A recombinant lactic acid bacteria vaccine and a preparation method and application thereof

Abstract

This invention belongs to the fields of genetic engineering and veterinary biological products technology, specifically relating to a recombinant lactic acid bacteria vaccine, its preparation method, and its application. The recombinant *Lactobacillus plantarum* expresses at least a dendritic cell-targeting peptide and an avian influenza virus HA1 antigen protein. The amino acid sequence of the dendritic cell-targeting peptide is shown in SEQ ID NO.1, and the amino acid sequence of the avian influenza virus HA1 antigen protein is shown in SEQ ID NO.7. This invention screens short peptides targeting dendritic cells as vaccine adjuvants and fuses the dendritic cell-targeting peptide sequence AT12-FITC with the H10 subtype AIV HA1 antigen to form an oral lactic acid bacteria vaccine. Through the adhesion properties of *Lactobacillus plantarum* and the targeting ability of dendritic cells, the immunogenicity and protective effect of the vaccine are enhanced, thus possessing good practical application value.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and veterinary biological products technology, specifically relating to a recombinant lactic acid bacteria vaccine, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Avian influenza is a highly contagious disease of birds caused by avian influenza virus (AIV). The H10N3 subtype, a typical low-pathogenic avian influenza virus, has become a significant threat to poultry farming and public health since it was confirmed in 2021 to have the potential for cross-species transmission. Due to its high genetic diversity and complex transmission routes, traditional biosecurity measures (such as quarantine and environmental disinfection) are insufficient to provide comprehensive protection against this virus, and the lack of commercial vaccines further exacerbates the control challenges. Currently, vaccine development for the H10N3 subtype is still lacking, and the entire H10 subtype AIV vaccine field is limited by traditional technological bottlenecks, exhibiting problems such as insufficient immunogenicity and weak mucosal immune induction, failing to meet the needs of efficient control.

[0004] Dendritic cells (DCs), as the most potent antigen-presenting cells in the immune system, are the core hub connecting innate and adaptive immunity. They not only efficiently take up, process, and present antigens to naive T cells, activating specific cellular immune responses, but also play an irreplaceable role in mucosal local immunity by secreting cytokines to regulate immune cell differentiation. Therefore, vaccine design targeting DCs has become a key direction for improving immunization efficacy. However, current screening of DC-targeting peptides is mostly focused on mammalian models; research on targeting efficiency and activation mechanisms in avian DCs is insufficient, and there is a lack of systematic integration with avian influenza virus antigens, preventing the development of feasible vaccine technology solutions.

[0005] Hemagglutinin (HA) is the core surface antigen of influenza A virus. Its HA1 subunit contains the viral receptor binding site and the main neutralizing epitope, making it a key target for inducing the production of specific neutralizing antibodies. Existing research has confirmed that recombinant vaccines expressing the HA1 protein can effectively stimulate humoral immune responses against influenza viruses. For example, Kamble et al. successfully enhanced host antiviral immunity by expressing the H1N1 subtype HA1 protein using an attenuated mutant of Salmonella Typhimurium. However, research on recombinant vaccines targeting the H10N3 subtype HA1 protein is still in its early stages, and traditional vaccine vectors (such as inactivated viruses and adenoviruses) have problems such as safety concerns, low mucosal delivery efficiency, and high production costs. Inactivated vaccines require large-scale virus culture, have a long preparation cycle, and cannot induce potent mucosal immunity; adenovirus vectors may cause interference from pre-existing antibodies, limiting the persistence of immune effects. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, the inventors, through long-term technical and practical exploration, have provided a recombinant lactic acid bacteria vaccine, its preparation method, and its application. Specifically, this invention screens short peptides targeting dendritic cells as vaccine adjuvants and fuses the dendritic cell-targeting peptide sequence AT12 with the H10 subtype AIV HA1 antigen to form an oral lactic acid bacteria vaccine. By leveraging the adhesion properties of *Lactobacillus plantarum* and the targeting ability of dendritic cells, the immunogenicity and protective effect of the vaccine are enhanced. Based on the above research results, this invention is thus completed.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a recombinant lactic acid bacteria vaccine comprising at least recombinant *Lactobacillus plantarum*, wherein the recombinant *Lactobacillus plantarum* expresses at least a dendritic cell targeting peptide and an avian influenza virus HA1 antigen protein, wherein the amino acid sequence of the dendritic cell targeting peptide is shown in SEQ ID NO.1, and the amino acid sequence of the avian influenza virus HA1 antigen protein is shown in SEQ ID NO.7.

[0009] In this invention, the avian influenza virus HA1 antigen protein can effectively induce the production of neutralizing antibodies, directly block viral infection, stimulate cross-immune protection, and initiate cellular immune responses. Compared with the complete HA protein, it has higher safety and expression efficiency, while reducing interference from non-specific immune responses.

