Recombinant bifunctional bacillus subtilis with surface display and tandem expression of PEDV and PDCoV S proteins and application of recombinant bifunctional bacillus subtilis

Abstract

The invention discloses a pDGT-CotY-EDoB expression vector as well as a construction method and application of the pDGT-CotY-EDoB expression vector. The invention also discloses a recombinant strain as well as a construction method and application thereof. The invention also discloses a genetic engineering live vaccine. The invention develops a novel live vector vaccine capable of targeting the intestinal mucosa key immune presentation cell function, and provides efficient intestinal mucosa immune protection for suckling piglets by enhancing the intestinal-mammary axis function of sows. The research result not only provides an effective strategy for effectively preventing and controlling the occurrence of porcine virus diarrhea clinically, but also provides a new thought for successfully realizing zero burden of piglet disease diarrhea in the pig industry in China in future.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and genetic engineering, and particularly relates to a recombinant bifunctional Bacillus subtilis with surface-displayed tandem expression of PEDV and PDCoV S proteins and its application. Background Art

[0002] Oral mucosal immunization, as a unique immune pathway, has many significant advantages. First of all, it can directly induce immune responses on the mucosal surface, effectively preventing the invasion and spread of pathogens. Through oral mucosal immunization, local mucosal immune responses can be stimulated to produce secretory IgA antibodies, preventing pathogens from colonizing and infecting on the mucosal surface. This local immune response can not only quickly respond to pathogen challenges but also provide protection throughout the body, achieving a dual protection effect. Secondly, oral mucosal immunization is non-invasive and convenient. Compared with traditional injectable vaccines, oral vaccines do not require needle injection, reducing the stress response of animals and improving the acceptance and safety of vaccination. This vaccination method not only avoids the risk of cross-infection caused by repeated use of needles but also reduces the dependence on professional personnel, allowing for more frequent booster immunizations. In addition, the storage and transportation of oral vaccines are also more convenient, without the need for strict cold chain conditions, which helps to promote their use in resource-limited areas. Moreover, oral mucosal immunization can induce both systemic and mucosal immune responses. Studies have shown that oral vaccines can simultaneously stimulate local mucosal immunity and systemic immunity, providing more comprehensive protection. This dual immune response can not only effectively prevent the occurrence of diseases but also reduce the spread of pathogens to a certain extent, which is of great significance for controlling the prevalence of infectious diseases. Finally, oral mucosal immunization can also induce immune tolerance in some cases, which has potential application value for the treatment of autoimmune diseases and allergic reactions. By oral administration of immunogens, intestinal mucosal and systemic immune tolerance can be established to inhibit excessive immune responses, providing new ideas for the treatment of related diseases.

[0003] In the field of livestock and poultry production, Bacillus subtilis can produce a large number of digestive enzymes after entering the digestive tract to make up for the deficiency and insufficiency of endogenous enzymes in the body. In addition, Bacillus subtilis can also antagonize anti-nutritional factors in feed, improve the absorption rate of nutrients by the body, and play a positive role in promoting the growth of livestock and poultry. Bacillus subtilis can increase the activity of immune cells, has an anti-inflammatory effect, and can enhance the resistance of livestock and poultry to various diseases. At the same time, Bacillus subtilis also plays a significant role in improving the balance of the intestinal flora of livestock and poultry and reducing the pollution caused by livestock and poultry manure. The research on Bacillus subtilis is relatively mature, its genome sequence has been completely decoded, and gene manipulation is simple. Bacillus subtilis has a high efficiency of exogenous protein expression and can directly secrete exogenous gene products into the extracellular space, which is beneficial to the purification and recovery of secreted proteins. Compared with the Escherichia coli expression system, Bacillus subtilis, which belongs to Gram-negative bacteria, has no endotoxin (lipopolysaccharide) in its cell wall, has higher safety, and does not require the step of removing endotoxin when processing the expressed protein subsequently, greatly simplifying the subsequent technology and operation.

