Recombinant lactobacillus plantarum for expressing porcine epidemic diarrhea virus S1 protein as well as preparation method and application of recombinant lactobacillus plantarum

Abstract

The invention provides recombinant lactobacillus plantarum for expressing porcine epidemic diarrhea virus S1 protein as well as a preparation method and application of the recombinant lactobacillus plantarum, and belongs to the technical field of preparation of porcine epidemic diarrhea virus vaccines. The recombinant lactobacillus plantarum provided by the invention contains a recombinant vector for expressing a PEDV tS1 fusion gene, and the PEDV tS1 fusion gene comprises a PEDV tS1 structural domain, an endogenous signal peptide 1320 and a dendritic cell targeting peptide DCpep. After the recombinant lactobacillus plantarum provided by the invention is used as a mucosal vaccine through nasal dripping, not only can a codon be prevented from being repeatedly optimized, but also strong cross-species immune response can be induced, pathogen specificity sIgA can be generated, mucosal immunity can be effectively activated, and the induced antibody has the activity of neutralizing PEDV (porcine epidemic diarrhea virus). The recombinant lactobacillus plantarum provided by the invention has a treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of porcine epidemic diarrhea virus vaccine preparation technology, specifically relating to a recombinant Lactobacillus plantarum expressing the S1 protein of porcine epidemic diarrhea virus, its preparation method and application. Background Technology

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious disease caused by porcine epidemic diarrhea virus (PEDV) of the Coronaviridae family. The main clinical manifestations are watery diarrhea, vomiting, and dehydration. Currently, widely used PEDV vaccines include live attenuated vaccines and inactivated vaccines, but both have limitations. Because PEDV can be efficiently transmitted through the air, and existing immunization regimens are insufficiently protective, immunized sows or gilts cannot provide adequate milk-derived immunity for their offspring. Traditional intramuscular injection primarily induces systemic immunity but fails to stimulate mucosal immunity, which is crucial for intestinal protection. This deficiency highlights the necessity of designing vaccines that consider both systemic and mucosal immunity. Furthermore, both live attenuated and inactivated vaccines pose potential biosafety risks: live attenuated vaccines may revert to virulence, while inactivated vaccines require adjuvants to enhance immunogenicity, introducing additional safety concerns. It is noteworthy that even using the same antigen, different immunization routes can induce drastically different immune responses, further emphasizing the importance of route optimization. At the same time, some nucleic acid vaccines and viral vector vaccines heavily rely on species-specific codon optimization, hindering cross-species evaluation and impeding their industrial application. Therefore, there is an urgent need to develop novel vaccine strategies and administration routes that can efficiently induce mucosal immunity in order to achieve effective prevention and control of PEDV.

[0003] PEDV primarily infects the intestinal mucosa of piglets, replicating within the epithelial cells of the small and colonic intestines. Studies have shown that antigen-specific secretory IgA (sIgA) induced by mucosal immunity is closely associated with enhanced protection against PEDV infection. Recent research further clarifies that PEDV can be transmitted through the air and invades the intestinal mucosa via the nasal cavity. After inhalation, the virus is first taken up by submucosal dendritic cells (DCs) in the nasal mucosa. Subsequently, DCs carrying the virus migrate to regional lymph nodes, where they transfer the virus to lymphocytes via immune synapses. These virus-carrying lymphocytes disseminate through the lymphatic and circulatory systems, ultimately reaching the intestinal mucosa and transferring the virus to intestinal epithelial cells, leading to clinical pathogenesis. This finding suggests that nasal mucosal immune cells could serve as a key target for vaccine development. Furthermore, there is evidence of airborne transmission for other enteric pathogens such as rotavirus and norovirus, although the mechanisms are not fully elucidated, further highlighting the potential applicability of intranasal immunization against enteric pathogens. Currently, there is a lack of research in this field regarding the preparation of PEDV mucosal vaccines. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a recombinant Lactobacillus plantarum, which, when used as nasal drops, can induce a strong cross-species immune response, produce pathogen-specific sIgA, effectively activate mucosal immunity, and the induced antibody has PEDV neutralizing activity, indicating that the recombinant Lactobacillus plantarum provided by the present invention has therapeutic effects.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a recombinant Lactobacillus plantarum containing a recombinant vector expressing a PEDV tS1 fusion gene. The PEDV tS1 fusion gene includes a PEDV tS1 domain with a nucleotide sequence as shown in SEQ ID NO.1, an endogenous signal peptide 1320 with a nucleotide sequence as shown in SEQ ID NO.2, and a dendritic cell targeting peptide DCpep with a nucleotide sequence as shown in SEQ ID NO.3.

[0006] Preferably, the PEDV tS1 fusion gene has an endogenous signal peptide 1320 attached to the 5' end of the PEDV tS1 domain and a dendritic cell targeting peptide DCpep attached to the 3' end of the PEDV tS1 domain.

[0007] Preferably, the nucleotide sequence of the PEDV tS1 fusion gene is shown in SEQ ID NO.4.

[0008] Preferably, the scaffold carrier of the recombinant vector is a pSIP411 carrier.

[0009] Preferably, the Lactobacillus plantarum is Lactobacillus plantarum LP12.

[0010] The present invention also provides a method for preparing the above-mentioned recombinant Lactobacillus plantarum, comprising the following steps: inserting the PEDV tS1 fusion gene into a backbone vector using a seamless cloning method to obtain a recombinant vector; and transferring the recombinant vector into Lactobacillus plantarum to obtain recombinant Lactobacillus plantarum.

[0011] The present invention also provides the application of the above-mentioned recombinant Lactobacillus plantarum in the preparation of a vaccine for the prevention and treatment of porcine epidemic diarrhea virus infection.