[0010] The dendritic cell-targeting peptide can directly deliver vaccine antigens to specific receptors on the surface of dendritic cells, bind to TLR4, induce dendritic cell activation, and further promote antigen-specific T cell proliferation, thereby enhancing the antigen-specific immune response. It also activates T cells and B cells during antigen presentation, thus enhancing the effectiveness of the immune response.

[0011] Furthermore, the recombinant *Lactobacillus plantarum* also expresses truncated poly-γ-glutamate synthase A (pgsA'), thereby stably anchoring the aforementioned target exogenous protein to the cell wall surface of the recombinant *Lactobacillus plantarum*, thus improving the accessibility of the exogenous protein and enhancing the immunogenicity and application efficiency of the antigen. The amino acid sequence of the truncated poly-γ-glutamate synthase A is shown in SEQ ID NO. 9.

[0012] In this invention, the recombinant lactic acid bacteria vaccine may also contain pharmaceutically acceptable excipients, particularly those used in vaccines, including but not limited to emulsifiers, stabilizers, preservatives, fillers, antioxidants, buffers, pH adjusters, and diluents. Furthermore, it can be formulated into oral, topical, suppository, and sterile injectable solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays, using conventional methods. In one specific embodiment of this invention, the recombinant lactic acid bacteria vaccine is an oral dosage form, thus facilitating administration.

[0013] In a second aspect, the present invention provides a method for preparing the above-mentioned recombinant lactic acid bacteria vaccine, the method comprising constructing recombinant Lactobacillus plantarum, the method comprising: constructing a recombinant expression vector containing a gene encoding a dendritic cell targeting peptide and an avian influenza virus HA1 antigen protein, and transforming the recombinant expression vector into Lactobacillus plantarum to obtain the vaccine.

[0014] The method for constructing the recombinant expression vector includes:

[0015] A gene fragment fused with a dendritic cell targeting peptide and an avian influenza virus HA1 antigen protein is ligated to an expression vector, which can be a plasmid. In one specific embodiment of the present invention, the plasmid can be a pSIP-409 plasmid. Further, the plasmid is a pSIP-409 plasmid (pSIP-409-pgsA') loaded with a truncated poly-γ-glutamate synthase A encoding gene.

[0016] The nucleotide sequence of the gene encoding the dendritic cell targeting peptide is shown in SEQ ID NO.2.

[0017] The nucleotide sequence of the gene encoding the HA1 antigen protein of avian influenza virus is shown in SEQ ID NO.8.

[0018] The nucleotide sequence of the gene encoding truncated poly-γ-glutamate synthase is shown in SEQ ID NO.10.

[0019] The starting strain, *Lactobacillus plantarum*, can specifically be *Lactobacillus plantarum* (… Lactobacillus plantarumNC8, the Lactobacillus plantarum NC8 has the advantages of high biosafety, strong mucosal adhesion ability and oral administration, and can effectively deliver exogenous antigens and immunomodulatory molecules to the host immune site, and efficiently induce immune response.

[0020] In this invention, the gene fragment fused with dendritic cell targeting peptide and avian influenza virus HA1 antigen protein is linked to the expression vector, specifically through homologous recombination.

[0021] A third aspect of the present invention provides the use of the above-mentioned recombinant lactic acid bacteria vaccine in the preparation of drugs for the prevention of avian influenza virus.

[0022] Specifically, the avian influenza virus in question is the H10N3 subtype avian influenza virus.

[0023] The avian influenza virus drug is to be administered to poultry, specifically chickens.

[0024] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial effects:

[0025] The above-mentioned technical solution screened dendritic cell-targeting peptides through cell experiments and bacterial growth curves. These peptides exhibit highly efficient DC targeting and activation capabilities, significantly enhancing the immunogenicity of the vaccine and solving the problem of poor immunization efficacy in traditional vaccines. Simultaneously, the recombinant lactic acid bacteria vaccine is administered orally, which is convenient, low-cost, and suitable for large-scale poultry immunization, overcoming the limitations of traditional injectable vaccines. Experimental verification shows that the vaccine can simultaneously induce humoral, cellular, and mucosal immune responses, providing comprehensive immune protection and offering a novel and efficient technical means for the prevention and control of H10N3 avian influenza, with broad application prospects. Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] Figure 1 This figure shows the binding of the dendritic targeting peptide to chicken dendritic cells at 4°C in Example 1 of this invention. **** indicates P < 0.0001; ns indicates P > 0.05.

[0028] Figure 2 This figure shows the binding of the dendritic targeting peptide to chicken dendritic cells at 37°C in Example 1 of this invention. *** indicates P < 0.001; **** indicates P < 0.0001.

[0029] Figure 3This figure shows the binding of the dendritic targeting peptide to chicken blood cells at 37°C in Example 1 of this invention. ** indicates P < 0.01.

