Construction method and application of mycobacterium tuberculosis lipoprotein labeled microvesicles

Abstract

The invention relates to a construction method and application of mycobacterium tuberculosis lipoprotein labeled microvesicles, and belongs to the technical field of biological medicines. According to the invention, an extracellular structural domain of mycobacterium tuberculosis lipoprotein LpqH is fused with a transmembrane region and an intracellular region of vesicular stomatitis virus G protein through a genetic engineering means, and an LpqH48-159-VSVG fusion expression vector is constructed. After the carrier is transfected to mammalian cells, the microvesicles of which the surfaces are specifically marked with LpqH48-159 can be separated from cell culture supernatant by combining with an optimized gradient centrifugation method. The prepared microvesicles specifically target macrophages, and can be used as a novel carrier of mRNA vaccines, and experiments prove that the immune effect and the protective efficacy of the carrier in prevention of EV71 virus infection are significantly superior to those of traditional lipid nanoparticles, which reveals that the microvesicles provided by the invention have wide application prospects in the field of immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for constructing and applying lipoprotein-labeled microvesicles of Mycobacterium tuberculosis. Background Technology

[0002] Based on their origin and size, extracellular vesicles (EVs) can be divided into two main subtypes: microvesicles (MVs) and exosomes. Exosomes are generally defined as multivesicle bodies (MVBs) with a size between 30 and 120 nm, originating from intraluminal vesicles (ILVs). Microvesicles are classified as having a diameter of 200-1000 nm and are released directly through the cell membrane via budding and division. Both exosomes and microvesicles can load and transfer specific substances (including RNA and proteins) to recipient cells, thereby influencing the physiological state of the cells. This characteristic makes them natural and ideal delivery vesicles for targeted drug delivery. In particular, extracellular vesicles are highly engineerable. They can be engineered by creating surface modifications to express proteins or peptides, thereby endowing them with cell and tissue targeting specificity.

[0003] In the prevention and treatment of infectious diseases, mRNA vaccines have shown great promise due to their low risk of insertional mutagenesis, strong immunogenicity, short development cycle, and cost-effective production potential. Among various vaccines, these have demonstrated high efficacy and played a crucial role in controlling pandemics. Importantly, mRNA is readily degraded by ribonucleases (RNases) in the body. Therefore, it is necessary to use suitable carriers to encapsulate mRNA to prevent its degradation during delivery. Lipid nanoparticles (LNPs) are widely used as a delivery system for mRNA vaccines, with several formulations approved and numerous clinical trials currently underway. However, LNPs and other synthetic nanocarriers still have limitations in targeting specific tissues, hindering their clinical application in certain indications. To overcome this limitation, novel nanocarrier systems need to be developed to facilitate the controllable delivery of encapsulated mRNA to specific organs and cell types. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention utilizes genetic engineering techniques to fuse the extracellular domain of Mycobacterium tuberculosis lipoprotein LpqH with the transmembrane and intracellular regions of vesicular stomatitis virus G protein, constructing a fusion expression vector. After transfecting this vector into mammalian cells, and using an optimized gradient centrifugation method, the surface-specific marker LpqH can be isolated from the cell culture supernatant. 48-159 Microvesicles.

[0005] The first objective of this invention is to provide a method for preparing Mycobacterium tuberculosis lipoprotein-labeled microvesicles, the steps of which include replacing the extracellular region of the vesicular stomatitis virus G protein with the extracellular domain of mycobacterial lipoprotein to obtain a fusion protein encoding gene, constructing a fusion protein expression vector, transferring the fusion protein expression vector into a host cell, collecting the supernatant after culture, and centrifuging to obtain the Mycobacterium tuberculosis lipoprotein-labeled microvesicles.

[0006] Furthermore, the nucleotide sequence of the extracellular domain of the mycobacterial lipoprotein is shown in SEQ ID NO.1.

[0007] Furthermore, the gene encoding the fusion protein is shown in SEQ ID NO.2.

