SPA / G-positive bacterium targeting vesicle as well as preparation method and application thereof
By fusing the VIS short peptide with the antibody Fc fragment, targeted vesicles that can specifically target SPA/G-positive bacteria were prepared, which solved the problems of high cost, low stability and poor targeting in the prior art, and achieved low cost, high stability and high targeting targeted vesicles preparation.
Patent Information
- Application Number
- CN202510079826.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-18
- Publication Date
- 2025-05-30
AI Technical Summary
The existing targeted vesicles have shortcomings such as high cost, low stability and poor targeting in actual applications, which limit their application scope.
By fusing the VIS short peptide to the Fc fragment of the antibody constant region, cells were induced to produce targeted vesicles that surface display Fc, using these vesicles to specifically target SPA/G-positive bacteria.
Low-cost, high-stability and high-targeting target vesicles are achieved, which can automatically load target proteins and specifically bind to protein A or protein G on the bacterial surface to enhance the antibacterial effect of antibiotics.
Smart Images
Figure CN120060363A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of vesicle preparation, and particularly relates to an SPA / G-positive bacteria-targeting vesicle, a preparation method thereof, and an application thereof. Background Art
[0002] Biological vesicles have characteristics such as non-toxicity and low immunogenicity, and can be used as carriers for biological nano-drug delivery. They are widely used in biomedical fields such as new drug delivery and vaccine development. Targeted drugs can precisely locate drugs to the lesion site, which can not only enhance the drug efficacy, but also weaken the drug side effects and reduce the damage to the body. Functional vesicles with targeting effects are usually obtained by using bioengineering techniques to secondarily process and modify vesicles with molecules such as functional short peptides, specific antigens, receptor proteins, and biotin. At present, most targeted vesicles still have deficiencies such as high cost, low stability, and poor targeting in practical applications, which greatly limits the application scope of targeted vesicles. Therefore, it is necessary to develop a new targeted vesicle preparation technology to further expand the application of targeted vesicles in different fields. Summary of the Invention
[0003] In view of the above problems, the present invention provides an SPA / G-positive bacteria-targeting vesicle, a preparation method thereof, and an application thereof. The vesicle is obtained by fusing the VIS short peptide with vesicle-inducing function with the Fc fragment of the antibody constant region, and inducing cells to produce targeted vesicles with Fc displayed on the surface through transfection and other means.
[0004] In order to achieve the above object, the technical solutions adopted by the present invention are as follows: An object of the present invention is to provide a preparation method of an SPA / G-positive bacteria-targeting vesicle. The vesicle is obtained by fusing the VIS short peptide with the antibody Fc fragment sequence, and expressing the VIS-Fc fusion protein through mammalian cells, which can induce cells to form vesicles carrying the VIS-Fc fusion protein; the vesicle can specifically target SPA / G-positive bacteria; the nucleotide sequence of the VIS-Fc fusion protein is shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:2.
[0005] Further, the antibody Fc fragment sequence is the Fc fragment of human antibody IgG.
[0006] Further, the cells are derived from mammalian cells.
[0007] Furthermore, the cells are BHK-21 cells.
[0008] Further, a protein tag is fused to the amino terminus of the VIS short peptide, the protein tag is a GFP tag, and the Fc sequence is fused to the carboxyl terminus of the VIS short peptide.
[0009] Furthermore, the SPA / G-positive bacteria-targeting vesicles are prepared by purifying VIS-Fc vesicles using magnetic beads conjugated with SPA / G on the surface.
[0010] Another object of the present invention is to provide SPA / G-positive bacteria-targeting vesicles prepared by a method for preparing SPA / G-positive bacteria-targeting vesicles, wherein the diameter of the vesicles is 100 - 500 nm.
[0011] Another object of the present invention is to provide the use of SPA / G-positive bacteria-targeting vesicles as a carrier in the preparation of pharmaceuticals and targeted drug delivery carriers.
[0012] Another object of the present invention is to provide the use of SPA / G-positive bacteria-targeting vesicles as a carrier in bioengineering, vaccine preparation, disease treatment, and virus-like particles.