[0030] Figure 4 The bacterial growth curves are for the three bacterial solutions NC8 / pSIP-409-pgsA'-HA1-AT12, NC8 / pSIP-409-pgsA'-HA1-HG21, and NC8 / pSIP-409-pgsA'-HA1-GW31 in Example 1 of this invention.

[0031] Figure 5 This is for the amplification of the target fragments H10N3 HA1-AT12 and H10N3 HA1 in Example 2 of the present invention.

[0032] Figure 6 This is obtained by enzyme digestion of the pSIP-409-pgsA' vector in Example 2 of the present invention.

[0033] Figure 7 The pSIP-409-pgsA'-HA1-AT12 and pSIP-409-pgsA'-HA1 stripes in Embodiment 2 of the present invention.

[0034] Figure 8 This invention provides an enzyme digestion identification of pSIP-409-pgsA'-HA1-AT12 and pSIP-409-pgsA'-HA1 plasmids in Example 2 of the present invention.

[0035] Figure 9 This invention provides PCR identification of pSIP-409-pgsA'-HA1-AT12 and pSIP-409-pgsA'-HA1 plasmids in Example 2 of the present invention.

[0036] Figure 10 The results show the protein expression of NC8 / pSIP-409-pgsA'-HA1-AT12, NC8 / pSIP-409-pgsA'-HA1, and NC8 / pSIP-409-pgsA' in Example 2 of this invention.

[0037] Figure 11 The results show the chicken serum IgG antibody levels in Example 3 of this invention. * indicates P < 0.05; ** indicates P < 0.01; **** indicates P < 0.0001; ns indicates P > 0.05.

[0038] Figure 12 The results of flow cytometry analysis of CD3 T cells in chicken spleen in Example 3 of this invention are shown. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001; ns indicates P > 0.05.

[0039] Figure 13 The results of flow cytometry analysis of CD4 T cells in chicken spleen in Example 3 of this invention are shown. ** indicates P < 0.01; **** indicates P < 0.0001; ns indicates P > 0.05.

[0040] Figure 14 The results of flow cytometry analysis of CD8 T cells in chicken spleen in Example 3 of this invention are shown. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; ns indicates P > 0.05.

[0041] Figure 15 The results of the virus excretion detection in chicken throat and pharyngeal swabs in Example 3 of this invention are shown. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; ns indicates P > 0.05. Detailed Implementation

[0042] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0043] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0044] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Any simple modifications, equivalent variations, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the present invention.

[0045] Example 1: Screening of short peptides targeting dendritic cells

[0046] 1.1 Isolation and Culture of Chicken Dendritic Cells

[0047] Bone marrow from the tibia and femur of chicks was collected, prepared into a single-cell suspension, and cultured until day 7. All suspended cells were then collected to identify immature bone marrow-derived dendritic cells, and their purity was determined using flow cytometry.

[0048] 1.2 Screening of AT-12, HG-21, and GW-31

[0049] 1.2.1 Flow cytometry detection of short peptide binding to DCs:

[0050] Bone marrow-derived dendritic cells were co-incubated with biotinylated peptides AT-12, HG-21, and GW-31 at 4°C for 1 hour. After staining with CD11c (APC-Cy7, 64x) and MHC II (Per-cp5.5, 256x) antibodies, flow cytometry was used for analysis. The results are as follows: Figure 1 As shown, the short peptide AT-12 binds better to dendritic cells at 4°C.

[0051] 1.2.2 Flow cytometry detection of short peptide activation of DCs:

[0052] Bone marrow-derived dendritic cells were incubated with biotinylated peptides AT-12, HG-21, and GW-31 at 37°C for 12 hours. After staining with CD11c (APC-Cy7, 64x) and MHC II (Per-cp5.5, 256x) antibodies, flow cytometry was used for analysis. The results are as follows: Figure 2 As shown, the short peptide AT-12 has a stronger activating effect on dendritic cells at 37°C.

[0053] 1.2.3 Flow cytometry detection of the binding of short peptides to chicken blood cells:

[0054] Chick blood cells were co-incubated with biotinylated peptide AT-12, and the test results were as follows: Figure 3 As shown, flow cytometry analysis revealed that the short peptide AT-12 can bind to chick blood cells.

[0055] 1.2.4 Determination of bacterial growth curve

[0056] OD values ​​of three bacterial cultures, NC8 / pSIP-409-pgsA'-HA1-AT12, NC8 / pSIP-409-pgsA'-HA1-HG21, and NC8 / pSIP-409-pgsA'-HA1-GW31, were measured at different time points. 600 Values, plot bacterial growth curves, such as Figure 4 As shown, NC8 / pSIP-409-pgsA'-HA1-AT12 exhibited the best growth.

[0057] Example 2: Preparation and validation of a recombinant lactic acid bacteria vaccine expressing avian influenza virus HA protein.

