A gene, a fusion protein, a recombinant vector, a recombinant cell, brain-targeted exosome and application thereof

Abstract

The application provides a fusion protein, a gene, a recombinant vector, a recombinant cell, brain-targeting exosomes and application thereof, and relates to the field of drug treatment. The exosomes can be precisely targeted to a target site as a drug delivery carrier to play a therapeutic role of genes and chemical drugs. In the application, a gene sequence of a highly expressed NCAM gene in microglial cells, a segment of a simulation peptide NMP2, and a gene sequence of an exosome membrane protein Lamp2b are connected, an expression vector is constructed, and the expression vector is transfected into a packaging cell, so that the NCAM simulation peptide NMP2 is presented on the surface of the exosomes, the exosomes have the ability of targeting microglial cells, the exosomes can carry therapeutic drugs to pass through the blood-brain barrier and enter the lesion area in the brain, regulate the microenvironment, promote the clearance of A beta deposition, and can be used for the treatment of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of drug therapy, specifically relating to a gene, fusion protein, recombinant vector, recombinant cell, brain-targeting exosome and their applications. Background Technology

[0002] Alzheimer's disease is a neurodegenerative disease and the most common type of dementia, accounting for 60% to 80% of all dementia cases. Microglia are one of the most important pathological features of Alzheimer's disease, acting as key mediators of neuroinflammation, participating in brain homeostasis, host defense against pathogens, and are related to the development of various central nervous system diseases. Pathologically, an increase in activated microglia has been found in neurodegenerative areas of Alzheimer's patients.

[0003] Currently, there are two promising approaches to treating Alzheimer's disease by regulating microglia: one is to inhibit the inflammatory response of microglia; the other is to enhance the anti-inflammatory and neuroprotective functions of microglia. Based on these two pathways, an increasing number of drugs are being developed to regulate microglia function. However, current drug therapy still has some limitations. First, drugs are inactivated through various metabolic processes after entering the body and are cleared by the body's immune system. Second, delivering drugs to the brain requires crossing the blood-brain barrier, which can prevent drugs from reaching microglia and exerting their effects. Third, the drugs have poor targeting, which can lead to them not effectively reaching microglia, thus weakening the therapeutic effect and even causing side effects due to off-target effects.

[0004] However, how to enable drugs to escape metabolic reactions and immune clearance while simultaneously targeting and regulating microglia function, reducing Aβ deposition, repairing neuronal damage, reversing neuroinflammation, and improving the pathological characteristics of Alzheimer's patients is a technical challenge that urgently needs to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide a fusion protein, gene, recombinant vector, recombinant cell, brain-targeting exosome and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] This invention connects the gene sequence of a mimic peptide NMP2, which is highly expressed in microglia, with the gene sequence of the exosome membrane protein Lamp2b. After constructing an expression vector and transfecting it into packaging cells, the NCAM mimic peptide NMP2 can be presented on the surface of exosomes, thereby enabling the exosomes to target microglia.

[0009] A fusion protein is encoded by the aforementioned gene.

[0010] The present invention also provides a recombinant vector comprising the above-mentioned genes.

[0011] The present invention also provides a recombinant cell comprising the above-described recombinant vector.

[0012] The present invention also provides a brain-targeting exosome extracted from the above-mentioned recombinant cells.

[0013] This invention also provides a method for preparing the above-mentioned brain-targeting exosomes, comprising:

[0014] S1. The gene sequence of the NCAM mimic peptide NMP2 was inserted into an expression vector containing the gene sequence of the exosome membrane protein Lamp2b to obtain a fusion protein expression vector.

[0015] S2. The fusion protein expression vector was transfected into packaging cells 293T, and stable cell lines were obtained after selection with puromycin.

[0016] S3. Tetracycline was added to the stable cell line to induce overexpression of the fusion protein expression vector. After culturing for 40-48 hours, the supernatant was collected and the target exosomes with NMP2 peptide modified on the surface were obtained by ultracentrifugation.

[0017] The present invention also provides the application of the above-mentioned brain-targeting exosomes as brain-targeting delivery carriers in the preparation of drugs for treating Alzheimer's disease.

