Porcine epidemic diarrhea virus bacterium-like particles and methods of making same

Abstract

The present application relates to the technical field of molecular markers, and particularly relates to a porcine epidemic diarrhea virus (PEDV) bacterium-like particle and a preparation method thereof, which takes S1 genes and COE genes of Guangxi PEDV epidemic strains as target genes, and successfully expresses fusion proteins S1-PA and COE-PA fused with anchor hook proteins PA by using an insect cell-baculovirus expression system; meanwhile, lactic acid lactobacillus MG1363 is treated by heat and acid to remove bacterial nucleic acids and proteins, and GEM particles are successfully prepared, and the fusion proteins S1-PA and COE-PA are successfully displayed on the surfaces of the GEM particles to obtain S1-GEM and COE-GEM particles, the two kinds of particles are mixed with GEL-01 adjuvant to prepare two kinds of bacterium-like particle vaccines, and the vaccines are used for immunization of mice, and it is confirmed through immunization routes, immunogens and immunization doses that 24 mu g COE-GEM can achieve the optimal immunization effect for the mice immunized by nose drops.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular markers, in particular to a porcine epidemic diarrhea virus bacterium-like particle and a preparation method thereof. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious enteric disease of pigs of all ages caused by porcine epidemic diarrhea virus (PEDV), characterized by watery diarrhea, vomiting and dehydration. The morbidity and mortality of suckling piglets can be as high as 100%. Since it first appeared in the United Kingdom in 1971, PEDV has spread worldwide and caused considerable economic losses. Although sporadic outbreaks have occurred in European countries, in Asia, PEDV has been the main cause of diarrhea outbreaks in pig farms.

[0003] PEDV is a member of the order Nidovirales, family Coronaviridae, genus Alphacoronavirus, which contains four structural proteins, i.e. spike (S), envelope (E), membrane (M) and nucleocapsid (N). The S protein is an important structural protein and immunogenic antigen of PEDV, carries the main B lymphocyte epitope, and is the main target for the development of PEDV vaccines and drugs. Deletion, insertion or amino acid mutation of the S gene is the main feature of PEDV mutants and the main reason for the global spread of PED. S1 is the main domain of the S protein, located on the surface of the virus, and contains three important neutralizing epitope regions of PEDV, which play an important role in the body's antigen immune response. COE is an important core epitope in the S1 gene discovered in 2002, and studies have confirmed that it also has good reactivity and can induce the body to produce neutralizing antibodies, and is considered to be the main target antigen for the development of PED subunit vaccines and antiviral drugs.

[0004] PEDV is highly infectious, and vaccination is an important measure to prevent PEDV. However, PED outbreaks have been reported in immunized pig farms in recent years. The main reasons are that the current PEDV epidemic strain is constantly mutating, and the existing vaccine cannot provide sufficient immune protection for the PEDV mutant strain. In addition, the existing vaccine may not be able to induce sufficient mucosal immunity in the animal body. Studies have shown that sIgA secreted by intestinal mucosal epithelial cells can effectively neutralize viruses at the invasion site, and vaccines administered via the mucosal route can induce mucosal immunity more effectively than other vaccination methods. Currently, there are two strategies for improving animal mucosal immune response. One is to use suitable carriers for mucosal vaccination, and the other is to add mucosal immune enhancers for conventional immunization. The development and application of mucosal immune enhancers and mucosal drug delivery carriers are of great significance for the development of traditional and new PED vaccines.

[0005] PED is an acute viral enteric infection. The traditional view is that PEDV is mainly transmitted through the fecal-oral route, but more and more studies have shown that air transmission can also lead to PEDV outbreaks. Current research has found that PEDV can cause typical diarrhea symptoms in piglets by nasal spray infection, and can develop into a short-term nasal epithelial infection, confirming that PEDV mainly infects the body through the nasal mucosa. Numerous studies have confirmed that sIgA antibodies produced by mucosal immunity have higher protection rates against viruses that infect the mucosa compared to IgG antibodies. Therefore, given the characteristics of PEDV, it is necessary to develop new vaccines to induce local reactions in the mucosal area through mucosal immunity to resist viral infection of the respiratory tract and gastrointestinal tract, and to improve the body's resistance to the virus. Since 2010, a new variant of PEDV with strong pathogenicity has emerged and rapidly spread in pig populations across the country, leading to large-scale epidemics. However, the existing PEDV vaccine cannot provide effective protection against it, so it is of great significance to develop a new PEDV vaccine against the mutant epidemic strain.

[0006] This study first used the antigen gene of the main epidemic strain of PEDV in Guangxi as the target gene and applied the GEM-PA surface display system to PEDV vaccine research. The prepared PEDV bacterial-like particle vaccine was used to immunize mice, and its immunogenicity was evaluated through humoral, cellular, and mucosal immunity, to explore the feasibility of PEDV bacterial-like particle vaccine and lay the foundation for the development of new PEDV vaccines. SUMMARY

[0007] In view of the above, it is necessary to provide a porcine epidemic diarrhea virus bacterial-like particle and a preparation method thereof, which takes the antigen gene of the main epidemic strain of Guangxi PEDV as a target gene, applies the GEM-PA surface display system to PEDV vaccine research, immunizes mice with the prepared PEDV bacterial-like particle vaccine, evaluates its immunogenicity from three aspects of humoral immunity, cellular immunity and mucosal immunity, and explores the feasibility of the PEDV bacterial-like particle vaccine.

