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Method for genetically modifying mesenchymal stem cells with trimer trail fusion protein and use thereof

A technology of mesenchymal stem cells and fusion protein, applied in the field of modification of mesenchymal stem cells, can solve the problem of poor stability of trimeric TRAIL protein

Active Publication Date: 2021-08-27
北京贝来生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Based on this, it is necessary to provide a stable trimeric TRAIL fusion protein for the technical problem of poor stability of the above-mentioned trimeric TRAIL protein

Method used

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  • Method for genetically modifying mesenchymal stem cells with trimer trail fusion protein and use thereof
  • Method for genetically modifying mesenchymal stem cells with trimer trail fusion protein and use thereof
  • Method for genetically modifying mesenchymal stem cells with trimer trail fusion protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] A trimeric TRAIL fusion protein, its nucleic acid sequence is as SEQ ID NO:1. The fusion protein includes:

[0049] Secretion signal protein, selected from tPA, IL-2 and SEC, preferably tPA, its nucleic acid sequence is as SEQID NO:2;

[0050] Pulmonary surfactant protein (pulmonary surfactant protein-D, SPD), its coding region is the amino acid amino group of 224-255, and its nucleic acid sequence is shown in SEQID NO:3;

[0051] Humanized tumor necrosis factor-related apoptosis-inducing ligand (homo sapiens tumor necrosis factor-related apoptosis inducing ligand, hTRAIL), its coding region is 114-281 amino acid residues, and its nucleic acid sequence is shown in SEQ ID NO:4.

[0052] The preparation method of trimeric TRAIL fusion protein, comprises the steps:

[0053] (1) Design and synthesize tPA, SPDcc, TRAIL and other gene sequences respectively.

[0054] (2) The sequences of tPA, SPDcc, TRAIL and the like were sequentially linked into tPA-SPDcc-TRAIL by using ...

Embodiment 2

[0059] A method for modifying mesenchymal stem cells with a trimeric TRAIA fusion protein, comprising the steps of:

[0060] S1, obtaining the trimeric TRAIL fusion protein;

[0061] Wherein, the preparation method of the trimeric TRAIL fusion protein is the same as that in Example 1.

[0062] S2. The fusion protein and the helper plasmid are mixed and cultured, and the cells are transfected;

[0063] Among them, the transfection methods include virus transfection, liposome transfection, electrotransfer transfection, gene editing transfection and mRNA transfection.

[0064] Trimeric TRAIL fusion protein and auxiliary plasmid mixed culture includes the following steps:

[0065] (1) Mix the fusion protein plasmid and helper plasmids pMDLg, pRSV / REV, and pMDNA2.G, dissolve the mixed plasmids in Opti-MEM medium, and let stand for 5 minutes;

[0066] (2) Add PLUS again TM Reagent, let stand for 5min;

[0067] (3) Will Mix the LTX reagent with the Opti-MEM solution, let it st...

Embodiment 3

[0079] Example 3. Stability detection of TRAIL protein secreted by gene-modified mesenchymal stem cells The two kinds of gene-modified mesenchymal stem cell culture supernatants obtained in Example 2 were used and placed in a warm bath at 37°C at different times (0 , 24h, 48h, 72h and 96h) aspirate a small amount of supernatant, and quickly store it in a -70°C refrigerator. After sampling at all time points, the TRAIL protein content of each sample was determined by the method described in Example 3.

[0080] Taking the protein content of each treatment group as %, calculate the TRAIL protein residual rate of each treatment group at each time point according to the following formula:

[0081] Residual rate = (TRAIL concentration at each time point / OhTRAIL concentration) x 100%

[0082] As a result, it was found that compared with the control group (TRAIL gene modified MSC), the TRAIL expressed by the SPDcc-TRAIL gene modified MSC constructed by the method of the present inven...

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Abstract

The present invention relates to a method for genetically modifying mesenchymal stem cells with a trimeric TRAIL fusion protein and an application thereof. The nucleic acid sequence of the trimeric TRAIL fusion protein is as shown in SEQ ID NO:1. The trimeric TRAIL fusion protein of the present invention can form a triple-helix parallel coiled coil structure domain through covalent combination of disulfide bonds in the molecular structure, thereby helping the protein to fold into a trimer. Because the trimer domain is covalently bonded, adding a domain at the N-terminal of the TRAIL protein can significantly enhance the stability of the TRAIL trimer and anti-tumor activity.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically, relates to a trimeric TRAIL fusion protein, a method for modifying mesenchymal stem cells by using the trimeric TRAIL fusion protein and use thereof. Background technique [0002] Malignant tumor is a disease that seriously endangers human health, and its morbidity and mortality are increasing year by year. However, clinical methods such as surgery, radiotherapy and chemotherapy cannot effectively treat tumors, and it is urgent to find other treatment methods. [0003] Stem cells have been widely studied as gene carriers for tumor transfer. Studies have found that mesenchymal stem cells have the characteristics of specifically tracking and migrating to tumor sites in the body under the action of inflammatory factors and chemokines. Mesenchymal stem cells are used as anti-cancer The targeting carrier of the drug, which carries exogenous tumor suppressor factors to act on...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/28A61P35/00
CPCA61K35/28A61P35/00C07K14/70575C07K14/785C07K2319/00C07K2319/02C12N5/0668C12N15/86C12N2740/15043C12N2800/107
Inventor 刘广洋刘拥军李欣张晨亮米一苗丽马静王皓
Owner 北京贝来生物科技有限公司
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