[0004] At present, the B. subtilis spore surface display exogenous protein system is more common and perfect. At the same time, recombinant expression of exogenous proteins does not affect the structure of B. subtilis spores and their performance of surviving in extremely harsh environments. And the fused protein can be surface-displayed without passing through the membrane barrier. Displaying antigens on the spore surface can also be used for immunization and can germinate in host phagocytes to produce an efficient antigen presentation process. In 2001, scholars such as Medaglini successfully fused CotB as a molecular carrier with the C unit of tetanus toxin for the first time, and this research result provided new ideas for the development of new drugs and vaccines. In 2007, scholar Seok Joon Kwon successfully fused β-galactosidase with the spore coat protein CotG and displayed it on the spore surface, and this recombinant spore had β-galactosidase catalytic activity in the water-organic phase reaction system.

[0005] Porcine epidemic diarrhea virus belongs to the order Nidovirales, family Coronaviridae, and is one of the alphacoronaviruses. Its genetic material is single-stranded positive-sense RNA, enclosed by an envelope, and the genome is approximately 28 kb in length. Among them, the S protein is closely related to the immune response of the body and can induce the production of neutralizing antibodies in the body. The S protein is a membrane protein located on the surface of the virus particle. During the process of invading host cells, it first recognizes and binds to the receptor on the surface of the host cell, stimulating the host cell to release neutralizing antibodies. Chang et al. named the 499-638aa region of the porcine epidemic diarrhea virus S protein as the COE region in 2002, which is an extremely important antigenic epitope. PDCoV, namely porcine deltacoronavirus, is a novel coronavirus that causes intestinal diarrhea in pigs. Woo et al. first detected this virus in a batch of fecal samples in 2012. The immunodominant neutralizing region of the PDCoV S protein has not been determined. Shang et al. found that the topological structure of the S1-CTD region of the PDCoV S protein is consistent with that of the S1-CTD regions of alpha and beta coronaviruses, and an unknown receptor on the surface of mammals can also bind to it. Thus, it is speculated that the immunodominant neutralizing region of the PDCoV S protein is also located in the S1-CTD segment. Chen et al. expressed three truncated PDCoV S proteins using the Escherichia coli expression system, namely the N-terminal domain (NTD, amino acids 50-286aa) of the S1 subunit, the C-terminal domain (CTD, 278-616aa) of the S1 subunit, and the S2 subunit (601-1087aa). Then, polyclonal antibodies against these three recombinant proteins were generated in rabbits and mice. The results showed that the virus neutralization effect of the CTD-specific antibody was the strongest among the three. Thus, it can also be speculated that the CTD region may contain the immunodominant neutralizing region of the PDCoV S protein.

[0006] Globally, PEDV and PDCoV are two enteroviruses that have a significant impact on the pig industry and have a certain prevalence. The antigens of these two viruses are mainly the S protein. However, due to the high variability of the S protein, this poses higher requirements for vaccine development. Therefore, it is particularly important to construct an efficient porcine diarrhea virus vaccine research system. In addition, due to the existence of multiple subtypes of porcine diarrhea virus, the vaccines on the market cannot guarantee good immune effects against all subtypes, which is also a limitation of existing vaccines. Therefore, it is particularly important to explore new vaccine prevention methods.

[0007] Both porcine deltacoronavirus disease and porcine epidemic diarrhea mainly occur in neonatal piglets. Immunizing neonatal piglets cannot generate protective immunity in a timely manner. Only by orally or nasally immunizing sows, and through the intestinal-mammary gland-SIgA axis or the intestinal-mammary gland-SIgA axis to produce specific antibodies and related cytokines, and transferring immunity to neonatal piglets through milk can the purpose of protecting neonatal piglets be achieved. 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 presentation carrier for oral immunization.

[0008] 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, the problem of protease degradation, and the negative impact of foreign proteins on the growth of the strain. Summary of the Invention

[0009] Object of the Invention: The technical problem to be solved by the present invention is to provide a pDGT-CotY-EDoB expression vector and its construction method.

[0010] Another technical problem to be solved by the present invention is to provide a recombinant strain containing the pDGT-CotY-EDoB expression vector and its construction method.

[0011] Another technical problem to be solved by the present invention is to provide the application of the expression vector or the recombinant strain in the preparation of drugs or vaccines for preventing or treating diseases related to PEDV and / or PDCoV infection.

