Gene, fusion protein, recombinant vector, recombinant cell, brain-targeted exosome and application thereof

Abstract

The invention provides a fusion protein, a gene, a recombinant vector, a recombinant cell, a brain-targeted exosome and application thereof, and relates to the field of drug therapy. As a drug delivery carrier, the exosome can accurately target a target site so as to play a therapeutic role of genes and chemical drugs. According to the present invention, the gene sequence of the mimic peptide NMP2 of the highly expressed NCAM gene in the microglia is connected with the gene sequence of the exosome membrane protein Lamp2b, and the expression vector is constructed and transfected into the packaging cell so as to present the NCAM mimic peptide NMP2 on the surface of the exosome, such that the exosome has the microglia targeting ability, the exosome can carry therapeutic drugs to penetrate through a blood brain barrier to enter a lesion area in the brain, the microenvironment is adjusted, A beta deposition clearing is promoted, and the exosome can be used for treating the Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of drug therapy, and specifically relates to a gene, a fusion protein, a recombinant vector, a recombinant cell, a brain-targeted exosome and applications thereof. Background Art

[0002] Alzheimer's disease is a neurodegenerative disease and the most common type of dementia, accounting for 60% to 80% of the total number of people with dementia. Microglia are one of the most important pathological features of Alzheimer's disease and are key mediators of neuroinflammation. They participate in brain homeostasis and host defense against pathogens and are related to the occurrence and development of a variety of central nervous system diseases. Pathologically, activated microglia have also been found to increase in neurodegenerative areas of Alzheimer's patients.

[0003] There are currently two promising pathways for 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, there are more and more drugs that regulate microglial function, but there are still some defects in current drug treatments. First, after entering the body, the drug will be inactivated through various metabolisms and will be cleared by the body's immune system; secondly, the delivery of drugs to the brain requires passing through the blood-brain barrier, which prevents the drug from reaching microglia to exert its effects; thirdly, the drug has poor targeting, which will result in its inability to effectively reach microglia and thus weaken the therapeutic effect, and may even produce side effects due to off-target effects.

[0004] However, how to make drugs escape metabolic reactions and immune clearance, while at the same time targetedly regulating microglial function, reducing Aβ deposition, repairing neuronal damage, and reversing neuroinflammation, and improving the pathological characteristics of Alzheimer's patients, is a technical problem that needs to be solved urgently. Summary of the invention

[0005] The object of the present invention is to provide a fusion protein, a gene, a recombinant vector, a recombinant cell, a brain-targeted exosome and applications thereof.

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

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

[0008] The present invention connects the gene sequence of a peptide NMP2 of the NCAM gene that is highly expressed in microglia and the gene sequence of the exosome membrane protein Lamp2b, and constructs an expression vector and transfects it into packaging cells. The NCAM peptide NMP2 can be presented on the surface of the exosomes, thereby enabling the exosomes to have the ability to target microglia.

[0009] A fusion protein is encoded by the above-mentioned gene.

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

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

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

[0013] The present invention also provides a method for preparing the above-mentioned brain-targeted exosome, comprising:

[0014] S1. Insert 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;

[0015] S2. Transfect the fusion protein expression vector into packaging cell 293T, and obtain a stable transfected cell line through puromycin screening;

[0016] S3. Add tetracycline to the stable transfected cell line to induce overexpression of the fusion protein expression vector, collect the supernatant after culturing for 40-48 h, and obtain target exosomes with NMP2 peptide modified on the surface by ultra-high speed centrifugation method.

[0017] The present invention also provides the application of the above-mentioned brain-targeted exosome as a brain-targeted delivery vector 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 co-incubating curcumin with the above-mentioned brain-targeted exosome at a mass ratio of 1-2:1-2 and then purifying.

[0019] Furthermore, the co-incubation conditions are: in a dark environment, mix evenly at room temperature and then stand still for 5-10 min.

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

[0021] In the present invention, the full length of NCAM mimetic peptide NMP2 and Lamp2b are fused as a complete gene. After the gene is transfected into cells by an expression vector, the NMP2 peptide can be presented on the surface of exosomes. The exosomes can target microglia, reduce the expression and secretion of inflammatory factors, promote the clearance of Aβ, repair nerve damage, and can be used for the treatment of Alzheimer's disease. Description of the Drawings

[0022] Figure 1 It is a plasmid map of the fusion protein expression vector pTRE-Tight-NMP2-Lamp2b.

