Recombinant bacillus subtilis with PEDV S protein and intestinal mammary gland axis key presenting cell targeting peptide displayed on surface and application of recombinant bacillus subtilis

Abstract

The invention discloses a recombinant expression vector as well as a construction method and application thereof. The invention also discloses a recombinant strain as well as a construction method and application thereof. The invention also discloses a genetic engineering live vector vaccine. According to the invention, probiotic bacillus subtilis is used as an antigen delivery carrier, and recombinant bacillus subtilis for recombinant integration tandem expression of porcine epidemic diarrhea virus antigen neutralizing epitopes and intestinal M cell and dendritic cell (DCs) targeting peptides is constructed. The recombinant live vector vaccine is orally taken and immunized by sows, and efficient intestinal mucosa immune protection is provided for piglets in the lactation period by enhancing the intestinal mammary axial function of the sows, so that a safe, simple and convenient immune way is provided for effectively preventing the occurrence of epidemic diarrhea of the piglets.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and genetic engineering, and particularly relates to a recombinant Bacillus subtilis displaying PEDV S protein on the surface and targeting peptides of key antigen-presenting cells of the gut-mammary axis, and its application. Background Art

[0002] Animal mucosa is the main gateway for pathogen invasion. Oral vaccines stimulate gut-associated lymphoid tissue (GALT) to induce the formation of "the first line of defense" by secretory IgA antibodies (sIgA) on the mucosal surface, while activating systemic IgG responses. For example, an oral vaccine against porcine epidemic diarrhea virus (PEDV) can increase the intestinal sIgA level by 4 - 8 times, block virus adsorption, and significantly reduce the morbidity and mortality of piglets. The intestinal immune system of ruminants shows stronger memory for oral antigens. Oral vaccination of cattle with Brucella vaccine can induce memory T cells in intestinal Peyer's patches and maintain protective antibody levels for more than 12 months, while traditional injectable vaccines only maintain for 6 - 8 months. Although oral mucosal immunization has broad application prospects in animals, challenges such as gastric acid degradation, dose standardization, and species differences still need to be overcome. Oral mucosal immunization shows significant advantages in animal immunization, especially in large-scale farming, wildlife protection, and pet health management. Its non-invasive, efficient, and economical features provide innovative solutions for animal disease prevention and control. In the livestock industry, traditional injectable immunization requires capturing animals one by one (such as pigs and poultry), which is time-consuming, labor-intensive, and prone to causing stress responses, resulting in a decline in growth performance. Oral vaccines can be directly mixed into feed or drinking water to achieve synchronous group immunization. For example, the oral vaccine against Newcastle disease in chickens can be administered through drinking water, increasing the immunization coverage rate of a flock of ten thousand chickens to over 95%, and the operation efficiency is 10 times higher than that of injection. For wild animals (such as foxes and raccoons) or aquatic animals (such as salmon and shrimps), it is extremely difficult to capture them. Oral vaccines can be delivered in the form of baits or microparticles to avoid contact risks. In Europe, the incidence of rabies in the fox population has decreased by 99% by distributing baits containing rabies vaccine. With the development of technologies such as 3D printing customized baits and CRISPR-edited probiotic vectors, it is expected to achieve "multiple defenses with one dose" and the delivery of environment-responsive intelligent vaccines in the future, further promoting the evolution of animal immunization towards high efficiency and welfare. This strategy not only improves the health level of animals but also provides key technical support for ensuring food safety and maintaining ecological balance.

[0003] Bacillus subtilis, as a Gram-positive, spore-forming probiotic, has become an important biological agent for replacing antibiotics and optimizing breeding efficiency in livestock and poultry production due to its characteristics such as high temperature resistance, acid and alkali resistance, and strong stability. The comprehensive advantages of Bacillus subtilis in improving animal growth performance, regulating intestinal health, enhancing immunity, and reducing environmental pollution have led to its widespread application in the breeding industry. Bacillus subtilis can secrete protease, amylase, lipase, and non-starch polysaccharide enzyme, directly decomposing anti-nutritional factors in feed and improving feed utilization rate. Research shows that adding 1×108 CFU / g of Bacillus subtilis to broiler diets can increase the crude protein digestibility by 12%, reduce the feed conversion ratio (FCR) by 6%-8%, and increase the daily weight gain by 5%-10%. Bacillus subtilis occupies intestinal adhesion sites by rapid proliferation and secretes antibacterial substances (such as surfactin and iturin) to directly inhibit the growth of pathogenic bacteria such as Escherichia coli and Salmonella. Experiments show that after broilers are infected with Salmonella, feeding them with feed containing Bacillus subtilis can reduce the intestinal Salmonella load by 3-4 logarithmic units and the diarrhea rate by 60%. Bacillus subtilis stimulates intestinal epithelial cells to secrete immune factors such as β-defensin, activates the TLR2 / MyD88 signaling pathway, and improves the phagocytic activity of macrophages. After the EU banned the addition of antibiotics to feed, Bacillus subtilis has been widely adopted as an alternative. Danish pig breeding data shows that the use of this bacterium can reduce the dosage of therapeutic antibiotics by 50% while maintaining the same growth performance. The application of Bacillus subtilis not only improves the economic benefits of livestock and poultry production, but also meets the global sustainable development needs of reducing antibiotics and environmental protection, becoming one of the core technologies for the transformation and upgrading of modern animal husbandry.

