CN117802024A - Three-spore-protein-fusion-stabilized-display three-key-antigen recombinant bacillus subtilis for porcine diarrhea virus, and construction method and application thereof - Google Patents

Abstract

The invention discloses three spore protein fusion and stable display pig diarrhea virus three key antigen recombinant bacillus subtilis, a construction method and application thereof, wherein the recombinant bacillus takes bacillus subtilis as an initial strain, and an expression vector PBE-Cas9 containing Cas9 protein and a key antigen part S1 protein fragment of three optimized pig diarrhea viruses of PDCOV, TGEV and PEDV are synthesized into any one or more of genes PDCOV-S1, TGEV-S1 and PEDV-S1, and the sequences of the PDCOV-S1, the TGEV-S1 and the PEDV-S1 are respectively shown as SEQ ID NO. 1-3. The invention combines the two vectors and transfers the two vectors into bacillus subtilis to construct three recombinant bacillus subtilis with porcine diarrhea virus antigen sites, and the mice experiments preliminarily prove that the recombinant bacillus has good immune efficacy. The invention provides a research foundation for the research of the novel oral vaccine of the porcine diarrhea virus.

Description

Recombinant Bacillus subtilis with three spore protein fusions to stably display three key antigens of porcine diarrhea virus and its construction method and application

Technical field

The invention belongs to the field of genetic engineering technology, and specifically relates to the recombinant Bacillus subtilis that fuses three spore proteins to stably display three key antigens of porcine diarrhea virus and its construction method and application.

Background technique

Bacillus subtilis is a Gram-positive bacterium that widely exists in the natural environment. It grows in irregular clusters or strains and is rod-shaped. Its cell wall structure contains polysaccharides, peptidoglycan and cross-linked products, and its glycosyl composition contains chondroitin (Glucosamine), mannose (Mannose), N-acetylglucosamine (N-Acetylglucosamine) and other ingredients. These sugar groups and their cross-linked structures give the cell wall better toughness and stability, allowing it to survive and reproduce in harsher environments. Bacillus subtilis spore surface display refers to the expression of the required enzyme or antigen (target protein) on the spore surface by fusion with the spore coat protein (anchoring motif), which has been proven to be an important factor in vaccine development, enzyme immobilization, and anticancer drugs and human A powerful tool in related protein delivery and other fields. B. subtilis spore surface display offers natural advantages, including high safety, excellent stability, and the unique ability to display a variety of biomolecules, such as antigens, tetrameric enzymes, and proteins.

Bacillus subtilis has been studied for more than 100 years at home and abroad. A variety of spore capsid proteins such as CotB, CotC, CotG and CotX have been successfully used in molecular carriers, which provides potential opportunities for the development of new applications. These target proteins include tetanus toxin fragment C (TTFC), streptomycin, green fluorescent protein (GFP), single-chain antibodies, phytase, b-galactosidase, x-aminotransferase and other enzymes, which have been successfully displayed on the surface of spores. In 2001, Medaglini et al. reported the first successful use of CotB as a molecular carrier to fuse tetanus toxin C units. This research result provides a new idea for the development of new drugs and vaccines. In 2007, scholar Seok Joon Kwon successfully fused β-galactosidase with spore capsid protein CotG and displayed it on the surface of spores, which can be used as a whole-cell biocatalyst for galactosyl transfer in a water-solvent biphasic reaction system. In 2014, Korean scholars Jae-Gu Pan and others successfully displayed the natural form of carboxymethyl cellulase (CMCase) on the surface of spores.

Bacillus subtilis, as an edible probiotic, is also the mainstream of current oral vaccine carrier research. Since the Italian scholar Medaglini reported in 2001 that he used spore CotB protein to fuse the tetanus toxin C unit to develop an oral vaccine for tetanus, more and more scholars have also conducted in-depth research on oral vaccines for Bacillus subtilis. In 2007, scholar Le H Duc used Bacillus subtilis spores to express the anthrax protective antigen PA in the spore capsid and spores in the germination stage, and conducted a mouse immune protection experiment, confirming that the immunized mice could obtain better results. High levels of protective immunity. In 2016, Sichuan Agricultural University used the Bacillus subtilis spore protein CotB as a spore-anchored protein to express the PEDVS protein on the spore surface and verified that it had good immunogenicity.

Since 2016, a variety of CRISPR-Cas9-based gene editing systems have been established in Bacillus subtilis. Since this type of system requires the expression of Cas9 and sgRNA targeting specific genes, it can be achieved through integrated expression of the genome, single plasmid expression, or dual plasmid expression. Among them, the integrated expression operation of the genome is more cumbersome and is not conducive to the elimination and continuous operation of related elements; while the plasmid of the single-plasmid system is larger, which is not conducive to the connection construction and transformation of sgRNA and homologous fragments, so Cas9 and sgRNA are expressed separately. Plasmid systems are currently the best choice. Among them, the expression vector of Cas9 does not need to make any modifications for gene editing, as long as it is directly transformed. It is only necessary to modify the expression vector of sgRNA according to the required genetic operations, and the homologous template can also be placed together in the second thus significantly improving the efficiency of gene editing.

Porcine Epidemic Diarrhea Virus (PEDV), Transmissible Gastroenteritis virus (TGEV) and Porcine Deltacoronavirus (PDCOV) are the three most important diseases in the global swine industry in recent years. One of the diarrheal viruses. Diseases caused by these viruses not only cause severe economic losses, but also pose challenges to the sustainable development of the livestock industry. Their virus particles are spherical or irregular in shape, with an envelope and a size between 80-120nm. The genome structure of these viruses is a single-stranded forward-sense RNA with a cap structure at the 5' end, followed by 6-11 open reading frames. 2/3 of the viral genome is encoded by the first reading frame for replicase, and the remaining 1/3 mainly encodes structural proteins, including spike protein (Spike, S), small envelope protein (Envelope, E), Envelope protein (Membrance, M) and nuclear protein (Nucleocapsid, N). The E and M proteins play an important role in the assembly process of the virus. The N protein packages the genome to form a nucleoprotein complex, while the S protein mediates virus invasion by binding to host cell receptors and determines the tissue or host tropism of the virus. . The main antigen of PEDV is S protein. In the early stage of PEDV infection, the antibodies produced by the body mainly neutralize the S protein, thereby inhibiting further PEDV infection. However, the high variability of S protein causes many immunogenic sites to constantly change, which increases the malignancy of PEDV. TGEV antigen is also S protein, which can induce the body to produce antibodies and cellular immune responses. In the early stages of TGEV infection, the antibodies produced by the body mainly target the S protein, thereby inhibiting further TGEV infection. PDCOV is a double-stranded negative-sense RNA virus with a genome length of 25 kb. PDCOV has S, N, M and other structural proteins, among which S protein is the most important antigen of PDCOV. PDCOV is a novel coronavirus that was first discovered in 2012 and has since spread globally. PDCOV poses a serious threat to the pig industry, especially in Asia. Although PDCOV has attracted great attention, effective prevention and control methods are still lacking.

