An organelle-targeted molecular in situ assembly chimera and its application

Abstract

The present invention provides an organelle-targeted molecular in situ assembly chimera and its application, belonging to the field of molecular technology. The chimera of the present invention comprises, from N-terminus to C-terminus, an organelle-residence structural unit, an assembly structural unit, a tracer structural unit, and a monomeric streptavidin structural unit, each of which is connected by a linker. The chimera constructed by the present invention assembles biological functional molecules in situ in the organelle region and is applied to various vaccine antigen models. It can significantly enhance antigen-specific immune responses in various vaccine antigen models, improve vaccine efficacy, and enhance the immune protection generated by the vaccine.

Description

Technical Field

[0001] The present invention belongs to the field of molecular technology, and in particular relates to an organelle-targeted molecular in situ assembly chimera and applications thereof. 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 microscopic imaging technology, spontaneous assembly behaviors at the organelle level in cells are gradually being discovered and revealed. For example, the assembly of receptors and kinases on the inner side of the T cell membrane mediates efficient transduction of immune signals; the assembly of clathrin / adapter proteins in the trans-Golgi network participates in efficient protein trafficking; and the biological condensates formed by the assembly of some proteins in the cytoplasm regulate cellular stress. Analyzing assembly behaviors at the organelle level: On the one hand, assembly can ensure high concentration enrichment of molecules in local areas, accelerating the speed of biochemical reactions; on the other hand, the occurrence of specific organelles or regions ensures that reactions in the region do not interfere with each other, thereby improving reaction efficiency. Therefore, the spontaneous assembly behaviors at the organelle level in cells demonstrate their unique advantages in the evolutionary process.

[0003] Although more and more organelle-level assembly processes have been revealed, artificially achieving the assembly of target molecules in specific organelle regions remains challenging. Currently, several assembly technologies have been developed that rely on specialized cellular environments (e.g., acidic environments, overexpressed phosphatases, high concentrations of reactive oxygen species, etc.) to achieve intracellular assembly. However, most cells and organelles lack such specialized microenvironments, significantly limiting their application. 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. The molecular in situ assembly chimera (ORBAC) realizes the in situ assembly of the immunostimulatory molecule cGAMP in the endoplasmic reticulum, greatly improving the immune response induced by it. It is applied to a variety of antigens and vaccine models, significantly enhancing the immune protection efficacy of the vaccine.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a molecular in situ assembly chimera, which comprises: an organelle resident structural unit, an assembly structural unit, a tracer structural unit and a monomeric streptomycin structural unit; 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, and the amino acid sequence of the monomeric streptomycin structural unit is shown in SEQ ID NO.3.

[0007] Preferably, the organelles in the organelle-residing structural unit include one or more of the endoplasmic reticulum, Golgi apparatus, endosome, lysosome and mitochondria.

[0008] Preferably, when the organelle is the endoplasmic reticulum, the amino acid sequence of the organelle resident structural unit is as shown in SEQ ID NO.4 and SEQ ID NO.5.

[0009] Preferably, the assembly structural unit, the tracer structural unit and the monomeric streptomycin structural unit are connected via a linker, and the amino acid sequence of the linker is shown in SEQ ID NO.6.

[0010] Preferably, the amino acid sequence of the molecular in situ assembled chimera is shown as SEQ ID NO.7.

[0011] The present invention also provides a recombinant plasmid encoding the molecular in situ assembly chimera, wherein the recombinant plasmid contains the nucleotide sequence of the molecular in situ assembly chimera.

[0012] The present invention also provides the use of the molecular in situ assembly chimera or the recombinant plasmid in preparing a molecular in situ targeted organelle product.

[0013] The present invention also provides the use of the molecular in situ assembly chimera or the recombinant plasmid in the preparation of a product for improving vaccine efficacy.

[0014] The present invention provides a product for improving vaccine efficacy, which is obtained by mixing the recombinant plasmid, biotin-labeled molecules and vaccine antigens.

[0015] Preferably, the vaccine antigens include one or more of the model antigen OVA, respiratory syncytial virus antigen and influenza virus hemagglutinin antigen.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention proposes for the first time to obtain a molecular in situ assembly chimera by connecting the organelle resident structural unit, the assembly structural unit, the tracer structural unit and the monomeric streptomycin structural unit through a linker. The chimera constructed by the present invention can assemble biological functional molecules in situ in the organelle region, and then be applied to a variety of vaccine antigen models. It can significantly enhance the antigen-specific immune response in a variety of vaccine antigen models, improve the efficacy of the vaccine, and enhance the immune protection produced by the vaccine.

[0018] Furthermore, the molecular in situ assembly chimeras described herein possess broad-spectrum properties, enabling targeted in situ assembly of organelles of varying types and sizes. Furthermore, they can also achieve in situ assembly of molecules within multiple organelle types within cells, thus possessing a wide range of applications. Furthermore, the molecular in situ assembly chimeras described herein do not require extensive modification of the functional molecules; only biotin labeling is required, resulting in a simple process and strong operability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Laser confocal microscopy images of A549 cells 24 hours after transfection (left) and fluorescence intensity analysis results of the endoplasmic reticulum and expressed ORBAC (right).

[0020] Figure 2 Figure 2 is the OVA antigen-specific IgG antibody titer in the sera of mice in different groups on days 10 and 21.

