Preparation method of novel PEDV (porcine epidemic diarrhea virus) subunit vaccine

Abstract

The invention provides a preparation method of a novel PEDV (porcine epidemic diarrhea virus) subunit vaccine, and belongs to the technical field of biology. The novel PEDV subunit vaccine is prepared by adopting a method comprising the following steps: (1) treating lactococcus lactis by using a high-pressure damp-heat method to obtain peptidoglycan particles; (2) treating the peptidoglycan particles by adopting an ultrahigh-pressure microjet technology to obtain a peptidoglycan carrier with the particle size of 280-850nm; and (3) mixing the peptidoglycan carrier with a PEDV subunit antigen of which the amino acid sequence is SEQ ID NO: 1 to obtain the novel PEDV subunit vaccine. The novel PEDV subunit vaccine comprises a peptidoglycan carrier with a specific size, can improve the in-vivo presentation efficiency of an antigen and rapidly induce efficient body fluid and mucous membrane immune response, and is of great significance to research and development of vaccines for porcine epidemic diarrhea diseases and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for preparing a novel PEDV subunit vaccine. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is an important pathogen causing diarrhea in piglets, causing huge economic losses to the global pig industry. After neonatal piglets are infected with PEDV, the lethality rate is as high as 80%-100%, seriously threatening the healthy development of the pig industry. Traditional vaccines (such as inactivated vaccines and attenuated vaccines) have obvious limitations in terms of protective efficacy, safety, and broad-spectrum, and it is difficult to effectively cope with the rapid mutation of PEDV. Therefore, the development of novel vaccines has become the focus of current research.

[0003] Subunit vaccines can induce immune responses against specific viral epitopes through precise design of antigen proteins, and have higher specificity and effectiveness. Compared with traditional vaccines, subunit vaccines can not only provide more effective immune protection, but also distinguish natural infection from vaccination, which is of great significance for the prevention and control of PEDV. However, the immunogenicity of subunit vaccines is relatively weak, and usually the immune response needs to be enhanced by optimizing the antigen presentation method or adding highly effective adjuvants.

[0004] The cell wall skeleton of Lactococcus lactis is composed of multiple layers of peptidoglycan, which is a reticular macromolecule formed by the cross-linking of a polysaccharide structure and a tetrapeptide side chain. Peptidoglycan can activate the innate immune response by binding to pattern recognition receptors of the host (such as Toll-like receptors and Nod-like receptors), and mediate the adaptive immune response through innate immune cells such as dendritic cells and macrophages. However, the antigen presentation efficiency of peptidoglycan particles is relatively low, restricting its application. Summary of the Invention

[0005] The object of the present invention is to provide a novel PEDV subunit vaccine, which contains a peptidoglycan carrier with a specific size, can improve the in vivo antigen presentation efficiency, rapidly induce high-efficiency humoral and mucosal immune responses, and is of great significance for the development of vaccines for porcine epidemic diarrhea diseases and the like.

[0006] The object of the present invention is achieved by adopting the following technical solutions:

[0007] A novel PEDV subunit vaccine is prepared by a method comprising the following steps:

[0008] (1) Treat Lactococcus lactis by high-pressure moist heat method to obtain peptidoglycan particles;

[0009] (2) Treat the peptidoglycan particles by ultra-high pressure microfluidization technology to obtain a peptidoglycan carrier with a particle size of 280-850 nm;

[0010] (3) Mix the peptidoglycan carrier with the PEDV subunit antigen having the amino acid sequence of SEQ ID NO: 1 to obtain a novel PEDV subunit vaccine.

[0011] In the present invention, in step (1), Lactococcus lactis is treated by high-pressure hydrothermal method and then soaked with acid to obtain the peptidoglycan particles.

[0012] In the present invention, in step (2), the concentration of the peptidoglycan particles is 10 - 20 mg / mL.

[0013] In the present invention, in step (2), a microfluidic homogenizer is used in the ultra-high pressure microfluidization technology, and the treatment conditions are: the sample injection speed is 160 - 200 mL / min, the treatment pressure is 20500 - 21500 Psi, and the cycle is 2 - 4 times to obtain the peptidoglycan carrier.

[0014] In the present invention, in step (3), the mass ratio of the peptidoglycan carrier to the PEDV subunit antigen having the amino acid sequence of SEQ ID NO: 1 is 3 - 4:1.

[0015] In the present invention, in step (3), after the peptidoglycan carrier is mixed with the PEDV subunit antigen having the amino acid sequence of SEQ ID NO: 1, it is incubated at 20 - 30 °C under shaking conditions for 20 - 40 min to obtain a novel PEDV subunit vaccine.

[0016] In the present invention, the PEDV subunit antigen having the amino acid sequence of SEQ ID NO: 1 is obtained by transfecting a eukaryotic expression vector carrying the nucleotide sequence encoding the PEDV subunit antigen into eukaryotic cells for expression.

[0017] In the present invention, the nucleotide sequence encoding the PEDV subunit antigen is as shown in SEQ ID NO: 2.