[0004] The Bacillus subtilis surface display protein system is an efficient bioengineering technology that enables the stable expression and directional display of functional proteins by anchoring target proteins on the surface of bacterial cells. In recent years, with the rapid development of gene editing technologies (such as CRISPR-Cas9) and synthetic biology, this system has demonstrated unique advantages in fields such as vaccine development, biocatalysis, environmental remediation, and diagnostic testing. Traditional anchor proteins (such as CotC, CotB, and CotG) fix target proteins to the spore or cell wall surface through the cell wall binding domain (CWBD). Recent studies have found that fusing the S-layer protein SlpA or the peptidoglycan binding domain (PGBD) can enhance the display efficiency. For example, a 2023 study reported that fusing SlpA with the green fluorescent protein GFP increased the display efficiency by three times compared to the traditional CotC system, with a fluorescence intensity of 85,000 AU / mg (dry cell weight). Displaying pathogen antigens on the surface of Bacillus subtilis can stimulate intestinal mucosal immunity. In 2022, a team from Wageningen University in the Netherlands displayed the avian influenza H5N1 hemagglutinin (HA) on the surface of spores. After oral immunization of chicken flocks, the intestinal sIgA level increased by six times, and the protection rate against challenge reached 90%, without the need for adjuvants. The spores formed by Bacillus subtilis can tolerate high temperatures, gastric acid (pH

[0005] 2.0), and bile salts, ensuring the stability of surface proteins in harsh environments. Experiments have shown that after the rotavirus VP6 antigen displayed on spores was treated in simulated gastric juice for 2 hours, the antigen retention rate was greater than 90%, while only 20% remained in the Escherichia coli system. The Bacillus subtilis surface display protein system has become an important tool in the fields of synthetic biology and biomanufacturing due to its high efficiency, safety, and versatility. With the integration of precision editing technologies and intelligent regulation strategies, the application of this system in areas such as new vaccines, green industrial enzymes, and environmental remediation will be accelerated. In the future, its cross-innovation with nanomaterials and artificial intelligence is expected to give rise to more disruptive biotechnological solutions.

[0006] Porcine epidemic diarrhea virus (PEDV) is an important member of the genus Alphacoronavirus in the family Coronaviridae. It was first identified in the UK in 1971 and has since spread widely around the world. The PEDV genome contains multiple open reading frames encoding structural proteins such as spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N), as well as various non-structural proteins involved in virus replication and immune regulation. The virus has multiple transmission routes and can be directly transmitted not only through the fecal-oral route but also spread in the form of aerosols through the fecal-nasal route. It shows strong stability in the environment and can infect pig herds of all ages. The mortality rate of suckling piglets infected with it can be as high as 100%. The feces of infected piglets are usually yellow or yellowish-white with a fishy smell. In contrast, the symptoms of nursery pigs, growing and finishing pigs, and adult sows are relatively mild after infection, but sows may show symptoms such as vomiting, diarrhea, and mammary gland edema, which affect reproductive performance. Currently, vaccination is the main strategy for preventing and controlling PEDV, but due to the high variability of the virus, the protective effect of traditional vaccines against new variant strains is often limited. Therefore, developing broad-spectrum and highly effective vaccines and improving prevention and control strategies are still the key research directions at present.

[0007] Porcine epidemic diarrhea mainly occurs in neonatal piglets. Immunizing neonatal piglets cannot generate protective immunity in time. Only by orally or nasally immunizing sows, the specific antibodies and related cytokines produced through the intestinal-mammary gland-SIgA axis can transfer immunity to neonatal piglets through milk to achieve the purpose of protecting neonatal piglets. Genetic engineering subunit vaccines have high efficiency and safety. Bacillus subtilis not only has the effect of enhancing immunity itself but also can be used as an antigen-presenting carrier for oral immunization. Although Bacillus subtilis has many advantages in expressing foreign proteins, there are still some unsolved problems in the existing technology. For example, it is difficult to find a suitable promoter, protease degradation problems, and the negative impact of foreign proteins on the growth of the strain. Summary of the Invention

[0008] Object of the Invention: The technical problem to be solved by the present invention is to provide a recombinant expression vector and its construction method.

