An ibv polyepitope recombinant bacillus subtilis nasal vaccine containing astragalus polysaccharide adjuvant, and a preparation method and application thereof

Abstract

The application discloses an IBV multi-epitope antigen recombinant bacillus subtilis nasal vaccine and a preparation method and application thereof. The application modularly connects four screened nucleotide sequences, such as cell antigenic determinants E1-E4 like SEQ ID NO. 4-7, with a CKS9 targeting peptide, a GPGPG / AAY connecting peptide and a 6xHis tag module to design a MeRSN antigen molecule, and the amino acid sequence of the MeRSN antigen molecule is shown as SEQ ID NO. 9. The nucleotide sequence encoding the MeRSN is transferred into bacillus subtilis WB800N to construct a recombinant bacillus subtilis, and after induced expression, the bacterial body is collected and mixed with an astragalus polysaccharide adjuvant to prepare a nasal instillation vaccine. Experiments show that the vaccine can significantly induce serum IgY and tracheal mucosa sIgA antibodies, improve IL-4 and IFN-gamma levels, reduce virus discharge amount and tissue virus load after an attack, and has good immunogenicity and protection effect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering vaccine technology, specifically to an IBV multi-epitope recombinant Bacillus subtilis nasal vaccine containing astragalus polysaccharide adjuvant, its preparation method, and its application. Background Technology

[0002] Infectious bronchitis (IB) is an acute, highly contagious respiratory infectious disease of poultry caused by the infectious bronchitis virus (IBV). It is characterized by rapid transmission and frequent mutations. IBV primarily affects the respiratory system of chickens, while some strains can also damage the kidneys and reproductive system, leading to a series of production losses such as respiratory inflammation, kidney lesions, decreased growth performance, reduced egg production, and decreased egg quality. It consistently causes significant and continuous economic losses to the broiler and egg-laying chicken farming industries both domestically and internationally. IBV belongs to the family Coronaviridae, genus Gamma Coronavirus, and is a single-stranded positive-sense RNA virus. Its genome is prone to mutation and recombination during replication and transmission, resulting in the continuous emergence of different genotypes and genetic lineages, forming a complex antigenic diversity. The QX type (GI-19 lineage) has become the dominant circulating strain in my country and Asia. The IBV spike protein (S protein) is the main structural protein on the viral surface, composed of two subunits, S1 and S2. The S1 subunit is the core neutralizing antigen mediating viral recognition of host receptors and initiating the invasion process. It is also a key determinant of viral genotype differentiation and serovariability. The S2 subunit mainly mediates the fusion of the viral envelope and cell membrane, and is an essential functional region for viral entry into host cells. Compared to the highly variable S1, S2 is relatively conserved among different IBV strains and contains antigenic epitopes with cross-reactivity potential. The N protein is a highly abundant structural protein of the virus, involved in the packaging, replication, and transcriptional regulation of viral genomic RNA, and is rich in conserved T-cell antigenic epitopes that can induce cellular immunity. Jointly screening for antigenic epitopes from S1, S2, and N proteins is beneficial for considering humoral immunity, cellular immunity, and antigen coverage among different strains in the same candidate vaccine molecule.

[0003] Vaccination remains the most economical and mainstream technical means for the prevention and control of IBV. Existing commercial IBV vaccines are mainly divided into two categories: live attenuated vaccines and inactivated vaccines. Both types of products have inherent technical limitations. Live attenuated IBV vaccines can be administered via nasal drops, eye drops, or drinking water, inducing local mucosal immunity. However, the vaccine strain has the ability to replicate in vivo, posing a dual safety risk of virulence reversion and the generation of novel variant strains through genetic recombination between the vaccine strain and the wild-type strain. Furthermore, its protective effect is closely related to the antigenic correlation between the vaccine strain and the circulating strain; when the antigenic differences between the two are significant, insufficient cross-protection may occur. Inactivated vaccines, on the other hand, have better safety, but primarily induce humoral immune responses, making it difficult to effectively induce local mucosal immunity in the respiratory tract. They also require individual intramuscular injection, which is cumbersome, costly for artificial immunization, and causes significant stress to animals, making them unsuitable for large-scale immunization. Therefore, current vaccine technology still struggles to simultaneously achieve high safety, broad antigenic coverage, effective respiratory mucosal immunity, and convenient large-scale vaccination.

[0004] In recent years, with the development of viral sequence databases and immunoinformatics analysis technologies, rational vaccine design based on epitopes has gradually become an important direction in the development of novel IBV vaccines. This type of technology typically combines previous immunological research data, viral antigen protein sequence alignment, and immunoinformatics predictions to screen for potentially immunogenic B-cell and T-cell epitopes from the major IBV antigen proteins. Combining multiple dominant epitopes from different antigen proteins is beneficial for addressing both humoral and cellular immunity in the same candidate vaccine molecule and provides a technical basis for improving the antigen coverage of vaccines against different IBV strains. However, some studies rely primarily on predictions from single immunoinformatics software or databases, lacking the combined support of previous experimental research evidence and actual viral sequence information, which can easily lead to inconsistencies between predicted results and actual immune activity. Furthermore, some studies focus mainly on the highly variable S1 protein, paying insufficient attention to cross-reactive epitopes in the relatively conserved S2 and N proteins. Therefore, the candidate antigen epitopes obtained by existing screening methods may have problems such as insufficient adaptability to target strains, incomplete safety evaluation, or limited applicability across strains, which in turn affect the immunization efficacy and application value of subsequent multi-epitope vaccines.

[0005] Bacillus subtilis is a well-established and applied Gram-positive bacterium, internationally recognized as a food-grade safe probiotic. It possesses advantages such as not producing endotoxins, simple culture conditions, a relatively complete genetic manipulation system, a mature exogenous protein expression system, and suitability for mucosal antigen delivery. Furthermore, Bacillus subtilis cell wall components (such as peptidoglycan and lipoteichoic acid) have natural adjuvant effects, activating the Toll-like receptor (TLR) signaling pathway and promoting the activation of antigen-presenting cells. However, despite these advantages, existing technologies for Bacillus subtilis as a vaccine vector still have significant limitations. First, as a Gram-positive bacterium, Bacillus subtilis has a dense cell wall structure, and the efficiency of exogenous gene introduction and homologous recombination is generally lower than that of Gram-negative bacteria such as Escherichia coli, posing a technical challenge to the construction of recombinant strains. Secondly, although Bacillus subtilis is often used in the research of oral mucosal vaccines, its colonization ability as a live vector and its induction mechanism of local respiratory immunity (especially sIgA secretion) in nasal mucosal immunity are not yet fully understood.

[0006] Astragalus polysaccharide (APS) is a natural active polysaccharide extracted from the traditional Chinese medicine Astragalus membranaceus. Extensive basic research has confirmed its immune-enhancing activity, promoting T cell proliferation, enhancing macrophage phagocytic function, and upregulating cytokine expression. It is a natural, green adjuvant with great application potential. Currently, the application of Astragalus polysaccharide in avian vaccines is limited to its use in combination with inactivated IBV vaccines as an injectable adjuvant, or as a feed additive to promote broiler growth and immune enhancement. A synergistic mucosal adjuvant system combining natural polysaccharide with live bacterial carriers expressing IBV antigen urgently needs development.

[0007] In summary, the existing technology lacks an IBV vaccine program that can organically integrate immunoinformatics multi-epitope design, recombinant Bacillus subtilis live vector delivery, and Astragalus polysaccharide nasal mucosal adjuvant to simultaneously address the three core issues of insufficient cross-protection of traditional vaccines, weak mucosal immunogenicity of live vector vaccines, and large-scale convenient immunization.

[0008] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0009] The purpose of this invention is to provide a recombinant IBV multi-epitope Bacillus subtilis containing Astragalus polysaccharide adjuvant and its preparation method.