[0021] Figure 3 For ELISPOT analysis on day 21, 10 mice in each group were 6 The secretion of OVA antigen-specific IFN-γ in spleen cells.

[0022] Figure 4 Flow cytometry analysis of CD4 + (Picture above), CD8 + (Bottom) Secretion of OVA antigen-specific IFN-γ, IL-2, and IFN-α in T cells.

[0023] Figure 5 The RSV antigen-specific IgG antibody titer in the serum of mice in different groups on the 35th day.

[0024] Figure 6 The RSV antigen-specific neutralizing antibody titers in the sera of mice in different groups on the 35th day.

[0025] Figure 7 For ELISPOT analysis on day 35, 10 mice in different groups were randomly divided into 6 The secretion of RSV antigen-specific IFN-γ in spleen cells.

[0026] Figure 8 Flow cytometry analysis of CD4 + (Picture above), CD8 + (Bottom) Secretion of RSV antigen-specific IFN-α, IFN-β, IFN-γ, TNF-α, IL-2, and IL-4 in T cells.

[0027] Figure 9 The figure shows the changes in body weight of mice in different groups for 10 consecutive days after infection.

[0028] Figure 10 These are lung tissue sections of mice in different groups on the 4th day after infection.

[0029] 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.

[0030] Figure 12 Laser confocal microscopy images (left) and fluorescence intensity analysis of the endoplasmic reticulum and ORBAC with partial structural unit deletions 24 hours after A549 cells were transfected with plasmids containing partial structural unit deletions (ORBAC ΔA, ORBAC ΔB, ORBAC ΔC) and endoplasmic reticulum marker plasmids (right).

[0031] Figure 13 The titers of OVA antigen-specific IgG antibodies in the sera of mice in different groups on the 21st day.

[0032] Figure 14 For ELISPOT analysis on day 21, 10 mice in each group were 6 The secretion of OVA antigen-specific IFN-γ in spleen cells.

[0033] Figure 15 The RSV antigen-specific IgG antibody titer in the serum of mice in different groups on the 35th day.

[0034] Figure 16 The RSV antigen-specific neutralizing antibody titers in the sera of mice in different groups on the 35th day.

[0035] Figure 17 For ELISPOT analysis on day 35, 10 mice in different groups were randomly divided into 6 The secretion of RSV antigen-specific IFN-γ in spleen cells. DETAILED DESCRIPTION

[0036] The present invention provides a molecular in situ assembly chimera, which includes: an organelle retention structure unit, an assembly structure unit, a tracer structure unit, and a monomeric streptomycin structure unit; preferably, the molecular in situ assembly chimera comprises, from N-terminus to C-terminus, a signal peptide sequence of the organelle retention structure unit that guides proteins into organelles, an assembly structure unit, a tracer structure unit, a monomeric streptomycin structure unit, and an organelle retention signal sequence of the organelle retention structure unit. The amino acid sequence of the assembly structure unit of the present invention is shown in SEQ ID NO.1, the amino acid sequence of the tracer structure unit is shown in SEQ ID NO.2, and the amino acid sequence of the monomeric streptomycin structure unit is shown in SEQ ID NO.3. The assembly structure unit, the tracer structure unit, and the monomeric streptomycin structure unit of the present invention are connected by a linker, the amino acid sequence of the linker is shown in SEQ ID NO.6. The organelle retention structure unit of the present invention is used to induce organelle retention, the assembly structure unit is used to drive assembly, the tracer structure unit is used for visualization, and the monomeric streptomycin structure unit is used to capture the target molecule to be assembled.

[0037] In the present invention, the organelles in the organelle-residence structural unit include one or more of the endoplasmic reticulum, lysosomes, ribosomes and mitochondria, preferably the endoplasmic reticulum. The organelle-residence structural unit of the present invention can be specifically adjusted and selected according to the specific target organelle. When the organelle of 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 proteins 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 of 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.

[0038] The present invention also provides a recombinant plasmid encoding the chimera, and the recombinant plasmid contains the nucleotide sequence of the molecular in situ assembled chimera. The recombinant plasmid of the present invention is prepared by the following method: reverse transcribing the amino acids of the molecular in situ assembled chimera into the chimera nucleotide sequence; linearizing the mammalian expression vector by the double enzyme digestion system KpnⅠ / XhoⅠ; and recombining the chimera nucleotide sequence into the linearized mammalian expression vector using homologous recombination technology to obtain a recombinant plasmid containing the chimera nucleotide sequence. The nucleotide sequence of the molecular in situ assembled chimera of the present invention is shown in SEQ ID NO.8. The mammalian expression vector of the present invention is pcDNA TM 3.1 (+).

[0039] The present invention also provides the use of the chimera or recombinant plasmid in the preparation of a product for molecular in situ targeting of organelles. The molecule of the present invention is preferably a cGAMP molecule, which is a small cyclic dinucleoside molecule that is considered a highly promising adjuvant molecule for enhancing vaccine efficacy due to its ability to activate the immune-related receptor STING and downstream signaling pathways.

[0040] The present invention provides a method for in situ assembly of cGAMP molecules in cellular organelles, comprising: mixing the recombinant plasmid and biotin-labeled cGAMP molecules and delivering them into cells, thereby achieving in situ assembly of the cGAMP molecules in the cellular organelles. The present invention utilizes a developed molecular in situ assembly chimera to in situ assemble cGAMP molecules in the endoplasmic reticulum region. Because the cGAMP receptor STING is localized in the endoplasmic reticulum, in situ assembly of cGAMP molecules in the endoplasmic reticulum is achieved.