[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: The SPA / G-targeting vesicles VIS-Fc prepared in the present invention can automatically load the target protein onto the surface of the vesicle membrane without secondary processing, and can specifically bind to protein A (SPA) or protein G (SPG) on the surface of bacteria, greatly reducing the production cost of the targeting vesicles. The functional vesicles constructed in the present invention are derived from biological cells, have good biocompatibility and stability, and can protect their payloads in circulation due to their low immunogenicity and ability to cross physiological barriers. In addition, the targeting vesicles of the present invention can, in principle, replace different functional sequences, specifically bind to any site of interest, and can enhance the antibacterial effect of antibiotics, that is, based on the present invention, its sequence has flexible variability, enhancing the functionality of the vesicles, and can be developed into different functional vesicles for development and use in targeted drug delivery, vaccine preparation, disease treatment, etc. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 is a diagram of the target protein of the VIS-Fc vesicles in the examples of the present invention; Figure 2 is a transmission electron micrograph of the VIS-Fc vesicles in the examples of the present invention; Figure 3 is a scanning electron micrograph of the targeted binding of the VIS-Fc vesicles in the examples of the present invention to protein A of Staphylococcus aureus; Figure 4 is a diagram of the enhancing effect of the VIS-Fc vesicles in the examples of the present invention on the antibacterial effect of antibiotics. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0015] In order to make the objectives and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0016] For the instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, they are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, they are all conventional experimental methods, detection methods, etc. existing in the prior art.
[0017] This embodiment provides a SPA / G-positive bacteria-targeting vesicle. This vesicle is formed by fusing the VIS short peptide with the antibody Fc fragment sequence and expressing the VIS-Fc fusion protein through mammalian cells, which can induce cells to form vesicles carrying the VIS-Fc fusion protein; this vesicle can specifically target SPA / G-positive bacteria; the nucleotide sequence of the VIS-Fc fusion protein is shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:2. The formation and function verification of this vesicle are specifically as follows: Example 1: Inducing the formation of vesicles with the expression of the VIS-Fc short peptide S1: According to the human Fc gene sequence (GenBank: JQ666008.1) published by NCBI, the target sequence was artificially synthesized using genetic engineering techniques and ligated to the carboxyl terminus of the VIS short peptide sequence, and then cloned into the pACGFP-C1 vector to construct the eukaryotic expression plasmid VIS-Fc.
[0018] S2: Transform the eukaryotic expression plasmid VIS-Fc into DH5α competent cells, and evenly coat them onto a solid LB medium supplemented with kanamycin, and culture at 37°C for 12 - 16 h.
[0019] S3: Pick a single colony into a liquid LB medium resistant to kanamycin, and culture at 37°C and 180 rpm for 12 - 16 h.
[0020] S5: Extract the VIS-Fc plasmid using an endotoxin-free plasmid miniprep kit and perform sequencing identification on it.
[0021] S6: One day in advance, inoculate BHK-21 cells on a cell culture plate. When the cells grow to 60% - 80%, use liposomes to transfect the recombinant plasmid and the empty vector plasmid into BHK-21 cells respectively.
[0022] S7: After 6 h of transfection, remove the cell culture supernatant, replace it with fresh medium, and continue to culture for 24 - 36 h. Observe the generation of virus-like vesicles under a fluorescence microscope.
[0023] S8: After washing the transfected cells twice with sterile PBS, adherent cells were harvested using a cell scraper and resuspended in a certain volume of sterile PBS.
[0024] S9: After lysing the cells, centrifuge at 12,000 rpm for 10 min, collect the supernatant, and discard the cell debris pellet.
[0025] S10: Purify the VIS-Fc vesicles using immunoaffinity technology. Add 60 μL of pre-washed magnetic beads to the vesicles and incubate by inversion for 3 h.
[0026] S11: Use a magnetic stand to collect the magnetic beads, add 1xPBST solution to wash the magnetic beads three times, elute the vesicles with elution buffer (0.2 M glycine, pH 2.5), and then add 35 μL of neutralization buffer (1 M Tris, pH 10.4) to neutralize the pH.
[0027] S12: Take 10 μL of the vesicle sample, use GFP monoclonal antibody as the primary antibody and HRP-labeled goat anti-mouse antibody as the secondary antibody, and identify the GFP protein in the collected vesicles using Western blot technology. The results are shown in Figure 1 。
[0028] S13: The collected vesicles were used immediately or slowly cooled to -80 °C for long-term storage.
