Active EDA-A1 loaded engineered small extracellular vesicle and preparation method thereof
Through gene editing and engineering transformation, small extracellular vesicles with active EDA-A1 components were successfully loaded, which solved the problem of low expression and easy degradation of EDA-A1 protein, achieved the function of activating downstream pathways, and had the advantage of rapid and large-scale production.
Patent Information
- Application Number
- CN202510245199.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-04
- Publication Date
- 2025-06-03
AI Technical Summary
In the prior art, the expression of EDA-A1 protein in epidermal stem cells is very low, the extracellular terminal ligand is easy to degrade, and it is difficult to fully bind to the receptor to activate the downstream EDAR/NF-κB signaling pathway, resulting in huge difficulties in its application in treatment.
Through gene editing and engineering modification, the EDA-A1 protein was overexpressed using lentiviral vectors, and Furin protease inhibitor was added during the culture process, and small extracellular vesicles were collected to extract small extracellular vesicles carrying active EDA-A1 fragments.
It has achieved stable loading of active EDA-A1 components in small extracellular vesicles, which can effectively activate downstream pathways, have great transformation application value, and has the advantage of being easy to produce rapidly and large-scale in large quantities compared with the production process of recombinant proteins.
Smart Images
Figure CN120082602A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of genetic engineering, and specifically refers to an engineered small extracellular vesicle loaded with active EDA-A1 and a preparation method thereof. Background Art
[0002] After the EDA-A1 protein is cleaved by Furin protease through the TNF homology domain in the extracellular domain, it forms a homotrimeric ligand, which binds to the EDAR receptor on the cell membrane to activate the downstream pathway and exert biological functions. However, the expression of the EDA-A1 protein in epidermal stem cells is very low, and the extracellular ligand is extremely easy to degrade, making it difficult to fully bind to the receptor and activate the downstream EDAR / NF-κB signaling pathway. Therefore, there are still great difficulties in its application in treatment at present.
[0003] Small extracellular vesicles (sEVs) are nanoscale bilayer phospholipid vesicles secreted by various types of cells, which can participate in cell-to-cell communication and regulate various physiological and pathological processes. As a natural carrier, sEVs can transfer a variety of endogenous bioactive molecules between cells. However, the bioactive components contained in natural sEVs are limited, and may not be able to play a sufficient therapeutic role.
[0004] Therefore, an engineered small extracellular vesicle loaded with active EDA-A1 is needed to solve the above technical problems. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to overcome the above technical defects and provide an engineered small extracellular vesicle loaded with active EDA-A1 and a preparation method thereof.
[0006] To solve the above technical problems, the technical solution provided by the present invention is a preparation method of an engineered small extracellular vesicle loaded with active EDA-A1, including the following steps:
[0007] Step 1) Overexpress the EDA-A1 protein in epidermal stem cells by infecting with lentivirus, and screen to obtain a stable transfected cell line that stably overexpresses EDA-A1;
[0008] Step 2) Amplify and culture the stable transfected cell line, and add Furin protease inhibitor during the culture process;
[0009] Step 3) After the amplification culture is completed, collect the cell culture supernatant and extract small extracellular vesicles to obtain engineered EDA-A1 small extracellular vesicles carrying the EDA-A1 active fragment.
[0010] Further, the lentivirus used in step 1 is packaged by the lentiviral vector EXU0031-Lv103. By using the Gateway technology, the complete coding region sequence of EDA-A1 is inserted behind the GFP coding region, thereby constructing a GFP-EDA-A1 fusion protein, and the GFP fluorophore is located at the N-terminus of the EDA-A1 protein, wherein the complete coding region sequence of EDA-A1 is shown as SEQ ID NO.1.
[0011] Further, the time for infecting the lentivirus in step 1 is 8 - 16 h, and the culture medium used for virus infection is Epilife medium without HKGS and containing 2 μg / mL polybrene.
[0012] Further, the method for screening stable transfected cells in step 1 includes drug screening using a medium containing puromycin and monoclonal amplification by picking single cells under a microscope.
