Targeted tuberculosis subunit antigen self-assembled ferritin nano vaccine as well as preparation method and application thereof

Abstract

The invention provides a self-assembled ferritin nano vaccine targeting tuberculosis subunit antigen as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention provides a subunit nano-particle, ESAT-6 (E6), CFP-10 (C10) and ESAT-6-CFP-10 (EC) proteins are selected as antigen targets, ferritin is used as a potential presentation carrier of TB epitope targets, E6, C10 and EC antigens are fused into ferritin, a uniform epitope nano-vaccine is successfully self-assembled, and the subunit nano-particle has better immunogenicity and can be used for preparing a TB epitope vaccine. And efficient cellular immune response and immune memory can be triggered. Expression and production of the subunit nanoparticles are completed by using a method of an expression system, and the subunit nanoparticles have economical efficiency and are suitable for large-scale production.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a self-assembled ferritin nano vaccine targeting tuberculosis subunit antigens, a preparation method and an application thereof. Background Art

[0002] Tuberculosis (TB) is a chronic, highly contagious disease caused by Mycobacterium tuberculosis (Mtb). It can infect the respiratory tract and seriously endanger human health worldwide. In recent years, with the emergence of drug-resistant / multidrug-resistant tuberculosis, human immunodeficiency virus (HIV) infection / AIDS combined with tuberculosis, and the use of immunosuppressants. As the only vaccine approved for the prevention of tuberculosis, Bacillus Calmette-Guerin (BCG) can effectively prevent tuberculous meningitis and miliary tuberculosis in children. BCG has slowed the spread of tuberculosis to a certain extent, but its preventive effect in adults is still unclear and varies greatly (0-80%).

[0003] Currently, self-assembled nanoparticles play an important role in vaccine development. Compared with monomeric antigens, the affinity between the surface of nanoparticles and cell membranes helps immune cells anchor antigens more efficiently. These nanoparticles can be used as vaccine delivery platforms and adjuvants. However, there have been no reports of fusion expression with TB antigens, self-assembly, and immunogenicity. Summary of the invention

[0004] The present invention provides a self-assembled ferritin nano vaccine targeting tuberculosis subunit antigens, a preparation method and an application thereof, and uses ferritin as a universal platform for TB candidate vaccines to improve the immunogenicity of BCG and existing subunit vaccines and induce strong cellular immunity.

[0005] The present invention provides a subunit nanoparticle, comprising a subunit antigen separated from tuberculosis and a self-assembly carrier, wherein the self-assembly carrier comprises ferritin.

[0006] In a preferred embodiment of the present invention, the subunit antigen separated from tuberculosis includes any one of the following: ESAT-6, CFP-10 and fusion protein, and the fusion protein is a fusion protein of ESAT-6 and CFP-10.

[0007] The present invention also provides a method for constructing the above-mentioned subunit nanoparticles, comprising the following steps: connecting the subunit antigens separated from tuberculosis to ferritin via a connecting peptide to construct the subunit nanoparticles.

[0008] In a preferred embodiment of the present invention, the ferritin includes ferritin derived from Helicobacter pylori.

[0009] The present invention also provides a recombinant vector for expressing the subunit nanoparticles.

[0010] The present invention also provides a recombinant engineering cell comprising the above recombinant vector and expressing the above subunit nanoparticles.

[0011] The present invention also provides a method for producing the above subunit nanoparticles, comprising inserting a gene encoding the subunit nanoparticles into a basic skeleton vector to obtain a recombinant vector;

[0012] Using the recombinant vector to transform host cells to construct recombinant engineered cells;

[0013] The recombinant engineered cells are induced to express the subunit nanoparticles.

[0014] In a preferred embodiment of the present invention, when the subunit nanoparticles are produced by a prokaryotic expression method, the basic skeleton vector includes pET-28a(+), and the host cell includes Escherichia coli.

[0015] The present invention also provides the use of the subunit nanoparticles or the subunit nanoparticles produced by the above production method in the preparation of tuberculosis nano vaccines.

[0016] The present invention also provides a vaccine for preventing and / or treating tuberculosis, wherein the effective ingredients include the above-mentioned subunit nanoparticles or the subunit nanoparticles produced by the above-mentioned production method.

[0017] Beneficial effects: The present invention provides a subunit nanoparticle, which selects ESAT-6 (E6), CFP-10 (C10) and ESAT-6-CFP-10 (EC) proteins derived from the Mtb gene difference region (Religion of difference, RD) as antigen targets, utilizes the efficient self-assembly ability of ferritin, and uses ferritin as a potential presentation carrier of TB epitope targets, fuses E6, C10 and EC antigens into ferritin, and successfully self-assembles into a uniform epitope nanovaccine.