[0008] Furthermore, the fusion protein expression vector uses pcDNA3.1 vector as its backbone.

[0009] Furthermore, the host cell is a 293T cell.

[0010] Furthermore, the centrifugal separation specifically includes the following steps:

[0011] Step S1: Centrifuge at 300-800g for 10 minutes at 4℃ and collect the supernatant;

[0012] Step S2: Centrifuge at 2000-3000g for 20 minutes at 4℃ and collect the supernatant;

[0013] Step S3, repeat step S2;

[0014] Step S3: Centrifuge at 12000g and 4℃ for 40 minutes, and collect the precipitate, which is the Mycobacterium tuberculosis lipoprotein-labeled microvesicle.

[0015] Furthermore, in step S1, the centrifugation conditions are 400g and 4℃.

[0016] Furthermore, in step S2, the centrifugation conditions are 2500g and 4℃.

[0017] Further, in step S3, the precipitate is resuspended in 100 times its volume of PBS and then centrifuged again at 12000g and 4°C for 40 minutes to remove soluble impurities. The resulting precipitate is the Mycobacterium tuberculosis lipoprotein-labeled microvesicle.

[0018] The second objective of this invention is to provide Mycobacterium tuberculosis lipoprotein-labeled microvesicles prepared by the above-described preparation method.

[0019] A third objective of the present invention is to provide a drug delivery system comprising the aforementioned Mycobacterium tuberculosis lipoprotein-labeled microvesicles.

[0020] A fourth object of the present invention is to provide the use of the above-described Mycobacterium tuberculosis lipoprotein-labeled microvesicles or the above-described drug delivery system in the preparation of products for the prevention and / or treatment of enterovirus 71.

[0021] A fifth object of the present invention is to provide a product for the prevention and / or treatment of enterovirus 71, the product comprising the above-described Mycobacterium tuberculosis lipoprotein-labeled microvesicles or the above-described drug delivery system.

[0022] Furthermore, the product also contains mRNA encoding the viral capsid protein VP1.

[0023] In one embodiment of the present invention, the sequence of the mRNA is shown in SEQ ID NO.3.

[0024] The beneficial effects of this invention are:

[0025] This invention utilizes LpqH 48-159 The surface-specific marker LpqH was constructed based on the principle of high affinity binding to mannose receptors on the surface of macrophages. 48-159 The microvesicles, by giving them the ability to target macrophages, do not alter their fundamental physical properties, ensuring their stability and reliability as a carrier. In vitro and in vivo experiments have confirmed that LpqH... 48-159 The labeled microvesicles showed significantly higher targeting efficiency and uptake on macrophages than the untargeted LNP vector, achieving highly efficient enrichment of mRNA vaccines into target immune cells. Compared to the LNP vector, delivery of mRNA encoding the capsid protein VP1 using LpqH-MV induced higher titers of neutralizing antibodies and a greater quantity of antigen-specific IFN-γ. + T cells. In a viral challenge model, mice immunized using the delivery system provided by this invention showed a significant reduction in serum viral load and a significant increase in survival rate, demonstrating its excellent performance in practical applications. Attached Figure Description

[0026] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0027] Figure 1 LpqH in Embodiment 1 of the present invention 48-159 - Schematic diagram of VSVG fusion gene construction;

[0028] Figure 2 LpqH in Embodiment 1 of the present invention 48-159 A schematic diagram of the preparation process of labeled extracellular vesicles;

[0029] Figure 3 LpqH in Embodiment 1 of the present invention 48-159 Results of Western blot assay for labeled extracellular vesicle proteins;

[0030] Figure 4 LpqH in Embodiment 1 of the present invention 48-159 The physical properties of labeled extracellular vesicles were characterized, where A represents the particle size distribution of unlabeled microvesicles, B represents the particle size distribution of LpqH-MV, C represents the comparison of microvesicle peak values, D represents the transmission electron microscopy results of microvesicles, E represents the particle size distribution of unlabeled exosomes, F represents the particle size distribution of LpqH-exo, G represents the comparison of exosome peak values, and H represents the transmission electron microscopy results of exosomes.