[0010] To achieve the above-mentioned objectives, the present invention provides a recombinant Bacillus subtilis, wherein the recombinant Bacillus subtilis expresses the multiepitope antigen MeRSN of chicken infectious bronchitis virus; The multi-epitope antigen MeRSN includes: M cell targeting peptide CKS9, B cell antigenic determinants E1 and E2, T cell antigenic determinants E3 and E4, linker peptide, and 6×His tag. The amino acid sequence of the B cell antigenic determinant E1 is shown in SEQ ID NO.4, and the amino acid sequence of E2 is shown in SEQ ID NO.5; the amino acid sequence of the T cell antigenic determinant E3 is shown in SEQ ID NO.6, and the amino acid sequence of E4 is shown in SEQ ID NO.7.

[0011] Preferably, in the recombinant Bacillus subtilis provided by the present invention, the full-length amino acid sequence of the multi-epitope antigen MeRSN is shown in SEQ ID NO.9.

[0012] The present invention also provides a nucleotide sequence encoding the multi-epitope antigen MeRSN, as shown in SEQ ID NO.10.

[0013] Preferably, the host bacterium of the recombinant Bacillus subtilis provided by the present invention is Bacillus subtilis WB800N.

[0014] Preferably, the expression vector of the recombinant Bacillus subtilis provided by the present invention is selected from pHT43.

[0015] The present invention also provides a recombinant Bacillus subtilis nasal vaccine with IBV multi-epitope antigen, comprising the recombinant Bacillus subtilis and Astragalus polysaccharide adjuvant.

[0016] Preferably, the concentration of recombinant Bacillus subtilis in the nasal vaccine is 1×10⁻⁶. 9 ~1×10 10 CFU / mL.

[0017] Preferably, the final concentration of the astragalus polysaccharide adjuvant in the nasal vaccine is 50 mg / mL.

[0018] The present invention also provides a method for preparing the nasal vaccine, comprising the following steps: (1) The MeRSN antigen gene with the nucleotide sequence shown in SEQ ID NO.10 was cloned into a prokaryotic expression vector to obtain the recombinant plasmid p-MeRSN; (2) The recombinant plasmid p-MeRSN was transformed into Bacillus subtilis WB800N, and positive transformants were screened to obtain recombinant Bacillus subtilis WB800N(p-MeRSN); (3) The recombinant Bacillus subtilis obtained in step (2) was induced and cultured, and the bacterial cells were collected; (4) Resuspend the bacterial cells until the bacterial concentration is 1×10⁻⁶. 9 ~1×10 10 CFU / mL; (5) Dissolve the Astragalus polysaccharide, filter it through a 0.22μm filter membrane to remove bacteria, and mix it with the bacterial solution obtained in step (4) to obtain the final product.

[0019] Preferably, the final concentration of Astragalus polysaccharide in the mixed system in step (5) of the preparation method is 50 mg / mL; the resuspended bacterial cells and Astragalus polysaccharide dissolution are selected from sterile PBS.

[0020] The recombinant Bacillus subtilis provided by this invention or the nasal vaccine prepared by the aforementioned method can be used in the preparation of drugs for the prevention of infectious bronchitis in chickens.

[0021] This invention first utilizes the complete coding sequences of S1, S2, and N of the prevalent IBV QX SCSN strain to conduct multi-level antigenic epitope screening. A systematic search of publicly available IBV-related literature initially screens for candidate peptides with excellent immunogenic activity. Then, sequence homology alignment is used to screen four highly conserved, non-cytotoxic, and non-sensitizing B / T cell antigenic determinants across strains. Subsequently, these are modularly tandemly coupled with a 6×His tag, a CKS9 M cell-targeting peptide, and a flexible linker peptide of GGPPG and AAY. This tandem antigenic polypeptide is named "MeRSN." Bioinformatics tools predict that it lacks transmembrane structures, exhibits high hydrophilicity overall, and that the antigenic epitopes within the polypeptide are concentrated in irregularly coiled regions.

[0022] This invention further constructs a recombinant Bacillus subtilis engineered strain capable of stably expressing the aforementioned MeRSN antigen. Using the pHT43 inducible expression vector, and based on the target gene sequence and expression vector map, Sangon Biotech (Shanghai) Co., Ltd. completed codon optimization, gene synthesis, and recombinant vector construction. The sequence accuracy was verified by double enzyme digestion and full-length sequencing, yielding the recombinant plasmid pHT43-MeRSN. Bacillus subtilis WB800N competent cells were prepared, and the recombinant plasmid pHT43-MeRSN was electroporated into them. Positive transformants were screened using chloramphenicol and neomycin double antibody plates. Western blotting confirmed that the engineered bacterium could stably secrete soluble MeRSN antigen under suitable induction conditions.

[0023] The preparation of the recombinant Bacillus subtilis nasal vaccine with Astragalus polysaccharide adjuvant described in this invention involves inducing culture of recombinant Bacillus subtilis WB800N (pHT43-MeRSN), collecting the bacterial cells by centrifugation, resuspending them in sterile PBS, and adjusting the bacterial concentration to 1×10⁻⁶. 9 ~1×10 10The astragalus polysaccharide was dissolved in sterile PBS and filtered through a 0.22 μm filter membrane for sterilization. It was then mixed with recombinant bacterial solution in the determined optimal ratio to prepare a nasal drop vaccine preparation.

[0024] The immunogenicity evaluation method of this invention involves randomly dividing SPF chickens into four groups: a PBS blank control group, an empty vector strain group, a recombinant strain without adjuvant, and a recombinant strain with astragalus polysaccharide adjuvant. These groups were immunized via eye drops or nasal drops at 7, 14, and 21 days of age, respectively, for three consecutive days. After immunization, serum and tracheal lavage fluid were collected periodically to detect serum IBV-specific IgY antibody levels and tracheal mucosal sIgA antibody levels, respectively. Simultaneously, changes in serum cytokine IFN-γ and IL-4 concentrations were measured. The challenge protection experiment method of this invention involves challenging chickens with the QX type IBV strain via eye drops or nasal drops after three immunizations. After challenge, the disease progression was continuously observed, and throat swabs and cloacal swabs were collected to detect viral shedding. Seven days after challenge, all chickens were euthanized, and tracheal, lung, and kidney tissues were collected to detect viral load and for histopathological observation.

[0025] The present invention has the following advantages: This invention screened four dominant antigenic determinants that simultaneously met the criteria of non-toxicity, non-allergenicity, antigenicity score ≥0.4, and cross-strain conservation ≥95%, and constructed a MeRSN antigen molecule, the amino acid sequence of which is shown in SEQ ID NO.9, and the nucleotide sequence encoding the antigen molecule is shown in SEQ ID NO.10.

[0026] This invention uses a safe strain of Bacillus subtilis as a vector to construct a recombinant Bacillus subtilis by introducing a sequence capable of expressing antigen molecules. After being combined with Astragalus polysaccharide, it can be used as a nasal vaccine, simultaneously inducing humoral immunity, cellular immunity, and respiratory mucosal sIgA responses.

[0027] The challenge protection experiment showed that the viral load in the trachea, lungs and kidneys of the combination of recombinant Bacillus subtilis and APS was significantly lower than that of the recombinant bacterial group, and no obvious lesions were observed in histopathological observation, indicating that the vaccine can effectively protect multiple target organs from viral damage. Attached Figure Description

[0028] Figure 1 This is the molecular design diagram of the MeRSN vaccine in Example 1 of the present invention.

[0029] Figure 2 This is the sensitization result of the vaccine molecule in Example 1 of the present invention.

[0030] Figure 3 The results show the immunogenicity and solubility of the vaccine molecule in Example 1 of this invention when overexpressed.

[0031] Figure 4 The results are the physicochemical property analysis and prediction results of the vaccine molecules in Example 1 of this invention.

[0032] Figure 5 This is the prediction result of the hydrophilicity / hydrophobicity of the vaccine molecules in Example 1 of the present invention.

[0033] Figure 6 The results show the predicted transmembrane domains of the vaccine molecule in Example 1 of this invention.

[0034] Figure 7 The results show the predicted phosphorylation sites of the vaccine molecule in Example 1 of this invention.