[0018] In the present invention, in step (3), the recombinant eukaryotic vector is obtained by the following method: inserting the sequence shown in SEQ ID NO: 2 between the restriction enzyme cleavage sites BamHⅠ and NotⅠ of the pcDNA3.1 vector.

[0019] The novel PEDV subunit vaccine prepared by the present invention has the following beneficial effects compared with the prior art: The novel PEDV subunit vaccine of the present invention can be rapidly enriched on the surface of immune cells, has high antigen presentation efficiency and speed, significantly promotes the maturation and activation of antigen-presenting cells, rapidly induces efficient humoral and mucosal immune responses, and is of great significance for the development of vaccines against porcine epidemic diarrhea and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1It is the result of detecting peptidoglycan particles PG0 and the particle size of peptidoglycan with specific sizes using dynamic light scattering technology. The vertical coordinate represents the percentage of detection intensity for different particle sizes, and the horizontal coordinate represents the particle size.

[0021] Figure 2 It is the result of identifying the expression of SD protein and PEDV S protein in eukaryotic cells using the Western-blotting method. Lane 1 is the secreted supernatant of the cell line expressing SD protein; Lane 2 is the cell pellet in the culture medium of the cell line expressing SD protein; Lane 3 is the secreted supernatant of the cell line expressing S protein; Lane 4 is the cell pellet in the culture medium of the cell line expressing S protein; M represents the standard protein molecular weight marker (Cat No: WJ107, Thermo Fisher).

[0022] Figure 3 It is the result of ELISA detection of antibodies in mice immunized with peptidoglycan carriers of different sizes in combination with PEDV antigen for 28 days. Among them, A is the detection result of serum IgG 28 days after the first immunization, and B is the detection result of intestinal IgA after the first immunization. Among them, "*" indicates that the difference between each control group and the experimental group is significant (p < 0.05), "**" indicates that the difference between each control group and the experimental group is very significant (p < 0.01), "***" indicates that the difference between each control group and the experimental group is extremely significant (p < 0.001), and "****" indicates that the difference between each control group and the experimental group is extremely significant (p < 0.0001).

[0023] Figure 4 It is the result of ELISA detection of antibodies in mice immunized with peptidoglycan PG3 in different ways in combination with PEDV antigen for 28 days. Among them, A is the detection result of serum IgG on the 28th day, and B is the detection result of intestinal IgA. Among them, "*" indicates that the difference between each group is significant (p < 0.05), "**" indicates that the difference between each group is very significant (p < 0.01), "***" indicates that the difference between each group is extremely significant (p < 0.001), and "****" indicates that the difference between each group is extremely significant (p < 0.0001).

[0024] Figure 5 It is the result of ELISA detection of antibodies in mice immunized with different delivery systems in combination with PEDV subunit antigen for 28 days. Among them, A is the detection result of serum IgG 28 days after immunization, and B is the detection result of intestinal IgA. Among them, "*" indicates that the difference between each group is significant (p < 0.05), "**" indicates that the difference between each group is very significant (p < 0.01), "***" indicates that the difference between each group is extremely significant (p < 0.001), and "****" indicates that the difference between each group is extremely significant (p < 0.0001).

[0025] In the present invention, the porcine epidemic diarrhea virus NJ strain (abbreviated as PEDV NJ strain) is disclosed in CN201611248419.4, and the deposit number is: CGMCC NO.13283.

[0026] In the present invention, the PB buffer solution is a buffer solution containing disodium hydrogen phosphate and sodium dihydrogen phosphate, and its concentration refers to the concentration of all phosphate radicals.

[0027] The PBS buffer solution is a buffer solution containing disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride, and its concentration refers to the concentration of all phosphate radicals. If the "concentration of sodium chloride" is not specifically indicated, the concentration of sodium chloride is 0.1 mol / L. For example, for the PBS buffer solution (concentration 0.05 mol / L, pH 7.4 - 7.5), 0.05 mol / L is the concentration of the PBS buffer solution, that is, the concentration of all phosphate radicals, and the concentration of sodium chloride therein is 0.1 mol / L.

[0028] Example 1 Preparation of a peptidoglycan carrier with a specific size

[0029] 1. Cultivation of Lactococcus lactis

[0030] ① Prepare a commercial GM17 liquid medium (purchased from BD Difco, USA) according to the instructions with sterile water, and autoclave at 110 °C for 15 min.

[0031] ② Take out the Lactococcus lactis MG1363 strain preserved at -80 °C (disclosed in CN201310065186.4), inoculate it into the GM17 liquid medium, and culture it statically in a sealed sterile Erlenmeyer flask at 37 °C for 14 h to obtain a bacterial mother liquor with a large number of proliferated bacteria.

[0032] ③ Inoculate the bacterial mother liquor into the freshly prepared GM17 liquid medium at a ratio of 1:500 (volume ratio), and culture it statically in a sealed sterile Erlenmeyer flask at 30 °C for 36 h to obtain a fermentation broth.