[0041] The present invention also provides the use of the described in situ assembled molecular chimera or recombinant plasmid in the preparation of products that enhance vaccine efficacy. This invention combines the described recombinant plasmid, biotin-labeled cGAMP molecules, and vaccine antigens, and then introduces them into animals. This significantly enhances antigen-specific immune responses (including cellular and humoral immunity), overall improving vaccine efficacy and enhancing the immune protection provided by the vaccine. The biotin-labeled cGAMP molecules of the present invention can be obtained using methods well known in the art or through commercial sources.

[0042] The present invention also provides a product for enhancing vaccine efficacy, obtained by mixing the recombinant plasmid, a biotin-labeled molecule, and a vaccine antigen. The vaccine antigens of the present invention include one or more of the model antigen OVA, respiratory syncytial virus antigen, and influenza virus hemagglutinin antigen. The biotin-labeled molecule of the present invention includes a biotin-labeled cGAMP molecule (Biotin-cGAMP).

[0043] 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.

[0044] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0045] The raw materials and sources used in this example are: cGAMP, trade name: Sigma, molecular weight: 674.41; Biotin-cGAMP, trade name: AAT Bioquest, molecular weight: 1434.51; pcDNATM 3.1(+), Supplier: Sangon; Opti-MEM™ Medium, Supplier: Gibco; Lipofectamine TM 3000 transfection kit, manufacturer: Invitrogen.

[0046] Example 1 Design of molecular in situ assembly chimera and synthesis of recombinant plasmid

[0047] Design of a molecular in situ assembly chimera: The amino acid sequences of the signal peptide sequence of the organelle retention domain unit that directs protein entry into the endoplasmic reticulum, the assembly unit, the tracer unit, the monomeric streptavidin unit, and the endoplasmic reticulum retention signal sequence of the organelle retention domain unit are sequentially connected from N-terminus to C-terminus. The assembly unit, the tracer unit, and the monomeric streptavidin unit are connected by a linker to obtain a molecular in situ assembly chimera. 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 monomeric streptavidin unit is shown in SEQ ID NO.3, the amino acid sequences of the organelle retention unit are shown in SEQ ID NO.4 (signal peptide sequence that directs protein entry into the endoplasmic reticulum) and SEQ ID NO.5 (endoplasmic reticulum retention 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.

[0048] Synthesis of recombinant plasmid containing molecular in situ assembly chimera gene (commissioned by Shenggong Biosynthesis): (1) The amino acid sequence of the molecular in situ assembly chimera synthesized above was reverse transcribed into a nucleotide sequence, and the nucleotide sequence was synthesized by chemical synthesis, as shown in SEQ ID NO.8; (2) the mammalian expression vector pcDNA3. TM 3.1(+) Linearization; (3) Using homologous recombination technology, the above nucleotide sequence is recombined into the linearized mammalian expression vector pcDNA TM 3.1(+), a recombinant plasmid containing the molecular in situ assembled chimera gene (ORBAC plasmid) was obtained.

[0049] Example 2 Observation of the formed endoplasmic reticulum resident assembly using laser confocal microscopy

[0050] 1. Place 4×10 5 A549 cells / well were evenly plated into a 12-well plate containing a cell slide and cultured overnight;

[0051] 2. Use 50 μL Opti-MEMTM 3 μL of Lipofectamine diluted in culture medium TM 3000 transfection reagent;

[0052] 3. Use 50 μL Opti-MEM TM 1 μL of ORBAC plasmid (1 μg / μL) and 1 μL of Sec-61B plasmid (1 μg / μL, synthesized by Sangon Biotech) that marks the endoplasmic reticulum were diluted in the culture medium, and 2 μL of the dyeing reagent P3000 was added thereto. TM , and obtain the plasmid in the dilution solution;

[0053] 4. Mix the diluted Lipofectamine™ 3000 transfection reagent (1.5 μL / well) and the diluted plasmid (1 μg / well), and then incubate at room temperature for 15 minutes to obtain the plasmid-lipid complex;

[0054] 5. Add the plasmid-lipid complex to the A549 cells cultured overnight and gently shake to mix. Place in a cell culture incubator at 37°C, 5% CO2 and incubate for 1 day.

[0055] 6. After 1 day, cells were washed three times with PBS and stimulated with sodium arsenite (500 μM) for 1 h;

[0056] 7. Wash the cells and fix them with 4% paraformaldehyde;

[0057] 8. Use antiquenching reagent containing DAPI to stain cell nuclei;

[0058] 9. Fix the slide with nail polish and observe it under a laser confocal microscope.

[0059] The results are as follows Figure 1 As shown in the figure, ORBAC particles appeared clearly in the cells and co-localized well with the endoplasmic reticulum, proving that ORBAC successfully achieved endoplasmic reticulum-targeted in situ assembly.

[0060] Example 3 Evaluation of the immunological properties of cGAMP molecules assembled in situ by ORBAC in the endoplasmic reticulum in the model antigen OVA model

[0061] 1. Immunization procedure of C57BL / 6 mice

[0062] Animal experimental groups: OVA group: each mouse was injected with OVA antigen (10 μg) only; OVA+cGAMP group: each mouse was injected with OVA antigen (10 μg) + GAMP molecule (2.5 μg, 0.0037 μmol) only; 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).