[0035] Figure 8 The results are the prediction results of the secondary structure of the vaccine in Example 1 of the present invention, where A is the result of the prediction of the secondary structure of the vaccine molecule using PSIPRED 4.0 in Example 1 of the present invention; and B is the result of the prediction of the secondary structure using SOPMA.

[0036] Figure 9 The results are the prediction results of the vaccine tertiary structure in Example 1 of the invention, where A is the result of the prediction of the tertiary structure of the vaccine molecule using SWISS-MODEL in Example 1; and B is the result of the prediction of the tertiary structure using AlphaFold3.

[0037] Figure 10 This is the visualization result of the tertiary structure of the vaccine molecule using PyMOL software in Example 1 of the present invention.

[0038] Figure 11 This is the plasmid map of the recombinant plasmid pHT43-MeRSN in Example 2 of the present invention.

[0039] Figure 12 The results of double digestion of recombinant plasmid pHT43-MeRSN with BamHI and SmaI in Example 2 of this invention are shown. M is BL 108A 1kb DNA Marker. Lanes 1, 2, 3, and 4 represent the double digestion products of recombinant plasmid pHT43-MeRSN, and lane 5 represents the double digestion product of empty vector plasmid pHT43.

[0040] Figure 13 The results of the PCR identification of recombinant Bacillus subtilis in Example 2 of this invention are shown. M is GimiRunDM2000 DNA Marker. Lanes 1, 2, 3, and 4 are the amplification products of the PCR of recombinant Bacillus subtilis. Lanes 5-12 are the amplification products of the PCR of the negative control empty vector Bacillus subtilis.

[0041] Figure 14The WB verification results of the antigen protein expression of recombinant Bacillus subtilis in Example 2 of this invention are shown. M is M5 Prestained Protein Ladder (10-180 kDa) with 45 kDa. Lane 1 shows the expression of the antigen protein of recombinant Bacillus subtilis, lane 2 is the positive control protein sample, and lane 3 shows the expression of empty Bacillus subtilis protein.

[0042] Figure 15 In Example 3 of this invention, the body weight of four groups of SPF chickens was dynamically monitored at 7, 14, 21, and 28 days of age during the immunization period. The horizontal axis represents the age of the chickens, and the vertical axis represents the average body weight, in g.

[0043] Figure 16 The relative levels of IBV-specific antibody IgY in the serum of four groups of SPF chickens were detected at different time points in Example 3 of this invention. The horizontal axis represents the age of the chickens, the vertical axis represents the antibody titer of the samples, and the S / P ratio is used to represent the relative levels.

[0044] Figure 17 In Example 3 of this invention, the relative levels of IBV-specific mucosal antibody sIgA in the tracheal lavage fluid of four groups of SPF chickens were detected at different time points. The horizontal axis represents the age of the chickens, and the vertical axis represents the absorbance (OD value) at 620 nm. The absorbance (OD value) is positively correlated with the sIgA antibody level.

[0045] Figure 18 In Example 3 of this invention, the concentration changes of cytokine IL-4 in the serum of four groups of SPF chickens were detected at different time points. The horizontal axis represents the age of the chickens, and the vertical axis represents the concentration of IL-4, with the unit being pg / ml.

[0046] Figure 19 The concentration changes of cytokine IFN-γ in four groups of SPF chicken serum were detected at different time points in Example 3 of this invention. The vertical axis represents the concentration of IFN-γ, and the unit is pg / mL.

[0047] Figure 20 In Example 3 of this invention, the viral shedding in pharyngeal swabs of four groups of SPF chickens was measured at different time points after challenge. The horizontal axis represents the age of the chickens, and the vertical axis represents the logarithm of the IBV viral copy number. 10 ).

[0048] Figure 21 In Example 3 of this invention, the viral shedding in cloacal swabs of four groups of SPF chickens was measured at different time points after challenge. The horizontal axis represents the age of the chickens, and the vertical axis represents the logarithm of the IBV viral copy number (log). 10 ).

[0049] Figure 22The trachea of ​​four groups of SPF chickens were examined after euthanasia following challenge in Example 3 of this invention. Figure 22 A) Lungs ( Figure 22 B) Kidney ( Figure 22 The C) tissue yield is represented by the horizontal axis, which indicates the immunization treatment method for each experimental group, and the vertical axis represents the logarithm of the IBV viral copy number. 10 ).

[0050] Figure 23 This is a pathological observation of the dissected organs (trachea, lungs, and kidneys) of chickens after being challenged and killed in Example 3 of the present invention. The horizontal row shows the trachea, lungs, and kidney tissues, and the vertical row shows the immunization treatment methods of the four experimental groups. Detailed Implementation

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only 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.

[0052] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0053] Example 1: Immunoinformatics Screening of Conserved IBV Antigenic Determinants and Vaccine Molecular Design I. Screening of IBV candidate antigen determinants A comprehensive search of domestic and international journal articles and patent documents related to the immunity of infectious bronchitis virus (IBV) was conducted to screen out multiple antigenic peptides that have been confirmed by multiple studies to have stable immune activation capabilities as initial candidate peptides. The complete coding sequences of the S1, S2, and N genes of the IBV QX SCSN strain (Tan X, Liu P, Jin Y, et al. Duckweed-based edible vaccine confers complete protection against avianinfectious bronchitis virus by inducing robust mucosal and systemic immunity[J]. Plant Biotechnology Journal, 2025, 23(11):4683.) isolated, purified, and sequenced in our laboratory were used as alignment templates (their nucleotide sequences are shown in SEQ ID NO. 1, 2, and 3, respectively). Global homology comparison was performed between the initial candidate peptides and the protein sequence of this strain, and four target peptides with high homology matching were located in the QX epidemic strain sequence. Multiple immunoinformatics analysis software programs were used to conduct multi-dimensional quantitative assessments of the four target peptides: ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / design.php) was used to predict peptide cytotoxicity, AllerTop2.0 (http: / / www.ddg-pharmfac.net / AllerTOP / ) was used to analyze peptide sensitization potential, and Vexigen 2.0 (http: / / www.ddg-pharmfac.net / vaxijen / VaxiJen / VaxiJen.html) was used to quantitatively assess peptide immunogenicity. Simultaneously, the sequences of the four peptides were globally homologous to over 100 IBV isolates of different genotypes in the NCBI database, and the maximum similarity of each epitope amino acid sequence was calculated to verify the broadly conserved nature of the peptides. Based on the above four-dimensional evaluation results, four dominant antigenic determinants that simultaneously meet the criteria of being non-toxic, non-allergenic, having an antigenicity score ≥0.4, and exhibiting cross-strain conservation ≥95% were finally selected and named E1, E2, E3, and E4, respectively. The specific sequences are as follows, and the screening results are detailed in Table 1.

[0054] Table 1. Antigenic determinants obtained by screening in this invention ; The specific sequence of the comparison template is as follows: S1 encoded sequence (SEQ ID NO.1): S2 encoded sequence (SEQ ID NO.2): N-coded sequence (SEQ ID NO.3): The specific sequences of the antigenic determinants are as follows: E1 (SEQ ID NO.4): QNVTSCSYVSYGRFCIEPDGSLKL; E2 (SEQ ID NO.5): QYNTGNFSDGFYPFTN; E3 (SEQ ID NO.6): QHGYWRRQARYKPGKGG; E4 (SEQ ID NO.7): LQPDGLHLKFEFTTVVPRDDPQFDNYVKICDECVDGVGTRPKDEVVRPKSRSNSRPATRANSPAPRQQRPKKEKKPKKQDDEVDKALTSDEERNNAQLEFDDEPKVINWGDSALGENEL.