As a widely spread swine viral disease, porcine diarrhea virus has a serious impact on the health management and disease control of pig farms. Therefore, it is of great significance to establish an efficient porcine diarrhea virus vaccine research system. Currently, there are two types of porcine diarrhea virus vaccines circulating on the market: inactivated vaccines and live vaccines, but both vaccines have some disadvantages. First, the immune effect of inactivated vaccines is relatively weak, and a large number of vaccinations are required to produce effective immunity. In addition, inactivated vaccines are easily inactivated during storage and transportation, which affects the immune effect of the vaccine. Secondly, although live vaccines can promote the immune system to produce a strong immune response, there are also some potential risks. Live vaccines may cause hybridization between vaccine strains and wild virus strains during vaccination, resulting in mutations of vaccine strains and even changes in their pathogenicity. In addition, since there are multiple subtypes of porcine diarrhea virus, the vaccines on the market cannot guarantee good immune effects on all subtypes, which is also a limitation of existing vaccines. Therefore, it is very important to seek new vaccine prevention methods.

Vaccination against PEDV and TGEV has become the main means of preventing and controlling infections caused by these two viruses. At present, there are a variety of PEDV and TGEV vaccines available on the market, including inactivated vaccines, attenuated live vaccines, genetically engineered vaccines, etc. Vaccination with these vaccines can effectively improve the body's immunity to PEDV and TGEV, thereby reducing the extent of their infection. However, due to the large variability of the S proteins of PEDV and TGEV, this poses a great challenge to the development of vaccines. Therefore, scientists are constantly working to develop safer and more effective PEDV and TGEV vaccines to cope with the constantly mutating virus strains. Compared with PEDV and TGEV, the development of vaccines for PDCOV is relatively lagging, and there is currently no available PDCOV vaccine. This is mainly because the genome structure of PDCOV is different from that of other coronaviruses, which makes its antigenic epitopes more complex and difficult to quickly and accurately identify effective antigens.

PEDV, TGEV and PDCOV are three important enteroviruses that have an impact on the pig industry, and they are all prevalent worldwide. The antigens of these viruses are mainly S proteins, but due to their high variability, higher requirements are put forward for vaccine development. Therefore, it is of great significance to establish an efficient porcine diarrhea virus vaccine research system. Since porcine diarrhea viruses exist in multiple subtypes, Types, therefore the vaccines on the market cannot guarantee good immune effects against all subtypes. This is also a limitation of existing vaccines. Therefore, it is very important to find new vaccine prevention methods.

Contents of the invention

Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a recombinant Bacillus subtilis that stably displays three key antigens of porcine diarrhea virus by fusing three spore proteins. The recombinant bacteria of the present invention can display three porcine diarrhea viruses on the surface of three different Bacillus spores, and can also display three different porcine diarrhea virus antigens on the surface of three different spore proteins of the same strain of Bacillus subtilis. The porcine diarrhea virus recombinant bacteria of the present invention can not only induce a systemic immune response in mice, but also play a role in regulating intestinal flora and colonization in the intestine. The porcine diarrhea virus recombinant bacteria constructed by the present invention have significant immune efficacy, and have the ability to induce IgG antibodies, neutralizing antibodies, humoral immunity and cellular immunity. Bacillus subtilis is used as a host bacteria for delivering proteins, and has the ability to regulate the intestinal flora of mice and improve the physical fitness of mice, laying a foundation for the research of a new type of triple oral vaccine for porcine diarrhea virus.

The present invention also provides the construction method and application of the recombinant Bacillus subtilis using the fusion of three spore proteins to stably display three key antigens of porcine diarrhea virus.

Technical solution: In order to achieve the above purpose, the three spore proteins of the present invention are fused to stably display the three key antigens of porcine diarrhea virus recombinant Bacillus subtilis. The recombinant bacterium uses Bacillus subtilis as the starting strain and uses the expression vector containing the Cas9 protein. PBE-Cas9 and any one or more of the optimized S1 protein fragment synthesis genes PDCOV-S1, TGEV-S1 and PEDV-S1, the key antigenic parts of the three porcine diarrhea viruses, PDCOV, TGEV and PEDV, were transferred into Bacillus subtilis. The sequences of PDCOV-S1, TGEV-S1 and PEDV-S1 are shown in SEQ ID NO. 1-3 respectively.

Among them, the recombinant bacteria uses Bacillus subtilis as the starting strain, and transfers the expression vector PBE-Cas9 containing the Cas9 protein and any one or more of the antigen expression vectors PTN-CotA-PDCOV, PTN-CotB-TGEV and PTC-CgeA-PEDV of porcine diarrhea virus PDCOV, TGEV and PEDV containing the key antigen S1 into Bacillus subtilis, and the sequences of the PTN-CotA-PDCOV, PTN-CotB-TGEV and PTC-CgeA-PEDV are shown in SEQ ID NO.4-6 respectively.

Among them, the expression vector PBE-Cas9 uses PBE as a basic vector and includes an Escherichia coli origin of replication (ori), a Bacillus subtilis thermosensitive origin of replication (rep-ts), a Bacillus subtilis P43 promoter, a Cas9 protein expression cassette, and The penicillin resistance gene (AmpR), a selection marker gene for E. coli, and the erythromycin resistance gene ErmR for Bacillus selection.

Among them, the antigen expression vector PTN-CotA-PDCOV is based on PTN, and contains the penicillin resistance gene (AmpR) for Escherichia coli screening marker gene, the neomycin resistance gene NeoR for Bacillus screening, the spore protein expression frame CotA, the S1 protein of porcine delta coronavirus PDCOV, the Escherichia coli replication origin (ori), the Bacillus subtilis thermosensitive replication origin (rep-ts), the Bacillus subtilis P43 promoter and the sgRNA1 with the target sequence.

Among them, the antigen expression vector PTN-CotB-TGEV is based on PTN, and contains the penicillin resistance gene (AmpR) for Escherichia coli screening marker gene, the neomycin resistance gene NeoR for Bacillus screening, the spore protein expression frame CotB, the S1 protein of porcine transmissible gastroenteritis virus TGEV, the Escherichia coli replication origin (ori), the Bacillus subtilis thermosensitive replication origin (rep-ts), the Bacillus subtilis P43 promoter and the sgRNA2 with the target sequence.

Among them, the antigen expression vector PTC-CgeA-PEDV uses PTC as the basic vector and includes the penicillin resistance gene (AmpR), a screening marker gene for E. coli, the chloramphenicol resistance gene ClR for Bacillus screening, and spores. Protein expression box CgeA, S1 protein of porcine epidemic diarrhea virus PEDV, Escherichia coli origin of replication (ori), Bacillus subtilis thermosensitive origin of replication (rep-ts), Bacillus subtilis P43 promoter and sgRNA3 with target sequence.

Among them, the S1 protein fragment synthesis genes of the key antigenic parts of three porcine diarrhea viruses, PDCOV, TGEV and PEDV, were selected to optimize the synthesis of PDCOV-S1, TGEV-S1 and PEDV-S1, and at the same time, the fluorescent protein expression vectors PTN-CotA-RFP and PTN were replaced. - Fluorescent protein genes in CotB-YFP and PTC-CgeA-GFP to construct porcine diarrhea virus expression vectors PTN-CotA-PDCOV, PTN-CotB-TGEV and PTC-CgeA-PEDV. The fluorescent protein expression vectors PTN-CotA-RFP, PTN-CotB-YFP and PTC-CgeA-GFP have been successfully constructed in the previous patent "A wild-type Bacillus subtilis JCL16 multi-plasmid gene editing system and its construction and application" , Chinese application number 2022117265678.7.