[0009] Another technical problem to be solved by the present invention is to provide a recombinant strain containing the above-mentioned recombinant expression vector and its construction method.

[0010] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned expression vector or the recombinant strain in the preparation of drugs or vaccines for preventing or treating diseases related to PEDV infection.

[0011] The last technical problem to be solved by the present invention is to provide a genetic engineering live vector vaccine.

[0012] Technical solution: To solve the above technical problems, the present invention provides a recombinant expression vector, which comprises a gene fragment of the P43 promoter, a gene fragment of the pCotY promoter, a gene fragment encoding the surface-displayed protein CotY, a gene fragment of the CoE region of the PEDV S protein, and a gene fragment encoding a targeting peptide, wherein the targeting peptide comprises a targeting peptide Col for intestinal M-like cells and a targeting peptide DCpep for dendritic cells.

[0013] Among them, the nucleotide sequences of the gene fragment of the CoE region of the PEDV S protein and the gene fragment encoding the targeting peptide are as shown in SEQ ID NO.7.

[0014] Among them, the nucleotide sequence of the gene fragment of the P43 promoter is as shown in SEQ ID NO.1, the nucleotide sequence of the gene fragment encoding the surface-displayed protein CotY is as shown in SEQ ID NO.2, and the nucleotide sequence of the gene fragment of the pCotY promoter is as shown in SEQ ID NO.3.

[0015] The present invention also includes a method for constructing the recombinant expression vector, which comprises the following steps: obtaining a recombinant gene fragment by overlapping PCR with the gene fragment of the P43 promoter, the gene fragment of the pCotY promoter, the gene fragment encoding the surface-displayed protein CotY, the CoE region of the PEDV S protein, the gene fragment encoding the targeting peptide including the targeting peptide Col for intestinal M-like cells, and the gene fragment encoding the targeting peptide DCpep for dendritic cells, and ligating the recombinant gene fragment into a vector.

[0016] Among them, in the overlapping PCR amplification, primers P7 and P10, the sequence of P7 is: 5’- ggagtgtcaaga atgtttgcaaaacgattcaaaa-3’, and the primer sequence of P10 is: 5’-tcaatggggaagagaaccgctt ctagccttgc cc -3’.

[0017] The present invention also includes a recombinant strain, which contains the recombinant expression vector. Preferably, the recombinant strain comprises recombinant Bacillus subtilis.

[0018] The present invention also includes a method for constructing the recombinant strain, which is obtained by introducing the recombinant expression vector into a host bacterium. Preferably, the host bacterium comprises Bacillus subtilis.

[0019] The present invention also includes the application of the recombinant expression vector or the recombinant strain in the preparation of drugs or vaccines for preventing or treating diseases related to PEDV infection.

[0020] The present invention also includes a genetically engineered live vector vaccine, which comprises the recombinant expression vector or the recombinant strain as described above.

[0021] Among them, the genetically engineered live vector vaccine is an oral vaccine.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention uses the probiotic Bacillus subtilis as an antigen delivery vector to construct a recombinant Bacillus subtilis that integratively and tandemly expresses the antigen neutralizing epitope of porcine epidemic diarrhea virus, as well as the targeting peptides of intestinal M cells and dendritic cells (DCs). By orally immunizing sows with this recombinant live vector vaccine, the function of the sow intestinal-mammary axis is enhanced, providing highly efficient intestinal mucosal immune protection for suckling piglets, and thus providing a safe and simple immunization route for effectively preventing the occurrence of porcine epidemic diarrhea in piglets. Description of the Drawings

[0023] Figure 1 It is a construction diagram of the pDG-CotY-EDB expression vector;

[0024] Figure 2 It is a PCR identification and Western-blot diagram of the expression product of recombinant Bacillus subtilis B.S.-EDB;

[0025] Figure 3 It is an immunization effect diagram of recombinant Bacillus subtilis B.S.-EDB in mice;

[0026] Figure 4 It is a result diagram of the PEDV challenge experiment on piglets after immunizing sows with recombinant Bacillus subtilis B.S.-EDB. Detailed Embodiments

[0027] The present invention will be further described below in conjunction with the drawings and embodiments. The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels without special instructions. The experimental methods without specific conditions noted in the embodiments usually follow conventional conditions or the conditions recommended by the manufacturer.