[0033] ④ Centrifuge at 6000 rpm for 5 min to collect the bacterial cells, weigh the centrifuge tube containing the bacterial cells with a ten-thousandth analytical balance, and calculate the wet weight of the bacterial cells according to the formula: wet weight of bacterial cells = weight of the centrifuge tube containing the bacterial cells - weight of the centrifuge tube. Add sterile water to resuspend the bacterial cells to 50 g / L, and shake well to obtain a Lactococcus lactis suspension.

[0034] 2. Preparation of peptidoglycan particles

[0035] ① Place the Lactococcus lactis suspension obtained in step ④ of item 1 in a sterile conical flask, put it into a high-pressure steam sterilizer (Systec V-100, Germany), adjust the pressure to 0.12 MPa and the temperature to 100 °C, and treat for 10 min.

[0036] ② Wait for the high-pressure autoclave to cool down to 70 °C, take out the conical flask, transfer the bacterial liquid into a sterile centrifuge cup, centrifuge at 10000 rpm for 5 min, and collect the precipitate.

[0037] ③ Add 0.1 mol / L hydrochloric acid (pre-warmed in a 65 °C water bath) to the precipitate obtained in step ② until the final concentration of the bacteria is 25 g / L, vortex and shake to disperse evenly, soak and wash under the conditions of constant temperature 65 °C and shaking at 120 r / min for 30 min, centrifuge at 10000 rpm for 5 min, and collect the bacterial precipitate.

[0038] ④ Add sterile water 0.4 times the volume of the fermentation broth corresponding to the bacterial precipitate obtained in step ③ to resuspend the precipitate, disperse and wash thoroughly, centrifuge at 10000 rpm for 5 min to remove the washing solution, and wash the bacteria again with sterile water according to the above method. Then add PBS buffer (concentration 0.01 mol / L, pH 7.2 - 7.5) 0.1 times the volume of the fermentation broth corresponding to the bacterial precipitate to resuspend, disperse and wash thoroughly, centrifuge at 10000 rpm for 5 min, take the precipitate, and obtain peptidoglycan particles, labeled as PG0.

[0039] ⑤ Disperse the peptidoglycan particles obtained in step ④ in sterile PBS buffer (concentration 0.05 mol / L, pH 7.4 - 7.5) to obtain a peptidoglycan particle suspension, and store it at -20 to 4 °C for later use.

[0040] 3. Quantification of peptidoglycan particles

[0041] ① Take three samples, each 10 mL, from the peptidoglycan particle suspension obtained in Title 2, place them in a rotary evaporator and dry overnight to remove excess water.

[0042] ② Weigh the dried samples three times on an analytical balance with a precision of one ten-thousandth, and take the average measurement as the mass of each sample. Calculate the average value of the masses of the three samples, and thus calculate the concentration of the peptidoglycan particle suspension obtained in step 2 and the total mass of the peptidoglycan particles.

[0043] 4. Preparation of peptidoglycan carriers with specific sizes

[0044] ① Dilute the peptidoglycan particle suspension obtained in Title 2 with sterile PBS buffer (concentration 0.05 mol / L, pH 7.4 - 7.5) to 15 mg / mL, disperse evenly, and process it with a microfluidic homogenizer (Shanghai Nozzle, Nano-Lab type) to obtain peptidoglycan PG3. The processing conditions of the microfluidic homogenizer are as follows: control the temperature at 10 °C, adjust the injection speed to 180 mL / min, the processing pressure is 21000 Psi, and cycle 3 times.

[0045] With other conditions remaining unchanged, the processing pressures of the microfluidic homogenizer in the preparation steps of peptidoglycan PG3 were adjusted to 15000, 18000, 24000, and 27000 Psi respectively, and peptidoglycans PG1, PG2, PG4, and PG5 were obtained successively. The corresponding preparation conditions for each sample are shown in Table 1.

[0046] Table 1 Particle size distribution and preparation conditions of peptidoglycan with specific size

[0047] Sample Name PG0 PG1 PG2 PG3 PG4 PG5 Processing Pressure / Psi - 15000 18000 21000 24000 27000 Peak Particle Size / nm 1100 1720 1280 459 190 13.5 Particle Size Distribution / nm 825-1400 1100-3000 825-2600 295-825 122-295 10-18 PDI 0.312 0.753 0.833 0.327 0.245 0.202

[0048] Note: 1. The processing pressure described in Table 1 refers to the pressure for processing peptidoglycan particles using a microfluidic homogenizer; 2. "-" in Table 1 indicates that no microfluidic treatment was performed; 3. The particle size distribution described in Table 1 refers to the particle size intensity distribution range where the proportion (percentage of intensity) is greater than 90%; 4. PDI in the table represents the dispersion coefficient. The smaller the value, the more uniform the solution dispersion and the more stable the system.