[0012] Preferably, the vaccine is an intranasal vaccine.

[0013] The present invention also provides the application of the above-mentioned recombinant Lactobacillus plantarum in the preparation of a drug for treating swine epidemic diarrhea virus infection.

[0014] The present invention also provides an intranasal vaccine for preventing and treating porcine epidemic diarrhea virus infection, wherein the intranasal vaccine contains the above-mentioned recombinant Lactobacillus plantarum.

[0015] The beneficial effects of this invention are: This invention provides a recombinant *Lactobacillus plantarum* as a mucosal vaccine. As a subunit vaccine, it not only avoids repeated codon optimization but also effectively induces mucosal immunity, rapidly triggering an antiviral response against PEDV infection in piglets. The recombinant *Lactobacillus plantarum* provided by this invention is designed as a cross-species vaccine, using probiotics as a delivery carrier, and can induce both mucosal and systemic immunity. During piglet immunization, intranasal administration for 5 consecutive days followed by challenge achieved 100% protection, indicating that the recombinant *Lactobacillus plantarum* provided by this invention not only functions as a vaccine but also acts as a preventative or therapeutic drug, providing protection without requiring a second immunization at least two weeks later. This invention is the first to propose a recombinant bacterial product suitable for use in piglets. Attached Figure Description

[0016] Figure 1 The construction of a recombinant *Lactobacillus plantarum* strain expressing the PEDV tS1 protein is shown in the diagram. A represents the antigenic structure of the PEDV tS1 protein; B is a schematic diagram of the inserted sequence 1320-PEDV tS1-DCpep; C is a schematic diagram of the recombinant plasmid LP12:PEDV tS1; D shows the gene-specific amplification results of 1320-PEDV tS1-DCpep; E shows the gene-specific amplification results of recombinant *Lactococcus lactis* NZ3900; and F shows the gene-specific amplification results of recombinant *Lactobacillus plantarum*. Figure 2 The following describes the identification of PEDV tS1 protein expression in LP12:PEDV tS1: A) Confirmation of PEDV tS1 protein expression in LP12:PEDV tS1 using an anti-HA-tagged rabbit polyclonal antibody; B) Confirmation of PEDV tS1 protein expression in LP12:PEDV tS1 using an anti-PEDV tS1 mouse monoclonal antibody; C) Imaging under a fluorescence microscope with a 100× objective, 488nm excitation wavelength, and 590nm emission wavelength; D) Evaluation of the display of S1 protein on the surface of LP12:PEDV tS1, using induced LP12:Vector and LP12 as controls. Figure 3 For the optimization and quantitative analysis of recombinant protein expression, A represents the expression effect evaluated by time gradient Western blot; B represents the expression effect evaluated by SppIP concentration Western blot; C represents the expression effect evaluated by temperature Western blot; D represents the standard curve for purifying PEDV protein established by ELISA; and E represents the expression level of the target protein at different time points after induction calculated using the equation of the standard curve. Figure 4Immunological evaluation of LP12:PEDV tS1 in mice, where A is the experimental design and immunization procedure; B is the detection of specific sIgA levels in feces of different groups of mice; C is the sIgA level in bronchoalveolar lavage fluid (BALF) on day 21; D is the sIgA level in nasal lavage fluid (NLF) on day 21; E is the neutralizing activity of sIgA in feces of immunized mice on day 21; statistical significance is marked as follows: P<0.05; P<0.01; P<0.001; P < 0.0001; ns indicates P > 0.05; Figure 5 To characterize mature dendritic cells (DCs) in mesenteric lymph nodes (MLNs) of mice induced by *Lactobacillus plantarum* expressing PEDV tS1, A shows representative flow cytometry analysis of DCs in MLNs of mice from different groups; B and C are respectively... and Subpopulation ratio changes in response to PBS (blue), LP12:Vector (green), and LP12:PEDV tS1 (red) stimulation. P<0.05; Figure 6 Immunological evaluation of LP12:PEDV tS1 in a pig model, where A represents the experimental design and immunization procedure; B and C represent the division of 10 piglets into two groups and the detection of PEDV gene using RT-PCR; D represents the detection of serum PEDV antibody levels by indirect ELISA, with a cutoff value of 1.10; E represents changes in piglet body temperature; F represents changes in body weight; G represents serum-specific IgG levels; H represents specific sIgA levels in nasal lavage fluid (NLF); and I represents specific sIgA levels in feces. P<0.05; Figure 7 This study measured the titer levels of neutralizing antibodies, where A represents the serum-specific neutralizing antibody titer level; B represents the specific neutralizing antibody titer level in nasal lavage fluid (NLF); and C represents the specific neutralizing antibody titer level in feces. P < 0.05, ns means P > 0.05; Figure 8 To demonstrate that LP12:PEDV tS1 provides effective protection against PEDV in a piglet infection model, where A represents the viral challenge and immunization program; and B represents the clinical symptoms of different groups. Figure 9The protective results of LP12:PEDV tS1 in a piglet infection model are shown, where A represents survival rate; B represents weight change; C represents viral shedding in feces; and D–G represent the RT-qPCR results of PEDV viral load in the duodenum, jejunum, ileum, and colon of piglets in each group, respectively. P<0.05; P<0.01; P < 0.0001, ns = P > 0.05. Detailed Implementation

[0017] This invention provides a recombinant Lactobacillus plantarum containing a recombinant vector expressing a PEDV tS1 fusion gene. The PEDV tS1 fusion gene includes a PEDV tS1 domain with a nucleotide sequence as shown in SEQ ID NO.1, an endogenous signal peptide 1320 with a nucleotide sequence as shown in SEQ ID NO.2, and a dendritic cell targeting peptide DCpep with a nucleotide sequence as shown in SEQ ID NO.3.

