CN120168630B - An organelle-targeted molecular in situ assembly chimera and its application - Google Patents
An organelle-targeted molecular in situ assembly chimera and its applicationInfo
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- CN120168630B CN120168630B CN202510668065.1A CN202510668065A CN120168630B CN 120168630 B CN120168630 B CN 120168630B CN 202510668065 A CN202510668065 A CN 202510668065A CN 120168630 B CN120168630 B CN 120168630B
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Abstract
The invention provides an in-situ assembled chimeric of a cell organelle targeting molecule and application thereof, and belongs to the technical field of molecular science. The chimeric body sequentially comprises a cell organelle resident structural unit, an assembly structural unit, a tracing structural unit and a monomer streptomycin structural unit from the N end to the C end, wherein the units are connected through a linker. The chimeric constructed by the invention is characterized in that the biological functional molecule unit is assembled in the organelle region and is applied to various vaccine antigen models, so that the immune response of antigen specificity can be obviously enhanced in the various vaccine antigen models, the vaccine efficacy is improved, and the immune protection generated by the vaccine is enhanced.
Description
Technical Field
The invention belongs to the technical field of molecular science, and particularly relates to an in-situ assembled chimeric of a cell organelle targeting molecule and application thereof.
Background
The assembly behavior of organelle hierarchy plays an important role in the normal execution of life activities and the efficient occurrence of biochemical reactions. At present, along with the rapid development of microscopic imaging technology, the spontaneous assembly behavior of organelle layers in cells is gradually discovered and revealed, for example, the assembly of receptors and kinases on the inner side of T cell membranes mediates the efficient transduction of immune signals, the assembly of clathrins/adapter proteins in trans-Golgi networks participates in the efficient transportation of proteins, and biological aggregates formed by the assembly of partial proteins in cytoplasm regulate cell stress. On one hand, the assembly can ensure the high concentration enrichment of molecules in a local area and quicken the speed of biochemical reaction, and on the other hand, the occurrence of specific organelles or areas ensures the mutual noninterference of the reaction in the areas and improves the reaction efficiency. Thus, the assembly behaviour at the organelle level, which occurs spontaneously in cells, demonstrates its unique advantage in the evolution process.
While more and more organelle-level assembly is disclosed, artificial realization of target molecule assembly in specific organelle regions is challenging. Currently, some assembly techniques that rely on specific environments (acidic environments, overexpressed phosphatases, high concentrations of reactive oxygen species, etc.) in cells are developed to achieve intracellular assembly, but the lack of such specific microenvironments for most cells, organelles, and thus greatly limit the scope of application.
Disclosure of Invention
In view of the above, the invention aims to provide an in-situ assembled chimeric molecule targeted by organelles and application thereof, wherein the in-situ assembled chimeric molecule (ORBAC) realizes in-situ assembly of an immune stimulating molecule cGAMP in an endoplasmic reticulum, greatly improves the immune response induced by the immune stimulating molecule cGAMP, and remarkably enhances the immune protection efficacy of vaccines when the immune stimulating molecule cGAMP is applied to various antigens and vaccine models.
In order to solve the technical problems, the invention provides the following technical scheme:
The invention provides a molecular in-situ assembled chimera, which comprises a cell organelle resident structural unit, an assembled structural unit, a tracer structural unit and a monomeric streptomycin structural unit, wherein the amino acid sequence of the assembled structural unit is shown as SEQ ID NO.1, the amino acid sequence of the tracer structural unit is shown as SEQ ID NO.2, and the amino acid sequence of the monomeric streptomycin structural unit is shown as SEQ ID NO. 3.
Preferably, the organelles in the organelle resident building block comprise one or more of endoplasmic reticulum, golgi apparatus, endosome, lysosome, and mitochondria.
Preferably, when the organelle is the endoplasmic reticulum, the amino acid sequence of the organelle resident structural unit is shown in SEQ ID NO.4 and SEQ ID NO. 5.
Preferably, the assembly structural unit, the tracer structural unit and the monomeric streptomycin structural unit are connected through a linker, and the amino acid sequence of the linker is shown as SEQ ID No. 6.
Preferably, the amino acid sequence of the molecule in-situ assembled chimera is shown in SEQ ID NO. 7.
The invention also provides a recombinant plasmid for encoding the molecule in-situ assembled chimera, wherein the recombinant plasmid contains a nucleotide sequence of the molecule in-situ assembled chimera.
The invention also provides application of the molecule in-situ assembled chimeric or the recombinant plasmid in preparing a molecule in-situ targeted organelle product.
The invention also provides application of the molecule in-situ assembled chimeric or the recombinant plasmid in preparing a product for improving vaccine efficacy.
The invention provides a product for improving vaccine efficacy, which is obtained by mixing the recombinant plasmid, biotin-labeled molecules and vaccine antigens.
Preferably, the vaccine antigen comprises one or more of the model antigen OVA, respiratory syncytial virus antigen and influenza virus hemagglutinin antigen.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides a molecule in-situ assembled chimera which is obtained by connecting an organelle resident structural unit, an assembled structural unit, a tracer structural unit and a monomeric streptomycin structural unit through a linker for the first time, and the chimera constructed by the invention can assemble biological functional molecules in situ in an organelle area and then is applied to various vaccine antigen models, so that the immune response of antigen specificity can be obviously enhanced in the various vaccine antigen models, the vaccine efficacy is improved, and the immune protection generated by the vaccine is enhanced.
