Organelle-targeted molecule in-situ assembly chimera and application thereof

Abstract

The invention provides an organelle-targeted molecule in-situ assembly chimera and application thereof, and belongs to the technical field of molecules. The chimera sequentially comprises an organelle residing structural unit, an assembling structural unit, a tracing structural unit and a monomer streptomycin structural unit from an N end to a C end, and all the units are connected through linkers. According to the chimera constructed by the invention, the biological functional molecules are assembled in an organelle area in situ and are applied to various vaccine antigen models, so that the immunoreaction of antigen specificity in the various vaccine antigen models can be remarkably enhanced, the vaccine efficacy is improved, and the immune protection generated by the vaccine is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular science, and particularly relates to an organelle-targeted molecular in-situ assembly chimera and its application. Background Art

[0002] Assembly behaviors occurring at the organelle level play an important role in the normal execution of life activities and the efficient occurrence of biochemical reactions. At present, with the rapid development of microscopy imaging technology, the spontaneous assembly behaviors occurring at the organelle level in cells have been gradually discovered and revealed. For example, the assembly of receptors and kinases on the inner side of the T cell membrane mediates the efficient transduction of immune signals; the assembly of clathrin / adaptor proteins occurring in the trans-Golgi network participates in the efficient trafficking of proteins; and the biomolecular condensates formed by the assembly of some proteins in the cytoplasm regulate cellular stress. Analyzing the assembly behaviors at the organelle level: on the one hand, assembly can ensure the high-concentration enrichment of molecules in a local area, accelerating the speed of biochemical reactions; on the other hand, the occurrence in specific organelles or regions ensures the non-interference of reactions in that area, improving the reaction efficiency. Therefore, the spontaneous assembly behaviors occurring at the organelle level in cells demonstrate their unique advantages in the process of evolution.

[0003] Although more and more assemblies at the organelle level have been revealed, artificially achieving the assembly of target molecules in specific organelle regions is full of challenges. Currently, some assembly technologies relying on special environments in cells (such as acidic environments, overexpressed phosphatases, high concentrations of reactive oxygen species, etc.) have been developed to achieve intracellular assembly. However, for most cells and organelles, such special microenvironments are lacking, which greatly limits their application scope. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an organelle-targeted molecular in-situ assembly chimera and its application. This molecular in-situ assembly chimera (ORBAC) realizes the in-situ assembly of the immune-stimulating molecule cGAMP in the endoplasmic reticulum, greatly enhancing the immune response it induces. When applied to various antigen and vaccine models, it significantly enhances the immune protection efficacy of the vaccine.

[0005] To solve the above technical problems, the present invention provides the following technical solutions: The present invention provides a molecular in-situ assembly chimera, which includes: an organelle residence structural unit, an assembly structural unit, a tracer structural unit, and a monomer streptomycin structural unit; the amino acid sequence of the assembly structural unit is as shown in SEQ ID NO.1, the amino acid sequence of the tracer structural unit is as shown in SEQ ID NO.2, and the amino acid sequence of the monomer streptomycin structural unit is as shown in SEQ ID NO.3.

[0006] Preferably, the organelles in the organelle retention structural unit include one or more of endoplasmic reticulum, Golgi apparatus, endosome, lysosome, and mitochondrion.

[0007] Preferably, when the organelle is endoplasmic reticulum, the amino acid sequence of the organelle retention structural unit is as shown in SEQ ID NO.4 and SEQ ID NO.5.

[0008] Preferably, the assembly structural unit, the tracer structural unit, and the monomer streptomycin structural unit are connected by a linker, and the amino acid sequence of the linker is as shown in SEQ ID NO.6.

[0009] Preferably, the amino acid sequence of the molecular in-situ assembly chimera is as shown in SEQ ID NO.7.

[0010] The present invention also provides a recombinant plasmid encoding the molecular in-situ assembly chimera, and the recombinant plasmid contains the nucleotide sequence of the molecular in-situ assembly chimera.

[0011] The present invention also provides the application of the molecular in-situ assembly chimera or the recombinant plasmid in the preparation of molecular in-situ targeting organelle products.

[0012] The present invention also provides the application of the molecular in-situ assembly chimera or the recombinant plasmid in the preparation of products for enhancing vaccine efficacy.

[0013] The present invention provides a product for enhancing vaccine efficacy, and the product is obtained by mixing the recombinant plasmid, the biotin-labeled molecule, and the vaccine antigen.

[0014] Preferably, the vaccine antigen includes one or more of model antigen OVA, respiratory syncytial virus antigen, and influenza virus hemagglutinin antigen.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention firstly proposes to obtain a molecular in-situ assembly chimera by connecting an organelle retention structural unit, an assembly structural unit, a tracer structural unit, and a monomer streptomycin structural unit through a linker. The chimera constructed by the present invention can in-situ assemble biofunctional molecules in the organelle region, and then be applied to various vaccine antigen models, which can significantly enhance the antigen-specific immune response in various vaccine antigen models, improve vaccine efficacy, and enhance the immune protection generated by the vaccine.

[0016] Furthermore, the molecular in-situ assembly chimera of the present invention has a broad-spectrum characteristic, enabling in-situ assembly targeting different types and sizes of organelles; in addition, it can also achieve in-situ assembly of molecules in various types of organelles within cells; it has a wide range of applications. In addition, in the application of the molecular in-situ assembly chimera of the present invention, there is no need to make excessive modifications to the functional molecules, and only biotin labeling is required to achieve it. The process is simple and highly operable. Brief Description of the Drawings

[0017] Figure 1 It is a laser confocal microscopy image (left figure) of A549 cells transfected for 24 hours and the analysis result of the fluorescence intensity of the endoplasmic reticulum and the expressed ORBAC (right figure).

[0018] Figure 2 It is the titer of OVA antigen-specific IgG antibodies in the sera of mice in different groups on the 10th and 21st days.

[0019] Figure 3 It is the analysis of the secretion of OVA antigen-specific IFN-γ by every 10 6 spleen cells of mice in different groups on the 21st day by ELISPOT.

