Recombinant lactic acid bacteria for preparing PDCoV oral live vector vaccine as well as construction method and application of recombinant lactic acid bacteria

Abstract

The invention discloses recombinant lactic acid bacteria for preparing a PDCoV oral live vector vaccine as well as a construction method and application of the recombinant lactic acid bacteria, and relates to the technical field of biology. The construction method of the recombinant lactic acid bacteria comprises the following steps: cloning and connecting a coding gene of a PDCoV-S protein to an expression vector, and converting lactobacillus plantarum to prepare the recombinant lactic acid bacteria of which the surface displays the PDCoV-S protein, the amino acid sequence of the PDCoV-S protein is as shown in SEQ ID NO. 2. The recombinant lactic acid bacteria provided by the invention have good tolerance in different environments, are safe and free of toxic effects, can effectively cause systemic immune response and intestinal local mucosa immune response, and have good immunogenicity. The invention provides powerful technical support for developing a PDCoV oral live vector vaccine, and has important significance for preventing and controlling PDCoV infection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a recombinant lactic acid bacteria for preparing an oral live vector vaccine for PDCoV, its construction method, and its application. Background Technology

[0002] Porcine deltacoronavirus (PDCoV) is a newly emerging and important enteropathogenic coronavirus in pigs, causing severe enteritis and dehydration. PDCoV infects pigs of all ages, but primarily newborn piglets. Studies indicate that PDCoV has a broad host tropism, and chickens, young turkeys, and cattle can also be infected. A 2021 report first demonstrated that PDCoV can infect humans, making it a potential zoonotic pathogen. The PDCoV genome is 25.4 kb in length, the smallest among coronavirus genomes, with a typical gene sequence of 5'UTR-ORF1a / 1b-SEM-NS6-N-NS7-3'UTR. PDCoV encodes four structural proteins and four non-structural proteins. The spike glycoprotein (S) forms a trimeric crown-like structure, mediating viral fusion and entry into the host cell membrane, and is a major surface protein of PDCoV and an important target of the host's humoral immunity.

[0003] Viruses evolve under selective pressure, and the overuse of antiviral drugs may lead to increased drug resistance. Therefore, the best solution is to develop PDCoV-specific antiviral drugs and vaccines. While some progress has been made in PDCoV vaccines, no fully approved vaccine has yet been identified. Mucosa, as the body's first line of defense, plays a crucial role in preventing viral infections transmitted via the fecal-oral route. Developing safe and effective novel mucosal immunotherapies is of great significance for controlling PDCoV infection. Summary of the Invention

[0004] The purpose of this invention is to provide a recombinant lactic acid bacteria for preparing an oral live vector vaccine for PDCoV, its construction method, and its application, thereby addressing the problems existing in the prior art. This recombinant lactic acid bacteria exhibits good tolerance in different environments, is safe and non-toxic, and can effectively induce systemic immune responses and local intestinal mucosal immune responses, demonstrating good immunogenicity.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for constructing recombinant lactic acid bacteria for preparing an oral live vector vaccine against PDCoV, comprising the steps of cloning and ligating the coding gene of PDCoV-S protein into an expression vector and transforming it into Lactiplantibacillus Plantarum to prepare the recombinant lactic acid bacteria displaying the PDCoV-S protein on its surface;

[0007] The amino acid sequence of the PDCoV-S protein is shown in SEQ ID NO.2.

[0008] Furthermore, the expression vector is pSIP409-pgsA'-EGFP.

[0009] Furthermore, the Lactobacillus plantarum is Lactobacillus plantarum NC8.

[0010] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0011] The present invention also provides a recombinant lactic acid bacteria for preparing an oral live vector vaccine for PDCoV, which is constructed according to the above-described construction method.

[0012] The present invention also provides the application of the above-mentioned recombinant lactic acid bacteria in the preparation of PDCoV vaccines.

[0013] Furthermore, the PDCoV vaccine is an oral live vector PDCoV vaccine.

[0014] The present invention also provides an oral live vector vaccine for PDCoV, comprising the above-mentioned recombinant lactic acid bacteria.

[0015] Furthermore, the PDCoV oral live vector vaccine also includes vaccine preparation excipients.