[0058] 1.1 Vectors, strains, and cells

[0059] Escherichia coli Trans-T1 competent cells were purchased from Beijing TransGen Biotech Co., Ltd., Lactobacillus plantarum NC8 competent cells were preserved in our laboratory, and the pSIP-409-pgsA' vector was constructed by the Animal Microecological Preparation Engineering Research Center of Jilin Agricultural University.

[0060] 1.2 Enzymes and Main Experimental Reagents

[0061] GelStain fluorescent nucleic acid staining reagent was purchased from TransGen Biotech Ltd.; SimplyP total RNA extraction kit was purchased from Beijing Huaxin Kangxin Biotechnology Co., Ltd.; DNA kit and agarose gel DNA recovery kit were purchased from Tiangen Biotech; DEPC (Rnase-Free) H2O was purchased from BD Biosciences (bdbiosciences.com); penicillin-streptomycin mixture was purchased from Hyclone; 180kDa Prestained Protein Marker was purchased from Vazyme; 4% paraformaldehyde universal tissue fixative was purchased from Biosharp Biotechnology Co., Ltd.; DMEM / HIGH GLUCOSE was purchased from Gibco; anti-fluorescence attenuation mounting medium was purchased from Beijing Solarbio Science & Technology Co., Ltd.; sodium citrate antigen retrieval solution was purchased from Beijing Solarbio Science & Technology Co., Ltd.; DL2000, DL 10000 DNA Marker, SDS-PAGE Loading Buffer (5×), endonucleases NcoⅠ, XhoⅠ, XbaⅠ and HindⅢ were all purchased from Takara; Albumin Bovine V bovine serum albumin V Purchased from Shanghai Yuanye Biotechnology Co., Ltd.; ProteinExt Mammalian Membrane Protein Extraction Kit

[0062] Trans1-T1 Phage Resistant Chemical Competent Cell was purchased from TransGen Biotech Ltd.; Omni-Easy™ One-Step PAGE Gel Preparation Kit and Coomassie Brilliant Blue Rapid Staining Solution were purchased from Yamei Biopharmaceutical Technology Co., Ltd.; PBS Phosphate Buffered (Powder) was purchased from Thermo Fisher Scientific (China) Co., Ltd.; Chicken CD3, CD4, and CD8 Antibodies were purchased from Southern Biotech, USA; HRP and Goat Anti-Chicken IgG were purchased from Wuhan Yacoin Biotechnology Co., Ltd.; and ClonExpress II One Step Cloning Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0063] 1.3 Main Instruments and Equipment for the Experiment

[0064] The following equipment was used: Leica DMi8 inverted fluorescence microscope (Germany); Eppendorf small high-speed centrifuge (Germany); Eppendorf 4°C refrigerated centrifuge (Centrifuge 5810R) (Germany); Eppendorf gradient PCR instrument (Germany); metal bath from Beijing Tiangen Biotech Co., Ltd. (Germany); pipettes from Eppendorf (Germany); Sigma GR85DA fully automated autoclave (Germany); Eppendorf constant temperature shaker incubator (Germany); BIO-RAD gel imaging analysis system (Universal Hood II) (USA); Mettler Toledo precision electronic balance (ME204) (Switzerland); and BIOTeK microplate spectrophotometer (Epoch2) (USA).

[0065] 1.4 Preparation method of recombinant lactic acid bacteria vaccine expressing avian influenza virus HA protein

[0066] 1.4.1 Obtaining the target gene H10N3HA1-AT12 and the target gene H10N3HA1

[0067] The nucleic acid gene sequence encoding AIV, H10N3 HA1, was found in GenBank. An XbaⅠ, HindⅢ, and AT12-FITC nucleic acid sequence (as shown in SEQ ID NO. 2) was added to the 5' end of the sequence (targeting the dendritic short peptide AT12) along with homologous arms at both ends. Another sequence was obtained without the dendritic cell-targeting short peptide, only with XbaⅠ, HindⅢ, and 5' and 3' homologous arms added to the 5' end. The primers involved are as follows: H10N3HA1-AT12-F (SEQ ID NO. 11), H10N3HA1-AT12-R (SEQ ID NO. 12), H10N3HA1-F (SEQ ID NO. 13), and H10N3HA1-R (SEQ ID NO. 14).

[0068] 1.4.2 Homologous recombination

[0069] (1) Using the above fragments H10N3 HA1-AT12 and H10N3 HA1 as templates, the target fragments were amplified by PCR and recovered, yielding bands of 1026 bp and 990 bp, respectively. Figure 5 As shown.

[0070] (2) The vector pSIP-409-pgsA', with a size of 6155 bp, was obtained by double digestion with XbaⅠ and HindⅢ. Figure 6 As shown.

[0071] (3) Add homologous recombinase, target fragment, buffer and pSIP-409-pgsA' vector, mix well and carry out homologous recombination reaction. After reacting at 37℃ for 30 min, cool to 4℃ or place on ice immediately to cool.