[0018] The present invention also uses an expression system method to complete the expression and production of the subunit nanoparticles. For example, in the embodiment, the subunit nanoparticles are efficiently expressed by a prokaryotic system, which is economical and suitable for large-scale production. Compared with conventional subunit vaccines, the subunit nanoparticles of the present invention have better immunogenicity and can trigger efficient cellular immune responses and immune memory. It is proved that the ferritin of the present invention can be used as a universal platform for TB candidate vaccines to further improve the immunogenicity of BCG and existing subunit vaccines.

[0019] The present invention adopts the BCG heterologous enhancement strategy, and subcutaneously injects subunit proteins and nanoparticles. HE staining results show that the nanovaccine has good safety. Compared with BCG and traditional subunit vaccines, the spleen cells of mice immunized with EFCF and ECF showed stronger cell proliferation levels after antigen stimulation. ELISpot and cytokine levels showed that the nanovaccine promoted IFN-γ recall response and strongly stimulated the secretion of various cytokines related to TB immunity, including Th1 type cytokines. The results of multicolor flow cytometry showed that nanoparticle vaccines represented by ECF induced the secretion of IFN-γ and Th1 type cytokines. + T CM / T EM , CD4 + IL-2 + T EM and CD8 + IL-2 + T CM In summary, in the context of heterologous enhanced immunity, mice immunized with EF, CF, and ECF showed acceptable safety and stronger cellular immune responses, induced immune memory dominated by IFNTM, and significantly stimulated the secretion of multiple tuberculosis immunity-related cytokines. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 1 is a diagram of recombinant protein expression and nanoparticle characterization analysis, in which A: design of target protein expression plasmid; B: SDS-PAGE electrophoresis analysis of purified recombinant protein, lane M: protein standard molecular weight; lane 1: ESAT-6; lane 2: CFP-10; lane 3: EC fusion protein; lane 4: Fer; lane 5: ESAT6-Fer; lane 6: CFP10-Fer; lane 7: EC-Fer; C: transmission electron microscopy (TEM) observation of nanoparticles; D: dynamic light scattering (DLS) analysis of nanoparticles; E: particle size and molecular weight; F: SDS-PAGE analysis of ferritin and antigen-coupled nanoparticles after 1, 3 and 5 freeze-thaw cycles; G: SDS-PAGE electrophoresis analysis of ferritin and antigen-coupled nanoparticles after storage at the specified temperature for 4 weeks;

[0021] Figure 2 Figure 1 shows the immunization process, immunization dose and lymphocyte proliferation test results. Figure 1 shows the immunization process, immunization dose and lymphocyte proliferation test results. Figure 1 shows the immunization process, immunization dose and lymphocyte proliferation test results.

[0022] Figure 3 Functional antigen-specific central memory T cells (T CM )Frequency diagram, A in the diagram: CD4 + IFN-γ + T CM ; B: CD4 + IL-2 + T CM ; C: CD8 + IFN-γ + T CM ;D:CD8 + IL-2 + T CM ;

[0023] Figure 4 Functional antigen-specific effector memory T cells (T EM ) frequency diagram, in which A: CD4 + IFN-γ + T EM ; B: CD4 + IL-2 + T EM ; C: CD8 + IFN-γ + T EM ;D:CD8 + IL-2 + T eM ;

[0024] Figure 5 The number of spot-forming cells (SFC) producing IFN-γ per million cells was determined by ELISpot. In the figure, A: ELISpot detection of IFN-γ secretion spot diagram; B: ELISpot detection of IFN-γ secretion statistical analysis;

[0025] Figure 6 The results of tuberculosis-related cytokine secretion levels are shown in Figure A: IFN-γ secretion expression level; B: TNF-α secretion expression level; C: IL-2 secretion expression level; D: IL-6 secretion expression level; E: IL-10 secretion expression level; F: IL-12 secretion expression level; G: IL-17 secretion expression level; H: IL-23 secretion expression level;

[0026] Figure 7The results of the transcriptional levels of tuberculosis-related cytokines are shown in Figure A: IFN-γ transcriptional expression level; B: TNF-α transcriptional expression level; C: IL-2 transcriptional expression level; D: IL-17 transcriptional expression level;

[0027] Figure 8 This is the result of H&E staining. DETAILED DESCRIPTION

[0028] The present invention provides a subunit nanoparticle, comprising a subunit antigen separated from tuberculosis and a self-assembly carrier, wherein the self-assembly carrier comprises ferritin.