[0008] To achieve the above object, the technical scheme adopted by the present application is:

[0009] The porcine epidemic diarrhea virus bacterial-like particle comprises a recombinant protein S1-PA or COE-PA displayed on the surface of a GEM particle; the amino acid sequence of the S1-PA is shown in SEQ ID NO. 3; and the amino acid sequence of the COE-PA is shown in SEQ ID NO. 4.

[0010] The present application also comprises the application of the porcine epidemic diarrhea virus bacterial-like particle in the preparation of a vaccine for preventing and treating porcine epidemic diarrhea caused by PEDV infection.

[0011] Further, the vaccine is immunized by injection, gavage and / or nasal instillation.

[0012] Further, the nasal instillation immunization route immunogen is a GEM particle displaying the COE-PA protein, named COE-GEM; and the concentration of the COE-GEM nasal instillation immunogen is 24 μg.

[0013] The present application also comprises a preparation method of the porcine epidemic diarrhea virus bacterial-like particle, which comprises the following steps:

[0014] (5) (1) Constructing a fusion gene and a recombinant expression plasmid thereof. The S1 gene or the COE gene of PEDV is subjected to insect cell codon optimization, and a Kozak sequence and a GP67 signal peptide sequence are introduced at the 5' end of the gene, then the S1 gene or the COE gene is fused with the codon-optimized PA gene of Lactococcus lactis MG1363 through a linker to construct a fusion gene S1-PA or COE-PA, and the fusion gene is directionally cloned into a pFastBacI vector to obtain a recombinant expression plasmid pFastBacI-S1-PA or pFastBacI-COE-PA; the nucleotide sequence of the fusion gene S1-PA is SEQ ID NO. 1; and the nucleotide sequence of the fusion gene COE-PA is SEQ ID NO. 2;

[0015] (2) Transform the recombinant plasmid pFastBacI-S1-PA or pFastBacI-COE-PA obtained in step (1) into DH10Bac competent cells to prepare recombinant bacmids Bacmid-S1-PA or Bacmid-COE-PA;

[0016] (3) Transfect the recombinant bacmids Bacmid-S1-PA or Bacmid-COE-PA into Sf9 cells respectively to rescue P1 generation of recombinant viruses, and obtain P1 generation of recombinant baculoviruses, which are named as Rbv-S1-PA and Rbv-COE-PA respectively; inoculate Sf9 cells with Rbv-S1-PA or Rbv-COE-PA at a ratio of MOI = 1 to express the recombinant proteins, and collect cell lysis supernatant after identification of the recombinant proteins;

[0017] (4) Prepare GEM particles: remove nucleic acids and self-proteins of Lactococcus lactis MG1363 after heat-acid treatment to obtain GEM particles;

[0018] (5) Take 0.1 U of the GEM particles of step (4) and add them into the cell lysis supernatant of step (3), take the maximum binding amount of the fusion protein to obtain S1-GEM or COE-GEM particle vaccines, and mix the two kinds of particles with GEL-01 adjuvant to prepare S1-GEM particle vaccines or COE-GEM particle vaccines; the S1-GEM particle vaccines display the recombinant protein S1-PA on the surface of the GEM particles; and the COE-GEM particle vaccines display the recombinant protein COE-PA on the surface of the GEM particles.

[0019] The present application has the following beneficial effects:

[0020] 1. The insect cell-baculovirus expression system is used to successfully obtain recombinant baculoviruses Rbv-S1-PA and Rbv-COE-PA expressing fusion proteins PEDV S1-PA and COE-PA, and the insect cells are infected to successfully express the fusion proteins S1-PA and COE-PA.

[0021] 2. The Lactococcus lactis MG1363 is treated by boiling trichloroacetic acid, and the bacterial nucleic acids and proteins are successfully removed through identification, and the GEM particles are successfully prepared.

[0022] 3. The S1-GEM and COE-GEM are successfully prepared by using the GEM-PA bacterial-like particle surface display system; and the detection results show that 1 U of GEM particles can bind 82.27 μg / U of PEDV S1-PA and 118.28 μg / U of COE-PA fusion proteins at most.

[0023] 4. The prepared S1-GEM and COE-GEM were used to immunize mice, and the optimal immune group was obtained by screening of the immune route, immunogen and immune dose: 24 μg COE-GEM was used to immunize mice by nasal instillation.

[0024] 5. The mouse immunization test results showed that 24 μg COE-GEM nasal instillation immunization of mice not only induced high levels of specific serum IgG antibodies, but also induced higher levels of sIgA antibodies in the intestinal mucosa (feces) of mice, and stimulated the proliferation of mouse spleen lymphocytes, and the expression levels of cytokines IFN-γ and IL-4 mRNA were significantly increased.

[0025] 6. The induced IgG and sIgA antibody levels in the body of mice immunized by 24 μg COE-GEM nasal instillation were significantly higher than those of the TGE-PED dual inactivated vaccine group. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 are transmission electron microscope observation diagrams of COE-GEM and S1-GEM; wherein, A and B are observation diagrams of COE-GEM under 7k and 40k microscopes, respectively; C and D are observation diagrams of S1-GEM under 7k and 40k microscopes, respectively;

[0027] Figure 2 is an SDS-PAGE detection diagram of the maximum binding amount of fusion protein COE-PA and GEM particles;

[0028] Figure 3 is an SDS-PAGE detection diagram of the maximum binding amount of fusion protein S1-PA and GEM particles; Figures 2-3 M is a protein marker; lane 1 is 0.5 mg / mL BSA standard protein; lanes 2-7 are respectively: 0 mL, 2 mL, 4 mL, 6 mL, 8 mL, 10 mL of 0.1 U GEM particles combined with cells;