[0023] Figure 2It is the electron microscopy detection image of NMP2 exosomes.

[0024] Figure 3 It is the detection image of the targeting peptide of NMP2 exosomes.

[0025] Figure 4 It is the detection image of the surface marker protein of exosomes.

[0026] Figure 5 It is the effect image of NMP2 exosomes targeting BV2 cells.

[0027] Figure 6 It is the treatment effect image after NMP2 exosomes are loaded with drugs. Among them, A is the expression level image of IL-1β, B is the expression level image of TNF-α, and C is the expression level image of IL-6. Detailed implementation manners

[0028] The present invention will be described in detail below with specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels 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 in the present invention, the NCAM mimetic peptide NMP2 and the exosome membrane protein Lamp2b are designed 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 operation is as follows: The gene sequence expressing the fusion protein NMP2-Lamp2b is ligated onto 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 pcDNA3.1-NMP2-Lamp2b vector and the pTRE-Tight plasmid are respectively double digested with SfaA I and Not I, and after the digestion products are ligated at 16 °C, the products are subjected to gel recovery to obtain the fusion protein expression vector pTRE-Tight-NMP2-Lamp2b, and its plasmid map is as shown in Figure 1 shown.

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

[0035] According to the instruction manual of the electroporator, after adding 8 μL of the fusion protein expression vector pTRE-Tight-NMP2-Lamp2b with a concentration of 500 ng / μL and 4 μL of the 293T cell suspension with a concentration of 1×10 5 cell / mL into the electroporation cuvette, electroporation is carried out under the conditions of 390 V and 30 ms. After electroporation, it is transferred to fresh medium and continuously cultured at 37 °C and 5% CO2 for 48 h, and then changed to the medium containing puromycin with a final concentration of 1 μg / mL and continuously cultured for 48 h to obtain the 293T-NMP2-Lamp2b stable cell line, which is then expanded in culture.

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

[0037] 1. Isolation and identification.

[0038] When the confluence of the 293T-NMP2-Lamp2b stable cell line reaches 70%, the medium is changed to the medium with a final concentration of 1 μg / mL tetracycline and continuously cultured for 48 h, and then the supernatant is collected. Under the condition of 4 °C, NMP2 exosomes are extracted by differential ultracentrifugation, specifically: 500 g, 10 min; 2000 g, 20 min; 10000 g, 30 min; 100000 g, 90 min to obtain the precipitate, which is resuspended with 500 μL of PBS to obtain NMP2 exosomes. The electron microscopy detection results of NMP2 exosomes are as shown in Figure 2 shown.

[0039] 2. Identification of the surface markers of NMP2 exosomes.

[0040] (1) Western blot was used to detect the expression of NMP2 in NMP2 exosomes.

[0041] Protein release from NMP2 exosomes by membrane disruption: 50 μL of NMP2 exosomes were taken and 20 μL of pre-cooled protein lysis buffer RIPA was added. The mixture was lysed on ice for 30 min, vortexed every 10 min, and then centrifuged at 12,000 g at 4 °C for 10 min. The supernatant was taken as the total protein of exosomes, and finally the protein concentration was determined by the BCA method.

[0042] 10 μL of the total protein of exosomes was added with 2 μL of 5×SDS loading buffer, boiled at 100 °C for 10 min and then loaded onto the gel, and run at a constant current of 45 mA. After running the gel, the proteins on the gel were transferred to a nitrocellulose membrane by transferring at 250 mA for 1 h. The nitrocellulose membrane was blocked with 5% skim milk for 1 h, incubated with Lamp2 primary antibody overnight, and incubated with fluorescently labeled IgG secondary antibody for 1 h and then developed. The experimental results are as Figure 3 shown. The exosomes without any modification were designated as the Unmod-Exo group, and the exosomes labeled with 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 NMP2 targeting peptide was successfully overexpressed in NMP2 exosomes.

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

[0044] Total protein of NMP2 exosomes was extracted, and the expression of marker proteins CD9, CD81 and TSG101 was detected by Western blot. The experimental results are as Figure 4 shown. The expression of the three marker proteins could be successfully detected in the NMP2 exosomes extracted in the NMP2-Exo group.