[0016] Furthermore, the vaccine preparation excipients include protective agents, stabilizers, or immune adjuvants.

[0017] The present invention discloses the following technical effects:

[0018] This invention utilizes molecular cloning technology to clone the coding gene of the PDCoV-S protein, ligate it into an expression vector, and transform it into *Lactobacillus plantarum*, thus preparing recombinant lactic acid bacteria displaying the PDCoV-S protein on their surface. This recombinant lactic acid bacteria exhibits good tolerance in different environments, is safe and non-toxic, and can effectively induce systemic immune responses and local intestinal mucosal immune responses, demonstrating good immunogenicity. This invention provides strong technical support for the development of oral live vector vaccines for PDCoV and is of great significance for the prevention and control of PDCoV infection. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 A schematic diagram of the recombinant plasmid construction route;

[0021] Figure 2 This is a schematic diagram of a mouse immunization program;

[0022] Figure 3 The image shows the construction and identification results of the pSIP409-pgsA'-PDCoV-S recombinant plasmid. In the image, A represents the amplification and identification results of the target fragment (M: 5000 Marker, 1: negative control, 2: PDCoV-S target fragment); B represents the double enzyme digestion identification results of the empty vector plasmid (M: 5000 Marker, 1: double enzyme digestion of the empty vector plasmid); and C represents the PCR identification results of the recombinant plasmid (M: 5000 Marker, 1: negative control, 2: PCR identification of the recombinant bacteria).

[0023] Figure 4 Figure 1 shows the identification results of recombinant Lactobacillus plantarum; where A is the PCR verification result of the NC8-pSIP409-pgsA'-PDCoV-S recombinant plasmid (M: 5000 Marker, 1: negative control, 2: PCR verification of recombinant plasmid); B is the double enzyme digestion identification result of the NC8-pSIP409-pgsA'-PDCoV-S recombinant plasmid (M: 5000 Marker, 1: double enzyme digestion of NC8-pSIP409-pgsA'-PDCoV-S).

[0024] Figure 5 Figure showing the results of recombinant protein expression validation;

[0025] Figure 6 The results are shown in the figure for detecting the expression of the target protein under different induction conditions;

[0026] Figure 7 The results are shown in the figure for detecting the expression of the target protein at different culture times;

[0027] Figure 8 The image shows the results of the passage stability assessment of recombinant lactic acid bacteria.

[0028] Figure 9 This is a growth curve of recombinant lactic acid bacteria;

[0029] Figure 10The figure shows the results of the recombinant lactic acid bacteria tolerance test; where A represents the results of the heat resistance analysis of the recombinant lactic acid bacteria; B represents the results of the acid resistance analysis of the recombinant lactic acid bacteria; and C represents the results of the bile salt resistance analysis of the recombinant lactic acid bacteria.

[0030] Figure 11 This is a graph showing the weight monitoring of immunized mice;

[0031] Figure 12 To detect CD3 in mouse spleen by flow cytometry + CD8 + Graph showing the results of T cell subsets;

[0032] Figure 13 To detect CD3 in mouse spleen by flow cytometry + CD4 + Graph showing the results of T cell subsets;

[0033] Figure 14 To detect B220 in mouse spleen by flow cytometry + CD19 + Results of B cell subsets;

[0034] Figure 15 To detect CD3 in mouse spleen by flow cytometry - CD49 + Resulting graph of NK cell subsets;

[0035] Figure 16 The figure shows the results of detecting the transcriptional levels of immune factors IL-4 (A) and IFN-γ (B) in mouse intestinal tissue;

[0036] Figure 17 The graph shows the results of detecting the secretion levels of immune factors IL-4 (A) and IFN-γ (B) in mouse serum;

[0037] Figure 18 The figure shows the results of serum neutralization titer detection in mice immunized with recombinant lactic acid bacteria. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] Example 1

[0044] 1. Materials and Methods

[0045] 1.1 Materials

[0046] 1.1.1 Strains, Viruses, and Laboratory Animals

[0047] The *Lactobacillus plantarum* NC8 and pSIP409-pgsA'-EGFP vector were provided by Professor Wang Chunfeng of Jilin Agricultural University and have been published in the literature "Cai Ruopeng. Construction and verification of recombinant *Lactobacillus plantarum* expression system based on pgsA as a surface display element [D]. Jilin Agricultural University, 2015"; the PDCoV strain was preserved in the laboratory of the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences, and 6-week-old female BALB / c mice were purchased from the Comparative Medicine Center of Yangzhou University.