[0028] The Bacillus subtilis WB800 strain and pDG expression plasmid (Lu Z, Yang S. Nucleic Acids Res, 2019) used in the present invention were kindly provided by Teacher Yan Xin of the College of Life Sciences, Nanjing Agricultural University; the pPZP222 plasmid (Jin H, Hong Z. Proc Natl Acad Sci U S A. 2009) used in the present invention was kindly provided by Teacher Gao Xuewen of the College of Plant Protection, Nanjing Agricultural University; the recombinant Bacillus subtilis B.S.-RCL expressing intestinal M-like cell targeting peptide (L-lectin-β-GF) and porcine epidemic diarrhea virus (PEDV) S protein (China Patent Application No. CN108265073B) was preserved in our laboratory; the gene fragment EDB (shown in SEQ ID NO. 7) containing the CoE region of PEDV S protein, M-like cell targeting peptide Col and dendritic cell targeting peptide DCpep, with a flexible amino acid sequence (GGGSTS) added between each fragment and a His tag added at the end, used in the examples of the present invention was synthesized by Nanjing Ainuodi Biotechnology Co., Ltd.

[0029] The data analysis of the present invention adopted the single-factor ANOVA variance test results of SPSS 29.0 software, and the experimental data were statistically analyzed. The statistical results were expressed as mean ± standard error (Mean ± SE) for error. The criteria for judging the significance of differences were as follows: P < 0.05, significant difference (*); P < 0.01, extremely significant difference (**); P < 0.001, extremely significant difference (***).

[0030] Example 1 Construction of pDG-CotY-EDB Expression Vector

[0031] 1. Cloning of P43 Promoter Gene

[0032] A pair of primers for amplifying the P43 promoter gene was designed with reference to the published P43 promoter gene sequence (GenBank accession number: CP170734.1). A homologous arm sequence of 12 bp at the 3' end of the homologous arm on the left side of the Bacillus subtilis amylolytic site was introduced at the 5' end of the upstream primer (the underlined part is the homologous arm sequence), and a homologous arm sequence of 12 bp at the 5' end of the pCotY promoter was introduced at the 3' end of the downstream primer. The primer sequences are as follows:

[0033] Upstream primer P1: 5'- tctgatcgctag tgataggtggtatgttttcgct-3'

[0034] Downstream primer P2: 5'-tggtaccgctatcactttatat tgcttttaggtc -3'

[0035] The above primers were all synthesized by Sangon Biotech (Shanghai) Co., Ltd.;

[0036] Using the whole genome of Bacillus subtilis 168 (GenBank accession number: NC_000964) as a template for PCR amplification; the PCR reaction system was 50 μL: 25 μL of 2×Phanta Max Master Mix, 2 μL each of the upstream and downstream primers (10 μM), 5 μL of the template (200 ng / μL), and 16 μL of ddH2O. The PCR reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 1 min, for a total of 35 cycles; extension at 72°C for 5 min. The PCR amplification products were identified by 1.0% agarose gel electrophoresis and recovered, and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the obtained sequences were aligned with the sequences published on GenBank. The homology of the cloned gene sequence of the P43 promoter, SEQ ID NO.1, with the sequence published on GenBank was 100%.

[0037] 2. Cloning of the surface-displayed protein CotY and the pCotY promoter of Bacillus subtilis

[0038] Referring to the published whole genome sequence of Bacillus subtilis 168, two pairs of primers were designed to amplify the surface-displayed protein CotY and the pCotY promoter of Bacillus subtilis (GenBank accession number: L10116.1). A homologous arm sequence of 12 bp at the 3'-end of the P43 promoter gene was introduced at the 3'-end of the upstream primer P3 of the pCotY promoter (the underlined part is the homologous arm sequence), and a homologous arm sequence of 12 bp at the 5'-end of the surface-displayed protein CotY was introduced at the 3'-end of the downstream primer P4; a homologous arm sequence of 12 bp at the 3'-end of the pCotY promoter gene was introduced at the 5'-end of the upstream primer P5 of the surface-displayed protein CotY, and a homologous arm sequence of 12 bp at the 5'-end of EDoB was introduced at the 3'-end of the downstream primer P6. The primer sequences are as follows:

[0039] Upstream primer P3: 5'- aatgtacacatg gacctaaaagcagagctaaaaa-3'

[0040] Downstream primer P4: 5'-tgatttcagctccttctttata tccgcagctcat -3'

[0041] Upstream primer P5: 5'- gagctgaaatca atgactttgccatcattcaatg-3'

[0042] Downstream primer P6: 5'-tctttgcaggcactgtggaact gctgccaccgcc-3’

[0043] The above primers were all synthesized by Sangon Biotech (Shanghai) Co., Ltd.;

[0044] Using the whole genome of Bacillus subtilis 168 as a template for PCR amplification; the PCR reaction system was 50 μL: 25 μL of 2×PhantaMax Master Mix, 2 μL each of the upstream and downstream primers (10 μM), 5 μL of the template (200 ng / μL), and 16 μL of ddH2O. The PCR reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 1 min, for a total of 35 cycles; extension at 72°C for 5 min. The PCR amplification products were identified by 1.0% agarose gel electrophoresis and recovered, and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the obtained sequences were aligned with the sequences published on GenBank. The homology of the cloned gene fragments SEQ ID NO.2 and SEQ ID NO.3 of the Bacillus subtilis surface display protein CotY and the pCotY promoter with the sequences published on GenBank was 100%.

