Rabies virus g protein extracellular region fusion polyepitope antigen and use thereof

Abstract

This invention discloses a fusion multi-epitope antigen of the extracellular region of the rabies virus G protein and its application, belonging to the field of vaccines. This invention uses an insect cell-baculovirus expression system to express a recombinant fusion protein containing the extracellular region of the rabies virus G protein, the dendritic cell targeting peptide DCpep, and the universal T cell epitope PADRE, and displays it on the surface of bacterial-like particles via the peptidoglycan binding domain PA to obtain a recombinant subunit vaccine. Comparative experiments with this vaccine and commercial inactivated vaccines and adjuvanted recombinant subunit vaccines show that the recombinant subunit vaccine provided by this invention has good immunogenicity, can induce specific immune responses in mice and increase neutralizing antibody levels, achieves 100% protection in lethal rabies virus standard strain challenge experiments, significantly reduces viral load in the brain of infected mice, and provides effective challenge protection.

Description

Technical Field

[0001] This invention relates to the field of vaccines, and more particularly to a multi-epitope antigen fused to the extracellular region of a rabies virus G protein and its application in a particulate subunit rabies vaccine. Background Technology

[0002] Rabies is a neurotropic zoonotic infectious disease caused by the rabies virus (RABV). Once clinical symptoms appear, it is almost untreatable, with a mortality rate approaching 100%. Statistics show that approximately 59,000 people die from rabies globally each year, primarily in developing countries. These regions face a particularly severe situation due to weak disease prevention systems and low vaccination coverage.

[0003] Vaccination is the most effective means of preventing rabies. Currently, commercially available vaccines are mainly inactivated vaccines, which typically require multiple doses and rely on adjuvants to enhance the immune response. This results in complex vaccination procedures, high costs, and insufficient duration of immunity. In contrast, next-generation recombinant subunit vaccines offer significant safety advantages, but their immunogenicity is relatively weak, requiring adjuvants to enhance the immune response. However, the use of adjuvants can cause local adverse reactions and is difficult to effectively induce cellular immune responses against the rabies virus, especially potent cytotoxic T lymphocyte responses. Therefore, developing novel genetically engineered vaccines that combine safety and immunogenicity using modern biotechnology has become an important direction in current rabies vaccine research.

[0004] Rabies virus (RABV) belongs to the genus Lyssavirus of the family Rhabdoviridae. It is a single-stranded, negative-sense RNA virus whose genome encodes five structural proteins sequentially: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and a large protein of RNA-dependent RNA polymerase (L). Among these, the G protein, located on the surface of the viral envelope, is the only viral protein exposed externally. During infection, it mediates the binding of the virus to host cell receptors and membrane fusion, and is a decisive factor in viral invasion. Simultaneously, the G protein is also the main immunogen that stimulates the host to produce neutralizing antibodies; the strength of its immunogenicity directly determines the protective efficacy of the vaccine. Therefore, due to its dual core functions in viral infection and immune response, the G protein has become the most critical candidate antigen in the development of novel rabies vaccines.

[0005] However, using G proteins alone as subunit vaccine antigens has drawbacks such as insufficient immunogenicity, difficulty in effectively inducing Th1-type cellular immune responses, and inability to maintain the native conformation, thus affecting the quality of neutralizing antibodies. Therefore, there is a need to develop new genetically engineered vaccines that are safer and have better immunogenicity targeting rabies virus G proteins. Summary of the Invention

[0006] One objective of this invention is to provide a rabies virus G protein extracellular region fusion multi-epitope antigen, comprising the rabies virus G protein extracellular region, baculovirus GP67 signal sequence, dendritic cell targeting peptide DCpep (amino acid sequence YPSYHSTPQRP), and universal T cell epitope PADRE (amino acid sequence AKFVAAWTLKAAA).

[0007] Furthermore, the rabies virus is the ERA strain of rabies virus (GenBank accession number EF206707.1).