[0049] ② Sample identification The peptidoglycan particles PG0 obtained in Title 2 and the peptidoglycans PG1, PG2, PG3, PG4, and PG5 with specific sizes obtained in Title 4 were respectively diluted to a peptidoglycan concentration (the concentration of particulate peptidoglycan or peptidoglycan with specific size) of 0.8 mg / mL with PBS buffer (concentration 0.05 mol / L, pH 7.4 - 7.5), and the particle sizes of the samples were analyzed using dynamic light scattering technology. The results show that ( Figure 1 ): PG0, as intact peptidoglycan particles ( Figure 1 shown by the blue broken line), presented as a milky white suspension state when observed with the naked eye. After standing for 4 hours, precipitation occurred, and it could be redispersed after shaking. Its particle size distribution was concentrated around 1100 nm, and more than 90% of the peptidoglycan was distributed in the range of 825 - 1400 nm. Peptidoglycan PG1 ( Figure 1 shown by the red broken line), also appeared as a milky white suspension when observed with the naked eye, and there were a large number of agglomerated suspended substances in the system. It could be dispersed after vigorous shaking, but it was extremely easy to aggregate and precipitate after standing. Its particle size intensity distribution range was relatively discrete, and more than 90% (about 94.2%) of the peptidoglycan was distributed in the range of 1100 - 3000 nm. Peptidoglycan PG2 ( Figure 1 shown by the green broken line), had similar appearance characteristics to PG1, also a milky white suspension with agglomerated suspended substances, could be dispersed by vigorous shaking, and was prone to aggregation and precipitation after standing. Its particle size intensity distribution range was relatively wide, and more than 90% (about 93.3%) of the peptidoglycan was distributed in the range of 825 - 2600 nm. Peptidoglycan PG3 ( Figure 1shown as the purple broken line), it appears as a whey-colored suspension to the naked eye. There are no flocculent aggregates in the system. Precipitation occurs after standing for 4 - 5 hours and can be dispersed by slight shaking. The particle size is mainly concentrated around 500 nm, and more than 90% (about 90.5%) of the peptidoglycan is distributed in the range of 295 - 825 nm. Peptidoglycan PG4( Figure 1 shown as the yellow broken line) and PG5( Figure 1 shown as the black broken line) are both light yellow transparent solutions to the naked eye and show good stability under centrifugation conditions. Their particle size intensity distributions are relatively concentrated, with peaks at 190 nm and 13.5 nm respectively. Among them, the proportion of PG4 with a particle size in the range of 122 - 295 nanometers is about 92%, and the proportion of PG5 with a particle size in the range of 10 - 18 nanometers is about 97%.

[0050] Based on the above experimental results, the high-pressure microfluidization pressure has a very significant effect on the size of peptidoglycan particles after treatment. When the pressure is at a relatively low level (such as 15000, 18000 Psi), the peptidoglycan particles cannot be fully destroyed, and a series of peptidoglycan fragments with uneven sizes are formed from the initial spherical particles. At this time, the particle size is large and the distribution range is wide, resulting in poor homogeneity and stability of the system. As the pressure gradually increases, the peptidoglycan particle size shows a gradually decreasing trend, the distribution range tends to be concentrated, and the stability and homogeneity of the solution are significantly improved.

[0051] Example 2 Preparation of PEDV Subunit Antigen

[0052] 1. Recombinant Vector Construction

[0053] The base sequence encoding the Spike protein (abbreviated as S protein) of the PEDV NJ strain (Accession No.: CGMCC NO.13283) was fused with the base sequence encoding the peptidoglycan recognition domain (Binding domain) (NCBI Accession NO: U17696, 1102 - 1293 bp) at the gene level through a flexible polypeptide. At the same time, 6 histidine tags were added to the carboxyl terminus of the fusion gene. After optimization, the fusion gene SD (spike-binding domain) encoding both the PEDV S protein and the peptidoglycan recognition domain was obtained. The nucleotide sequence of the fusion gene SD is shown in SEQ ID NO:2, and the encoded protein is named SD protein, and the amino acid sequence is shown in SEQ ID NO:1. The fusion gene SD was sent to Shanghai Bioengineering Co., Ltd. for full sequence synthesis. After sequence verification, it was inserted between the restriction enzyme sites BamHⅠ and NotⅠ of the eukaryotic expression vector pcDNA3.1 (CatNo.V790 - 20, Thermo Fisher Scientific) to obtain the recombinant eukaryotic expression plasmid pcDNA - SD.

[0054] 2. Construction and Screening of Transfected Cells

[0055] ① Cell Preparation: Culture CHO cells in Opti-MEM medium (CatNo. 31985070, Thermofisher) containing 10% (v / v) fetal bovine serum at 37 °C and 5% CO 2 conditions. 24 h before transfection, adjust the cell density to 5.5×10 5 cells / mL, passage and culture overnight, and control the cell density to reach 1.2 - 1.5×10 6 cells / mL before transfection.

[0056] 1 h before transfection, dilute the cells to 1×10 6 cells / mL with serum-free and antibiotic-free Opti-MEM, and culture in an incubator at 37 °C and 5% CO 2 for standby.