[0029] The plasmid PACGFP-VIS-Fc was constructed and transfected into BHK-21 cells. After culturing for a period of time, the cells were lysed using a hypotonic lysis buffer. The cell lysate was co-incubated with magnetic beads conjugated with SPA / G on the surface to purify the VIS-Fc vesicles. The purified VIS-Fc vesicles were identified by Western blot by incubating with GFP-tag antibody; Figure 1 The results showed that the VIS-Fc vesicles were successfully purified.
[0030] Example 2 Transmission Electron Microscopy Imaging of VIS-Fc Vesicles S1: One day in advance, inoculate healthy BHK-21 cells on a cell culture dish. When the cells grow to 60% - 80%, use lipo8000 TM transfection reagent to transfect the VIS-Fc plasmid into BHK-21 cells.
[0031] S2: After 6 h of transfection, remove the cell culture medium supernatant, replace it with fresh medium, and continue culturing. After culturing for 24 h, observe the transfection efficiency using a fluorescence microscope.
[0032] S3: Discard the culture medium in the dish after transfection. Add 2 mL of PBS solution to the cell culture dish to wash the cells in the dish. Subsequently, use a cell scraper to scrape the cells in the dish, add 500 μL of hypotonic lysis solution, and grind and lyse the cells with a homogenizer for 10 min. Centrifuge and collect the supernatant containing vesicles.
[0033] S4: Add 25 μL of pre-washed magnetic beads to the collected supernatant, incubate by inversion at room temperature for 3 h, and purify VIS-Fc vesicles using immunoaffinity technology.
[0034] S5: Use a magnetic stand to collect the magnetic beads, add 1 mL of 1xPBST solution to wash the magnetic beads 3 times, elute the vesicles with 65 μL of elution solution (0.2 M glycine, pH 2.5), and then add 35 μL of neutralization solution (1 M Tris, pH 10.4) to neutralize the pH.
[0035] S6: Take 10 μL of the vesicle sample and adsorb it on the surface of a copper mesh (150 mesh). Use filter paper to absorb the excess liquid along the edge of the copper mesh. Float the copper mesh adsorbed with vesicles in distilled water and wash for 3 min, repeating three times.
[0036] S7: Float the copper mesh on 1% phosphotungstic acid staining solution, let it stand for staining for 30 s, air-dry it with the front side up, and then observe it under a transmission electron microscope. The results are shown in Figure 2 .
[0037] The VIS-Fc vesicles were purified by the method in Example 1 and observed under a transmission electron microscope after negative staining; Figure 2 The results showed that VIS-Fc vesicles with a plump morphology and a surrounding phospholipid bilayer were successfully purified using magnetic beads conjugated with SPA / G on the surface.
[0038] Example 3 Targeting of VIS-Fc Vesicles to Staphylococcus aureus S1: Take 100 μL of the frozen bacterial solution and add it to 10 mL of liquid medium. Culture it overnight in a shaker at 37 °C and 180 rpm.
[0039] S2: Take 150 μL of the bacterial solution, centrifuge at low speed to discard the supernatant, and add 100 μL of PBS solution to resuspend the bacterial cells.
[0040] S3: Add 100 μL of 4% paraformaldehyde and fix at room temperature for 10 min. After fixation, wash with 100 μL of PBS, and then add 100 μL of distilled water for washing.
[0041] S4: Centrifuge at low speed to collect the bacterial cell precipitate, and add 100 μL of PBS to resuspend the bacterial cells.
[0042] S5: Take 10 μL of the resuspended bacteria and drop it onto a pre-treated silicon wafer sized 5 mm × 5 mm. After it dries naturally, sputter it with gold and observe it under a scanning electron microscope.
[0043] S6: Add 100 μL of 5% BSA blocking solution and block for 1 h at room temperature. After blocking, blow and mix with 100 μL of PBS for washing, and then add 100 μL of distilled water and blow and mix for washing.
[0044] S7: Add the purified vesicles to the above-treated bacterial cell pellet and invert and incubate for 2 h at room temperature.