[0018] In this invention, tS1 refers to a truncated S1 fragment. The PEDV tS1 fusion gene preferably has an endogenous signal peptide 1320 linked to the 5' end of the PEDV tS1 domain and a dendritic cell targeting peptide DCpep linked to the 3' end of the PEDV tS1 domain. The nucleotide sequence of the PEDV tS1 fusion gene is preferably as shown in SEQ ID NO. 4. More preferably, the PEDV tS1 fusion gene carries an HA tag with a nucleotide sequence as shown in SEQ ID NO. 5. The nucleotide sequence of the PEDV tS1 fusion gene with the HA tag is preferably as shown in SEQ ID NO. 6. In this invention, the endogenous signal peptide 1320 is preferably derived from *Lactobacillus plantarum*. In this invention, the backbone vector of the recombinant vector is preferably the pSIP411 vector; the *Lactobacillus plantarum* is preferably *Lactobacillus plantarum* LP12.

[0019] This invention, supported by the National Key Research and Development Program of China (No. 2022YFC2604204), provides a novel strategy for PEDV mucosal vaccines based on a recombinant *Lactobacillus plantarum* expression system: The key antigenic domain gene sequence of PEDV tS1 is synthesized, integrated, and anchored for expression on the surface of *Lactobacillus plantarum* LP12, yielding recombinant *Lactobacillus plantarum* LP12:PEDV tS1. Subsequently, the immunogenicity was evaluated by intranasal immunization of mice and piglets. Results showed that LP12:PEDV tS1 induced a strong cross-species immune response, producing pathogen-specific sIgA, indicating effective activation of mucosal immunity. In vitro neutralization assays further confirmed that the induced antibody possessed PEDV neutralizing activity, demonstrating its functional effectiveness. In summary, this invention demonstrates that the expression system using *Lactobacillus plantarum* as a vector can serve as a feasible platform for developing mucosal vaccines against intestinal pathogens, providing a new approach for the prevention and control of diseases such as PEDV that rely on mucosal immunity.

[0020] To prevent potential viral spillover, vaccine design principles should consider interspecies differences. Traditional nucleic acid vaccines have significant limitations in the design of novel cross-species vaccines, often requiring codon optimization in the target host to improve translation efficiency and immunogenicity. The recombinant *Lactobacillus plantarum* (recombinant lactic acid bacteria mucosal vaccine) designed in this invention, as a subunit vaccine, not only avoids repeated codon optimization but also effectively induces mucosal immunity, rapidly triggering an antiviral response against PEDV infection in piglets. The recombinant *Lactobacillus plantarum* vaccine provided by this invention uses *Lactobacillus plantarum* LP12 as the delivery vector. Its inherent immunomodulatory properties and mucosal adhesion ability make it an ideal vector for delivering heterologous protective antigens. By expressing antigen proteins itself, it not only solves the problem of immunity across different species but also reduces the cost of purification. Traditional intramuscular injection mainly induces systemic immunity, making it difficult to stimulate mucosal immunity, which is crucial for intestinal protection. The recombinant *Lactobacillus plantarum* provided by this invention, as an intranasal vaccine, can induce both mucosal and systemic immunity, achieving effective control of PEDV.

[0021] This invention also provides a method for preparing the above-mentioned recombinant *Lactobacillus plantarum*, comprising the following steps: inserting the PEDV tS1 fusion gene into a backbone vector using a seamless cloning method to obtain a recombinant vector; and transforming the recombinant vector into *Lactobacillus plantarum* to obtain recombinant *Lactobacillus plantarum*. This invention does not specifically limit the method of transforming the recombinant vector into *Lactobacillus plantarum*; conventional transformation methods in the art are acceptable.

[0022] This invention also provides the application of the above-mentioned recombinant *Lactobacillus plantarum* in the preparation of a vaccine for the prevention and treatment of porcine epidemic diarrhea virus infection. In this invention, the vaccine is preferably an intranasal vaccine. The immunogenicity of the recombinant *Lactobacillus plantarum* provided by this invention has been effectively verified in different species (mice and piglets); intranasal administration can activate mucosal immunity and systemic immunity, enhancing the antiviral effect. Existing nasal mucosal vaccines in the art suffer from low vaccine delivery system efficiency, difficulty in activating the required immune response, and compromises on safety. The recombinant *Lactobacillus plantarum* provided by this invention, however, has high delivery efficiency, can activate mucosal and systemic immunity, and is highly safe. This invention deeply studies and develops the potential of *Lactobacillus plantarum* as a natural mucosal vaccine delivery carrier, using mouse and piglet animal models, making the animal vaccine safety assessment system of this invention specific and complete, providing strong evidence for the practical application of the recombinant *Lactobacillus plantarum* of this invention.

[0023] This invention also provides the application of the above-mentioned recombinant *Lactobacillus plantarum* in the preparation of a drug for combating porcine epidemic diarrhea virus infection. In this invention, the animals protected against porcine epidemic diarrhea virus infection include piglets. During piglet immunization, intranasal administration followed by challenge with the virus for 5 consecutive days achieves 100% protection, indicating that the recombinant *Lactobacillus plantarum* provided by this invention not only functions as a vaccine but also acts as a preventative or therapeutic drug, providing protection without requiring a second immunization more than two weeks later.

[0024] This invention also provides an intranasal vaccine for preventing and treating porcine epidemic diarrhea virus (PEDV) infection, wherein the intranasal vaccine contains the aforementioned recombinant *Lactobacillus plantarum*. Traditional intramuscular injection primarily induces systemic immunity, failing to stimulate mucosal immunity crucial for intestinal protection. However, the intranasal vaccine provided by this invention can induce both mucosal and systemic immunity, thereby achieving effective control of PEDV. Verification has shown that the recombinant *Lactobacillus plantarum* LP12:PEDV tS1 provided by this invention can rapidly induce a protective immune response in piglets.