[0072] 1.4.3 Connection Conversion

[0073] The recombinant product was transformed into TransⅠ competent cells, amplified on plates, and single colonies were picked and cultured. Plasmids pSIP-409-pgsA'-HA1-AT12 and pSIP-409-pgsA'-HA1 were extracted, with sizes of 7181 bp and 7145 bp, respectively. Figure 7 As shown. Double enzyme digestion identification revealed the target fragments to be 1026 bp and 990 bp, respectively, as shown. Figure 8 As shown, plasmids with the correct bands were sent to a sequencing company for sequencing.

[0074] 1.4.4 Electroconversion to NC8 lactic acid bacteria

[0075] The plasmids pSIP-409-pgsA'-HA1-AT12 and pSIP-409-pgsA'-HA1 were electroconverted to [a specific structure / condition - likely a specific process or feature] under conditions of 2000 V, 400 Ω, and 25 μF. L. plantarum In strain NC8, recombinant bacteria were selected for erythromycin plate culture.

[0076] PCR identification, such as Figure 9 As shown, the band sizes were 1026 bp and 990 bp, respectively. The positive recombinant bacteria were named NC8 / pSIP-409-pgsA'-HA1-AT12 and NC8 / pSIP-409-pgsA'-HA1, respectively.

[0077] 1.4.5 Protein purification

[0078] NC8 / pSIP-409-pgsA'-HA1-AT12, NC8 / pSIP-409-pgsA'-HA1, and NC8 / pSIP-409-pgsA' bacterial cultures were activated, respectively. SppIP was added to induce expression. After collecting the bacterial cells, they were lysed by sonication, purified and eluted using a His Trap pre-packed column, and the resulting samples and positive controls were analyzed by Western blotting.

[0079] like Figure 10 As shown, the bands of NC8 / pSIP-409-pgsA'-HA1-AT12, NC8 / pSIP-409-pgsA'-HA1, and the positive control are of the same size, at 37.6 kDa.

[0080] Example 3 Animal experiments on recombinant lactic acid bacteria NC8 / pSIP-409-pgsA'-HA1-AT12 and pSIP-409-pgsA'-HA1 expressing avian influenza virus HA1 protein.

[0081] In Example 2, a recombinant lactic acid bacteria expressing the HA1 protein of avian influenza virus was successfully constructed and expressed in vitro. In this example, chicks were selected as experimental animals for preliminary animal experiments. The chicks were immunized orally and nasally. Changes in humoral immunity, cellular immunity, and mucosal immunity of the immunized chicks were detected by flow cytometry, neutralizing antibody test, etc., so as to study the effect of the recombinant lactic acid bacteria on the immunity of immunized animals.

[0082] 1.1 Instruments

[0083] The equipment included: a Leica RM2245 fully automated paraffin embedding machine (Germany); a Thermo Scientific HERACELL 240i CO2 incubator (USA); a Bio-Rad electrophoresis apparatus (USA); a BD LSRFortessa™ flow cytometer (USA); a low-temperature ultracentrifuge (Eppendorf 5810R); a SW-CJ-2FD single-sided double-person clean bench (Shanghai Boxun); an HRLM-80 fully automated autoclave; and a BCD-649WDCE refrigerator (Haier).

[0084] 1.2 Laboratory Animals

[0085] One hundred two-day-old chicks were purchased from a private chicken farm in Changchun; after two weeks of acclimatization at the experimental animal breeding base of Jilin Agricultural University, the experiment was conducted.

[0086] 1.3 Main Reagents

[0087] Chick leg bands, scissors, forceps, 1.5 mL EP tubes, 2 mL EP tubes, 5 mL EP tubes, 10 mL EP tubes, 50 mL centrifuge tubes, gavage syringes, and lymphocyte separation medium were purchased from Beijing Solarbio Biotechnology Co., Ltd.; PBS, 24-well cell culture plates, and Cell 1640 culture medium were purchased from Beijing TransGen Biotech Co., Ltd.; flow cytometry antibodies CD3, CD4, CD8, IgA, IFN-γ, and IL-4 were purchased from BD Biosciences.

[0088] 1.4 Methods

[0089] 1.4.1 Experimental Grouping

[0090] One hundred chicks were randomly divided into 5 groups of 20 each. The groups were: NC8 / pSIP-409-pgsA'-HA1-AT12, NC8 / pSIP-409-pgsA'-HA1, NC8 / pSIP-409-pgsA', PBS, and inactivated virus. Each chick was labeled with a leg band, with 20 natural numbers for each group.

[0091] 1.4.2 Blood collection from the subwing vein

[0092] 0.5 mL of blood was collected from the subwing vein of chicks and placed in a 1.5 mL EP tube. Venous blood was collected every 14 days.

[0093] 1.4.3 Fecal collection

[0094] Feces are collected every 14 days.