[0013] Further, the culture medium used for amplification culture in step 2 is Epilife medium containing 1% (by mass) HKGS and 0.125 - 2 μg / mL HDa.
[0014] Further, the method for extracting small extracellular vesicles in step 3 includes the following steps: collecting cell supernatant, collecting the supernatant after low-speed centrifugation at 4°C to remove dead cells and cell debris, performing ultrafiltration membrane filtration, and ultracentrifuging the obtained filtrate to obtain small extracellular vesicles.
[0015] Further, the rotational speed for removing dead cells by low-speed centrifugation is 3000g, and the time for low-speed centrifugation is 15 minutes.
[0016] Further, the rotational speed for removing cell debris by low-speed centrifugation is 10000g, and the time for low-speed centrifugation is 60 minutes. The pore size of the ultrafiltration membrane used is 0.22 μm.
[0017] Further, the rotational speed for ultracentrifugation is 200000g, and the centrifugation time is 90 minutes. Taking the precipitate and resuspending it with sterile PBS to obtain engineered small extracellular vesicles.
[0018] The second object of the present invention is to provide an engineered small extracellular vesicle loaded with active EDA-A1 prepared by the above preparation method.
[0019] The advantages of the present invention compared with the prior art are as follows:
[0020] 1) By means of gene editing and engineering transformation, the present invention enables small extracellular vesicles to carry the component of active EDA-A1, and can exert the function of the EDA-A1 protein acting on cell surface receptors to activate downstream pathways.
[0021] 2) The preparation method of the present invention can produce engineered small extracellular vesicles loaded with active EDA-A1 fragments, which are more stable than EDA-A1 recombinant proteins. Compared with the complex process of producing recombinant proteins, the engineered small extracellular vesicles provided by the present invention have the advantages of being easy to produce rapidly in large quantities, and at the same time have the ability to stimulate the activation of downstream cell pathways, which has great potential for translational application. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 It is a schematic diagram of the backbone pattern of the lentiviral vector EX-U0031-Lv103 used to infect epidermal stem cells provided in Example 1 of the present invention.
[0023] Figure 2 After the epidermal stem cells were infected with lentivirus in Example 1 of the present invention, it was observed under a fluorescence microscope that green fluorescent fusion protein was expressed on the cell surface. Scale bar: 50μm.
[0024] Figure 3 It is a schematic diagram of the collection and extraction process of the engineered small extracellular vesicles EDA-sEVs provided in Example 1 of the present invention.
[0025] Figure 4 It is a transmission electron microscope ultrastructure diagram and nanoparticle analysis diagram of the engineered small extracellular vesicles provided in Example 2 of the present invention. Scale bar: 200nm.
[0026] Figure 5 It is a result diagram of the expression of specifically labeled proteins on the surface of the engineered small extracellular vesicles provided in Example 2 of the present invention.
[0027] Figure 6 It is a result diagram of the expression of active EDA-A1 protein in the engineered small extracellular vesicles provided in Example 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0028] The following further elaborates on an engineered small extracellular vesicle loaded with active EDA-A1 and its preparation method of the present invention in conjunction with examples.
[0029] Example 1
[0030] A preparation method of an engineered small extracellular vesicle loaded with active EDA-A1, comprising the following steps:
[0031] Step 1) Overexpress EDA-A1 protein in epidermal stem cells by infecting with lentivirus, and screen to obtain a stable transfected cell line that stably overexpresses EDA-A1;
[0032] Among them, the lentivirus for over-infection was constructed and provided by GeneCopoeia. It utilized the lentiviral vector EXU0031-Lv103 and applied Gateway technology to insert the complete coding region sequence of EDA-A1 behind the GFP coding region, as shown in SEQ ID NO.1, thereby constructing the GFP-EDA-A1 fusion protein, and the GFP fluorophore was located at the N-terminus of the EDA-A1 protein, as Figure 1 shown;
[0033] Then, the lentivirus was added to the EpiLife medium (Gibco) without antibiotics, without HKGS and containing 2 μg / mL polybrene (thermo fisher) to infect epidermal stem cells. After 12 h, it was replaced with the EpiLife medium containing 1% (mass fraction) of HKGS (Gibco). After 48 h, it was replaced with the EpiLife medium containing puromycin and HKGS for drug screening culture. After 2 weeks, a stable cell line stably expressing EDA-A1 was obtained. And under the microscope, it was observed that the cells expressed green fluorescence on the cell surface, as Figure 2 shown;
[0034] Step 2) Amplify the cells using the EpiLife medium (Gibco) containing 1% (mass) HKGS (Gibco) and 0.125 - 2 μg / mL HDa (Merck).