[0063] The OVA antigen (purchased from Sigma) was mixed evenly with cGAMP, the ORBAC plasmid synthesized in Example 1, and Biotin-cGAMP, or aluminum hydroxide, and then immunized into the aforementioned groups of SPF-grade, 6-8-week-old female C57BL / 6 mice. The OVA antigen and aluminum hydroxide were mixed evenly and allowed to stand overnight; the other components were mixed immediately before use. Specifically, injections were made intramuscularly in the hind legs, with half injected in each leg. Immediately after injection, electroporation was performed using an electroporator.

[0064] Mouse immunization timeline: C57BL / 6 mice were purchased and raised for one week to acclimate to the environment. After acclimatization, each group was immunized on days 0 and 14, for a total of two immunizations. Blood was collected from the tail vein on days 10 and 21 to detect OVA-specific IgG antibody titers in the serum. At the same time, the mice were dissected on day 21, and spleen cells were isolated for cellular immune analysis.

[0065] 2. ELISA determination of OVA-specific IgG antibody titer in the serum of immunized mice

[0066] The antigen-specific antibody titer produced in the serum of mice after immunization directly reflects the ability of different adjuvants and immune molecules to enhance humoral immunity; sera at different time points are selected in the OVA model, and the OVA-specific IgG antibody titer in the serum at different time points is measured by ELISA to reveal the effectiveness of different immune stimulatory molecules in enhancing humoral immunity.

[0067] Specific steps of ELISA assay: (1) Coat a 96-well plate with 10 μg / mL OVA antigen protein (100 μL per well) at 4°C overnight; (2) Discard the antigen coating solution, add blocking solution (2% BSA in PBS) to the 96-well plate, 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), and then wash with 2% The mouse serum was diluted with BSA in PBS to different concentration gradients, 100 μL per well, and incubated at 37°C for 1 hour; (4) The mouse serum was discarded, and the cells were washed three times with PBST (300 μL / well). HRP-labeled goat anti-mouse secondary antibody was diluted (1:15000). After dilution, 100 μL of secondary antibody diluent was added to each well and incubated at 37°C for 1 hour; (5) The secondary antibody diluent was discarded, and the cells were washed three times with PBST (300 μL / well) and drained. TMB colorimetric solution (100 μL / well) was then added. After 15 minutes, 2M H2SO4 was added to each well to stop colorimetric development. The OD value was detected by enzyme-linked immunosorbent assay at wavelengths of 450 nm and 630 nm. Negative serum was used as the control. Negative serum was the serum of mice injected with saline only. 2.1 times the OD value of negative serum was used as the threshold value, and the antibody titer was determined based on the sample serum dilution corresponding to the threshold value (antibody titer = the reciprocal of the highest dilution factor that was higher than 2.1 times the OD value of negative serum).

[0068] The results are as follows Figure 2 As shown, at different time points (days 10 and 21), ORBAC-driven endoplasmic reticulum in situ assembled cGAMP molecules induced significantly enhanced OVA-specific IgG antibody titers and significantly enhanced humoral immune responses; in addition, in terms of enhancing humoral immunity, endoplasmic reticulum in situ assembled cGAMP molecules were also significantly better than commercial aluminum adjuvants.

[0069] 3. ELISPOT determination of OVA-specific IFN-γ secretion by spleen cells of different groups of mice after immunization

[0070] The level of antigen-specific IFN-γ secreted by mouse spleen cells is an important evaluation indicator of the strength of its cellular immunity. Therefore, the ability of different immunostimulants to enhance cellular immunity can be evaluated by isolating the level of OVA-specific IFN-γ secreted by spleen cells from different groups of mice.

[0071] Specific steps of ELISPOT assay: (1) Add 100 μL sample / well to the IFN-γ ELISPOT bottom plate: the sample is 50 μL stimulus diluent (OVA epitope peptide SIINFEKL) + 50 μL cell diluent (10 6(2) Drying: Wash the plate 3 times with PBS (300 μL / well), pat dry on absorbent paper. (3) Add Detection AB (100 μL / well), seal the plate with sealing film, and incubate at room temperature for 2 hours. (4) Drying: Wash the plate 3 times with PBS (300 μL / well), pat dry on absorbent paper. (5) Add Diluted Streptavidin-AP Conjugate (100 μL / well), seal the plate with sealing film, and incubate at room temperature for 1 hour. (6) Drying: Wash the plate 3 times with PBS (300 μL / well), pat dry on absorbent paper. (7) Add ready-to-use BCIP / NBT buffer (100 μL / well) and incubate at room temperature for 20 min to develop color. (8) Control drying: Rinse the plate three times with a wash bottle filled with distilled water, pat dry on absorbent paper to remove excess water, and air dry naturally before detecting the reading spot using the German AID ELISPOT READER (German AID enzyme-linked spot analyzer).

[0072] The results are as follows Figure 3 As shown, ORBAC-driven endoplasmic reticulum in situ assembled cGAMP molecules can significantly induce spleen cells to secrete more cytokine IFN-γ and enhance cellular immunity levels.