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Unless otherwise specified, the following embodiments are all conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] The statistical analysis of the data in the following examples was performed using GraphPad Prism software. Statistical significance was analyzed through one-way or two-way ANOVA combined with Tukey or Bonferroni multiple comparison tests. Data are expressed as mean ± standard error. Significance markers are as follows: p<0.05; p<0.01; p<0.001; p < 0.0001, ns means p > 0.05.

[0029] Example 1 (1) Construction of recombinant Lactobacillus plantarum expressing PEDV tS1 protein Select the PEDV tS1 domain (SEQ ID NO.1) (e.g.) Figure 1 As shown in Figure A), the endogenous signal peptide 1320 (SEQ ID NO.2) derived from Lactobacillus plantarum LP12 is attached to its 5' end, and the dendritic cell targeting peptide DCpep (SEQ ID NO.3) and the HA tag (SEQ ID NO.5) are sequentially added to its 3' end (as shown in Figure A). Figure 1 (As shown in B in the figure). The gene was synthesized after codon optimization of Lactobacillus plantarum LP12 and named 1320-PEDV tS1-DCpep (SEQ ID NO.6). Homologous recombination arms (nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8, respectively) were added to both ends of 1320-PEDV tS1-DCpep (SEQ ID NO.6) to obtain a fusion gene with an HA tag and homologous recombination arms (nucleotide sequence shown in SEQ ID NO.9).

[0030] Using the fusion gene with the HA tag and homologous recombination arm as a template, amplification was performed using primers F01 / R01. A total reaction volume of 30 μL was used, comprising 1 μL template, 15 μL KOD enzyme, 1 μL each of the upper and lower primers, with the remainder made up with sterile water. The reaction program was: pre-denaturation at 98℃ for 5 min; 98℃ for 10 sec, 60℃ for 10 sec, 68℃ for 30 sec, and 72℃ for 4 min, for 35 cycles. The PCR amplification products were subjected to 1% agarose gel electrophoresis. The results showed that a specific band of 1858 bp was obtained from the fusion gene, consistent with the expected size (see [link to PCR results]). Figure 1 (D in the middle).

[0031] The target gene fragment was inserted into the pSIP411 vector via seamless cloning. To obtain the linear plasmid pSIP411, a double enzyme digestion and gel purification method was used. The reaction system was: Xho I 0.5 μL, Nco I 0.5 μL, buffer 10 μL, pSIP411 < 2 μg, and ddH2O to a final volume of 20 μL. Based on the principle of seamless cloning, homologous recombination arms (nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8, respectively, as above) were designed to ensure that the vector pSIP411:insertion fragment ratio was 1:3. These arms were added to the reaction cloning system. The reaction system was: 5×CE MultiS Buffer 4 μL, Exnase MultiS 2 μL, DNA fragment vector 50–200 ng, DNA fragment 20–200 ng, and ddH2O to a final volume of 20 μL. The reaction was carried out at 37°C for 30 min and then stored on ice. The recombinant vector was obtained.

[0032] Electroporation of the positive recombinant vector into *Lactococcus lactis* NZ3900: Strain resuscitation: The glycerol-preserved NZ3900 strain was resuscitated using the four-zone streak method and incubated at 30°C for 24 hours until single colony formation was observed. Single colonies were picked and inoculated into M17 liquid medium for expansion culture to ensure the strain regained its typical growth characteristics. Competent cell preparation: Samples were taken from the resuscitated NZ3900 bacterial culture and inoculated into competent cell liquid medium at a ratio of 2%, and incubated for approximately 6 hours to allow the OD of the culture medium to reach the required level. 600 The concentration was approximately 0.4-0.6. The bacterial pellet was then collected by centrifugation, washed twice with sterile solution A, and finally resuspended in solution B for further electroporation. Electroporation: Seamless clone products were added to prepared NZ3900 competent cells and incubated on ice for 45 min. The cells were then transferred to a pre-chilled electroporation cuvette. The pulse parameters were set to 2000 V for 5 ms. After the pulse was complete, GM17 recovery medium was added, and the cells were inverted at 30°C for recovery culture. The glycerol-preserved NZ3900 strain was recovered using the four-zone streak method. Single colonies were isolated, cultured at 30°C, and then picked and expanded in M17 liquid medium to restore the strain's typical characteristics. Positive clone PCR screening: After 3 h of recovery culture, the bacterial pellet was collected by centrifugation and spread onto GM17 solid medium containing 20 ng / mL erythromycin. The cells were incubated inverted at 30°C until visible and pickable single colonies appeared. Single colonies were picked and subjected to bacterial PCR using identification primers (verified by PCR and sequencing using primer 411-test-F / R). The results are as follows. Figure 1As shown in E, potential positive clones were initially screened. PCR products with the correct band size were sent to a biotechnology company for Sanger sequencing verification.

[0033] Recombinant plasmid extraction: Positive bacterial strains were inoculated into GM17 liquid medium (containing 20 ng / mL erythromycin) and cultured for approximately 8 hours. Subsequently, the bacterial pellet was collected by centrifugation, and the recombinant plasmid was extracted according to the instructions of the plasmid miniprep kit. The extracted plasmid was amplified using specific primers F01 / F02 to amplify the target fragment. After confirming that the band size met the expected PCR product, the extracted plasmid could be stored at -20°C for subsequent experiments.