Furthermore, the molecule in-situ assembled chimera has the characteristic of broad spectrum, can realize the in-situ assembly of cell organelles of different types and sizes in a targeted manner, can realize the in-situ assembly of the molecules in various types of organelles in cells, and has wide application range. In addition, in the in-situ assembled chimera of the application molecules, the invention can be realized by only carrying out biotin labeling without excessive modification on the functional molecules, and has simple process and strong operability.
Drawings
FIG. 1 shows the results of fluorescence intensity analysis of the laser confocal images (left panel) and endoplasmic reticulum, expressed ORBAC (right panel) 24 hours after A549 cell transfection.
Figure 2 shows OVA antigen-specific IgG antibody titers in serum from different groups of mice on days 10 and 21.
FIG. 3 shows secretion of OVA antigen specific IFN-gamma per 10 6 spleen cells from mice of different groups on day 21 of ELISPOT analysis.
FIG. 4 shows the secretion of OVA antigen specific IFN-. Gamma.IL-2 and IFN-. Alpha.in T cells of different groups of mice on day 21 of flow cytometry analysis (upper panel), CD8 + (lower panel).
Figure 5 shows RSV antigen-specific IgG antibody titers in sera of mice of different groups on day 35.
Figure 6 shows RSV antigen-specific neutralizing antibody titers in sera of mice of different groups on day 35.
FIG. 7 shows secretion of RSV antigen specific IFN-gamma per 10 6 spleen cells from mice of different groups on day 35 of ELISPOT analysis.
FIG. 8 is a flow cytometry analysis of secretion of RSV antigen specific IFN- α, IFN- β, IFN- γ, TNF- α, IL-2 and IL-4 in T cells of different groups of mice on day 35 (upper panel), CD8 + (lower panel).
FIG. 9 shows the body weight change of mice of different groups 10 consecutive days after challenge.
FIG. 10 is a lung tissue section of mice of different groups on day 4 after challenge.
FIG. 11 is a graph showing the ratio of center B cells (GC B) generated in lymph nodes of mice of different groups on day 10 post immunization.
FIG. 12 shows the fluorescence intensity analysis of 24 hours after A549 cells were transfected with a plasmid with partial structural unit deletion (ORBAC. DELTA. A, ORBAC. DELTA. B, ORBAC. DELTA.C) and endoplasmic reticulum marker plasmid with laser confocal images (left panel) and endoplasmic reticulum, partial structural unit deletion ORBAC (right panel).
FIG. 13 shows the titers of OVA antigen-specific IgG antibodies in the sera of mice of different groups on day 21.
FIG. 14 shows secretion of OVA antigen specific IFN-. Gamma.by ELISPOT analysis on day 21 in different groups of mice per 10 6 spleen cells.
Figure 15 shows RSV antigen-specific IgG antibody titers in sera of mice of different groups on day 35.
Figure 16 shows RSV antigen-specific neutralizing antibody titers in sera of mice of different groups on day 35.
FIG. 17 shows secretion of RSV antigen specific IFN-gamma per 10 6 spleen cells from mice of different groups on day 35 of ELISPOT analysis.
Detailed Description
The invention provides a molecular in-situ assembled chimeric body, which comprises an organelle resident structural unit, an assembled structural unit, a tracer structural unit and a monomer streptomycin structural unit, wherein the molecular in-situ assembled chimeric body is preferably a signal peptide sequence of the organelle resident structural unit for guiding protein into organelles, an assembled structural unit, the tracer structural unit, the monomer streptomycin structural unit and an organelle resident signal sequence of the organelle resident structural unit from an N end to a C end. The amino acid sequence of the assembly structural unit is shown as SEQ ID NO.1, the amino acid sequence of the tracing structural unit is shown as SEQ ID NO.2, and the amino acid sequence of the monomeric streptomycin structural unit is shown as SEQ ID NO. 3. The assembly structural unit, the tracer structural unit and the monomeric streptomycin structural unit are connected through a linker, and the amino acid sequence of the linker is shown as SEQ ID NO. 6. The organelle residence structural unit is used for inducing organelle residence, the assembly structural unit is used for driving assembly, the tracing structural unit is used for visualization, and the monomeric streptomycin structural unit is used for capturing target molecules to be assembled.
In the present invention, the organelles in the organelle resident building block include one or more of endoplasmic reticulum, lysosome, ribosome and mitochondria, preferably endoplasmic reticulum. The organelle resident building blocks of the invention may be specifically tailored and selected according to the specific target organelle. When the organelle is an endoplasmic reticulum, the amino acid sequence of the organelle resident structural unit is shown as SEQ ID NO.4 and SEQ ID NO.5, the amino acid sequence shown as SEQ ID NO.4 is a signal peptide sequence for guiding protein to enter the endoplasmic reticulum, and the amino acid sequence shown as SEQ ID NO.5 is an endoplasmic reticulum resident signal sequence. When the organelle is an endoplasmic reticulum, the amino acid sequence of the molecule in-situ assembled chimera is shown as SEQ ID NO. 7.
The invention also provides a recombinant plasmid for encoding the chimera, wherein the recombinant plasmid contains a nucleotide sequence of the molecule in-situ assembled chimera. The recombinant plasmid is prepared through reverse transcription of amino acid of the in-situ assembled chimeric molecule into chimeric nucleotide sequence, linearization of mammal expression vector with double enzyme cutting system KpnI/XhoI, and recombination of the chimeric nucleotide sequence onto the linearized mammal expression vector with homologous recombination technology. The nucleotide sequence of the molecule in-situ assembled chimeric is shown as SEQ ID NO. 8. The mammalian expression vector of the invention is pcDNA TM 3.1.1 (+).