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

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

[0047] The experimental results are as Figure 5As shown in the figure, according to the image analysis of the cell uptake of exosomes for 1 - 5 hours: 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 period, indicating that the exosomes modified with NMP2 could be taken up by BV2 cells faster and in larger amounts. The above experiments showed that NMP2 exosomes had good targeting to BV2 cells.

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

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

[0050] 5 mg of curcumin was dissolved in 1.3573 mL of dimethyl sulfoxide to obtain a curcumin stock solution with a concentration of 10 mM. The solubility of curcumin in PBS was determined. The specific operation was as follows: 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of the curcumin stock solution were slowly added drop by drop to EP tubes containing 1 mL of PBS respectively. The OD values of curcumin at different concentrations were measured at 450 nm by an enzyme-linked immunosorbent assay (ELISA) reader, and the concentration standard curve was obtained as y = 228.6x - 10.77, R 2 = 0.9982.

[0051] 2. Loading of curcumin onto NMP2 exosomes.

[0052] 36.8 μg of the curcumin stock solution was slowly added drop by drop to 1 mL of PBS containing 36.8 μg of exosomes, and the mixture was mixed well while adding. Then it was placed in a dark environment at room temperature for 10 min for loading.

[0053] The OD value of the remaining curcumin in the PBS solution was measured at 450 nm by an ELISA reader, and the content of the remaining curcumin was calculated through the concentration standard curve. Then it was calculated by the formula:

[0054]

[0055] where Wtotal is the total amount of the added drug, in g, and Wfree is the amount of the remaining drug, in g. The encapsulation efficiency of curcumin encapsulated by NMP2 exosomes was obtained as: 36.58%.

[0056] 3. Inhibition of the release of inflammatory factors by microglia after NMP2 exosomes are loaded with curcumin.

[0057] The experiment was divided into 6 groups. The control group was BV2 cells growing normally without adding any drugs.

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

[0059] The supernatant was assayed using an enzyme-linked immunosorbent assay kit, and IL-1β, TNF-α, and IL-6 were detected according to the manufacturer's instructions. The experimental results are as Figure 6 shown. Compared with the control group cells, the amounts of IL-1β, TNF-α, and IL-6 secreted by BV2 cells treated with lipopolysaccharide were significantly increased. However, after treating the cells with curcumin, unmodified exosomes loaded with curcumin, and NMP2 exosomes loaded with curcumin, the secretion of IL-1β, TNF-α, and IL-6 could be reduced. Among them, the secretion amounts of IL-1β, TNF-α, and IL-6 were reduced the most after treatment with NMP2-Exo-Cur. The above results indicate that treating BV2 cells with curcumin loaded in exosomes modified with NMP2 peptide has the best effect on inhibiting the release of inflammatory factors.

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

[0061] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0062] Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

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

1.

2. A fusion protein, characterized in that: It is encoded by the gene according to claim 1.

3. A recombinant vector, characterized in that: Comprising the gene according to claim 1.

4. A recombinant cell, characterized in that Comprising the recombinant vector according to claim 3.

5. A brain-targeted exosome, characterized in that: Extracted from the recombinant cell according to claim 4.

6. The method for preparing brain-targeted exosomes according to claim 5, characterized in that: include: S1. Inserting the gene sequence of NCAM mimetic peptide NMP2 into the 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 after puromycin selection; S3. Tetracycline was added to the stably transfected cell line to induce overexpression of the fusion protein expression vector. The supernatant was collected after culturing for 40 to 48 hours, and the targeted exosomes modified with NMP2 peptide on the surface were obtained by ultrahigh-speed centrifugation.

7. Use of the brain-targeted exosomes according to claim 5 as brain-targeted delivery vectors in the preparation of drugs for treating Alzheimer's disease.

8. A drug for treating Alzheimer's disease, characterized in that: The drug is obtained by co-incubating curcumin and the brain-targeted exosomes according to claim 5 at a mass ratio of 1-2:1-2 and then purifying them.

9. The drug for treating Alzheimer's disease according to claim 8, characterized in that: The co-incubation conditions are: mixing at room temperature in a dark environment and then letting it stand for 5 to 10 minutes.

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