[0012] The last technical problem to be solved by the present invention is to provide a genetic engineering live vaccine.

[0013] Technical Solution: To solve the above technical problems, the present invention provides a pDGT-CotY-EDoB expression vector. The pDGT-CotY-EDoB expression vector includes the CoE region of the PEDV S protein, the CTD region of the PDCoV S protein, the targeting peptide Col for intestinal M-like cells, and the targeting peptide DCpep for dendritic cells. The nucleotide sequence of the CoE region of the PEDV S protein is as shown in SEQ ID NO.1, the nucleotide sequence of the CTD region of the PDCoV S protein is as shown in SEQ ID NO.2, the nucleotide sequence of the targeting peptide DCpep for dendritic cells is as shown in SEQ ID NO.6, and the nucleotide sequence of the targeting peptide Col for intestinal M-like cells is as shown in SEQ ID NO.7.

[0014] Among them, the pDGT-CotY-EDoB expression vector further comprises a promoter P43, a gene encoding a surface-displayed protein CotY, and a promoter pCotY. The nucleotide sequence of the promoter P43 is as shown in SEQ ID NO.3, the nucleotide sequence of the gene encoding the surface-displayed protein CotY is as shown in SEQ ID NO.4, and the nucleotide sequence of the promoter pCotY is as shown in SEQ ID NO.5.

[0015] The present invention also includes a method for constructing the pDGT-CotY-EDoB expression vector, which comprises the following steps: introducing the CoE region of the PEDV S protein, the CTD region of the PDCoV S protein, the intestinal M-like cell targeting peptide Col, and the dendritic cell targeting peptide DCpep into a plasmid.

[0016] Specifically, the method for constructing the pDGT-CotY-EDoB expression vector specifically comprises the following steps:

[0017] 1) Obtaining the gene fragments of the CoE region of the PEDV S protein, the CTD region of the PDCoV S protein, the intestinal M-like cell targeting peptide Col, the dendritic cell targeting peptide DCpep, the promoter P43, the gene encoding the surface-displayed protein CotY, and the promoter pCotY respectively;

[0018] 2) Using the gene fragments obtained in step 1) as templates, performing overlapping PCR amplification to obtain a recombinant gene fragment;

[0019] 3) Connecting the recombinant gene fragment to a vector.

[0020] Among them, in the overlapping PCR amplification, the primers P7 and P10, the sequence of P17 is: 5’-atgtttgcaaaacgattcaaaa-3’, and the primer sequence of P10 is: 5’-tcaatggggaagagaaccgctt-3’.

[0021] The present invention also includes a recombinant strain, which contains the pDGT-CotY-EDoB expression vector. Preferably, the recombinant strain includes recombinant Bacillus subtilis.

[0022] The present invention also includes a method for constructing the recombinant strain, which comprises introducing the pDGT-CotY-EDoB expression vector into a host bacterium. Preferably, the host bacterium includes Bacillus subtilis.

[0023] The present invention also includes the application of the expression vector or the recombinant strain in the preparation of a drug or vaccine for preventing or treating diseases related to PEDV and / or PDCoV infection.

[0024] The present invention also includes a genetically engineered live vaccine, which comprises the pDGT-CotY-EDoB expression vector or the recombinant strain as described above.

[0025] Among them, the genetically engineered live vaccine is an oral vaccine.

[0026] 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 integrated tandem expression of the antigen neutralizing epitopes of porcine epidemic diarrhea virus and porcine deltacoronavirus, as well as a recombinant Bacillus subtilis targeting intestinal M cells and dendritic cells (DCs). By orally immunizing sows with this recombinant live vector vaccine, by enhancing the function of the sow intestinal-mammary axis, it provides high-efficiency intestinal mucosal immune protection for suckling piglets, and further provides a safe, simple and immunization route for effectively preventing the occurrence of clinical viral porcine diarrhea diseases, and also provides new ideas for successfully achieving "zero burden" of piglet diarrhea in the future pig farming industry in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a construction diagram of the pDGT-CotY-EDoB expression vector;