[0055] II. Selection of Vaccine Units and Vaccine Molecular Design CKS9 peptide (CKSTHPLSC, SEQ ID NO. 8) was selected as the mucosal targeting functional element. This peptide can specifically recognize M cell surface receptors in nasal mucosa-associated lymphoid tissue, enhancing the efficiency of antigen molecule recognition and uptake by the mucosal immune system. Multiple schemes were compared and screened by changing the type of flexible linker and adjusting the arrangement of the four antigenic peptides E1, E2, E3, and E4 to determine the optimal tandem arrangement. Finally, GGPPG and AAY flexible amino acid linkers were selected as linkers, sequentially linking the CKS9 targeting peptide with the four antigenic determinants E1, E2, E3, and E4 obtained from the screening. Specifically, GGPPG linkers were used between CKS9 and E1, E1 and E2, and E2 and E3, while AAY linkers were used between E3 and E4. His tags were introduced at both the N-terminus and C-terminus of the complete polypeptide sequence to facilitate subsequent expression detection and identification of the recombinant protein. The complete antigen molecule obtained by modular tandem assembly was named MeRSN, which serves as the exogenous expression target for the recombinant Bacillus subtilis of this invention. Its vaccine molecule design is as follows: Figure 1 As shown in SEQ ID NO.9, the full-length amino acid sequence is as follows:

[0056] Full-length sequence of the vaccine molecule (SEQ ID NO.9): HHHHHHCKSTHPLSCGPGPGQNVTSCSYVSYGRFCIEPDGSLKLGPGPGQYNTGNFSDGFYPFTNGPGPGQHGYWRRQARYKPGKGGAAYLQPDGLHLKFEFTTVVPR DDPQFDNYVKICDECVDGVGTRPKDEVVRPKSRSNSRPATRANSPAPRQQRPKKEKKPKKQDDEVDKALTSDEERNNAQLEFDDEPKVINWGDSALGENELHHHHHH.

[0057] III. Physicochemical Properties Analysis and Tertiary Structure Prediction of Vaccines To evaluate the feasibility of the MeRSN vaccine molecule designed in this invention as a candidate antigen, a systematic physicochemical property analysis and structural prediction were performed, as detailed below: AllerTop2.0 (http: / / www.ddg-pharmfac.net / AllerTOP / ) was used to predict the potential sensitization risk of peptides. The sensitization results are shown below. Figure 2 As shown, the results indicate that the vaccine molecules are non-allergenic.

[0058] The overall immunogenicity of the peptide and its solubility level under overexpression in host bacteria were assessed using Scratch Protein Predictor (http: / / scratch.proteomics.ics.uci.edu / ). Results are shown in [link to results]. Figure 3 As shown, the results indicate that the predicted solubility during overexpression is 0.947708, suggesting that the vaccine is soluble and has good solubility; the predicted antigenicity probability is 0.827877, indicating good immunogenicity potential.

[0059] The basic physicochemical parameters of MeRSN, including amino acid composition, molecular weight, isoelectric point, instability coefficient, and hydrophilicity, were fully analyzed using the ExPASy ProtParam tool (https: / / web.expasy.org / protparam / ). The results are shown in [link to results]. Figure 4 As shown, the results indicate that MeRSN encodes 215 amino acids with a molecular weight of 24095.48, a theoretical isoelectric point (pI) of 7.83, and an instability coefficient of 34.89. It is classified as a stable protein, and its hydrophilicity is -1.211, indicating that the vaccine is inherently hydrophilic and readily interacts with water molecules.

[0060] Hydrophilicity and hydrophobicity values ​​were calculated site-by-site along the polypeptide sequence using ProtScale (https: / / web.expasy.org / cgi-bin / protscale / protscale.pl#opennewwindow). The results are shown below. Figure 5 As shown, the results indicate that the most hydrophilic amino acid is arginine (Arg) at position 159, with a value of -3.644; while the most hydrophobic amino acid is glutamic acid (Glu) at position 101, with a value of 1.000.

[0061] TMHMM-2.0 (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ) was used to predict transmembrane domains in peptide sequences to determine the presence of hydrophobic transmembrane regions. Results are shown below. Figure 6 As shown, the results indicate that the protein has 0 transmembrane helices, does not have any transmembrane helical domains, and its entire sequence (1-215 amino acids) is located outside the cell membrane.

[0062] The phosphorylation sites of peptides were predicted using NetPhos-3.1 (https: / / services.healthtech.dtu.dk / service.php?NetPhos-3.1), and the results are shown in [link to results]. Figure 7 As shown in the figure. The results show that there are 9, 6 and 3 phosphorylation sites for serine (S), threonine (T) and tyrosine (Y), respectively, for a total of 18 phosphorylation sites.

[0063] Each was tested using PSIPRED 4.0 (http: / / bioinf.cs.ucl.ac.uk / psipred) (see...) Figure 8 In A), SOPMA (https: / / npsa.lyon.inserm.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_sopma.html) (see...) Figure 8 In section B), two algorithms predict the secondary structure composition of MeRSN, and statistically analyze the amino acid proportions and distribution positions of each structural unit in the α-helix, extended chain, and random coil. (See section B). Figure 8 As shown in the figure. The results show that the MeRSN protein consists of an α-helix of 51 amino acids (23.72%), an extended β-sheet of 38 amino acids (17.67%), and a random coil of 126 amino acids (58.60%).

[0064] Based on the SWISS-MODEL homology modeling tool (https: / / swissmodel.expasy.org) (see...) Figure 9A) and the AlphaFold3 AI prediction platform (https: / / alphafoldserver.com / ) (see Figure 9 In section B), a complete three-dimensional spatial structure model of MeRSN was constructed, and the results are shown in [the table]. Figure 9 As shown in the figure. The results show that the core region is mainly composed of α-helices as secondary structural elements, with long flexible irregular curled regions and end extensions. The structural domains are tightly connected with no obvious spatial steric hindrance, which is consistent with the secondary structure prediction.

[0065] The three-level structural model was graphically rendered and its structure visualized using PyMOL software. (See...) Figure 10 As shown in the figure. The results show that the MeRSN multi-epitope fusion protein can theoretically fold correctly into a stable three-dimensional conformation, forming a complete three-dimensional entity with well-defined surface topological features. Each antigenic epitope region is fully exposed on the surface of the protein molecule, which is beneficial for the recognition and binding of antigens by the body's immune cells.

[0066] Example 2: Construction and Identification of Recombinant Bacillus subtilis Expressing IBV Tandem Antigenic Determinants I. Construction and Identification of Recombinant Expression Plasmid pHT43-MeRSN Based on the complete amino acid sequence of MeRSN obtained in Example 1, codon optimization was performed using the codon usage preference of the host strain Bacillus subtilis WB800N to obtain the target gene nucleotide sequence adapted to the vaccine molecule, as shown in SEQ ID NO.10; the Bacillus subtilis-specific inducible expression vector pHT43 was selected as the backbone vector, and the multiple cloning site of the vector was selected. BamH I, Sma I was used as the insertion site. The optimized MeRSN target gene was directionally inserted into the vector via homologous recombination. The gene synthesis and in vitro construction of the recombinant vector were completed by Sangon Biotech (Shanghai) Co., Ltd., resulting in the recombinant plasmid pHT43-MeRSN. The recombinant vector map is shown below. Figure 11 As shown, the finished product is delivered in the form of 4 μg of plasmid dry powder.

[0067] Vaccine molecule nucleotide sequence (SEQ ID NO.10): .

[0068] Take 4 μg of recombinant plasmid powder, centrifuge at 12000 rpm for 30-60 seconds, add 15 μL of sterile ultrapure water, vortex to dissolve completely, and determine the concentration to be 266 ng / μL; use the empty vector pHT43 as a negative control, and employ... BamH I, Sma I. Double digestion with two restriction endonucleases was performed for verification. The 20 μL double digestion reaction system was prepared as follows: 10x QuickCut Green Buffer, 2μL; Plasmid pHT43-MeRSN, 3μL; QuickCut BamH I, 1 μL; QuickCut Sma 1 μL; add ultrapure water to bring the total to 20 μL.