[0045] 3. Cloning of the left and right homologous arms of the amylase locus in Bacillus subtilis

[0046] Referring to the published whole genome sequence of Bacillus subtilis 168 (GenBank accession number: NC_000964), two pairs of primers were designed to amplify the left and right homologous arms of the amylase locus in Bacillus subtilis (GenBank accession number: AP019714.1). Primers P7 and P8 were designed to amplify the left homologous arm sequence of the amylase locus in Bacillus subtilis; primers P9 and P10 were designed to amplify the right homologous arm sequence of the amylase locus in Bacillus subtilis.

[0047] At the 5' end of the upstream primer P7 for the left homologous arm of the amylase locus in Bacillus subtilis, a 12-bp homologous arm sequence with the 3' end of the multiple cloning site of the pDG vector was introduced (the underlined part is the homologous arm sequence), and at the 3' end of the downstream primer P8, a 12-bp homologous arm sequence with the 5' end of the P43 promoter was introduced; at the 5' end of the upstream primer P9 for the right homologous arm of the amylase locus in Bacillus subtilis, a 12-bp homologous arm sequence with the 3' end of the spectinomycin resistance sequence was introduced, and at the 3' end of the downstream primer P10, a 12-bp sequence with the 5' end of the pDG vector multiple cloning site was introduced. The primer sequences are as follows:

[0048] Upstream primer P7: 5'- ggagtgtcaaga atgtttgcaaaacgattcaaaa-3’

[0049] Downstream primer P8: 5'-ctagcgatcagaccagtttttataccacctatca -3'

[0050] Forward primer P9: 5'- aaaaaattataa atgagcgatgatgatatccgtt-3'

[0051] Reverse primer P10: 5'-tcaatggggaagagaaccgctt ctagccttgccc -3'

[0052] The above primers were all synthesized by Sangon Biotech (Shanghai) Co., Ltd.;

[0053] Using the whole genome of Bacillus subtilis 168 as a template for PCR amplification; the PCR reaction system was 50 μL: 25 μL of 2×PhantaMax Master Mix, 2 μL each of the forward and reverse primers (10 μM), 5 μL of the template (200 ng / μL), and 16 μL of ddH2O. The PCR reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 1 min, for a total of 35 cycles; extension at 72°C for 5 min. The PCR amplification products were identified by 1.0% agarose gel electrophoresis and recovered, and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the obtained sequences were aligned with the sequences published on GenBank. The homology of the cloned left and right homologous arm sequences SEQ ID NO.4 and SEQ ID NO.5 of the amylase locus of Bacillus subtilis with the sequences published on GenBank was 100%.

[0054] 4. Cloning of the spectinomycin resistance gene

[0055] Referring to the published spectinomycin resistance gene sequence (GenBank accession number AB459504.1), primers for amplifying the spectinomycin resistance gene sequence were designed. A homologous arm sequence of 12 bp at the 3' end of the EDoB fragment sequence was introduced at the 5' end of the forward primer (the underlined part is the homologous arm sequence), and a homologous arm sequence of 12 bp at the 5' end of the right homologous arm sequence of the amylase locus of Bacillus subtilis was introduced at the 3' end of the reverse primer. The primer sequences are as follows:

[0056] Forward primer P11: 5'- catcatcattga atgtttggatcaggagttgaga-3'

[0057] Reverse primer P12: 5'-ttataatttttttaatctgtta atcatcgctcat -3'

[0058] The above primers were all synthesized by Sangon Biotech (Shanghai) Co., Ltd.;

[0059] PCR amplification was performed using the pPZP222 plasmid as a template; the PCR reaction system was 50 μL: 25 μL of 2×Phanta Max MasterMix, 2 μL each of the upstream and downstream primers (10 μM), 5 μL of the template (200 ng / μL), and 16 μL of ddH2O. The PCR reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 1 min, for a total of 35 cycles; extension at 72°C for 5 min. The PCR amplification products were identified by 1.0% agarose gel electrophoresis and recovered, and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the obtained sequences were aligned with the sequences published on GenBank. The homology of the cloned spectinomycin resistance gene sequence SEQ ID NO.6 with the sequence published on GenBank was 100%.