[0028] Figure 2 It is a PCR identification and Western-blot diagram of the expression product of recombinant Bacillus subtilis B.S.-EDoB;

[0029] Figure 3 It is an immunization effect diagram of recombinant Bacillus subtilis B.S.-EDoB in mice;

[0030] Figure 4 It is an immunization effect diagram of recombinant Bacillus subtilis B.S.-EDoB in sows;

[0031] Figure 5 It is a result diagram of the PEDV challenge experiment in piglets after immunizing sows with recombinant Bacillus subtilis B.S.-EDoB;

[0032] Figure 6 It is a result diagram of the PDCoV challenge experiment in piglets after immunizing sows with recombinant Bacillus subtilis B.S.-EDoB. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The present invention will be further described below with reference to 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 in the embodiments usually follow conventional conditions or the conditions recommended by the manufacturer.

[0034] The Bacillus subtilis WB800 strain and pDG expression plasmid (Lu Z, Yang S. Nucleic Acids Res, 2019) used in this invention were kindly provided by Teacher Yan Xin of the College of Life Sciences, Nanjing Agricultural University; the pJOE-8999 plasmid (Yu C, Chen H. Microbiol Spectr. 2023) and pPZP222 plasmid (Jin H, Hong Z. Proc Natl Acad Sci U S A. 2009) used in this invention were kindly provided by Teacher Gao Xuewen of the College of Plant Protection, Nanjing Agricultural University; the gene fragment EDoB (shown in SEQ ID NO. 12) containing the CoE region of the PEDV S protein, the CTD region of the PDCoV S protein, the M-like cell targeting peptide (Col), and the dendritic cell targeting peptide (DCpep), with flexible amino acid sequences (GGGSTS) added between each fragment and a His tag added at the end, used in the examples of this invention was synthesized by Nanjing Ainuodi Biotechnology Co., Ltd.

[0035] The data analysis of this invention adopted the single-factor ANOVA variance test results of SPSS 29.0 software, and statistical analysis was performed on the experimental data. The statistical results were expressed as mean ± standard error (Mean ± SE) for error. The criteria for judging significant differences were: P < 0.05, significant difference (*); P < 0.01, extremely significant difference (**); P < 0.001.

[0036] Example 1 Construction of the pDGT-CotY-EDoB expression vector

[0037] 1. Cloning of the P43 promoter gene

[0038] 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 amylo-lytic 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:

[0039] Upstream primer P1: 5'- tctgatcgctag tgataggtggtatgttttcgct-3'

[0040] Downstream primer P2: 5'-tggtaccgctatcactttatat tgcttttaggtc -3'

[0041] All of the above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.;

[0042] Using the complete 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. 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, 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 gene sequence SEQ ID NO.3 of the cloned P43 promoter had 100% homology with the sequences already published on GenBank.

[0043] 2. Cloning of the surface-displayed protein CotY and promoter pCotY of Bacillus subtilis

[0044] Two pairs of primers for amplifying the surface-displayed protein CotY and promoter pCotY (GenBank accession number: L10116.1) of Bacillus subtilis were designed with reference to the published complete genome sequence of Bacillus subtilis 168. 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 promoter pCotY (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 P43 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:

[0045] Upstream primer P3: 5'- aatgtacacatg gacctaaaagcagagctaaaaa-3'

[0046] Downstream primer P4: 5'-tgatttcagctccttctttata tccgcagctcat -3'

[0047] Upstream primer P5: 5'- gagctgaaatca atgactttgccatcattcaatg-3'

[0048] Downstream primer P6: 5'-tctttgcaggcactgtggaact gctgccaccgcc -3'

[0049] All of the above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.;

[0050] PCR amplification was carried out using the whole genome of Bacillus subtilis 168 as a template; the PCR reaction system was 50 μL: 25 μL of 2×PhantaMax Master Mix, 2 μL each of upstream and downstream primers (10 μM), 5 μL of template (200 ng / μL), 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, 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 gene fragment sequences SEQ ID NO.4 and SEQ ID NO.5 of the surface-displayed protein CotY and the promoter pCotY of Bacillus subtilis cloned was 100% with the sequences published on GenBank.