[0029] In a preferred embodiment of the present invention, the subunit antigen separated from tuberculosis includes any one of the following: ESAT-6 (E6), CFP-10 (C10) and fusion protein (EC), and the fusion protein is a fusion protein of ESAT-6 and CFP-10. The ESAT-6 described in the present invention is the main T cell immunodominant antigen in the RD region purified from the short-term culture filtrate of Mtb, which is closely related to the increase and mutation of Mtb virulence, inhibits the degradation and killing of Mtb by immune cells through various pathways, and promotes Mtb to establish a chronic migration infection for a long time in vivo. The GenBank of E6 described in the present invention: NP_178023, and the GenBank of C10: NP_218391. The fusion protein of E6 and C10 described in the present invention is connected by a flexible linker, and the connection order is N-terminus-E6-linker-C10-C-terminus. In one embodiment, the linker is (G4S)3.

[0030] The ferritin of the present invention is composed of 24 subunits arranged in an octahedral symmetry, and can be used as a protein nanoplatform for antigen presentation and immune stimulation, and can induce an efficient immune response, because it has significant thermal stability, chemical stability and low heterogeneity. In a preferred embodiment of the present invention, the ferritin includes ferritin derived from Helicobacter pylori, and its GenBank: NP_223316.

[0031] The present invention also provides a method for constructing the above-mentioned subunit nanoparticles, comprising the following steps: connecting the subunit antigens separated from tuberculosis to ferritin via a connecting peptide to construct the subunit nanoparticles.

[0032] In one embodiment of the present invention, ESAT-6, CFP-10 and EC are fused to Helicobacter pylori ferritin through a flexible linker, wherein the flexible linker may be (G4S) 3. In one embodiment of the present invention, the subunit antigen is fused to the N-terminus of ferritin, and then the flexible linker is added to the C-terminus of ferritin.

[0033] The present invention also provides a recombinant vector for expressing the subunit nanoparticles.

[0034] The basic skeleton vector of the recombinant vector of the present invention can be a prokaryotic expression vector, such as the pET-28a(+) used in the embodiment, or a common vector of other expression systems. In one embodiment of the present invention, the coding gene of the subunit nanoparticle is connected to the Nco I and Xho I restriction sites of the pET-28a(+).

[0035] The present invention also provides a recombinant engineering cell comprising the above recombinant vector and expressing the above subunit nanoparticles.

[0036] In a preferred embodiment of the present invention, prokaryotic bacteria are used as host cells, such as Escherichia coli in one embodiment, and the above-mentioned recombinant vector is transformed into the host bacteria to obtain recombinant Escherichia coli that can express the subunit nanoparticles.

[0037] The present invention also provides a method for producing the above subunit nanoparticles, comprising inserting a gene encoding the subunit nanoparticles into a basic skeleton vector to obtain a recombinant vector;

[0038] Using the recombinant vector to transform host cells to construct recombinant engineered cells;

[0039] The recombinant engineered cells are induced to express the subunit nanoparticles.

[0040] In a preferred embodiment of the present invention, when the subunit nanoparticles are produced by a prokaryotic expression method, the basic skeleton vector includes pET-28a (+), and the host cell includes Escherichia coli. In an embodiment of the present invention, the prokaryotic expression vector pET-28a (+) containing a His tag at the C-terminus is double-digested using restriction endonucleases Nco I and Xho I, and the E6, C10 and EC sequences are fused with Helicobacter pylori ferritin (GenBank: NP_223316) through GSG linker, and the target sequence and pET-28a (+) are connected with DNA ligase, and are named ESAT6-Fer (EF), CFP10-Fer (CF) and ESAT6-CFP10-Fer (ECF), respectively.

[0041] The present invention also provides the use of the subunit nanoparticles or the subunit nanoparticles produced by the above production method in the preparation of tuberculosis nano vaccines.

[0042] The present invention fuses E6, C10 and EC antigens into ferritin and successfully constructs subunit nanoparticles for preparing epitope nanovaccines; in the context of heterologous enhanced immunity, mice immunized with EF, CF and ECF showed acceptable safety and stronger cellular immune response, induced immune memory mainly based on IFNTM and significantly stimulated the secretion of multiple tuberculosis immunity-related cytokines.

[0043] The present invention also provides a vaccine for preventing and / or treating tuberculosis, wherein the effective ingredients include the above-mentioned subunit nanoparticles or the subunit nanoparticles produced by the above-mentioned production method.