[0073] 4. Flow cytometry comprehensively analyzes the ability of immune stimulatory molecules to enhance cellular immunity

[0074] In order to more comprehensively analyze the enhancement of cellular immunity by immunostimulatory molecules, flow cytometry was used to detect the levels of IFN-γ, IL-2 and TNF-α secreted by spleen cells of different groups of mice after immunization.

[0075] Flow cytometry assay specific steps: (1) Spread mouse spleen cells into a round-bottom 96-well plate (10 6(2) Stimulate spleen cells with 1 μg / μL of OVA epitope peptide SIINFEKL and incubate at 37°C in a 5% CO2 incubator for 5 h. (3) Centrifuge cells at 1800 rpm for 5 min, drain, wash the plate twice with PBS (200 μL / well), and pat dry on absorbent paper. (4) Block cells with Fc blocker at room temperature for 15 min. (5) Centrifuge cells at 1800 rpm for 5 min, drain, wash the plate once with PBS (200 μL / well), and pat dry on absorbent paper. (6) Resuspend cells in Zombie NIR dead dye (PBS) and incubate at room temperature in the dark for 15 min. (7) Centrifuge cells at 1800 rpm for 5 min, drain, wash the plate once with 2% FBS / PBS (200 μL / well), and pat dry on absorbent paper. (8) Surface 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 at 4°C in the dark 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 pat dry on absorbent paper. (10) Resuspend the cells in fixation / permeabilization buffer (100 μL / well) and incubate at 4°C in the dark 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 pat dry on absorbent paper. (12) Intracellular staining of cells with intracellular dyes (anti-mouse IFN-γ (Biolegend, #505825), anti-mouse IL-2 (Biolegend, #503825) and anti-mouse TNF-α (Biolegend, #506339) antibodies) was performed and incubated at 4°C in the dark for 30 min. (13) Cells were centrifuged at 1800 rpm for 5 min, drained, and the plate was washed once with Perm / Wash buffer (200 μL / well) and gently patted dry on absorbent paper. (14) Cells were resuspended in 2% FBS / PBS (100 μL / well) and loaded onto a flow cytometer.

[0076] The results are as follows Figure 4 As shown, ORBAC-driven in situ assembly of cGAMP molecules in the endoplasmic reticulum also enhanced CD4 + and CD8 +The secretion of multiple pro-inflammatory cytokines IFN-γ, IL-2 and TNF-α in T cells significantly improved the level of cellular immunity.

[0077] Example 4 Evaluation of the immunogenicity of cGAMP molecules assembled in situ by ORBAC in the endoplasmic reticulum in a respiratory syncytial virus (RSV) vaccine model

[0078] 1. Immunization procedure of C57BL / 6 mice

[0079] Animal experimental groups: RSV group: each mouse was injected with RSV pre-F antigen (5 μg, purchased from Sino-Bio; 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).

[0080] Respiratory syncytial virus (RSV) pre-F antigen was mixed with cGAMP, ORBAC plasmid, and Biotin-cGAMP, or aluminum hydroxide, respectively. SPF-grade female C57BL / 6 mice, aged 6-8 weeks, were immunized according to the groupings described above. The OVA antigen was mixed with the aluminum hydroxide and allowed to stand overnight. The other components were mixed immediately before use. Specifically, injections were given intramuscularly in the hind legs, with half injected in each leg. Immediately after injection, electroporation was performed using an electroporator.

[0081] Mouse immunization timeline: C57BL / 6 mice were purchased and housed for one week to acclimate to the environment. After acclimatization, they were immunized twice on days 0 and 21, respectively. On day 35, blood was collected from the tail vein and the mice were dissected to isolate spleen cells for detection of RSV-specific IgG antibodies in the serum, neutralizing antibody titers, and cellular immunity analysis.

[0082] 2. ELISA determination of RSV-specific IgG antibody titer in the serum of immunized mice

[0083] The steps are the same as those in step 2 of Example 3. Figure 5 As shown, compared with ordinary cGAMP molecules and commercial aluminum adjuvants, cGAMP assembled in situ in the endoplasmic reticulum driven by ORBAC can promote the production of higher levels of RVA-specific IgG antibodies and significantly enhance the level of humoral immune response.

[0084] 3. Neutralization test to detect the neutralizing antibody titer of different groups of mice after immunization

[0085] Neutralizing antibodies refer to antibodies that prevent pathogens from binding to host cell surface receptors and invading target cells to replicate and multiply during pathogen infection. Therefore, the induced neutralizing antibody titer is of great significance for evaluating the performance of vaccines and adjuvants.

[0086] The specific steps of the neutralization test are as follows: (1) Heat-inactivate the serum and incubate it with RSV virus at 37°C for 90 minutes; (2) Use Vero cells to spread on 96-well plates and wait until the cells grow into a monolayer; (3) Dilute the incubated RSV virus-serum mixture in a 1:2 dilution gradient and spread it on a 96-well plate containing a monolayer of Vero cells and incubate for 90 minutes; (4) Wash the plate three times with PBS (300 μL / well) and pat dry on absorbent paper; (5) Control dryness, pat dry on absorbent paper to wipe off excess water, and after natural air drying, read the plate with a CTL Immunospot Analyzer and determine the neutralizing antibody titer based on fluorescence.

[0087] The results are as follows Figure 6 As shown, compared with cGAMP, ORBAC-driven endoplasmic reticulum assembly can significantly enhance RSV-specific neutralizing antibody production.