[0051] 3. Cloning of the left and right homologous arms of the amylase locus of Bacillus subtilis

[0052] Two pairs of primers were designed to amplify the left and right homologous arms of the amylase locus (GenBank accession number: AP019714.1) of Bacillus subtilis with reference to the published whole genome sequence of Bacillus subtilis 168 (GenBank accession number: NC_000964). Primers P7 and P8 were designed to amplify the left homologous arm sequence of the amylase locus of Bacillus subtilis; primers P9 and P10 were designed to amplify the right homologous arm sequence of the amylase locus of Bacillus subtilis.

[0053] A homologous arm sequence of 12 bp at the 3' end of the multiple cloning site of the pDG vector was introduced at the 5' end of the upstream primer P7 for the left homologous arm of the amylase locus of Bacillus subtilis (the underlined part is the homologous arm sequence), and a homologous arm sequence of 12 bp at the 5' end of the P43 promoter was introduced at the 3' end of the downstream primer P8; a homologous arm sequence of 12 bp at the 3' end of the spectinomycin resistance sequence was introduced at the 5' end of the upstream primer P9 for the right homologous arm of the amylase locus of Bacillus subtilis, and a homologous arm sequence of 12 bp at the 5' end of EDoB was introduced at the 3' end of the downstream primer P10. The primer sequences are as follows:

[0054] Upstream primer P7: 5'- ggagtgtcaaga atgtttgcaaaacgattcaaaa-3'

[0055] Downstream primer P8: 5'-ctagcgatcagaccagttttta taccacctatca -3'

[0056] Upstream primer P9: 5'- aaaaaattataaatgagcgatgatgatatccgtt-3’

[0057] Downstream primer P10: 5’-tcaatggggaagagaaccgctt ctagccttgccc -3’

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

[0059] Using the whole genome of Bacillus subtilis 168 as a template for PCR amplification; The PCR reaction system was 50 μL: 2×PhantaMax Master Mix 25 μL, 2 μL each of upstream and downstream primers (10 μM), 5 μL of template (200 ng / μL), 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, 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 left and right homologous arm sequences SEQ ID NO.8 and SEQ ID NO.9 of the amylase locus of the cloned Bacillus subtilis with the sequences published on GenBank was 100%.

[0060] 4. Cloning of the spectinomycin resistance gene

[0061] 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 upstream 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 downstream primer. The primer sequences are as follows:

[0062] Upstream primer P11: 5’- catcatcattga atgtttggatcaggagttgaga-3’

[0063] Downstream primer P12: 5’-ttataatttttttaatctgtta atcatcgctcat -3’

[0064] The above primers were all synthesized by Sangon Biotech (Shanghai) Co., Ltd.;

[0065] PCR amplification was carried out 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. 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, 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, 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.10 with the sequence already published on GenBank was 100%.

[0066] 7. Construction of the pDGT-CotY-EDoB expression vector

[0067] Using the gene fragments of EDoB shown in SEQ ID NO.12, the gene fragment shown in SEQ ID NO.3, the gene fragment shown in SEQ ID NO.4, the gene fragment shown in SEQ ID NO.5, the gene fragment shown in SEQ ID NO.8, the gene fragment shown in SEQ ID NO.9, and the gene fragment of SEQ ID NO.10 synthesized by Nanjing Ainuodi Biotechnology Co., Ltd., which contain the CoE region of the PEDV S protein, the CTD region of the PDCoV 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 carried out 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. 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, 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 cloned into the pDG vector using the One Step Cloning Kit site-directed cloning kit. After sequencing, the expression vector pDGT-CotY-EDoB was obtained, as Figure 1As shown. Among them, the full-length sequence of the gene fragment after combining the gene fragment of EDoB in the expression vector pDGT-CotY-EDoB, the gene fragment shown in the SEQ ID NO.3 sequence, the gene fragment shown in the SEQ ID NO.4 sequence, the gene fragment shown in the SEQ ID NO.5 sequence, the gene fragment shown in the SEQ ID NO.8 sequence, the gene fragment shown in the SEQ ID NO.9 sequence, and the gene fragment of the SEQ ID NO.10 sequence is as shown in SEQ ID NO.11.