[0008] Furthermore, the amino acid sequence of the extracellular region of the rabies virus G protein fused with a multi-epitope antigen is shown in SEQ ID NO 1.

[0009] Furthermore, the gene sequence of the extracellular region of the rabies virus G protein fused with a multi-epitope antigen is shown in SEQ ID NO2.

[0010] The second objective of this invention is to provide the application of the above-mentioned rabies virus G protein extracellular region fusion multi-epitope antigen in rabies particulate subunit vaccines.

[0011] A third objective of this invention is to provide a rabies particulate subunit vaccine comprising bacterial-like particles, wherein the surface of the bacterial-like particles is anchored to display the aforementioned rabies virus G protein extracellular region fused with a multi-epitope antigen.

[0012] Furthermore, the bacterial-like particles are the peptidoglycan backbone of lactic acid bacteria MG1363.

[0013] Furthermore, the rabies virus G protein extracellular region fused with a multi-epitope antigen is displayed on the surface of bacterial-like particles via peptidoglycan binding domain PA gene anchoring.

[0014] Furthermore, the peptidoglycan binding domain PA gene is the C-terminal cell wall binding active site of the peptidoglycan hydrolase AcmA of lactic acid bacteria MG1363, which consists of three autolysin motifs separated by heterologous sequences. The AcmA sequence is numbered U17696 in GenBank.

[0015] The fourth objective of this invention is to provide a method for preparing the rabies particulate subunit vaccine described in any of the above claims, comprising the following steps: S1. A recombinant gene fragment containing the baculovirus GP67 signal sequence, dendritic targeting peptide DCpep, universal T cell epitope PADRE, peptidoglycan binding domain PA, and extracellular domain of rabies virus G protein was constructed and cloned into an expression vector. The recombinant fusion protein was obtained by expression using an expression system. The expression vector was pFastBac1, and the expression system was an insect cell-baculovirus expression system. S2. Lactic acid bacteria are treated with hot acid to obtain bacterial-like particles containing only a peptidoglycan backbone. S3. Co-incubate the recombinant fusion protein obtained in step S1 with the bacterial-like particles obtained in step S2. The binding amount of the two is 30 μg / U, where 1U = 2.5 × 10⁻⁶. 9 Each particle, containing a recombinant fusion protein, is displayed on the surface of a bacterial-like particle via the peptidoglycan binding domain (PA), resulting in a rabies particulate subunit vaccine.

[0016] Compared with the prior art, the technical effects of the present invention are as follows: This invention provides a rabies virus G protein extracellular region fused with multiple epitope antigens, and incorporates a baculovirus GP67 signal sequence to promote efficient secretion and correct folding of the protein in eukaryotic expression systems; it also conjugates the dendritic cell targeting peptide DCpep to guide antigen targeting to antigen-presenting cells, improving antigen uptake and presentation efficiency; and introduces the universal T cell epitope PADRE (AKFVAAWTLKAAA) to potently activate T cell responses, overcoming the limitations of single antigen epitopes and promoting Th1-type immune responses. These synergistic technical features effectively address the technical problems of insufficient immunogenicity and weak cellular immune responses in G protein vaccines.