[0020] Figure 4 It is the analysis of the secretion of OVA antigen-specific IFN-γ, IL-2, and IFN-α by CD4 + (upper figure), CD8 + (lower figure) T cells of mice in different groups on the 21st day by flow cytometry.

[0021] Figure 5 It is the titer of RSV antigen-specific IgG antibodies in the sera of mice in different groups on the 35th day.

[0022] Figure 6 It is the titer of RSV antigen-specific neutralizing antibodies in the sera of mice in different groups on the 35th day.

[0023] Figure 7 It is the analysis of the secretion of RSV antigen-specific IFN-γ by every 10 6 spleen cells of mice in different groups on the 35th day by ELISPOT.

[0024] Figure 8 It is the analysis of the secretion of RSV antigen-specific IFN-α, IFN-β, IFN-γ, TNF-α, IL-2, and IL-4 by CD4 + (upper figure), CD8 + (lower figure) T cells of mice in different groups on the 35th day by flow cytometry.

[0025] Figure 9 It is the change in the body weights of mice in different groups for 10 consecutive days after virus challenge.

[0026] Figure 10 Lung tissue sections of mice in different groups on the 4th day after administration of the toxic substance.

[0027] Figure 11 On the 10th day after immunization, flow cytometry was used to analyze the proportion of germinal center B cells (GCB) in the lymph nodes of mice in different groups.

[0028] Figure 12 Confocal laser microscopy images (left panel) of A549 cells transfected with plasmids lacking some structural units (ORBAC ΔA, ORBAC ΔB, ORBAC ΔC) and an endoplasmic reticulum marker plasmid, and fluorescence intensity analysis of the endoplasmic reticulum and ORBAC lacking some structural units (right panel) 24 hours after transfection.

[0029] Figure 13 OVA antigen-specific IgG antibody titers in the sera of mice in different groups on the 21st day.

[0030] Figure 14 For ELISPOT analysis of the secretion of OVA antigen-specific IFN-γ by every 10 6 spleen cells of mice in different groups on the 21st day.

[0031] Figure 15 RSV antigen-specific IgG antibody titers in the sera of mice in different groups on the 35th day.

[0032] Figure 16 RSV antigen-specific neutralizing antibody titers in the sera of mice in different groups on the 35th day.

[0033] Figure 17 For ELISPOT analysis of the secretion of RSV antigen-specific IFN-γ by every 10 6 spleen cells of mice in different groups on the 35th day. Detailed implementation manners

[0034] The present invention provides a molecular in-situ assembly chimera, which includes: an organelle residence structural unit, an assembly structural unit, a tracer structural unit, and a monomer streptomycin structural unit; preferably, the molecular in-situ assembly chimera sequentially includes, from the N-terminus to the C-terminus, a signal peptide sequence that guides a protein into an organelle of the organelle residence structural unit, an assembly structural unit, a tracer structural unit, a monomer streptomycin structural unit, and an organelle residence signal sequence of the organelle residence structural unit. The amino acid sequence of the assembly structural unit in the present invention is as shown in SEQ ID NO.1, the amino acid sequence of the tracer structural unit is as shown in SEQ ID NO.2, and the amino acid sequence of the monomer streptomycin structural unit is as shown in SEQ ID NO.3. In the present invention, the assembly structural unit, the tracer structural unit, and the monomer streptomycin structural unit are connected by a linker, and the amino acid sequence of the linker is as shown in SEQ ID NO.6. The organelle residence structural unit in the present invention is used to induce organelle residence, the assembly structural unit is used to drive assembly, the tracer structural unit is used for visualization, and the monomer streptomycin structural unit is used to capture a target molecule to be assembled.

[0035] In the present invention, the organelle in the organelle residence structural unit includes one or more of endoplasmic reticulum, lysosome, ribosome, and mitochondrion, preferably the endoplasmic reticulum. The organelle residence structural unit in the present invention can be specifically adjusted and selected according to a specific target organelle. When the organelle in the present invention is the endoplasmic reticulum, the amino acid sequence of the organelle residence structural unit is as shown in SEQ ID NO.4 and SEQ ID NO.5. The amino acid sequence shown in SEQ ID NO.4 is a signal peptide sequence that guides a protein into the endoplasmic reticulum, and the amino acid sequence shown in SEQ ID NO.5 is an endoplasmic reticulum residence signal sequence. When the organelle in the present invention is the endoplasmic reticulum, the amino acid sequence of the molecular in-situ assembly chimera is as shown in SEQID NO.7.

[0036] The present invention also provides a recombinant plasmid encoding the chimera, and the recombinant plasmid contains the nucleotide sequence of the molecular in-situ assembly chimera. The recombinant plasmid in the present invention is prepared by the following method: reverse transcribing the amino acid of the molecular in-situ assembly chimera into a chimera nucleotide sequence; linearizing a mammalian expression vector through a double digestion system of KpnⅠ / XhoⅠ; and recombining the chimera nucleotide sequence onto the linearized mammalian expression vector by using homologous recombination technology to obtain a recombinant plasmid containing the chimera nucleotide sequence. The nucleotide sequence of the molecular in-situ assembly chimera in the present invention is as shown in SEQ ID NO.8. The mammalian expression vector in the present invention is pcDNA TM 3.1 (+).

[0037] The present invention also provides the use of the chimeric body or the recombinant plasmid in the preparation of a product for in situ molecular targeting of organelles. The molecule in the present invention is preferably a cGAMP molecule, which is a small molecule of cyclic dinucleotide. Because it can activate the immune-related receptor STING and downstream signaling pathways, it is considered a highly potential adjuvant molecule for improving vaccine efficacy.

[0038] The method for in situ assembling the cGAMP molecule on organelles in the present invention includes: mixing the recombinant plasmid and the biotin-labeled cGAMP molecule, and delivering them into cells, so that the cGAMP molecule can be in situ assembled on the organelles. The present invention uses the developed chimeric body for in situ molecular assembly to in situ assemble the cGAMP molecule in the endoplasmic reticulum region. Because the receptor STING of cGAMP is located in the endoplasmic reticulum, the cGAMP molecule is in situ assembled on the endoplasmic reticulum.