[0068] Example 2 Transformation of Recombinant Plasmid

[0069] Take 100 μL of electrocompetent cells of Bacillus subtilis WB800 and mix with 1 μL (100 ng / μL) of the recombinant expression vector pDGT-CotY-EDoB plasmid, add them to an electroporation cuvette, incubate on ice for 5 min, and then perform electroporation. 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.-EDoB.

[0070] Example 3 Verification of Recombinant Bacterial Genome PCR and Protein Expression

[0071] Pick a single colony from the transformation plate and transfer it to a liquid LB medium with spectinomycin resistance (100 μg / mL), place it in a constant temperature shaker at 37 °C and 200 rpm / min, and shake and culture for 6 h. Use primers P5 and P8 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-denature at 95 °C for 3 min; denature at 95 °C for 15 s, anneal at 60 °C for 15 s, extend at 72 °C for 1 min, for a total of 35 cycles; extend at 72 °C for 5 min. The results are as Figure 2 shown in A: Lanes 1 to 4 are the bacterial solutions, and lane 5 is the blank medium control. It can be seen that single bands appear between 2000 - 3000 bp in lanes 1, 3, and 4, which is consistent with the size of the tandem PEDV and PDCoV S proteins, indicating that the recombinant Bacillus subtilis B.S.-EDoB is successfully constructed.

[0072] The normal Bacillus subtilis WB800 strain (without exogenous genes in its genetic material) (B.S.) and the recombinant Bacillus subtilis WB800 strain (B.S.-EDoB) displaying surface proteins CotY, intestinal M-like cell targeting peptide Col, dendritic cell targeting peptide DCpep, the CoE region of PEDV S protein, and the CTD region of PDCoV 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. 200 μL of the extracted spore coat protein solution was taken, 5 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 products. 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 chemiluminescent solution was used for exposure and photography. The results are as Figure 2 shown in Figure B: 1 is the whole cell control of Bacillus subtilis WB800, 2 is the whole cell of recombinant Bacillus subtilis B.S.-EDoB, 3 is the supernatant after sonication and centrifugation of the whole cell of recombinant Bacillus subtilis B.S.-EDoB, and 4 is the precipitate after sonication and centrifugation of the whole cell of recombinant Bacillus subtilis B.S.-EDoB. It can be seen from the figure that there is an obvious protein blot band at 70 kD in lanes 2 and 4, and no non-specific bands appear, proving that the surface-displayed proteins CotY, intestinal M-like cell targeting peptide Col, dendritic cell targeting peptide DCpep, the CoE region of PEDV S protein, and the CTD region of PDCoV S protein were successfully expressed in the recombinant Bacillus subtilis B.S.-EDoB.

[0073] Example 4 Recombinant Bacillus subtilis can effectively induce virus-specific intestinal mucosal immune responses and systemic immune responses

[0074] Recombinant Bacillus subtilis WB800 strain (B.S.-EDoB) expressing PEDV and PDCoV S proteins in tandem, as well as targeting intestinal M-like cells and dendritic cells, and amplifying the promoter P43, surface display protein CotY, intestinal M-like cell targeting peptide Col, dendritic cell targeting peptide DCpep, PEDV S protein CoE region, and PDCoV S protein CTD region using pDG-CotY-EDoB as a template, and ligating them to the pJOE8999 vector containing sgRNA using T4 DNA Ligase, and transforming them into Bacillus subtilis WB800 to obtain the control recombinant Bacillus subtilis WB800 strain (B.S.-pJOE-EDoB) and performing an oral immunization test on 6-week-old mice with Bacillus subtilis WB800. The results showed that B.S.-EDoB could effectively induce virus-specific intestinal mucosal immune responses and systemic immune responses. The animal experiment and immunodetection methods are as follows:

