoptosis ligand fusion protein and its preparation method and application
  • A tumor necrosis factor-related apoptosis ligand fusion protein and its preparation method and application
  • A tumor necrosis factor-related apoptosis ligand fusion protein and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0091] Design, expression, preparation and identification of tumor necrosis factor-related apoptosis ligand fusion protein

[0092] Using computer-aided structural simulation and molecular design, on the SGI computer workstation, using MSI's molecular design software (InsightII, Discover and other modules), based on the crystal structure of tumor necrosis factor-related apoptosis ligand and annexin V, Molecular modeling and molecular design were carried out for tumor necrosis factor-related apoptosis ligand and annexin V fusion protein, and the amino acid length of the connecting peptide was determined.

[0093]In the computer-aided molecular design of the tumor necrosis factor-related apoptosis ligand fusion protein, the inventors carried out structural simulation and molecular design on the tumor necrosis factor-related apoptosis ligand fusion protein, in the tumor necrosis factor-related apoptosis ligand and Based on the crystal structure of annexin V, molecular modeling an...

Embodiment 2

[0102] Detection of Sensitivity of Different Human Tumor Cell Lines to Tumor Necrosis Factor-related Apoptosis Ligand Fusion Protein TP8

[0103]Different human tumor cell lines: colon cancer Colo-205, breast cancer MDA-MB-231, cervical cancer Hela, small cell lung cancer H446, liver cancer PLC, non-small cell lung cancer H1229, liver cancer Bel7402, non-small cell lung cancer A549, Breast cancer MCF-7, lymphoma U937, liver cancer HepG2, colon cancer HT29, breast cancer highly metastatic cell MDA-MB-435, tongue squamous cell carcinoma TCA8113, colon cancer HCT116, pancreatic cancer SW1990, K562 cells, chronic myelogenous leukemia K562, etc. In DMEM containing 10% newborn bovine serum (containing 100 U / ml penicillin and 100 μg / ml streptomycin), and placed in an incubator at 37°C and 5% CO2. K562 and Colo205 were grown in RPMI1640 (containing 100 U / ml penicillin and 100 μg / ml streptomycin) containing 10% newborn bovine serum in an incubator at 37°C and 5% CO2. All cells were pu...

Embodiment 3

[0115] Mechanism Analysis of Tumor Necrosis Factor-related Apoptosis Ligand Fusion Protein

[0116] Western Blot detects the expression of important proteins related to the apoptotic pathway from the protein level: A549 cells are placed in a 6-well cell culture plate to grow to a density of 80%, and treated with an equimolar amount of drugs as required, and after continuing to culture for 6 hours, Collect the cells, wash the cell pellet once with pre-cooled PBS, add an appropriate amount of cell lysate (containing protease inhibitors, Roche Company) to suspend and mix well, place on ice for 30 minutes, centrifuge at 12,000 rpm for 15 minutes at 4°C, and pipette Clear, use the Coomassie brilliant blue method (Bradford method) to measure the protein concentration, adjust the protein concentration, add 4×loading buffer and boil the sample in a boiling water bath for 5 minutes, and store the sample at -20°C for later use.

[0117] Prepare 12% SDS-PAGE gel according to the formula ...

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Abstract

The invention belongs to the field of biological technology, and specifically discloses a tumor necrosis factor family cell apoptosis protein fusion protein, a preparation method and application thereof. The fusion protein is composed of annexin, connecting peptide and tumor necrosis factor family cell apoptosis protein, and the coding gene of the fusion protein is constructed by cloning. The tumor necrosis factor family cell apoptosis protein fusion protein has significantly enhanced effect of inducing apoptosis, can induce apoptosis of tumor cells insensitive to cell apoptosis, and can reduce protein dosage required for obtaining therapeutic effect.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a tumor necrosis factor-related apoptosis ligand fusion protein and its preparation method and application. Background technique [0002] Cancer is the second leading cause of death in the world. In addition to surgery, the current methods of treating cancer mainly include chemotherapy and radiotherapy, but these two methods lack specificity and have obvious cytotoxicity. An ideal antitumor drug is one that can selectively kill tumor cells without harming normal cells. Many chemotherapeutic drugs kill tumor cells by inducing apoptosis, and tumor cells also produce the mechanism of inhibiting apoptosis, which leads to the failure of chemotherapy. Therefore, the apoptotic pathway has become the most attractive target for the development of antitumor drugs. [0003] Since Aggarwal et al. isolated and identified the first tumor necrosis factor (TNF) in 1984, this protein family ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21C12P21/02A61K47/62A61K38/17A61P35/00C12R1/19
Inventor 华子春邱樊胡敏进
Owner JIANGSU TARGET BIOMEDICINE RES INST
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