[0029] Figure 4 is a determination diagram of the amount of GEM particles combined with fusion protein S1-PA;

[0030] Figure 5 is a determination diagram of the amount of GEM particles combined with fusion protein COE-PA; Figures 4-5 M is a protein marker; lanes 1-5 are respectively 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL of S1 protein ( Figure 4 ) or COE protein ( Figure 5 ); lane 6 is GEM particles, and lane 7 is S1-GEM protein ( Figure 4 ) or COE-GEM protein (Figure 5

[0031] Figure 6 is a detection result graph of mouse serum PEDV specific IgG antibody;

[0032] Figure 7 is a detection result graph of mouse feces PEDV specific sIgA antibody;

[0033] Figure 8 is a detection graph of mouse serum PEDV specific IgG antibody;

[0034] Figure 9 is a detection graph of mouse feces PEDV specific sIgA antibody;

[0035] Figure 10 is a detection graph of mouse serum PEDV specific IgG antibody;

[0036] Figure 11 is a detection graph of mouse feces PEDV specific sIgA antibody;

[0037] Figure 12 is a detection graph of mouse serum PEDV specific IgG antibody;

[0038] Figure 13 is a detection graph of mouse feces PEDV specific sIgA antibody;

[0039] Figure 14 is a detection result graph of in vitro proliferation of spleen lymphocytes of COE-GEM (24 μg) immunized mice under antigen stimulation;

[0040] Figure 15 is a detection result graph of IFN-γ (A) and IL-4 (B) qPCR of COE-GEM (24 μg) immunized mice.

DETAILED DESCRIPTION

[0041] In order to make the above objectives, features and advantages of the present application more apparent, specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without some or all of these specific details. In other instances, well known process steps have not been described in detail in order not to unnecessarily obscure the present application.

[0042] Example 1:

[0043] Recombinant protein expression, specific experimental methods are as follows:

[0044] 1. Cells and strains

[0045] ​Sf9 (Spodoptera frugiperda 9) insect cell line, Lactococcus lactis MG1363 were gifted by Institute of Veterinary, Military Medical Academy and preserved in our laboratory; DH5a E. coli was purchased from Baobioengineering (Dalian) Co., Ltd.

[0046] 2. Plasmid

[0047] pFastBacI vector plasmid was preserved in our laboratory.

[0048] 3. Main reagents:

[0049] Sf9 cell serum-free SF900II medium (gibco company), insect cell transfection reagent CellfectinII Reagent (gibco company), liquid trichloroacetic acid (Sigma company), mouse His monoclonal antibody (Biunten company), M17 culture medium (Hibio company), Tks Gflex TM DNAPolymerase high-fidelity DNA polymerase (TAKARA company), In-Fusion seamless cloning kit (TAKARA company), FastPure Cell / Tissue Total RNA isolation kit (Vazyme company), bacterial genome DNA extraction kit (Tiangen Biotech Co., Ltd.), mouse epidemic diarrhea virus antibody IgG (PEDV-IgG) kit (Enzyme Immune Biotechnology Co., Ltd.), mouse epidemic diarrhea virus antibody sIgA (PEDV sIgA) kit (Enzyme Immune Biotechnology Co., Ltd.).

[0050] 4. Construction and identification of recombinant plasmids pFastBacI-S1-PA and pFastBacI-COE-PA

[0051] (1) Optimized synthesis of genes

[0052] The S1 gene sequence of the Guangxi PEDV-GXNN epidemic strain (NCBI accession number: MW762534) isolated in the laboratory and the PA sequence of the Lactococcus lactis MG1363 strain were used as templates, and were synthesized after baculovirus codon optimization by Nanjing Zhongding Biotechnology Co., Ltd. The GXNN strain of the present application is GII-b subtype, which is in the same branch as most of the domestic isolates in recent years, and has a distant genetic relationship with the classic vaccine strain and early epidemic strains at home and abroad, which is a different subtype. Moreover, the S gene has large variation, with 4 unique amino acid site mutations in the COE region and the main antigen epitope region, and a potential recombination region at the high variable region 826-3142 nt.

[0053] (2) Construction of recombinant plasmid pFastBacI-S1-PA

[0054] The construction of the recombinant plasmid pFastBacI-S1-PA is based on the following points:

[0055] ① When using the insect cell-baculovirus system to express foreign proteins, the foreign proteins are mostly expressed intracellularly. In order to improve the transcription and translation efficiency of the foreign proteins and make them expressed secretively, the Kozak sequence and the GP67 signal peptide are introduced at the 5' end of the fusion gene.

[0056] ② The PEDV S1 gene is fused with the PA gene of Lactococcus lactis MG1363 to construct a fusion gene and express it. In order to ensure that the expressed fusion protein has the correct spatial conformation, a linker sequence is added between the S1 gene and the PA gene.

[0057] ③ In order to facilitate the identification of the fusion protein in the later stage, a his tag sequence is added at the 3' end of the fusion gene.

[0058] The designed S1-PA fusion gene (nucleotide sequence is SEQ ID NO. 1) is sent to Nanjing Zhongding Biotechnology Co., Ltd. for sequence synthesis, and then directionally cloned into the pFastBacI vector to construct the recombinant plasmid pFastBacI-S1-PA.