[0069] The reaction was carried out at a constant temperature of 37℃ in a metal bath for 1 hour. The reaction products were identified by 1% agarose gel electrophoresis, showing two bands of 8032 bp and 656 bp. The empty control was 8032 bp. The electrophoresis results are shown in [Figure number missing]. Figure 12 As shown, lanes 1, 2, 3, and 4 contain recombinant plasmids, and lane 5 contains an empty vector. It can be seen that the target band is consistent with the theoretical gene fragment size. The recombinant plasmids that passed enzyme digestion identification were sent to Sangon Biotech (Shanghai) Co., Ltd. for full-length bidirectional sequencing. The qualified recombinant plasmids with correct sequencing results were preserved for subsequent Bacillus subtilis transformation experiments.

[0070] II. Construction and Identification of Recombinant Bacillus subtilis WB800N (pHT43-MeRSN) 1. Preparation of Bacillus subtilis WB800N competent cells (1) Preparation of electroporation solution: 1) Prepare electroporation growth medium GM: Sodium chloride 0.5g, tryptone 0.5g, yeast extract powder 0.25g, sorbitol 4.5g, ddH2O to a final volume of 50mL, autoclave at 121℃ for 15 minutes; 2) Prepare electroporation medium ETM: sorbitol 9g, mannitol 9.25g, trehalose 17.1g, glycerol 10mL, ddH2O to a final volume of 100mL, autoclave at 121℃ for 15 minutes; 3) Prepare electroporation recovery solution RM: 1g sodium chloride, 1g tryptone, 0.5g yeast extract powder, 9g sorbitol, 7g mannitol, and ddH2O to a final volume of 100mL. Autoclave at 121℃ for 15 minutes.

[0071] (2) Steps for preparing competent Bacillus subtilis cells: 1) Bacillus subtilis WB800N (neomycin Neo 50 μg / mL) was streaked onto a plate. The next day, a single colony was picked and inoculated into 3 mL of LB liquid medium and cultured overnight to prepare a seed culture. 2) The next day, take the seed culture from the overnight culture, take 2.6 mL of the overnight culture and transfer it to 50 mL of electroporation growth medium GM, and incubate at 37℃ and 220 r / min until OD600 = 0.85~0.95; 3) In a clean bench, transfer the above bacterial culture to two pre-cooled 50 mL centrifuge tubes at 4°C and incubate on ice for 30 min. Centrifuge at 4°C, 6,000 r / min for 10 min, discard the supernatant, and collect the bacterial cells;

[0072] 4) Resuspend the bacterial cells in 20 mL of pre-cooled electroporation medium (ETM), centrifuge and discard the supernatant, centrifuge at 4°C, 6,000 r / min for 10 min, collect the bacterial cells, and repeat the washing 5 times. 5) Take 1 mL of electroporation medium (ETM) to resuspend the washed bacterial cells, aliquot 80 μL into 1.5 mL centrifuge tubes (operate on ice), and store at -80℃ for later use.

[0073] 2. Electroporation of recombinant plasmid pHT43-MeRSN into Bacillus subtilis WB800N competent cells 1) Place 80 μL of prepared Bacillus subtilis WB800N competent cells on ice, and add 8 μL of recombinant plasmid pHT43-MeRSN (total plasmid amount is about 800 ng, and the added volume does not exceed 8 μL) to the system. Simultaneously set up a control group for transformation with empty pHT43 plasmid. Gently tap the wall of the centrifuge tube to mix the plasmid with the competent cells thoroughly, and incubate on ice for 5 min throughout the process. 2) Transfer all the mixed bacterial solution to a pre-cooled 2mm electroporation cup. After thoroughly wiping the outer wall of the electroporation cup dry, place it in the slot of the electroporator. Set the electroporation parameters: voltage 2500V, resistance 200Ω, capacitance 25μF. Execute the electroporation program, and control the duration of each pulse to 5.0 msec. 3) After the electroporation operation is completed, immediately add 1 mL of preheated RM solution (37°C) to the electroporation cup. Gently mix the system by pipetting, avoiding the generation of air bubbles during the operation. Transfer all the bacterial culture to a sterile 1.5 mL centrifuge tube and incubate at 37°C with shaking for 3 hours. 4) After resuscitation, centrifuge at 5000 rpm for 5 min, discard 900 μL of supernatant, and resuspend the remaining 100 μL of bacterial solution evenly on LB solid double antibody plates containing chloramphenicol (10 μg / mL) and neomycin (10 μg / mL). After spreading, invert the plate and incubate overnight in a 37℃ constant temperature incubator to obtain transformed single colonies.

[0074] 3. Validation of transformant WB800N (pHT43-MeRSN) Single colonies grown on the above-mentioned double-antibiotic plates were picked and inoculated into 1 mL of LB liquid medium containing chloramphenicol (10 μg / mL) and neomycin (10 μg / mL), and cultured at 37℃ and 200 r / min for 6 h with shaking. Using the above culture as a template, a 20 μL bacterial culture PCR identification system was prepared, with the following components:

[0075] Bacterial template, 1 μL; pHT43-F (10 μM), 1 μL; pHT43-R (10 μM), 1 μL; 2×Phanta FlashMaster Mix (Dye Plus), 10 μL; ultrapure water to bring the total to 20 μL.

[0076] pHT43-F / R was synthesized by Shanghai Sangon Biotech, and its 5'-3' sequence is as follows: pHT43-F (SEQ ID NO. 11): taacataatcggtacggggg; pHT43-R (SEQ ID NO. 12): gcctcgtatgtttcaaccat.

[0077] The PCR amplification program was set as follows: 98℃ pre-denaturation for 30 s; followed by 35 cycles, with single-cycle parameters of 98℃ denaturation for 10 s, 53℃ annealing for 5 s, and 72℃ extension for 10 s; after all cycles, a final extension at 72℃ for 1 min was performed. The amplified products were identified by 1.0% agarose gel electrophoresis. Positive transformants containing the pHT43-MeRSN recombinant plasmid showed a specific amplification band at 1048 bp, while the control strain containing the empty pHT43 plasmid showed a band at approximately 406 bp. See [link to electrophoresis results] for more details. Figure 13 Lanes 1-4 show the amplification results of transformants, and lanes 5-12 show the amplification results of strains containing empty plasmids. The results show that the molecular weight of the bands is consistent with the theoretical expectation.

[0078] The PCR products of the transformants that were correctly identified by bacterial culture PCR were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. The positive recombinant strain with correct sequencing results was named WB800N(pHT43-MeRSN), and after adding 15%~20% glycerol, it was stored at -80℃ for later use.

[0079] III. Induction and Identification of the Antigen Protein MeRSN 1) Take the glycerol-preserved recombinant strain WB800N (pHT43-MeRSN) and the empty vector control strain WB800N (pHT43) respectively, and inoculate them into LB solid double antibody plates containing 10 μg / mL chloramphenicol and 10 μg / mL neomycin using the triple streak method. Invert the plates and place them in a 37℃ constant temperature incubator for overnight culture.

[0080] 2) The next day, pick a single intact colony from the plate and inoculate it into 500 μL of the same component double-antibiotic LB liquid medium. Incubate at 37°C and 200 rpm for 6 hours with shaking.

[0081] 3) Subsequently, the bacterial culture was transferred to 10 mL of the same double-antibiotic LB liquid medium at a volume ratio of 1:100, and cultured overnight at 37°C and 200 r / min for about 12 h with shaking to obtain the seed culture.

[0082] 4) Take 1 mL of recombinant bacterial seed solution and empty vector control bacterial seed solution respectively, and inoculate them into 50 mL of the same component double-antibiotic LB liquid medium, and continue to culture at 37℃ and 200 r / min.

[0083] 5) Measure the absorbance of the bacterial solution at a wavelength of 600 nm during the culture period. When the OD of the bacterial solution... 600 When the concentration reaches 0.6~0.8, 100mM IPTG stock solution is added to the bacterial culture under aseptic conditions to control the final IPTG concentration in the system to 1 mM. The culture temperature is then adjusted to 28℃ and induced by continuous shaking for 20h.