[0075] Mice were orally immunized with recombinant Bacillus subtilis (B.S.-EDoB), and Bacillus subtilis WB800 and the control recombinant Bacillus subtilis WB800 strain (B.S.-pJOE-EDoB) constructed using the pJOE-8999 vector were used as controls to explore the induction effect of recombinant Bacillus subtilis on intestinal mucosal immunity and systemic immunity. Seven days after the first immunization, the mice were given a second immunization, and 14 days later, a third immunization. Each mouse was gavaged with 200 μL of recombinant Bacillus subtilis containing 1×10 10 CFU each time, and the sera and intestinal lavage fluids of the mice were collected for detection. The COE region of the PEDV S protein and the CTD region of the PDCoV S protein expressed in prokaryotes were used as coating antigens, and the levels of specific IgG in the sera of the mice and specific SIgA in the intestinal lavage fluids were detected by the indirect ELISA method. The results of the immunization experiment are as Figure 3 shown. Recombinant Bacillus subtilis could rapidly induce the levels of specific IgG and specific SIgA in the sera. It was shown that the expressed intestinal M cell targeting peptide (Col) and dendritic cell targeting peptide (DCpep) could effectively bind to intestinal M cells and dendritic cells, enhancing the level of intestinal local mucosal immune responses.

[0076] Example 5: Recombinant bifunctional Bacillus subtilis (B.S.-EDoB) expressing the core antigen regions and targeting peptides of PEDV and PDCoV S proteins induces highly efficient virus-specific milk-derived immune protection

[0077] The recombinant Bacillus subtilis WB800 strain (B.S.-EDoB) expressing PEDV and PDCoV S proteins in tandem and targeting intestinal M-like cells and dendritic cells, and its control recombinant Bacillus subtilis WB800 strain (B.S.) significantly increased the level of specific antibodies in colostrum after immunizing sows, and effectively protected neonatal piglets from PEDV and PDCoV infections. The animal experiment and immunoassay methods are as follows:

[0078] 1. The recombinant Bacillus subtilis significantly increased the level of specific antibodies in colostrum

[0079] Twenty healthy sows randomly selected from a pig farm were divided into 4 groups, with 5 sows in each group: blank control group; B.S.-EDoB group, in which sows were fed 2 mL of the recombinant Bacillus subtilis expressing PEDV and PDCoV S proteins (1×10 10 CFU / mL) on the 80th day of pregnancy, and repeated feeding once after 14 days; B.S group, in which sows were fed 2 mL of Bacillus subtilis WB800 (1×10 10 CFU / mL) on the 80th day of pregnancy, and repeated feeding once after 14 days; PBS group, in which sows were orally administered 2 mL of PBS on the 80th day of pregnancy, and repeated feeding once after 14 days.

[0080] After sows were orally administered the recombinant Bacillus subtilis, specific IgA+ and IgG+ plasmablasts migrated to the mammary tissue through the gut-mammary-SIgA axis. After reaching the mammary gland, the antibodies secreted by these cells were transported into the milk through epithelial cells. In this study, colostrum within one day after sow parturition was collected, filtered through a nylon mesh (210 μm) to remove impurities, centrifuged to discard the fat layer and precipitate, and the supernatant was collected for the detection of specific antibodies. The defatted milk collected was used for the detection of specific SIgA and IgG antibodies by a kit coated and assembled in our laboratory. The results are as Figure 4 shown. Compared with the group fed with blank Bacillus subtilis, the virus-specific SIgA produced in the colostrum of sows fed with B.S.-EDoB increased significantly. Secondly, the content of virus-specific IgG in colostrum also increased significantly.

[0081] 2. The recombinant Bacillus subtilis effectively protected neonatal piglets from PEDV and PDCoV infections