[0048] 1.1.2 Main Reagents

[0049] Mouse spleen lymphocyte isolation kit was purchased from Tianjin Haoyang Biological Products Co., Ltd.; FITC anti-mouse CD3e, PE anti-mouse CD4, APC anti-mouse CD8a, FITC anti-mouse CD19, and APC anti-mouse CD49b were all purchased from Biolegend; Percp-cy5.5 anti-mouse B220 was purchased from BD Biosciences; MRS broth and modified MRS medium were purchased from Guangdong Huankai Microbial Technology Co., Ltd.; FastDigest Xba I and FastDigest Hind III were purchased from Thermo Fisher Scientific.

[0050] 1.1.3 Main Instruments

[0051] The Centrifuge 5424R refrigerated centrifuge was purchased from Eppendorf. The Mastercycler X50a gradient PCR amplification system was purchased from Eppendorf. The DK-8D water bath was purchased from Shanghai Jinghong Experimental Equipment. The BPX-272 constant temperature incubator was purchased from Suzhou Jiangdong Precision Instrument Co., Ltd. The GC-100 metal bath was purchased from Hangzhou Youning Instrument. The BIO-RADXR gel imaging system and the Sub-Cell nucleic acid electrophoresis system were both purchased from Bio-Rad Laboratories, USA. The LSM 880 laser confocal microscope was purchased from ZEISS. The ZQWY-200 benchtop constant temperature shaker was purchased from Shanghai Anting Scientific Instrument Factory.

[0052] 1.2 Methods

[0053] 1.2.1 Primer Design

[0054] Based on the target gene and vector sequence information, primers for amplifying the target fragment were designed. The expected amplification size of the target fragment is 2049 bp (nucleotide sequence as shown in SEQ ID NO.1; amino acid sequence of the protein it encodes as shown in SEQ ID NO.2). Simultaneously, universal primers for detecting the recombinant plasmid, as well as primers for detecting mouse β-actin, IL-4, and IFN-γ, were designed on the vector. The primer sequences are shown in Table 1. All primers were synthesized by Nanjing Qingke Biotechnology Co., Ltd.

[0055] Table 1 Primer Sequences

[0056]

[0057] Note: The underlined locations in this table are restriction enzyme sites.

[0058]

[0059] SEQ ID NO.2: MDDLLDLLTFPGAHRFLHKLTSNSSSLYSRANNFDVGVLPGYATKNVNLFSPLTNSTLPINGLHRSYQPLMLNCFTKITNHTLSMYLLPSDVQTYSCGGAMVKYQTHDAVRIILDLTATDHISVEVVGQHGENYVFVCSEQFTYTTALHKSTFFSLNSELYCFTNNTYLGILPPDLTDFTVYRTGQFYANGYLLGTLPITVNYVRLYRGQLAANSAHFALANLTDTLITLTNTTISQITYCDKSVVDSIACQRSSHEVEDGFYSDPKSAVRARQRTIVTLPKLPELEVVQLNISAHMDFGEARLDSVTINGNTSYCVTKPYFRLETNFMCTGCTMNLRTDTCSFDLSAVNNGMSFSQFCLSTESGACEMKIIVTYVWNYLLRQRLYVTAVEGQTHTGTTSVHATDTSSVITDVCTDYTIYGVSGTGIIKPSDLLLHNGIAFTSPTGELYAFKNITTGKTLQVLPCETPSQLIVINNTVVGAITSSNSTENNRFTTTIVTPTFFYSTNATTFNCTKPVLSYGPISVCSDGAIVGTSTLQNTRPSIVSLYDGEIEIPSAFSLSVQTEYLQVQAEQVIVDCSQYVCNGNSRCLQLLAQYTSACSNIEAALHSSAQLDSREIINMFQTSTQSLQLANITNFKGDYNFSSIITPRIGGRSAIEDLLFNKVVTSGLGTHHHHHH.

