CN113801853B - Exendin-4 fusion gene modified MSC and application thereof - Google Patents
Exendin-4 fusion gene modified MSC and application thereof Download PDFInfo
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Abstract
The invention provides an MSC modified by an Exendin-4 fusion gene, wherein the Exendin-4 fusion gene is obtained by connecting Exendin-4 with one of IgG2, Albumin-1, Albumin and Ig kappa chain in series; the invention also provides an application of the MSC modified by the Exendin-4 fusion gene, and an application of the MSC modified by the Exendin-4 fusion gene in preparation of GLP-1 protein related medicines. The invention can improve the Exendin-4 expression quantity of MSC modified by Exendin-4 fusion gene; the fusion gene is obtained by one of Exendin-4, IgG2, Albumin-1, Albumin and Ig kappa chain, and the half-life period of the Exendin-4 protein can be prolonged.
Description
Technical Field
The invention relates to an Exendin-4 fusion gene modified MSC and application thereof, belonging to the technical field of genetic engineering.
Background
Type 2 diabetes (T2D) is a chronic progressive disease characterized by deficiencies in blood glucose regulation caused by factors such as insulin resistance, impaired cell function that declines year by year, hypercoagulability, and inappropriate hepatic galactose production. Eating stimulates the secretion of a variety of gastrointestinal hormones, including insulin, glucagon, GIP, GLP-1, etc., which are involved in regulating intestinal motility, secretion of gastric and pancreatic enzymes, contraction of the gallbladder and nutrient absorption, and which also stimulate the digestive absorption of glucose by stimulating secretion of insulin from the pancreas.
GLP-1 functions by binding to structurally distinct G protein-coupled receptors (GPCRs). The GLP-1 receptor (GLP-1R) is expressed in islet alpha and beta cells and peripheral tissues, including the central and peripheral nervous system, heart, kidney, lung, and gastrointestinal tract. GLP-1 also inhibits glucagon secretion, gastric emptying and food intake, and promotes enhanced processing of glucose by neural mechanisms. At present, hypoglycemic drugs developed aiming at GLP1 are mainly DPP4 inhibitors and GLP1 analogues which inhibit GLP1 degradation, but both have the problem of short half-life and are frequently taken by patients. While infusion of native GLP-1 is very effective in lowering blood glucose in type 2 diabetic patients, a single subcutaneous injection of this native peptide will rapidly degrade and disappear from circulation within minutes. Therefore, most pharmaceutical approaches to the development of GLP-1 mimetics have focused on the development of long-acting degradation resistant peptides.
In recent years, transplantation of Mesenchymal Stem Cells (MSCs), which are easily obtained, easily proliferated and survive for a long time after transplantation, has received increasing attention for the treatment of diabetes. Given that such transplants have proven safe in a variety of applications, and that MSCs possess potent immunomodulatory and chemotactic properties, the use of these cells as a vehicle for the delivery or production of beneficial proteins for therapeutic purposes has been the focus of much research attention.
CN109929806A provides a double-gene modified stem cell, and the adopted genes are FGF21 and GLP-1 or variants thereof; CN104805058A also used are GLP-1 gene modified mesenchymal stem cells; the patent WO2021/042321A1 discloses Exendin-4 gene modified mesenchymal stem cells alone, which are not linked to an Fc domain to form a fusion protein to prolong the half-life.
The development of a GLP-1 protein related medicine with long half-life period and reduction of medication for diabetics is a technical problem to be solved urgently.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an Exendin-4 fusion gene modified MSC and application thereof, and the following aims are achieved: the expression quantity of the Exendin-4 of the MSC modified by the Exendin-4 fusion gene is high, the half-life period is long, the medication of a diabetic patient is reduced, and the medication time is prolonged.
In order to solve the technical problems, the invention adopts the following technical scheme:
the Exendin-4 fusion gene modified MSC is obtained by connecting Exendin-4 with one of IgG2, Albumin-1, Albumin and Ig kappa chain in series.
The following is a further improvement of the above technical solution:
the nucleic acid human process sequence of the Exendin-4 is shown as SEQ ID NO. 3; the nucleic acid human process sequence of the IgG2 is shown as SEQ ID NO.4, the nucleic acid human process sequence of the Albumin-1 is shown as SEQ ID NO.5, the nucleic acid human process sequence of the Albumin is shown as SEQ ID NO.6, and the nucleic acid human process sequence of the Ig kappa chain is shown as SEQ ID NO. 7.
The Exendin-4 fusion gene is one of Exendin-4-IgG2, Exendin-4-Albumin-1, Exendin-4-Albumin and Exendin-4-Ig kappa chain; the nucleic acid sequence of the Exendin-4-IgG2 is shown in SEQ ID NO. 1; the nucleic acid sequence of the Exendin-4-Albumin-1 is shown in SEQ ID NO. 8; the nucleic acid sequence of the Exendin-4-Albumin is shown in SEQ ID NO. 9; the nucleic acid sequence of the Exendin-4-Ig kappa chain is shown in SEQ ID NO. 10.
The preparation method of the MSC modified by the Exendin-4 fusion gene comprises the steps of synthesizing the fusion gene, preparing recombinant plasmids, transfecting mesenchymal stem cells by the recombinant plasmids and screening.
The preparation method comprises the steps of preparing a recombinant plasmid, loading an Exendin-4 fusion gene by using a vector pIRES2-eGFP, transforming the fusion gene to E.coli, and extracting to obtain the recombinant plasmid with the concentration of 855-956 ng/mu L.