[0088] 4. ELISPOT determination of RSV antigen-specific IFN-γ secretion levels in splenocytes of mice in different groups after immunization

[0089] The steps are the same as those in step 3 of Example 3. The results are as follows Figure 7 As shown, ORBAC-driven endoplasmic reticulum in situ assembled cGAMP molecules can significantly promote spleen cells to secrete more antigen-specific IFN-γ and enhance cellular immunity levels.

[0090] 5. Flow cytometry comprehensively analyzes the ability of immune stimulatory molecules to enhance cellular immunity

[0091] The steps are the same as step 4 of Example 3. The results are as follows Figure 8 As shown, compared with common cGAMP molecules and commercial aluminum adjuvants, ORBAC-driven endoplasmic reticulum in situ assembled cGAMP can induce CD4 + and CD8 + T cells secrete higher levels of antigen-specific IFN-α, IFN-β, IFN-γ, INF-α, IL-2 and IL-4; the intensity of cellular immunity is significantly enhanced.

[0092] Example 5 Evaluation of the immune protection induced by cGAMP molecules assembled in situ on the endoplasmic reticulum in an influenza virus vaccine model

[0093] 1. Immunization procedure of C57BL / 6 mice

[0094] Animal experimental groups: Vacc group: each mouse was injected with hemagglutinin antigens derived from H1N1, H3N2 and BV only (20 μg, purchased from Sino-Bio; Vacc+cGAMP group: each mouse was injected with hemagglutinin antigens derived from H1N1, H3N2 and BV (20 μg) + cGAMP molecules (2.5 μg, 0.0037 μmol); Vacc+cGAMP+ORBAC group: each mouse was injected with hemagglutinin antigens derived from H1N1, H3N2 and BV (20 μg) + Biotin-cGAMP molecules (5.32 μg, 0.0037 μmol) + ORBAC plasmid (80 μg).

[0095] Influenza virus hemagglutinin antigen was mixed with cGAMP or ORBAC plasmid and Biotin-cGAMP, respectively, and then immunized into SPF-grade, 6-8-week-old female C57BL / 6 mice. Injection was performed intramuscularly in the hind legs, with half injected in each leg. Immediately after injection, electroporation was performed using an electroporator.

[0096] 2. Influenza virus challenge test

[0097] On day 28 after immunization, mice were challenged with influenza virus. Each mouse was inhaled with 50 μL of 10 8 EID 50 / mLA / Victoria / 2570 / 2019(H1N1), 50μL 10 8 EID 50 / mL A / Darwin / 9 / 2021(H3N2) and 50μL10 8 EID 50 After challenge, the body weight of mice was recorded and the damage to their lung tissue was analyzed.

[0098] The results are as follows 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 weight loss in mice, the level of weight loss in mice was also milder, and it produced better immune protection. Figure 10 The results showed that by observing the inflammatory cell infiltration and interstitial edema in the lung tissue, it can be judged that the cGAMP molecules assembled in situ by the endoplasmic reticulum driven by ORBAC can significantly reduce lung damage in mice and produce stronger immune protection against influenza virus.

[0099] 3. Germinal center (GC) response evaluation

[0100] High-quality germinal center reactions are important for the production of high-affinity antibodies and efficient immune protection. Therefore, the ability of adjuvant molecules to induce germinal reactions was evaluated by analyzing the number of B cells in the germinal centers using flow cytometry.

[0101] Specific steps: (1) Lymphocytes of mice 10 days after immunization were plated into a round-bottom 96-well plate (10 6 Cells were centrifuged at 1800 rpm for 5 min, drained, and washed twice with PBS (200 μL / well). The plates were gently patted dry on absorbent paper. Cells were blocked with Fc blocker at room temperature for 15 min. Cells were centrifuged at 1800 rpm for 5 min, drained, and washed once with PBS (200 μL / well). The plates were gently patted dry on absorbent paper. Cells were resuspended in Zombie NIR dead-alive dye (PBS) and incubated at room temperature in the dark for 15 min. (4) Centrifuge the cells at 1800 rpm for 5 min, drain, wash the plate once with 2% FBS / PBS (200 μL / well), and pat dry on absorbent paper. (5) Surface stain the cells with dyes (anti-mouse CD45R (Biolegend, #103247), anti-mouseCD95 (Biolegend, #152607), and anti-mouse GL7 (Biolegend, #144619) and incubate at 4°C in the dark for 30 min. (6) Centrifuge the cells at 1800 rpm for 5 min, drain, wash the plate once with 2% FBS / PBS (200 μL / well), and pat dry on absorbent paper. (7) Resuspend the cells in 2% FBS / PBS (100 μL / well) and load on flow cytometer.

[0102] The results are as follows Figure 11 As shown, ORBAC-driven production of cGAMP assembled in situ by the endoplasmic reticulum induces the generation of a larger number of germinal center B cells and promotes the germinal center reaction.

[0103] Example 6 Design of missing structural units in molecular in situ assembly chimeras and synthesis of recombinant plasmids

[0104] 1. Design of a molecular in situ assembly chimera lacking the organelle-resident structural unit (ORBAC ΔA): The amino acid sequences of the assembly structural unit, the tracer structural unit, and the monomeric streptavidin structural unit were linked sequentially from N-terminus to C-terminus via a linker to obtain ORBAC ΔA. 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 monomeric 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.