[0045] S8: Centrifuge the mixture of vesicles and bacteria at low speed and resuspend the bacteria with 100 μL of PBS.
[0046] S9: Take 10 μL of the resuspended bacteria and drop it onto a pre-treated silicon wafer sized 5 mm × 5 mm. After it dries naturally, sputter it with gold and observe it under a scanning electron microscope. The results are shown in Figure 3 the VIS-Fc + S. aureus (-αSPA) group.
[0047] S10: Take the bacteria blocked with 5% BSA blocking solution and add 100 μL of protein A antibody of Staphylococcus aureus diluted with BSA blocking solution (1:2000), and block it on a shaker for 1 h at room temperature.
[0048] S11: After blocking, blow and mix with 100 μL of PBS for washing, and then add 100 μL of distilled water and blow and mix for washing.
[0049] S12: Add the purified vesicles to the above-treated bacterial cell pellet and invert and incubate for 2 h at room temperature.
[0050] S13: Centrifuge the mixture of vesicles and bacteria at low speed and resuspend it with 100 μL of PBS. Pipette 10 μL of the sample and drop it onto a pre-treated silicon wafer sized 5 mm × 5 mm. After it dries naturally, sputter it with gold and observe it under a scanning electron microscope. The results are shown in Figure 3 VIS-Fc + S. aureus (+αSPA).
[0051] In the experiment, Staphylococcus aureus was pretreated with 5% BSA blocking solution. The purified VIS-Fc vesicles were added to the bacterial cell pellet and inverted and incubated. At the same time, the bacterial cell pellet after binding with protein A antibody of Staphylococcus aureus was also inverted and incubated with the purified VIS-Fc vesicles as a control, and observed by scanning electron microscope; Figure 3 The results showed that VIS-Fc vesicles could bind to Staphylococcus aureus, and this phenomenon disappeared after treating Staphylococcus aureus with protein A antibody of Staphylococcus aureus, indicating that VIS-Fc vesicles could specifically bind to SPA / G.
[0052] Example 4: VIS-Fc vesicles promote the antibacterial effect of antibiotics S1: One day in advance, inoculate healthy BHK-21 cells on a cell culture dish. When the cells grow to 60% - 80%, use lipo8000 TM transfection reagent to transfect the VIS-Fc plasmid into BHK-21 cells.
[0053] S2: After 6 hours of transfection, remove the supernatant of the cell culture medium, replace it with fresh medium, and continue culturing. After culturing for 24 hours, use a fluorescence microscope to observe the transfection efficiency.
[0054] S3: Discard the medium in the dish after transfection. Add 2 mL of PBS solution to the cell culture dish to wash the cells in the dish. Then use a cell scraper to scrape the cells in the dish, add 500 μL of hypotonic lysis solution, and grind and lyse the cells with a homogenizer for 10 minutes. Centrifuge and collect the supernatant containing vesicles.
[0055] S4: Add 25 μL of pre-washed magnetic beads to the collected supernatant, incubate by inversion at room temperature for 3 hours, and purify the VIS-Fc vesicles using immunoaffinity technology.
[0056] S5: Use a magnetic stand to collect the magnetic beads, add 1 mL of 1xPBST solution to wash the magnetic beads 3 times, elute the vesicles with 65 μL of elution solution (0.2 M glycine, pH 2.5), and then add 35 μL of neutralization solution (1 M Tris, pH 10.4) to neutralize the pH.
[0057] S6: Take 10 μL of the vesicle sample, use anti-GFP monoclonal antibody as the primary antibody and HRP-labeled goat anti-mouse antibody as the secondary antibody, and identify the collected vesicles using Western blot technology.
[0058] S7: Store the collected vesicles for later use.
[0059] S8: Take 100 μL of the frozen bacterial solution, add it to 10 mL of liquid medium, and culture it overnight in a shaker at 37 °C and 180 rpm.
[0060] S9: Add the linezolid solution to the purified vesicles and incubate them together for 2 hours.
[0061] S10: Take 100 μL of the resuscitated bacterial solution and spread it evenly on a culture dish.
[0062] S11: Use a punch with a diameter of about 7 mm to make three small holes with similar spacing in the culture dish.