[0039] The present invention also provides the use of the chimeric body for in situ molecular assembly or the recombinant plasmid in the preparation of a product for improving vaccine efficacy. The present invention mixes the recombinant plasmid, the biotin-labeled cGAMP molecule and the vaccine antigen, and transfers them into an animal body, which can significantly enhance the antigen-specific immune response (including cellular immunity and humoral immunity), overall improve the vaccine efficacy, and enhance the immune protection generated by the vaccine. The biotin-labeled cGAMP molecule in the present invention can be obtained by methods well known in the art or through commercial channels.

[0040] The present invention also provides a product for improving vaccine efficacy, which is obtained by mixing the recombinant plasmid, the biotin-labeled molecule and the vaccine antigen. The vaccine antigen in the present invention includes one or more of the model antigen OVA, respiratory syncytial virus antigen and influenza virus hemagglutinin antigen. The biotin-labeled molecule in the present invention includes the biotin-labeled cGAMP molecule (Biotin-cGAMP).

[0041] In the present invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0042] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] Raw materials and sources used in this example: cGAMP, merchant: Sigma, molecular weight: 674.41; Biotin-cGAMP, merchant: AAT Bioquest, molecular weight: 1434.51; pcDNATM 3.1(+), Supplier: Sangon; Opti-MEMTM Medium, Supplier: Gibco; Lipofectamine TM 3000 Transfection Reagent Kit, Supplier: Invitrogen.

[0044] Example 1 Design of Molecular In-situ Assembly Chimera and Synthesis of Recombinant Plasmid Design of Molecular In-situ Assembly Chimera: The amino acid sequences of the signal peptide sequence that guides the protein into the endoplasmic reticulum of the organelle resident domain unit, the assembly unit, the tracer unit, the monomer streptomycin unit, and the endoplasmic reticulum resident signal sequence of the organelle resident domain unit are sequentially linked from the N-terminus to the C-terminus. Among them, the assembly unit, the tracer unit, and the monomer streptomycin unit are connected by a linker to obtain the molecular in-situ assembly chimera. Among them, the amino acid sequence of the assembly unit is shown in SEQ ID NO.1, the amino acid sequence of the tracer unit is shown in SEQ ID NO.2, the amino acid sequence of the monomer streptomycin unit is shown in SEQ ID NO.3, the amino acid sequences of the organelle resident domain unit are shown in SEQ ID NO.4 (the signal peptide sequence that guides the protein into the endoplasmic reticulum) and SEQ ID NO.5 (the endoplasmic reticulum resident signal sequence), and the amino acid sequence of the linker is shown in SEQ ID NO.6. The amino acid sequence of the designed molecular in-situ assembly chimera is shown in SEQ ID NO.7.

[0045] Synthesis of Recombinant Plasmid Containing the Molecular In-situ Assembly Chimera Gene (Entrusted to Sangon Biotech for Synthesis): (1) The amino acid sequence of the above-synthesized molecular in-situ assembly chimera is reverse transcribed into a nucleotide sequence, and this nucleotide sequence is synthesized by chemical synthesis method, as shown in SEQ ID NO.8; (2) The mammalian expression vector pcDNA TM 3.1(+) is linearized by a double digestion system (KpnⅠ / XhoⅠ); (3) The above nucleotide sequence is recombined onto the linearized mammalian expression vector pcDNA TM 3.1(+) by means of homologous recombination technology to obtain a recombinant plasmid (ORBAC plasmid) containing the molecular in-situ assembly chimera gene.

[0046] Example 2 Observation of the Formed Endoplasmic Reticulum Resident Assembly by Laser Confocal Microscopy 1. Uniformly seed A549 cells at 4×10 5 / well into a 12-well plate containing cell slides and culture overnight; 2. Dilute 3 μL of Lipofectamine with 50 μL of Opti-MEM TM Medium TM3000 transfection reagent; 3. Dilute 1 μL of ORBAC plasmid (1 μg / μL) and 1 μL of Sec-61B plasmid (1 μg / μL, synthesized by Sangon Biotech) labeled with endoplasmic reticulum in 50 μL of Opti-MEM medium, and add 2 μL of the co-transfection reagent P3000 TM to obtain the plasmid in the dilution; TM 4. Mix the diluted LipofectamineTM 3000 transfection reagent (1.5 μL / well) and the diluted plasmid (1 μg / well), and then incubate at room temperature for 15 min to obtain the plasmid-lipid complex; 5. Add the plasmid-lipid complex to the A549 cells cultured overnight above and gently shake well, place in a cell culture incubator, and incubate at 37 °C and 5% CO2 for 1 day; 6. After 1 day, wash the cells three times with PBS and add sodium arsenite (500 μM) to stimulate for 1 h; 7. Wash the cells and fix the cells with 4% paraformaldehyde; 8. Stain the cell nuclei with a quenching agent containing DAPI; 9. Fix the coverslip with nail polish and observe under a laser confocal microscope. The results are as

[0047] shown. It can be seen from the figure that obvious ORBAC particles appear in the cells and there is also good co-localization with the endoplasmic reticulum, proving that ORBAC has successfully achieved in-situ assembly of endoplasmic reticulum targeting. Figure 1 Example 3 Evaluate the immunogenicity of cGAMP molecules induced by ORBAC-mediated in-situ assembly of endoplasmic reticulum in the model antigen OVA model

[0048] 1. Immunization protocol for C57BL / 6 mice Grouping of animal experiments: OVA group: Each mouse was only injected with OVA antigen (10 μg); OVA + cGAMP group: Each mouse was only injected with OVA antigen (10 μg) + GAMP molecule (2.5 μg, 0.0037 μmol); OVA + cGAMP + ORBAC group: Each mouse was injected with OVA antigen (10 μg) + Biotin-cGAMP molecule (5.32 μg, 0.0037 μmol) + ORBAC plasmid (80 μg); OVA + aluminum adjuvant group: Each mouse was injected with OVA antigen (10 μg) + aluminum hydroxide adjuvant (35 μg).