The invention also provides application of the chimera or the recombinant plasmid in preparing a molecular in-situ targeting organelle product. The molecule of the invention is preferably a cGAMP molecule, a small molecule of the cyclic dinucleotide class, which is considered to be a very potent adjuvant molecule for increasing vaccine efficacy due to its ability to activate the immune-related receptor STING and downstream signaling pathways.
The method for assembling the cGAMP molecular site on the organelle comprises the steps of mixing the recombinant plasmid and the biotin-marked cGAMP molecule, and delivering the mixture into the cell, so that the cGAMP molecular site can be assembled on the organelle. The invention utilizes developed molecule in-situ assembled chimeras to assemble the cGAMP molecules in situ in the endoplasmic reticulum region, and the receptor STING of the cGAMP is positioned in the endoplasmic reticulum, so that the cGAMP molecules are assembled in situ on the endoplasmic reticulum.
The invention also provides application of the molecule in-situ assembled chimeric or the recombinant plasmid in preparing a product for improving vaccine efficacy. The recombinant plasmid, the biotin-marked cGAMP molecules and vaccine antigens are mixed and transferred into an animal body, so that the antigen-specific immune response (including cellular immunity and humoral immunity) can be obviously enhanced, the vaccine efficacy is integrally improved, and the immune protection generated by the vaccine is enhanced. The biotin-labeled cGAMP molecules of the invention can be obtained by methods well known in the art, and are also commercially available.
The 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 comprises one or more of model antigen OVA, respiratory syncytial virus antigen and influenza virus hemagglutinin antigen. The Biotin-labeled molecules of the present invention include Biotin-labeled cGAMP molecules (Biotin-cGAMP).
In the present invention, all components or reagents or media are commercially available as known to those skilled in the art unless specifically indicated.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The raw materials used in this example were cGAMP, merchant Sigma, molecular weight 674.41, biotin-cGAMP, merchant AAT Bioquest, molecular weight 1434.51, pcDNA TM 3.1.1 (+), merchant, manufacturer (Sangon), opti-MEMTM medium, merchant, gibco, lipofectamine TM 3000 transfection kit, merchant, invitrogen.
Example 1 design of molecular in situ assembled chimeras and recombinant plasmid Synthesis
The design of the molecule in-situ assembled chimeric body comprises the steps of connecting the amino acid sequences of a signal peptide sequence of a guide protein of an organelle resident structural domain unit into an endoplasmic reticulum, an assembly structural unit, a tracer structural unit, a monomeric streptomycin structural unit and an endoplasmic reticulum resident signal sequence of the organelle resident structural domain unit from N end to C end in sequence, wherein the assembly structural unit, the tracer structural unit and the monomeric streptomycin structural unit are connected through a linker, and the molecule in-situ assembled chimeric body. The amino acid sequence of the assembled structural unit is shown as SEQ ID NO.1, the amino acid sequence of the tracer structural unit is shown as SEQ ID NO.2, the amino acid sequence of the monomeric streptomycin structural unit is shown as SEQ ID NO.3, the amino acid sequence of the organelle resident structural unit is shown as SEQ ID NO.4 (signal peptide sequence for guiding protein into endoplasmic reticulum) and SEQ ID NO.5 (endoplasmic reticulum resident signal sequence), the amino acid sequence of the linker is shown as SEQ ID NO.6, and the amino acid sequence of the designed molecule in-situ assembled chimeric is shown as SEQ ID NO. 7.
The recombinant plasmid synthesis (entrusting biological synthesis) of the molecule in-situ assembled chimeric gene comprises (1) reverse transcription of the amino acid sequence of the synthesized molecule in-situ assembled chimeric gene into a nucleotide sequence, synthesis of the nucleotide sequence by chemical synthesis method, as shown in SEQ ID NO.8, (2) linearization of a mammalian expression vector pcDNA TM 3.1.1 (+) by a double enzyme digestion system (KpnI/XhoI), and (3) recombination of the nucleotide sequence onto the linearized mammalian expression vector pcDNA TM 3.1.1 (+) by means of homologous recombination technology to obtain the recombinant plasmid (ORBAC plasmid) containing the molecule in-situ assembled chimeric gene.
Example 2 formed endoplasmic reticulum resident Assembly Using laser confocal microscopy
1. Uniformly spreading 4×10 5/well of A549 cells on a 12-well plate containing a cell climbing sheet, and culturing overnight;
2. mu.L of Lipofectamine TM 3000 transfection reagent was diluted with 50. Mu.L of Opti-MEM TM medium;
3. mu.L of ORBAC plasmid (1. Mu.g/. Mu.L) and 1. Mu.L of Sec-61B plasmid (1. Mu.g/. Mu.L, a biological construction synthesis) of the labeled endoplasmic reticulum were diluted with 50. Mu.L of Opti-MEM TM medium, and 2. Mu.L of a counterstain reagent P3000 TM was added thereto to obtain diluted plasmids;
4. Mixing diluted Lipofectamine 3000 transfection reagent (1.5. Mu.L/well) and diluted plasmid (1. Mu.g/well), and incubating at room temperature for 15min to obtain a plasmid-lipid complex;
5. the plasmid-lipid complex was added to the above-described overnight a549 cells and gently shaken, placed in a cell incubator, and incubated at 37 ℃ for 1 day with 5% CO 2;
After 6.1 days, cells were washed three times with PBS and stimulated for 1h with the addition of sodium arsenite (500 μm);
7. washing the cells, fixing the cells with 4% paraformaldehyde;
8. Nuclear staining with anti-quencher containing DAPI;
9. The climbing plate is fixed by nail polish and is placed under a laser confocal microscope for observation.
The results are shown in FIG. 1. From the figure, it can be seen that the apparent ORBAC particles appear in the cells and also have good co-localization with the endoplasmic reticulum, demonstrating that ORBAC successfully achieves in situ assembly of endoplasmic reticulum targeting.