Wherein, the Bacillus subtilis is Bacillus subtilis JCL16.

The method for constructing recombinant Bacillus subtilis that fuses three spore proteins and stably displays three key antigens of porcine diarrhea virus according to the present invention includes the following steps:

(1) Transfer the constructed pBE-Cas9 plasmid and the expression plasmids PTN-CotA-PDCOV, PTN-CotB-TGEV and PTC-CgeA-PEDV containing the key antigen S1 protein of porcine diarrhea virus into E. coli competent cells DH5a respectively. , large-scale amplification and extraction of plasmids;

(2) Transfer the extracted pBE-Cas9 plasmid and any one or more of the PTN-CotA-PDCOV, PTN-CotB-TGEV and PTC-CgeA-PEDV plasmids into the competent state of Bacillus subtilis, culture them, and obtain the recombinant bacteria .

Further, the genome of the transformed recombinant bacteria was extracted, and genomic PCR was performed to verify whether the recombinant bacteria had been successfully transformed; the spore protein solution of the recombinant bacteria was extracted, and Western-blot and IFA were performed to verify whether the three key antigenic proteins of the porcine diarrhea virus were successfully expressed.

The present invention uses three spore proteins to fuse and stably display three key antigens of porcine diarrhea virus recombinant Bacillus subtilis in the preparation of porcine diarrhea virus vaccine.

Among them, the use of the recombinant Bacillus subtilis that utilizes the fusion of three spore proteins to stably display three key antigens of porcine diarrhea virus is used in the preparation of oral vaccines for porcine diarrhea virus.

Furthermore, the application of the recombinant Bacillus subtilis that fuses the three spore proteins of the present invention and stably displays the three key antigens of the porcine diarrhea virus in the preparation of a vaccine that induces the production of specific antibodies against the key antigens of the porcine diarrhea virus.

Furthermore, the antigens are key antigens of three porcine diarrhea viruses: PDCOV, TGEV and PEDV. The present invention first constructs an expression vector PBE-Cas9 containing Cas9 protein (Chinese patent application number 2022117265678.7); then constructs porcine diarrhea viruses (PDCOV, TGEV and PEDV) containing the P43 strong promoter, sgRNA, spore protein CotR, and key antigen S1 Antigen expression vectors PTN-CotA-PDCOV, PTN-CotB-TGEV and PTC-CgeA-PEDV; then transfer any of the above antigen expression vectors together with the expression vector PBE-Cas9 into Bacillus subtilis to construct expression of porcine diarrhea virus antigen subtilis; further prepared mouse immune recombinant bacteria, and conducted a preliminary evaluation of the immunogenicity of the recombinant bacteria.

Further, mouse immune recombinant bacteria were prepared, and a preliminary evaluation of the immunogenicity of the recombinant bacteria was performed. The immunogenicity evaluation included the following steps:

(1) Preparing immune recombinant bacteria, spore culture the recombinant bacteria whose expression has been verified in spore medium, adjust the bacterial concentration to a final concentration of 7×10 ¹⁰ CFU, collect the bacterial cells by centrifugation, and suspend them in 10% skim milk powder;

(2) Inject the resuspended recombinant bacteria into the stomach of Balb/c mice and set up a PBS control group. The immunization cycle is divided into three times, each time for three consecutive days of immunization. The mice are collected after 14, 28, and 42 days of immunization. feces and serum, and the mice were sacrificed 14 days after the third immunization to collect spleen lymphocytes and small intestinal contents;

(3) Conduct ELISA, neutralizing antibody detection and cytokine detection on the blood and spleen lymphocytes collected in step (2), analyze the detection data, and evaluate the immunogenicity of the recombinant bacteria.

The present invention uses gene editing technology to improve the construction efficiency of dual plasmids and the antigen expression rate of vaccines. By constructing a dual plasmid gene editing system, CRISPR-Cas9 technology is used to display three types of porcine diarrhea viruses (PEDV, PEDV, PDCOV and TGEV) key antigen S1 gene fragment (shown in SEQ ID NO. 1-3). The immunogenicity evaluation of the recombinant bacterium constructed in the present invention on mice and the detection of mouse serum IgG, neutralizing antibodies, lymphocyte factors and other immune indicators show that intragastric immunization with Bacillus subtilis can induce specific immune responses in mice. Antibodies produced in mice against porcine diarrhea virus provide new ideas and methods for the development of triple oral vaccines, help develop new prevention and control strategies, and improve disease prevention and treatment capabilities. This lays the foundation for the following research on oral vaccines for porcine viral diarrhea.

The present invention selects Bacillus subtilis spore capsid proteins CgeA, CotA and CotB to respectively display the S1 fragments of porcine diarrhea viruses PEDV, PDCOV and TGEV. The present invention successfully constructs a Bacillus subtilis double-plasmid gene editing system using CRISPR-Cas9, successfully expresses fluorescent proteins on different spore capsid proteins, and further constructs three porcine diarrhea virus recombinant bacteria of PEDV, PDCOV and TGEV. The recombinant bacteria of the present invention can display the three porcine diarrhea viruses on the surfaces of three different Bacillus spores, and can also display the three different porcine diarrhea virus antigens on the surfaces of three different spore proteins of the same Bacillus subtilis.

In the present invention, the constructed Escherichia coli-Bacillus subtilis shuttle plasmid vectors PBE, PTN, and PTC are used to provide a basic framework for the establishment of a dual-plasmid gene editing system. Through the CRISPR-Cas9 gene editing system, the surface-displayed fluorescent protein expression vectors PTN-CotA-RFP, PTC-CgeA-GFP, and PTN-CotB-YFP of Bacillus subtilis spore capsid proteins were successfully constructed, and the correct surface-expressed fluorescent proteins were screened. The recombinant bacteria provide a strong basis for testing the work of the dual-plasmid gene editing system, and then successfully constructed the Bacillus subtilis surface-displayed porcine diarrhea virus key antigen expression vectors PTN-CotA-PDCOV, PTC-CgeA-PEDV, and PTN-CotB-TGEV, and Recombinant bacteria displaying porcine diarrhea virus antigens on the spore surface were successfully screened, and mice were immunized with three recombinant porcine diarrhea virus Bacillus subtilis, and mouse serum IgG, neutralizing antibodies, lymphocyte factors and other immune indicators were detected. The recombinant bacteria of the present invention The bacteria have good immune effects in animal immunity experiments and provide a research basis for the research of new triple oral vaccines related to porcine diarrhea virus. Compared with traditional porcine diarrhea vaccines, the method of displaying porcine diarrhea virus on the surface of Bacillus subtilis in the present invention has good thermal stability, and the spores can be stored at room temperature for a long time without the need for cold chain storage and transportation; it is injection-free and can be administered orally. to immunize; compared with traditional vaccines, the production cost of spore display technology is lower, the shelf life of spores is long, and it is easy to store; the surface of Bacillus subtilis can display the antigens of different pathogens, which is suitable for vaccine research on different pathogens. The recombinant strain of porcine diarrhea virus of the present invention can not only induce systemic immune response in mice, but also regulate intestinal flora and colonize in the intestine. Moreover, the recombinant bacterium of the present invention can display three kinds of porcine diarrhea viruses on the surface of three different Bacillus spore spores, and can also display three different porcine diarrhea virus antigens on the surface of three different spore proteins of the same strain of Bacillus subtilis.