[0034] The verified recombinant plasmid was transferred into *Lactobacillus plantarum* LP12. The resuscitation and culture methods for *Lactococcus plantarum* were similar to those for *Lactococcus lactis*, but MRS medium was used, and the culture conditions were 37°C. All other steps followed the *Lactococcus lactis* protocol. The preparation of competent cells was similar to that for *Lactococcus lactis*. *Lactobacillus plantarum* was cultured at 37°C until OD... 600 When the pH reaches 0.3-0.5, centrifuge to collect the bacterial pellet. Resuspend the pellet in sterile Wash buffer and wash three times. Finally, add an appropriate amount of Wash buffer to resuspend the pellet again, preparing competent cells. Add the extracted recombinant plasmid to *Lactobacillus plantarum* LP12 competent cells and process them according to the electroporation procedure for *Lactococcus lactis*. Specific parameters are set to 1750V, 5ms electroporation. Immediately after electroporation, transfer the competent cells to MRS liquid medium for recovery culture.

[0035] Screening and identification of recombinant *Lactobacillus plantarum*: Transformed *Lactobacillus plantarum* was plated on MRS solid medium containing 20 ng / mL erythromycin and incubated at 37°C until single colonies were visible to the naked eye; these colonies were considered suspected positive clones. Single colonies were picked from the plates and preliminarily screened by PCR amplification. The target fragment was amplified using specific primers 411-test-F / R, and the results are as follows: Figure 1 As shown in Figure F, a specific band of 2486 bp is displayed, consistent with the expected result. After confirming that the band size matches the expected PCR product, it was sent to a professional sequencing company for further verification to ensure that the target gene has been successfully integrated into the Lactobacillus plantarum genome. Sequencing analysis confirmed the successful acquisition of the nucleotide-free recombinant PEDV tS1 sequence, and the obtained strain was named LP12:PEDV tS1 (e.g., ...). Figure 1 (As shown in C). The empty vector control strain LP12:Vector was constructed using the same method.

[0036] The primer pairs used in the above process are shown in Table 1. For amplification of the target fragment, different bacterial cultures were used as templates to prepare a 20 μL PCR reaction system: 1 μL template DNA, 10 μL Taq enzyme, 1 μL upstream primer, 1 μL downstream primer, and sterile water to make up the volume. The reaction was extended at 72℃ for 5 min. The results showed that all experimental strains amplified the expected size of specific bands, and were preliminarily identified as positive. Furthermore, the PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the sequencing results were analyzed using NCBI BLAST to verify gene sequence consistency.

[0037] Table 1 Primer pairs

[0038] (2) Identification of S1 protein expression in LP12:PEDV tS1 recombinant strain To verify the expression of the target protein, Western blot analysis was performed on the LP12:PEDV tS1 engineered strain, as follows: Positive clones were inoculated into MRS liquid medium containing erythromycin (10 μg / mL) and cultured at 37°C until OD 0.3–0.5. Then, 50 ng / mL SppIP was added for induction for 8 h. The cells were collected by centrifugation at 8000×g for 3 min at 4°C, washed once with PBS, homogenized with 0.1 μm glass beads for 20 min, and centrifuged again to collect the supernatant for subsequent detection.

[0039] Western blot: Mix 100 μL of protein supernatant with 5× Loading Buffer, boil for 7 min, and separate by 10% SDS-PAGE; after transfer, block with 5% skim milk powder, add rabbit anti-HA primary antibody (1:1000, Proteintech, USA) and incubate for 2 h, then add HRP-labeled goat anti-rabbit secondary antibody (1:5000, Beyotime, China) and incubate for 40 min, followed by ECL staining.

[0040] IFA and FCM: After induction, the bacterial cells were washed with PBS, incubated overnight at 4°C with rabbit anti-HA primary antibody (1:100, Proteintech, USA), and then incubated at room temperature in the dark for 2 hours with FITC-labeled goat anti-rabbit secondary antibody (1:5000, Beyotime, China). Finally, the positive expression rate was calculated by fluorescence microscopy and flow cytometry.

[0041] The results showed that using rabbit-derived HA-tagged polyclonal antibodies (see...) Figure 2 A) and mouse-derived PEDV tS1 domain monoclonal antibody (see A) Figure 2In section B), specific bands of approximately 60 kDa were detected, consistent with the expected size of the S1 protein. To further confirm the localization of the S1 protein on the bacterial cell surface, immunofluorescence and flow cytometry were used for verification. Immunofluorescence results showed a green fluorescent signal, confirming the successful display of the S1 protein on the surface of LP12 bacteria (see section B). Figure 2 (C) ; Flow cytometry analysis showed a significant rightward shift in the fluorescence peak of the experimental group, with a positive rate of 88.52% (see C). Figure 2 The PEDV tS1 protein was successfully anchored to the surface of LP12 cells, achieving surface display of the recombinant protein. The empty vector and wild-type control group showed no significant fluorescence shift. These results collectively confirm that the PEDV tS1 protein was successfully anchored to the surface of LP12 cells, achieving surface display of the recombinant protein.

[0042] (3) Optimization of PEDV tS1 protein expression conditions To obtain optimal expression of LP12:PEDV tS1, the induction conditions were optimized as follows: induction doses of 50, 100, 150, and 200 ng / mL SppIP were used, with induction at 37℃ for 6 h; induction times of 4, 6, 8, 10, 12, 16, 20, and 24 h were used, all with 100 ng / mL SppIP at 37℃; and induction temperatures of 33, 37, and 42℃ were used, with induction at 100 ng / mL SppIP for 6 h. Protein samples were prepared according to the aforementioned method, and PEDV tS1 expression levels were detected by Western blotting after BCA quantification.