Example 3 evaluation of the immune Properties of ORBAC-induced in situ assembled cGAMP molecules of the endoplasmic reticulum in a model antigen OVA model
1. C57BL/6 mouse immunization program
Animal experiment groups were OVA group in which each mouse was injected with only OVA antigen (10. Mu.g), OVA+cGAMP group in which each mouse was injected with only OVA antigen (10. Mu.g) + GAMP molecules (2.5. Mu.g, 0.0037. Mu. Mol), OVA+cGAMP+ ORBAC group in which each mouse was injected with OVA antigen (10. Mu.g) +Biotin-cGAMP molecules (5.32. Mu.g, 0.0037. Mu. Mol) + ORBAC plasmid (80. Mu.g), and OVA+aluminum adjuvant group in which each mouse was injected with OVA antigen (10. Mu.g) +aluminum hydroxide adjuvant (35. Mu.g).
Antigen OVA (purchased from Sigma) was homogeneously mixed with cGAMP molecule or ORBAC plasmid synthesized in example 1 and Biotin-cGAMP molecule or aluminum hydroxide, respectively, and each group of SPF-class, 6-8 week female C57BL/6 mice was immunized. Wherein the antigen OVA and the aluminum hydroxide are mixed uniformly and then are stood overnight, and other components are mixed for use. Specifically, the hind leg is intramuscular injected and the left and right legs are half injected respectively, and the electroporation instrument is used for electric shock immediately after the injection is completed.
Mice were immunized twice in total on days 0 and 14, and on days 10 and 21, tail vein blood was collected for detection of OVA-specific IgG antibody titer in serum, while on day 21, mice were dissected and spleen cells were isolated for cellular immunoassay.
2. ELISA determination of OVA-specific IgG antibody titres in mice serum after immunization
The antigen-specific antibody titer generated in serum after mice immunization directly reflects the enhancement capability of different adjuvants and immune molecules on humoral immunity, serum at different time points is selected in an OVA model, and the ELISA is used for measuring the OVA-specific IgG antibody titer in serum at different time points, so that the efficacy of different immune stimulating molecules on humoral immunity enhancement is revealed.
The ELISA assay comprises (1) coating 96-well plates (100. Mu.L per well) with 10. Mu.L of OVA antigen protein, and standing overnight at 4 ℃, (2) removing antigen coating solution, adding blocking solution (PBS of 2% BSA) to 96-well plates, adding 250. Mu.L per well, incubating at 37 ℃ for 2 hours, (3) removing blocking solution, washing PBST three times (300. Mu.L per well), sequentially diluting mouse serum to different concentration gradients with PBS containing 2% BSA, incubating at 100. Mu.L per well for 1 hour at 37 ℃, and (4) removing mouse serum, washing PBST three times (300. Mu.L per well), diluting the labeled goat anti-mouse secondary antibody (1:15000), adding 100. Mu.L secondary anti-diluent per well after dilution, incubating at 37 ℃ for 1 hour, (5) removing secondary anti-diluent, washing PBST three times (300. Mu.L per well), and drying, adding TMB chromogenic solution (100. Mu.L per well), adding 2-M H 2SO4 after 15min, and detecting the negative serum at the same time when the serum is negative serum is detected by an ELISA tester at the wavelength of 630nm, and the serum is detected by using a physiological saline. With 2.1 times of the OD value of the negative serum as a threshold, the antibody titer was determined from the sample serum dilutions corresponding to the threshold (antibody titer=reciprocal of the highest dilution 2.1 times higher than the OD value of the negative serum).
The results are shown in figure 2, at different time points (days 10, 21), the induction of ORBAC-driven in-situ assembled cGAMP molecules significantly enhanced OVA-specific IgG antibody titers and significantly enhanced humoral immune responses, and in addition, the enhancement of humoral immunity, the in-situ assembled cGAMP molecules were also significantly superior to commercial aluminum adjuvants.
3. ELISPOT assay of spleen cells from different groups of mice post-immunization secrete OVA-specific IFN-gamma levels
The level of the spleen cells of mice secreting antigen specific IFN-gamma is an important evaluation index of the cellular immunity intensity of the mice, so that the capability of different immunostimulants to enhance the cellular immunity can be evaluated by separating the level of the spleen cells secreting OVA specific IFN-gamma from different groups of mice.
ELISPOT assay specific procedure (1) IFN-. Gamma.ELISPOT bottom plate 100. Mu.L sample/well was added to 50. Mu.L of the stimulus dilution (OVA epitope peptide SIINFEKL) +50. Mu.L of the cell dilution (10 6 cells, specific volumes based on cell count concentration), 1640 complete medium was added to 50. Mu.L, cells were added after addition of the stimulus, the assay plate was sealed with sealing film, and incubated for 24h at 37℃in CO 2 incubator. (2) And (3) drying, namely washing the plate 3 times (300 mu L/hole) by using PBS, and gently beating the plate on absorbent paper. (3) Detection AB (100. Mu.L/well) was added, the Detection plate was sealed with a sealing film, and incubated at room temperature for 2 hours. (4) And (3) drying, namely washing the plate 3 times (300 mu L/hole) by using PBS, and gently beating the plate on absorbent paper. (5) Diluted Streptavidin-AP Conjugate (100. Mu.L/well) was added, the assay plate was sealed with sealing film and incubated for 1h at room temperature. (6) And (3) drying, namely washing the plate 3 times (300 mu L/hole) by using PBS, and gently beating the plate on absorbent paper. (7) Add ready-to-use BCIP/NBT buffer (100. Mu.L/well) and incubate for 20min at room temperature to develop color. (8) And (3) drying, namely flushing the plate 3 times by using a bottle with distilled water, gently beating the plate on absorbent paper, drying excessive water, and detecting reading points by using German AID ELISPOT READER (German AID enzyme-linked spot analyzer) after natural air drying.