The recombinant plasmid transfects mesenchymal stemCells, every 5X 105Adding 2 mug of recombinant plasmid into the umbilical cord mesenchymal stem cells, performing electric transfer, immediately transferring the cells into a preheated mesenchymal stem cell culture medium after the electric transfer is completed, putting the cells back into a 37 ℃ incubator for continuous culture for 24 hours, performing liquid change, and discarding the culture medium after the continuous culture for 24 hours; the mesenchymal stem cell culture medium is Hyclone mesenchymal culture medium containing platelet lysate.
After the screening and the electrotransfer are completed, 2mL of screening culture medium containing 400 mug/mL G418 is added into each hole, the screening culture medium is replaced once every 3 days until the cell fusion degree reaches 60%, the cells are passed to a small bottle, 10mL of maintenance culture medium containing 200 mug/mL G418 is replaced, and the cells are subjected to amplification culture.
The screening culture medium is a Hyclone mesenchymal culture medium added with 400 mug/mL G418 and containing platelet lysate; the maintenance culture medium is Hyclone mesenchymal culture medium added with 200 mug/mL G418 and containing platelet lysate.
The application of the MSC modified by the Exendin-4 fusion gene and the application of the MSC modified by the Exendin-4 fusion gene in preparing GLP-1 protein related medicaments.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention optimizes amino acid and codon of the Exendin-4 gene, and can improve the Exendin-4 expression quantity of MSC modified by Exendin-4 fusion gene.
(2) The fusion gene is obtained by using one of Exendin-4, IgG2, Albumin-1, Albumin and Ig kappa chain, so that the half-life of Exendin-4 protein can be prolonged, and the high concentration of Exendin-4 protein in vivo can be still maintained at 7 days after administration.
Drawings
FIG. 1 is a structural diagram of an Exendin-4 fusion gene;
FIG. 2 is an electrophoretogram of the recombinant expression vector pIRES2-eGFP-Exendin-4-IgG2 verified by double digestion in example 2;
FIG. 3 is a microscope picture of a P3 generation umbilical cord mesenchymal stem cell;
fig. 4 is a flow chart of flow cytometry for detecting CD105 markers of mesenchymal stem cells;
fig. 5 is a flow chart of flow cytometry to detect CD90 marker of mesenchymal stem cells;
fig. 6 is a flow chart of flow cytometry to detect CD73 marker of mesenchymal stem cells;
fig. 7 is a flow chart of flow cytometry to detect CD34 marker of mesenchymal stem cells;
FIG. 8 is a flow chart of flow cytometry for detection of HLA-DR markers of mesenchymal stem cells;
FIG. 9 is a flow chart of flow cytometry for detecting the expression rate of GFP in Exendin-4-IgG 2-MSC;
FIG. 10 is a flow chart of flow cytometry for detecting the expression rate of GFP in Exendin-4-Albumin-1-MSC;
FIG. 11 is a flow chart of flow cytometry for detecting the expression rate of GFP in Exendin-4-Albumin-MSC;
FIG. 12 is a flow chart of flow cytometry for detecting the expression rate of GFP in Exendin-4-Ig kappa chain-MSC;
FIG. 13 is a flow chart of flow cytometry for detecting the expression rate of GFP in Exendin-4-MSC;
FIG. 14 is a bar graph showing the relative expression amount of RNA of Exendin-4;
fig. 15 is a line graph showing the amount of insulin secretion from MIN6 cells after glucose stimulation.
Detailed Description
Example 1 Structure of fusion Gene
The fusion gene (Exendin-4-IgG 2) for modifying the mesenchymal stem cells comprises a signal peptide nucleic acid artificial sequence, an Exendin-4 nucleic acid artificial sequence, a Linker nucleic acid artificial sequence and an IgG2 nucleic acid artificial sequence, wherein the IgG2 nucleic acid artificial sequence can be replaced by Albumin-1, Albumin and Ig kappa chain to obtain an Exendin-4-Albumin-1 fusion gene, an Exendin-4-Albumin fusion gene and an Exendin-4-Ig kappa chain fusion gene, which are shown in figure 1. The Exendin-4-IgG2 fusion gene is formed by connecting a signal peptide nucleic acid artificial sequence, an Exendin-4 nucleic acid artificial sequence, a Linker nucleic acid artificial sequence and an IgG2 nucleic acid artificial sequence in series in sequence, wherein the nucleic acid sequence is SEQ ID NO. 1. Wherein the signal peptide nucleic acid human process sequence is SEQ ID NO. 2; the human process sequence of the Exendin-4 nucleic acid is SEQ ID NO. 3; the IgG2 nucleic acid human process sequence is SEQ ID NO.4, the Albumin-1 nucleic acid human process sequence is SEQ ID NO.5, the Albumin nucleic acid human process sequence is SEQ ID NO.6, and the Ig kappa chain nucleic acid human process sequence is SEQ ID NO. 7; the nucleic acid sequence of the Exendin-4-Albumin-1 fusion gene is SEQ ID NO. 8; the nucleic acid sequence of the Exendin-4-Albumin fusion gene is SEQ ID NO. 9; the nucleic acid sequence of the Exendin-4-Ig kappa chain fusion gene is SEQ ID NO. 10.
EXAMPLE 2 preparation of recombinant plasmid
In this embodiment, IgG2 is taken as an example for illustration, and when any of Albumin-1, Albumin and Ig kappa chain is adopted, the preparation method is basically the same, and will not be described in detail herein.