[0060] 7. Construction of the pDG-CotY-EDB expression vector

[0061] Using the gene fragments of EDB shown in SEQ ID NO.7, the gene fragments shown in SEQ ID NO.1, the gene fragments shown in SEQ ID NO.2, the gene fragments shown in SEQ ID NO.3, the gene fragments shown in SEQ ID NO.4, the gene fragments shown in SEQ ID NO.5, and the gene fragments of SEQ ID NO.6 synthesized by Nanjing Ainuodi Biotechnology Co., Ltd., which contain the CoE region of the PEDV S protein, M cell and dendritic cell targeting peptides, with flexible amino acid sequences (GGGSTS) added between each fragment and a His tag added at the end, as templates, overlapping PCR amplification was performed with primers P7 and P10. The PCR reaction system was 50 μL: 25 μL of 2×Phanta Max Master Mix, 2 μL each of the upstream and downstream primers (10 μM), 1 μL each of the templates (200 ng / μL), and 14 μL of ddH2O. The PCR reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 1 min, for a total of 35 cycles; extension at 72°C for 5 min. The PCR amplification products were identified by 1.0% agarose gel electrophoresis and recovered, and then cloned into the pDG vector using the One Step Cloning Kit site-directed cloning kit. After sequencing, the expression vector pDG-CotY-EDB was obtained, as Figure 1As shown. Among them, the full-length sequence of the gene fragment after combining the gene fragment of EDB in the expression vector pDG-CotY-EDB, the gene fragment shown by the SEQ ID NO.1 sequence, the gene fragment shown by the SEQ ID NO.2 sequence, the gene fragment shown by the SEQ ID NO.3 sequence, the gene fragment shown by the SEQ ID NO.4 sequence, the gene fragment shown by the SEQ ID NO.5 sequence, the gene fragment shown by the SEQ ID NO.6 sequence and the gene fragment of the SEQ ID NO.7 sequence is as shown by the SEQ ID NO.8.

[0062] Example 2 Transformation of Recombinant Plasmid

[0063] Take 100 μL of electrotransformation-competent cells of Bacillus subtilis WB800 and mix with 1 μL (100 ng / μL) of the recombinant expression vector pDG-CotY-EDB plasmid, add them to an electroporation cuvette, and perform electroporation after ice-bathing for 5 min. Electroporation conditions: 22 KV / cm, 25 μF, 200 Ω, electroporate once. Add 1 mL of electroporation recovery solution, incubate at 37 °C and 100 rpm for 3 h, and spread on a solid medium LB plate with spectinomycin resistance. Positive colonies can be seen after 12 h - 16 h, which are the recombinant Bacillus subtilis B.S.-EDB.

[0064] Example 3 Verification of Recombinant Bacterial Genome PCR and Protein Expression

[0065] Pick a single colony B.S.-EDB from the transformation plate into a liquid LB medium with spectinomycin resistance (100 μg / mL), and place it in a constant temperature shaker at 37 °C and 200 rpm / min for shaking culture for 6 h. Design two pairs of primers P13: 5’-atgactttgccatcattcaatg-3’ and P14: 5’-tctttgcaggcactgtggaact-3’ according to the inserted PEDV S protein CoE region, and use primers P13 and P14 for PCR verification. The PCR reaction system is 50 μL: 2×Phanta Max Master Mix 25 μL, 2 μL each of the upstream and downstream primers (10 μM), 5 μL of the bacterial solution, and 16 μL of ddH2O. PCR reaction conditions: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 1 min, a total of 35 cycles; extension at 72 °C for 5 min. The results are as Figure 2 shown in A: Lanes 1 - 3 are the bacterial solutions, lane 4 is the blank medium control. It can be seen that single bands appear between 500 - 750 bp in lanes 2 and 3, which is consistent with the size of the PEDV S protein CoE region, indicating that the recombinant Bacillus subtilis B.S.-EDB is successfully constructed.