[0049] ​The antigen OVA (purchased from Sigma) was uniformly mixed with cGAMP molecules or the ORBAC plasmid synthesized in Example 1 and Biotin-cGAMP molecules or aluminum hydroxide, respectively, to immunize female C57BL / 6 mice at the SPF level, 6 - 8 weeks old in each group. Among them, after the antigen OVA was uniformly mixed with aluminum hydroxide, it was left standing overnight; the other components were mixed and used immediately after preparation. Specifically, during injection, intramuscular injection was performed in the hind legs, and half of the injection volume was injected into each leg. Immediately after injection, electroshock was performed using an electroporator.

[0050] Mouse immunization timeline: After the C57BL / 6 mice were purchased, they were first raised for one week to adapt to the environment. After adaptation, each group was immunized on day 0 and day 14, for a total of two immunizations. Blood was collected from the tail vein on day 10 and day 21 respectively to detect the titer of OVA-specific IgG antibodies in the serum; meanwhile, on day 21, the mice were dissected and spleen cells were isolated for cellular immune analysis.

[0051] 2. ELISA determination of the titer of OVA-specific IgG antibodies in the serum of immunized mice The titer of antigen-specific antibodies produced in the serum of immunized mice directly reflects the ability of different adjuvants and immune molecules to enhance humoral immunity; sera at different time points were selected in the OVA model, and the titer of OVA-specific IgG antibodies in the sera at different time points was determined by ELISA to reveal the efficacy of different immune-stimulating molecules in enhancing humoral immunity.

[0052] Specific steps for ELISA determination: (1) Coat a 96-well plate (100 μL per well) with 10 μg / mL OVA antigen protein and leave it overnight at 4°C; (2) Discard the antigen coating solution, add blocking solution (PBS with 2% BSA) to the 96-well plate for blocking, add 250 μL per well, and incubate at 37°C for 2 h; (3) Discard the blocking solution, wash three times with PBST (300 μL / well), then serially dilute mouse sera to different concentration gradients with PBS containing 2% BSA, add 100 μL per well, and incubate at 37°C for 1 h; (4) Discard the mouse sera, wash three times with PBST (300 μL / well), dilute the HRP-labeled goat anti-mouse secondary antibody (1:15000), add 100 μL of the diluted secondary antibody solution per well after dilution, and incubate at 37°C for 1 h; (5) Discard the diluted secondary antibody solution, wash three times with PBST (300 μL / well) and drain, then add TMB chromogenic solution (100 μL / well), add 2 M H2SO4 to terminate the color development in each well after 15 min, and detect the OD value with an enzyme-linked immunosorbent assay detector at wavelengths of 450 nm and 630 nm. Among them, negative serum was used as a control, and negative serum was the serum of mice injected with only normal saline. Take 2.1 times the OD value of the negative serum as the threshold, and determine the antibody titer according to the sample serum dilution corresponding to the threshold (antibody titer = reciprocal of the highest dilution multiple higher than 2.1 times the OD value of the negative serum).

[0053] The results were as follows Figure 2 shown. At different time points (day 10 and day 21), the cGAMP molecules assembled in situ in the endoplasmic reticulum driven by ORBAC significantly enhanced the OVA-specific IgG antibody titer, significantly enhancing the humoral immune response. In addition, for the enhancement of the humoral immune response, the cGAMP molecules assembled in situ in the endoplasmic reticulum were also significantly superior to the commercial aluminum adjuvant.

[0054] 3. ELISPOT assay for the level of OVA-specific IFN-γ secreted by splenocytes of mice in different groups after immunization The level of antigen-specific IFN-γ secreted by mouse spleen cells is an important evaluation index of the intensity of its cellular immunity. Therefore, the ability of different immunostimulants to enhance cellular immunity can be evaluated by separating the level of OVA-specific IFN-γ secreted by splenocytes from mice in different groups.

[0055] Specific steps of the ELISPOT assay: (1) Add 100 μL of sample per well to the IFN-γ ELISPOT plate: The sample is 50 μL of stimulator diluent (OVA epitope peptide SIINFEKL) + 50 μL of cell diluent (10 6 cells, and the specific volume is calculated according to the cell counting concentration, and 1640 complete medium is added to make up to 50 μL). After adding the stimulator, add the cells, seal the detection plate with a sealing film, and incubate in a 37 °C CO2 incubator for 24 h. (2) Drain: Wash the plate 3 times with PBS (300 μL / well), and gently pat dry on absorbent paper. (3) Add Detection AB (100 μL / well), seal the detection plate with a sealing film, and incubate at room temperature for 2 h. (4) Drain: Wash the plate 3 times with PBS (300 μL / well), and gently pat dry on absorbent paper. (5) Add Diluted Streptavidin-AP Conjugate (100 μL / well), seal the detection plate with a sealing film, and incubate at room temperature for 1 h. (6) Drain: Wash the plate 3 times with PBS (300 μL / well), and gently pat dry on absorbent paper. (7) Add ready-to-use BCIP / NBT buffer (100 μL / well) and incubate at room temperature for 20 min for color development. (8) Drain: Rinse the plate 3 times with a wash bottle filled with distilled water, gently pat dry on absorbent paper, dry the excess water, and detect and read the spots with a German AID ELISPOT READER (German AID enzyme-linked immunosorbent spot analyzer) after natural air drying.

[0056] The results were as follows Figure 3 shown. The cGAMP molecules assembled in situ in the endoplasmic reticulum driven by ORBAC could significantly induce splenocytes to secrete more cytokine IFN-γ, enhancing the cellular immune level.

[0057] 4. Comprehensive analysis of the ability of immune-stimulating molecules to enhance cellular immunity by flow cytometry To more comprehensively analyze the enhancement of cellular immunity by immune-stimulating molecules, the levels of multiple cytokines, such as IFN-γ, IL-2, and TNF-α, secreted by splenocytes from different groups of immunized mice were detected by flow cytometry.