The fusion gene (Exendin-4-IgG 2) was synthesized by Shanghai Czeri bioengineering GmbH, and the synthesized fusion gene sequence was digested with EcoRI and BamHI to obtain a target fragment having a cohesive end. Meanwhile, carrying out EcoRI and BamHI double enzyme digestion on a vector pIRES2-eGFP (purchased from Shanghai leaf Biotechnology Co., Ltd.) to obtain a linearized vector fragment, connecting the target fragment and the linearized vector fragment by using T4 ligase, transforming the linearized vector fragment into E.coli (Top10), carrying out enzyme digestion verification (see figure 2), extracting plasmids by using a plasmid extraction kit of an OMEGA company after the sequencing is correct, and obtaining recombinant expression vectors pIRES2-eGFP-Exendin-4-IgG2, pIRES2-eGFP-Exendin-4-Albumin-1, pIRES2-eGFP-Exendin-4-Albumin, pIRES2-eGFP-Exendin-4-Ig kappa chain and pIRES 2-eGFP-Exendin-4. The concentration of the recombinant expression vector pIRES2-eGFP-Exendin-4-IgG2 extracted in the invention is 950 ng/muL, the concentration of pIRES2-eGFP-Exendin-4-Albumin-1 is 870 ng/muL, the concentration of pIRES2-eGFP-Exendin-4-Albumin is 924 ng/muL, the concentration of pIRES2-eGFP-Exendin-4-Ig kappa chain is 855 ng/muL, and the concentration of RESpIRE 2-eGFP-Exendin-4 is 956 ng/muL.
Example 3 preparation of umbilical cord mesenchymal Stem cells
Collecting umbilical cord of newborn donated in hospital, sterilizing twice with 75wt% alcohol in clean bench, placing in culture dish, and removing Fahrenheit gelatin group with forcepsWeaving, cutting with scissors to 0.5mm2Small pieces of size. Sheared Fahrenheit tissue was transferred to culture flasks and cultured in Hyclone mesenchymal medium (purchased from Hyclone) containing platelet lysate and observed daily with a microscope. When 80% of the grown stem cells are paved on the bottom of the bottle, passage is carried out, the cell growth speed is accelerated after passage, one passage is carried out every 2-3 days, the cell growth speed is transmitted to P3 for substituting for an experiment (see figure 3), and markers CD105, CD73, CD90, CD34 and HLA-DR of umbilical cord mesenchymal stem cells are detected by a flow cytometer (see figures 4-8).
EXAMPLE 4 transfection of mesenchymal Stem cells with recombinant plasmids and selection
Preparation before electric transfer: umbilical cord mesenchymal stem cells in log phase (umbilical cord mesenchymal stem cells of P3 generation obtained in example 3) in T75 flask were digested with TrypLE-Select enzyme (purchased from gibco, cat. No. 12563029), collected in a 50mL centrifuge tube, centrifuged at 400g for 5min, and the supernatant was removed for use. Taking out the electrotransformation liquid (P1 Primary Cell 4D X Kit L nuclear transfer Kit from LONZA, V4 XP-1012) from a refrigerator at 4 deg.C, placing 1.5mL of the electrotransformation liquid in a centrifuge tube, returning to room temperature, and returning the rest of the electrotransformation liquid to the refrigerator at 4 deg.C; take 7.5X 106And the cells are resuspended by using 1.5mL of electrotransfer liquid, the cells are divided into five parts on average, 6 mu g of pIRES2-eGFP-Exendin-4-IgG2, pIRES2-eGFP-Exendin-4-Albumin-1, pIRES2-eGFP-Exendin-4-Albumin, pIRES2-eGFP-Exendin-4-Ig kappa chain and pIRES2-eGFP-Exendin-4 are respectively added and uniformly mixed, the number of mesenchymal stem cells added into each electrotransfer cup is 5 multiplied by 105Each, 3 electric tumblers are required.
Placing the electric rotary cup into LONZA 4D electric rotary instrument, selecting mesenchymal stem cell electric rotary procedure to make electric rotary, after the electric rotary is completed, transferring the cell into preheated mesenchymal stem cell culture medium, adding 5X 10 to every hole5The cells were returned to the 37 ℃ incubator for further incubation for 24 hours, and the medium was changed.
The preheated mesenchymal culture medium is added into a six-hole plate in advance, 2mL of culture medium is added into each hole, and the mixture is preheated to 37 ℃;
the mesenchymal stem cell medium was Hyclone mesenchymal medium (purchased from Hyclone) containing platelet lysate as in example 3.
After the culture is continued for 24h after the liquid change, the culture medium in the six-well plate is discarded, a screening culture medium containing 400 mug/mL G418 is added (2 mL of screening culture medium is added to each well, namely Hyclone mesenchymal culture medium containing platelet lysate added with G418), and the screening culture medium is replaced every 3 days. Until the cell fusion degree reaches 60%, the cells are passaged into a small bottle, 10mL of maintenance medium containing 200 mug/mL G418 (200 mug/mL G418 added Hyclone mesenchymal medium containing platelet lysate) is replaced, and the cells are subjected to amplification culture. Meanwhile, a flow cytometer is utilized to detect the GFP expression rate of the screened mesenchymal stem cells, wherein the GFP expression rate of the Exendin-4-IgG2-MSC is 93.1%, the GFP expression rate of the Exendin-4-Albumin-1-MSC is 94.2%, the GFP expression rate of the Exendin-4-Albumin-MSC is 94.5%, the GFP expression rate of the Exendin-4-Ig kappa chain-MSC is 93.6%, and the GFP expression rate of the Exendin-4-MSC is 95.6% (see fig. 9-13).
Example 5 Gene expression level of Exendin-4 in MSC modified with fusion Gene
Respectively inoculating the same number of different cells into T75 bottles, adding 10mL of mesenchymal stem cell culture medium into each bottle, culturing for 48h, and collecting the cells. Total RNA of cells was extracted using RNeasy Mini Kit (cat # 74104) from QIAGEN, reverse transcription was performed according to the instructions of the reverse transcription Kit (purchased from Ecori, cat # AG 11728), real-time fluorescent quantitative PCR was performed using SYBR green Kit (purchased from Solebao, cat # SR 2110-50), and the gene expression levels of Exendin-4 in 5 kinds of genetically modified mesenchymal stem cells and normal mesenchymal stem cells were examined.