[0084] 6) After induction, centrifuge the bacterial culture at 4℃ and 12000 rpm for 10 min, and collect the supernatant culture medium as the secreted protein sample to be detected. Take 100 μL of recombinant bacterial culture supernatant, empty vector control bacterial culture supernatant, and positive control sample (the positive control is a protein sample expressed in the same expression system with a molecular weight of 19.7 kDa), add 25 μL of 5×SDS protein loading buffer, vortex to mix, and heat in a 98℃ metal bath for 10 min to fully denature the protein. After heating, briefly centrifuge the sample and use it directly for subsequent detection; unused samples should be stored at −20℃.

[0085] 7) Western blot was used to specifically identify denatured protein samples. Electrophoresis, transfer, blocking, primary antibody incubation (1:5000 dilution), secondary antibody incubation (1:10000 dilution), and chemiluminescence imaging were all performed according to standard Western blot procedures. Results of WB detection of antigen protein expression are shown below. Figure 14 As shown, lane 1 represents the recombinant bacteria, lane 2 represents the positive control sample, and lane 3 represents the empty vector control strain. The results showed that the recombinant engineered bacterial sample exhibited a specific protein band at 27.9 kDa, the positive control sample showed a control band at 19.7 kDa, and the control group carrying only the empty vector showed no specific band, demonstrating that the MeRSN tandem antigen can be effectively expressed in the WB800N host bacteria.

[0086] Example 3: Immunogenicity evaluation of recombinant Bacillus subtilis nasal vaccine with Astragalus polysaccharide adjuvant I. Preparation of immunogen, grouping of SPF chickens and immunization program 1. Preparation of immunogens Glyceryl-preserved recombinant strain WB800N (pHT43-MeRSN) and empty vector control strain WB800N (pHT43) were respectively used. Bacterial cell amplification and antigen induction were performed according to the experimental procedures described above. After induction, the bacterial suspension was centrifuged at 4℃ and 5000 rpm for 10 min, the supernatant was discarded, and the bacterial pellet was resuspended in sterile 1×PBS buffer. The bacterial concentration was adjusted to 1×10⁻⁶ using a plate count method. 9 ~1×10 10 CFU / mL was used to obtain the vaccine formulations for groups 2 and 3.

[0087] Weigh 1.11 g of Astragalus polysaccharide solid powder (90% purity), dissolve it in 20 mL of sterile PBS buffer, and stir thoroughly to obtain an Astragalus polysaccharide stock solution with a concentration of 50 mg / mL. The stock solution was then filtered through a 0.22 μm sterile filter for sterilization. To prepare recombinant bacterial immunogen containing Astragalus polysaccharide adjuvant, the recombinant bacterial cells WB800N (pHT43-MeRSN) collected after centrifugation and discarding the supernatant were resuspended in the above Astragalus polysaccharide solution, and the bacterial concentration was adjusted to 1 × 10⁻⁶. 9 ~1×10 10 The final concentration of the astragalus polysaccharide solution was 50 mg / mL, yielding the vaccine preparation for group 4.

[0088] 2. SPF Chicken Grouping Forty-eight healthy SPF chicks, aged one day, were purchased. On the first day, they were not given feed or water to avoid gastrointestinal stress. On the second day, they were provided with sterilized drinking water and sterilized feed. After seven days of acclimatization to the experimental environment, they were randomly divided into four experimental groups of 12 chicks each. The treatment protocols for each group are as follows:

[0089] Group 1: Blank control group, with sterile PBS buffer applied via eye drops or nasal drops; Group 2: Negative control group of empty-carry bacteria, with empty-carry WB800N (pHT43) bacterial suspension applied via eye drops or nasal drops; Group 3: Simple recombinant bacterial vaccine group, WB800N (pHT43-MeRSN) bacterial solution for eye and nasal drops; Group 4: Recombinant bacterial vaccine with adjuvant, consisting of an eye drop / nasal drop of Astragalus polysaccharide APS combined with recombinant bacterial vaccine WB800N (pHT43-MeRSN).

[0090] Each group of chicks was kept in a separate pen and isolated, with the same temperature, humidity, light, and feeding conditions.

[0091] 3. Immunization program All four groups of chicks were immunized via eye drops or nasal drops, with a single immunization dose of 100 μL per chick. The experiment consisted of three immunization cycles, with a 7-day interval between adjacent cycles. Within each cycle, chicks were immunized daily for three consecutive days. The first immunization cycle began at 7 days of age, with the second and third cycles starting at 14 and 21 days of age, respectively. After a 14-day interval following the completion of the entire immunization program, chicks were challenged with the IBV QX SCSN strain at 35 days of age. Clinical morbidity was observed for 7 days post-challenge. All chicks were euthanized at 42 days of age for subsequent testing.

[0092] II. Monitoring of SPF chicken growth and weight dynamics during immunization To evaluate the effects of recombinant Bacillus subtilis and Astragalus polysaccharide adjuvant on the growth status and immune safety of SPF chickens, chicks were weighed at 7, 14, 21, and 28 days of age, and the weight changes of each group were recorded. The results are as follows: Figure 15 As shown in the figure. The results showed that during the immunization period, the weight of SPF chicks in each experimental group steadily increased with age, and the growth performance of chicks supplemented with recombinant bacterial vaccine (especially the recombinant bacterial vaccine combined with adjuvant) was slightly better than that of the PBS control group, indicating that the immunization program had no negative impact on the growth and development of chicks and had a certain growth-promoting potential.

[0093] III. ELISA detection of IBV-specific antibody IgY in serum Blood samples were collected from the subwing vein of SPF chicks at four time points: 7, 14, 21, and 28 days of age. The collected whole blood samples were incubated at 37°C for 1 hour, followed by incubation at 4°C for 2 hours or overnight. The serum separated from the blood was aspirated and centrifuged at 3000 rpm for 10 minutes at 4°C. The clear serum supernatant was carefully aspirated, aliquoted, and stored at -80°C for later use.

[0094] The IBV-specific antibody IgY in serum was detected using the commercially available IDEXX IBV Ab ELISA kit. The specific steps are as follows: 1) The kit should be allowed to equilibrate to room temperature for 1 hour beforehand, allowing all reagents to warm up to 18-26℃; 2) Add 100 μL of the test serum, the positive control serum provided with the kit, and the negative control serum, diluted 500 times, to each well of the IBV antigen pre-coated ELISA plate. After adding the samples, incubate at 25°C for 30 min. 3) After incubation, discard all liquid in the wells, add 350 μL of distilled water to each well and wash thoroughly. After washing, invert the microplate onto absorbent paper and pat dry any remaining washing solution. Repeat this washing operation 5 times. 4) After washing the plate, add 100 μL of enzyme-labeled secondary antibody to each well and incubate at 25°C for 30 min; 5) Discard the liquid in the hole again, and repeat the above washing with distilled water and patting dry with absorbent paper. The washing cycle is repeated a total of 5 times. 6) Add 100 μL of TMB substrate solution to each well and incubate at 25 °C for 15 min to develop the color. 7) After color development, add 100 μL of stop solution to each well to terminate the enzymatic reaction. Immediately afterward, use a microplate reader to measure the absorbance of each well at a wavelength of 650 nm and record it as OD. 650 .

[0095] The S / P ratio method is used to determine the antibody positivity or positivity of a sample. The calculation formula is: S / P = (OD of the sample to be tested) / ... 650 - Negative control OD 650 ) / (Positive control OD650 - Negative control OD 650 The criteria for judgment are: an S / P value > 0.2 is considered positive, and an S / P value < 0.2 is considered negative.

[0096] Test results as follows Figure 16 As shown, at 7 days of age, the levels of IBV-specific IgY antibodies in the serum of the four groups of chickens were low and largely consistent. With increasing immunization frequency, the S / P values ​​of the two vaccine groups continuously increased, while the PBS group and the empty vector group remained at low levels, indicating that the MeRSN recombinant bacteria can induce a clear IBV antigen-specific humoral immune response. At 21 and 28 days of age, the S / P values ​​of the recombinant bacteria combined with APS adjuvant group were 0.60 and 0.78, respectively, higher than the 0.47 and 0.61 of the recombinant bacteria vaccine group, with increases of approximately 26%–28%. This result indicates that, under the same conditions of recombinant bacterial antigen, bacterial dose, and immunization schedule, APS can further enhance the level of systemic antibodies induced by the recombinant bacterial vaccine.