[0031] Figure 5 LpqH in Embodiment 2 of the present invention 48-159 The results of the specific targeting ability verification of labeled extracellular vesicles are as follows: A is the uptake of PKH67 labeled extracellular vesicles by macrophages under different treatments; B is the statistical results of uptake; C is the immunofluorescence results of macrophages in mouse muscle sections; D is the statistical results of macrophage uptake in muscle tissue by flow cytometry; and E is the statistical results of macrophage uptake in lung tissue by flow cytometry.

[0032] Figure 6 LpqH in Embodiment 3 of the present invention 48-159 The results of the labeled microvesicle delivery of mRNA vaccine validation are shown in the following figures: A is the Western blot validation of VP1 mRNA expression; B is a schematic diagram of the mouse immunization process; C is the VP1 / IgG antibody titer after three immunizations in mice; D is the result of the enzyme-linked immunospot assay; E is the statistical result of the enzyme-linked immunospot assay; and F is the CD4+ expression in spleen cells. + Percentage of IFN-γ T cells, G represents CD8+ in spleen cells. + Percentage of IFN-γ T cells, H represents viral load in mouse serum, and I represents mouse survival rate. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0034] Example 1: LpqH 48-159 Preparation and characterization of labeled extracellular vesicles

[0035] Replace the extracellular region of the vesicular stomatitis virus glycoprotein G (VSVG) gene with the extracellular domain of Mycobacterium tuberculosis lipoprotein LpqH. 48-159 (nucleotide sequence as shown in SEQ ID NO.1), preserving both the transmembrane and intracellular regions of the VSVG gene. LpqH was synthesized by a gene synthesis company.48-159 -VSVG fusion gene (nucleotide sequence as shown in SEQ ID NO.2), construction process as follows: Figure 1 As shown. LpqH 48-159 The VSVG fusion gene was ligated and cloned into the expression vector pcDNA3.1 to construct pcDNA-LpqH. 48-159 -VSVG fusion gene vector, sequenced for verification.

[0036] pcDNA-LpqH 48-159 293T cells were transfected with the VSVG fusion gene vector and cultured in cell-free vesicle medium for 48 hours. The cell culture supernatant was collected and centrifuged twice at 300g for 10 min and 2500g for 20 min. The supernatant was then collected, cell debris was removed, and the precipitate was centrifuged at 12000g at 4℃ for 40 min. The precipitate was LpqH. 48-159 Labeled microvesicles LpqH-MV were processed by filtering the supernatant through a 0.22-micron membrane, centrifuging at 100,000 g, 4 °C for 90 minutes, discarding the supernatant, adding an appropriate amount of PBS, mixing well, and centrifuging again at 100,000 g, 4 °C for 90 minutes. The supernatant was discarded, and the mixture was resuspended in an appropriate amount of PBS to obtain LpqH. 48-159 The preparation process of the labeled exosome LpqH-exo is as follows: Figure 2 As shown.

[0037] LpqH 48-159 Labeled microvesicles (LpqH-MV) and LpqH 48-159 Labeled exosomes (LpqH-exo) were subjected to Western blotting analysis for protein expression, and the results are as follows: Figure 3 As shown, LpqH can be detected in both microvesicles and exosomes isolated from transfected cells. 48-159 Protein expression. This protein was not detected in microvesicles (nc-MV) and exosomes (nc-exo) isolated from untransfected cells. Exosome markers (CD9 and TSG101) and the microvesicle marker Annexin A1 were used for identification to verify LpqH. 48-159 -VSVG fusion protein has been integrated into microvesicles and exosomes.