[0043] The results are as follows Figure 3 As shown in the figure, the optimization results of the induction time indicate that the highest protein expression level can be obtained 8 hours after induction (see figure). Figure 3 (A) The inducing agent concentration experiment showed that 50 ng / mL was the optimal inducing concentration (see A). Figure 3 (B in the text); Temperature optimization experiments showed that the protein expression level was highest at 33℃ (see...). Figure 3 (C in the text). In summary, the optimal expression conditions were determined to be: induction at 33℃ with an inducer concentration of 50 ng / mL for 8 hours. This combination of parameters ensures that the recombinant protein achieves the highest yield under the experimental conditions.

[0044] Target protein quantification analysis The expression level of the target protein in recombinant LP12:PEDV tS1 bacteria was quantitatively analyzed by indirect enzyme-linked immunosorbent assay (ELISA). A standard curve was established using purified PEDV tS1 protein, with the horizontal axis representing absorbance at 450 nm and the vertical axis representing the target protein expression level (see [link to standard curve]). Figure 3 (D) in 1×10 9CFU LP12:PEDV tS1 cells were induced and cultured to obtain a total protein of 410 μg. Substituting the OD450 values ​​at different time points into the regression equation, the target protein expression level reached its highest value of 1.7656 μg after 8 hours of induction. Substituting the OD450 values ​​at each time point into the regression equation, the target protein expression level at 8 hours of induction was calculated to be 0.00176 μg. Similarly, the expression levels at 8, 10, 12, 16, and 20 hours were obtained using the same equation (see...). Figure 3 (See E in Table 2). The results showed that the expression of the target protein reached a peak of 1.7656 μg after 8 hours of induction, and then gradually decreased over time, dropping to 1.0476 μg after 20 hours, indicating that the expression of the target protein decreased in a time-dependent manner after the initial peak.

[0045] Table 2. Expression levels of target proteins at different time points

[0046] Example 2 Mice were immunized with the recombinant Lactobacillus plantarum LP12:PEDV tS1 constructed in Example 1. Thirty mice were randomly divided into three groups: PBS group (n=10), LP12:Vector group (n=10), and LP12:PEDVtS1 group (n=10). Immunization regimens are detailed below. Figure 4 In group A, the three groups of mice received PBS via intranasal route, 1×10 9 CFU was treated with LP12:Vector or LP12:PEDV tS1. The immunization regimen consisted of a primary immunization for 3 consecutive days, followed by a booster immunization for 3 consecutive days two weeks later. Samples were collected on days 7, 14, 21, 28, and 35.

[0047] The specific method is as follows: LP12:PEDV tS1 and LP12:Vector strains were prepared under the optimized conditions described in Example 1 and resuspended in PBS. After anesthesia, depending on the group, each mouse was intranasally inoculated with 20 μL of PBS (negative control) or 20 μL of PBS containing 1×10⁻⁶ ppm. 9 CFU in LP12:Vector in PBS, or 20 μL containing 1×10 9CFU of LP12:PEDV tS1 bacterial suspension in PBS. Mice were immunized for 3 consecutive days on day 0 and day 14. Fecal, bronchoalveolar lavage fluid (BALF), and nasal lavage fluid (NLF) samples were collected on days 7, 14, 21, 28, and 35. The samples were resuspended in PBS containing BSA (Sigma-Aldrich, USA) and 1 mM PMSF (Beyotime, China), homogenized, and centrifuged. The supernatant was immediately analyzed or stored at -80°C for later use. Mesenteric lymph nodes (MLNs) were removed after sacrifice, milled through a 70 μm filter to prepare single-cell suspensions, washed three times with PBS, and used for subsequent analysis.

[0048] To investigate the activating effect of LP12:PEDV tS1 on humoral immunity in mice, fecal-specific sIgA levels were measured on days 7, 14, 21, 28, and 35 post-immunization, and sIgA levels in bronchoalveolar lavage fluid and nasal lavage fluid were measured on day 21. Results showed that compared with the PBS group, fecal-specific sIgA levels in the LP12:PEDV tS1 group significantly increased starting on day 7 (P<0.05) (see [link to study]. Figure 4 (B) Compared with the PBS and empty vector groups (LP12:Vector), the fecal sIgA level in the experimental group (LP12:PEDV tS1) was significantly increased (P<0.0001), peaking on day 21 and remaining elevated until day 28 and day 35. In bronchoalveolar lavage fluid, the sIgA level in the experimental group (LP12:PEDV tS1) was significantly higher than that in the two control groups (P<0.05 or P<0.01) (see B). Figure 4 In the nasal irrigation fluid, although the sIgA level in the experimental group (LP12:PEDV tS1) was slightly increased, there was no statistically significant difference (see C). Figure 4 (D in the text). The results showed that LP12:PEDV tS1 successfully induced mice to produce specific sIgA antibodies via the nasal immunization pathway.

[0049] Mouse neutralizing antibody detection Neutralizing antibodies in mouse feces were detected using a virus neutralization assay on days 21 and 28 post-immunization. Neutralizing antibodies in mouse serum and feces were also detected using the virus neutralization assay. Samples were filtered through a 0.22 μm filter and irradiated with ultraviolet light for 40 minutes. Serum, feces, and nasal lavage fluid samples were inactivated by heat treatment at 56°C for 30 minutes, followed by serial 2-fold dilutions in DMEM medium, from 1:2 to 1:256, 50 μL per well. Simultaneously, 200 mg TCID₂ was added to each well. 5050 μL of PEDV viral solution was incubated at 37°C in a 5% CO2 incubator for 1 hour. The mixture was then inoculated into Vero cells and cultured for another 72 hours at 37°C in a 5% CO2 incubator to observe the virus-induced cytopathic effect. Results showed that compared with the control group, the LP12:PEDV tS1 group had an anti-PEDV neutralizing antibody titer of 1:8 (P<0.001 or P<0.0001) (see...). Figure 4 The difference in E values ​​was statistically significant, indicating that the recombinant bacterial vaccine of this invention can effectively induce the production of specific neutralizing antibodies in the intestinal mucosa of mice.