[0044] To further illustrate the present invention, a self-assembled ferritin nanovaccine targeting tuberculosis subunit antigens, a preparation method and an application thereof provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0045] The materials and reagents used in the embodiments of the present invention are all conventional commercially available reagents in the art, such as experimental animals: SPF-grade female C57BL / 6 mice (6-8 weeks old, weighing 18-20 g) were purchased from Jiangsu Jicui Pharmaceutical Co., Ltd., and the mice were randomly divided into cages. The mice were placed in individual ventilated cages with an ambient temperature of 22-25°C, a relative humidity of 45-65%, and a light / dark cycle of 12h / 12h. The animals were free to eat and drink throughout the experiment. The animal experiments involved in the article were approved by the Animal Ethics Committee of Bengbu Medical University (approval number: 2022127).

[0046] Experimental strains: E. coli Top10 clone was used for recombinant plasmid cloning and BL21 (DE3) expression strain was used for protein expression. E. coli was inoculated in LB agar medium containing kanamycin (50 μg / mL, Kan+) and cultured at 37°C and 5% CO2.

[0047] Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using Tukey's multiple comparison test in SPSS Version 29.0. Data were visualized in GraphPadPrism software (version 9.0). Pairwise comparisons were performed in each group. P<0.05 was considered statistically significant (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05).

[0048] Example 1

[0049] 1.1 Plasmid construction

[0050] The ESAT-6, CFP-10 and EC gene sequences of the Mtb H37Rv standard strain (ATCC 27294) were synthesized by Nanjing Zhongding Biological Company. The prokaryotic expression vector pET-28a(+) containing a His tag at the C-terminus was double-digested with restriction endonucleases Nco I and Xho I. The ESAT-6, CFP-10 and EC sequences were fused with Helicobacter pylori ferritin (GenBank: NP_223316) through GSG linker, and the target sequences and pET-28a(+) were connected with DNA ligase, and named ESAT6-Fer(EF), CFP10-Fer(CF) and ESAT6-CFP10-Fer(ECF), respectively.

[0051] In the present invention, E6, C10 or EC fusion protein is fused to the N-terminus of ferritin as antigen-ferritin (connected by a flexible linker (G4S)3), and then GSG and 6×His-tag are added to the C-terminus of ferritin to construct an expression plasmid as shown in the schematic diagram. Figure 1 As shown in A. The constructed expression vector was transformed into competent cells, induced by IPTG, and then purified by Ni-NTA column to obtain purified ESAT-6, CFP-10, EC, Fer, ESAT-6-Fer, CFP-10-Fer and EC-Fer proteins. The results of SDS-PAGE electrophoresis are shown in Figure 1 As shown in B, after purification, the protein band is single and consistent with the predicted size of the target protein ( Figure 1 Middle E).

[0052] 1.2 Expression and purification of recombinant proteins and nanoparticles

[0053] The constructed plasmid was transformed into the competent E. coli expression strain BL21 (DE3), evenly spread on the Kan+LB plate, and cultured in a 37°C incubator for 16 hours. Then, the monoclonal positive strain was selected and cultured in 600 mL of LB liquid medium containing 50 μg / mL kanamycin, placed in a constant temperature shaker, and cultured at 37°C until the bacterial solution OD 600 When the value reached 0.6-0.8, 0.2 mM IPTG was added to the culture medium and the expression was induced overnight at 16°C. The induced bacterial solution was collected and disrupted by ultrasound, and the supernatant solution was loaded onto a Ni-IDA-Sepharose Cl-6B affinity chromatography column pre-equilibrated with Ni-IDA Binding-Buffer, and washed with Ni-IDA Binding-Buffer and Ni-IDA Washing-Buffer (20 mM Tris-HCl, 30 mM imidazole, 0.15 M NaCl, pH 8.0) respectively until the effluent OD 280When the value reaches the baseline, slowly elute the target protein with Ni-IDA Elution-Buffer (20mMTris-HCl, 250mM imidazole, 0.15M NaCl, pH 8.0) and collect the effluent. Add the collected protein solution into the dialysis bag and dialyze with PBS overnight. Collect the protein and determine the protein concentration using the BCA method, and store at -80°C for later use.

[0054] 1.3 Protein identification and characterization

[0055] The recombinant proteins and nanoparticles were verified by 12% SDS-PAGE and Western-blot for protein purity and specificity. The morphology and particle size distribution of self-assembled nanoparticles were observed and detected by TEM and DLS. In order to determine the temperature / freeze-thaw stability of the nanoparticles, each assembled nanoparticle was placed in a -80°C environment until the solution was completely frozen, and then taken out and incubated at room temperature until it was completely thawed. This process was defined as one freeze-thaw cycle. After the above solutions were subjected to 1, 3 and 5 freeze-thaw cycles, each sample was analyzed by SDS-PAGE. In order to study the storage stability, all self-assembled nanoparticle solutions were stored at -80°C, -20°C, 4°C and 24°C for 4 weeks, and SDS-PAGE was used to preliminarily evaluate whether there was protein degradation.