[0017] The rabies particulate subunit vaccine provided by this invention is based on particle surface display technology using a Gram-positive enhancer matrix (GEM). Food-grade strains such as lactic acid bacteria are treated with heat and acid to remove intracellular proteins and nucleic acids, retaining the cell wall skeleton composed of peptidoglycan to obtain bacterial-like particles (BLPs). These particles then specifically bind to the C-terminal anchoring domain (PA) of bacterial cell wall hydrolases, achieving efficient display of rabies virus G protein extracellular region fusion multi-epitope antigens on the particle surface. BLPs exhibit good biocompatibility and a simple preparation process, significantly reducing vaccine production costs. Its main component, peptidoglycan, is a key ligand for Toll-like receptor 2 (TLR2), which can promote the maturation and activation of antigen-presenting cells such as dendritic cells and macrophages by activating the TLR2 signaling pathway. It possesses both adjuvant activity and antigen delivery function, successfully constructing a novel genetically engineered vaccine that is safer and has better immunogenicity against the rabies virus G protein. By comparing this vaccine with commercial inactivated vaccines and recombinant subunit vaccines with adjuvants, the recombinant subunit vaccine provided by this invention has good immunogenicity, can induce specific immune responses in mice and increase the level of neutralizing antibodies, achieves 100% protection in the challenge experiment of lethal rabies virus standard strain, significantly reduces the viral load in the brain of infected mice, and provides effective challenge protection. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0019] Figure 1 This is a schematic diagram of a recombinant gene construction model; Figure 2 The image shows the results of double enzyme digestion identification of the recombinant plasmid. Figure 3 To identify the expression of recombinant fusion proteins using Western blotting; Figure 4 The results were identified by indirect immunofluorescence assay. Figure 5 A schematic diagram of a transmission electron microscope image of a recombinant subunit vaccine; Figure 6 Western blot identification results for recombinant subunit vaccine; Figure 7This is a schematic diagram showing the results of neutralizing antibody detection in mouse serum after immunization. Figure 8 Schematic diagram of the results of flow cytometry detection of mouse dendritic cell activation after immunization; Figure 9 This is a schematic diagram showing the survival rate of mice in a standard rabies virus challenge experiment. Figure 10 This is a schematic diagram of an immunofluorescence section of mouse brain tissue during a challenge experiment with a standard strain of rabies virus. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores. Unless otherwise specified, all experimental methods used in the following examples are conventional methods.

[0022] Example 1: Preparation of multi-epitope antigen fused with the extracellular region of rabies virus G protein 1. Recombinant plasmid construction strategy Based on the nucleotide sequence encoding the glycoprotein of the NCBI rabies virus ERA strain (GenBank: EF206707.1), sequence analysis was performed using biological software. The extracellular antigen domain was selected, and the following sequences were added: baculovirus GP67 signal sequence, dendritic cell targeting peptide DCpep (YPSYHSTPQRP), universal T cell epitope PADRE (AKFVAAWTLKAAA), linker (GGGGS), and protein anchor (PA) sequence (AcmA from Lactococcus lactis MG1363, GenBank: U17696). An 8×His tag was added to the 3' end, and restriction enzyme sites BamHI and HindIII were added to the 5' and 3' ends, respectively. A recombinant gene fragment was designed and constructed, and after codon optimization, it was synthesized. The construction schematic is shown in [Figure number missing]. Figure 1 The amino acid sequence of the extracellular region of the rabies virus G protein fused with the multi-epitope antigen is shown in SEQ ID NO 1, and the gene sequence is shown in SEQ ID NO 2. Simultaneously, the primer sequences required for amplifying the target fragment were synthesized, as shown in Table 1.

[0023] Table 1 Primer Sequences

[0024] 2. Construction of recombinant plasmids The G-PA gene fragment was amplified by PCR using the synthesized plasmid as a template. The product was recovered after agarose gel electrophoresis. The recovered product and the pFastbac1 vector plasmid were double-digested with BamH I and Hind III, respectively. The digestion system is shown in Table 2. After purification, the target fragment was ligated into the pFastbac1 vector overnight. The ligation system is shown in Table 3. The ligation product was transformed into E. coli DH5α competent cells and plated to construct the plasmid pFastBac1-G-PA. Single colonies were picked and cultured for colony expansion, and then subjected to PCR identification. Positive plasmids were extracted and digested for identification. The product was analyzed by agarose gel electrophoresis. The band size was consistent with the size of the G-PA gene. The results are shown in Table 3. Figure 2 .