The recombinant bacterium of the porcine diarrhea virus of the present invention can not only induce the systemic immune response of the mouse body, but also play a role in regulating intestinal flora and colonization in the intestine; and the recombinant bacterium of the present invention can display three kinds of porcine diarrhea viruses in the intestine. Three different Bacillus spore surfaces can also display three different porcine diarrhea virus antigens on the surface of three different spore proteins of the same strain of Bacillus subtilis, and have good immunity. The present invention successfully displays foreign proteins on the surface of Bacillus subtilis through the CRISPR-Cas9 gene editing system, confirming the successful establishment of a dual-plasmid gene editing system. In addition to the proteins in the present invention, other Bacillus subtilis gene editing technologies can be subsequently performed. Research.

By constructing a dual plasmid gene editing system, the present invention uses CRISPR-Cas9 technology to display the key antigen S1 gene fragments of three types of porcine diarrhea viruses (PEDV, PDCOV and TGEV) on different spore surfaces of Bacillus subtilis for the first time. The present invention utilizes Bacillus subtilis spores as a carrier of porcine diarrhea virus S1 antigen to be administered to mice. The porcine diarrhea virus-specific IgG antibodies can be detected through indirect ELISA detection methods and indirect immunofluorescence IFA, and the immunized mice can be detected through neutralizing antibodies. The highest serum neutralization titer can reach 1:64; through the detection of spleen lymphocytes of immunized mice, the recombinant bacteria can induce lymphocyte proliferation and increase the secretion levels of cytokines IL-4 and IFN-γ, and can also increase T, B lymphocyte number ratio; by monitoring the general physical conditions such as the weight of immune mice, it was found that Bacillus subtilis can increase the body's intake of nutrients and increase body weight, and the retention of immune strains was detected in the feces and intestines of mice. In summary, the recombinant strain of porcine diarrhea virus can not only induce systemic immune response in mice, but also regulate and colonize the intestinal flora in the intestine.

The invention uses specific Bacillus subtilis as a new type of nanomaterial, which is resistant to high temperature, acid and alkali and other extreme environments. The use of surface proteins of spores to express antigens is resistant to degradation by acids and proteases in the gastrointestinal tract of animals. Compared with intra-spore and extra-spore antigen expression, the spore surface has the advantages of good expression stability, high expression activity and safety, and the ability to display a variety of biomolecules.

Beneficial effects: Compared with the existing technology, the present invention has the following advantages:

The present invention uses gene editing technology to improve the construction efficiency of dual plasmids or multiple plasmids and the antigen expression rate of the vaccine. By constructing a dual plasmid or multiple plasmid gene editing system, the present invention uses CRISPR-Cas9 technology to respectively Displaying the key antigen S1 gene fragments of three porcine diarrhea viruses (PEDV, PDCOV and TGEV) provides new ideas and methods for the development of triple oral vaccines. It will also help develop new prevention and control strategies and improve disease prevention and treatment capabilities. .

The present invention successfully constructs Escherichia coli-Bacillus subtilis shuttle plasmid vectors PBE, PTN, and PTC, providing a basic framework for the establishment of a dual-plasmid gene editing system. Through the CRISPR-Cas9 gene editing system, the key antigen expression vectors PTN-CotA-PDCOV, PTC-CgeA-PEDV, and PTN-CotB-TGEV of Bacillus subtilis surface-displayed porcine diarrhea virus were successfully constructed, and the spore surface-displayed porcine diarrhea virus was successfully screened. The recombinant bacterium of the antigen uses Bacillus subtilis spores as the carrier of porcine diarrhea virus S1 antigen and is administered to mice. Porcine diarrhea virus-specific IgG antibodies can be detected by indirect ELISA detection method and indirect immunofluorescence IFA, and neutralizing antibodies can be detected. The highest neutralizing titer of the serum of immunized mice can reach 1:64; through the detection of spleen lymphocytes of immunized mice, the recombinant bacteria can induce lymphocyte proliferation and increase the secretion levels of cytokines IL-4 and IFN-γ, and can also Increase the ratio of the number of T and B lymphocytes; by monitoring the weight and other general physical conditions of immune mice, it was found that Bacillus subtilis can increase the body's intake of nutrients and increase body weight, and the number of immune strains was detected in the feces and intestines of mice. Stay.

In summary, the recombinant bacteria of the present invention can not only induce systemic immune responses in mice, but also regulate intestinal flora and colonize the intestines. This research result is a promising candidate for a new oral vaccine for porcine diarrhea virus. Exploration lays the foundation for research.

Description of drawings

Figure 1 shows the enzyme digestion verification results of recombinant plasmids PTC-CgeA-PEDV, PTN-CotA-PDCOV, and PTN-CotB-TGEV from left to right;

Figure 2 shows the genome PCR verification results of recombinant Bacillus subtilis ΔBS-PEDV, ΔBS-PDCOV and ΔBS-TGEV from left to right;

Figure 3 shows the Western-blot identification of recombinant Bacillus subtilis ΔBS-PEDV, ΔBS-PDCOV and ΔBS-TGEV from left to right;

Figure 4 shows the IFA identification results of recombinant Bacillus subtilis ΔBS-PEDV, ΔBS-PDCOV and ΔBS-TGEV;

Figure 5 shows the statistical results of serum IgG antibody levels (S/N) in mice immunized with recombinant bacteria;

Figure 6 shows the results of serum IFA detection of mice immunized with recombinant bacteria ΔBS-TGEV;

Figure 7 shows the neutralizing titer level results of serum from mice immunized with recombinant bacteria. Figure A shows the neutralizing titer of serum from mice immunized with recombinant bacteria ΔBS-PEDV, and Figure B shows the neutralizing titer of serum from mice immunized with recombinant bacteria ΔBS-PDCOV. , Picture C shows the neutralization titer of serum from mice immunized with the recombinant strain ΔBS-TGEV;

FIG8 is the proliferation index of spleen lymphocytes in mice immunized with recombinant bacteria;

Figure 9 is an analysis of the proportion of T lymphocytes in mice immunized with recombinant bacteria (A) CD3+

Analysis of the proportion of CD4+T lymphocytes; (B) Analysis of the proportion of CD3+CD8+T lymphocytes in mice immunized with recombinant bacteria;

Figure 10 is an analysis of the proportion of B220+CD19+B lymphocytes in mice immunized with recombinant bacteria;

Figure 11 shows the observation of the growth types of colonies spread on the cecal contents of mice immunized for 42 days. Picture A shows the wild bacteria JCL16 group, Picture B shows the ΔBS-Mix group, Picture C shows the ΔBS-PEDV group, and Picture D shows the ΔBS-PDCOV group. , Figure E shows the ΔBS-TGEV group.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and examples.