[0035] Step 3) When the cell density reached about 80%, the cell culture supernatant was collected, centrifuged at 3000 g for 15 minutes at low speed to remove dead cells, and the supernatant was collected; and the collected supernatant was centrifuged at 10000 g for 60 minutes to remove cell debris. The supernatant was filtered using a 0.22 μm filter; subsequently, it was ultracentrifuged at 200000 g for 90 minutes, the precipitate was taken, and after resuspension with sterile PBS, engineered small extracellular vesicles were obtained, as Figure 3 shown.
[0036] Example 2
[0037] Identify the engineered small extracellular vesicles obtained in Example 1:
[0038] 1) Use a transmission electron microscope to observe the morphology of the small extracellular vesicles and take pictures for recording, as Figure 4 shown in a. Detect the concentration and particle size of the small extracellular vesicles through a nanoparticle analysis system, as Figure 4 shown in b.
[0039] Culture normal epidermal stem cells with the EpiLife medium containing HKGS, collect their cell culture supernatant, and extract the small extracellular vesicles secreted by epidermal stem cells as the control group. The small extracellular vesicles of the control group were named ESC-sEVs, and the exosomes overexpressing EDA-A1 were named EDA-sEVs.
[0040] As can be seen from Figure 4 a, both groups of small extracellular vesicles have the classic bilayer membrane-like structure of small extracellular vesicles, and the overall appearance is spherical or cup-shaped. There is no obvious difference in diameter between the two groups;
[0041] As can be seen from Figure 4 b, the average and peak particle sizes of the two groups of engineered exosomes are concentrated in the range of 50 - 150 nm, and there is no obvious difference between the groups, which is in line with the particle size distribution characteristics of small extracellular vesicles.
[0042] 2) After lysing epidermal stem cells to extract total cellular proteins, and extracting the total proteins of small extracellular vesicles derived from epidermal stem cells and engineered small extracellular vesicles constructed by infecting with lentivirus, use a BCA kit to detect the protein concentration. For western blot experiments, prepare a 10% concentration gel, load the samples, and then perform protein electrophoresis, membrane transfer, blocking, and incubation with primary and secondary antibodies. Finally, use chemiluminescent imaging analysis for exposure and photography to detect the expression levels of surface specific marker proteins of small extracellular vesicles such as CD63, CD9, TSG101 and the cell marker protein Calnexin in the cell lysate proteins of epidermal stem cells (Cell lysis group), small extracellular vesicles derived from epidermal stem cells (ESC-sEVs group) and engineered small extracellular vesicles group (EDA-sEVs group), as shown in Figure 5 .
[0043] As can be seen from Figure 5 the figure, both ESC-sEVs and EDA-sEVs express the marker proteins CD63, CD9, TSG101 of small extracellular vesicles, while Calnexin is only expressed in the Cell lysis group. The results prove that the extracted ESC-sEVs and EDA-sEVs belong to small extracellular vesicles.
[0044] 3) After extracting the proteins of engineered small extracellular vesicles, use western blot to detect the expression level of EDA-A1 in engineered small extracellular vesicles and compare it with small extracellular vesicles derived from epidermal stem cells (ESC-sEVs group). The results are shown in Figure 6 .
[0045] The molecular weight of endogenously expressed EDA-A1 protein in cells is about 41 kDa, while the molecular weight of the GFP-EDA-A1 fusion protein translated from the exogenously introduced genome should be the molecular weight of EDA-A1 protein + the molecular weight of GFP fluorescent group protein, that is, 41 + 27 = 68 kDa.