[0060] 1.2.2 Recombinant plasmid construction

[0061] Using the synthesized target fragment as a template, the target fragment was amplified and purified using PDCoV-SF / R primers. Both the empty vector pSIP409-pgsA'-EGFP and the purified target fragment were double-digested with Xba I / Hind III. The digested target fragment and linear vector were recovered, and the target gene and linear vector were ligated using T4 ligase. The resulting cells were transformed into E. coli EPI400 competent cells, plated on LB solid medium containing erythromycin, and cultured for 16 h. Single colonies were picked, and positive colonies were detected and screened using universal primers. Plasmids of the correctly identified positive clones were extracted and sequenced for verification. The correctly identified plasmid was named pSIP409-pgsA'-PDCoV-S. The plasmid construction route is described in [link to plasmid construction instructions]. Figure 1 .

[0062] 1.2.3 Construction of recombinant lactic acid bacteria

[0063] NC8 competent cells were prepared. The correctly identified pSIP409-pgsA'-PDCoV-S plasmid was electroporated into the NC8 competent cells at 3000 V, 200 Ω, and 3-5 ms. The electroporated competent cells were then transferred to MRS medium containing 0.05 mol / L sucrose and cultured at 37℃ for 2.5 h. An appropriate amount of bacterial culture was spread on MRS medium containing erythromycin resistance and cultured anaerobically for 16 h. Single colonies were picked and propagated. The cells were treated with 0.5 g / L lysozyme bacteria for 1 h. The plasmid was extracted and identified by PCR and sequencing, yielding the correctly identified recombinant lactic acid bacteria.

[0064] Meanwhile, the empty pSIP409-pgsA'-PDCoV-S plasmid was electroporated into NC8 competent cells as a control for subsequent experiments.

[0065] 1.2.4 Validation and Optimization of Recombinant Lactic Acid Bacteria Expression

[0066] 1.2.4.1 Validation of Recombinant Lactic Acid Bacteria Protein Expression

[0067] Recombinant lactic acid bacteria were inoculated at a 1% inoculum in 1 mL of fresh MRS broth and cultured overnight with the addition of inducible peptides during the logarithmic phase. The recombinant lactic acid bacteria were washed three times with PBS, resuspended in 100 μL of PBS, and 25 μL of 5× Loading Buffer was added. The culture was incubated in a metal bath at 100 °C for 10 min. Protein expression was verified by Western blot using anti-His-tagged mouse polyclonal antibody as the primary antibody and mouse monoclonal antibody as the secondary antibody.

[0068] 1.2.4.2 Optimization of inducible expression and passage stability of recombinant lactic acid bacteria

[0069] During the logarithmic growth phase of recombinant lactic acid bacteria, 10, 50, 100, and 200 ng / mL of SppIP inducible peptide were added, and the bacteria were cultured for 4, 8, 12, and 16 h, respectively. Western blot analysis was performed as described above to select the optimal induction conditions. Recombinant lactic acid bacteria were continuously passaged under the optimal induction conditions, and the stability of the PDCoV-S protein expressed by the recombinant lactic acid bacteria was evaluated by Western blot analysis.

[0070] 1.2.5 Physicochemical properties of recombinant lactic acid bacteria

[0071] 1.2.5.1 Determination of growth curve

[0072] Resuscitated recombinant lactic acid bacteria were inoculated into MRS broth at a 1% inoculum and cultured anaerobically at 37°C. OD values ​​were measured using a UV spectrophotometer every 2 hours. 600nm Values, with incubation time as the x-axis, OD 600nm The value is used as the ordinate to plot the growth curve.

[0073] 1.2.5.2 Acid resistance, heat resistance and bile salt resistance tests

[0074] The pH of MRS broth medium was adjusted to 2.5 using HCl. One mL of bacteria induced overnight was centrifuged, and the supernatant was discarded. The culture was then treated at different temperatures (37℃, 50℃) for 20 min, at different pH values ​​(pH=2.5, 7.0) for 30 min, and at different concentrations of porcine bile salts (0%, 0.2%, 0.5%) for 2 h. All treated bacteria were plated onto MRS solid medium and cultured for 16 h. Colony counts were recorded, and changes in protein activity were detected by Western blot.