[0018] The present invention also provides a drug for treating Alzheimer's disease, which is obtained by purifying curcumin and the above-mentioned brain-targeting exosomes after co-incubation at a mass ratio of 1-2:1-2.

[0019] Furthermore, the co-incubation conditions are as follows: in a dark environment, mix at room temperature and let stand for 5-10 minutes.

[0020] The present invention has the following beneficial effects:

[0021] This invention fuses the full-length NCAM mimic peptides NMP2 and Lamp2b into a single gene. After transfection of cells with an expression vector, this gene can present the NMP2 peptide on the surface of exosomes. These exosomes can target microglia, reduce the expression and secretion of inflammatory factors, promote the clearance of Aβ, and repair nerve damage, which can be used for the treatment of Alzheimer's disease. Attached Figure Description

[0022] Figure 1 Image of the fusion protein expression vector pTRE-Tight-NMP2-Lamp2b plasmid.

[0023] Figure 2Electron micrograph of NMP2 exosomes.

[0024] Figure 3 This is a graph showing the detection of NMP2 exosome-targeting peptides.

[0025] Figure 4 This is a graph showing the detection of exosome surface marker proteins.

[0026] Figure 5 This image shows the effect of NMP2 exosomes targeting BV2 cells.

[0027] Figure 6 The image shows the therapeutic effect of NMP2 exosomes loaded with drugs. In the image, A represents the expression level of IL-1β, B represents the expression level of TNF-α, and C represents the expression level of IL-6. Detailed Implementation

[0028] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0029] Example 1: Exosome preparation and drug loading.

[0030] I. Construction of the fusion protein expression vector pTRE-Tight-NMP2-Lamp2b.

[0031] Figure 1 The plasmid map shows that the present invention designs the NCAM mimic peptide NMP2 and the exosome membrane protein Lamp2b as a fusion protein, and the gene sequence encoding the fusion protein NMP2-Lamp2b is shown in SEQ ID NO.1.

[0032]

[0033] The specific procedure is as follows: The gene sequence expressing the fusion protein NMP2-Lamp2b is ligated into the pcDNA3.1 plasmid to construct the pcDNA3.1-NMP2-Lamp2b fusion expression vector; the NMP2-Lamp2b fragment is synthesized by a gene synthesis company and introduced into the pcDNA3.1 vector to obtain the pcDNA3.1-NMP2-Lamp2b vector; then, the above-mentioned pcDNA3.1-NMP2-Lamp2b vector and pTRE-Tight plasmid are double-digested with SfaI and NotI, respectively. The digestion products are ligated at 16℃, and the products are then gel-cleaved to obtain the fusion protein expression vector pTRE-Tight-NMP2-Lamp2b, the plasmid image of which is shown below. Figure 1 As shown.

[0034] II. Construction of a stable 293T-NMP2-Lamp2b cell line secreting NMP2 exosomes.

[0035] According to the electroporator's instruction manual, 8 μL of the fusion protein expression vector pTRE-Tight-NMP2-Lamp2b at a concentration of 500 ng / μL and 4 μL of a fusion protein expression vector at a concentration of 1×10⁻⁶ ng / μL were added. 5 After adding 293T cell suspension (cell / mL) to an electroporation cuvette, electroporation was performed at 390V for 30ms. After electroporation, the cells were transferred to fresh culture medium and cultured at 37℃ and 5% CO2 for 48h. Then, the medium was changed to a medium containing puromycin at a final concentration of 1μg / mL and cultured for another 48h. Stable 293T-NMP2-Lamp2b cell line was obtained and expanded.

[0036] III. Isolation, purification and identification of NMP2 exosomes.

[0037] 1. Separation and identification.

[0038] When the confluence of the 293T-NMP2-Lamp2b stable cell line reached 70%, the culture medium was replaced with medium containing tetracycline at a final concentration of 1 μg / mL. After 48 h of culture, the supernatant was collected, and NMP2 exosomes were extracted using differential ultracentrifugation at 4°C: 500 g, 10 min; 2000 g, 20 min; 10000 g, 30 min; 100000 g, 90 min. The precipitate was resuspended in 500 μL of PBS to obtain NMP2 exosomes. Electron microscopy results of NMP2 exosomes are shown below. Figure 2 As shown.