The experimental result is shown in fig. 14, compared with the normal mesenchymal stem cell, the gene expression amount of the Exendin-4 of the genetically modified mesenchymal stem cell is obviously increased, wherein the expression amount of the Exendin-4 in the Exendin-4-IgG2-MSC is 2958 times that of the normal mesenchymal stem cell, the expression amount of the Exendin-4 in the Exendin-4-Albumin-1-MSC is 2832 times that of the normal mesenchymal stem cell, the expression amount of the Exendin-4 in the Exendin-4-Albumin-MSC is 2896 times that of the normal mesenchymal stem cell, the expression amount of the Exendin-4 in the Exendin-4-Ig kappa chain-MSC is 2780 times that of the normal mesenchymal stem cell, and the expression amount of the Exendin-4 in the Exendin-4-MSC is 3050 times that of the normal mesenchymal stem cell.
Example 6 protein expression amount of Exendin-4 in MSC modified by fusion Gene
MSCs modified by different fusion genes at 1X 105Per cm2The cell culture supernatant was inoculated into a T75 flask, 10mL of a medium was added to a control group of normally cultured MSCs, the cells were cultured at 37 ℃ for 24 hours, 48 hours, 72 hours, and 96 hours, cell culture supernatants of different cells were collected at different time periods, the concentration of Exendin-4 in the cell culture supernatants was measured with an Exendin-4 ELISA kit (purchased from Jianglai Bio, cat # JL 18186), and the cell culture supernatants were diluted 10-fold and 100-fold with a diluent, respectively, for detection. The specific detection steps are as follows:
(1) the required laths were taken out of the aluminum foil bag after equilibration for 60min at room temperature, and the remaining laths were sealed with a valve bag and placed back at 4 ℃.
(2) A standard well, a blank well and a sample well are provided, and 50 μ L of standard with different concentrations is added to each standard well.
(3) Adding 50 mu L of sample to be detected into the sample hole; blank wells were loaded with 50. mu.L of this dilution.
(4) Each of the blank well, the standard well and the sample well was filled with 100. mu.L of detection antibody labeled with horseradish peroxidase (HRP), the reaction wells were sealed with a sealing plate film, and incubated in a 37 ℃ water bath or incubator for 60 min.
(5) Discarding the liquid, patting dry on absorbent paper, filling 350 μ L of washing solution into each hole, standing for 1min, throwing off the washing solution, patting dry on absorbent paper, and repeating the plate washing for 5 times.
(6) 50. mu.L of substrate A, B was added to each well and incubated at 37 ℃ for 15min in the absence of light.
(7) Add stop solution 50. mu.L to each well, measure the OD value of each well at a wavelength of 450nm within 15 min.
TABLE 1 amount of Exendin-4 secretion from MSCs modified with different fusion genes
As shown in the table 1, the amount of the Exendin-4 protein secreted by the mesenchymal stem cells modified by different fusion genes is increased along with the increase of time, and at 96h, the amount of the Exendin-4-IgG2-MSC secreted by the Exendin-4 protein reaches 356.54 ng/mL.
Example 7 Effect of fusion Gene-modified MSCs on insulin secretion
MIN6 cells (purchased from Shanghai Tong Seiko Co., Ltd.) were plated in six well plates for a total of 36 wells, and the plates were divided into six groups of 6 wells each having a seeding density of 1X 105Each well of each cell was supplemented with 2mL of complete medium (low-sugar DMEM medium containing 10% Vol of FBS and 50 uM. beta. -mercaptoethanol), and cultured in an incubator at 37 ℃ for 24 hours. 1mL of the medium was aspirated, and 1X 10 of the medium was added to each group51mL of mesenchymal stem cell culture medium is added into the MSC modified by Exendin-4-IgG2, the MSC modified by Exendin-4-Albumin-1, the MSC modified by Exendin-4-Albumin, the MSC modified by Exendin-4-Ig kappa chain, the MSC modified by Exendin-4 and the common MSC for mixed culture, wherein the common MSC cell is used as a control group.
After 24h, glucose with a final concentration of 10mM was added to all wells to stimulate cell secretion, and each well was tested at 30min, 60min, 12h, 1d, 3d, and 5d, and the amount of insulin secreted from the cell culture supernatant was determined using a mouse insulin ELISA kit (purchased from SeKM0141, a Biotech Co., Ltd., Beijing Sorbombo).
As shown in fig. 15, the amount of insulin secretion began to increase after 0.5h, and reached the maximum at 24h, and slowly decreased with time. Compared with the control group of MIN6 treated with normal MSC cells, the fusion gene-modified mesenchymal stem cells promoted the secretion of insulin. At 24h, the secretion amount of insulin in the Exendin-4-IgG2-MSC experimental group is 446ng, the secretion amount of insulin in the Exendin-4-Albumin-1-MSC experimental group is 432ng, the secretion amount of insulin in the Exendin-4-Albumin-MSC experimental group is 421ng, the secretion amount of insulin in the Exendin-4-Ig kappa chain-MSC experimental group is 438ng, and the secretion amount of insulin in the Exendin-4-MSC experimental group is 426 ng.
Example 8 Effect of fusion Gene-modified MSCs on apoptosis of mouse insulinoma cells
The MSCs and MIN6 cells were plated separately in six well plates, 18 wells for each cell, at a seeding density of 1 × 10 per well5Each of which is 1X 10 in each cell5Co-culturing Exendin-4-IgG2-MSC, Exendin-4-Albumin-1-MSC, Exendin-4-Albumin-MSC, Exendin-4-Ig kappa chain-MSC, Exendin-4-MSC and 1mL PBS, repeating 3 wells, and adding 2mL of culture medium into each well.