[0058] Specific steps for flow cytometry determination: (1) Spread mouse spleen cells onto round-bottom 96-well plates (10 6Cells / well), and add 1640 medium to a final volume of 100 μL. (2) Stimulate splenocytes with 1 μg / μL OVA epitope peptide SIINFEKL and incubate in a 37 °C, 5% CO2 incubator for 5 h. (3) Centrifuge the cells at 1800 rpm for 5 min, drain, wash the plate twice with PBS (200 μL / well), and gently pat dry on absorbent paper. (4) Block the cells with Fc blocker at room temperature for 15 min. (5) Centrifuge the cells at 1800 rpm for 5 min, drain, wash the plate once with PBS (200 μL / well), and gently pat dry on absorbent paper. (6) Resuspend the cells with Zombie NIR live / dead dye (PBS) and incubate in the dark at room temperature for 15 min. (7) Centrifuge the cells at 1800 rpm for 5 min, drain, wash the plate once with 2% FBS / PBS (200 μL / well), and gently pat dry on absorbent paper. (8) Stain the cells with surface dyes (anti-mouse CD3 (Biolegend, #100217), anti-mouse CD4 (Biolegend, #100406) and anti-mouse CD8 (Biolegend, #100730) antibodies) and incubate in the dark at 4 °C for 30 min. (9) Centrifuge the cells at 1800 rpm for 5 min, drain, wash the plate once with 2% FBS / PBS (200 μL / well), and gently pat dry on absorbent paper. (10) Resuspend the cells with fixation / permeabilization solution (100 μL / well) and incubate in the dark at 4 °C for 30 min. (11) Centrifuge the cells at 1800 rpm for 5 min, drain, wash the plate once with Perm / Wash buffer (200 μL / well), and gently pat dry on absorbent paper. (12) Stain the cells with intracellular dyes (anti-mouse IFN-γ (Biolegend, #505825), anti-mouse IL-2 (Biolegend, #503825) and anti-mouse TNF-α (Biolegend, #506339) antibodies) and incubate in the dark at 4 °C for 30 min. (13) Centrifuge the cells at 1800 rpm for 5 min, drain, wash the plate once with Perm / Wash buffer (200 μL / well), and gently pat dry on absorbent paper. (14) Resuspend the cells with 2% FBS / PBS (100 μL / well) and analyze by flow cytometry.

[0059] The results are as Figure 4 shown. The cGAMP molecules assembled in situ in the endoplasmic reticulum driven by ORBAC simultaneously enhanced CD4 + and CD8 +The secretion of various pro-inflammatory cytokines IFN-γ, IL-2, and TNF-α in T cells has significantly enhanced the cellular immune level.

[0060] Example 4 Evaluate the immunogenicity of ORBAC-induced endoplasmic reticulum in-situ assembled cGAMP molecules in a respiratory syncytial virus (RSV) vaccine model 1. Immunization protocol for C57BL / 6 mice Grouping for animal experiments: RSV group: Each mouse was injected only with RSV pre-F antigen (5 μg, purchased from Sino Biological); RSV + cGAMP group: Each mouse was injected with RSV pre-F (5 μg) + cGAMP molecule (2.5 μg, 0.0037 μmol); RSV + cGAMP + ORBAC group: Each mouse was injected with RSV pre-F antigen (5 μg) + Biotin-cGAMP molecule (5.32 μg, 0.0037 μmol) + ORBAC plasmid (80 μg); RSV + Alu group: Each mouse was injected with RSV pre-F antigen (5 μg) + aluminum hydroxide adjuvant (35 μg).

[0061] The respiratory syncytial virus antigen RSV pre-F was mixed evenly with cGAMP molecule or ORBAC plasmid and Biotin-cGAMP molecule or aluminum hydroxide respectively, and SPF-grade, 6-8-week-old female C57BL / 6 mice were immunized according to the above grouping. After the antigen OVA was mixed evenly with aluminum hydroxide, it was left standing overnight; other components were mixed and used immediately. Specifically, when injecting, it was intramuscularly injected into the hind legs, and half of the injection was given to each leg. Immediately after injection, it was electroshocked with an electroporator.

[0062] Immunization timeline for mice: After C57BL / 6 mice were purchased, they were first raised for one week to adapt to the environment. After adaptation, they were immunized on day 0 and day 21 respectively, for a total of two immunizations. On day 35, blood was collected from the tail vein and the spleen cells of the mice were isolated for detecting the RSV-specific IgG antibody, neutralizing antibody titer in the serum and cellular immune analysis.

[0063] 2. ELISA determination of RSV-specific IgG antibody titer in the serum of immunized mice The steps were the same as those in step 2 of Example 3. The results were as Figure 5 shown. Compared with ordinary cGAMP molecules and commercial aluminum adjuvants, ORBAC-driven endoplasmic reticulum in-situ assembled cGAMP can promote the production of higher levels of RVA-specific IgG antibodies, and significantly enhance the humoral immune response level.

[0064] 3. Neutralization test to detect the neutralizing antibody titers of different groups after mouse immunization Neutralizing antibodies refer to antibodies that prevent pathogens from binding to the surface receptors of host cells and invading target cells for replication and reproduction during the process of pathogen infection. Therefore, the induced neutralizing antibody titer is of great significance for evaluating the performance of vaccines and adjuvants.

[0065] Specific steps for neutralization test detection: (1) Heat-inactivate the serum and incubate it with RSV virus at 37 °C for 90 minutes; (2) Seed Vero cells into a 96-well plate and wait for the cells to grow into a monolayer; (3) Dilute the incubated RSV virus-serum mixture at a dilution gradient of 1:2 and seed it onto the 96-well plate containing monolayer Vero cells, and incubate for 90 minutes; (4) Wash the plate 3 times with PBS (300 μL / well), and gently pat dry on absorbent paper; (5) Drain, gently pat dry on absorbent paper, dry off the excess moisture, and after natural air drying, read the plate with a CTL Immunospot Analyzer and determine the neutralizing antibody titer according to fluorescence.