[0075] 1.2.6 Oral immunization of mice with recombinant lactic acid bacteria

[0076] Balb / c mice were randomly divided into three groups of four each: a PBS group, an empty vector group (Vector group), and a recombinant lactic acid bacteria group (PDCoV-S group). Immunization was performed via gavage. The PDCoV-S and Vector groups were administered recombinant lactic acid bacteria (recombinant lactic acid bacteria transformed with the pSIP409-pgsA'-PDCoV-S plasmid as constructed in 1.2.3 or recombinant lactic acid bacteria transformed with the empty vector plasmid) via gavage. The gavage volume per mouse was 1 × 10⁻⁶. 9 CFU. The PBS group served as a blank control, receiving an equal volume of sterile PBS solution via gavage. The immunization cycle consisted of three immunizations, each lasting three days. Mice were observed for their mental state and body weight daily. Serum was collected 28 and 42 days after immunization, and spleen lymphocytes and small intestinal tissue were collected 14 days after the third immunization. The immunization procedure is detailed below. Figure 2 .

[0077] 1.2.7 Detection of the effect of orally immunizing mice with recombinant lactic acid bacteria

[0078] Isolate mouse spleen lymphocytes, and use flow cytometry to detect the proportions of T, B, and NK cells. Weigh 0.2 g of small intestinal tissue, grind it, and extract RNA. Relatively quantify the transcriptional levels of IL-4 and IFN-γ factors; use IL-4 and IFN-γ ELISA kits to detect the contents of immune factors in the serum; and use a neutralization test to detect the level of neutralizing antibodies in the serum.

[0079] 1.2.8 Data statistical analysis

[0080] Use GraphPad 9.5 for statistical analysis. The data are expressed as the mean ± standard deviation (SD) of at least three replicates. A P-value less than 0.05 is defined as the threshold for statistical significance. 0.01 < P < 0.05 is indicated by one asterisk *, 0.001 < P < 0.01 is indicated by two asterisks **, and P < 0.001 is indicated by three asterisks ***.

[0081] 2 Experimental results

[0082] 2.1 Construction and identification of the pSIP409-pgsA’-PDCoV-S recombinant plasmid

[0083] Use PDCoV-S fragment-specific primers to amplify the target fragment. The results show that the electrophoretic verification band is about 2049 bp, which is consistent with the expected size ( Figure 3 in A). Double digest the empty plasmid pSIP409-pgsA’-EGFP with Xba Ⅰ / Hind Ⅲ. The results show that the EGFP fragment is successfully cut off, and a linear vector is obtained ( Figure 3 in B). Recover and purify the target fragment and the linear vector, and use T4 ligase to construct the recombinant plasmid. Extract the plasmid of monoclonal colonies for PCR verification. The results show that the amplified fragment is 2402 bp, and the sequencing verification shows that the sequence is correct, proving that the recombinant plasmid is successfully constructed ( Figure 3 in C).

[0084] 2.2 Construction and identification of recombinant Lactobacillus plantarum

[0085] Electrotransform the verified recombinant plasmid into Lactobacillus plantarum NC8. Extract the plasmid of monoclonal colonies for PCR verification. The results show that the detected band size is about 2402 bp ( Figure 4 in A), which is consistent with the expectation. Double digest and identify the recombinant lactic acid bacteria plasmid. The results show that the band sizes are all consistent with the expectation ( Figure 4 in B). The sequencing alignment shows that the sequence is correct. Name the recombinant plasmid NC8-pSIP409-pgsA’-PDCoV-S.

[0086] 2.3 Expression Validation of Recombinant Lactobacillus plantarum

[0087] To verify protein expression, Western blot analysis was performed on the recombinant lactic acid bacteria protein. The results showed that the recombinant lactic acid bacteria had a distinct band around 105 kDa, while the empty vector control did not show a protein band of the target size, proving that the PDCoV-S protein was successfully expressed in the recombinant lactic acid bacteria. Figure 5 ).

[0088] To achieve better protein expression in recombinant lactic acid bacteria, the induction conditions were optimized. Results showed that the highest protein expression level was observed in recombinant lactic acid bacteria cultured for 12 hours after the addition of 50 ng / mL inducible peptide during the logarithmic growth phase. Figures 6-7 To verify the stability of recombinant lactic acid bacteria protein expression, the recombinant lactic acid bacteria were passaged for 10 consecutive generations, and Western blot analysis was performed. The results showed that the expression of PDCoV-S protein in recombinant lactic acid bacteria was relatively stable with increasing passage number. Figure 8 ).