[0025] Table 2 Composition of the enzyme digestion system

[0026] Table 3 Connection Reaction System

[0027] 3. Construction of recombinant rod particles The recombinant plasmid pFastBac1-G-PA was transformed into E. coli DH10 Bac competent cells. Blue-white screening was performed on solid plates containing kanamycin (25 μg / mL), tetracycline (5 μg / mL), x-gal (3.5 μg / mL), and IPTG (20 μg / mL). After PCR identification, the plasmid was named rBacmid-G-PA. Positive bacteria were inoculated onto LB medium containing tetracycline (10 μg / mL), kanamycin (50 μg / mL), and gentamicin (7 μg / mL) antibodies. After expansion culture, recombinant bacmids were extracted, and the bacmid concentration was determined. The bacmids were then stored at -80℃.

[0028] 4. Rescue and identification of recombinant baculoviruses Following the instructions for the Bac-to-Bac® baculovirus expression system, rBacmid-G-PA was transfected into Sf9 cells using Cellfectin® II transfection reagent. The specific steps are as follows: (1) After the Sf9 cells have grown to a confluence, they are seeded into 6-well plates and transfected when the cells reach a density of 80%. (2) Add 6 μg of rod particles and 8 μL of Cellfectin II Reagent to 100 μL of Grace medium, mix gently, and let stand at room temperature for 5 min. (3) Add the solution containing rod particles to the solution containing Cellfectin II Reagent, mix gently, and let stand at room temperature for 30 min. (4) Gently add Grace medium to the 6-well plate, wash the Sf9 cells twice, and add 1.5 mL of Grace medium. (5) Add the mixture of rod particles and transfection reagent evenly to the cell culture medium in a 6-well plate, and set up untransfected control wells at the same time; (6) Incubate at 27℃ for 5 h, discard the supernatant medium, and replace it with a complete medium containing 10% FBS and 1% penicillin and streptomycin; (7) After culturing at 27℃ for about 5 days, the cells showed pathological phenomena such as increased volume, partial cell fragmentation, and shedding. The cell supernatant was collected and centrifuged to remove cell debris, thus rescuing the recombinant baculovirus, which was named rBac-G-PA. The first generation (P1) virus was continuously amplified in Sf9 cells to the P3 generation, and the viral supernatant was collected for later use.

[0029] 5. Identification of recombinant baculoviruses A. Identification of the expression of recombinant fusion proteins The harvested P3 generation recombinant baculovirus was used to infect Sf9 cells at an MOI of 0.5 for 72 hours. The cell culture supernatant was then harvested for Western blot experiments, as follows: (1) Add 5× protein loading buffer to each sample and incubate in boiling water for 10 min; (2) Use a 10% concentration SDS-PAGE gel, maintain a constant voltage of 100 V, and turn off the power when the sample reaches the bottom of the gel; (3) Transfer the sample in the gel to a nitrocellulose membrane at a current of 300 mA for 30 min; (4) Prepare 5% skim milk powder with TBST solution and seal at room temperature for 2 h; (5) Incubate with mouse anti-rabies virus glycoprotein monoclonal antibody for 1 h; (6) Wash TBST 3 times, 5 min / time; (7) Use HRP-labeled goat anti-mouse IgG as a secondary antibody and incubate at room temperature for 1 h; (8) Wash TBST 3 times, 5 min / time; (7) A chemiluminescent substrate was added for color development, and the results were observed and photographed. The results showed that a specific band was detected between 70-100 kDa, and the theoretical molecular weight of the target protein was approximately 81 kDa, indicating that the G-PA fusion protein was successfully expressed and secreted into the supernatant after the recombinant baculovirus infected the cells. Figure 3 ).

[0030] B. Indirect immunofluorescence identification Sf9 cells cultured in 24-well plates were infected with P3 generation recombinant baculovirus rBac-G-PA and cultured at 27°C for 24-36 hours. When obvious pathological changes such as cell enlargement and fragmentation were observed, the cells were removed from the incubator and indirect immunofluorescence assay was performed according to the following steps: (1) Discard the culture medium, add 300 μL PBS to each well, and wash once, 5 min / time; (2) Add 300 μL of 80% acetone pre-cooled at -20℃ to each well and fix at room temperature for 30 min; (3) Discard the supernatant, add PBS to each well and wash 3 times, 5 min each time; (4) Add 300 μL of 2% BSA to each well and incubate at room temperature for 30 min; (5) After washing again, add mouse anti-rabies virus glycoprotein monoclonal primary antibody, incubate at 37°C for 30 min, and set up negative control wells; (6) Discard the supernatant, add 300 μL PBS to each well, and wash 3 times, 5 min each time; (7) Add Alexa Fluor™ Plus 488-labeled goat anti-mouse IgG (H+L) secondary antibody to each well and incubate at 37°C for 30 min in the dark; (8) Discard the supernatant, add 300 μL PBS to each well, and wash 3 times, 5 min each time; (9) Add 300 μL of DAPI to each well to counterstain the cell nuclei.