[0095] 1.4.4 Immunization Procedure

[0096] One hundred chicks were randomly divided into 5 groups of 20 each. Each group was orally immunized with NC8 / pSIP-409-pgsA'-HA1-AT12, pSIP-409-pgsA'-HA1, NC8 / pSIP-409-pgsA', and PBS on days 0, 1, and 2, respectively. Each group was re-immunized intranasally on days 14, 15, and 16. The fifth group was immunized intranasally with inactivated virus. The specific immunization challenge protocol is shown in Table 1.

[0097] Table 1 Immune Challenge Protocol

[0098]

[0099] 1.4.5 Study on the immune effect of recombinant lactic acid bacteria on chicks

[0100] 1.4.5.1 ELISA measurement of IgG levels:

[0101] Serum samples were collected 14 days after booster immunization, and specific serum IgG antibodies in each group were detected by ELISA to assess the ability of the recombinant candidate vaccine to stimulate humoral immunity throughout the body.

[0102] The results are as follows Figure 11As shown, the inactivated virus group had the highest IgG antibody level, followed by the NC8 / pSIP-409-pgsA'-HA1-AT12 group. Significant differences were found between the inactivated virus group, the NC8 / pSIP-409-pgsA'-HA1 group, the NC8 / pSIP-409-pgsA'-HA1 group, and the NC8 / pSIP-409-pgsA' group and the PBS group. Differences were also found between the inactivated virus group, the NC8 / pSIP-409-pgsA'-HA1 group, and the NC8 / pSIP-409-pgsA'-HA1 group. No significant difference was found between the inactivated virus group and the NC8 / pSIP-409-pgsA'-HA1 group.

[0103] 1.4.5.2 Immunohistochemistry

[0104] Lung tissue from chickens before challenge was fixed and prepared into paraffin sections. After dewaxing and antigen retrieval, the sections were stained with CD3, CD4, CD3, CD8, and IgA antibodies, and then stained with DAPI. Fluorescence microscopy showed that the positive results of PBS and NC8 / pSIP-409-pgsA' group were weaker, while the positive results of NC8 / pSIP-409-pgsA'-HA1-AT12 and inactivated vaccine group were stronger than those of NC8 / pSIP-409-pgsA'-HA1 group.

[0105] 1.4.5.3 Attacking the virus

[0106] (1) Fourteen days after booster immunization, all chickens were intranasally infected with H10N3 AIV (5×10⁻⁶). 6.5 EID50 (N=10). Changes in T cell levels in the spleen of chickens in each group were detected by flow cytometry on day 5 post-challenge. The immune responses induced by the recombinant vaccine and the inactivated virus group were compared.

[0107] (2) On the 5th day after viral infection, pharyngeal swabs and anal swabs were taken from each group of chickens to detect viral shedding and to compare the immune response induced by the recombinant vaccine and the inactivated virus group.

[0108] 1.4.5.4 Flow cytometry

[0109] (1) Preparation of spleen cell suspension

[0110] Five days after the viral challenge, the chicks were euthanized by drawing blood from their hearts and injecting air. The spleens were then ground on a small petri dish with a copper mesh. Lymphocytes were separated using a lymphocyte separation solution to prepare a spleen lymphocyte suspension.

[0111] (2) Splenic lymphocyte count: Splenic lymphocytes were counted after dilution.

[0112] 1.4.5.5 Detection of T cells in the germinal centers of chick spleens

[0113] Take 24 1.5 EP tubes and label 20 samples, 1 ISO tube, and 3 single-standard tubes. Add 5 × 10⁻⁶ ppm to each tube. 5 Cell stock solution, 3 μL of corresponding antibody added to 3 single-labeled tubes, and 9 μL of premixed antibody solution for staining was added to each of the remaining 20 sample tubes, and then detected by flow cytometry.

[0114] like Figure 12 As shown, CD3 in chicken spleen T cells + The levels of the inactivated virus group and NC8 / pSIP-409-pgsA'-HA1-AT12 were significantly different from those of the PBS and NC8 / pSIP-409-pgsA' groups; the inactivated virus group was stronger than the NC8 / pSIP-409-pgsA'-HA1-AT12 group; the NC8 / pSIP-409-pgsA'-HA1 group was weaker than the inactivated virus group and the NC8 / pSIP-409-pgsA'-HA1-AT12 group.

[0115] like Figure 13 As shown, CD4 in chicken spleen T cells + At different levels, there were significant differences between the inactivated virus group, the NC8 / pSIP-409-pgsA'-HA1-AT12 group, and the PBS group and the NC8 / pSIP-409-pgsA' group; there were no significant differences between the inactivated virus group and the NC8 / pSIP-409-pgsA'-HA1-AT12 group, but the inactivated virus group was stronger than the NC8 / pSIP-409-pgsA'-HA1-AT12 group; while... Figure 14 As shown, CD8 in chicken spleen T cells + Level and CD4 + The levels are in the opposite state.