[0039] 2. Identification of NMP2 exosome surface markers.

[0040] (1) Detection of NMP2 expression in NMP2 exosomes by Western blotting.

[0041] NMP2 exosome lysis and protein release: Take 50 μL of NMP2 exosomes, add 20 μL of pre-cooled protein lysis buffer RIPA, lyse on ice for 30 min, vortex once every 10 min, then centrifuge at 12000g, 4℃ for 10 min, take the supernatant, which is the total exosome protein, and finally determine the protein concentration by BCA method.

[0042] 10 μL of total exosome protein was added to 2 μL of 5×SDS loading buffer, boiled at 100℃ for 10 min, and then loaded onto the gel. The gel was run at a constant current of 45 mA. After gel running, the protein was transferred to a nitrocellulose membrane via transfer at 250 mA for 1 h. The nitrocellulose membrane was blocked with 5% skim milk powder for 1 h, incubated overnight with Lamp2 primary antibody, and then incubated with fluorescently labeled IgG secondary antibody for 1 h before color development. The experimental results are as follows: Figure 3 As shown, exosomes without any modification were designated as the Unmod-Exo group, and exosomes labeled with the NMP2 peptide were designated as the NMP2-Exo group. Compared with the Unmod-Exo group, the expression level of Lamp2 protein in NMP2 exosomes in the NMP2-Exo group was significantly increased, indicating that the NMP2 targeting peptide was successfully overexpressed in NMP2 exosomes.

[0043] (2) Identification of NMP2 exosome surface markers CD9, CD81 and TSG101.

[0044] Total protein was extracted from NMP2 exosomes, and the expression of marker proteins CD9, CD81, and TSG101 was detected by Western blotting. The results are as follows: Figure 4 As shown, the NMP2 exosomes extracted from the NMP2-Exo group can be successfully detected to express the three marker proteins.

[0045] IV. Validation of the targeting of NMP2 exosomes to microglia.

[0046] NMP2 exosomes were stained using Sigma's PKH26 dye kit. The PKH26-stained NMP2 exosomes were added to microglial BV2 culture medium, and the cells were transferred to a high-content cell imaging analysis system for culture. Simultaneously, the uptake of NMP2 exosomes by the cells was captured in real time from 1 to 5 hours.

[0047] Experimental results are as follows Figure 5As shown in the image analysis from 1 to 5 hours after exosome uptake, compared with the Unmod-Exo group, the fluorescence signal of NMP2 exosomes in BV2 cells was stronger in the NMP2-Exo group at any time point, indicating that the exosomes modified with NMP2 can be taken up by BV2 cells more quickly and in greater quantities. These experiments demonstrate that NMP2 exosomes have good targeting properties for BV2 cells.

[0048] V. Drug loading of NMP2 exosomes and its therapeutic effects.

[0049] 1. Preparation and standard curve determination of curcumin.

[0050] 5 mg of curcumin was dissolved in 1.3573 mL of dimethyl sulfoxide to obtain a 10 mM curcumin stock solution. The solubility of curcumin in PBS was determined by slowly adding 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of the curcumin stock solution dropwise to EP tubes containing 1 mL of PBS. The OD values ​​of different concentrations of curcumin were measured at 450 nm using a microplate reader, and the concentration standard curve was obtained as y = 228.6x - 10.77, R0. 2 =0.9982.

[0051] 2. NMP2 exosomes loaded with curcumin.

[0052] 36.8 μg of curcumin stock solution was slowly added dropwise to 1 mL of PBS containing 36.8 μg of exosomes, while mixing. The mixture was then placed in a dark environment at room temperature for 10 min to load the exosomes.

[0053] The OD value of the remaining curcumin in the PBS solution was measured at 450 nm using a microplate reader. The content of the remaining curcumin was calculated using a concentration standard curve. Then, the content was calculated using the following formula:

[0054]

[0055] Where Wtotal represents the total amount of drug added, in grams, and Wfree represents the amount of remaining drug, in grams. The encapsulation efficiency of curcumin encapsulated by NMP2 exosomes was found to be 36.58%.

