Fusion protein and its prepared drug and coding gene and msc containing the gene

A fusion protein and gene technology, applied in the field of genetic engineering, can solve problems such as poor therapeutic effect, achieve the effect of increasing the cell ratio and reducing the incidence of aGVHD

Active Publication Date: 2020-06-23
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the treatment recommendation for aGVHD is mainly the prevention after allo-HSCT, and the drugs include cyclosporine A, methotrexate, antithymocyte globulin, etc.; for the aGVHD that has occurred, the first-line drug recommendation is methyl strong Songlong, second-line treatment includes mycophenolate mofetil capsules, methotrexate, tacrolimus, anti-TNF antibody, mesenchymal stem cell (MSC) infusion, etc., and the treatment effect is still poor

Method used

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  • Fusion protein and its prepared drug and coding gene and msc containing the gene
  • Fusion protein and its prepared drug and coding gene and msc containing the gene
  • Fusion protein and its prepared drug and coding gene and msc containing the gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The connection of embodiment 1 target gene and carrier

[0021] 1. Design primers

[0022] Upstream primer: 5'-CGGAATTCGGATCCAGGCCTAAGCT-3'; (SEQ ID NO.5)

[0023] Downstream primer: 5'-CGGGATCCGAATTCGAAGTTGAGCTC-3'; (SEQ ID NO.6)

[0024] 2. Target gene PCR, the reaction system is: 1 μL DNA template (IFNG-IFNGR1, as shown in SEQ ID NO.4), 2 μL Primer-F (10 μM), 2 μL Primer-R (10 μM), 10 μL 2×MasterMix, Finally with ddH 2 O supplemented to 50 μL;

[0025] The reaction program was: preheating at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, a total of 25 cycles, and finally extension at 72°C for 10 min.

[0026] 3. Respectively digest the target gene and the vector, wherein the enzyme digestion system of the target gene includes: 25 μL PCR product, 5 μL Buffer EcoRI, 2.5 μL EcoRI, 2.5 μL BamHI, and finally use ddH 2 O supplemented to 50 μL;

[0027] The vector digestion system includes: 20 μL pWPXLD, 5 μL Bu...

Embodiment 2

[0031] Example 2 Lentiviral Packaging of Recombinant Plasmid pWPXLd-IFNG-IFNGR1

[0032] 1. Use the TIANGEN endotoxin-free plasmid extraction kit to extract the recombinant plasmid. The specific process is as follows:

[0033] 1. Put the adsorption column CP6 into a 50mL collection tube first, then add 2.5mL of balance solution BL, centrifuge at 8000r / min at room temperature for 2 minutes, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0034] 2. Take 100mL of overnight cultured bacterial solution into a centrifuge tube, centrifuge at room temperature at 8000r / min for 3 minutes to collect the bacteria, try to absorb the supernatant, and use clean absorbent paper to absorb the water droplets on the bottle wall;

[0035] 3. Add 8 mL of RNase A-containing solution P1 to the centrifuge tube with bacterial sediment, and vortex to completely suspend the bacterial sediment;

[0036] 4. Add 8 mL of solution P2 to the cen...

Embodiment 3

[0077] Example 3 Infection of human mesenchymal stem cells

[0078] 1. The mesenchymal stem cells were divided into 1.0×10 5 Inoculate 10% FBS, 100U / L double-antibody DMEM medium 2mL / well in 5% CO 2 , 37 ℃ cell incubator culture;

[0079] 2. When the inoculated mesenchymal stem cells adhere to the wall evenly and the confluence reaches 30%, calculate the required virus volume according to the measured virus titer according to the Moi value of 50;

[0080] 3. Add the required virus volume to 2 mL of DMEM medium with 10% FBS and 100 U / L double antibody;

[0081] 4. Suck off the original medium of mesenchymal stem cells in the 6-well plate, add 2 mL of virus-containing medium to each well; in 5% CO 2 , 37 ℃ cell incubator culture, need to observe the state of the cells during the culture;

[0082] 5. Change the medium after culturing for 12-16 hours;

[0083] 6. After 72 hours of culture, the cells were harvested, the expression of Ifn-γ and Ifn-γR1 in the membrane was detec...

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PUM

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Abstract

The invention discloses a fusion protein, a drug prepared therefrom, a coding gene and MSC containing the gene. The amino acid sequence of the fusion protein is shown in SEQ ID NO.1; the nucleotide sequence of the gene is shown in SEQ ID NO.4. The mesenchymal stem cells prepared by the present invention can overexpress IDO and iNOS hundreds of thousands of times, can significantly inhibit the proportion of Th17 cells in vivo and in vitro, increase the proportion of Treg cells, can significantly regulate the Th17 / Treg axis, and significantly reduce the proportion of allogeneic hematopoietic stem cells Incidence of aGVHD and aGVHD-related mortality in transplanted mice.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a fusion protein, a medicine prepared therefrom, a coding gene and an MSC containing the gene. Background technique [0002] The main feature of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the activation of T lymphocytes and dendritic cells of the donor, and through the immune mechanism, the remaining leukemia cells of the recipient are eliminated to the greatest extent, in order to achieve the goal of transplantation. Graft-versus-leukemia (GVL), while the activation of donor T cells and dendritic cells in the recipient often causes acute graft-versus-host disease (aGVHD), which is the most Severe complications often increase the mortality of allo-HSCT. The main mechanism is that the proportion of T helper 17 cells (Th17) is increased, while the proportion of T regulatory cells (Treg) is decreased, which leads to the imbalance of Th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K38/21A61K47/64A61P37/06
CPCA61K38/00A61P37/06C07K14/555C07K14/7156C07K2319/00C12N5/0662C12N15/86C12N2510/00C12N2740/15043
Inventor 贾永前吴鹏强王甫珏王永生
Owner SICHUAN UNIV
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