After overnight cell culture, the cells were treated with glucose at a final concentration of 25mM for 5 days. Then using H with a final concentration of 0.125 mM2O2Cells were treated for 6 hours and then treated with Annexin V-FITC Apoptosis Detection Kit I (from BD) and PI (Propidium Iodide, from BD) as follows, and the Apoptosis rate was measured by flow cytometry (from Beckman).
(1) Diluting the 10 multiplied Binding Buffer into 1 multiplied Binding Buffer by deionized water;
(2) and (4) collecting the cells. Digesting and collecting with TrypLE (trypsin) -like Select enzyme, centrifuging at room temperature and 2000rpm for 5 minutes, and collecting cells;
(3) washing the cells, resuspending the cells once in precooled 1 × PBS (4 ℃), centrifuging at 2000rpm for 5 minutes, and washing the cells;
(4) adding 300 mu L of 1 × Binding Buffer suspension cells;
(5) annexin V-FITC labeling: adding 5 mu L Annexin V-FITC, mixing uniformly, keeping out of the sun, and incubating for 30 minutes at room temperature;
(6) PI marking: 5 μ L of PI was added for staining 5 minutes before loading.
(7) Before loading, 200. mu.L of 1 XBinding Buffer is added.
TABLE 2 apoptosis rates of MIN6 and MSC cells
The results are shown in table 2, after the fusion gene modified MSC cells were co-cultured with MIN6 cells, MIN6 apoptosis was inhibited and the survival rate of MIN6 cells was increased, but no effect was observed on normal MSC cells, indicating that Exendin-4 gene inhibited islet cell apoptosis and increased survival rate.
Example 9 expression level of Exendin-4 in mice by MSC modified with fusion gene
18-22g male BLAB/C mice (purchased from Shenyang blue Spectrum Dars laboratory science and technology Co., Ltd.) were housed in animal houses (room temperature 23 + -2 deg.C, humidity 50% + -10%), and fed with high-fat diet, and 1 × 10 mice were collected6The MSC cells modified by different fusion genes and the common MSC cells are injected into a mouse body through tail veins, and the expression quantity of Exendin-4 in serum at different time points in the mouse body is detected by adopting an ELISA method.
TABLE 3 expression level of Exendin-4 in mouse serum
As a result, as shown in Table 3, the expression level of Exendin-4 in mice injected with the fused gene-modified MSC reached a peak at day 6, and the expression level of Exendin-4 in mice injected with Exendin-4-MSC reached a peak at day 4, and then gradually decreased. However, the expression level of Exendin-4 in the Exendin-4-IgG2-MSC group was significantly lower than that of Exendin-4 in the Exendin-4-MSC group. The degradation speed of the Exendin-4 added with fusion genes such as IgG2 is obviously slower than that of Exendin-4 single gene, and the existence time of Exendin-4 protein is prolonged.
Sequence listing
<110> Shandong Xingyi Biotechnology Ltd
<120> Exendin-4 fusion gene modified MSC and application thereof
<130> 2021
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1209
<212> DNA
<213> Homo sapiens
<400> 1
atgaaaatca tcctgtggct gtgtgttttt gggctgttcc ttgcaacttt attccctatc 60
agctggcaac atggtgaagg cacatttacc tctgacttgt caaagcagat ggaggaggaa 120
gcagtgcggt tatttattga gtggcttaag aacggaggac caagtagcgg ggcacctccg 180
tctaagaaaa agaaaaagaa aggaggagga ggaagcggag gaggaggaag cgcttcaact 240
aaaggcccgt ctgtgttccc tctggctcct tgttcaagat ctacatcaga atcaaccgcc 300
gcactcggct gtctggtgaa ggactatttt cctgaacccg ttaccgtttc ctggaacagc 360
ggtgcgctca ccagcggcgt gcacaccttc ccagctgtcc tacagtcctc aggactctac 420
tccctcagca gcgtggtgac cgtgccctcc agcaacttcg gcacccagac ctacacctgc 480
aacgtagatc acaagcccag caacaccaag gtggacaaga cagttgagcg caaatgttgt 540
gtcgagtgcc caccgtgccc agcaccacct gtggcaggac cgtcagtctt cctcttcccc 600
ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacgtg cgtggtggtg 660
gacgtgagcc acgaagaccc cgaggtccag ttcaactggt acgtggacgg cgtggaggtg 720
cataatgcca agacaaagcc acgggaggag cagttcaaca gcacgttccg tgtggtcagc 780
gtcctcaccg ttgtgcacca ggactggctg aacggcaagg agtacaagtg caaggtctcc 840
aacaaaggcc tcccagcccc catcgagaaa accatctcca aaaccaaagg gcagccccga 900
gaaccacagg tgtacaccct gcccccatcc cgggaggaga tgaccaagaa ccaggtcagc 960
ctgacctgcc tggtcaaagg cttctacccc agcgacatcg ccgtggagtg ggagagcaat 1020
gggcagccgg agaacaacta caagaccaca cctcccatgc tggactccga cggctccttc 1080
ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1140
tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1200
ccgggtaaa 1209
<210> 2
<211> 69
<212> DNA
<213> Homo sapiens
<400> 2
atgaaaatca tcctgtggct gtgtgttttt gggctgttcc ttgcaacttt attccctatc 60
agctggcaa 69
<210> 3
<211> 132
<212> DNA