[0066] The normal Bacillus subtilis WB800 strain (without exogenous genes in its genetic material) (B.S.) and the recombinant Bacillus subtilis WB800 strain (B.S.-EDB) expressing surface-displayed protein CotY, intestinal M-like cell-targeting peptide Col, dendritic cell-targeting peptide DCpep, and the CoE region of PEDV S protein were treated with lysozyme (10 mg / mL) at 37 °C for 30 min to break the cell walls, and then centrifuged to collect the precipitate. The precipitate was resuspended with spore lysis buffer, placed on ice, sonicated, and then centrifuged at low temperature for 10 min. After centrifugation, the precipitate was resuspended with PBS to obtain the spore coat protein solution. 200 μL of the extracted spore coat protein solution was taken, 50 μL of 5X SDS-PAGE Loading buffer was added, mixed well, boiled at 100 °C for 10 min, and then Western-blot analysis was performed on the expression product. After SDS electrophoresis, protein transfer was carried out; the transferred NC membrane was blocked with 5% skim milk, incubated overnight with mouse anti-His tag antibody; after washing, appropriately diluted horseradish peroxidase-labeled rabbit anti-mouse IgG was added, and ECL chemiluminescence solution was used for exposure and photography. The results are as Figure 2 shown in Figure B: 1 is the whole cell of recombinant Bacillus subtilis B.S.-EDB, 2 is the supernatant after sonication and centrifugation of the whole cell of recombinant Bacillus subtilis B.S.-EDB, 3 is the precipitate after sonication and centrifugation of the whole cell of recombinant Bacillus subtilis B.S.-EDB, and 4 is the whole cell control of Bacillus subtilis WB800. It can be seen from the figure that there is an obvious protein blot band at 40 kD in lanes 1 and 3, and no non-specific bands appear, indicating that the surface-displayed protein CotY, intestinal M-like cell-targeting peptide Col, dendritic cell-targeting peptide DCpep, and the CoE region of PEDV S protein were successfully expressed in recombinant Bacillus subtilis B.S.-EDB.

[0067] Example 4 Recombinant Bacillus subtilis can effectively induce virus-specific intestinal mucosal immune responses and systemic immune responses

[0068] The recombinant Bacillus subtilis WB800 strain (B.S.-EDB) expressing PEDV S protein in tandem and targeting intestinal M-like cells and dendritic cells, the recombinant Bacillus subtilis B.S.-RCL (Chinese Patent Application No. CN108265073B) expressing intestinal M-like cell-targeting peptide (L-lectin-β-GF) and porcine epidemic diarrhea virus (PEDV) S protein constructed in our laboratory before, and Bacillus subtilis WB800 were used for oral immunization experiments on 6-week-old mice. The results showed that B.S.-EDB could effectively induce virus-specific intestinal mucosal immune responses and systemic immune responses. The animal experiments and immune detection methods are as follows:

[0069] Mice were orally immunized with recombinant Bacillus subtilis (B.S.-EDB), and Bacillus subtilis WB800 (B.S.) and recombinant Bacillus subtilis B.S.-RCL expressing intestinal M-like cell-targeting peptide (L-lectin-β-GF) and porcine epidemic diarrhea virus (PEDV) S protein constructed in our laboratory previously were used as control bacteria to explore the induction effects of recombinant Bacillus subtilis on intestinal mucosal immunity and systemic immunity. Mice were boosted 7 days after the first immunization, and boosted again 14 days later. Each mouse was intragastrically administered 200 μL of recombinant Bacillus subtilis containing 1×10 10 CFU each time. Mouse sera and intestinal lavage fluids were collected for detection respectively. The levels of specific IgG in mouse sera and specific SIgA in intestinal lavage fluids were detected by indirect ELISA method. The results of the immunization test are as Figure 3 shown. Recombinant Bacillus subtilis can rapidly induce the levels of specific SIgA and specific IgG in sera. It is indicated that the expressed intestinal M cell-targeting peptide Col and dendritic cell-targeting peptide DCpep can effectively bind to intestinal M cells and dendritic cells, enhancing the level of local intestinal mucosal immune response. At the same time, the results show that the immune effect of the modified recombinant Bacillus subtilis B.S.-EDB is more significant than that of the recombinant Bacillus subtilis B.S.-RCL constructed in our laboratory previously.

[0070] Example 5 Recombinant bifunctional Bacillus subtilis (B.S.-EDB) expressing the core antigen region of PEDV S protein and targeting peptides induces highly efficient virus-specific milk-derived immune protection

[0071] After immunizing sows with recombinant Bacillus subtilis WB800 strain (B.S.-EDB) expressing PEDV S protein in tandem and targeting intestinal M-like cells and dendritic cells and its control recombinant Bacillus subtilis WB800 strain (B.S.), the levels of specific antibodies in colostrum were significantly increased, and at the same time, neonatal piglets were effectively protected against PEDV infection. The animal experiment and immunological detection methods are as follows:

[0072] Fifteen healthy sows randomly selected from a pig farm were divided into 3 groups, with 5 sows in each group: blank control group; B.S.-EDB group, the sows were fed 2 mL of recombinant Bacillus subtilis expressing PEDV S protein (1×10 10 CFU / mL) on the 80th day of pregnancy, and repeated feeding once 14 days later; B.S group, the sows were fed 2 mL of Bacillus subtilis WB800 (1×10 10 CFU / mL) on the 80th day of pregnancy, and repeated feeding once 14 days later.