[0089] 2.4 Physicochemical properties of recombinant lactic acid bacteria

[0090] 2.4.1 Growth Curve

[0091] With time as the horizontal axis, OD 600 The values ​​are plotted on the ordinate to create a growth curve for lactic acid bacteria. For example... Figure 9 As shown, the growth curves of recombinant lactic acid bacteria and Lactobacillus plantarum containing empty vector plasmids are relatively consistent. They enter the logarithmic growth phase at 2-3 h and reach the end of the logarithmic growth phase at 10-12 h. The subsequent induction time for expression of recombinant lactic acid bacteria is selected at 2-3 h.

[0092] 2.4.2 Results of acid resistance, heat resistance, and bile salt resistance

[0093] Recombinant lactic acid bacteria were heat-treated at 50℃ for 20 min. The survival rate of the empty vector control group was 95.9%, while the survival rate of the PDCoV-S group was 89.9%. Figure 10 (A). After adjusting the pH to simulate a gastric acid environment for 30 min, the survival rate of the empty control group was 85.5%, and the survival rate of the PDCoV-S group was 81.4%. Figure 10 (B) Simulating the small intestinal environment, recombinant lactic acid bacteria were treated for 2 h in a medium containing 0.2% and 0.5% porcine bile salts. The survival rates of the empty control group were 77.9% and 22.5%, respectively, while the survival rates of the PDCoV-S group were 20.4% and 11.9%, respectively. The activity decreased, but the number of viable bacteria remained within the same order of magnitude. Figure 10 (C)

[0094] Western blot analysis confirmed that the recombinant protein was stable under heat and simulated gastric acid conditions, with no significant difference between the treated and normal groups. Under different concentrations of bile salts, the protein activity decreased with decreasing viable bacterial count, but still maintained a certain level of protein activity.

[0095] 2.5 Immunogenicity evaluation of recombinant lactic acid bacteria

[0096] 2.5.1 Safety Evaluation

[0097] Mice were prepared by gavage with recombinant lactic acid bacteria according to the immunization program. The mice's mental state, fur, and body weight changes were observed daily. Results are shown below. Figure 11 The results showed that the weight of mice immunized with recombinant lactic acid bacteria increased steadily, with no significant change compared to the control group. During the immunization process, the general health indicators of the mice, such as their mental state, remained normal, proving that the recombinant lactic acid bacteria were safe and non-toxic to the mice.

[0098] 2.5.2 Flow cytometry detection

[0099] Fourteen days after the third immunization of mice, spleen lymphocytes were isolated and prepared into a suspension. CD3 levels were detected by flow cytometry. + CD4 + T cells, CD3 + CD8 + T cells, B220 + CD19 + B cells and CD3 - CD49 + NK lymphocyte count, results are shown in Figures 12-15 The results showed that, compared with the PBS group and the recombinant lactic acid bacteria group containing empty vector (Vector group), the number of T cells and B cells in the recombinant lactic acid bacteria NC8-pSIP409-pgsA'-PDCoV-S (PDCoV-S group) was significantly increased (P<0.05), while the number of NK cells did not change significantly.

[0100] 2.5.3 Detection of mouse immune factors

[0101] Immunoglobulin tissue was collected, and the secretion index and transcription level of immune factors in serum and intestinal tissue were detected using an ELISA kit. Results are shown below. Figures 16-17 The results showed that, compared with the control group, the transcriptional levels of IL-4 and IFN-γ factors in the intestinal tissue of the immunized mice were significantly increased (P<0.05), while the serum secretion levels of IL-4 and IFN-γ factors were significantly increased compared with the PBS group, but there was no significant difference compared with the Vector group.

[0102] 2.5.4 Detection of Neutralizing Antibody Levels

[0103] Mice were immunized with recombinant lactic acid bacteria. Serum samples were collected on days 14, 28, and 42. The neutralization antibody titer in the immunized mouse serum was detected using a neutralization assay. Results are shown below. Figure 18 The results showed that the neutralizing antibody titer increased 14 days after the second immunization with recombinant lactic acid bacteria, and the antibody titer was significantly higher than that of the PBS group 14 days after the third immunization, with the highest neutralizing antibody titer reaching 1:32.