[0105] The synthesis steps of the recombinant plasmid containing the ORBAC ΔA gene (ORBAC ΔA plasmid) were the same as those of the recombinant plasmid containing the ORBAC gene in Example 1, except that the organelle-resident structural unit was missing.

[0106] 2. Design of a molecular in situ assembly chimera lacking an assembly building block (ORBAC ΔB): The amino acid sequences of the organelle-resident building block's signal peptide sequence for protein entry into the organelle, the tracer building block, the monomeric streptavidin building block, and the endoplasmic reticulum retention signal sequence of the organelle-resident domain building block were connected in sequence from N-terminus to C-terminus via a linker to produce ORBAC ΔB. The amino acid sequence of the tracer building block is shown in SEQ ID NO. 2, the amino acid sequence of the monomeric streptavidin building block is shown in SEQ ID NO. 3, the amino acid sequences of the organelle-resident building block are shown in SEQ ID NO. 4 (signal peptide sequence for protein entry 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.

[0107] The synthesis steps of the recombinant plasmid containing the ORBAC ΔB gene (ORBAC ΔB plasmid) were the same as those of the recombinant plasmid containing the ORBAC gene in Example 1, except that the assembly structural unit was missing.

[0108] 3. Design of a molecular in situ assembly chimera lacking the monomeric streptavidin structural unit (ORBAC ΔC): The amino acid sequences of the organelle-resident structural unit's signal peptide sequence for protein entry into the organelle, the assembly structural unit, and the endoplasmic reticulum retention signal sequence of the organelle-resident structural unit were connected sequentially from N-terminus to C-terminus via a linker to produce ORBAC ΔC. 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-resident structural unit are shown in SEQ ID NO.4 (signal peptide sequence for protein entry 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.

[0109] 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 monomeric streptomycin structural unit.

[0110] Example 7 Observation of the endoplasmic reticulum-resident assembly of chimeras with missing structural units using laser confocal microscopy

[0111] The steps are the same as in Example 2. Figure 12 As shown in the figure, when the organelle retention domain unit is missing (ORBAC ΔA), the chimeric protein expressed by the cell can assemble in situ but cannot co-localize with the endoplasmic reticulum; when the assembly structural unit is missing (ORBAC ΔB), the chimeric protein expressed by the cell can co-localize with the endoplasmic reticulum but loses the assembly function; for the monomeric streptavidin structural unit missing (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 organelle residence.

[0112] Example 8 Validation of cGAMP Immunity in Chimeras with Deletions of Structural Units (ORBAC ΔA, ORBAC ΔB, and ORBAC ΔC) in the Model Antigen OVA Model

[0113] 1. Immunization procedure of C57BL / 6 mice

[0114] Animal experimental groups: OVA+cGAMP+ORBAC ΔA group: each mouse was injected with 10 μg OVA antigen, 5.32 μg Biotin-cGAMP molecule (0.0037 μmol) and 79 μg ORBAC ΔA (7.9 kbp); OVA+cGAMP+ORBAC ΔB group: each mouse was injected with 10 μg OVA antigen, 5.32 μg Biotin-cGAMP molecule (0.0037 μmol) and 65 μg ORBAC ΔB (6.5 kbp); OVA+cGAMP+ORBAC ΔC group: each mouse was injected with 10 μg OVA antigen, 5.32 μg Biotin-cGAMP molecule (0.0037 μmol) and 76 μg ORBAC ΔC (7.6 kbp); OVA+cGAMP+ORBAC group: each mouse was injected with 10 μg OVA antigen, 5.32 μg Biotin-cGAMP molecule (0.0037 μmol) and 76 μg ORBAC ΔC (7.6 kbp). Biotin-cGAMP molecule (0.0037 μmol) and 80 μg ORBAC (8 kbp).

[0115] The OVA antigen was mixed with the Biotin-cGAMP molecule and various ORBAC chimeras and used to immunize SPF-grade, 6-8-week-old female C57BL / 6 mice. The injection was injected intramuscularly into the hind legs, with half injected into each leg. Immediately after the injection, the mice were electroporated using an electroporator.

[0116] Mouse immunization timeline: same as Example 3.

[0117] 2. ELISA determination of OVA-specific IgG antibody titer in the serum of immunized mice

[0118] The steps are the same as in Example 3. Figure 13 As shown, compared with cGAMP assembled in situ at the endoplasmic reticulum driven by ORBAC, the levels of OVA-specific antibodies induced by cGAMP that resides only in the endoplasmic reticulum (ORBAC ΔA) or is assembled in situ at the endoplasmic reticulum (ORBAC ΔB) were reduced, and the humoral immune response was reduced; for the chimera (ORBAC ΔC) that could not capture cGAMP in the cytoplasm and assembled in situ at the endoplasmic reticulum, cGAMP induction also relatively reduced the level of humoral immunity.

[0119] 3. ELISPOT determination of OVA-specific IFN-γ secretion by spleen cells of different groups of mice after immunization

[0120] The steps are the same as in Example 3. Figure 14As shown, compared with cGAMP assembled in situ in the endoplasmic reticulum driven by ORBAC, cGAMP molecules that were only assembled in situ (ORBAC ΔA), retained in the endoplasmic reticulum (ORBAC ΔB), or could not be captured (ORBAC ΔC) induced relatively low levels of antigen-specific IFN-γ secretion and weakened the cellular immune response.