[0050] To investigate the effect of LP12:PEDV tS1 on the activation of dendritic cells in mouse mesenteric lymph nodes, the expression of the dendritic cell surface activation markers CD80 and CD86 was detected. Flow cytometry analysis of mesenteric lymph node cell subsets: Single-cell suspensions of prepared mesenteric lymph nodes were counted, and 100 μL of cell suspension (containing 1 × 10⁻⁶ cells) was collected. 6 (Number of cells) were placed in 1.5 mL centrifuge tubes, with blank controls and single-stained samples set up simultaneously. Samples were stained with FITC-labeled mouse CD80 antibody, PE / Cy5-labeled mouse CD86 antibody, and PE-labeled mouse CD11c antibody, respectively. 10 μL of the corresponding diluted antibody was added to each experimental group, and after thorough mixing, the samples were incubated at 4°C in the dark for 30 minutes. The samples were then washed twice with PBS, resuspended in 500 μL of PBS solution, filtered through a 0.22 μm filter, and analyzed by flow cytometry. The gating strategy and results of flow cytometry are shown below. Figure 5 As shown in A, the results indicate that the experimental group (LP12:PEDV tS1) CD11c + CD80 + The proportion of cell subsets was significantly higher in the PBS control group than in the control group (P<0.05), while there was no significant difference between the empty vector group (LP12:Vector) and the experimental group (LP12:PEDV tS1) (P>0.05). Figure 5 The proportions of CD11c+CD86+ cell subsets showed the same trend (B in the text). Figure 5 (C in the original text). The results show that the recombinant Lactobacillus plantarum of the present invention can activate dendritic cells of mouse mesenteric lymph nodes.

[0051] Example 3 Piglets were immunized using the recombinant Lactobacillus plantarum LP12:PEDV tS1 constructed in Example 1. Blood samples were collected from piglets before immunization. RNA was extracted from samples containing anticoagulants and detected by RT-PCR using primers PEDV-F and PEDV-R (Table 1). Serum from samples containing procoagulants was separated by centrifugation, and specific antibodies were detected by an optimized indirect ELISA method. PEDV-negative piglets were then screened.

[0052] Ten 25-day-old, PEDV-negative crossbred piglets (Large White pigs) were selected from a local breeding farm; two piglets were placed in each stainless steel isolator and given sterile drinking water and piglet feed. After one week of environmental acclimatization, they were... Figure 6 LP12:PEDV tS1 was administered according to regimen A. Piglets were randomly divided into the LP12:Vector group (n=5) and the LP12:PEDV tS1 group (n=5). The LP12:PEDV tS1 and LP12:Vector bacterial cells cultured under optimized conditions were resuspended in PBS and inoculated intranasally with 1×10⁻⁶ cells / day. 10 CFU was treated with LP12:Vector or LP12:PEDV tS1. The immunization regimen consisted of a primary immunization for 3 consecutive days (days 1-3), followed by a booster immunization for another 3 consecutive days (days 14-16) two weeks later. Body temperature and weight were measured every two days. Fecal, bronchoalveolar lavage fluid (BALF), and nasal lavage fluid (NLF) samples were collected from piglets on days 7, 14, 21, 26, and 31. The samples were resuspended in PBS containing BSA (Sigma-Aldrich, USA) and 1 mM PMSF (Beyotime, China), homogenized, and centrifuged. The supernatant was immediately analyzed or stored at -80°C for later use. Mesenteric lymph nodes (MLNs) were removed after euthanasia, ground through a 70 μm filter to prepare a single-cell suspension, washed three times with PBS, and used for subsequent analysis.

[0053] Using piglets as a model, the ability of LP12:PEDV tS1 to induce systemic and mucosal immune responses was evaluated by detecting anti-PEDV specific sIgA and IgG. Ten piglets were screened by RT-PCR and indirect ELISA, confirming that both nucleic acid and antibody tests were negative (see...). Figure 6 (See B, C, and D in the original text). Monitoring data showed that the piglets' body temperature remained within the normal range of 38.5-39.5℃, and the immunization treatment did not cause abnormal fluctuations in body temperature (see...). Figure 6 (E in the text); the weight of piglets in each group maintained a steady increase (see...). Figure 6 The F in the figure indicates that the recombinant strain and immunogen did not affect the normal growth and development of piglets, and the safety was good.

[0054] Seven days after immunization, serum IgG levels significantly increased (P<0.05). Although they decreased slightly at day 14, they remained significantly higher than those in the empty vector group (LP12:Vector) (P<0.05). There was no significant difference in antibody levels between days 21 and 31 (P>0.05) (see...). Figure 6 (G in the text). On day 7 after nasal instillation of sIgA, nasal lavage fluid showed a significant increase (P<0.05). From days 14 to 31, the experimental group showed an increasing trend compared to the control group, but the difference was not statistically significant (P>0.05) (see...). Figure 6 (H in the text). Fecal sIgA was significantly higher than that in the control group at 21 and 26 days post-immunization (P<0.05) (see H). Figure 6 (I in the text). The results showed that LP12:PEDV tS1 could simultaneously induce systemic and mucosal immune responses in piglets.