[0057] ② Preparation of Transfection Complex: At room temperature, dilute the recombinant plasmid pcDNA-SD (4 - 5 μg / μL, endotoxin-free) and the transfection reagent Lipofectamine TM 2000 (Invitrogen, CatNo11668 - 019) with serum-free Opti-MEM medium respectively. The specific operations are as follows: Add 2 μg of the recombinant plasmid pcDNA-SD to 100 μL of serum-free Opti-MEM medium for dilution, and gently mix well for standby; Dilute 4 μL of Lipofectamine TM 2000 to 100 μL with serum-free Opti-MEM medium, and gently mix well. Gently add the above diluted Lipofectamine TM 2000 solution to the diluted pcDNA-SD, gently invert and mix. At this time, the final concentration of Lipofectamine TM 2000 is 2 μL / μg DNA, and let it stand at 25 °C for 30 min to obtain the pcDNA-SD-Lipofectamine TM 2000 transfection complex.

[0058] ③ Cell Transfection: Add the prepared pcDNA-SD-Lipofectamine TM 2000 transfection complex (within 1 h after preparation) to the cells prepared in step ①, and gently shake to make the transfection complex evenly distributed and mixed. 4 - 6 hours after transfection, replace it with Opti-MEM containing 10% fetal bovine serum to provide sufficient nutrients for transfection. Place the cells in an incubator at 37 °C and 5% CO 2 for culture.

[0059] 3. Screening of stable expression cell lines and identification of protein expression

[0060] ① Screening after transfection: 48 hours after transfection in Example 2 of this title, add G418 antibiotic (Thermo Fisher, CatNo10131035) with a final concentration of 600 μg / mL to the transfection system to screen for stably transfected cells. Observe the cell growth status every day, and change the screening medium containing G418 every 3 - 5 days to maintain the G418 concentration at 600 μg / mL.

[0061] ② Monoclonal screening: 6 days after transfection, inoculate a small number of surviving resistant clones into a 96 - well plate by limited dilution, with 1 cell per well. Culture for 5 - 7 days. When the cells are distributed in clusters, mark the monoclonal cells, and gradually amplify and passage the monoclonal cells. Collect the cell supernatant, and analyze the monoclonal protein expression content by Western - blotting to screen for the high - expression stable cell line CHO - SD18.

[0062] 4. Preparation of PEDV subunit antigen

[0063] ① Purification of SD protein: Culture the obtained high - expression stable cell line CHO - SD18 in DMEM medium (11966025, Thermo Fisher) containing 10% fetal bovine serum at 37°C and 5% CO 2Cultivate for 56 hours under the above conditions, collect the above cells and culture supernatant, centrifuge the supernatant at 12,000 rpm for 5 min to separate the supernatant, and purify the SD protein in the culture supernatant by nickel ion affinity chromatography. The specific operations are as follows: Equilibrate the nickel affinity column (Lanxiao, NTA-Ni affinity column) to room temperature, and successively add 5 column volumes of deionized water and 5 column volumes of binding buffer (PB buffer with pH 8.0 and a concentration of 50 mmol / L containing 0.3 mol / L NaCl and 20 mmol / L imidazole) to wash the column body, and control the flow rate at 1 mL / min. Centrifuge the collected cell secretory supernatant at 12,000 r / min for 5 min, recover the supernatant and mix it with an equal volume of binding buffer, and add the mixed solution to the equilibrated nickel affinity column, and control the flow rate at 0.4 mL / min. After the sample loading is completed, add 5 column volumes of washing buffer (PB buffer with pH 8.0 and a concentration of 50 mmol / L containing 0.3 mol / L NaCl and 50 mmol / L imidazole) to wash the column body to remove non-specific attachment on the column body, and then add 1.5 column volumes of Elution buffer (PB buffer with pH 8.0 and a concentration of 50 mmol / L containing 0.3 mol / L NaCl and 300 mmol / L imidazole) to collect the target protein eluted. Add a new Elution buffer to repeat the above elution steps three times, successively collect the target protein eluted, and use SDS-PAGE to determine the protein purity.

[0064] ② Protein desalting and quantification: Mix the target protein solutions eluted multiple times above, place them in a low-power ultrasonic oscillator, and ultrasonically treat them at a power of 30 KHz for 10 min to remove the gas in the protein solution. Then filter with a 0.25 μm filter to remove large particle impurities. Subsequently, perform desalting treatment using a G25 gel filtration chromatography desalting column (Zhongke Senhui) to remove imidazole, and collect the effluent after elution. The obtained protein is the purified SD protein. Quantify the SD protein using a BCA protein quantification kit, adjust the protein concentration to 2 mg / mL, and store it at -80 °C for later use.

[0065] In the link of constructing the expression vector, according to the preparation method of the SD protein, replace the sequence of the fusion gene SD in the expression vector with the nucleotide sequence encoding the PEDV S protein, and keep the other conditions unchanged, and obtain the PEDV S protein using the eukaryotic expression system. Then purify the PEDV S protein using nickel ion affinity chromatography technology to obtain the purified PEDV S protein antigen, and store it for later use after quantification.