[0116] 1.4.5.6 Detection of Toxin Shedding Using Immunotherapy

[0117] On day 5 post-infection, swabs were taken from the throat and cloaca of chickens. These swabs were mixed and then inoculated into the allantoic cavity of five 10-day-old SPF chicken embryos (0.2 ml per embryo). The embryos were incubated at 37°C for 120 hours, and the HA titer of the embryonic fluid was determined. Figure 15 As shown, the PBS group and the NC8 / pSIP-409-pgsA' group had the highest number of positive virus results, and the NC8 / pSIP-409-pgsA'-HA1 group had more positive virus results than the NC8 / pSIP-409-pgsA'-HA1-AT12 group.

[0118] Nucleotide / amino acid sequence information involved in this invention

[0119] The amino acid sequence of AT12-FITC:

[0120] AWSFACKTANGT (SEQ ID NO.1)

[0121] Nucleotide sequence of AT12-FITC:

[0122] GCCTGGTCATTCGCCTGTAAAACCGCCAATGGTACA (SEQ ID NO.2)

[0123] The amino acid sequence of HG21-FITC:

[0124] HNDYPETITDYVTLQRGSAYG (SEQ ID NO.3)

[0125] The nucleotide sequence of HG21-FITC:

[0126] CATAACGATTATCCGGAAACATTACAGACTATGTCACACTGCAACGAGGCTCGGCTTATGGC (SEQ ID NO.4)

[0127] The amino acid sequence of GW31-FITC:

[0128] GTVKYSGSSYPFPTTSETPRVVYNSRTDKPW (SEQ ID NO.5)

[0129] Nucleotide sequence of GW31-FITC:

[0130] GGGACCGTAAAATATAGTGGCAGTAGCTATCCATTTCCTACCACCAGCGAAACGCCGCCGTTGTTTATAATTCGAGAACGGATAAGCCGTGG (SEQ ID NO.6)

[0131] Amino acid sequence of avian influenza virus H10N3 HA1:

[0132] LDKICLGHHAVANGTIVKTLTNEQEEVTNATETVESKGLNKLCMKGRNHKDLGNCHPIGMLIGTPACDLHLTGTWDTLIERENAIAYCYPGATINEEALRQKIMESGGISKISTGFTYGSSINSAGTTKACMRNGENSFYAELKWLVSKNKGQNFPQTTNTYRNTDTAEHLIMWGIHHPSSTQEKNDLYGTQSLSISVGSSTYYSNFVPVVGARPRVNGQSSRIDFHWTLVQPGDNITFSHNGGLIAPSRVSKLIGRGLGIQSDAPIDNNCESKCFWREGSINTRLPFQNLSPRTVGQCPKYVNKKSLMLATGMRNVPEIIQGR (SEQ ID NO.7)

[0133] Nucleotide sequence of avian influenza virus H10N3 HA1:

[0134] CTTGATAAGATCTGCCTGGGACATCATGCAGTGGCCAATGGGACCATTGTAAAGACTCTCACAAATGAACAGGAAGAGGTGACAAATGCTACTGAGACAGTGGAGAGCAAAGGCCTAAACAAATTATGTATGAAGGGAAGGAACCATAAAGACCTGGGCAACTGCCATCCAATAGGAATGCTAATAGGAACACCAGCTTGTGACCTGCACCTTACAGGGACATGGGACACTCTCATTGAGCGAGAAAATGCTATTGCTTATTGCTACCCTGGAGCTACTATAAATGAAGAAGCACTGAGGCAGAAAATAATGGAAAGTGGGGGAATCAGCAAAATAAGCACCGGATTTACTTATGGATCTTCCATAAATTCAGCCGGGACCACTAAAGCATGCATGAGAAATGGAGAAAATAGCTTTTATGCAGAGCTTAAGTGGCTAGTATCAAAGAACAAGGGACAAAATTTCCCTCAGACCACGAACACTTACAGAAACACAGACACGGCTGAACATCTCATAATGTGGGGAATTCATCACCCCTCTAGCACTCAAGAGAAGAATGACTTGTATGGGACACAATCACTGTCCATATCAGTCGGAAGTTCCACTTACTATAGCAATTTTGTACCAGTTGTTGGAGCAAGACCCCGGGTCAATGGACAGAGTAGCAGAATCGAYTTTCACTGGACATTGGTACAGCCAGGTGATAATATCACCTTCTCACACAATGGGGGCCTGATAGCACCGAGCCGAGTTAGCAAATTAATTGGGAGAGGCTTGGGGATTCAATCTGATGCACCAATAGACAATAATTGTGARTCCAAATGTTTTTGGAGAGAAGGTTCCATAAACACAAGGCTTCCCTTTCAGAATTTGTCACCAAGAACAGTTGGCCAATGTCCTAAATATGTGAACAAAAAGAGCTTGATGCTTGCAACAGGGATGAGAAACGTGCCAGAGATAATACAGGGGAGA(SEQ IDNO.8)