[0082] Next, we investigated the effect of oral administration of recombinant Bacillus subtilis to sows on the resistance of neonatal piglets to PEDV and PDCoV infections. 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 PDCoV. The clinical symptoms of the piglets were observed, and all piglets were euthanized 36 h after infection. Blood, duodenum, jejunum, and ileum were collected for relevant detections. It was found that piglets in the Bacillus subtilis WB800 group (B.S. group) showed typical clinical symptoms of PEDV and PDCoV at 36 h, including acute watery diarrhea, depression, and decreased appetite, while piglets in the recombinant Bacillus subtilis group (B.S.-EDoB 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, as a negative control) and the B.S.-EDoB group. The intestinal contents of the piglets were full, and the intestinal wall showed no changes. However, there were no intestinal contents in the intestines of piglets in the B.S. group, and the intestinal wall was thin and transparent( Figure 5 A, 6A). Immunofluorescence results showed that no PEDV-positive cells and a small number of PDCoV-positive cells were observed in the jejunal villus epithelial cells of piglets in the B.S.-EDoB group, while a large number of PEDV and PDCoV-positive cells were found in the jejunum of piglets in the B.S group( Figure 5 B, 6B). Finally, the virus distribution in the intestines of piglets was detected by RT-qPCR. The results showed that PEDV and PDCoV were mainly present in the jejunum and ileum of piglets. The viral RNA content in the jejunum and ileum of piglets in the B.S.-EDoB group was significantly lower than that in the B.S. group( Figure 5 C, 6C).

Claims

1. The pDGT-CotY-EDoB expression vector, characterized in that, The pDGT-CotY-EDoB expression vector includes the CoE region of the PEDV S protein, the CTD region of the PDCoV S protein, the targeting peptide Col for intestinal M-like cells, and the targeting peptide DCpep for dendritic cells. The nucleotide sequence of the CoE region of the PEDV S protein is as shown in SEQ ID NO.1, the nucleotide sequence of the CTD region of the PDCoV S protein is as shown in SEQ ID NO.2, the nucleotide sequence of the targeting peptide Col for intestinal M-like cells is as shown in SEQ IDNO.6, and the nucleotide sequences of the targeting peptide DCpep for dendritic cells are as shown in EQ ID NO.6 and SEQ ID NO.

7.

2. The pDGT-CotY-EDoB expression vector according to claim 1, characterized in that, The pDGT-CotY-EDoB expression vector also contains the promoter P43, the gene encoding the surface-display protein CotY, and the promoter pCotY. The nucleotide sequence of the promoter P43 is as shown in SEQ ID NO.3, the nucleotide sequence of the gene encoding the surface-display protein CotY is as shown in SEQID NO.4, and the nucleotide sequence of the promoter pCotY is as shown in SEQ ID NO.

5.

3. The construction method of the pDGT-CotY-EDoB expression vector according to claim 1, characterized in that, It includes the following steps: introducing the genes of the CoE region of the PEDV S protein, the CTD region of the PDCoV S protein, the targeting peptide Col for intestinal M-like cells, and the targeting peptide DCpep for dendritic cells into a plasmid to obtain it.

4. The method for constructing the pDGT-CotY-EDoB expression vector according to claim 2, characterized in that, It includes the following steps: 1) Obtaining the gene fragments of the CoE region of the PEDV S protein, the CTD region of the PDCoV S protein, the targeting peptide Col for intestinal M-like cells, the targeting peptide DCpep for dendritic cells, the promoter P43, the gene encoding the surface-display protein CotY, and the promoter pCotY respectively; 2) Using the gene fragments obtained in step 1) as templates, performing overlapping PCR amplification to obtain recombinant gene fragments; 3) Connecting the recombinant gene fragments into a vector to obtain it.

5. The method for constructing the pDGT-CotY-EDoB expression vector according to claim 4, characterized in that, The primers P7 and P10 in the overlapping PCR amplification, the sequence of P7 is: 5’-atgtttgcaaaacgattcaaaa-3’, and the primer sequence of P10 is: 5’-tcaatggggaagagaaccgctt-3’.

6. A recombinant strain, characterized in that, It contains the pDGT-CotY-EDoB expression vector described in claim 1 or 2. 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 includes obtaining by introducing the pDGT-CotY-EDoB expression vector described in claim 1 or 2 into a host bacterium. Preferably, the host bacterium includes Bacillus subtilis.

8. Use of the expression vector described in claim 1 or 2 or the recombinant strain described in claim 5 in the preparation of a drug or vaccine for preventing or treating diseases related to PEDV and / or PDCoV infection.

9. A genetically engineered live vaccine, characterized in that, The genetically engineered live vaccine includes the pDGT-CotY-EDoB expression vector described in claim 1 or 2 or the recombinant strain described in claim 5.

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

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