[0066] ③Identification of PEDV SD and S proteins Collect the eukaryotic expression cell culture supernatants of SD protein and S protein prepared above, centrifuge at 12,000 r / min for 5 min to separate the protein supernatant and cell precipitate. Take 80 μL of the protein supernatant and 80 μL of the cell precipitate resuspended in an equal volume respectively, add 20 μL of 5×loading buffer, and place in a metal bath at 100 °C for 10 min. Separate the proteins by 6% polyacrylamide gel electrophoresis, and then transfer the protein bands to a PVDF membrane by wet protein blotting for Western-blotting identification. The specific method is as follows: Block the PVDF membrane transferred with bands with PBST (PBS buffer with a pH of 7.4, a concentration of 50 mmol / L containing 0.5% Tween-20) containing 5% skim milk at room temperature for 1 h; then wash once with the above PBST, add mouse anti-PEDV S protein monoclonal antibody (purchased from Qianxun Biotech, Guangzhou) diluted at a ratio of 1:2000 with PBST, and incubate overnight (12 - 16 h) at 4 °C; wash 5 times with PBST, add goat anti-mouse HRP-IgG (product number: HS201-01, purchased from TransGen Biotech, Beijing) diluted at a ratio of 1:5000 with PBST, and react at 37 °C for 45 min. Wash 5 times with PBST, and add DAB reagent for protein staining.

[0067] It can be seen from Figure 2 that the size of SD protein is about 161 KD, and almost all of it is secreted into the cell culture supernatant ( Figure 2 , white arrow in lane 1). The size of PEDV S protein is about 151 KD, and most of the protein is in the cell secretion supernatant ( Figure 2 , dark blue arrow in lane 3), and a small part is expressed intracellularly ( Figure 2 , light blue arrow in lane 4). The Western-blotting results show that the SD protein and S protein obtained by eukaryotic expression both have good reactivity with PEDV S monoclonal antibody.

[0068] ④Preparation of PEDV antigen - peptidoglycan carrier complex Dilute the peptidoglycan PG3 prepared in Example 1 with sterile PBS buffer (pH 7.4, concentration 0.05 mol / L) to 6.5 mg / mL, vortex and shake thoroughly to disperse evenly, add it to an equal volume of purified SD protein at 2 mg / mL, and incubate with shaking at 25 °C and a rotation speed of 120 r / min for 30 min to obtain the PEDV antigen - peptidoglycan carrier complex SD - PG3.

[0069] According to the preparation method of the PEDV antigen - peptidoglycan carrier complex SD - PG3, replace PG3 with PG0, PG1, PG2, PG4, and PG5 respectively, and keep other conditions unchanged to prepare the PEDV antigen - peptidoglycan carrier complexes SD - PG0, SD - PG1, SD - PG2, SD - PG4, and SD - PG5.

[0070] According to the preparation method of the PEDV antigen - peptidoglycan carrier complex SD - PG3, replace the SD protein with the prepared PEDV S protein, and keep other conditions unchanged to prepare a mixture of the PEDV S antigen and peptidoglycan PG3, labeled as S + PG3.

[0071] Example 3 Preparation and Efficacy Verification of PEDV Subunit Vaccine

[0072] 1. Preparation of PEDV Subunit Vaccine

[0073] Before immunization, dilute the PEDV antigen - peptidoglycan carrier complex SD - PG3 prepared in Example 2 with PBS buffer at a concentration of 50 mmol / L and pH of 7.4 to a protein concentration of 100 μg / mL (at this time, the peptidoglycan concentration is 325 μg / mL) to prepare the experimental group vaccine.

[0074] According to the preparation method of the experimental group vaccine, replace the PEDV antigen - peptidoglycan carrier complex SD - PG3 with the PEDV antigen - peptidoglycan carrier complex SD - PG0, and keep other conditions unchanged to prepare control vaccine 1. According to the preparation method of the experimental group vaccine, replace the PEDV antigen - peptidoglycan carrier complex SD - PG3 with the PEDV antigen - peptidoglycan carrier complex SD - PG1, and keep other conditions unchanged to prepare control vaccine 2. According to the preparation method of the experimental group vaccine, replace the PEDV antigen - peptidoglycan carrier complex SD - PG3 with the PEDV antigen - peptidoglycan carrier complex SD - PG2, and keep other conditions unchanged to prepare control vaccine 3. According to the preparation method of the experimental group vaccine, replace the PEDV antigen - peptidoglycan carrier complex SD - PG3 with the PEDV antigen - peptidoglycan carrier complex SD - PG4, and keep other conditions unchanged to prepare control vaccine 4. According to the preparation method of the experimental group vaccine, replace the PEDV antigen - peptidoglycan carrier complex SD - PG3 with the PEDV antigen - peptidoglycan carrier complex SD - PG5, and keep other conditions unchanged to prepare control vaccine 5.

[0075] According to the preparation method of the experimental group vaccine, replace the PEDV antigen - peptidoglycan carrier complex SD - PG3 with the mixture of the PEDV S antigen and peptidoglycan PG3, S + PG3, and keep other conditions unchanged to prepare control vaccine 6.

[0076] According to the vaccine preparation method of the experimental group, replace the PEDV antigen - peptidoglycan carrier complex SD - PG3 with the purified PEDV S protein, and keep the others unchanged to prepare the antigen control vaccine.

