MSC modified by exendin-4 fusion gene and its application

A technology of fusion gene and albumin-1, applied in the field of genetic engineering, can solve problems such as prolonging half-life, and achieve the effect of prolonging half-life

Active Publication Date: 2022-03-15
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] CN109929806A provides a double-gene modified stem cell, which uses FGF21 and GLP-1 or its variants; CN104805058A also uses GLP-1 gene-modified mesenchymal stem cells; WO2021 / 042321A1 patent uses Exendin- 4. Genetically modified mesenchymal stem cells without linking with Fc domain to form fusion protein to prolong their half-life

Method used

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  • MSC modified by exendin-4 fusion gene and its application
  • MSC modified by exendin-4 fusion gene and its application
  • MSC modified by exendin-4 fusion gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 The structure of the fusion gene

[0038] Modified mesenchymal stem cell fusion gene (Exendin-4-IgG2), including signal peptide artificial nucleic acid sequence, Exendin-4 nucleic acid artificial sequence, Linker nucleic acid artificial sequence and IgG2 nucleic acid artificial sequence, wherein the IgG2 nucleic acid artificial sequence can be produced by Albumin-1 , Albumin, Ig kappa chain instead, get Exendin-4-Albumin-1 fusion gene, Exendin-4-Albumin fusion gene, Exendin-4-Ig kappa chain fusion gene, such as figure 1shown. The Exendin-4-IgG2 fusion gene is composed of a signal peptide artificial nucleic acid sequence, an Exendin-4 nucleic acid artificial sequence, a Linker nucleic acid artificial sequence and an IgG2 nucleic acid artificial sequence in sequence, and its nucleic acid sequence is SEQ ID NO.1. Wherein, the artificial sequence of the signal peptide nucleic acid is SEQ ID NO.2; the artificial sequence of the Exendin-4 nucleic acid is SEQ ID NO....

Embodiment 2

[0039] Example 2 Preparation of recombinant plasmid

[0040] In this embodiment, IgG2 is taken as an example for illustration. When any of Albumin-1, Albumin, and Ig kappa chain is used, its preparation method is basically the same, and details will not be repeated here.

[0041] The fusion gene (Exendin-4-IgG2) was entrusted to Shanghai Jierui Bioengineering Co., Ltd. to synthesize, and the synthetic fusion gene sequence was double-digested with EcoRI and BamHI to obtain the target fragment with sticky ends. At the same time, the carrier pIRES2-eGFP (purchased from Shanghai Zeye Biotechnology Co., Ltd.) was digested with EcoRI and BamHI to obtain a linearized vector fragment, and the target fragment and the linearized vector fragment were connected with T4 ligase, and transformed into E.coli ( Top10), carry out enzyme digestion verification (see figure 2 ), after the sequencing is correct, use the plasmid extraction kit from OMEGA to extract the plasmids to obtain recombina...

Embodiment 3

[0042] Example 3 Preparation of umbilical cord mesenchymal stem cells

[0043] Take the neonatal umbilical cord donated by the hospital, disinfect it twice with 75wt% alcohol in the ultra-clean workbench, put it in a petri dish, use tweezers to peel off the Fahrenheit jelly tissue, and cut it to 0.5mm with scissors 2 sized nubs. The shredded Wahrenheit jelly tissue was transferred to a culture bottle, added with Hyclone mesenchymal medium (purchased from Hyclone) containing platelet lysate for culture, and observed with a microscope every day. When the grown stem cells covered 80% of the bottom of the bottle, passage was carried out. After passage, the growth rate of the cells was accelerated. Passage was carried out every 2-3 days, and passed to P3 passage for experiment (see image 3 ), and the markers CD105, CD73, CD90, CD34, HLA-DR of umbilical cord mesenchymal stem cells were detected by flow cytometry (see Figure 4-8 ).

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Abstract

The present invention provides MSC modified by Exendin-4 fusion gene, the Exendin-4 fusion gene is obtained by connecting Exendin-4 with one of IgG2, Albumin-1, Albumin, and Ig kappa chain; the present invention also provides Exendin-4 fusion gene modification The application of the MSC, the application of the Exendin-4 fusion gene modified MSC in the preparation of GLP-1 protein-related drugs. The present invention can improve the Exendin-4 expression level of MSC modified by Exendin-4 fusion gene; the present invention adopts one of Exendin-4 and IgG2, Albumin-1, Albumin, Ig kappa chain to obtain fusion gene, which can prolong the expression of Exendin-4 protein half life.

Description

technical field [0001] The invention relates to MSC modified by Exendin-4 fusion gene and application thereof, belonging to the technical field of genetic engineering. Background technique [0002] Type 2 diabetes mellitus (T2D) is a chronic progressive disease characterized by glycemic regulation resulting from a combination of factors including insulin resistance, impaired cellular function, hyperhemagglutinemia, and inappropriate hepatic galactose production defect. Eating stimulates the secretion of various gastrointestinal hormones, including insulin, glucagon, GIP, GLP-1, etc. These hormones are involved in the regulation of intestinal motility, secretion of gastric acid and pancreatic enzymes, gallbladder contraction and nutrient absorption. The hormone also promotes the digestion and absorption of glucose by stimulating the pancreatic endocrine secretion of insulin. [0003] GLP-1 acts by binding to structurally distinct G protein-coupled receptors (GPCRs). The GL...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/62A61K38/26A61K47/64A61K47/68A61K48/00A61P3/10
CPCC12N5/0662C07K14/605A61K38/26A61K47/68A61K47/643A61K48/0008A61K48/005A61P3/10C07K2319/00
Inventor 刘明录张传鹏冯建海金海锋王立新强邦明王亮许淼
Owner SHANDONG XINRUI BIOTECH CO LTD
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