[0073] After the sows were orally administered recombinant Bacillus subtilis, specific IgA + and IgG +Plasmablasts migrate to the mammary gland tissue through the gut-mammary-SIgA axis. After reaching the mammary gland, the antibodies secreted by these cells are transported into the milk through epithelial cells. This invention explored the effect of orally administered recombinant Bacillus subtilis in sows on the resistance of neonatal piglets to PEDV infection. Piglets suckled breast milk freely after birth. After 3 days of age, 3 piglets were randomly selected from each group and orally infected with PEDV, and the clinical symptoms of the piglets were observed. All piglets were euthanized 36 h after infection. Blood, duodenum, jejunum and ileum were collected for related detections. It was found that piglets in the Bacillus subtilis WB800 group (B.S. group) showed typical PEDV clinical symptoms at 36 h, including acute watery diarrhea, depression and decreased appetite, while piglets in the recombinant Bacillus subtilis group (B.S.-EDB group) and the Mock group did not show diarrhea symptoms. By dissection, it was found that there were no obvious pathological symptoms in the intestines of piglets in the Mock group (no virus infection, used as a negative control) and the B.S.-EDB group. The intestinal contents of the piglets were full and the intestinal wall showed no change, but there was no content in the intestines of piglets in the B.S. group and the intestinal wall became thin and transparent( Figure 4 A). Immunofluorescence results showed that no PEDV-positive cells were observed in the jejunal villus epithelial cells of piglets in the B.S.-EDB group, while a large number of PEDV-positive cells were found in the jejunum of piglets in the B.S group( Figure 4 A). Finally, the virus distribution in the intestines of piglets was detected by RT-qPCR. The results showed that PEDV-positive cells were mainly present in the jejunum and ileum of piglets, and the viral RNA content in the jejunum and ileum of piglets in the B.S.-EDB group was significantly lower than that in the B.S. group( Figure 4 B).

Claims

1. A recombinant expression vector, characterized in that, The recombinant expression vector comprises a gene fragment of the P43 promoter, a gene fragment of the pCotY promoter, a gene fragment encoding the surface-displayed protein CotY, a gene fragment of the CoE region of the PEDV S protein, and a gene fragment encoding a targeting peptide, wherein the targeting peptide comprises a gut M-like cell targeting peptide Col and a dendritic cell targeting peptide DCpep.

2. The recombinant expression vector according to claim 1, wherein The nucleotide sequences of the gene fragment of the CoE region of the PEDV S protein and the gene fragment encoding the targeting peptide are as shown in SEQ ID NO.

7.

3. The recombinant expression vector according to claim 1, wherein The nucleotide sequence of the gene fragment of the P43 promoter is as shown in SEQ ID NO.1, the nucleotide sequence of the gene fragment encoding the surface-displayed protein CotY is as shown in SEQ ID NO.2, and the nucleotide sequence of the gene fragment of the pCotY promoter is as shown in SEQ ID NO.

3.

4. The method for constructing the recombinant expression vector according to any one of claims 1 to 3, characterized in that, It includes the following steps: Obtain a recombinant gene fragment by overlapping PCR with the gene fragment of the P43 promoter, the gene fragment encoding the surface-displayed protein CotY, the gene fragment of the pCotY promoter, the CoE region of the PEDV S protein, the gene fragment encoding the targeting peptide including the gut M-like cell targeting peptide Col, and the gene fragment encoding the dendritic cell targeting peptide DCpep, and ligate the recombinant gene fragment into a vector.

5. The construction method of the recombinant expression vector according to claim 4, wherein, The primers P7 and P10 in the overlapping PCR amplification, the sequence of P7 is: 5'- ggagtgtcaaga atgtttgcaaaacgattcaaaa-3’, and the primer sequence of P10 is: 5’-tcaatggggaagagaaccgctt ctagccttgccc -3’.

6. A recombinant strain, characterized in that, It contains the recombinant expression vector according to any one of claims 1 to 3. Preferably, the recombinant strain includes recombinant Bacillus subtilis.

7. The method for constructing the recombinant strain according to claim 6, characterized in that, The construction method is obtained by introducing the recombinant expression vector according to any one of claims 1 to 3 into a host bacterium. Preferably, the host bacterium includes Bacillus subtilis.

8. Use of the recombinant expression vector according to any one of claims 1 to 3 or the recombinant strain according to claim 6 in the preparation of a drug or vaccine for preventing or treating diseases related to PEDV infection.

9. A genetically engineered live vector vaccine, characterized in that, The genetically engineered live vector vaccine includes the recombinant expression vector according to any one of claims 1 to 3 or the recombinant strain according to claim 6.

10. The genetically engineered live vector vaccine according to claim 9, wherein, The genetically engineered live vector vaccine is an oral vaccine.

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