[0077] Dilute the PEDV S protein with PBS buffer (concentration: 50 mmol / L, pH 7.4) to a concentration of 218 μg / mL, and mix it with the ISA201 adjuvant from SEPPIC Company, France, at a volume ratio of 46:54, and emulsify to prepare the adjuvant control vaccine.

[0078] Another set of PBS buffer (concentration: 50 mmol / L, pH 7.4) is used as the blank control vaccine.

[0079] The numbers of each vaccine and the doses of peptidoglycan carrier contained are shown in Table 1.

[0080] 2. Verification of the efficacy of PEDV subunit vaccine

[0081] Select 80 5 - week - old specific - pathogen - free BALB / c mice (about 25 g per mouse), and randomly divide them into 10 groups: Control Group 1, Control Group 2, Control Group 3, Control Group 4, Control Group 5, Control Group 6, Experimental Group, Antigen Control Group, Control Group 7, and Blank Control Group. Immunize them with the corresponding vaccines respectively. For the immunization grouping, refer to Table 1. Conduct the second immunization 14 days after the first immunization for the above - mentioned vaccines. The immunization method is: subcutaneous injection in the lower back, and the volume of each immunization is 100 μL per mouse. The immunization doses are shown in Table 2. Collect serum samples and intestinal samples 28 days after the first immunization, and analyze the secretion levels of serum IgG and intestinal IgA by ELISA method.

[0082] Table 2 Immunization grouping of mice and vaccine components

[0083]

[0084]

[0085] 3. Detection of serum IgG

[0086] Coat the ELISA plate with the virus culture solution of inactivated PEDV NJ strain (preservation number: CGMCC NO.13283) after purification by sucrose density gradient centrifugation. The specific operation is as follows:

[0087] ① Dilute the purified virus with coating buffer (sodium carbonate - sodium bicarbonate buffer, pH 9.6) to 2 μg / mL, add it to the blank ELISA plate (Shanghai Bioengineering Co., Ltd.), add 100 μL to each well, and coat it overnight (12 - 16 h) at 4℃.

[0088] ② Discard the solution, wash three times with PBST buffer (PBS buffer with a concentration of 50 mmol / L and pH 7.4 containing 0.5% Tween-20). Add 200 μL of blocking solution (PBST buffer containing 3% skim milk powder) to each well and block at 37 °C for 1 h.

[0089] ③ Wash three times with PBST buffer, air dry and store at 4 °C for later use.

[0090] ④ Dilute mouse serum at a dilution ratio of 1:8000 with PBS buffer (concentration of 50 mmol / L and pH 7.4), add it to the coated well plate, and react at 37 °C for 1 h.

[0091] ⑤ Then wash 5 times with PBST, and then add goat anti-mouse HRP-IgG (product number: HS201-01, TransGen Biotech, Beijing) diluted at a dilution ratio of 1:5000 with PBST buffer, 100 μL per well, and react at 37 °C for 45 min.

[0092] ⑥ Then wash 5 times with PBST, add 100 μL of TMB chromogenic solution per well, develop color at room temperature (20 °C) for 10 min, then add 100 μL of 2% sulfuric acid aqueous solution per well to terminate the reaction, and detect the OD value at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0093] Among them, the method for purifying the virus of the PEDVNJ strain (accession number: CGMCC NO.13283) is disclosed in the following literature: Hofmann M, Wyler R. Quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (PEDV). Vet Microbiol. 1989;20:131–42.

[0094] 4. Intestinal IgA detection

[0095] ① Twenty-eight days after the first immunization, randomly select 5 mice from each group, sacrifice them by cervical dislocation, dissect them, collect the middle segment of the small intestine of 3 - 5 cm, add 400 μL of PBS buffer (concentration of 50 mmol / L and pH 7.4), grind it with a 3 mm steel bead in a tissue homogenizer, centrifuge the obtained suspension at 12000 r / min for 5 min, take the supernatant, and dilute it with PBS buffer (concentration of 50 mmol / L and pH 7.4) at a dilution ratio of 1:50;

[0096] ② Add it to the ELISA plate coated with the PEDVNJ strain in Title 3 of this example, 100 μL per well, and react at 37 °C for 1 h;

[0097] ③ Wash 5 times with PBST, add 100 μL / well of goat anti-mouse HRP-IgA (Purigen C2219, Beijing, dilute the antibody with PBST at a ratio of 1:5000), and react at 37 °C for 1 h.