[0059] (2) Construction of recombinant plasmid pFastBacI-COE-PA

[0060] ① According to the construction strategy of the recombinant plasmid pFastBacI-S1-PA, the Kozak sequence and the GP67 signal peptide sequence are introduced at the 5' end of the pFastBacI vector, the codon-optimized PA sequence is added at the 3' end, and appropriate enzyme digestion sites are added to construct the pFastBac1-PA vector. The required synthesized sequence is sent to Nanjing Zhongding Biotechnology Co., Ltd. for synthesis, and the synthesized sequence is directionally cloned into the pFastBacI vector to successfully construct the recombinant plasmid pFastBac1-PA;

[0061] ② The COE gene is PCR specifically amplified using the codon-optimized S1 gene as the template and the COE-F1 and COE-R1 primers. The COE-F1 primer sequence is as follows: 5'-CGCCTTCGCTCATATGGTCACCCCTCCTTCCTTCAAC-3'; and the COE-R1 primer sequence is as follows: 5'-AGCCGCCGCCACCGGTCACGTCGGTCACACCCTCCAGA-3';

[0062] The reaction system of the PCR amplification reaction is as follows: 0.2 μL of template, 1 μL of COE-F1 (10 μmol·L -1 ), 1 μL of COE-R1 (10 μmol·L -1 ), 1 μL of Tks Gflex DNA Polymerase, 25 μL of 2×Gflex PCR Buffer, and ddH2O to 50 μL. The PCR reaction conditions are as follows: pre-denaturation at 94°C for 1 min; denaturation at 98°C for 10 s, extension at 68°C for 20 s, for a total of 30 cycles; finally, 72°C for 5 min, and storage at 4°C.

[0063] After the PCR reaction is completed, the reaction product is subjected to agarose gel electrophoresis analysis, and the COE gene is recovered and purified according to the operation steps of the FastPure Gel DNA Extraction Mini Kit;

[0064] The pFastBac1-PA backbone vector after modification is subjected to Nde I and Age I double enzyme digestion according to the instructions of the restriction endonuclease. The double enzyme digestion reaction system is prepared as follows: 0.2 μL of Nde I enzyme, 0.4 μL of Age I enzyme, 2 μL of pFastBac1-PA plasmid, 1 μL of 10×Cutsmart buffer, and ddH2O to 10 μL; the reaction temperature is 37°C for 3 h.

[0065] After the enzyme digestion reaction is completed, the linearized pFastBac1-PA is recovered and purified. The recovered product COE and pFastBac1-PA are used for In-Fusion connection. The connection system is as follows: 2 μL of 5×In-Fusion HD Enzyme Premix, 1 μL of pFastBac1-PA linear vector, 7 μL of COE-PA, and ddH2O to 10 μL; the reaction temperature is 50°C for 15 min.

[0066] The connection product is transformed. 10 μL of the connection product is added to E. coli DH5α competent cells, placed on ice for 30 min, subjected to 42°C heat shock for 70 s, immediately placed in ice water for 2 min, then 800 μL of LB liquid medium is added, and placed in a 37°C 200 rpm shaking incubator for 1 h. 100 μL of the cultured bacterial liquid is uniformly coated on an LB solid culture dish containing ampicillin, and the culture dish is placed in a 37°C incubator and incubated for 12-18 h;

[0067] (4) Recombinant bacmid preparation: the DH10Bac competent cells into which the pFast TAT-VP2 recombinant plasmid is transformed are diluted by 100 times for plating culture, and white colonies are picked up for expansion culture after blue-white spot screening. Recombinant bacmid extraction is performed according to the bacmid small-scale extraction kit instruction, and the recombinant bacmids Bacmid-S1-PA or Bacmid-COE-PA are obtained.

[0068] (3) Recombinant plasmid transformation of DH10Bac competent cells: the correctly identified recombinant plasmids pFastBacI-S1-PA, pFastBacI-COE-PA and pFastBac I empty vector are respectively transformed into DH10Bac competent cells. Under the action of the helper plasmid, the recombinant plasmid and the baculovirus backbone vector undergo homologous recombination, and finally the foreign genes S1-PA or COE-PA are inserted into the baculovirus backbone vector to obtain the recombinant bacmids of the target genes.

[0069] (4) Recombinant bacmid preparation: the DH10Bac competent cells into which the pFast TAT-VP2 recombinant plasmid is transformed are diluted by 100 times for plating culture, and white colonies are picked up for expansion culture after blue-white spot screening. Recombinant bacmid extraction is performed according to the bacmid small-scale extraction kit instruction, and the recombinant bacmids Bacmid-S1-PA or Bacmid-COE-PA are obtained.

[0070] (5) Recombinant bacmid identification: universal primers M13F / M13R are respectively used for PCR amplification identification with the extracted recombinant bacmids as templates; specific primers DL-S1 / COE-F1 and DL-S1 / COE-R1 are respectively used for qPCR amplification identification with the extracted recombinant bacmids as templates; wherein the M13F primer sequence is as follows: 5'-GTTTTCCCAGTCACGAC-3', the M13R primer sequence is as follows: 5'-CAGGAAACAGCTATGAC-3'; the DL-S1 / COE-F1 primer sequence is as follows: 5'-AGTCCGTCAACGACTACCTCTC-3'; and the DL-S1 / COE-R1 primer sequence is as follows: 5'-CCCTTGGTGAACTGGAAGTAGA-3'.

[0071] (6) Rescue of recombinant baculovirus: Wild type bacmid and recombinant bacmids Bacmid-S1-PA and Bacmid-COE-PA were transfected into Sf9 cells according to the instructions of Cellfectin II transfection reagent to rescue P1 generation of recombinant virus. During culture, the CPE state of cells was observed every day. When the cells became large and round, stopped growing, and even some cells fell off, the virus was collected. The cells were broken by repeated freeze-thawing, and the supernatant was collected by centrifugation at 1500 rpm for 5 min at 4°C. The P1 generation of recombinant baculovirus was obtained, and was named as Rbv-S1-PA and Rbv-COE-PA, respectively.