As shown in fig. 3, ORBAC-driven in situ assembled cGAMP molecules of the endoplasmic reticulum can significantly induce spleen cells to secrete more cytokine IFN- γ, enhancing cellular immune levels.
4. Flow cytometry to fully analyze the enhancing capacity of immunostimulatory molecules on cellular immunity
To more fully analyze the enhancement of cellular immunity by immunostimulatory molecules, levels of various cytokines such as IFN-gamma, IL-2 and TNF-alpha were measured by flow cytometry in different groups of spleen cells of immunized mice.
Flow cytometry assay specific steps (1) mouse spleen cells were plated into round bottom 96 well plates (10 6 cells/well), and 1640 medium was added to a final volume of 100 μl. (2) Spleen cells were stimulated with 1. Mu.g/. Mu.L of OVA epitope peptide SIINFEKL and incubated for 5h in a 37℃5% CO 2 incubator. (3) The cells were centrifuged at 1800rpm for 5min, drained, plates washed 2 times with PBS (200. Mu.L/well) and gently blotted on absorbent paper. (4) blocking cells with Fc blocker at room temperature for 15min. (5) The cells were centrifuged at 1800rpm for 5min, drained, the plates were washed 1 time (200. Mu.L/well) with PBS and gently blotted on absorbent paper. (6) Cells were resuspended with Zombie NIR dead dye (PBS) and incubated at room temperature for 15min in the dark. (7) The cells were centrifuged at 1800rpm for 5min, drained, plates were washed 1 time (200. Mu.L/well) with 2% FBS/PBS and gently blotted on absorbent paper. (8) The cells were surface stained with surface dye （anti-mouse CD3 (Biolegend, #100217), anti-mouse CD4 (Biolegend, #100406) and anti-mouse CD8 (Biolegend, #100730) antibodies） and incubated at 4℃for 30min in the absence of light. (9) The cells were centrifuged at 1800rpm for 5min, drained, plates were washed 1 time (200. Mu.L/well) with 2% FBS/PBS and gently blotted on absorbent paper. (10) Cells were resuspended (100 μl/well) with fixation/membrane-rupture fluid and incubated at 4 ℃ for 30min protected from light. (11) The cells were centrifuged at 1800rpm for 5min, drained, the plates were washed 1 time (200. Mu.L/well) with Perm/Wash buffer and gently blotted on absorbent paper. (12) Cells were stained intracellular with intracellular dye （anti-mouse IFN-γ (Biolegend, #505825), anti-mouse IL-2 (Biolegend, #503825) and anti-mouse TNF-α (Biolegend, #506339) antibodies ） and incubated at 4 ℃ for 30min in the dark. (13) The cells were centrifuged at 1800rpm for 5min, drained, the plates were washed 1 time (200. Mu.L/well) with Perm/Wash buffer and gently blotted on absorbent paper. (14) Cells were resuspended (100. Mu.L/well) with 2% FBS/PBS and flow-on-machine.
As a result, as shown in FIG. 4, ORBAC-driven, in situ assembled cGAMP molecules in the endoplasmic reticulum enhanced secretion of a variety of pro-inflammatory cytokines IFN-gamma, IL-2 and TNF-alpha in CD4 + and CD8 + T cells, and the cellular immune levels were significantly improved.
Example 4 evaluation of the immune Properties of ORBAC-induced in situ assembled cGAMP molecules of the endoplasmic reticulum in a Respiratory Syncytial Virus (RSV) vaccine model
1. C57BL/6 mouse immunization program
The animals were grouped into RSV groups, in which each mouse was injected with only RSV pre-F antigen (5. Mu.g, available from Yinqiao, china), RSV+cGAMP groups, in which each mouse was injected with only RSV pre-F (5. Mu.g) +cGAMP molecule (2.5. Mu.g, 0.0037. Mu.mol), and RSV+cGAMP+ ORBAC groups, in which each mouse was injected with RSV pre-F antigen (5. Mu.g) +Biotin-cGAMP molecule (5.32. Mu.g, 0.0037. Mu.mol) + ORBAC plasmid (80. Mu.g), and RSV+alu groups, in which each mouse was injected with RSV pre-F antigen (5. Mu.g) +aluminum hydroxide adjuvant (35. Mu.g).
Respiratory syncytial virus antigen RSVpre-F was homogeneously mixed with cGAMP molecule or ORBAC plasmid and Biotin-cGAMP molecule or aluminum hydroxide, respectively, and SPF-grade, 6-8 week female C57BL/6 mice were immunized according to the above group. Wherein the antigen OVA and the aluminum hydroxide are mixed uniformly and then are stood overnight, and other components are mixed for use. Specifically, the hind leg is intramuscular injected and the left and right legs are half injected respectively, and the electroporation instrument is used for electric shock immediately after the injection is completed.