[0046] Detect the expression of EDA-A1 protein by Western Blot. As can be seen from Figure 6It can be seen that there are no protein bands at either 41 kDa or 68 kDa for ESC-sEVs, while EDA-sEVs show double bands at 41 kDa and 68 kDa. This indicates that EDA-sEVs successfully carried the GFP-EDA-A1 fusion protein, and the TNF homology domain in the extracellular domain of EDA-A1 was not cleaved by Furin protease.
[0047] Since HDa was not added during the culture of ESCs, the EDA-A1 protein was easily hydrolyzed; moreover, the endogenous expression of EDA-A1 in ESCs itself was low, and it was difficult to be encapsulated into small vesicles during the formation of sEVs. Therefore, no protein bands were detected in the ESC-sEVs group.
[0048] The above describes the present invention and its implementation manners. Such description is not restrictive. What is shown in the drawings is only one of the implementation manners of the present invention, and the actual structure is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar structural manners and embodiments to this technical solution without creative efforts without departing from the gist of the present invention, they should all fall within the protection scope of the present invention.
Claims
1. A method for preparing engineered small extracellular vesicles loaded with active EDA-A1, characterized in that: The following steps are involved: Step 1) overexpressing EDA-A1 protein in epidermal stem cells by infecting them with lentivirus, and screening to obtain stably transfected cells that stably overexpress EDA-A1; Step 2) amplifying and culturing the stably transfected cells, and adding Furin protease inhibitor during the culturing process; Step 3) After the expansion culture is completed, the cell culture supernatant is collected to extract small extracellular vesicles to obtain engineered EDA-A1 small extracellular vesicles carrying the EDA-A1 active fragment.
2. The preparation method according to claim 1, characterized in that: In the step 1, the superinfected lentivirus is packaged by the lentiviral vector EXU0031-Lv103, and the complete coding region sequence of EDA-A1 is inserted behind the GFP coding region by using Gateway technology, thereby constructing a GFP-EDA-A1 fusion protein, and the GFP fluorescent group is located at the N-terminus of the EDA-A1 protein, wherein the complete coding region sequence of EDA-A1 is shown in SEQ ID NO.
1.
3. The preparation method according to claim 2, characterized in that: The time for infecting the lentivirus in step 1 is 8-16 hours, and the culture medium used for virus infection is Epilife culture medium containing no HKGS and 2 μg / mL polybrene.
4. The preparation method according to claim 1, characterized in that: The method for screening stable transgenic cells in step 1 includes using a culture medium containing puromycin for drug screening and selecting single cells under a microscope for monoclonal amplification.
5. The preparation method according to claim 1, characterized in that: The culture medium used for the amplification culture in step 2 is Epilife culture medium containing 1% by weight HKGS and 0.125-2 μg / mL HDa.
6. The preparation method according to claim 1, characterized in that: The method for extracting small extracellular vesicles in step 3 comprises the following steps: collecting cell supernatant, removing dead cells and cell debris by low-speed centrifugation at 4° C., collecting supernatant, filtering with an ultrafiltration membrane, and ultracentrifuging the obtained filtrate to obtain small extracellular vesicles.
7. The preparation method according to claim 6, characterized in that: The rotation speed of the low-speed centrifugation to remove dead cells is 3000 g, and the time of the low-speed centrifugation is 15 minutes.
8. The preparation method according to claim 6, characterized in that: The rotation speed of the low-speed centrifugation to remove cell debris is 10000 g, and the time of the low-speed centrifugation is 60 minutes. The pore size of the ultrafiltration membrane used is 0.22 μm.
9. The preparation method according to claim 6, characterized in that: The ultracentrifugation speed is 200000 g, and the centrifugation time is 90 minutes. The precipitate is taken and resuspended in sterile PBS to obtain engineered small extracellular vesicles. 10 . An engineered small extracellular vesicle loaded with active EDA-A1 prepared by the preparation method according to any one of claims 1 to 9 .