[0072] (7) Passage of recombinant baculovirus: The P1 generation of recombinant baculovirus was inoculated into Sf9 cells in logarithmic growth phase at a ratio of 10%, and the cells were cultured at 27°C for 3-5 days. When the CPE was obvious, the virus was collected. The cells were broken by repeated freeze-thawing, and the supernatant was collected by centrifugation at 1500 rpm for 5 min at 4°C. The P2 generation of recombinant baculovirus was obtained. The same method was used to pass the recombinant baculovirus to obtain P3 and P4 generations of recombinant baculovirus.

[0073] (8) Observation of cytopathic effect: Sf9 cells in logarithmic growth phase were inoculated into a T25 sealed cell bottle and cultured at 27°C for 12 h. Then, the P2 generation of recombinant baculovirus was inoculated into the cells at a ratio of 10%, and the cells were cultured at 27°C for 72 h. The CPE was observed, and Sf9 cells without virus were used as a control.

[0074] (9) Identification of recombinant baculovirus genome

[0075] To ensure that the recombinant baculovirus can express the fusion protein, it is necessary to detect whether the recombinant baculovirus genome contains the target gene. Since the virus may lose the exogenous gene during passage, the P3 generation of recombinant baculovirus was subjected to qPCR identification of the target gene. The viral DNA / RNA co-extraction kit was used to extract the genomic DNA of Rbv-S1-PA, Rbv-COE-PA and wild type baculovirus according to the instructions. The genomic DNA was used as a template for qPCR identification using the specific primers DL-S1 / COE-F1 and DL-S1 / COE-R1.

[0076] Meanwhile, the Sf9 cells after infection were collected, total RNA of the cells was extracted by FastPure Cell / Tissue Total RNA Isolation Kit, and cDNA was prepared by reverse transcription with the total RNA as a template; qPCR was performed by using the specific primers DL-S1 / COE-F1 and DL-S1 / COE-R1 with the cDNA as a template, so as to further determine whether the target gene was transcribed in the Sf9 cells. After the reaction, the recombinant baculoviruses with correct identification were named as Rbv-S1-PA and Rbv-COE-PA respectively.

[0077] (10) Expression of S1-PA and COE-PA fusion proteins

[0078] The recombinant baculoviruses Rbv-S1-PA and Rbv-COE-PA were inoculated into Sf9 cells at an MOI of 1, and a wild-type baculovirus infection group was set as a negative control, and the cells were cultured at 27°C for 3-5 days. When the CPE reached 80%, the supernatant and cell precipitate were collected, and an appropriate amount of PBS was added to the cells, and the cells were lysed by ultrasonic, and the supernatant and cell precipitate were collected and stored at -70°C for standby.

[0079] 5. Preparation and identification of GEM particles

[0080] (1) Preparation of GEM particles:

[0081] ① 1 mL of Lactococcus lactis MG1363 strain was added into 500 mL of sterile GM17 broth (M17 broth + 0.5% glucose with a final concentration), and cultured at 30°C and 145 rpm for 19 h.

[0082] ② When OD 600 = 2, centrifugation was performed at 6000 rpm for 15 min at room temperature, and the supernatant was removed, and Lactococcus lactis MG1363 was collected.

[0083] ③ Lactococcus lactis MG1363 was resuspended in sterile PBS, and centrifugation was performed at 6000 rpm for 15 min at room temperature, and the bacteria were washed twice.

[0084] ④ 10% trichloroacetic acid was boiled in boiling water for 30 min, and centrifugation was performed at 6000 rpm for 15 min at room temperature to collect the precipitate.

[0085] ⑤ The precipitate was resuspended in sterile PBS, and centrifugation was performed at 6000 rpm for 15 min at room temperature, and the bacteria were washed five times.

[0086] ⑥ The precipitate was resuspended in an appropriate amount of sterile PBS, and the resuspended bacteria were counted under a microscope. According to the counting result, the concentration of GEM particles was adjusted to 2.5×10 91U. GEM particles were prepared and stored at -70℃ for later use.

[0087] (2) qPCR identification of GEM particles:

[0088] According to the published MG1363 gene sequence on GenBank, a pair of specific primers MG1363-F1 / R1 were designed using Primer 5.0 software. The MG1363 genomic DNA was extracted using a bacterial genomic DNA extraction kit and subjected to PCR amplification. The amplified target fragment was purified and ligated to the pMD18-T vector. The recombinant plasmid was sequenced and identified. After the sequence was correct, it was used as a positive plasmid standard. The MG1363-F1 primer sequence was 5'-CAGATGAACTTGTAGCGGACTC-3', and the MG1363-R1 primer sequence was 5'-AGTAGCCTTTTGGATGACTTTGA-3'.

[0089] (3) Activity identification of GEM particles:

[0090] MG1363 and GEM particles were inoculated on GM17 solid medium (M17 broth + 0.5% glucose + 2% agar) respectively, and cultured at 30℃ for 19h. The bacterial growth was observed.

[0091] (4) Transmission electron microscope observation of GEM particles: MG1363 and prepared GEM particles were treated with 2% glutaraldehyde, and placed at 4℃ overnight. They were sent to Wuhan Saiver Biological Technology Co., Ltd. for transmission electron microscope observation and photography.