[0097] IV. ELISA detection of IBV-specific mucosal antibodies (sIgA) in tracheal lavage fluid At two time points, 14 days and 28 days of age, 3 chicks were randomly selected from each group for euthanasia. A 5cm tracheal tissue was collected from each chick, and the inner wall was repeatedly rinsed with 1mL PBS 3-5 times. Then, the tissue was centrifuged at 12000rpm and 4℃ for 10min, and the supernatant was collected and stored at -80℃ for later use.

[0098] The same ELISA kit as described above was used to detect IBV-specific mucosal antibody sIgA in tracheal lavage fluid. The basic incubation, washing, color development, and termination procedures were consistent with those for serum IgY detection. The only difference was that the 500-fold diluted serum in step 2) was replaced with tracheal lavage fluid (PBS as a control), and the goat anti-chicken IgY enzyme-labeled antibody in step 4) was replaced with a 2000-fold diluted HRP-labeled goat anti-chicken IgA antibody. Finally, the absorbance (OD value) at 620 nm wavelength in each well was compared.

[0099] Test results as follows Figure 17As can be seen, at 14 days of age, the overall level of IBV-specific IgA in the tracheal lavage fluid of each group was relatively low, while the recombinant bacterial vaccine group and the recombinant bacterial combined with APS adjuvant group showed a certain upward trend. By 28 days of age, the changes in the PBS group and the empty vector bacterial group were small, while the recombinant bacterial vaccine group showed a significant increase, and the recombinant bacterial combined with APS adjuvant group further reached the highest level. At 28 days of age, the OD620 of the recombinant bacterial combined with APS adjuvant group was 0.60, and that of the recombinant bacterial vaccine group was 0.49, an increase of about 25%. Since IBV mainly invades through the respiratory mucosa, APS further enhances the local antibody level in the trachea on the basis of the same MeRSN recombinant bacteria, which is one of the most targeted effects of this invention. This indicates that the effect of APS is not only manifested in the increase of serum antibodies, but also extends to the local mucosal immunity of the respiratory tract corresponding to nasal administration. This local synergistic effect is directly related to the nasal compound form of this invention, and can serve as an important inventive support that distinguishes it from the application of general injectable adjuvants.

[0100] V. ELISA detection of changes in serum cytokine (IL-4, IFN-γ) concentrations Serum samples were collected from chicks at 14 and 28 days of age (serum collection method as above). The levels of IL-4 and IFN-γ cytokines in the serum of each group were measured using the Yunclone Chicken Interleukin-4 (IL-4) and Chicken Interferon-γ (IFN-γ) ELISA kits. The detection procedures for IL-4 and IFN-γ kits are detailed in the kit instructions.

[0101] The test results are shown below. Figure 18 , 19 As shown. Figure 18 The results showed that at 14 and 28 days of age, the IL-4 levels in the PBS group and the empty vector bacterial group were low and the difference was small. The IL-4 levels in the recombinant bacterial vaccine group were significantly higher, with the recombinant bacterial vaccine combined with APS adjuvant group reaching the highest levels at both time points. At 14 days of age, the IL-4 level in the recombinant bacterial vaccine combined with APS adjuvant group was 97.27 pg / mL, and in the recombinant bacterial vaccine group it was 66.03 pg / mL; at 28 days of age, they were 120.83 pg / mL and 82.94 pg / mL, respectively, with the increase due to APS being approximately 45%–50%. This result is consistent with the increasing trend of IgY and tracheal mucosal IgA, suggesting that APS can further enhance the Th2-related immune response induced by the recombinant bacterial vaccine, providing a more favorable immune environment for the formation of humoral and mucosal antibodies. Figure 19The results showed that the IFN-γ levels in the PBS group and the empty vector group were generally low at 14 and 28 days of age. The recombinant bacterial vaccine group increased IFN-γ levels, and this level increased further after the addition of APS. At 14 days of age, the IFN-γ level in the recombinant bacterial vaccine combined with APS adjuvant group was 102.73 pg / mL, and that in the recombinant bacterial vaccine group was 55.66 pg / mL; at 28 days of age, the levels were 108.96 pg / mL and 61.53 pg / mL, respectively, representing an increase of approximately 77%–85%. Figure 18 The simultaneous increase in IL-4 shown indicates that the APS combination does not merely enhance a single humoral immune response, but rather simultaneously enhances both Th1-related IFN-γ and Th2-related IL-4 responses, exhibiting a more balanced immune activation profile. This 'bidirectional enhancement' phenomenon better reflects the comprehensive immune effect of the combination of specific recombinant bacteria and APS than simply increasing a single antibody or cytokine.

[0102] VI. IBV challenge protection experiment and evaluation of protection effect At 35 days of age, the remaining SPF chicks in each experimental group were challenged with IBV QX SCSN strain via eye drops or nasal drops. The challenge dose was 200 μL per chick (containing 10... 5.3 EID50 virus).

[0103] 1. Detection of in vitro toxin excretion To quantify the in vitro viral shedding levels in the respiratory and digestive tracts of chicks after challenge, throat swabs and cloacal swabs were collected from three chicks randomly selected from each experimental group at two time points: day 4 and day 7 after challenge. The swabs were immersed in 1 mL of sterile PBS buffer and vigorously shaken to wash away the virus. The eluent was centrifuged at 4°C and 5000 rpm for 5 min, and the supernatant was collected and stored at -80°C for later use.

[0104] Total RNA was extracted from the swab supernatant using the TRIzol method. Using the extracted total RNA as a template, cDNA was synthesized by reverse transcription using the All-In-One 5X RT MasterMix Reverse Transcription Kit (abm).

[0105] The viral copy number in the sample was detected using qRT-PCR: 1) Amplification primers specifically synthesized for the N protein gene of IBV QX SCSN strain were prepared. The upstream primer F sequence (SEQ ID NO.13): CGCTCAAATTCTAGACCTGCTA; the downstream primer R sequence (SEQ ID NO.14): CATCATCCTGCTTCTTTGGCT.

[0106] 2) Using the laboratory-constructed and preserved pcDNA 3.1-N recombinant plasmid as a quantitative standard, the plasmid was serially diluted 10-fold, with four replicates for each dilution. qRT-PCR reactions were performed according to the following system: 2×Taq Pro Universal SYBR qPCR Master Mix, 10μL; pcDNA 3.1-NF (10um), 0.4μL; pcDNA 3.1-NR (10um), 0.4μL; template cDNA, 2μL; sterile enzyme-free water to a final volume of 20μL.

[0107] 3) The amplification program is set as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 10s; 60℃ annealing / extension for 30s (signal acquisition stage), for a total of 40 cycles. Finally, the instrument's default program is run to acquire the melting curve.

[0108] 4) The formula for calculating the copy number is: Plasmid copy number (copies / μL) = (6.02 × 10⁻⁶) / (6.02 × 10⁻⁶) 14 × concentration (ng / μL) / plasmid molecular weight; using the logarithmic copy number of plasmids at each dilution gradient as the x-axis and the corresponding Ct value as the y-axis, a linear regression equation was fitted to establish the IBV N gene standard curve.

[0109] 5) The samples to be tested are tested according to the same reaction procedure as in 2), and the obtained Ct values ​​are substituted into the standard curve in 3) to calculate the viral copy number (copies / μL) in each sample.