[0038] To investigate LpqH 48-159 Whether protein integration into microvesicles and exosome membranes affects their overall physical properties was investigated using nanoparticle tracking analysis (NTA) to examine LpqH. 48-159 The particle size distribution of labeled extracellular vesicles. For example... Figure 4As shown in A, B, and C, the particle size of LpqH-MV is similar to that of unlabeled microvesicles (nc-MV), with a peak particle size of 163 nm for nc-MV and 162 nm for LpqH-MV. Furthermore, similar results were obtained by transmission electron microscopy (TEM) analysis, as shown in... Figure 4 As shown in Figure D, both nc-MV and LpqH-MV exhibit similar small, round vesicle morphologies. Additionally, unlabeled exosomes (nc-exo) and LpqH... 48-159 The labeled exosomes (LpqH-exo) had peak particle sizes of 142 nm and 144 nm, respectively, indicating that LpqH... 48-159 The presence of exosomes did not change their particle size. Figure 4 G, F, G). TEM images also show that nc-exo and LpqH-exo have similar morphology and structure (G, F, G). Figure 4 H).

[0039] Example 2: LpqH 48-159 Validation of the targeting ability of labeled extracellular vesicles

[0040] To detect LpqH 48-159 The specific targeting ability of labeled extracellular vesicles to macrophages was demonstrated by staining extracellular vesicles with the lipophilic dye PKH67 and incubating them in human macrophage culture medium for 12 hours. The results were as follows: Figure 5 A and Figure 5 As shown in B, macrophages uptake LpqH-MV and LpqH-exo at significantly higher rates than commercially available lipid nanoparticles LNP.

[0041] To further determine LpqH 48-159 Whether the labeled extracellular vesicles specifically target tissue macrophages in vivo was determined by administering equal volumes (5 × 10⁻⁶) of the vesicles to mice via intramuscular injection and intranasal inhalation. 8 (Vesicles) were labeled with the lipophilic dye PKH67 using LpqH-MV, LpqH-exo, or LNP. Immunofluorescence of muscle sections showed that macrophages (labeled with F4 / 80) taking up LpqH-labeled vesicles were significantly more numerous than macrophages taking up LNP. Figure 5 C). Flow cytometry further confirmed the corresponding results. Figure 5 D). Meanwhile, using LNP as a control, flow cytometry analysis revealed that macrophages (labeled with F4 / 80) in mouse lung tissue had significantly higher uptake efficiency for LpqH-MV and LpqH-exo than LNP, and the uptake efficiency for LpqH-MV was higher than that for LpqH-exo. Therefore, LpqH-MV was selected for further experiments.

[0042] Example 3: LpqH 48-159Application of labeled extracellular vesicles in mRNA delivery

[0043] Evaluation of LpqH 48-159 Labeled microvesicles were used as an efficient delivery system for the prevention of enterovirus 71 (EV71) infection. This virus is the main pathogen causing hand-foot-and-mouth disease (HFMD). Since neutralizing epitopes are most concentrated on the viral capsid protein (VP1), mRNA encoding the EV71 capsid protein VP1 gene (nucleotide sequence shown in SEQ ID NO. 3) was constructed and packaged into LpqH-MV or LNP via electroporation. Specifically, the vesicles were diluted with electroporation buffer at a ratio of 1:9. 5 μg of VP1 mRNA was added to the electroporation buffer system and transferred to a pre-chilled 0.2 cm gene pulse electroporation cuvette. The cuvette was then placed in an electroporator for five electroporation cycles (parameters: 400 V, 125 μF, 5 ms). After electroporation, the sample was placed on ice for 10 minutes, followed by ultrafiltration using a 100 kDa molecular weight cutoff membrane to remove unloaded mRNA.

[0044] The expression of VP1-encoding mRNA delivered to recipient cells by LpqH-MV or LNP was verified by Western blotting, and the results are as follows: Figure 6 As shown in Figure A, mice were immunized intramuscularly with LpqH-MV or LNP, which encapsulates the mRNA encoding VP1, with PBS injection serving as a negative control. Forty-two days after the third vaccination, all immunized mice were challenged intravenously with EV71, as detailed in the following procedure. Figure 6 As shown in Figure B, after the initial immunization (day 14), serum anti-VP1 / IgG antibody titers in mice immunized with both LpqH-MV and LNP increased significantly, but there was no significant difference between the two. However, after the second (day 28) and third (day 42) booster immunizations, the neutralizing antibody titers induced by LpqH-MV were higher than those induced by LNP. Figure 6 C).