[0055] Neutralizing antibodies in piglet serum, feces, and nasal lavage fluid were detected using a virus neutralization assay. Samples were filtered through a 0.22 μm filter and irradiated with ultraviolet light for 40 minutes. Serum, feces, and nasal lavage fluid samples were inactivated by heat treatment at 56°C for 30 minutes, followed by serial 2-fold dilutions in DMEM medium, from 1:2 to 1:256, 50 μL per well. Simultaneously, 200 TCID50 solutions were added to each well. 50 50 μL of PEDV viral solution was incubated at 37°C in a 5% CO2 incubator for 1 hour. The mixture was then inoculated into Vero cells and cultured at 37°C in a 5% CO2 incubator for another 72 hours to observe the virus-induced cytopathic effect. Neutralization assays showed that the average titer of neutralizing antibodies in the immunized group was 1:63, with a maximum of 1:81, significantly higher than that in the control group (LP12:Vector) (P<0.05) (see...). Figure 7 In the A section, the neutralizing antibody titers in feces and nasal lavage fluid were both 1:4, with no significant difference compared to the control group (P>0.05) (see section A). Figure 7 (B and C in the text). It was confirmed that the vaccine can effectively induce serum IgG neutralizing antibodies, but the mucosal sIgA neutralizing antibody response is weak, which may be related to insufficient immunization dose.

[0056] Example 4 In nasal inoculation Example 1: Recombinant Lactobacillus plantarum LP12:PEDV tS1 constructed was used to challenge piglets. Nineteen 3-day-old piglets were divided into four groups: a blank control group (Mock, n=4), a PEDV challenge group (n=5), an LP12:empty vector group (LP12:Vector, n=5), and an LP12:PEDV tS1 group (n=5). After a 3-day acclimatization period, they were intranasally vaccinated daily for 5 consecutive days with 1×10 10 CFU LP12:Vector or LP12:PEDV tS1 bacterial suspension. Then, at a titer of 5 × 10⁻⁶. 6 PEDV virus solution at a concentration of copies / mL was administered via gavage for challenge, followed by treatment with recombinant bacteria for 5 consecutive days after challenge (see [link to relevant documentation]). Figure 8 (A) The antiviral efficacy of LP12:Vector or LP12:PEDV tS1 was evaluated by survival rate, weight change and viral shedding, and viral load in duodenal, jejunal, ileal and colon tissue samples was detected by RT-qPCR.

[0057] To enhance antigen exposure, the frequency of immunizations in piglets was increased. Notably, compared with the virus challenge control group, piglets in the LP12:PEDV tS1 immunization group showed significantly improved clinical manifestations, with marked reductions in diarrhea and dehydration (see...). Figure 8 (B in the text). The survival rate of the immunized group was significantly increased to 100%, significantly higher than the 60% of the challenge control group (PEDV) and the empty vector group (LP12: Vector) (see B). Figure 9 (A) The weight gain of piglets in the immunized group was comparable to that of the blank control group (MOCK) and significantly better than that of the challenge control group (PEDV) and the empty vector group (LP12: Vector) (see A). Figure 9 (B in the text). Anal swab testing showed a significant reduction in viral shedding in the feces of the immunized group, indicating a stronger viral clearance capacity (see [reference]). Figure 9 (C) Quantitative analysis of intestinal tissue samples by RT-qPCR revealed that the viral load in all small intestinal segments of the immunized group was significantly lower than that of the control group (PEDV) and the empty vector group (LP12: Vector) (see C). Figure 9 D- Figure 9 In the F segment, viral load in the colon also showed a decreasing trend (see F). Figure 9 (G in the text). These results collectively demonstrate that LP12:PEDV tS1 can rapidly induce a protective immune response in piglets.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant Lactobacillus plantarum, characterized in that, The recombinant Lactobacillus plantarum contains a recombinant vector expressing the PEDV tS1 fusion gene, which includes the PEDV tS1 domain as shown in SEQ ID NO.1, the endogenous signal peptide 1320 as shown in SEQ ID NO.2, and the dendritic cell targeting peptide DCpep as shown in SEQ ID NO.

3.

2. The recombinant Lactobacillus plantarum according to claim 1, characterized in that, The PEDV tS1 fusion gene has an endogenous signal peptide 1320 attached to the 5' end of the PEDV tS1 domain and a dendritic cell targeting peptide DCpep attached to the 3' end of the PEDV tS1 domain.

3. The recombinant Lactobacillus plantarum according to claim 1, characterized in that, The nucleotide sequence of the PEDV tS1 fusion gene is shown in SEQ ID NO.

4.

4. The recombinant Lactobacillus plantarum according to claim 1, characterized in that, The backbone vector of the recombinant vector is the pSIP411 vector.

5. The recombinant Lactobacillus plantarum according to claim 1, characterized in that, The Lactobacillus plantarum is Lactobacillus plantarum LP12.

6. The method for preparing recombinant Lactobacillus plantarum according to any one of claims 1 to 5, characterized in that, The process includes the following steps: inserting the PEDV tS1 fusion gene into a backbone vector using a seamless cloning method to obtain a recombinant vector; and transforming the recombinant vector into *Lactobacillus plantarum* to obtain recombinant *Lactobacillus plantarum*.

7. The use of the recombinant Lactobacillus plantarum according to any one of claims 1 to 5 in the preparation of a vaccine for the prevention and treatment of porcine epidemic diarrhea virus infection.

8. The application according to claim 7, characterized in that, The vaccine in question is an intranasal vaccine.

9. The use of the recombinant Lactobacillus plantarum according to any one of claims 1 to 5 in the preparation of a drug for treating swine epidemic diarrhea virus infection.

10. An intranasal vaccine for preventing and treating porcine epidemic diarrhea virus infection, characterized in that, The intranasal vaccine contains the recombinant Lactobacillus plantarum as described in any one of claims 1 to 5.

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