[0121] Example 9 Validation of cGAMP Immunity in Respiratory Syncytial Virus (RSV) Vaccine Models with Chimeras Deleting Building Blocks (ORBAC ΔA, ORBAC ΔB, and ORBAC ΔC)

[0122] 1. Immunization procedure of C57BL / 6 mice

[0123] Animal experimental groups: RSV+cGAMP+ORBAC ΔA group: each mouse was injected with 5 μg RSV pre-F antigen, 5.32 μg Biotin-cGAMP molecule (0.0037 μmol) and 79 μg ORBAC ΔA (7.9 kbp); RSV+cGAMP+ORBAC ΔB group: each mouse was injected with 5 μg RSV pre-F antigen, 5.32 μg Biotin-cGAMP molecule (0.0037 μmol) and 65 μg ORBAC ΔB (6.5 kbp); RSV+cGAMP+ORBAC ΔC group: each mouse was injected with 5 μg RSV pre-F antigen, 5.32 μg Biotin-cGAMP molecule (0.0037 μmol) and 76 μg ORBAC ΔC (7.6 kbp); OVA+cGAMP+ORBAC group: each mouse was injected with 10 μg OVA antigen, 5.32 μg Biotin-cGAMP molecules (0.0037 μmol) and 80 μg ORBAC (8 kbp).

[0124] The anti-RSV pre-F antigen was mixed with the Biotin-cGAMP molecule and various ORBAC chimeras and used to immunize SPF-grade, 6-8-week-old female C57BL / 6 mice. The injection was injected intramuscularly into the hind legs, half of each leg, followed by immediate electroporation.

[0125] Mouse immunization timeline: The steps are the same as Example 4.

[0126] 2. ELISA determination of RSV-specific IgG antibody titer in the serum of immunized mice

[0127] The steps are the same as in Example 4. Figure 15As shown, compared with the endoplasmic reticulum-assembled cGAMP driven by ORBAC, the endoplasmic reticulum-resident in situ assembled cGAMP molecules that failed to reside in the endoplasmic reticulum or could not be captured (ORBAC ΔC) induced significantly lower RSV-specific antibody titers.

[0128] 3. Neutralization test to detect RSV-specific neutralizing antibody titer in the serum of immunized mice

[0129] The steps are the same as in Example 4. Figure 16 As shown, compared with cGAMP produced by ORBAC-driven in situ assembly in the ER, only cGAMP molecules that were in situ assembled (ORBAC ΔA), ER-resident (ORBAC ΔB), or unable to be captured (ORBAC ΔC) induced the production of relatively lower levels of RSV-specific neutralizing antibodies.

[0130] 4. ELISPOT determination of RSV-specific IFN-γ secretion levels in splenocytes of mice in different groups after immunization

[0131] The steps are the same as in Example 4. Figure 17 As shown, compared with cGAMP assembled in situ in the ER (ORBAC driven), the forms of in situ assembly (ORBAC ΔA), ER retention (ORBAC ΔB), or non-capture (ORBAC ΔC) significantly reduced the secretion of RSV-specific IFN-γ and weakened the cellular immune response.

[0132] From the above experiments, it can be seen that in the in situ assembly system of molecules for organelle residence designed by the present invention, the lack of organelle residence domain unit cannot achieve endoplasmic reticulum residence; the lack of assembly structure unit cannot achieve in situ assembly; the lack of monomeric streptomycin structure unit cannot capture the target molecule; further animal experimental multi-model evaluation shows that the developed organelle-residence molecular in situ assembly chimera has shown great advantages in improving the performance of vaccines and adjuvants.

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A molecular in situ assembled chimera, characterized in that The chimera comprises: an organelle resident structural unit, an assembly structural unit, a tracer structural unit and a monomeric streptomycin structural unit; 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, and the amino acid sequence of the monomeric streptomycin structural unit is shown in SEQ ID NO.3; When the cell organelle is the endoplasmic reticulum, the amino acid sequence of the cell organelle resident structural unit is as shown in SEQ ID NO.4 and SEQ ID NO.5; The assembly structural unit, the tracer structural unit and the monomeric streptomycin structural unit are connected by a linker, and the amino acid sequence of the linker is shown in SEQ ID NO.6; The amino acid sequence of the molecular in situ assembled chimera is shown in SEQ ID NO.

7.

2. A recombinant plasmid encoding the molecular in situ assembly chimera according to claim 1, characterized in that: The recombinant plasmid contains the nucleotide sequence of the molecular in situ assembly chimera.

3. Use of the molecular in situ assembly chimera according to claim 1 or the recombinant plasmid according to claim 2 in the preparation of a cGAMP molecule in situ targeted endoplasmic reticulum product.

4. Use of the molecular in situ assembly chimera according to claim 1 or the recombinant plasmid according to claim 2 in the preparation of a product for improving the efficacy of a vaccine containing a cGAMP molecule.

5. A product for improving vaccine efficacy, characterized in that: The product is obtained by mixing the recombinant plasmid according to claim 2, biotin-labeled cGAMP molecules and vaccine antigens.

6. The product according to claim 5, characterized in that The vaccine antigens include one or more of the model antigen OVA, respiratory syncytial virus antigen and influenza virus hemagglutinin antigen.

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