[0092] (5) S1-PA, COE-PA fusion protein binding to GEM particles:

[0093] 1U of GEM particles from step (4) were added to the supernatant of Rbv-S1-PA and Rbv-COE-PA fusion protein obtained in step 4(10), and shaken at room temperature for 1h. They were centrifuged at 6000rpm for 10min, and the supernatant was discarded. They were resuspended and washed with sterile PBS for 3 times, to obtain S1-PA and COE-PA fusion protein loaded GEM particles, which were named S1-GEM particles and COE-GEM particles respectively. The S1-GEM particles displayed recombinant protein S1-PA (amino acid sequence as shown in SEQ ID NO. 3) on the surface of GEM particles, and the COE-GEM particles displayed recombinant protein COE-PA (amino acid sequence as shown in SEQ ID NO. 4) on the surface of GEM particles.

[0094] Example 2:

[0095] Evaluation of S1-GEM and COE-GEM particles

[0096] 1. Test whether S1-PA and COE-PA proteins are successfully fused to GEM particles

[0097] After glutaraldehyde treatment, S1-GEM and COE-GEM were observed by transmission electron microscopy. It can be seen that the surface of GEM particles after fusion of the fusion protein appears rough, with a large number of obvious small flocculent substances fixed on the surface, proving that S1-PA and COE-PA fusion proteins are successfully displayed on the surface of GEM particles. Figure 1

[0098] 2. Determination of the maximum binding amount of GEM particles to S1-PA and COE-PA fusion proteins

[0099] 0.1 U of GEM particles were combined with 0, 2, 4, 6, 8, and 10 mL of cell lysate supernatant infected with Rbv-COE-PA, and an equal amount of sample was analyzed by SDS-PAGE gel electrophoresis. 0.1 U of GEM particles were combined with 0, 4, 8, 12, and 16 mL of cell lysate supernatant infected with Rbv-S1-PA, and an equal amount of sample was analyzed by SDS-PAGE gel electrophoresis. The results are shown in Figure 2 and 3 As the volume of the combined GEM particles increased, the color of the target band gradually deepened. When the volume of the cell lysate supernatant was 8 mL or more, the binding amount of COE-GEM tended to saturate. When the volume of the cell lysate supernatant was 12 mL or more, the binding amount of S1-GEM tended to saturate.

[0100] 3. Determination of the anchoring binding amount of S1-PA and COE-PA fusion proteins

[0101] S1-GEM and COE-GEM loaded with the maximum binding amount of fusion proteins were subjected to SDS-PAGE electrophoresis with different concentrations of BSA standard (0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL) to prepare a BSA standard curve, which was analyzed by quantity one software. The results are shown in Figure 4 , 5 According to the calculation by the quantity one gray scale analysis software, the binding amount of S1-PA was 82.27 μg / U, and the binding amount of COE-PA was 118.28 μg / U.

[0102] 4. Immunization route screening

[0103] S1-GEM particles and COE-GEM particles prepared in Example 2 were mixed with GEL-01 adjuvant by low shear force to prepare S1-GEM particle vaccine and COE-GEM particle vaccine, and the specific steps were performed according to the GEL-01 adjuvant instructions.​

[0104] COE-GEM particle vaccine was selected for immune route exploration experiment, 4 mice in each group, the immune dose was 24 μg per mouse, the F group blank control group was immunized with PBS (30 μL per mouse) by nose drop, immunized once every two weeks, a total of 3 times. According to the above dose, COE-GEM particle vaccine was used to immunize mice by muscle injection, nose drop, intraperitoneal injection and gavage, respectively. On the 0th, 14th, 28th and 42nd day after the initial immunization, blood and feces were collected to detect specific IgG and sIgA antibodies, and the production of PEDV specific antibodies in mice stimulated by different immune routes was analyzed.

[0105] The serum IgG antibody detection results are shown in Figure 6 After 14 days of immunization, the specific IgG antibody level of the intraperitoneal injection group was significantly higher than that of the gavage immunization group, the nose drop immunization group and the PBS control group (p<0.05), and there was no significant difference with the muscle injection group (p>0.05). After 28 days of immunization, the IgG antibody level of each immunization group continued to rise, which was extremely significantly higher than that of the PBS control group (p<0.001), and the IgG antibody level of the muscle injection group was the highest, which was extremely significantly different from that of the nose drop immunization group and the gavage immunization group (p<0.001), but there was no significant difference with the intraperitoneal injection group (p>0.05). After 42 days of immunization, the IgG antibody level of each immunization group reached the maximum, which was extremely significantly higher than that of the PBS control group (p<0.001), among which the muscle injection group reached 729.1 U / L, which was significantly higher than that of the intraperitoneal injection group (669.6 U / L) (p<0.05), and extremely significantly higher than that of the nose drop immunization group (467.1 U / L) and the gavage immunization group (298.9 U / L) (p<0.001).

[0106] The fecal sIgA antibody detection results are shown in Figure 7As shown in Table 2, after 14 days of immunization, the sIgA antibody levels of each immunization group had no significant difference compared with the PBS control group (p<0.05). After 28 days of immunization, the sIgA antibody levels of each immunization group increased, and were extremely significantly higher than the PBS control group (p<0.001). Among the four immunization groups, the sIgA antibody level of the nose drop group was the highest, and the difference was extremely significant (p<0.001). After 42 days of immunization, the sIgA antibody levels of each immunization group continued to rise, and the nose drop group had the highest sIgA antibody level of 21.6 U / L, which was extremely significantly higher than the intraperitoneal injection group (15.5 U / L), the gavage group (13.7 U / L) and the intramuscular injection group (11.3 U / L) (p<0.001). The above results showed that the COE-GEM granular vaccine could stimulate the mice to produce specific IgG and sIgA antibodies by different immunization routes. From the monitoring results, the antibody levels of each test group showed a continuous upward trend after immunization for 42 days. Considering the infection characteristics of PEDV and the production of IgG and sIgA antibodies by different immunization routes, the nose drop immunization route which could produce high levels of sIgA antibodies and stimulate the production of IgG antibodies was selected for subsequent experiments.