The materials, reagents, etc. used in the examples of the present invention can all be obtained from commercial sources unless otherwise specified. Experimental methods without specifying specific conditions in the examples usually follow conventional conditions or conditions recommended by the manufacturer.

The Bacillus subtilis JCL16 (deposit number: CCTCC NO: M2018336) used in the present invention has been publicly deposited in the prior patent CN111411053A, is a conventional wild-type strain provided by Huaiyin Institute of Technology, and is prepared into Bacillus subtilis competent cells by conventional methods.

The sequences of the basic vector plasmid PBE, plasmid PTN, and plasmid vector PTC used in the present invention are detailed in the patent application "Escherichia coli-Bacillus shuttle plasmid vector and its construction method and application (Chinese application number 202111297649.0). The expression vector PBE-Cas9 containing Cas9 protein used in the present invention, the modified vector plasmid PTN-CotA-RFP, PTN-CotB-YFP and PTC-CgeA-GFP sequences, and the preparation method of the competent state of Bacillus subtilis JCL16 are detailed in the patent application "A wild-type Bacillus subtilis JCL16 multi-plasmid gene editing system and its construction and application (application number 2022117265678.7).

PDCOV-S1, TGEV-S1 and PEDV-S1 used in the examples of the present invention were synthesized by biological companies.

The RFP gene fragment in PTN-CotA-RFP was replaced with PDCOV-S1 to construct the PDCOV expression vector PTN-CotA-PDCOV. The sequence of the expression plasmid PTN-CotA-PDCOV is shown in SEQ ID NO.4.

The YFP gene fragment in PTN-CotB-YFP was replaced with TGEV-S1 to construct the TGEV expression vector PTN-CotA-TGEV. The sequence of the expression vector PTN-CotA-TGEV is shown in SEQ ID NO.5.

The GFP gene fragment in PTC-CgeA-GFP was replaced with PEDV-S1 to construct a PEDV expression vector PTC-CgeA-PEDV. The sequence of the expression vector PTC-CgeA-PEDV is shown in SEQ ID NO. 6.

Example 1

Construction of recombinant porcine diarrhea virus from Bacillus subtilis

(1) Transformation of porcine diarrhea virus recombinant expression plasmid

Take a prepared 100uL Bacillus subtilis JCL16 competent cell and place it on ice to thaw for 5 minutes. Add any one of the purified expression plasmids PTN-CotA-PDCOV, PTN-CotB-TGEV and PTC-CgeA-PEDV and plasmid PBE. -Add 10ul (about 2ug) of each Cas9 into the competent cells, add 10ul 100X EGTA, add 1ml LB aseptically, put it into a 37°C shaker and shake slowly at 100rpm for 30 minutes, then increase the speed to 200rpm, shake and culture for 1.5h, and then collect the cells. , take 150ul of the culture supernatant, resuspend the pellet and spread it on an erythromycin Erm ^R 10ug/mL and neomycin sulfate Neo ^R 10ug/mL or chloramphenicol (Cl ^R 12.5ug/mL) double-antibody LB plate, 37 Incubate in a ℃ incubator for 24-48 hours, and take out the transformed colonies after they grow on the plate.

(2) Recombinant bacterial genome PCR verification

Pick a single colony from the transformation plate to the corresponding double-antibody LB liquid medium (PTC-CgeA-PEDV: chloramphenicol + erythromycin double-antibody LB plate; PTN-CotA-PDCOV and PTN-CotB-TGEV: neomycin sulfate + Erythromycin double-antibody LB plate; chloramphenicol Cl ^R 12.5ug/mL; neomycin sulfate Neo ^R 10ug/mL; erythromycin Erm ^R 10ug/mL), extract the genomic DNA of the recombinant bacteria grown, and according to the spore coat The primers designed for the shell Cot protein gene fragment amplify the spore capsid protein fragment, and the wild-type Bacillus subtilis JCL16 genomic DNA is used as a control. The total size of the amplified band of the recombinant bacteria is the Cot gene + the antigen fragment gene size is the correct recombinant bacteria. The amplified fragments were purified and sent to a gene company for sequencing. If the sequencing results were correct, three recombinant strains of Bacillus subtilis for porcine diarrhea virus were successfully constructed, namely ΔB.S-PEDV, ΔB.S-PDCOV, and ΔBS-TGEV.

Verification methods used for three recombinant strains. Spore capsid protein primers: Using the Bacillus subtilis JCL16 genome as the template, COTAUF/COTAUR and COTADF/COTADR were used as primers to amplify the upstream and downstream of CotA protein through ordinary PCR technology; using the Bacillus subtilis JCL16 genome as the template, using COTAUF/COTAUR and COTADF/COTADR as primers respectively. COTBUF/COTBUR and COTBDF/COTBDR were used as primers to amplify the upstream and downstream parts of the CotB protein through ordinary PCR technology; the Bacillus subtilis JCL16 genome was used as the template, and CGEAUF/CGEAUR and CGEADF/CGEADR were used as primers to amplify the CgeA protein through ordinary PCR technology. The upstream and downstream primers are as follows.

(3) Extraction of recombinant bacterial spore capsid protein

Pick a single colony of the recombinant bacteria that has been confirmed by PCR and place it in 5 mL of LB double-antibody liquid medium at 37°C with shaking at 180 rpm overnight. The next day, transfer 1% of the overnight culture to 50 mL of DSM double-antibody liquid medium at 37°C with 180 rpm shaking. Incubate the bed for 48 hours, centrifuge the spore culture medium, discard the supernatant and retain the precipitate, add an appropriate amount of lysozyme (10mg/mL) to break the wall at 37°C for 30 minutes, then centrifuge to retain the sediment, then wash the sediment once with 1M NaCl and 1M KCl and centrifuge to retain the sediment. , add an appropriate amount of SDS-DTT solution and treat at 37°C for 1 hour, then centrifuge the pellet, resuspend the pellet in spore lysate, place it on ice, ultrasonically disrupt it, and centrifuge it at low temperature for 10 minutes. After centrifugation, the pellet is resuspended in PBS. The centrifugation speed is 12000rpm during the extraction process. .

(4) Wsetern-blot identification of recombinant bacterial spore capsid protein

First, take 45 μL of the spore capsid protein solution extracted in step (3), add 5 μL of 5X SDS-PAGE Loadingbuffer, mix well, boil at 100°C for 10 minutes, and then perform 12.5% SDS-PAGE electrophoresis. Add 25 μL of sample to each lane, electrophoresis at 90V for 2 hours, and then perform Wsetern. -blot identification. SDS-PAGE gel is configured with Yazy's PAGE gel kit. Please refer to the kit instructions for the operating steps. The Wsetern-blot steps are as follows:

a. Transfer: Soak the PAGE gel after electrophoresis in the transfer buffer for 30 seconds, then lay it flat on the PVDF membrane activated with methanol and drive away the air bubbles. Sandwich it between the sponge pads soaked in the transfer buffer and use a roller. After getting rid of the bubbles in each layer, use a rapid transfer machine to transfer for 15 minutes.

b. Blocking: After the transfer is completed, remove the PVDF membrane and place it in a blocking box. Add 5% skimmed milk powder and shake at room temperature for 2 hours to block.

c. Incubate the primary antibody: After blocking, pour out the blocking solution, wash 3 times with PBST, and add the positive serum diluted with PBS for 5 minutes each time (the primary antibodies used in this experiment are all corresponding positive mouse serum for porcine diarrhea virus), 37°C Incubate for 1 hour or at room temperature for 2 to 3 hours, and wash 5 times with PBST, 5 minutes each time.

d. Incubate secondary antibody: Add HRP-labeled goat anti-mouse secondary antibody (1:10000) diluted in PBST, incubate at 37°C for 50 minutes, and wash 5 times with PBST, 5 minutes each time.

e. Color development: Prepare the color development solution according to the kit, lay the incubated PVDF membrane flat on the storage plate in the cassette of the chemical light instrument, add color development solution dropwise to the PVDF membrane and drive away the air bubbles, close the cassette for exposure .