<213> Homo sapiens
<400> 3
catggtgaag gcacatttac ctctgacttg tcaaagcaga tggaggagga agcagtgcgg 60
ttatttattg agtggcttaa gaacggagga ccaagtagcg gggcacctcc gtctaagaaa 120
aagaaaaaga aa 132
<210> 4
<211> 978
<212> DNA
<213> Homo sapiens
<400> 4
gcttcaacta aaggcccgtc tgtgttccct ctggctcctt gttcaagatc tacatcagaa 60
tcaaccgccg cactcggctg tctggtgaag gactattttc ctgaacccgt taccgtttcc 120
tggaacagcg gtgcgctcac cagcggcgtg cacaccttcc cagctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc 240
tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 300
aaatgttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 360
ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc 420
gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 480
gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt 540
gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga gtacaagtgc 600
aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 660
cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 720
caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 780
gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac 840
ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 900
gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 960
tccctgtctc cgggtaaa 978
<210> 5
<211> 576
<212> DNA
<213> Homo sapiens
<400> 5
cgtggtgtgt tccgtcgtga tgctcacaaa tctgaagtag cacaccgttt caaagatctg 60
ggtgaggaaa actttaaagc gctggtcctg atcgccttcg cccagtatct gcagcagtgc 120
ccattcgagg accacgttaa gttggttaac gaggttactg agttcgctaa gacttgtgtt 180
gctgacgaat ccgctgagaa ctgtgataag tccttgcaca ctttgttcgg tgacaagttg 240
tgtactgttg ctactttgag agaaacttac ggtgagatgg ctgactgttg tgctaagcaa 300
gagcctgaga gaaacgagtg tttcttgcaa cacaaggacg acaacccaaa cttgccaaga 360
ttggttagac cagaggttga cgttatgtgt actgctttcc acgacaacga agagactttc 420
ttgaagaagt acttgtacga gatcgctaga agacacccat acttctacgc tccagagttg 480
ttgttcttcg ctaagagata caaggctgct ttcactgagt gttgtcaggc tgctgataag 540
gctgcttgtt tgttgccaaa gttggacgag ttgaga 576
<210> 6
<211> 1773
<212> DNA
<213> Homo sapiens
<400> 6
cgtggtgtgt tccgtcgtga tgctcacaaa tctgaagttg cgcaccgttt taaagacctg 60
ggcgaagaga actttaaagc cctggtactg atcgctttcg ctcaatacct gcaacagtgc 120
ccgttcgaag accacgtgaa actggttaac gaagtaactg aatttgcaaa aacctgcgtc 180
gctgacgaat ctgctgaaaa ctgtgacaag tctttgcaca ctttgttcgg tgacaagttg 240
tgtactgttg ctactttgag agaaacttac ggtgaaatgg ctgactgttg tgctaagcaa 300
gaaccagaaa gaaacgaatg tttcttgcaa cacaaggacg acaacccaaa cttgccaaga 360
ttggttagac cagaagtcga cgttatgtgt actgctttcc acgacaacga agaaactttc 420
ttgaagaagt acttgtacga aattgctaga agacacccat acttctacgc tccagaattg 480
ttgttcttcg ctaagagata caaggctgct ttcactgaat gttgtcaagc tgctgacaag 540
gctgcttgtt tgttgccaaa gttggacgaa ttgagagacg aaggtaaggc ttcttctgct 600
aagcaaagat tgaagtgtgc ttctttgcaa aagttcggtg aaagagcttt caaagcttgg 660
gctgttgcta gattgtctca aagattccca aaggctgaat ttgctgaagt ttctaagttg 720
gttactgact tgactaaggt tcacactgaa tgttgtcacg gtgacttgtt ggaatgtgct 780
gacgacagag ctgacttggc taagtacatt tgtgaaaacc aagactctat ttcttctaag 840
ttgaaggaat gttgtgaaaa gccattgttg gaaaagtctc actgtattgc tgaagttgaa 900
aacgacgaaa tgccagctga cttgccatct ttggctgctg acttcgttga atctaaggac 960
gtttgtaaga actacgctga agctaaggac gttttcttgg gtatgttctt gtacgaatac 1020
gctagaagac acccagacta ctctgttgtt ttgttgttga gattggctaa gacttacgaa 1080
actactttgg aaaagtgttg tgcggccgct gacccacacg aatgttacgc taaggttttc 1140
gacgaattta agccattggt tgaagaacca caaaacttga ttaagcaaaa ctgtgaattg 1200
ttcgaacaat tgggtgaata caagttccaa aacgctttgt tggttagata cactaagaag 1260
gttccacaag tttctactcc aactttggtt gaagtttcta gaaacttggg taaggttggt 1320
tctaagtgtt gtaagcaccc agaagctaag agaatgccat gtgctgaaga ctacttgtct 1380
gttgttttga accaattgtg tgttttgcac gaaaagactc cagtttctga cagagttact 1440
aagtgttgta ctgaatcttt ggttaacaga agaccatgtt tctctgcttt ggaagttgac 1500
gaaacttacg ttccaaagga atttaacgct gaaactttca ctttccacgc tgacatttgt 1560
actttgtctg aaaaggaaag acaaattaag aagcaaactg ctttggttga attggttaag 1620
cacaagccaa aggctactaa ggaacaattg aaggctgtta tggacgactt cgctgctttc 1680
gttgagaaat gctgcaaagc ggatgacaaa gaaacgtgct ttgcggaaga aggtaaaaaa 1740
ctggtagcgg cgtcccaagc agctctgggt ctg 1773
<210> 7
<211> 300
<212> DNA
<213> Homo sapiens
<400> 7
gctccgtctg ttttcatctt cccgccgtcc gatgaacagc tgaaatccgg tactgcgagc 60
gttgtttgtc tgctgaataa cttctatccc agagaggcca aagtacagtg gaaggtggat 120
aacgccctcc aatcgggtaa ctcccaggag agtgtcacag agcaggacag caaggacagc 180
acctacagcc tcagcagcac cctgacgctg agcaaagcag actacgagaa acacaaagtc 240
tacgcctgcg aagtcaccca tcagggcctg agctcgcccg tcacaaagag cttcaacagg 300
<210> 8
<211> 807
<212> DNA
<213> Homo sapiens
<400> 8