[0107] 5. Immunogen screening

[0108] To screen the immunogen with relatively good immunogenicity, the prepared S1-GEM and COE-GEM granular vaccines were used to immunize mice by nose drop at the same dose. The serum and feces of mice in each group were collected on the 0th, 14th, 28th and 38th days after the first immunization, and the production levels of specific IgG and sIgA antibodies were detected.

[0109] The serum IgG antibody detection results are shown in Table 3. Figure 8 As shown in Table 3, after 14 days of the first immunization, the IgG antibody levels of the two immunization groups were significantly higher than the PBS control group (p<0.05), but the antibody levels between the two immunization groups had no significant difference (p>0.05). After 28 days and 38 days of immunization, the IgG antibody levels of the two immunization groups continued to rise, and were extremely significantly higher than the PBS control group at the same period (p<0.001), but the difference between the two immunization groups at the same period was not significant (p>0.05).

[0110] The fecal sIgA antibody detection results are shown in Table 4. Figure 9 As shown in Table 4, after 14 days of the first immunization, the sIgA antibody levels of the two immunization groups had no significant difference compared with the PBS control group (p>0.05). After 28 days and 38 days of immunization, the sIgA antibody levels of the two immunization groups continued to rise rapidly, and were extremely significantly higher than the PBS control group at the same period (p<0.01), but the difference between the two immunization groups at the same period was not significant (p>0.05).

[0111] The above results show that both S1-GEM and COE-GEM particle vaccines can stimulate the body to produce specific IgG and sIgA antibodies, and there is no significant difference between the two, indicating that there is no difference in immunogenicity of the two antigens by nasal immunization.

[0112] 6. Screening of immunization dose

[0113] To further screen the best immunization dose, three different concentrations (4 μg, 12 μg, 24 μg) of COE-GEM particle vaccines were designed for nasal immunization of mice, and the serum and feces of each group of mice were collected on the 0th, 14th, 28th and 38th day after the first immunization to compare the levels of PEDV-specific IgG and sIgA antibodies produced by different doses of COE-GEM particle vaccines.

[0114] The serum IgG antibody detection results are shown in Table 2. Figure 10 As shown in Table 2, 14 days after the first immunization, the IgG antibody levels of each dose group were not significantly different from the PBS control group (p>0.05). 28 days after immunization, the IgG antibody levels of each immunization group rapidly increased and were extremely significantly higher than the PBS control group (p<0.001); and the 24 μg dose group was extremely significantly higher than the other two dose groups (p<0.001). 38 days after immunization, the IgG antibody levels of the 4 μg and 12 μg dose groups still rapidly increased, while the 24 μg dose group increased at a slower rate; although the antibody level of the 24 μg dose group was still the highest, there was no significant difference compared with the 12 μg dose group (p>0.05).

[0115] The fecal sIgA antibody detection results are shown in Table 3. Figure 11 As shown in Table 3, 14 days after the first immunization, the sIgA antibody levels of each dose group were not significantly different from the PBS control group (p>0.05). 28 days after immunization, the sIgA antibody levels of each immunization group increased, of which the 24 μg dose group was extremely significantly higher than the 4 μg dose group (p<0.01) and significantly higher than the 12 μg dose group (p<0.05). 38 days after immunization, the sIgA antibody levels of each immunization group were extremely significantly higher than the PBS control group (p<0.001), the 24 μg dose group was extremely significantly higher than the 4 μg dose group (p<0.001), and there was no significant difference compared with the 12 μg dose group (p>0.05).

[0116] The above results show that the 4 μg, 12 μg and 24 μg dose COE-GEM particle vaccines immunized by nasal method can stimulate the body to produce specific IgG and sIgA antibodies, and considering the production of IgG and sIgA at different immunization times, 24 μg is selected as the best immunization dose.

[0117] 7. Comparison of the best immunization group with the commercial vaccine

[0118] The best immune dose, the best immunogen and the best immune route screened by the above test were used for the comparative test of the vaccine immune effect. COE-GEM granular vaccine (24 μg of intranasal immunization) and commercial PEDV vaccine (TGE-PED double inactivated vaccine, used according to the instruction) were used to immunize mice, and the serum and feces of mice in each group were collected on the 0th day, 14th day, 28th day and 38th day after the initial immunization, so as to compare the levels of PEDV specific IgG and sIgA antibodies produced after the immunization of mice.

[0119] The serum IgG antibody detection results are shown in Table 2. Figure 12 As shown in Table 2, after the initial immunization for 14 days, the IgG antibody levels of the two immunization groups had no significant difference with the PBS control group (p>0.05). After the immunization for 28 days, the IgG antibody levels of the two immunization groups were increased, and the IgG antibody level of the COE-GEM immunization group was extremely significantly higher than that of the TGE-PED double inactivated vaccine immunization group (p<0.001). After the immunization for 38 days, the IgG antibody levels of the two immunization groups were continuously increased, and the IgG antibody level of the COE-GEM group reached 401 U / L, which was significantly higher than that of the TGE-PED group (332 U / L) (p<0.05).