Mice were immunized twice on days 0 and 21 after being housed for one week after purchase, and were pooled and spleen cells were dissected for detection of RSV-specific IgG antibodies in serum, neutralizing antibody titer, and cellular immunoassay, respectively, on day 35 tail vein blood collection.
2. ELISA determination of RSV-specific IgG antibody titres in mice serum after immunization
The procedure is as in example 3, step 2. As shown in figure 5, ORBAC driven in situ assembly of the produced endoplasmic reticulum with cGAMP promotes the production of higher levels of RVA-specific IgG antibodies, with significantly elevated humoral immune response levels compared to the common cGAMP molecule and commercial aluminum adjuvants.
3. Neutralization assay to detect neutralizing antibody titres in different groups after immunization of mice
Neutralizing antibodies refer to antibodies that prevent a pathogen from binding to host cell surface receptors and invading target cells for replication and reproduction during infection by the pathogen, and thus the induced neutralizing antibody titer is of great importance for evaluating the performance of vaccines and adjuvants.
The neutralization test comprises the specific steps of (1) heating and inactivating serum, incubating the serum and RSV at 37 ℃ for 90 minutes, (2) spreading the RSV-serum mixture to a 96-well plate with Vero cells until the cells are full of a monolayer, (3) diluting the incubated RSV-serum mixture in a dilution gradient of 1:2, spreading the mixture to the 96-well plate with the monolayer Vero cells, incubating for 90 minutes, (4) washing the plate 3 times (300 mu L/hole) with PBS, gently beating on a piece of absorbent paper, and (5) controlling the water to be light-beaten and dry on the piece of absorbent paper, wiping the redundant water, reading the plate with CTL Immunospot Analyzer after natural air drying, and judging the neutralizing antibody titer according to fluorescence.
As shown in fig. 6, the orcac driven endoplasmic reticulum assembly thereof significantly enhanced RSV-specific neutralizing antibody production compared to cGAMP.
4. ELISPOT assay of specific IFN-gamma levels secreted by spleen cells of different groups of mice after immunization
The procedure is as in example 3, step 3. As shown in fig. 7, ORBAC-driven in situ assembled cGAMP molecules of the endoplasmic reticulum significantly promoted secretion of more antigen-specific IFN- γ by spleen cells, enhancing cellular immune levels.
5. Flow cytometry to fully analyze the enhancing capacity of immunostimulatory molecules on cellular immunity
The procedure is as in example 3, step 4. As shown in fig. 8, ORBAC driven in situ assembly of the produced endoplasmic reticulum induced 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 than common cGAMP molecules and commercial aluminum adjuvants, with significantly enhanced cellular immunity.
Example 5 evaluation of the immunoprotection properties induced by in situ assembled cGAMP molecules of the endoplasmic reticulum in an influenza vaccine model
1. C57BL/6 mouse immunization program
Animal experiment groups Vacc+cGAMP groups each injected with H1N1, H3N2 and BV-derived hemagglutinin antigen alone (20. Mu.g, available from Yizhushen), vacc+cGAMP groups each injected with H1N1, H3N2 and BV-derived hemagglutinin antigen (20. Mu.g) +cGAMP molecules (2.5. Mu.g, 0.0037. Mu. Mol), vacc+cGAMP+ ORBAC groups each injected with H1N1, H3N2 and BV-derived hemagglutinin antigen (20. Mu.g) +Biotin-cGAMP molecules (5.32. Mu.g, 0.0037. Mu. Mol) + ORBAC plasmid (80. Mu.g).
Influenza virus hemagglutinin antigen was mixed homogeneously with cGAMP molecule or ORBAC plasmid and Biotin-cGAMP molecule, respectively, and SPF-grade, 6-8 week female C57BL/6 mice were immunized. The injection is performed by muscle injection of the hind leg and half of the injection is performed by the left and right legs, and the electroporation instrument is used for electric shock immediately after the injection is finished.
2. Influenza virus challenge test
Influenza virus challenge was performed 28 days after mice immunization, each mouse inhaling 50μL 108EID50/mL A/Victoria/2570/2019(H1N1),50μL 108EID50/mL A/Darwin/9/2021(H3N2) by nasal drops and 50 μl of 10 8EID50/mL B/Victoria lineage (BV) virus cocktail. After challenge, the mice were recorded for changes in body weight and analyzed for lung tissue damage.
The results are shown in fig. 9 and 10. The results in fig. 9 show that the endoplasmic reticulum assembly delays the weight loss time of mice compared to cGAMP molecules, and that the weight loss level of mice is also more slight, resulting in better immune protection. The results of fig. 10 show that, by observing inflammatory cell infiltration and interstitial edema of lung tissue, it can be judged that the cGAMP molecule assembled in situ by the endoplasmic reticulum generated by ORBAC can significantly reduce lung injury of mice and generate stronger immune protection against influenza virus.
3. Evaluation of germinal center (GERMINAL CENTER, GC) response
High quality germinal center reactions are important for the production of high affinity antibodies and for efficient immunoprotection, and therefore the ability of adjuvant molecules to induce germinal reactions is assessed by analyzing the number of germinal center B cells by flow cytometry.
