Method for gene modification of mesenchymal stem cells through tripolymer TRAIL fusion protein, and application

A fusion protein, stem cell technology, applied to cells modified by introducing foreign genetic material, animal/human proteins, receptors/cell surface antigens/cell surface determinants, etc., can solve the problem of poor stability of trimeric TRAIL proteins, etc. question

Active Publication Date: 2020-01-10
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 gene modification of mesenchymal stem cells through tripolymer TRAIL fusion protein, and application
  • Method for gene modification of mesenchymal stem cells through tripolymer TRAIL fusion protein, and application
  • Method for gene modification of mesenchymal stem cells through tripolymer TRAIL fusion protein, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] A trimeric TRAIL fusion protein whose nucleic acid sequence is as SEQID NO:1. The fusion protein includes:

[0049] The secretion signal protein is selected from tPA, IL-2 and SEC, preferably tPA, the nucleic acid sequence of which is as SEQ ID NO: 2;

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

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

[0052] The preparation method of the trimeric TRAIL fusion protein includes the following steps:

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

[0054] (2) Use molecular biology methods to connect tPA, SPDcc, TRAIL and other sequences in sequence into tPA-SPDcc-...

Embodiment 2

[0059] A method for modifying mesenchymal stem cells with trimer TRAIA fusion protein, including the following steps:

[0060] S1. Obtain the trimer TRAIL fusion protein;

[0061] Among them, the preparation method of the trimer TRAIL fusion protein is the same as in Example 1.

[0062] S2, the fusion protein is mixed with the auxiliary plasmid, and the cells are transfected;

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

[0064] The mixed culture of trimer TRAIL fusion protein and helper plasmid includes the following steps:

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

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

[0067] (3) Put Mix the LTX reagent with the Opti-MEM solution, let stand for 5 minutes, and mix th...

Embodiment 3

[0079] Example 3. Detection of the stability of TRAIL protein secreted by genetically modified mesenchymal stem cells. The two genetically modified mesenchymal stem cell culture supernatants obtained in Example 2 were used and placed in a 37°C warm bath at different time intervals (0 , 24h, 48h, 72h and 96h) aspirate a small amount of supernatant and quickly store it in a refrigerator at -70℃. After sampling at all time points was completed, 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 residual rate of TRAIL protein at each time point in each treatment group according to the following formula:

[0081] Residual rate = (TRAIL concentration at each time point / 0h TRAIL concentration) x 100%

[0082] The results showed that compared with the control group (TRAIL gene modified MSC), the SPDcc-TRAIL gene modified MSC constructed by the method of the present inve...

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Abstract

The invention relates to a method for gene modification of mesenchymal stem cells through a tripolymer TRAIL fusion protein, and application. A nucleic acid sequence of the tripolymer TRAIL fusion protein is shown as SEQ ID NO:1. The tripolymer TRAIL fusion protein can form a triple-helix parallel coiled coil structure domain in a molecular structure through disulfide bond covalent binding, and thus the protein is assisted in being folded into a tripolymer. The tripolymer structure domain is subjected to covalent bond binding, therefore, the N tail end of the TRAIL protein is additionally provided with the structure domain, the stability and the antineoplastic activity of the TRAIL tripolymer can be enhanced significantly.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and more specifically to a trimeric TRAIL fusion protein, a method and application for modifying mesenchymal stem cells using the trimeric TRAIL fusion protein. Background technique [0002] Malignant tumor is a disease that seriously endangers human health, and its morbidity and fatality rate are increasing year by year. However, clinical methods such as surgery, radiotherapy and chemotherapy cannot effectively treat tumors, and other treatment methods are urgently needed. [0003] Stem cells have been extensively studied as gene carriers for tumor metastasis. Studies have found that mesenchymal stem cells have the characteristics of 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 Targeted drug carriers, carrying exogenous tumor suppressor factors to act on tumors, are currently more effective ...

Claims

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Application Information

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Patent Type & Authority Applications(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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