[0066] The results are as Figure 6 shown. Compared with cGAMP, the endoplasmic reticulum assembly driven by ORBAC can significantly enhance the production of RSV-specific neutralizing antibodies.

[0067] 4. ELISPOT assay to determine the level of RSV antigen-specific IFN-γ secreted by splenocytes of mice in different groups after immunization The steps are the same as step 3 of Example 3. The results are as Figure 7 shown. The cGAMP molecule with in-situ endoplasmic reticulum assembly driven by ORBAC can significantly promote splenocytes to secrete more antigen-specific IFN-γ and enhance the cellular immune level.

[0068] 5. Flow cytometry to comprehensively analyze the ability of immune stimulatory molecules to enhance cellular immunity The steps are the same as step 4 of Example 3. The results are as Figure 8 shown. Compared with ordinary cGAMP molecules and commercial aluminum adjuvants, the cGAMP with in-situ endoplasmic reticulum assembly driven by ORBAC can induce CD4 + and CD8 + T cells to secrete higher levels of antigen-specific IFN-α, IFN-β, IFN-γ, INF-α, IL-2 and IL-4; the cellular immune intensity is significantly enhanced.

[0069] Example 5 Evaluation of the immune protection performance induced by cGAMP molecules with in-situ endoplasmic reticulum assembly in an influenza virus vaccine model 1. Immunization protocol for C57BL / 6 mice Animal experiment grouping: Vacc group: Each mouse was injected only with hemagglutinin antigens from H1N1, H3N2, and BV (20 μg, purchased from Sino Biological); Vacc + cGAMP group: Each mouse was injected with hemagglutinin antigens from H1N1, H3N2, and BV (20 μg) + cGAMP molecule (2.5 μg, 0.0037 μmol); Vacc + cGAMP + ORBAC group: Each mouse was injected with hemagglutinin antigens from H1N1, H3N2, and BV (20 μg) + Biotin-cGAMP molecule (5.32 μg, 0.0037 μmol) + ORBAC plasmid (80 μg).

[0070] The influenza virus hemagglutinin antigens were respectively mixed evenly with cGAMP molecule or ORBAC plasmid and Biotin-cGAMP molecule, and then used to immunize SPF-grade female C57BL / 6 mice aged 6 - 8 weeks. During injection, the muscles of the hind legs were used for injection, and half of the dose was injected into each leg. Immediately after injection, electropermeation was performed using an electroporator.

[0071] 2. Influenza virus challenge test 28 days after mouse immunization, influenza virus challenge was carried out. Each mouse was intranasally inoculated with 50 μL of a virus mixture containing 10 8 EID 50 / mL A / Victoria / 2570 / 2019 (H1N1), 50 μL of 10 8 EID 50 / mL A / Darwin / 9 / 2021 (H3N2) and 50 μL of 10 8 EID 50 / mL B / Victoria lineage (BV). After challenge, the changes in mouse body weight were recorded and the lung tissue damage was analyzed.

[0072] The results are as Figure 9 and Figure 10 shown. Figure 9 The results showed that compared with the cGAMP molecule, its endoplasmic reticulum assembly delayed the time of mouse body weight loss, and the level of mouse body weight loss was also milder, resulting in better immune protection. Figure 10 The results showed that by observing the infiltration of inflammatory cells and interstitial edema in lung tissue, it could be judged that the cGAMP molecule in situ assembled in the endoplasmic reticulum driven by ORBAC could significantly reduce lung injury in mice and generate stronger immune protection against influenza virus.

[0073] 3. Evaluation of germinal center (GC) reaction A high-quality germinal center reaction is crucial for the production of high-affinity antibodies and efficient immune protection. Therefore, by means of flow cytometry, the ability of adjuvant molecules to induce the germinal reaction was evaluated by analyzing the number of germinal center B cells.

[0074] Specific steps: (1) Spread the lymphocytes of mice 10 days after immunization into a round-bottom 96-well plate (10 6 cells / well), and supplement with PBS to a final volume of 100 μL. (2) Centrifuge the cells at 1800 pm for 5 min, drain, wash the plate with PBS twice (200 L / well), gently pat dry on the absorbent paper, and block the cells with Fc blocker at room temperature for 15 min. (3) Centrifuge the cells at 1800 rpm for 5 min, drain, wash the plate with PBS once (200 μL / well), gently pat dry on the absorbent paper; resuspend the cells with Zombie NIR live / dead dye (PBS) and incubate in the dark at room temperature for 15 min. (4) Centrifuge the cells at 1800 rpm for 5 min, drain, wash the plate with 2% FBS / PBS once (200 μL / well), gently pat dry on the absorbent paper; (5) Stain the surface of the cells with dyes (anti-mouse CD45R (Biolegend, #103247), anti-mouse CD95 (Biolegend, #152607) and anti-mouse GL7 (Biolegend, #144619)), and incubate in the dark at 4 °C for 30 min. (6) Centrifuge the cells at 1800 rpm for 5 min, drain, wash the plate with 2% FBS / PBS once (200 μL / well), gently pat dry on the absorbent paper. (7) Resuspend the cells with 2% FBS / PBS (100 μL / well) and analyze by flow cytometry.

[0075] The results Figure 11 showed that the endoplasmic reticulum in situ-assembled cGAMP driven by ORBAC induced a larger number of germinal center B cells, promoting the germinal center reaction.

[0076] Example 6 Design of the missing structural unit in the molecular in situ-assembled chimera and synthesis of the recombinant plasmid 1. Design of the molecular in situ-assembled chimera lacking the organelle retention structural unit (ORBAC ΔA): The amino acid sequences of the assembly structural unit, the tracer structural unit, and the monomer streptavidin structural unit were connected from the N-terminus to the C-terminus through a linker in sequence to obtain ORBAC ΔA. Among them, the amino acid sequence of the assembly structural unit is shown in SEQ ID NO.1, the amino acid sequence of the tracer structural unit is shown in SEQ ID NO.2, the amino acid sequence of the monomer streptavidin structural unit is shown in SEQ ID NO.3, and the amino acid sequence of the linker is shown in SEQ ID NO.5.