[0120] The feces sIgA antibody detection results are shown in Table 3. Figure 13 As shown in Table 3, after the initial immunization for 14 days, the sIgA antibody levels of the two immunization groups had no significant difference with the PBS control group (p>0.05). After the immunization for 28 days, the sIgA antibody level of the COE-GEM group was rapidly increased, and the sIgA antibody level of the TGE-PED immunization group had no obvious change. After the immunization for 38 days, the sIgA antibody levels of the two immunization groups were increased, and the sIgA antibody level of the COE-GEM group reached 21.1 U / L, which was extremely significantly higher than that of the TGE-PED immunization group (9.62 U / L) (p<0.001).

[0121] 8. Mouse spleen T lymphocyte proliferation test

[0122] In order to further understand the stimulation of the COE-GEM granular vaccine to the immune system of mice, the MTT method was used to detect the spleen lymphocyte proliferation of mice after the intranasal immunization of COE-GEM (24 μg). The detection results are shown in Table 4. Figure 14 As shown in Table 4, the in vitro proliferation index (SI) of the spleen lymphocytes of the immunization group mice had significant difference compared with the PBS control group (*p<0.05). It is indicated that the intranasal immunization of mice with COE-GEM (24 μg) can promote the proliferation of spleen lymphocytes, and further promote the immune response.

[0123] ① Detection of IFN-γ and IL-4 mRNA

[0124] The qPCR method was used to detect the production of IFN-γ and IL-4 of mice after the intranasal immunization of COE-GEM (24 μg). The detection results are shown in Table 5.Figure 15 As shown, the levels of IFN-γ and IL-4 mRNA in the immunized mice were significantly different from those in the PBS control group (*p<0.05).

[0125] In summary, the S1 gene and COE gene of the PEDV epidemic strain in Guangxi were used as target genes, and the insect cell-baculovirus expression system was used to successfully express the fusion protein S1-PA and COE-PA fused with the anchor protein PA. At the same time, the lactic acid bacteria MG1363 was treated with heat and acid to remove the bacterial nucleic acid and protein, and GEM particles were successfully prepared. The fusion proteins S1-PA and COE-PA were successfully displayed on the surface of the GEM particles to prepare S1-GEM and COE-GEM. Further, the two kinds of PEDV bacterial-like particles prepared were used to immunize mice, and the immunization route, immunogen, and immunization dose were screened to confirm that 24 μg of COE-GEM intranasally immunized mice could not only induce the production of high levels of specific serum IgG antibodies in mice, but also induce the production of high levels of sIgA antibodies in the intestinal mucosa (feces) of mice, and the levels of IgG and sIgA antibodies induced were significantly higher than those in the TGE-PED bivalent inactivated vaccine group. At the same time, 24 μg of COE-GEM intranasally immunized mice could stimulate the proliferation of mouse spleen lymphocytes, and the expression levels of cytokines IFN-γ and IL-4 mRNA were significantly increased.

[0126] The above-described embodiments only express several embodiments of the present application, which are described in more detail and in more detail, but should not be understood as limiting the scope of the present application. It should be noted that for those skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims.

Claims

1. The application of porcine epidemic diarrhea virus (PEDV) bacterial-like particles in the preparation of vaccines for the prevention and treatment of porcine epidemic diarrhea caused by PEDV infection, characterized in that, The particles include recombinant proteins S1-PA or COE-PA displayed on the surface of GEM particles; the amino acid sequence of S1-PA is shown in SEQ ID NO. 3; the amino acid sequence of COE-PA is shown in SEQ ID NO. 4; The vaccine is administered via nasal drops. The PEDV mentioned is the Guangxi PEDV-GXNN prevalent strain.

2. The method for preparing porcine epidemic diarrhea virus bacterial-like particles as described in claim 1, characterized in that, The method includes the following steps: (1) Construction of fusion genes and their recombinant expression plasmids: The S1 gene or COE gene of PEDV was codon optimized for insect cells, and the Kozak sequence and GP67 signal peptide sequence were introduced at its 5' end. Then, the gene was fused with the codon-optimized PA gene of Lactococcus lactis to construct fusion genes S1-PA or COE-PA. The gene was then directionally cloned into the pFastBacI vector to obtain recombinant expression plasmids pFastBacI-S1-PA or pFastBacI-COE-PA. The nucleotide sequence of the fusion gene S1-PA is SEQ ID NO.

1. The nucleotide sequence of the fusion gene COE-PA is SEQ ID NO.

2. (2) Transform DH10Bac competent cells with the recombinant plasmid pFastBacI-S1-PA or pFastBacI-COE-PA obtained in step (1) to prepare recombinant bamboo plasmid Bacmid-S1-PA or Bacmid-COE-PA. (3) Recombinant baculoviruses Bacmid-S1-PA or Bacmid-COE-PA were transfected into Sf9 cells to rescue P1 generation recombinant viruses and obtained P1 generation recombinant baculoviruses, which were named Rbv-S1-PA and Rbv-COE-PA, respectively. Rbv-S1-PA or Rbv-COE-PA was inoculated into Sf9 cells at an MOI=1 ratio to express recombinant proteins. After the recombinant proteins were identified, the cell lysate supernatant was collected. (4) Preparation of GEM particles: Lactococcus lactis MG1363 was treated with hot acid to remove Lactococcus lactis nucleic acid and its own protein to obtain GEM particles. (5) Take 0.1U of the GEM particles from step (4) and add them to the cell lysis supernatant from step (3). Take the fusion protein with the maximum binding amount to obtain S1-GEM or COE-GEM particles. Mix the two particles with GEL-01 adjuvant to prepare S1-GEM particle vaccine or COE-GEM particle vaccine. The S1-GEM particle vaccine displays the recombinant protein S1-PA on the surface of the GEM particles. The COE-GEM particle vaccine displays the recombinant protein COE-PA on the surface of the GEM particles.

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