[0031] (10) Discard the supernatant, wash, and observe and acquire images under a fluorescence microscope. The results are as expected, and the recombinant baculovirus rBac-G-PA was successfully expressed. Figure 4 ).

[0032] Example 2: Preparation of Rabies Particulate Subunit Vaccine 1. Preparation of exposed BLP particles (1) The laboratory-frozen Lactococcus lactis MG1363 was inoculated into M17 medium containing 0.5% glucose and cultured overnight at 30°C with shaking at 220 r / min. (2) The revived Lactococcus lactis was inoculated into M17 medium containing 0.5% glucose and cultured at 30°C and 180 rpm for 18 h. The bacterial culture was then collected. (3) Centrifuge at 3000 rpm for 5 min, discard the supernatant, add PBS solution and resuspend, centrifuge at 3000 rpm for 5 min, and wash a total of 3 times; (4) Prepare a 10% trichloroacetic acid solution with 0.2 times the volume of the culture medium using double-distilled water, add it and resuspend the precipitate; (5) Transfer the solution to an Erlenmeyer flask and heat it in boiling water for 30 min; (6) After cooling, centrifuge at 7500 rpm for 5 min, discard the supernatant, and wash the precipitate thoroughly with PBS 5 times. (7) Add an appropriate amount of PBS and mix the solution by pipetting. Then count the cells using a cell counting chamber. (8) Adjust the particle density in the solution with PBS, ultimately to 2.5 × 10⁻⁶. 9 One BLP is defined as one unit (U).

[0033] 2. The fusion protein binds to naked BLP particles. Mix 1 U of BLPs particles with the supernatant of Sf9 cells infected with rBac-G-PA, and incubate at room temperature with gentle inversion for 60 min to allow the G-PA fusion protein to bind to the BLPs surface via the anchoring protein PA. After the reaction, centrifuge at 8000 r / min for 10 min to collect the precipitate, wash five times with PBS to remove unbound free protein, and finally obtain the G-BLPs complex.

[0034] 3. Identification of recombinant subunit vaccines Lactococcus lactis MG1363, naked BLPs, and G-BLPs samples were fixed with 20% glutaraldehyde and ultrathin sections were prepared. Transmission electron microscopy was used to observe the particle morphology. A row of cotton-like proteins was observed on the surface of G-BLPs, while the interior of BLPs showed a smooth surface with regular morphology and characteristic hollow vesicle structures. The results indicate that the recombinant rabies virus G protein antigen was successfully displayed on the surface of BLPs through anchoring protein (PA). Figure 5 G-BLPs samples were treated with 5×SDS-PAGE loading buffer and then subjected to 10% SDS-PAGE electrophoresis. Unbound BLPs served as a negative control. Western blotting analysis was performed using mouse anti-RABV monoclonal antibody as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody. The results showed specific bands detected in the 70-100 kDa range, further confirming that the fusion protein was specifically anchored to the BLPs vector. Figure 6 ).