[0056] 3. NMP2 exosomes loaded with curcumin inhibited the release of inflammatory factors from microglia.

[0057] The experiment was divided into 6 groups, with the control group consisting of normally growing BV2 cells that were not treated with any drugs.

[0058] The LPS group consisted of BV2 cells pre-treated with 2 mL of 500 ng / mL lipopolysaccharide (LPS) for 12 h, followed by another 12 h of LPS treatment, and then centrifugation at 500 g for 10 min to collect the supernatant. The DMSO group consisted of BV2 cells pre-treated with 0.1% dimethyl sulfoxide (DMSO) for 12 h, followed by another 12 h of LPS treatment with 2 mL of 500 ng / mL LPS, and then centrifugation at 500 g for 10 min to collect the supernatant. The Curcumin group consisted of BV2 cells pre-treated with 20 μM curcumin for 12 h, followed by another 12 h of LPS treatment with 2 mL of 500 ng / mL LPS, and then centrifugation at 500 g for 10 min to collect the supernatant. The Unmod-Exo-Cur group consisted of BV2 cells pretreated with unmodified exosomes loaded with curcumin (3880 ng / mL) for 12 h, followed by treatment with 2 mL of lipopolysaccharide (LPS) at a concentration of 500 ng / mL for another 12 h, and then centrifuged at 500 g for 10 min to collect the supernatant. The NMP2-Exo-Cur group consisted of BV2 cells pretreated with NMP2 exosomes loaded with curcumin (3880 ng / mL) for 12 h, followed by treatment with LPS for another 12 h, and then centrifuged at 500 g for 10 min to collect the supernatant.

[0059] The supernatant was analyzed using an enzyme-linked immunosorbent assay (ELISA) kit, and IL-1β, TNF-α, and IL-6 were detected according to the manufacturer's instructions. The experimental results are as follows: Figure 6 As shown, compared with the control group, BV2 cells treated with lipopolysaccharide (LPS) showed a significant increase in the secretion of IL-1β, TNF-α, and IL-6. However, treatment with curcumin, unmodified exosomes loaded with curcumin, and NMP2 exosomes loaded with curcumin reduced the secretion of IL-1β, TNF-α, and IL-6. NMP2-Exo-Cur treatment resulted in the greatest reduction in the secretion of IL-1β, TNF-α, and IL-6. These results indicate that treatment with NMP2 peptide-modified exosomes loaded with curcumin effectively inhibits the release of inflammatory factors in BV2 cells.

[0060] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0061] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0062] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A gene, characterized in that, The nucleotide sequence is shown as SEQ ID NO.

1.

2. A fusion protein, characterized in that, encoded by the gene of claim 1.

3. A recombinant vector, characterized in that, comprising the gene of claim 1.

4. A recombinant cell, characterized in that, comprising the recombinant vector of claim 3.

5. A brain-targeting exosome, characterized in that, extracted from the recombinant cell of claim 4.

6. The method of claim 5, wherein the brain-targeting exosomes are prepared by, comprising: S1, inserting the gene sequence of NCAM mimetic peptide NMP2 into an expression vector containing the gene sequence of exosome membrane protein Lamp2b to obtain a fusion protein expression vector; S2, transfecting the fusion protein expression vector into packaging cells 293T, and obtaining a stable cell line through puromycin screening; S3, adding tetracycline to the stable cell line to induce overexpression of the fusion protein expression vector, collecting the supernatant after 40-48h of culture, and obtaining target exosomes with NMP2 peptide surface modification using ultracentrifugation.

7. The brain-targeting exosome of claim 5 as a brain-targeting delivery carrier for use in the preparation of a medicament for treating Alzheimer's disease.

8. A medicament for treating Alzheimer's disease, characterized by comprising a compound of the formula (I) as an active ingredient. The medicament is obtained by co-incubating curcumin with the brain-targeting exosome of claim 5 at a mass ratio of 1-2:1-2, and then purifying.

9. The medicament for treating Alzheimer's disease according to Claim 8, wherein The co-incubation conditions are: mixing at room temperature in a dark environment, and then standing for 5-10min.

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