Western blot analysis was performed on the total capsid protein of wild-type and recombinant Bacillus subtilis to identify whether CgeA-PEDV, CotA-PDCOV, CotB-TGEV, and fusion genes are expressed on the spore surface. The results are shown in Figure 3. In wild-type Bacillus subtilis, No specific band appeared in the spore capsid protein component, but one specific band of about 105KD, 132KD and 116KD appeared in the recombinant Bacillus subtilis capsid protein component. Western-blot analysis results showed that CgeA-PEDV, CotA-PDCOV, and CotB-TGEV fusion genes were fused and expressed under the induction of P43 promoter. The porcine diarrhea virus antigen fragment was successfully displayed on the surface of Bacillus subtilis spores and was immunogenic. , can be recognized and bound by porcine diarrhea virus-specific antibodies.

(5) Immunofluorescence microscopy observation of recombinant bacterial spores

Extract the genomic DNA of the recombinant bacteria, wash it 3 times with PBS, centrifuge at 10000 rpm to retain the precipitate, add 1:500 diluted primary antiserum (same as W-B primary antibody) and mix slowly at 37°C for 1 hour, centrifuge the spores and wash 5 times with PBST. Wash slowly for 5 minutes with slow shaking for 5 minutes. Add FITC goat anti-mouse fluorescent secondary antibody diluted 1:500 in PBST in the dark. Shake slowly for 45 minutes at 37°C. Wash with PBST 5 times for 5 minutes each time. Finally, resuspend the spores in PBS and take 10 μL of resuspended spores. The suspended spores were dropped onto a glass slide, covered with a coverslip and air-dried naturally. They were placed under a fluorescence microscope to first observe the field of view with an ordinary microscope, and then to observe the fluorescence of the spores under a fluorescence microscope.

Fluorescence results of spore fluorescence observed under a fluorescence microscope: As shown in Figure 4, wild-type Bacillus subtilis JCL 16 was used as a control, and specific antibodies were used to observe the recombinant Bacillus subtilis spores under an immunofluorescence microscope. Under fluorescence, the surface of the recombinant Bacillus subtilis spores had Green fluorescence, under the same conditions, wild-type Bacillus subtilis JCL16 showed no fluorescence. The results showed that the porcine diarrhea virus antigen fragment was successfully displayed on the surface of Bacillus subtilis spores and was immunogenic, and could be recognized and combined by porcine diarrhea virus-specific antibodies. That is, the key antigenic site S1 protein of PEDV, TGEV and PDCOV can be displayed and expressed on the surface of recombinant Bacillus subtilis CgeA, CotB and CotA spores.

Example 2

Preliminary evaluation of immunogenicity of recombinant Bacillus subtilis against porcine diarrhea virus

(1)Animal immunity

Balb/c mice were randomly divided into 6 groups, with 6 mice in each group. The grouping information is shown in Table 1. Immunization was performed by gavage. Each mouse was administered 200uL of recombinant bacteria containing 1×10 ¹⁰ CFU each time (ΔB.S-PEDV, ΔB.S-PDCOV, ΔBS-TGEV, respectively corresponding to PTC-CgeA- in the table). PEDV, PTN-CotA-PDCOV, PTN-CotB-TGEV), and the PBS group was administered with 200 μL of sterile PBS solution as a blank control. The immunization cycle was divided into three times, with each immunization lasting for three days. Feces and serum were collected from mice 14, 28, and 42 days after immunization. The mice were sacrificed 14 days after the third immunization to collect spleen lymphocytes and small intestinal contents.

Table 1 Mouse immune grouping

(2) Detection of IgG in the serum of mice immunized with recombinant bacteria ΔB.S-PEDV and ΔB.S-PDCOV

a. Coating: Use carbonate coating solution to dilute PEDVS protein 0.25μg/mL and PDCOV S1 protein 0.3μg/mL (according to the S gene sequence of porcine diarrhea virus (PEDV, PDCOV, TGEV), select the S1 gene with a size of about 2000bp fragment), coat 96-well enzyme plate with 100 μL per well, and coat in a refrigerator at 4°C overnight;

b. Blocking: discard the coating solution, wash the enzyme plate with 200 μL of PBST per well, wash three times, 3 minutes each time, add 200 μL of 5% skim milk powder per well, and let stand at 37°C for 1.5-2 hours;

c. Primary antibody: Wash the blocked enzyme plate with 200 μL of PBST per well 3 times, 3 minutes each time, and add 1:100 diluted serum from mice immunized with the recombinant bacteria ΔB.S-PEDV or ΔB.S-PDCOV. 100 μL, incubate at 37°C for 1 hour, and use pre-immune mouse serum as a negative control;

d. Secondary antibody: Wash the primary antibody 5 times with 200 μL of PBST per well, 5 min each time, add 100 μL of HRP-labeled goat anti-mouse enzyme-labeled secondary antibody diluted 1:20000, and incubate at 37°C for 45-50 min;

e. Color development: Wash the secondary antibody 5 times with 200 μL of PBST per well, 5 minutes each time, add 50 μL of TMB chromogenic solution, and develop color for 10 minutes at 37°C in the dark;

f. Termination: When the negative serum is about to turn blue, add 50 μL of 2M sulfuric acid to terminate the color development;

g. Reading: Place the color-developed enzyme plate under the microplate reader and read at OD=450.

(3) Indirect immunofluorescence detection of recombinant bacteria ΔBS-TGEV

Indirect immunofluorescence is used to detect IgG in immunized mice. The specific operation method is as follows:

a. Use a monolayer of ST cells to spread into a 96-well cell plate and wait until the cells grow into a monolayer (24-48 hours);

b. Inoculate TGEV virus at moi = 0.01, dilute 1000 times of 10*8TCID50 virus stock solution and inoculate 100 μL per well; (10 μL + 10 mL DMEM containing 1% trypsin without serum)

c. After incubating with the virus for 12 hours, discard the supernatant, wash twice with PBS, fix the cells with ice ethanol (or methanol), 200 μL per well, and fix in a -20°C refrigerator for 30 minutes;

d. The fixed 96-well plate is blocked with 0.1% BSA or 0.5% skimmed milk powder for 2 hours (4°C);

e. Wash the sealed plate three times with PBS, add the diluted recombinant bacterial ΔBS-TGEV immune serum (1:100), 100 μL per well, and make negative and positive controls at the same time, and incubate in a 37°C incubator for 1 hour;

f. Wash with PBS 3 times/5min, add diluted goat anti-mouse fluorescent secondary antibody (1:500), 50μL per well, and incubate in a 37°C incubator in the dark for 45 minutes;

g. Wash with PBS 3 times/5min, and avoid light for observation (observation under an inverted fluorescence microscope).

