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A tumor necrosis factor-related apoptosis-inducing ligand variant and its preparation method and use

A technology of apoptosis-inducing ligand and tumor necrosis factor, which is applied in the field of genetic engineering to achieve the effect of excellent curative effect and good clinical application prospect.

Active Publication Date: 2020-02-04
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] F3 peptide is composed of 17-48 amino acids of human high mobility group nucleosomalbinding protein 2 (high mobility group nucleosomalbinding protein 2), which can specifically bind tumor cells and tumor vascular endothelial cells (Porkka K et al.Proc Natl Acad Sci U S A.2002, 99(11): 7444-9), can enhance the targeting of some antitumor drugs, but there is no report of using it to enhance the tumor targeting of TRAIL and thereby improve its antitumor activity

Method used

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  • A tumor necrosis factor-related apoptosis-inducing ligand variant and its preparation method and use
  • A tumor necrosis factor-related apoptosis-inducing ligand variant and its preparation method and use
  • A tumor necrosis factor-related apoptosis-inducing ligand variant and its preparation method and use

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Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1 Preparation of TRAIL variant of the present invention

[0043] 1. Design and gene cloning of TRAIL variants

[0044] 1) Design of TRAIL variants

[0045] The F3 peptide consists of 31 amino acids. TRAIL is a fragment that intercepts the amino acid composition of human TRAIL114-281. F3 can be connected to the N-terminal or C-terminal of TRAIL, and a flexible linker is added between the two fragments, such as (G4S) 3 wait. like figure 1 As shown, the fusion proteins formed by linking F3 at the N-terminus or C-terminus of TRAIL were named TRAIL-F3-N and TRAIL-F3-C, respectively. At the same time, the control peptide PC was selected and connected to the N-terminus of TRAIL to construct TRAIL-PC-N as a control.

[0046] Using nucleic acid analysis software, the coding genes of each fragment were spliced ​​to obtain the coding genes of TRAIL variant proteins TRAIL-F3-N, TRAIL-F3-C and TRAIL-PC-N, and then submitted to Nanjing GenScript Company for artificia...

experiment example 1

[0060] Experimental example 1 Effect of the covalent linking method of functional fragments of TRAIL variants on the activity of the variants

[0061] 1. Experimental method

[0062] The cell-killing activity of the TRAIL variant protein was detected by an in vitro cytotoxicity test model.

[0063] Experiments were carried out with human liver cancer cell line SMMC-7721 and human lung cancer cell line A549: the cells were placed in RPMI 1640 containing 10% calf serum, 2mM L-glutamine, 100μg / ml streptomycin and 100U / ml penicillin. 37°C, 5% CO 2 cultivated under conditions. Will 1×10 4 Cells were seeded in a 96-well plate and adhered to the wall overnight, and then the medium was replaced with 1640 medium containing 2% calf serum, and proteins of different concentrations were added at the same time. After acting overnight, CCK-8 solution was added and reacted for 2-4 hours. Afterwards, the absorbance at 495 nm was measured with a microplate reader. The cell survival rate of...

experiment example 2

[0075] Experimental Example 2 Detection of the Selective Killing Properties of Variant Proteins on Tumor Cells

[0076] 1. Experimental method

[0077] The in vitro cytotoxicity test model was used to compare the killing activity of TRAIL variant protein on tumor cells and normal cells, so as to determine its cell killing selectivity. Will 1×10 4 Cells (100 μl) were inoculated in a 96-well plate to adhere to the wall overnight, and then the culture medium was replaced with 1640 medium containing 2% calf serum, and the TRAIL and TRAIL variant proteins obtained in different concentrations of Example 1 were added simultaneously to act overnight Afterwards, 10 μl of CCK-8 solution was added, and the absorbance at 495 nm was measured with a microplate reader after reacting for 2-4 hours. The cell survival rate of the protein treatment group was calculated as the cell survival rate of the non-protein treatment group as 100%.

[0078] 2. Experimental results

[0079] The result i...

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Abstract

The invention discloses a variant of tumor necrosis factor-related apoptosis-inducing ligand. The variant is a fusion protein of the tumor necrosis factor-related apoptosis-inducing ligand and F3 peptide, wherein the F3 peptide is connected with the N terminal or C terminal of the tumor necrosis factor-related apoptosis-inducing ligand through connexon. The invention also discloses a nucleotide sequence, a recombinant vector and recombinant bacterium containing the nucleotide sequence, and a preparation method and application of the variant. According to the invention, the variant of the tumor necrosis factor-related apoptosis-inducing ligand is prepared by using a genetic engineering manner; the variant is obviously superior in affinity to tumor cells, capability of inducing apoptosis of tumor cells, tumor targeting performance and in-vivo antineoplastic activity compared with the tumor necrosis factor-related apoptosis-inducing ligand; and the variant has excellent curative effect on tumors and has good clinical application prospects.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a tumor necrosis factor-related apoptosis-inducing ligand variant and its preparation method and application. Background technique [0002] Cancer is one of the major diseases threatening human life. Surgery, radiotherapy, and chemotherapy are currently the mainstays of cancer treatment. Drugs that can penetrate deep into the tumor to kill tumor cells and cause less damage to normal cells are ideal chemotherapy drugs. However, most traditional chemotherapeutic drugs lack selectivity for killing cells. With the in-depth understanding of the molecular mechanism of tumorigenesis and development and the discovery of tumor markers, targeted therapy drugs have become a new trend in the development of tumor chemotherapy drugs (Nero TL et al.Nat Rev Cancer.2014,14:248–62; Goel HL et al. Nat Rev Cancer. 2013, 13:871-82). [0003] Tumor necrosis factor-related apoptosis-inducing liga...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/17A61K47/64A61P35/00A61P37/02
Inventor 卢晓风杨浩万琳程惊秋
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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