[0035] Example 3: Evaluation of Vaccine Efficacy 1. Immunization and challenge experiments To evaluate the immunogenicity and protective efficacy of G-BLPs, 100 female 6-8 week old BALB / c mice were randomly divided into 5 groups (n=20), and immunized with 1U G-BLPs, Adj-G-BLPs (a mixture of 100µL 1U G-BLPs and 5µL AddaS03 oil-in-water adjuvant) or BLPs via intramuscular injection. After the initial immunization, two booster immunizations were administered at two-week intervals with the same dose. A group of mice was given an intramuscular injection of a commercially available inactivated vaccine (HCP-SAD) as a positive control, and another group was given PBS as a negative control. Blood samples were collected at weeks 2, 4, and 6 after the initial immunization, and serum was separated for virus-neutralizing antibody (VNA) detection. One week after the second booster immunization, spleens were harvested for flow cytometry to detect dendritic cell activation. On day 14 after the final immunization, all mice received 50LD... 50 Mice were challenged with a dose of CVS-24 virus via intramuscular injection. They were observed for 14 consecutive days post-challenge, during which time clinical symptoms and survival rates were continuously monitored and recorded. Three mice from each group were randomly selected and euthanized on day 7 post-challenge for the preparation of immunofluorescent sections of brain tissue.

[0036] 2. Rabies virus neutralizing antibody level detection Rabies virus neutralizing antibody titers were detected using the fluorescent antibody virus neutralization assay (FAVN). Serum was heat-inactivated at 56°C for 30 minutes. The inactivated serum was serially diluted three-fold (50 μL / well) and added to each well of a 96-well plate. Then, 50 μL of 100 TCID50 RABV-ERA-EGFP virus solution was added to each well. After mixing by pipetting, the plates were incubated at 37°C in a 5% CO2 incubator for 1 hour. Finally, 2 × 10⁻⁶ mmol / L of the virus solution was added to each well. 4 BHK cells were cultured for 48 hours, and the fluorescence signal was observed using a fluorescence microscope. The endpoint for determining the virus neutralizing antibody (VNA) titer was defined as the reciprocal of the highest serum dilution that completely suppressed the fluorescence signal, and the units were converted to IU / mL. A serum positivity threshold of 0.5 IU / mL was used, and the results were compared with reference standard serum. Figure 7 The results showed that VNA was detectable during the first booster immunization after G-BLP vaccination, and reached a peak of 13.25 IU / mL after two booster immunizations. This indicates that G-BLP-vaccinated mice induced the production of a large number of rabies virus neutralizing antibodies, and the antibody level was far higher than the protective threshold of 0.5 IU / mL.

[0037] 3. Flow cytometry-based analysis of dendritic cell activation The activation rate of dendritic cells (DCs) in the spleen of mice was determined by flow cytometry. On day 7 after booster immunization, mice receiving G-BLPs, Adj-G-BLPs, BLPs, commercial vaccine, and the PBS control group (n=3) were sacrificed. Spleen tissue was collected, homogenized, and the resulting suspension was filtered through a 100 μm nylon cell filter to remove impurities and separate single-cell suspensions. Cells were washed twice with PBS containing 2% fetal bovine serum, followed by erythrocyte lysis buffer lysis. Cells were incubated with blocking buffer containing CD16 / 32 antibody for 20 minutes, washed, and stained with APC-labeled CD11c antibody, PE-labeled CD80 antibody, BV421-labeled CD86 antibody, and APC-Cy7-labeled MHC II antibody, respectively. After washing, cell samples were analyzed using flow cytometry. CD11c detection was performed. + CD80 + DCs, CD11c + CD86 + DCs and CD11c + MHCII + The proportion of dendritic cells (DCs) was measured. Results showed that, compared to the negative control group, the G-BLPs group significantly recruited and activated dendritic cells (DCs), stimulating the surface co-stimulatory molecules CD80 and CD86 (CD86). Figure 8 (A, B, D) and T cell co-stimulatory molecule MHC II ( Figure 8 The expression of C and D in the middle.