The fluorescence in the cytoplasm of the recombinant bacteria ΔBS-TGEV immune group and the recombinant bacteria mixed immunization ΔBS-Mix group is shown in Figure 6, that is, the presence of immunized mouse antibodies was detected, and the fluorescence of the recombinant bacteria ΔBS-TGEV immune group was stronger than that of the ΔBS-Mix group. This shows that immunizing mice with recombinant bacteria can induce mice to produce specific antibodies.

(4) Neutralizing antibody detection

a. First place the serum to be tested in a 56°C water bath for 30 minutes to inactivate it to reduce the influence of non-specific factors in the serum;

b. Plate a 96-well cell plate with a monolayer of Vero cells, grow into a monolayer and wait for use (24-48 hours);

c. Add the serum to be tested to a 96-well plate in a 2-fold gradient dilution, 50 μL per well, add the diluted virus solution 50 (200 TCID50), mix and neutralize in a 37°C incubator for 1 hour;

d. For a 96-well cell plate with a full monolayer, discard the original culture medium, wash twice with PBS, transfer the neutralized serum and virus mixture to the corresponding cell wells, and set up a virus control well to determine whether the virus titer is correct;

e. After incubating the cells with the neutralizing mixture for 1.5 to 2 hours, discard the liquid in the wells and wash them three times with PBS. Add a maintenance solution containing trypsin, place them in a 37°C 5% CO2 cell incubator for 72 hours, and observe the results of cell lesions. statistical results.

(5)Statistical analysis

GraphPad Prism9 and Microsoft Excel 2010 were used for statistical analysis and graphing of the data. Each group of experimental data were statistically analyzed using one-way ANOVA or two way ANOVA, and the T test method was used to analyze the differences in the data. P<0.005 means significant difference between each group, P<0.001 means extremely significant difference between each group.

Statistical analysis of experimental data from the initial evaluation of immunogenicity of recombinant Bacillus subtilis of porcine diarrhea virus:

The IgG antibody detection results of mice immunized with recombinant bacteria ΔB.S-PEDV and ΔB.S-PDCOV are shown in Figure 5.

After immunization with the recombinant bacteria, blood was collected from the eyeball vein of mice at 14 days, 28 days, and 42 days after the first immunization, and the serum was separated. The IgG antibody in the serum of the immunized mice was detected by the indirect ELISA method established in the laboratory. The results are shown in Figure 5. 42 days after the first immunization, the IgG antibody of mice in all immunized groups was higher than that in the PBS group (P<0.001). As can be seen from the bar graph, the IgG antibody of mice was not fully stimulated 14 days after the first immunization. The serum IgG of immunized mice increased significantly 28 days after the first immunization, that is, 14 days after the second immunization. The IgG antibody level of immunized mice increased significantly 42 days after the first immunization. There was no significant difference in the IgG antibody of mice immunized with the recombinant bacteria ΔBS-PEDV group and the ΔBS-Mix group, but both were significantly higher than the wild bacteria BS-JCL16 group, and the difference was significant (P<0.005). Compared with the serum IgG antibody of mice immunized with the recombinant bacteria ΔBS-PDCOV group and the ΔBS-Mix group, the ΔBS-Mix group was higher than the ΔBS-PDCOV group and the wild bacteria BS-JCL16 group, and the difference was significant (P<0.005), and the ΔBS-PDCOV group was better than the BS-JCL16 group. In summary, the recombinant bacteria can induce mice to produce IgG antibodies, and the IgG antibody level of the recombinant bacteria is significantly higher than that of the wild bacteria JCL16.

(2) The results of neutralizing antibody detection in mice immunized with recombinant bacteria are shown in Figure 7:

After immunization with the recombinant bacteria, blood was collected from the eyeball veins of the mice on 14 days, 28 days, and 42 days after the first immunization, and the serum was separated to detect the neutralizing antibody titer in the serum of the immunized mice. The results are shown in Figure 7. Similar to the trend in the production of IgG antibody levels in immunized mice, neutralizing antibodies began to be produced 14 days after the second immunization, and reached a higher level 14 days after the third immunization. The neutralizing antibody titers produced by mice immunized with recombinant bacteria were significantly higher than those in the PBS group ( P<0.001), but the difference in serum neutralizing antibody titers between mice immunized with recombinant bacteria alone and mixed immunization was not significant. The highest neutralizing titer of serum from mice immunized with recombinant ΔBS-PEDV and recombinant ΔBS-PDCOV could reach 1:64, but the highest neutralizing titer of serum from mice immunized with recombinant ΔBS-TGEV could only reach 1:32. It shows that the recombinant Bacillus subtilis constructed in the present invention can effectively induce mice to produce neutralizing antibodies as a carrier to present porcine diarrhea virus antigen, and the effect is good.

(3) Spleen lymphocyte proliferation index is shown in Figure 8:

After the spleen cells were isolated from the mice, the spleen lymphocytes were stimulated with concanavalin protein ConA (10ug/mL). After 72 hours, the CCK-8 method was used to detect the proliferation of spleen lymphocytes. The stimulation results are shown in Figure 8. The immune group was The proliferation index of mouse spleen lymphocytes was higher than that of the PBS group, and the difference was significant (P<0.005). Among them, the spleen lymphocyte proliferation index of the ΔBS-PDCOV group was more prominent, and it was extremely different from the PBS group (P<0.001), and it was also significantly higher than that of the PBS group. wild bacteria JCL16, and there was a significant difference (P<0.05); the lymphocyte proliferation index of mice in other immune groups was better than the PBS group and BS-JCL16 group.

(4) Flow cytometry detection

T lymphocytes are shown in Figure 9:

Flow cytometry was used to detect the proportion of CD3 ⁺ CD4 ⁺ CD8 ⁺ T lymphocytes in the spleens of immunized mice 14 days after the third vaccination. The results are shown in Figure 9 below. The proportions of CD3 ⁺ CD4 ⁺ and CD3 ⁺ CD8 ⁺ T lymphocytes in the immunized mice were significantly higher than those in the PBS group (P<0.001). There was no significant difference between the mouse groups immunized with recombinant bacteria, but the proportions of CD3 ⁺ CD4 ⁺ and CD3 ⁺ CD8 ⁺ T lymphocytes in mice immunized with recombinant bacteria were higher than those immunized with wild bacteria JCL16. groups, and also have significant differences.

B lymphocyte subpopulations are shown in Figure 10:

Flow cytometry was used to detect the proportion of B220 ⁺ CD19 ⁺ B lymphocyte subpopulations in the spleens of immunized mice 14 days after the third immunization. The results are shown in Figure 10. The proportion of B220 ⁺ CD19 ⁺ B lymphocytes in the immunized mice was significantly higher than that in PBS. group (P<0.001), there was no significant difference between the groups of mice immunized with recombinant bacteria, but the proportion of B220 ⁺ CD19 ⁺ B lymphocytes in the mice immunized with recombinant bacteria was higher than that of the wild bacteria immunized group, and the difference was significant.