The method comprises (1) plating lymphocytes from mice 10 days after immunization into round bottom 96-well plates (10 6 cells/well), and adding PBS to a final volume of 100. Mu.L. (2) 1800pm of the cells were centrifuged for 5min, drained, plates were washed 2 times (200L/well) with PBS, gently blotted dry on absorbent paper, and the cells were blocked with Fc blocking for 15min at room temperature. (3) The cells were centrifuged at 1800rpm for 5min, drained, washed 1 Xwith PBS (200. Mu.L/well), gently blotted on absorbent paper, resuspended with Zombie NIR dead-live dye (PBS) and incubated 15min at room temperature in the absence of light. (4) The cells were centrifuged at 1800rpm for 5min, drained, plates were washed 1 time (200. Mu.L/well) with 2% FBS/PBS, gently patted dry on absorbent paper, and surface stained with dye （anti-mouse CD45R (Biolegend, #103247), anti-mouse CD95 (Biolegend, #152607) and anti-mouse GL7 (Biolegend, #144619） and incubated at 4℃for 30min in the dark. (6) The cells were centrifuged at 1800rpm for 5min, drained, plates were washed 1 time (200. Mu.L/well) with 2% FBS/PBS and gently blotted on absorbent paper. (7) Cells were resuspended (100. Mu.L/well) with 2% FBS/PBS and flow-on.
As shown in fig. 11, ORBAC driven in situ assembly of the endoplasmic reticulum cGAMP induced a greater number of germinal center B cells, promoting germinal center response.
EXAMPLE 6 design of deletion building blocks in molecular in situ assembled chimeras and recombinant plasmid Synthesis
1. The design of the molecular in-situ assembled chimera (ORBAC delta A) with the cell-resident structural unit deleted comprises the steps of connecting the amino acid sequences of the assembled structural unit, the tracer structural unit and the monomeric streptomycin structural unit from the N end to the C end through a linker in sequence to obtain ORBAC delta A. The amino acid sequence of the assembly structural unit is shown as SEQ ID NO.1, the amino acid sequence of the tracer structural unit is shown as SEQ ID NO.2, the amino acid sequence of the monomeric streptomycin structural unit is shown as SEQ ID NO.3, and the amino acid sequence of the linker is shown as SEQ ID NO. 5.
The procedure for synthesizing the recombinant plasmid containing ORBAC Δa gene (ORBAC Δa plasmid) was the same as that of example 1 containing ORBAC gene, except that the organelle resident structural unit was deleted.
2. The design of the molecule in-situ assembled chimeric (ORBAC delta B) with the missing assembled structural unit comprises the steps of connecting the amino acid sequences of a signal peptide sequence for leading a protein of an organelle resident structural unit to enter an organelle, a tracer structural unit, a monomeric streptomycin structural unit and an endoplasmic reticulum resident signal sequence of the organelle resident structural unit from an N end to a C end through a linker in sequence, so as to obtain ORBAC delta B. The amino acid sequence of the tracer structural unit is shown as SEQ ID NO.2, the amino acid sequence of the monomeric streptomycin structural unit is shown as SEQ ID NO.3, the amino acid sequence of the organelle resident structural unit is shown as SEQ ID NO.4 (signal peptide sequence for leading protein into organelles) and SEQ ID NO.5 (organelle resident signal sequence), and the amino acid sequence of the linker is shown as SEQ ID NO. 6.
The procedure for synthesizing the recombinant plasmid containing ORBAC Δb gene (ORBAC Δb plasmid) was the same as that of example 1 containing ORBAC gene, except that the assembled structural unit was deleted.
3. The design of the molecular in-situ assembled chimera (ORBAC delta C) with the deletion of the monomeric streptomycin structural unit comprises the steps of connecting the amino acid sequences of a signal peptide sequence for leading the protein of the cell-resident structural unit to enter the cell, an assembly structural unit and an endoplasmic reticulum-resident signal sequence of the cell-resident structural unit from N end to C end through a linker in sequence to obtain ORBAC delta C. Wherein the amino acid sequence of the assembly structural unit is shown as SEQ ID NO.1, the amino acid sequence of the tracing structural unit is shown as SEQ ID NO.2, the amino acid sequence of the cell organelle resident structural unit is shown as SEQ ID NO.4 (signal peptide sequence for guiding protein into the organelle) and SEQ ID NO.5 (signal sequence for cell organelle resident), and the amino acid sequence of the linker is shown as SEQ ID NO. 6.
The procedure for synthesizing the recombinant plasmid containing ORBAC ΔC gene (ORBAC ΔC plasmid) was the same as that of example 1 containing ORBAC gene, except for the monomeric streptomycin structural unit.
EXAMPLE 7 examination of the Inset Assembly of chimeric endoplasmic reticulum with separate deletions of structural units by laser confocal microscopy
The procedure is as in example 2. As a result, as shown in FIG. 12, when the cell-associated domain unit was deleted (ORBAC. DELTA.A), the cell-expressed chimeric protein could not be co-localized with the endoplasmic reticulum although it could be assembled in situ, when the assembled domain unit was deleted (ORBAC. DELTA.B), the cell-expressed chimeric protein could be co-localized with the endoplasmic reticulum but the function of assembly was lost, and when the monomeric streptomycin domain unit was deleted (ORBAC. DELTA.C), it could not capture molecules and could not achieve assembly of the target molecule cell-associated site, although it could be co-localized with the endoplasmic reticulum and assembled in situ.