[0056] TEM, DLS and SDS-PAGE were used to characterize and verify the nanoparticles (NPs). Uniformly dispersed NPs were observed by transmission electron microscopy, confirming that the structure of all NPs was similar to that of purified ferritin particles, indicating that they can self-assemble into ferritin nanoparticles of uniform size ( Figure 1 DLS measured the particle size distribution of NPs ( Figure 1 D), the results show that the size of the antigen-loaded nanoparticles is larger than the size of the ferritin monomer ( Figure 1 Middle E).

[0057] The present invention performed SDS-PAGE analysis on proteins under multiple freeze-thaw cycles and storage conditions at different temperatures. The gel results showed that after 1, 3, and 5 freeze-thaw cycles and storage at different temperatures for 4 weeks, the corresponding bands of the nanoparticles remained clear and did not disperse significantly, indicating that the protein may not have been degraded ( Figure 1 F and G).

[0058] 1.4 Mouse immunization protocol

[0059] The mice were acclimated for one week before the experiment. All immunogens were diluted in sterile PBS to keep the total volume at 200 μL. The mice were divided into eight groups (5 mice per group) and immunized once every two weeks for a total of 3 times ( Figure 2 At the beginning of the experiment (i.e., week 0), the first group of mice was subcutaneously injected with 2×106 CFU of BCG or self-assembled nanoparticles ( Figure 2 (B) Other mice using the “basal-boost” strategy were subcutaneously inoculated with protein antigens or corresponding self-assembled nanoparticles at weeks 2 and 4 after primary immunization with BCG.

[0060] 1.5 Lymphocyte proliferation assay

[0061] Mice were euthanized 42 days after the first vaccination, and spleens were collected aseptically. Spleen lymphocytes were separated using mouse spleen lymphocyte separation medium. The separated spleen lymphocytes were diluted to 3×10 in RPMI-1640 medium containing 10% fetal bovine serum. 6 cells / mL, and 2×10 5 Cells were inoculated into 96-well plates at a concentration of cells / well and stimulated with 20 μg / mL recombinant ESAT-6, CFP-10 and EC fusion proteins, respectively. 2 mg / mL ConA stimulated cells and untreated cells (RPMI-1640 medium containing FBS only) were used as positive and negative controls. The cells were incubated in an incubator at 37°C and 5% CO2 for 48 hours, and then tested using a cell proliferation detection kit, and then continued to be cultured for 4 hours. OD was read within 10 minutes after the end of incubation. 490 SI is used to represent the proliferation response of individual mouse spleen lymphocytes: SI = (average OD of stimulation wells 490 Value - Average OD of blank control wells 490 value) / (average OD of negative control wells 490 Value - Average OD of blank control wells 490 value).

[0062] Two weeks after the last immunization, cells were stimulated with ESAT-6, CFP-10, and EC recombinant proteins, respectively, and SI was introduced to evaluate the level of lymphocyte proliferation. Figure 2As shown in C: Regardless of the type of stimulator used, the lowest proliferation capacity was observed only in the Fer group. After recombinant protein stimulation, the proliferation levels of lymphocytes in the monomeric antigen group and the NPs group were higher than those in the BCG group. In summary, the cell proliferation capacity of the NPs-vaccinated group was better than that of the monomeric antigen group. However, when CFP-10 was used as a stimulus, the level of the BEF group was similar to that of the BC10 group and the EC fusion protein group, but not higher than that of the BC10 group. This does not mean that NPs cannot effectively promote cell proliferation. The classical immunological view is that when the immune function is normal and immune exemption is not considered, when the body encounters the same antigen stimulation again, the cells will be rapidly activated or proliferate. This result may be because this effect induced by CFP-10 is stronger, so it can be observed that the BEF group is similar to the BC10 group. Importantly, in this group, the proliferation level of the BCF group was significantly higher than that of the BC10 group, indicating that after the immunogen is determined, the ferritin-loaded immunogen will further promote cell proliferation. When mice were stimulated with ESAT-6, EC and ConA, the SI of the BEF and BCF groups was significantly higher than that of mice vaccinated with EC fusion protein, which still indicated that the proliferation induced by single antigen-loaded Fer was stronger than that of common fusion antigen subunit vaccine.