[0077] The synthesis steps of the recombinant plasmid containing the ORBAC ΔA gene (ORBAC ΔA plasmid) are the same as those of the recombinant plasmid containing the ORBAC gene in Example 1, except that the organelle retention structural unit is deleted.

[0078] 2. Design of the molecular in-situ assembly chimera with the assembly structural unit deleted (ORBAC ΔB): The amino acid sequences of the signal peptide sequence that guides the protein into the organelle of the organelle retention structural unit, the tracer structural unit, the monomer streptomycin structural unit, and the endoplasmic reticulum retention signal sequence of the organelle retention domain unit are linked from the N-terminus to the C-terminus through a linker in sequence to obtain ORBAC ΔB. Among them, the amino acid sequence of the tracer structural unit is shown in SEQ ID NO.2, the amino acid sequence of the monomer streptomycin structural unit is shown in SEQ ID NO.3, the amino acid sequences of the organelle retention structural unit are shown in SEQ ID NO.4 (signal peptide sequence that guides the protein into the organelle) and SEQ ID NO.5 (organelle retention signal sequence), and the amino acid sequence of the linker is shown in SEQ ID NO.6.

[0079] The synthesis steps of the recombinant plasmid containing the ORBAC ΔB gene (ORBAC ΔB plasmid) are the same as those of the recombinant plasmid containing the ORBAC gene in Example 1, except that the assembly structural unit is deleted.

[0080] 3. Design of the molecular in-situ assembly chimera with the monomer streptomycin structural unit deleted (ORBAC ΔC): The amino acid sequences of the signal peptide sequence that guides the protein into the organelle of the organelle retention structural unit, the assembly structural unit, and the endoplasmic reticulum retention signal sequence of the organelle retention domain unit are linked from the N-terminus to the C-terminus through a linker in sequence to obtain ORBAC ΔC. Among them, the amino acid sequence of the assembly structural unit is shown in SEQ ID NO.1, the amino acid sequence of the tracer structural unit is shown in SEQ ID NO.2, the amino acid sequences of the organelle retention structural unit are shown in SEQ ID NO.4 (signal peptide sequence that guides the protein into the organelle) and SEQ ID NO.5 (organelle retention signal sequence), and the amino acid sequence of the linker is shown in SEQ ID NO.6.

[0081] The synthesis steps of the recombinant plasmid containing the ORBAC ΔC gene (ORBAC ΔC plasmid) are the same as those of the recombinant plasmid containing the ORBAC gene in Example 1, except for the monomer streptomycin structural unit.

[0082] Example 7 Observation of the endoplasmic reticulum retention and assembly of chimeras with structural units deleted respectively by laser confocal microscopy The steps are the same as those in Example 2. The results are asFigure 12 As shown, when the organelle retention domain unit is deleted (ORBAC ΔA), although the chimeric protein expressed by the cell can be assembled in situ, it cannot co-localize with the endoplasmic reticulum; when the assembly structure unit is deleted (ORBAC ΔB), the chimeric protein expressed by the cell can co-localize with the endoplasmic reticulum, but loses the assembly function; for the deletion of the monomer streptomycin structure unit (ORBAC ΔC), although it can co-localize with the endoplasmic reticulum and assemble in situ at the same time, it cannot capture molecules and cannot achieve the assembly of the target molecule in the organelle retention.

[0083] Example 8 Verify the immunological performance of cGAMP in chimeras with structural unit deletions (ORBAC ΔA, ORBAC ΔB, and ORBAC ΔC) in the model antigen OVA model 1. Immunization protocol for C57BL / 6 mice Grouping of animal experiments: OVA + cGAMP + ORBAC ΔA group: Each mouse was injected with 10 μg of OVA antigen, 5.32 μg of Biotin-cGAMP molecule (0.0037 μmol), and 79 μg of ORBAC ΔA (7.9 kbp); OVA + cGAMP + ORBAC ΔB group: Each mouse was injected with 10 μg of OVA antigen, 5.32 μg of Biotin-cGAMP molecule (0.0037 μmol), and 65 μg of ORBAC ΔB (6.5 kbp); OVA + cGAMP + ORBAC ΔC group: Each mouse was injected with 10 μg of OVA antigen, 5.32 μg of Biotin-cGAMP molecule (0.0037 μmol), and 76 μg of ORBAC ΔC (7.6 kbp); OVA + cGAMP + ORBAC group: Each mouse was injected with 10 μg of OVA antigen, 5.32 μg of Biotin-cGAMP molecule (0.0037 μmol), and 80 μg of ORBAC (8 kbp).

[0084] Mix the antigen OVA with the Biotin-cGAMP molecule and different ORBAC chimeras evenly, and immunize SPF-grade female C57BL / 6 mice at 6 - 8 weeks old. During injection, inject intramuscularly into the hind legs, with half injected into each leg, and immediately use an electroporator for electroshock after injection.

[0085] Timeline of mouse immunization: The same as in Example 3.

[0086] 2. ELISA determination of the titer of OVA-specific IgG antibodies in the sera of immunized mice The steps are the same as in Example 3. The results are as Figure 13As shown, compared with the cGAMP assembled in situ in the endoplasmic reticulum driven by ORBAC, the levels of OVA-specific antibodies induced by cGAMP with only endoplasmic reticulum residence (ORBAC ΔA) or in situ assembly (ORBAC ΔB) were both decreased, and the humoral immune response declined. For the chimera (ORBAC ΔC) that could not capture the cGAMP assembled in situ in the cytoplasm, the induction of cGAMP also relatively decreased the humoral immune level.

[0087] 3. ELISPOT assay for the level of OVA-specific IFN-γ secreted by splenocytes of mice in different groups after immunization The procedure was the same as that in Example 3. The results were as Figure 14 shown. Compared with the cGAMP assembled in situ in the endoplasmic reticulum driven by ORBAC, the cGAMP molecules with only in situ assembly (ORBAC ΔA), endoplasmic reticulum residence (ORBAC ΔB), or those that could not be captured (ORBAC ΔC) induced a relatively lower level of antigen-specific IFN-γ secretion, and the cellular immune response was weakened.