[0098] ④ Then wash 5 times with PBST, and successively add the chromogenic solution and the termination solution according to the method in Title 3 of this example, and detect the OD reading at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0099] The effects of peptidoglycan carriers of different sizes in combination with PEDV antigen on the humoral immune effect of the aqueous vaccine are shown in Figure 3 A, and the effects on the mucosal immune effect are shown in Figure 3 B. 28 days after immunization, the mean serum antibody values of each immunized group were as follows: Control Group 1 was 1.54, Control Group 2 was 1.30, Control Group 3 was 1.44, Control Group 4 was 1.71, Control Group 5 was 1.47, the experimental group was 2.28, the antigen control group was 1.17, and the blank control group was 0.03. The mean intestinal mucosal antibody values of each immunized group were as follows: Control Group 1 was 0.76, Control Group 2 was 0.74, Control Group 3 was 0.68, Control Group 4 was 0.82, Control Group 5 was 0.45, the experimental group was 1.18, the antigen control group was 0.43, and the blank control group was 0.01. The results showed that under the aqueous dosage form conditions, the antibody uniformity in Control Groups 2 and 3 was poor and the mean antibody values were low, indicating that too large a size and uneven particle size of the peptidoglycan carrier would affect the vaccine immune response; among Control Groups 1, 4, 5 and the experimental group with better antibody uniformity, the mean antibody value of Control Group 5 was the lowest, indicating that under the aqueous dosage form conditions, too small a size of peptidoglycan would affect its immune enhancement efficacy; while the antibody uniformity in the experimental group was good and the mean antibody value was much higher than that of the other groups, indicating that the size of PG3 in the experimental group was conducive to the peptidoglycan carrier to fully exert its immune enhancement effect and could promote the induction of high-level antibodies by the SD antigen.

[0100] The effects of the compatibility mode of peptidoglycan PG3 and PEDV antigen on the humoral immune effect of the aqueous vaccine are shown in Figure 4 A, and the effects on the mucosal immune effect are shown in Figure 4 B. The results showed that 28 days after immunization, for the experimental group vaccine, that is, the vaccine prepared by covalently binding peptidoglycan PG3 and PEDV antigen into one entity, the mean antibody values of the immunized mice were 2.28 for serum antibody and 1.18 for mucosal antibody, while for Control Group 6, that is, the vaccine prepared by mixing peptidoglycan PG3 and PEDV antigen, the mean antibody values of the immunized mice were 1.45 for serum antibody and 0.66 for mucosal antibody. This indicates that compared with simple mixing, preparing the vaccine by integrating the carrier peptidoglycan PG3 and the antigen into one entity is more conducive to the antigen presentation process after immunization and can effectively enhance the humoral immune and mucosal immune responses.

[0101] The effects of different delivery systems on the immune efficacy of PEDV subunit antigen vaccines are shown in Figure 5 Figures A and 5B. The results showed that 28 days after immunization, the humoral immune level in the experimental group was similar to that in Control Group 7, but the intestinal mucosal immune level was significantly higher than that in Control Group 7, indicating that using peptidoglycan PG3 as the PEDV antigen delivery system could not only induce a humoral immune level similar to that of the water-in-oil-in-water ISA201 adjuvant system, but also induce a high-level mucosal immune response simultaneously.

[0102] In summary, the vaccine containing the PEDV antigen - peptidoglycan carrier complex SD-PG3 can rapidly induce a high-level humoral immune response and mucosal immune response in the body.

Claims

1. A novel PEDV subunit vaccine, characterized in that The method is prepared by the following steps: (1) Treating Lactococcus lactis with high pressure wet heat to obtain peptidoglycan particles; (2) treating the peptidoglycan particles using ultrahigh pressure microfluidization technology to obtain a peptidoglycan carrier with a particle size of 280-850 nm; (3) Mixing the peptidoglycan carrier with a PEDV subunit antigen having an amino acid sequence of SEQ ID NO: 1 to obtain a new PEDV subunit vaccine.

2. The vaccine according to claim 1, characterized in that In step (1), the lactococcus lactis is treated by high pressure wet heat method and then soaked in acid to obtain the peptidoglycan particles.

3. The vaccine according to claim 2, characterized in that The concentration of the peptidoglycan particles in step (2) is 10-20 mg / mL.

4. The vaccine according to any one of claims 1 to 3, characterized in that In step (2), a microfluidizer is used in the ultrahigh pressure microfluidization technology, and the processing conditions are: injection speed is 160-200 mL / min, processing pressure is 20500-21500 Psi, and circulation is 2-4 times to obtain the peptidoglycan carrier.

5. The vaccine according to claim 4, characterized in that The mass ratio of the peptidoglycan carrier to the PEDV subunit antigen having an amino acid sequence of SEQ ID NO: 1 in step (3) is 3-4:

1.

6. The vaccine according to claim 5, characterized in that In step (3), the peptidoglycan carrier is mixed with the PEDV subunit antigen having an amino acid sequence of SEQ ID NO: 1, and then incubated at 20-30° C. with shaking for 20-40 min to obtain a novel PEDV subunit vaccine.

7. The vaccine according to claim 6, characterized in that The PEDV subunit antigen with the amino acid sequence of SEQ ID NO: 1 is expressed by transfecting a eukaryotic expression vector carrying a nucleotide sequence encoding the PEDV subunit antigen into a eukaryotic cell.

8. The vaccine according to claim 7, characterized in that The nucleotide sequence encoding the PEDV subunit antigen is shown in SEQ ID NO:

2.

9. The vaccine according to claim 8, characterized in that The recombinant eukaryotic vector in step (3) is obtained by the following method: inserting the sequence shown in SEQ ID NO: 2 between the restriction sites BamH Ⅰ and Not Ⅰ of the pcDNA3.1 vector.

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