Example 8 validation of cGAMP immune performance in a model antigen OVA model with structural unit deleted chimeras (ORBAC Δ A, ORBAC Δb and ORBAC Δc)
1. C57BL/6 mouse immunization program
The animal experiment group consisted of 10. Mu.g of OVA antigen, 5.32. Mu.g of Biotin-cGAMP molecule (0.0037. Mu. Mol) and 79. Mu.3724ΔA (7.9 kbp) per mouse, the OVA+cGAMP+ ORBAC ΔB group consisted of 10. Mu.g of OVA antigen, 5.32. Mu.g of Biotin-cGAMP molecule (0.0037. Mu. Mol) and 65. Mu. g ORBAC ΔB (6.5 kbp) per mouse, the OVA+cGAMP+527ΔC group consisted of 10. Mu.g of OVA antigen, 5.32. Mu.g of Biotin-cGAMP molecule (0.0037. Mu. Mol) and 76. Mu. g ORBAC ΔC (7.6 kbp) per mouse, and the OVA+cGAMP+ ORBAC group consisted of 10. Mu.g of OVA antigen, 5.32. Mu.g of Biotin-cGAMP molecule (0.0037. Mu. Mol) and 80. Mu. g ORBAC (8 kbp).
Antigen OVA was mixed with Biotin-cGAMP molecules and different ORBAC chimeras uniformly and SPF-grade, 6-8 week female C57BL/6 mice were immunized. The injection is performed by muscle injection of the hind leg and half of the injection is performed by the left and right legs, and the electroporation instrument is used for electric shock immediately after the injection is finished.
Mice immunization time line same as in example 3.
2. ELISA determination of OVA-specific IgG antibody titres in mice serum after immunization
The procedure is as in example 3. As shown in fig. 13, the levels of OVA-specific antibodies induced by cGAMP in endoplasmic reticulum resident (ORBAC Δa) or in situ assembled ORBAC Δb) alone were reduced compared to cGAMP in endoplasmic reticulum in situ assembled generated by ORBAC drive, and the humoral immune response was decreased, and the humoral immune level was also relatively decreased for chimeras in which cGAMP in the cytoplasm could not be captured (ORBAC Δc) in situ assembled in the endoplasmic reticulum.
3. ELISPOT assay of spleen cells from different groups of mice post-immunization secrete OVA-specific IFN-gamma levels
The procedure is as in example 3. The results are shown in fig. 14, where cGAMP molecules that are assembled in situ (ORBAC Δa), endoplasmic reticulum resident (ORBAC Δb) alone, or cannot be captured (ORBAC Δc) induced relatively low levels of antigen-specific IFN- γ secretion and impaired cellular immune responses compared to cGAMP that was produced by ORBAC drive.
Example 9 validation of cGAMP immune performance in structural unit deleted chimeras (ORBAC a A, ORBAC a B and ORBAC a C) in Respiratory Syncytial Virus (RSV) vaccine models
1. C57BL/6 mouse immunization program
The animal experiment groups were 5. Mu.g of RSV pre-F antigen, 5.32. Mu.g of Biotin-cGAMP molecule (0.0037. Mu. Mol) and 79. Mu. g ORBAC. Delta. A (7.9 kbp) per mouse, 5. Mu.g of RSV pre-F antigen, 5.32. Mu.g of Biotin-cGAMP molecule (0.0037. Mu. Mol) and 65. Mu. g ORBAC. Delta. B (6.5 kbp) per mouse, 5.32. Mu.g of RSV pre-F antigen, 5.32. Mu.g of Biotin-cGAMP molecule (0.0037. Mu. Mol) and 76. Mu. g ORBAC. Delta. C (7.6 kbp) per mouse, and the group of OVA+cGAMP+ ORBAC were 10. Mu.g of OVA antigen, 5.32. Mu.g of Biotin-cGAMP molecule (0.0037. Mu. Mol) and 80. Mu.42 kbp (8).
Anti-RSV pre-F antigen was mixed with Biotin-cGAMP molecules and different ORBAC chimeras to immunize SPF-grade, 6-8 week female C57BL/6 mice. The injection is performed by muscle injection of the hind leg and half of the injection is performed by the left and right legs, and the electroporation instrument is used for electric shock immediately after the injection is finished.
Mice immunization time line procedure was as in example 4.
2. ELISA determination of RSV-specific IgG antibody titres in mice serum after immunization
The procedure is as in example 4. As shown in fig. 15, the in situ assembled cGAMP molecules that were either not endoplasmic reticulum resident or that could not be captured (ORBAC Δc) were significantly reduced in induced RSV-specific antibody titers compared to the ORBAC drive-generated endoplasmic reticulum-assembled cGAMP.
3. Neutralization assay detection of RSV-specific neutralizing antibody titers in post-immunization mouse serum
The procedure is as in example 4. As shown in fig. 16, cGAMP molecules that were assembled in situ alone (ORBAC Δa), endoplasmic reticulum resident (ORBAC Δb), or incapable of being captured (ORBAC Δc) induced the production of relatively low levels of RSV-specific neutralizing antibodies compared to the ORBAC-driven production of endoplasmic reticulum assembled in situ.
4. ELISPOT assay of RSV-specific IFN-gamma levels secreted by spleen cells of mice of different groups after immunization
The procedure is as in example 4. The results are shown in figure 17, where secretion of RSV-specific IFN- γ is significantly reduced and cellular immune responses are impaired compared to the in situ assembled cGAMP (ORBAC driven) of the endoplasmic reticulum, in situ assembled (ORBAC a), endoplasmic reticulum resident (ORBAC a B), or non-captured (ORBAC a C) forms.
In conclusion, experiments show that in the molecular in-situ assembly system for designing the organelle resident in the invention, the cytoplasmic network resident can not be realized by the missing organelle resident structural domain unit, in-situ assembly can not be realized by the missing assembly structural unit, the target molecule can not be captured by the missing monomeric streptomycin structural unit, and further animal experiment multi-model evaluation shows that the developed molecular in-situ assembly chimera for the organelle resident has great advantages in improving the performances of vaccines and adjuvants.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
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