[0045] Since cellular immunity is crucial for resisting viral infection, the cellular immune response and systemic cytokine status were investigated after two (day 28) and three (day 42) immunizations. Spleen cells from mice in each immunization group underwent an enzyme-linked immunospot assay (ELISpots) targeting interferon-γ (IFN-γ). Following restimulation with a VP1 peptide library, the number of Th1-biased IFN-γ spleen cells induced by LpqH-MV vaccination was significantly greater than that induced by LNP. Figure 6 D and Figure 6 E). Furthermore, LpqH-MV vaccination promotes the production of higher frequencies of CD4+. + and CD8 +IFN-γ T cells elicited a strong Th1 cell immune response in spleen cells. Figure 6 F and Figure 6 These results indicate that the mRNA vaccine delivered by LpqH-MV can induce a more effective VP1-specific T-cell immune response.

[0046] Viral load in mouse serum was measured on days 1, 3, 5, and 7 after challenge with a lethal dose of EV71. The results are as follows: Figure 6 As shown in Figure H, compared with LNP-immunized mice, LpqH-MV-immunized mice showed a significant decrease in EV71 load from day 3. Mouse survival rates were statistically analyzed, and the results are as follows: Figure 6 As shown in Figure I, the survival rate of mice immunized with LpqH-MV was approximately 80%, while the survival rate of mice immunized with LNP was approximately 50%, demonstrating that LpqH... 48-159 Labeled extracellular vesicles can effectively deliver mRNA vaccines, with significantly improved efficacy compared to LNP.

[0047] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing Mycobacterium tuberculosis lipoprotein-labeled microvesicles, characterized in that: The steps include replacing the extracellular region of the vesicular stomatitis virus G protein with the extracellular domain of mycobacterial lipoprotein to obtain the fusion protein encoding gene, constructing a fusion protein expression vector, transferring the fusion protein expression vector into host cells, collecting the supernatant after culture, and centrifuging to obtain the microvesicles labeled with the Mycobacterium tuberculosis lipoprotein.

2. The preparation method according to claim 1, characterized in that: The nucleotide sequence of the extracellular domain of the mycobacterial lipoprotein is shown in SEQ ID NO.

1.

3. The preparation method according to claim 2, characterized in that: The gene encoding the fusion protein is shown in SEQ ID NO.

2.

4. The preparation method according to claim 1, characterized in that: The fusion protein expression vector uses pcDNA3.1 vector as its backbone.

5. The preparation method according to claim 1, characterized in that: The host cell was a 293T cell.

6. The preparation method according to claim 1, characterized in that, The centrifugal separation specifically includes the following steps: Step S1: Centrifuge at 300-800g for 10 minutes at 4℃ and collect the supernatant; Step S2: Centrifuge at 2000-3000g for 20 minutes at 4℃ and collect the supernatant; Step S3, repeat step S2; Step S3: Centrifuge at 12000g and 4℃ for 40 minutes, and collect the precipitate, which is the Mycobacterium tuberculosis lipoprotein-labeled microvesicle.

7. Mycobacterium tuberculosis lipoprotein-labeled microvesicles prepared by any of the preparation methods described in claims 1-6.

8. A drug delivery system, characterized in that: The drug delivery system comprises Mycobacterium tuberculosis lipoprotein-labeled microvesicles as described in claim 7.

9. The use of the Mycobacterium tuberculosis lipoprotein-labeled microvesicles of claim 7 or the drug delivery system of claim 8 in the preparation of products for the prevention and / or treatment of enterovirus 71.

10. A product for the prevention and / or treatment of enterovirus 71, characterized in that: The product comprises the Mycobacterium tuberculosis lipoprotein-labeled microvesicles of claim 7 or the drug delivery system of claim 8.

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