[0063] S12: Add the incubated antibiotic vesicle mixture to the holes and label it as 1. The results are shown inFigure 4 。
[0064] S13: Add an antibiotic solution with the same final concentration as the previous group into another well, labeled as 2. The results are shown in Figure 4 。
[0065] S14: Add normal saline into the last well, labeled as 3. The results are shown in Figure 4 。
[0066] S15: Place the culture dish in an incubator at 37 °C and observe the promoting effect of the vesicles on the antibacterial effect of the antibiotic. The results are shown in Figure 4 。
[0067] In this experiment, the purified VIS-Fc vesicles were incubated and combined with linezolid. A linezolid solution with the same final concentration was used as a control, and its antibacterial effect was compared by the hole punching method; Figure 4 The results showed that antibacterial zones were normally produced in both groups. However, the antibacterial zone produced after the incubation of VIS-Fc vesicles and linezolid was significantly larger than that produced by the antibiotic alone, and the difference between the two groups was significant and statistically significant, indicating that VIS-Fc vesicles can enhance the antibacterial effect of antibiotics.
[0068] In this example, the VIS peptide with vesicle induction function was fused and expressed with the antibody constant region Fc fragment, and cells were induced to produce targeted vesicles displaying Fc on the surface through transfection and other methods; the isolated and purified targeted vesicles can not only carry drugs (such as antibiotics), but also specifically bind to SPA / G-positive bacteria, enhancing the killing effect of antibiotic drugs on SPA / G bacteria; the targeted vesicles can be used as drug delivery carriers, with the functions of precisely killing pathogenic bacteria, prolonging the drug half-life, and enhancing the drug efficacy. The SPA / G-positive bacteria targeted vesicles prepared in this example can specifically bind to SPA / G-positive bacteria, and in principle, other proteins with targeting functions can be displayed, developed into targeted vesicles or drug carriers with different functions for use.
[0069] The vesicles developed in this example can be expanded to have different functions by designing different functional sequences, enabling them to target any desired tissue or site. This technology has great potential in improving drug efficacy and reducing systemic side effects, providing a novel and effective strategy for the treatment of various diseases, and also providing a reference for the future development of bioengineering and drug delivery carriers.
[0070] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.
Claims
1. A method for preparing SPA / G positive bacteria targeting vesicles, characterized in that: The vesicle is expressed by fusing a VIS short peptide with an antibody Fc fragment sequence, and the VIS-Fc fusion protein is expressed by mammalian cells, which can induce the cells to form vesicles carrying the VIS-Fc fusion protein; the vesicle can specifically target SPA / G positive bacteria; the nucleotide sequence of the VIS-Fc fusion protein is shown in SEQ ID NO: 1, and the amino acid sequence thereof is shown in SEQ ID NO:
2.
2. The method for preparing a SPA / G positive bacteria targeting vesicle according to claim 1, characterized in that: The antibody Fc fragment sequence is the Fc fragment of human antibody IgG.
3. The method for preparing a SPA / G positive bacteria targeting vesicle according to claim 1, characterized in that: The cells are derived from mammalian cells.
4. The method for preparing a SPA / G positive bacteria targeting vesicle according to claim 3, characterized in that: The cells are BHK-21 cells.
5. The method for preparing a SPA / G positive bacteria targeting vesicle according to claim 1, characterized in that: The amino terminal of the VIS short peptide is fused with a protein tag, which is a GFP tag, and the carboxyl terminal of the VIS short peptide is fused with an Fc sequence.
6. The method for preparing a SPA / G positive bacteria targeting vesicle according to claim 1, characterized in that: The SPA / G positive bacteria targeting vesicles are prepared by purifying VIS-Fc vesicles using magnetic beads with SPA / G coupled to their surfaces.
7. The SPA / G-positive bacteria targeting vesicles prepared by the method for preparing SPA / G-positive bacteria targeting vesicles as described in any one of claims 1 to 6.
8. Use of the SPA / G-positive bacteria targeting vesicle as claimed in claim 7 as a carrier in the preparation of medicines and targeted drug delivery carriers.
9. Use of a SPA / G-positive bacteria targeting vesicle as described in claim 7 as a carrier in bioengineering, vaccine preparation, disease treatment, and virus-like particles.