atgaaaatca tcctgtggct gtgtgttttt gggctgttcc ttgcaacttt attccctatc 60
agctggcaac atggtgaagg cacatttacc tctgacttgt caaagcagat ggaggaggaa 120
gcagtgcggt tatttattga gtggcttaag aacggaggac caagtagcgg ggcacctccg 180
tctaagaaaa agaaaaagaa aggaggagga ggaagcggag gaggaggaag ccgtggtgtg 240
ttccgtcgtg atgctcacaa atctgaagta gcacaccgtt tcaaagatct gggtgaggaa 300
aactttaaag cgctggtcct gatcgccttc gcccagtatc tgcagcagtg cccattcgag 360
gaccacgtta agttggttaa cgaggttact gagttcgcta agacttgtgt tgctgacgaa 420
tccgctgaga actgtgataa gtccttgcac actttgttcg gtgacaagtt gtgtactgtt 480
gctactttga gagaaactta cggtgagatg gctgactgtt gtgctaagca agagcctgag 540
agaaacgagt gtttcttgca acacaaggac gacaacccaa acttgccaag attggttaga 600
ccagaggttg acgttatgtg tactgctttc cacgacaacg aagagacttt cttgaagaag 660
tacttgtacg agatcgctag aagacaccca tacttctacg ctccagagtt gttgttcttc 720
gctaagagat acaaggctgc tttcactgag tgttgtcagg ctgctgataa ggctgcttgt 780
ttgttgccaa agttggacga gttgaga 807
<210> 9
<211> 2004
<212> DNA
<213> Homo sapiens
<400> 9
atgaaaatca tcctgtggct gtgtgttttt gggctgttcc ttgcaacttt attccctatc 60
agctggcaac atggtgaagg cacatttacc tctgacttgt caaagcagat ggaggaggaa 120
gcagtgcggt tatttattga gtggcttaag aacggaggac caagtagcgg ggcacctccg 180
tctaagaaaa agaaaaagaa aggaggagga ggaagcggag gaggaggaag ccgtggtgtg 240
ttccgtcgtg atgctcacaa atctgaagtt gcgcaccgtt ttaaagacct gggcgaagag 300
aactttaaag ccctggtact gatcgctttc gctcaatacc tgcaacagtg cccgttcgaa 360
gaccacgtga aactggttaa cgaagtaact gaatttgcaa aaacctgcgt cgctgacgaa 420
tctgctgaaa actgtgacaa gtctttgcac actttgttcg gtgacaagtt gtgtactgtt 480
gctactttga gagaaactta cggtgaaatg gctgactgtt gtgctaagca agaaccagaa 540
agaaacgaat gtttcttgca acacaaggac gacaacccaa acttgccaag attggttaga 600
ccagaagtcg acgttatgtg tactgctttc cacgacaacg aagaaacttt cttgaagaag 660
tacttgtacg aaattgctag aagacaccca tacttctacg ctccagaatt gttgttcttc 720
gctaagagat acaaggctgc tttcactgaa tgttgtcaag ctgctgacaa ggctgcttgt 780
ttgttgccaa agttggacga attgagagac gaaggtaagg cttcttctgc taagcaaaga 840
ttgaagtgtg cttctttgca aaagttcggt gaaagagctt tcaaagcttg ggctgttgct 900
agattgtctc aaagattccc aaaggctgaa tttgctgaag tttctaagtt ggttactgac 960
ttgactaagg ttcacactga atgttgtcac ggtgacttgt tggaatgtgc tgacgacaga 1020
gctgacttgg ctaagtacat ttgtgaaaac caagactcta tttcttctaa gttgaaggaa 1080
tgttgtgaaa agccattgtt ggaaaagtct cactgtattg ctgaagttga aaacgacgaa 1140
atgccagctg acttgccatc tttggctgct gacttcgttg aatctaagga cgtttgtaag 1200
aactacgctg aagctaagga cgttttcttg ggtatgttct tgtacgaata cgctagaaga 1260
cacccagact actctgttgt tttgttgttg agattggcta agacttacga aactactttg 1320
gaaaagtgtt gtgcggccgc tgacccacac gaatgttacg ctaaggtttt cgacgaattt 1380
aagccattgg ttgaagaacc acaaaacttg attaagcaaa actgtgaatt gttcgaacaa 1440
ttgggtgaat acaagttcca aaacgctttg ttggttagat acactaagaa ggttccacaa 1500
gtttctactc caactttggt tgaagtttct agaaacttgg gtaaggttgg ttctaagtgt 1560
tgtaagcacc cagaagctaa gagaatgcca tgtgctgaag actacttgtc tgttgttttg 1620
aaccaattgt gtgttttgca cgaaaagact ccagtttctg acagagttac taagtgttgt 1680
actgaatctt tggttaacag aagaccatgt ttctctgctt tggaagttga cgaaacttac 1740
gttccaaagg aatttaacgc tgaaactttc actttccacg ctgacatttg tactttgtct 1800
gaaaaggaaa gacaaattaa gaagcaaact gctttggttg aattggttaa gcacaagcca 1860
aaggctacta aggaacaatt gaaggctgtt atggacgact tcgctgcttt cgttgagaaa 1920
tgctgcaaag cggatgacaa agaaacgtgc tttgcggaag aaggtaaaaa actggtagcg 1980
gcgtcccaag cagctctggg tctg 2004
<210> 10
<211> 531
<212> DNA
<213> Homo sapiens
<400> 10
atgaaaatca tcctgtggct gtgtgttttt gggctgttcc ttgcaacttt attccctatc 60
agctggcaac atggtgaagg cacatttacc tctgacttgt caaagcagat ggaggaggaa 120
gcagtgcggt tatttattga gtggcttaag aacggaggac caagtagcgg ggcacctccg 180
tctaagaaaa agaaaaagaa aggaggagga ggaagcggag gaggaggaag cgctccgtct 240
gttttcatct tcccgccgtc cgatgaacag ctgaaatccg gtactgcgag cgttgtttgt 300
ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 360
caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 420
ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 480
gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag g 531
Claims (8)
- An Exendin-4 fusion gene modified MSC characterized by: the Exendin-4 fusion gene is obtained by connecting Exendin-4 with one of IgG2, Albumin-1, Albumin and Ig kappa chain in series;the nucleic acid human process sequence of the Exendin-4 is shown as SEQ ID NO. 3; the nucleic acid human process sequence of the IgG2 is shown as SEQ ID NO.4, the nucleic acid human process sequence of the Albumin-1 is shown as SEQ ID NO.5, the nucleic acid human process sequence of the Albumin is shown as SEQ ID NO.6, and the nucleic acid human process sequence of the Ig kappa chain is shown as SEQ ID NO. 7.