[0063] To verify whether the nanoparticle vaccine has the ability to provide early or sustained protective immunity, the present invention compared the number of specific memory cells in the spleen of each group based on the secreted effector cytokines and memory T cell subsets. Figure 3 Shown: In CD4 + IFN-γ + T CM ( Figure 3 A) and CD4 + IFN-γ + T EM ( Figure 4 In A), regardless of which protein was used as the stimulator, the antigen-specific memory T cells induced by the nanovaccine group were significantly higher than those induced by the corresponding common antigen group. When CFP-10 stimulated the cells, the most CD8 + IFN-γ + T CM ( Figure 3 C) and CD4 + IL2 + T EM ( Figure 4 Similarly, the present invention also detected that when ESAT-6 was stimulated, the BCF group CD4 + IL2 + T EM Highest level ( Figure 4 Middle B). It is speculated that the cross immunity may be caused by the co-transcription of ESAT-6 and CFP-10 at the gene level.

[0064] As the most classic tuberculosis vaccine, the advantages of BCG can still be reflected in the flow cytometry results. For example, when ESAT-6 and EC stimulated cells, BCG group CD8 + IFN-γ + T CM ( Figure 3 C) and CD8 + IL-2 + T EM ( Figure 4 D) dominates. In addition to CD8 + IL-2 + T EM ( Figure 4 In addition to the above, the present invention still harvested the highest level of antigen-specific memory T cells in the Fer nanovaccine group, and this phenomenon was particularly evident in the BECF group. + IL-2 + T EM The frequency is not the highest in the BECF group ( Figure 4 B), but it is at least one of the BEF or BCF groups, which also illustrates the advantage of Fer as a TB antigen carrier.

[0065] In summary, under the immunization program of the present invention, no matter what kind of recombinant protein stimulation is used, ferritin nanoparticle vaccine can significantly stimulate CD4 + IFN-γ + T M (Including T CM and T EM )、CD8 + IFN-γ + T EM proliferation and increased CD4 + IL-2 + T CM and CD8 + IL-2 + T CM The frequency ( Figure 3 (B and D).

[0066] 1.6 ELISpot assay

[0067] The number of antigen-specific immune cells secreting IFN-γ was detected using a commercial mouse IFN-γ pre-coated ELISPOT kit. The PVDF membrane was first activated with a universal serum-free medium, and then 3×10 5Cells were stimulated with 15 μg / mL recombinant ESAT-6, CFP-10 and EC fusion proteins, respectively. Untreated cell suspension and a mixture containing 500 ng / mL PMA and 10 ng / mL ionomycin were used as negative and positive controls. The ELISpot plate was incubated at 37°C and 5% CO2 for 16 h, then operated according to the instructions and observed for antigen-specific IFN-γ secreted spot forming cells (SFC), and finally the number of SFC was counted using an IRISTM ELISpot reader.

[0068] ELISpot results Figure 5 As shown in the figure, after stimulation with relevant recombinant antigens, the empty Fer group had almost no response, and the BCG group had a weak response. Regardless of the degree of protein stimulation, the BECF group reached the highest level. The number of spot-forming cells (SFC) in the BEF group that produced IFN-γ induced by ESAT-6 was higher than that in the other protein groups, and the secretion level was significantly stronger than that in the BCG group ( Figure 5 Similarly, similar results were observed with CFP-10 and EC stimulation. Overall, the number of IFN-γ-secreting cells in the spleen of mice in the EF-Fer, C10-Fer, or EC-Fer booster immunization groups was generally higher than that in the subunit vaccine immunization groups, and this trend was particularly evident in the BECF group ( Figure 5 The above results indicate that the heterologous boosting vaccination strategy using nano-TB antigens can significantly enhance the ability of BCG-stimulated cells to secrete IFN-γ, and that the TB nano-vaccine exhibits a higher degree of specificity and efficacy in response to antigen restimulation, and has the potential to secrete more effector molecules compared to traditional subunit vaccines.

[0069] 1.7 ELISA

[0070] In order to fully verify the ability of spleen lymphocytes of each group of mice to secrete TB immune-related cytokines under the stimulation of TB antigens ESAT-6, CFP-10 and EC, the secretion levels of various TB-related cytokines in the culture supernatant of mouse spleen lymphocytes were detected using a commercial ELISA kit. 6 cells / well were incubated with 15 μg / mL recombinant protein at 37°C and 5% CO2 for 24 h to detect IL-2, and for 48 h to detect the secretion levels of IFN-γ, TNF-α, IL-6, IL-10, IL-12, IL-17 and IL-23. After the culture, the cell culture supernatant was collected and tested using the corresponding commercial pre-coated ELISA kit. Within the first 15 min after the color reaction, the OD was detected using a SpectraMax Mini microplate reader. 450, and the secretion levels of cytokines IFN-γ, TNF-α, IL-6, IL-2, IL-10, IL-12, IL-17, and IL-23 were analyzed.