[0088] Example 9 Verification of the immunological performance of cGAMP under chimeras with structural unit deletions (ORBAC ΔA, ORBAC ΔB, and ORBAC ΔC) in a respiratory syncytial virus (RSV) vaccine model 1. Immunization protocol for C57BL / 6 mice Grouping of animal experiments: RSV + cGAMP + ORBAC ΔA group: Each mouse was injected with 5 μg of RSV pre-F antigen, 5.32 μg of Biotin-cGAMP molecule (0.0037 μmol), and 79 μg of ORBAC ΔA (7.9 kbp); RSV + cGAMP + ORBAC ΔB group: Each mouse was injected with 5 μg of RSV pre-F antigen, 5.32 μg of Biotin-cGAMP molecule (0.0037 μmol), and 65 μg of ORBAC ΔB (6.5 kbp); RSV + cGAMP + ORBAC ΔC group: Each mouse was injected with 5 μg of RSV pre-F antigen, 5.32 μg of Biotin-cGAMP molecule (0.0037 μmol), and 76 μg of ORBAC ΔC (7.6 kbp); OVA + cGAMP + ORBAC group: Each mouse was injected with 10 μg of OVA antigen, 5.32 μg of Biotin-cGAMP molecule (0.0037 μmol), and 80 μg of ORBAC (8 kbp).

[0089] Mix the anti-RSV pre-F antigen, Biotin-cGAMP molecules, and different ORBAC chimeras evenly, and immunize SPF-grade female C57BL / 6 mice aged 6-8 weeks. During injection, inject intramuscularly into the hind legs, with half injected into each leg, and immediately perform electroshock with an electroporator after injection.

[0090] Mouse immunization timeline: The steps are the same as in Example 4.

[0091] 2. ELISA determination of the RSV-specific IgG antibody titer in the sera of immunized mice The steps are the same as in Example 4. The results are as Figure 15 shown. Compared with the cGAMP assembled in situ in the endoplasmic reticulum driven by ORBAC, the RSV-specific antibody titers induced by the in situ assembled cGAMP molecules that failed to reside in the endoplasmic reticulum or could not be captured (ORBAC ΔC) were significantly reduced.

[0092] 3. Neutralization assay to detect the RSV-specific neutralizing antibody titer in the sera of immunized mice The steps are the same as in Example 4. The results are as Figure 16 shown. Compared with the cGAMP assembled in situ in the endoplasmic reticulum driven by ORBAC, only the in situ assembled (ORBAC ΔA), endoplasmic reticulum-resident (ORBAC ΔB), or non-capturable (ORBAC ΔC) cGAMP molecules induced relatively lower levels of RSV-specific neutralizing antibody production.

[0093] 4. ELISPOT determination of the level of RSV-specific IFN-γ secreted by splenocytes of mice in different groups after immunization The steps are the same as in Example 4. The results are as Figure 17 shown. Compared with the cGAMP assembled in situ in the endoplasmic reticulum (ORBAC-driven), the in situ assembled (ORBAC ΔA), endoplasmic reticulum-resident (ORBAC ΔB), or non-capturable (ORBAC ΔC) forms secreted significantly less RSV-specific IFN-γ, and the cellular immune response was weakened.

[0094] In summary, from the above experiments, it can be seen that in the molecular in situ assembly system with organelle residence designed in the present invention, the absence of the organelle residence domain unit cannot achieve endoplasmic reticulum residence; the absence of the assembly structural unit cannot achieve in situ assembly; the absence of the monomer streptomycin structural unit cannot capture the target molecule; further multi-model evaluations of animal experiments show that the developed molecular in situ assembly chimeras with organelle residence have shown great advantages in improving the performance of vaccines and adjuvants.

[0095] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A molecular in-situ assembly chimera, characterized in that, The chimeras include: an organelle retention structural unit, an assembly structural unit, a tracer structural unit, and a monomer streptomycin structural unit; the amino acid sequence of the assembly structural unit is as shown in SEQ ID NO.1, the amino acid sequence of the tracer structural unit is as shown in SEQ ID NO.2, and the amino acid sequence of the monomer streptomycin structural unit is as shown in SEQ ID NO.

3.

2. The molecular in-situ assembly chimera according to claim 1, characterized in that, The organelles in the organelle retention structural unit include one or more of endoplasmic reticulum, Golgi apparatus, endosome, lysosome, and mitochondrion.

3. The molecular in-situ assembly chimera according to claim 2, characterized in that, When the organelle is endoplasmic reticulum, the amino acid sequence of the organelle retention structural unit is as shown in SEQ ID NO.4 and SEQ ID NO.

5.

4. The molecular in-situ assembly chimera according to claim 1, characterized in that, The assembly structural unit, the tracer structural unit, and the monomer streptomycin structural unit are connected by a linker, and the amino acid sequence of the linker is as shown in SEQ ID NO.

6.

5. The molecular in-situ assembly chimera according to any one of claims 1-4, characterized in that, The amino acid sequence of the in-situ molecular assembly chimera is as shown in SEQ ID NO.

7.

6. A recombinant plasmid encoding the molecular in-situ assembly chimera according to any one of claims 1-5, characterized in that, The recombinant plasmid contains the nucleotide sequence of the in-situ molecular assembly chimera.

7. Use of the molecular in-situ assembly chimera according to any one of claims 1-5 or the recombinant plasmid according to claim 6 in the preparation of a molecular in-situ targeted organelle product.

8. Use of the molecular in-situ assembly chimera according to any one of claims 1-5 or the recombinant plasmid according to claim 6 in the preparation of a product for enhancing vaccine efficacy.

9. A product for enhancing vaccine efficacy, characterized in that, The product is obtained by mixing the recombinant plasmid according to claim 6, a biotin-labeled molecule, and a vaccine antigen.

10. The product according to claim 9, characterized in that, The vaccine antigen includes one or more of a model antigen OVA, a respiratory syncytial virus antigen, and an influenza virus hemagglutinin antigen.

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