[0104] In summary, this invention selects Lactobacillus plantarum NC8 as the antigen protein expression vector and constructs a recombinant lactic acid bacterium that anchors the expression of PDCoV-S protein on the surface of NC8, thereby obtaining an economical, simple and efficient live vector vaccine.

[0105] This invention conducted an immunization experiment on mice via oral administration of recombinant lactic acid bacteria. Compared with the control group, the immunized mice showed stable weight gain and no toxic effects. After three immunizations, the level of neutralizing antibodies significantly increased, indicating that the recombinant lactic acid bacteria constructed in this invention can effectively induce a systemic immune response against specific antigens. Antibodies have been shown to play a key role in preventing coronavirus infection, and the immune response of T cells is indispensable for virus clearance, reducing disease progression, and improving prognosis. T cells are important guardians of the immune system in the fight against infectious diseases. When the T cell receptor (TCR) recognizes the specific peptide presented on the major histocompatibility complex (MHC-II) expressed by antigen-presenting cells (APCs), T cells are activated and can differentiate into two cell subsets, Th1 and Th2, which produce the marker cytokines IFN-γ and IL-4, respectively. This invention isolated mouse spleen lymphocytes and found that, compared with the control group, the PDCoV-S immunization group induced CD4+. + CD8 + T cells and B220 + CD19 + B cells were significantly increased, while CD3 - CD49 + No significant changes were observed in NK cells. Simultaneously, the transcriptional levels of small intestinal immune factors IL-4 and IFN-γ were measured. Compared with the control group, the transcriptional levels of immune factors in the immunized group were significantly increased, while the levels of immune factors secreted in serum were slightly increased, but not significantly. During T cell immune responses, epitope-specific cell populations are activated, acquiring and expressing multiple cytokines and migrating to various tissues to activate downstream target cells. This invention demonstrates that the recombinant lactic acid bacteria vaccine simultaneously activates CD4+. + CD8 + T cells and B220 + CD19 + B cell immune response, which induces a strong local immune reaction in the gut.

[0106] Compared to traditional injection immunization methods, oral live vector vaccination is simpler and safer, reducing stress responses in animals. Furthermore, recombinant lactic acid bacteria vaccines are inexpensive, suitable for mass production, and effectively stimulate the intestinal mucosa to produce specific antibodies. Lactic acid bacteria themselves can enhance the body's immunity and promote mucosal immunity, offering significant advantages over traditional vaccines.

[0107] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing recombinant lactic acid bacteria for the preparation of PDCoV oral live vector vaccine, characterized by, The step of preparing the recombinant lactic acid bacteria displaying PDCoV-S protein on the surface comprises cloning and connecting the coding gene of PDCoV-S protein to an expression vector and transforming Lactiplantibacillus Plantarum. The amino acid sequence of the PDCoV-S protein is shown as SEQ ID NO.

2.

2. The construction method of claim 1, wherein, The expression vector is pSIP409-pgsA'-EGFP.

3. The construction method of claim 1, wherein, The Lactiplantibacillus Plantarum is Lactiplantibacillus Plantarum NC8.

4. The construction method of claim 1, wherein, The nucleotide sequence of the coding gene is shown as SEQ ID NO.

1.

5. A recombinant lactic acid bacteria for preparing PDCoV oral live vector vaccine, which is constructed by the construction method according to any one of claims 1-4.

6. Use of the recombinant lactic acid bacteria according to claim 5 in the preparation of PDCoV vaccine.

7. Use according to claim 6, characterized in that, The PDCoV vaccine is PDCoV oral live vector vaccine.

8. A PDCoV oral live vector vaccine, characterized in that, The recombinant lactic acid bacteria according to claim 5.

9. The PDCoV oral live-carrier vaccine according to claim 8, characterized in that, The PDCoV oral live vector vaccine further comprises vaccine preparation adjuvant.

10. The PDCoV oral live-carrier vaccine according to claim 9, characterized in that, The vaccine preparation adjuvant comprises protective agent, stabilizer or immunoadjuvant.

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