[0071] The secretion levels of various cytokines in the cell culture supernatant were as follows Figure 6 As shown, lymphocytes from mice vaccinated with TB nanoantigens stimulated IFN-γ and IL-2 responses more significantly than BCG and monomeric proteins after stimulation ( Figure 6 A and C). A stronger secretion trend similar to that of IL-2 was also detected in TNF-α ( Figure 6 (B) Therefore, ferritin nanocarriers can help TB antigens stimulate a stronger Th1 immune response and thus significantly improve the immunogenicity of BCG.

[0072] In addition, when stimulated with ESAT-6, the present invention observed that only the BECF group had the highest levels of IL-6 and IL-12, while the IL-6 levels of all booster groups were higher than those of the BCG group, which was similar to the IL-10 secretion under EC stimulation conditions ( Figure 6 For IL-6 stimulated by CFP-10 and IL-10 stimulated by ESAT-6, the BEF, BCF and BECF of the three nanoantigen groups were significantly higher than those of the BCG group and the booster immunization group with the corresponding common antigen ( Figure 6 D and E).

[0073] The IL-17 levels obtained after booster immunization induced by conventional subunit vaccines in the present invention were almost the same as those obtained by BCG vaccination alone, while after the antigen was fused with Fer, the IL-17 levels of the BEF, BCF and BECF groups were more ideal or significantly higher than those observed in the other groups, regardless of ESAT6, CFP-10 or EC stimulation ( Figure 6 However, each group secreted only a small amount of IL-23, and there was no difference between the groups ( Figure 6 H). It shows that when the spleen cells of mice immunized with ferritin nano-TB vaccine encounter the relevant Mtb antigen, they not only produce appropriate amounts of IL-6 and IL-10, but more importantly, they can show an immune response dominated by Th1 type, and cytokines related to Th17 response may be secreted through IL-23-independent pathways.

[0074] 1.8 Flow cytometric analysis

[0075] Splenic lymphocytes were collected at 3×10 6cells / well were inoculated in a 24-well plate, incubated with 15 μg / mL recombinant protein at 37°C, 5% CO2 for 16 h, and monensin was added for 5 h. The cells were collected by centrifugation, washed with pre-cooled PBS, and 1 μL TruStain FcXTMPLUS labeled anti-mouse CD16 / 32 monoclonal antibody was used to block Fc receptors, 2 μL FITC labeled anti-mouse CD3ε, 1.5 μL PE labeled anti-mouse CD4, 1.5 μL APC / FireTM 750 labeled anti-mouse CD8α, 2.5 μL Brilliant Violet 605 TM Surface staining was performed with anti-mouse CD44 and 1.5 μL PE / Cyanine 7-labeled anti-mouse CD62L monoclonal antibody. TM Buffer was used for fixation, and the cell membrane was permeabilized with Perm Wash Buffer. Then, 5 μL APC anti-mouse IFN-γ and 1 μL Brailiant Violet 421 anti-mouse IL-2 mAb were used for intracellular staining. Subsequently, the cells were washed with PBS and resuspended in FACS buffer. TM Flow cytometry was used for detection. Flowjo software was used to analyze antigen-specific CD4 + / 8 + IFN-γ + / IL-2 + Central memory T(T CM , CD44 + CD62L high ) / Effect memory T(T EM , CD44 + CD62L low ) cell number.

[0076] The results of multicolor flow cytometry were as follows Figure 3 and Figure 4 As shown, nanoparticle vaccines represented by ECF induced IFN-γ + T CM / T EM , CD4 + IL-2 + T EM and CD8 + IL-2 + T CM Mainly immune memory.

[0077] 1.9 Real-time fluorescence quantitative PCR

[0078] TRIzol extraction was used for 10 7The total RNA of each cell was analyzed by Nanodrop UV analyzer according to OD 260 / 280 and OD 260 / 230 Check the concentration and purity of RNA. One-Step gDNA Removal and cDNASynthesis SuperMix was used for reverse transcription PCR and cDNA synthesis. Green qPCRSuperMix was used to perform qPCR to assess the transcriptional levels of cytokines IFN-γ, TNF-α, IL-2, and IL-17. -ΔΔCT Methods The mRNA expression level was calculated.