- 2. The Exendin-4 fusion gene modified MSC of claim 1, wherein: the Exendin-4 fusion gene is one of Exendin-4-IgG2, Exendin-4-Albumin-1, Exendin-4-Albumin and Exendin-4-Ig kappa chain; the nucleic acid sequence of the Exendin-4-IgG2 is shown in SEQ ID NO. 1; the nucleic acid sequence of the Exendin-4-Albumin-1 is shown in SEQ ID NO. 8; the nucleic acid sequence of the Exendin-4-Albumin is shown in SEQ ID NO. 9; the nucleic acid sequence of the Exendin-4-Ig kappa chain is shown in SEQ ID NO. 10.
- 3. The Exendin-4 fusion gene modified MSC of claim 1, wherein: the preparation method of the MSC modified by the Exendin-4 fusion gene comprises the steps of synthesizing the fusion gene, preparing recombinant plasmids, transfecting mesenchymal stem cells by the recombinant plasmids and screening.
- 4. The Exendin-4 fusion gene modified MSC of claim 3, characterized in that: the preparation method comprises the steps of preparing a recombinant plasmid, loading an Exendin-4 fusion gene by using a vector pIRES2-eGFP, transforming the fusion gene to E.coli, and extracting to obtain the recombinant plasmid with the concentration of 855-956 ng/mu L.
- 5. The Exendin-containing material as claimed in claim 34 a fusion gene modified MSC characterized by: the recombinant plasmid transfects mesenchymal stem cells, and each 5 multiplied by 105Adding 2 mug of recombinant plasmid into the umbilical cord mesenchymal stem cells, performing electric transfer, immediately transferring the cells into a preheated mesenchymal stem cell culture medium after the electric transfer is completed, putting the cells back into a 37 ℃ incubator for continuous culture for 24 hours, performing liquid change, and discarding the culture medium after the continuous culture for 24 hours; the mesenchymal stem cell culture medium is Hyclone mesenchymal culture medium containing platelet lysate.
- 6. The Exendin-4 fusion gene modified MSC of claim 3, characterized in that: after the screening and the electrotransfer are completed, 2mL of screening culture medium containing 400 mug/mL G418 is added into each hole, the screening culture medium is replaced once every 3 days until the cell fusion degree reaches 60%, the cells are passed to a small bottle, 10mL of maintenance culture medium containing 200 mug/mL G418 is replaced, and the cells are subjected to amplification culture.
- 7. The Exendin-4 fusion gene modified MSC of claim 6, characterized in that: the screening culture medium is a Hyclone mesenchymal culture medium added with 400 mug/mL G418 and containing platelet lysate; the maintenance culture medium is Hyclone mesenchymal culture medium added with 200 mug/mL G418 and containing platelet lysate.
- The application of MSC modified by Exendin-4 fusion gene is characterized in that: the use of the Exendin-4 fusion gene modified MSC of claim 1 in the preparation of GLP-1 protein related drugs.
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| AU2002226897A1 (en) * | 2000-12-07 | 2002-08-22 | Eli Lilly And Company | GLP-1 fusion proteins |
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| AU2015202305A1 (en) * | 2011-09-26 | 2015-06-25 | Irm Llc | Dual function proteins for treating metabolic disorders |
| CN109929806A (en) * | 2017-12-19 | 2019-06-25 | 北京吉源生物科技有限公司 | A kind of stem cell and application thereof of dual-gene modification |
| CN110628723A (en) * | 2019-09-05 | 2019-12-31 | 清华大学 | Gene modified MSCs for treating type 2 diabetes |
| CN112138025A (en) * | 2020-09-27 | 2020-12-29 | 东北师范大学 | Long-acting GLP-1 gene modified stem cell preparation, preparation method and application thereof |
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|---|---|---|---|---|
| PL393178A1 (en) * | 2000-12-07 | 2011-02-14 | Eli Lilly And Company | Heterogeneous fusion protein, pharmaceutical composition for the treatment of diabetic patients with insulin-independent diabetes and pharmaceutical composition for the treatment of obese patients |
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| AU2002226897A1 (en) * | 2000-12-07 | 2002-08-22 | Eli Lilly And Company | GLP-1 fusion proteins |
| CN101891823A (en) * | 2010-06-11 | 2010-11-24 | 北京精益泰翔技术发展有限公司 | Exendin-4 and analog fusion protein thereof |
| AU2015202305A1 (en) * | 2011-09-26 | 2015-06-25 | Irm Llc | Dual function proteins for treating metabolic disorders |
| CN109929806A (en) * | 2017-12-19 | 2019-06-25 | 北京吉源生物科技有限公司 | A kind of stem cell and application thereof of dual-gene modification |
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