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Preparation and application of gene-modified mesenchymal stem cell exosomes

A technology of mesenchymal stem cells and gene modification, applied in the field of biopharmaceuticals, to achieve the effect of enhancing therapeutic effect, strengthening immune regulation function, and improving bioavailability

Active Publication Date: 2021-08-24
赛浦生物科技(长春)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve one or more technical problems in the prior art, the applicant found that the use of IL-27 gene to modify the exosomes of mesenchymal stem cells has a good effect on the treatment of allergic rhinitis, by secreting and expressing IL-27 factors , enhance the immune regulation and anti-allergic functions of mesenchymal stem cell exosomes, can well repair the nasal microenvironment, improve the symptoms of allergic rhinitis, and overcome the side effects and therapeutic effects of existing allergic rhinitis treatment drugs Bad question

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  • Preparation and application of gene-modified mesenchymal stem cell exosomes
  • Preparation and application of gene-modified mesenchymal stem cell exosomes
  • Preparation and application of gene-modified mesenchymal stem cell exosomes

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Embodiment approach

[0043] According to another preferred embodiment of the present invention, there is also provided a method for preparing gene-modified mesenchymal stem cell exosomes, which is characterized by the following steps:

[0044] a. Design and obtain the nucleic acid sequence for highly expressing IL-27 in mammalian cells, the nucleic acid sequence for highly expressing IL-27 in mammalian cells includes tPA signal peptide coding sequence, IL-27B chain coding sequence, linker sequence and IL-27A chain coding Sequence, as shown in SEQ ID NO.1;

[0045] b. transferring the nucleic acid sequence of the mammalian cell highly expressing IL-27 into mesenchymal stem cells to obtain genetically modified mesenchymal stem cells;

[0046] c. culturing the genetically modified mesenchymal stem cells to obtain exosomes of the genetically modified mesenchymal stem cells.

[0047] According to another preferred embodiment of the present invention, in step a, the nucleic acid sequence for highly exp...

Embodiment 1

[0080] Example 1, artificially synthesized DNA sequence encoding IL-27 (as shown in SEQ ID NO.1)

[0081] The mature protein sequence of the B chain of IL-27 contains 209 amino acid residues, and the mature protein sequence of the A chain of IL-27 contains 215 amino acid residues. The link between the B chain and the A chain consists of GGGS GGGS GGGS. The codons preferred by human cells were artificially synthesized for the above amino acid sequence. During synthesis, tPA signal peptide (MDAMKRGLCCVLLLCGAVFVSP), Kozak sequence and BamHI site were introduced upstream of the gene, and stop codon and EcoRI site were introduced downstream of the gene.

Embodiment 2

[0082] Embodiment 2, construction expression vector

[0083]The synthesized gene fragment and the pHBAd adenovirus expression vector were digested with BamHI and EcoRI, respectively, and kept at 37°C overnight. After the digested product was cut out by agarose gel electrophoresis, the target fragment was recovered using an agarose gel DNA recovery kit. Mix the recovered target gene with the carrier at a molar ratio of 3:1, and use T4 DNA Ligase ligase to ligate overnight at a constant temperature of 16°C in a PCR machine to construct the pHBAd-IL27 recombinant plasmid. The ligation product was transformed into competent Escherichia coli DH5α, and the transformed competent cells were spread evenly on the LB plate medium containing ampicillin (100 μg / mL). After the bacterial solution was completely absorbed, cultured upside down at 37°C for 12-16 hours. Pick 4-8 ​​well-growth single colonies from the transformation plate with a sterile tip, inoculate them in 5 mL of LB medium c...

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Abstract

The invention discloses a gene-modified mesenchymal stem cell exosome and its preparation method and its application in the treatment of allergic rhinitis. The gene sequence expressed by IL-27 is mainly constructed by means of molecular biology, and mammalian cells are constructed. expression vector, further transfected mesenchymal stem cells, and obtained gene-modified mesenchymal stem cell exosomes. The invention has remarkable therapeutic effect on allergic rhinitis, has great economic and social benefits, and has good market prospect.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to the preparation and application of a gene-modified mesenchymal stem cell exosome, in particular to the preparation of a gene-modified mesenchymal stem cell exosome and its application in the treatment of allergic rhinitis in the application. Background technique [0002] Allergic rhinitis, also known as allergic rhinitis, is a type I allergic disease of nasal mucosa induced by the release of histamine, kinins and leukotrienes mediated by IgE. The main symptoms of the onset are nasal itching, nasal congestion, sneezing, runny nose, and olfactory disturbance. The disease is one of the common chronic diseases in human beings. In recent years, due to the impact of global climate change, environmental pollution, social and psychological pressure, etc., its incidence has been increasing year by year, and it is regarded as an epidemic in the "21st century". About 25% of the w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/24C12N15/861A61K35/28A61K35/51A61K35/50A61K9/12A61K47/38A61P11/02A61P37/08
CPCC07K14/54C12N15/86C12N5/0665C12N5/0668C12N5/0605A61K35/28A61K35/51A61K35/50A61K9/0048A61K47/38A61P11/02A61P37/08C12N2800/22C12N2710/10043C12N2510/00
Inventor 马杰焦平王伟李佳霖
Owner 赛浦生物科技(长春)有限公司
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