[0079] The mRNA transcription of IFN-γ, TNF-α, IL-2 and IL-17 is shown in Figure 7 As shown in Figures AD, the results of cytokine (IFN-γ, TNF-α, IL-2, and IL-17) transcription and secretion levels in the BE6, BC10, and BEC groups were similar, indicating that ESAT-6 and CFP-10 failed to enhance BCG-induced cytokine transcription under the immunization regimen before TB protein stimulation. The results obtained in the BEF, BCF, and especially BECF groups were significantly higher than those in mice receiving BCG or conventional subunit protein vaccines, indicating that ferritin nanoparticles help antigens enhance Th1 cytokine mRNA transcription levels in the absence of TB protein stimulation. Therefore, the cytokine transcription levels verified by qPCR are roughly similar to the secretion trends after protein stimulation.

[0080] 1.10 H&E staining

[0081] The hearts, livers, spleens, lungs, and kidneys of mice in each group were fixed with 4% paraformaldehyde, dehydrated with gradient ethanol of different concentrations, embedded in paraffin, and evenly cut into 6 μm slices. H&E staining was performed to evaluate the safety of ferritin vaccine.

[0082] In order to detect the safety of the vaccine and whether it will cause damage to the important organs of mice, the present invention not only records the behavioral activities of mice before and after vaccination, but also removes the heart, liver, spleen, lungs, kidneys and other related organs of euthanized mice, and observes the pathological state of each organ section under a HE staining microscope. After two rounds of booster immunization, no inflammatory reaction was observed at the injection site and surrounding areas, breathing was normal, hair was in good condition, eating and drinking were normal, and no mice died during the whole process. This shows that the vaccine did not cause systemic adverse reactions in mice. Next, the gross condition and pathological sections of each organ were observed continuously and closely. The morphology of each organ was generally normal when observed with the naked eye. Microscopy showed that there was no obvious inflammatory cell infiltration in each organ ( Figure 8). Specifically, the myocardial cell fibers of mice in each group were normal, and no eosinophils and plaque formation were observed. No basement membrane thickening, inflammatory exudation, or cell proliferation was observed in the glomeruli and renal tubules. The number of central veins in the hepatic lobule and portal area was single and non-biased; the hepatocytes were normal in morphology, without ballooning, eosinophilic degeneration, or abnormal substance deposition. There were no abnormalities in the lung parenchyma and interstitium; there was no effusion in the alveolar cavity, the alveolar septum was normal, the bronchial tissue structure at all levels was clear, and no granulomatous inflammatory lesions were observed. The red pulp area and the white pulp area in the spleen were clearly demarcated; there were a large number of red blood cells and megakaryocytes in the red pulp area, accompanied by a small number of lymphoid follicles; the white pulp area contained lymphoid follicles, with a germinal center visible in the center, and obvious cell proliferation in the lymphoid sheath around the artery. These results indicate that the vaccine does not cause systemic symptoms in mice, and does not show toxic effects on important organs, confirming that the nano-tuberculosis vaccine constructed by the present invention is tolerable and safe in mice.

[0083] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A subunit nanoparticle, characterized in that: It comprises a subunit antigen isolated from tuberculosis and a self-assembling carrier, wherein the self-assembling carrier comprises ferritin.

2. The subunit nanoparticle according to claim 1, characterized in that: The subunit antigen separated from tuberculosis includes any one of the following: ESAT-6, CFP-10 and fusion protein, and the fusion protein is a fusion protein of ESAT-6 and CFP-10.

3. The method for constructing subunit nanoparticles according to claim 1 or 2, characterized in that: The method comprises the following steps: connecting the subunit antigen separated from tuberculosis to ferritin via a connecting peptide to construct the subunit nanoparticle.

4. The construction method according to claim 3, characterized in that: The ferritin includes ferritin derived from Helicobacter pylori.

5. A recombinant vector expressing the subunit nanoparticles according to claim 1 or 2.

6. A recombinant engineered cell comprising the recombinant vector of claim 5 and expressing the subunit nanoparticles of claim 1 or 2.

7. The method for producing subunit nanoparticles according to claim 1 or 2, characterized in that: The method comprises inserting a gene encoding the subunit nanoparticle into a basic backbone vector to obtain a recombinant vector; Using the recombinant vector to transform host cells to construct recombinant engineered cells; The recombinant engineered cells are induced to express the subunit nanoparticles.

8. The production method according to claim 7, characterized in that: When the subunit nanoparticles are produced by a prokaryotic expression method, the basic backbone vector includes pET-28a(+), and the host cell includes Escherichia coli.

9. Use of the subunit nanoparticles according to claim 1 or 2 or the subunit nanoparticles produced by the production method according to claim 7 or 8 in the preparation of tuberculosis nanovaccines.

10. A vaccine for preventing and / or treating tuberculosis, characterized in that: The active ingredients include the subunit nanoparticles described in claim 1 or 2 or the subunit nanoparticles produced by the production method described in claim 7 or 8.

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