[0038] 4. Virus attack protection effect During the 14-day observation period after challenge, all mice in the control group and 4 mice in the BLPs-inoculated group died from viral infection within 10 days. In contrast, all mice in the G-BLPs group, Adj-G-BLPs group, and the commercial inactivated vaccine group survived. Figure 9 Immunofluorescence sections of mouse hippocampal CA3 and DG regions were prepared to observe the distribution of the virus in the brain. Mouse brain tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned. The sections were treated with 3% hydrogen peroxide for 30 minutes to quench endogenous peroxidase activity, followed by incubation with mouse anti-rabies virus monoclonal antibody. After washing with PBST, the sections were incubated with Alexa Fluor™ 488-labeled goat anti-mouse IgG secondary antibody for 50 minutes in the dark. Finally, the cell nuclei were stained with DAPI, and the sections were mounted and observed under a fluorescence microscope. The results showed that the deposition of rabies virus G protein in the brain of mice immunized with G-BLPs was significantly reduced. Figure 10 This indicates that G-BLPs can effectively reduce viral load in the brain while providing 100% protection against lethal doses of viral attack.

[0039] The embodiments described above are merely specific implementations of this application, used to illustrate the technical solutions of this application, and not to limit them. The protection scope of this application is not limited thereto. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the scope of the technology disclosed in this application, or make equivalent substitutions for some of the specific technologies; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application. All should be covered within the protection scope of this application. Therefore, the protection scope of this application should be determined by the protection scope of the claims.

Claims

1. A rabies virus G protein extracellular region fused with a multi-epitope antigen, characterized in that, It contains the extracellular region of the rabies virus G protein, the baculovirus GP67 signal sequence, the dendritic cell targeting peptide DCpep, and the universal T cell epitope PADRE.

2. The rabies virus G protein extracellular region fused with a multi-epitope antigen as described in claim 1, characterized in that, The rabies virus in question is the ERA strain of rabies virus.

3. The rabies virus G protein extracellular region fusion multi-epitope antigen as described in claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO 1.

4. The rabies virus G protein extracellular region fusion multi-epitope antigen as described in claim 1, characterized in that, Its gene sequence is shown in SEQ ID NO 2.

5. The application of the rabies virus G protein extracellular region fusion multi-epitope antigen as described in claim 1 in the preparation of rabies particulate subunit vaccine.

6. A rabies particulate subunit vaccine, characterized in that, It contains bacterial-like particles whose surface is anchored to display the extracellular region fusion multiepitope antigen of the rabies virus G protein as described in claim 1.

7. The rabies particulate subunit vaccine as described in claim 6, characterized in that, The bacterial-like particles are the peptidoglycan backbone of lactic acid bacteria MG1363.

8. The rabies particulate subunit vaccine as described in claim 6, characterized in that, The rabies virus G protein extracellular region fused with a multi-epitope antigen is displayed on the surface of bacterial-like particles via peptidoglycan binding domain PA gene anchoring.

9. The rabies particulate subunit vaccine as described in claim 8, characterized in that, The peptidoglycan binding domain PA gene is the C-terminal cell wall binding active site of the peptidoglycan hydrolase AcmA of lactic acid bacteria MG1363, and consists of three autolysin motifs separated by heterologous sequences.

10. The method for preparing a rabies particulate subunit vaccine according to any one of claims 6-9, characterized in that, Includes the following steps: S1. A recombinant gene fragment containing the baculovirus GP67 signal sequence, dendritic targeting peptide DCpep, universal T cell epitope PADRE, peptidoglycan binding domain PA, and extracellular domain of rabies virus G protein was constructed and cloned into an expression vector. The recombinant fusion protein was obtained by expression using an expression system. The expression vector was pFastBac1, and the expression system was an insect cell-baculovirus expression system. S2. Lactic acid bacteria are treated with hot acid to obtain bacterial-like particles containing only a peptidoglycan backbone. S3. Co-incubate the recombinant fusion protein obtained in step S1 with the bacterial-like particles obtained in step S2. The binding amount of the two is 30 μg / U, where 1U = 2.5 × 10⁻⁶. 9 Each particle, containing a recombinant fusion protein, is displayed on the surface of a bacterial-like particle via the peptidoglycan binding domain (PA), resulting in a rabies particulate subunit vaccine.

Images