[0135] The amino acid sequence of truncated poly-γ-glutamate synthase A:

[0136] GKKELSFHEKLLKLTKQQKKKTNKHVFIAIPIVFVLMFAFMWAGKAETPKVKTYSDDVLSASFVGDIMMGRYVEKVTEQKGADSIFQYVEPIFRAS DYVAGNFENPVTYQKNYKQADKEIHLQTNKESVKVLKDMNFTVLNSANNHAMDYGVQGMKDTLGEFAKQNLDIVGAGYSLSDAKKKISYQKV (SEQ ID NO.9)

[0137] The nucleotide sequence of truncated poly-γ-glutamate synthase A:

[0138] (SEQ ID NO.10)

[0139] H10N3HA1-AT12-F:

[0140] AGTTACCAGAAAGTGTCTAGATCTAGAATGCTTGATAAGATCTGCCT (SEQ ID NO.11)

[0141] H10N3HA1-AT12-R:

[0142] ACGTGCTGTAATTTGAAGCTTAGCTTCTATGTACCATTGGCGG (SEQ ID NO.12)

[0143] H10N3HA1-F:

[0144] AGTTACCAGAAAGTGTCTAGATCTAGAATGCTTGATAAGATCTGCCT (SEQ ID NO.13)

[0145] H10N3HA1-R:

[0146] ACGTGCTGTAATTTGAAGCTTAAGCTTCTATCTCCCCTGTATTATCTC (SEQ ID NO.14)

[0147] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant lactic acid bacteria vaccine, characterized in that, The recombinant lactic acid bacteria vaccine contains at least recombinant Lactobacillus plantarum, which expresses at least a dendritic cell targeting peptide and an avian influenza virus HA1 antigen protein; wherein the amino acid sequence of the dendritic cell targeting peptide is shown in SEQ ID NO.1, and the amino acid sequence of the avian influenza virus HA1 antigen protein is shown in SEQ ID NO.7; The recombinant Lactobacillus plantarum also expresses truncated poly-γ-glutamate synthase A, the amino acid sequence of which is shown in SEQ ID NO.9; The preparation method of recombinant lactic acid bacteria vaccine includes constructing recombinant Lactobacillus plantarum. The method of constructing recombinant Lactobacillus plantarum includes: constructing a recombinant expression vector containing a gene encoding a dendritic cell targeting peptide and an avian influenza virus HA1 antigen protein, and transforming the recombinant expression vector into Lactobacillus plantarum to obtain the vaccine. The method for constructing the recombinant expression vector includes: PCR amplification was performed using primers SEQ ID NO.11 and SEQ ID NO.12 to obtain a gene fragment fused with dendritic cell targeting peptide and avian influenza virus HA1 antigen protein. The gene fragment was then ligated to an expression vector, which was a plasmid, specifically the pSIP-409 plasmid loaded with a truncated poly-γ-glutamic acid synthase A encoding gene. The gene fragment, from the 5' end to the 3' end, encodes the avian influenza virus HA1 antigen protein and the dendritic cell targeting peptide, respectively.

2. The recombinant lactic acid bacteria vaccine as described in claim 1, characterized in that, The recombinant lactic acid bacteria vaccine is an oral formulation.

3. The method for preparing the recombinant lactic acid bacteria vaccine according to any one of claims 1-2, characterized in that, The preparation method includes constructing recombinant Lactobacillus plantarum. The method for constructing recombinant Lactobacillus plantarum includes: constructing a recombinant expression vector containing a gene encoding a dendritic cell targeting peptide and an avian influenza virus HA1 antigen protein, and transforming the recombinant expression vector into Lactobacillus plantarum to obtain the product. The method for constructing the recombinant expression vector includes: PCR amplification was performed using primers SEQ ID NO.11 and SEQ ID NO.12 to obtain a gene fragment fused with dendritic cell targeting peptide and avian influenza virus HA1 antigen protein. The gene fragment was then ligated to an expression vector, which was a plasmid, specifically the pSIP-409 plasmid loaded with a truncated poly-γ-glutamic acid synthase A encoding gene.

4. The preparation method according to claim 3, characterized in that, The nucleotide sequence of the gene encoding the dendritic cell targeting peptide is shown in SEQ ID NO.2; The nucleotide sequence of the gene encoding the HA1 antigen protein of avian influenza virus is shown in SEQ ID NO.

8.

5. The preparation method according to claim 3, characterized in that, The starting strain, *Lactobacillus plantarum*, was *Lactobacillus plantarum* NC8.

6. The use of the recombinant lactic acid bacteria vaccine according to any one of claims 1-2 in the preparation of a drug for preventing H10N3 subtype avian influenza virus, wherein the avian influenza virus drug is administered to chickens.

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