[0110] The results of throat swabs and cloacal swabs on detoxification are as follows: Figure 20 , 21 As shown, Figure 20 The results showed that on days 4 and 7 post-challenge, the viral shedding from pharyngeal swabs in both the PBS group and the empty vector group was at a high level, while the viral shedding from the respiratory tract in the recombinant bacterial vaccine group was significantly reduced, with the lowest levels observed in the recombinant bacterial vaccine combined with APS adjuvant group at both time points. On day 4 post-challenge, the viral shedding in the recombinant bacterial vaccine group and the recombinant bacterial vaccine combined with APS adjuvant group were 3.75 and 2.56 log, respectively. 10 APS further reduced toxin excretion by approximately 1.19 log. 10 This translates to approximately 15 times; on the 7th day after the viral challenge, the two groups showed log values ​​of 2.69 and 1.81 respectively. 10 This further reduces the log by approximately 0.88. 10 This translates to approximately an 8-fold increase. This result indicates that APS, especially in the early stages of infection, significantly reduces respiratory viral shedding, thus helping to reduce virus transmission and providing key evidence of the innovativeness of the compound system. Figure 21The results showed that on days 4 and 7 post-challenge, the cloacal viral shedding in the recombinant vaccine group was lower than that in the PBS group, and further decreased to the lowest level in the recombinant vaccine combined with APS adjuvant group. On day 4, the recombinant vaccine group showed a viral shedding rate of 3.15 log [value missing]. 10 The recombinant bacteria combined with APS adjuvant group had a yield of 2.32 log [value missing]. 10 The difference is 0.83 log. 10 This is equivalent to a further reduction of approximately 7 times; on day 7, the two groups were 2.38 and 2.04 log, respectively. 10 It continues to show a downward trend of about 2 times.

[0111] 2. Organize viral load testing All chickens were euthanized on the 7th day after infection (42 days old), and tissue samples such as trachea, lungs, and kidneys were collected and stored at -80℃ for later use.

[0112] Weigh 0.1g of tracheal, lung, and kidney tissue into a 1.5mL EP tube, add 1mL of PBS buffer, and homogenize using a tissue homogenizer. Centrifuge the homogenate at 12000rpm and 4℃ for 5min, and take 200μL of the supernatant as the tissue virus solution. The subsequent extraction of viral RNA and calculation of viral load are carried out in the same way.

[0113] Test results as follows Figure 22 RT-qPCR results showed that on day 7 post-challenge, the viral load of IBV in the trachea, lungs, and kidneys exhibited a continuous gradient: highest in the PBS group, followed by the empty vector group, decreased in the recombinant vaccine group, and lowest in the recombinant bacteria combined with APS adjuvant group. Compared with the recombinant vaccine group, the viral load in the trachea of ​​the recombinant bacteria combined with APS adjuvant group decreased from 2.80 log [missing value]. 10 Reduced to 2.15 log 10 This further reduces the log value by 0.65. 10 This is approximately equivalent to 4 to 5 times; the lungs increased from 1.55 log... 10 Dropped to 0.79 log 10 , reduced by 0.76 log 10 This is approximately equivalent to 6 times; the kidneys are composed of approximately 2.10 log... 10 Dropped to 1.25 log 10 This reduces the log by approximately 0.85. 10 The effect was approximately seven times greater. The synergistic effect of APS was observed as a decrease in both the primary respiratory target tissue and distal target organs such as the kidneys, indicating that this combination system not only locally increased antibody levels but also further limited viral replication and spread in multiple target tissues. From a tissue distribution perspective, the viral load in the trachea and kidneys was generally higher than in the lungs, indicating that the recombinant bacterial vaccine combined with APS had a good inhibitory effect on IBV replication in the respiratory tract and kidney tissues.

[0114] 3. Pathological observation of dissected organs in chickens Tracheal, lung, and kidney tissue samples were collected from chickens euthanized by the virus and fixed in centrifuge tubes containing 4% paraformaldehyde fixative. After HE staining, pathological tissue sections were prepared, and the histopathological changes of each tissue and organ in each group of chickens were observed under an optical microscope.

[0115] Staining results as follows Figure 23 As shown, the tracheal tissues of the control PBS group and the empty vector group showed significant lesions after viral challenge, including a significant decrease in the number of goblet cells, accompanied by inflammatory cell infiltration, necrotic cell shrinkage and fragmentation, cytoplasmic dissolution, and vacuolation. In contrast, the lesions in the recombinant bacterial vaccine group and the recombinant bacterial combined with APS group were milder or showed no obvious lesion characteristics. Lung tissue morphology analysis showed epithelial cell proliferation after viral challenge in the PBS group, with disordered arrangement of proliferating epithelial cells and enlarged nuclei, and a large amount of eosinophilic exudate. No obvious histopathological damage was observed in other groups. Kidney tissue morphology analysis showed significant lesions after viral challenge in the PBS group and the empty vector group, including varying degrees of degeneration and necrosis of renal tubular epithelial cells, with inflammatory cell infiltration in the necrotic areas, predominantly neutrophils. No obvious histopathological damage was observed in other vaccine groups. This indicates that both the recombinant bacteria and the recombinant bacteria combined with APS can protect the body's tissues from lesions caused by viral infection.

[0116] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A recombinant Bacillus subtilis, characterized in that, The recombinant Bacillus subtilis expressed the multi-epitope antigen MeRSN of chicken infectious bronchitis virus; The multi-epitope antigen MeRSN includes: M cell targeting peptide CKS9, B cell antigenic determinants E1 and E2, T cell antigenic determinants E3 and E4, linker peptide, and 6×His tag. The amino acid sequence of the B cell antigenic determinant E1 is shown in SEQ ID NO.4, and the amino acid sequence of E2 is shown in SEQ ID NO.5; the amino acid sequence of the T cell antigenic determinant E3 is shown in SEQ ID NO.6, and the amino acid sequence of E4 is shown in SEQ ID NO.

7.

2. The recombinant Bacillus subtilis according to claim 1, characterized in that, The full-length amino acid sequence of the multi-epitope antigen MeRSN is shown in SEQ ID NO.

9.

3. The recombinant Bacillus subtilis according to claim 1, characterized in that, The nucleotide sequence encoding the multi-epitope antigen MeRSN is shown in SEQ ID NO.

10.

4. The recombinant Bacillus subtilis according to claim 1, characterized in that, The host strain of the recombinant Bacillus subtilis is Bacillus subtilis WB800N.

5. A recombinant Bacillus subtilis nasal vaccine containing IBV multi-epitope antigen, characterized in that, It contains recombinant Bacillus subtilis and Astragalus polysaccharide adjuvant as described in any one of claims 1-4.

6. The nasal vaccine according to claim 5, characterized in that, The concentration of the recombinant Bacillus subtilis was 1×10⁻⁶. 9 ~1×10 10 CFU / mL.

7. The nasal vaccine according to claim 5, characterized in that, The final concentration of the astragalus polysaccharide adjuvant in the vaccine is 50 mg / mL.

8. The method for preparing the nasal vaccine according to any one of claims 5 to 7, characterized in that, Includes the following steps: (1) The MeRSN antigen gene with the nucleotide sequence shown in SEQ ID NO.10 was cloned into a prokaryotic expression vector to obtain the recombinant plasmid p-MeRSN; (2) The recombinant plasmid p-MeRSN was transformed into Bacillus subtilis WB800N, and positive transformants were screened to obtain recombinant Bacillus subtilis WB800N(p-MeRSN); (3) The recombinant Bacillus subtilis obtained in step (2) was induced and cultured, and the bacterial cells were collected; (4) Resuspend the bacterial cells until the bacterial concentration is 1×10⁻⁶. 9 ~1×10 10 CFU / mL; (5) Dissolve the Astragalus polysaccharide, filter it through a 0.22μm filter membrane to remove bacteria, and mix it with the bacterial solution obtained in step (4) to obtain the final product.

9. The preparation method according to claim 8, characterized in that, The final concentration of Astragalus polysaccharide in the mixed system in step (5) is 50 mg / mL; the resuspended bacterial cells and Astragalus polysaccharide dissolution are selected from sterile PBS.

10. The use of the recombinant Bacillus subtilis according to any one of claims 1 to 4, or the nasal vaccine according to any one of claims 5 to 7, or the nasal vaccine prepared by the preparation method according to any one of claims 8 to 9, in the preparation of a drug for the prevention of infectious bronchitis in chickens.

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