(6) Bacillus subtilis colonizes the intestinal tract

Mouse feces were collected into 1.5 mL centrifuge tubes before immunization on the 14th, 28th, and 42nd days after mouse immunization, and 300 μL of sterile PBS was added and soaked in a 4°C refrigerator for 2 h. After the feces were softened, they were fully homogenized with a pipette tip on a clean bench and centrifuged using gradient centrifugation. The homogenate was centrifuged at 500 rpm for 15 minutes, and the supernatant was aspirated and centrifuged at 12000 rpm for 10 minutes. The precipitate was resuspended with 100 uL PBS, then diluted in multiple ratios, and spread on the corresponding double-antibody LB plates for culture to observe the bacterial morphology and colony number.

The results are shown in Figure 11. The cecal contents of the culled mice 42 days after three immunizations were diluted and spread on the corresponding resistance plates of the non-antibiotic and recombinant bacteria, and the morphology of the colonies was observed. The test results were: in the anti-resistant plate More colonies grew. Most of the colonies in the experimental group were Bacillus subtilis, and there were more miscellaneous bacteria in the PBS group, indicating that Bacillus successfully colonized the mouse intestines and existed in larger numbers.

The results of the present invention illustrate the importance and potential of the constructed specific recombinant bacteria for the preparation of oral vaccines. The use of recombinant Bacillus subtilis to prepare porcine diarrhea virus intragastric vaccines can establish a line of defense at the mucosal invasion site, induce immune responses, resist infection, and thereby treat pigs. The prevention and control of enteric coronavirus is of great significance. The recombinant bacteria can induce the production of specific antibodies against porcine diarrhea virus antigens, and the immune response has been significantly improved. It is used to prevent African porcine diarrhea virus infection. For surface expression using Bacillus subtilis Vaccine research provides a reference.

Claims (10)

1. Three spore proteins are fused to stably display the three key antigens of porcine diarrhea virus recombinant Bacillus subtilis, which is characterized in that the recombinant Bacillus subtilis uses Bacillus subtilis as the starting strain, and combines the expression vector PBE-Cas9 containing the Cas9 protein and Any one or more of the S1 protein fragment synthesis genes PDCOV-S1, TGEV-S1 and PEDV-S1, the key antigenic parts of the three optimized porcine diarrhea viruses PDCOV, TGEV and PEDV, are transferred into Bacillus subtilis, and the PDCOV- The sequences of S1, TGEV-S1 and PEDV-S1 are shown in SEQ ID NO. 1-3 respectively. 2. The recombinant Bacillus subtilis fused with three spore proteins to stably display three key antigens of porcine diarrhea virus according to claim 1, characterized in that the recombinant Bacillus subtilis uses Bacillus subtilis as the starting strain and preferably contains Cas9 Protein expression vector PBE-Cas9 and any one or more of the key antigen S1 porcine diarrhea virus PDCOV, TGEV and PEDV antigen expression vectors PTN-CotA-PDCOV, PTN-CotB-TGEV and PTC-CgeA-PEDV. Into Bacillus subtilis, the sequences of PTN-CotA-PDCOV, PTN-CotB-TGEV and PTC-CgeA-PEDV are shown in SEQ ID NO.4-6 respectively. 3. The three spore proteins fused to stably display the three key antigens of porcine diarrhea virus recombinant Bacillus subtilis according to claim 2, characterized in that the expression vector PBE-Cas9 uses PBE as the basic vector and contains an Escherichia coli replication origin. (ori), Bacillus subtilis thermosensitive origin of replication (rep-ts), Bacillus subtilis P43 promoter, Cas9 protein expression cassette, screening marker gene penicillin resistance gene (AmpR) for E. coli and for Bacillus screening The erythromycin resistance gene ErmR. 4. The three spore protein fusion stably displays the three key antigens of porcine diarrhea virus recombinant Bacillus subtilis according to claim 1, characterized in that the antigen expression vector PTN-CotA-PDCOV is based on PTN, comprising a selection marker gene penicillin resistance gene (AmpR) for Escherichia coli, a neomycin resistance gene NeoR for Bacillus screening, a spore protein expression frame CotA, S1 protein of porcine delta coronavirus PDCOV, an Escherichia coli replication origin (ori), a Bacillus subtilis thermosensitive replication origin (rep-ts), a Bacillus subtilis P43 promoter and a sgRNA1 with a target sequence. 5. The recombinant Bacillus subtilis that fuses three spore proteins to stably display three key antigens of porcine diarrhea virus according to claim 1, characterized in that the antigen expression vector PTN-CotB-TGEV uses PTN as the basic vector and contains Screening marker gene penicillin resistance gene (AmpR) for Escherichia coli, neomycin resistance gene NeoR for Bacillus selection, spore protein expression cassette CotB, S1 protein of porcine transmissible gastroenteritis virus TGEV, Escherichia coli replication Origin (ori), Bacillus subtilis thermosensitive origin of replication (rep-ts), Bacillus subtilis P43 promoter and sgRNA2 with target sequence. 6. The recombinant Bacillus subtilis that stably displays three key antigens of porcine diarrhea virus with three spore protein fusions according to claim 1, characterized in that the antigen expression vector PTC-CgeA-PEDV is based on PTC, comprising a selection marker gene penicillin resistance gene (AmpR) for Escherichia coli, a chloramphenicol resistance gene ClR for Bacillus screening, a spore protein expression frame CgeA, S1 protein of porcine epidemic diarrhea virus PEDV, an Escherichia coli replication origin (ori), a Bacillus subtilis thermosensitive replication origin (rep-ts), a Bacillus subtilis P43 promoter and a sgRNA3 with a target sequence. 7. The recombinant Bacillus subtilis that stably displays three key antigens of porcine diarrhea virus by fusion of three spore proteins according to claim 1, characterized in that the Bacillus subtilis is Bacillus subtilis JCL16. 8. A method for constructing a recombinant Bacillus subtilis that fuses three spore proteins and stably displays three key antigens of porcine diarrhea virus according to claim 1, characterized in that it includes the following steps: (1) Transfer the constructed pBE-Cas9 plasmid and the expression plasmids PTN-CotA-PDCOV, PTN-CotB-TGEV and PTC-CgeA-PEDV containing the key antigen S1 protein of porcine diarrhea virus into E. coli competent cells DH5a respectively. , large-scale amplification and extraction of plasmids; (2) Transfer the extracted pBE-Cas9 plasmid and any one or more of the PTN-CotA-PDCOV, PTN-CotB-TGEV and PTC-CgeA-PEDV plasmids into the competent state of Bacillus subtilis, culture them, and obtain the recombinant bacteria . 9. Application of a recombinant Bacillus subtilis that fuses three spore proteins and stably displays three key antigens of porcine diarrhea virus according to claim 1 in the preparation of a vaccine that induces the production of specific antibodies against the key antigens of porcine diarrhea virus. 10. The use according to claim 9, characterized in that the three spore protein fusions stably display the three key antigens of porcine diarrhea virus recombinant Bacillus subtilis in the preparation of porcine diarrhea virus oral vaccine.