Composite peptidoglycan immunopotentiator and application thereof

Abstract

The invention provides a composite peptidoglycan immunopotentiator and application thereof, and belongs to the technical field of biology. The composite peptidoglycan immunopotentiator is prepared by adopting a method comprising the following steps: (1) treating peptidoglycan particles prepared from lactococcus lactis by adopting an ultrahigh pressure microjet technology to obtain peptidoglycan particle lysis buffer; (2) adding calcium chloride and manganese chloride into the peptidoglycan particle lysis solution, and treating by adopting an ultrahigh-pressure microjet technology to obtain a peptidoglycan nano suspension; and (3) mixing the peptidoglycan particles with a peptidoglycan nano suspension to obtain the composite peptidoglycan immunopotentiator. The composite peptidoglycan immunopotentiator disclosed by the invention can activate innate immunity and adaptive immunity response, improve body fluid and mucous membrane immune levels and shorten an immune window period, and has important significance on development of vaccines for porcine epidemic diarrhea diseases and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, it relates to a preparation method of a composite peptidoglycan immune enhancer. Background Art

[0002] Peptidoglycan is a macromolecule with a reticular structure formed by cross-linking glycan chains formed by alternating N-acetylmuramic acid and N-acetylglucosamine with tetrapeptide side chains. It can recognize host pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and Nod-like receptors (Nod1 and Nod2), and initiate innate immune responses. The synergistic effect of Nod1 and TLRs not only enhances TLR-mediated IL-12 release, but also promotes the initiation of Th1 and Th17 immune responses, which is crucial for adaptive immune responses. In addition, peptidoglycan mediates humoral and mucosal immune responses by activating innate immune cells such as dendritic cells (DCs) and macrophages, promoting the activation and differentiation of T cells and the antibody production of B cells.

[0003] Lactococcus lactis is a food-grade safe Gram-positive bacterium with simple culture conditions and easy large-scale culture. The main component of its cell wall skeleton is multi-layer peptidoglycan. Preparing peptidoglycan from Lactococcus lactis has good biological safety and economic benefits. In production, bacterial nucleic acids, surface proteins, and lipids are mainly removed by acid boiling treatment to obtain particulate peptidoglycan, and further lysozyme is added to prepare peptidoglycan fragments from the particles. Peptidoglycan fragments are mainly used as feed additives with the effect of enhancing immunity. Peptidoglycan particles bind to foreign proteins through specific functional domains, can be used as biosorbents or biocatalysts to treat harmful substances, and can also be used as antigen delivery carriers and mucosal immune enhancers for vaccine development.

[0004] However, in the prior art, when using peptidoglycan as an immune enhancer for vaccine development, the immune enhancement effect is not ideal. Summary of the Invention

[0005] The purpose of the present invention is to provide a composite peptidoglycan immune enhancer, which can activate innate and adaptive immune responses, and at the same time improve the levels of humoral and mucosal immunity.

[0006] Another purpose of the present invention is to provide the application of the composite peptidoglycan immune enhancer in the preparation of veterinary vaccines. This veterinary vaccine can induce rapid and efficient humoral and mucosal immune responses, which is of great significance for the development of vaccines against diseases such as porcine epidemic diarrhea.

[0007] The purpose of the present invention is achieved by adopting the following technical solutions:

[0008] A composite peptidoglycan immune enhancer is prepared by a method including the following steps:

[0009] (1) Treat the peptidoglycan particles prepared from Lactococcus lactis with the ultra-high pressure microfluidization technology to obtain a peptidoglycan particle lysate;

[0010] (2) Add calcium chloride and manganese chloride to the peptidoglycan particle lysate and treat it with the ultra-high pressure microfluidization technology to obtain a peptidoglycan nano-suspension;

[0011] (3) Mix the peptidoglycan particles with the peptidoglycan nano-suspension to obtain a composite peptidoglycan immune enhancer.

[0012] In the present invention, Lactococcus lactis is treated by the high-pressure wet heat method and then acid-soaked to obtain peptidoglycan particles.

[0013] In the present invention, in the ultra-high pressure microfluidization technology in step (1), a microfluidizer is used, and the treatment conditions are: treating at 18000 - 22000 Psi for 2 - 4 cycles; then treating at 23000 - 27000 Psi for 2 - 4 cycles.

[0014] In the present invention, the concentration of calcium chloride in the peptidoglycan nano-suspension is 0.06 - 1.0 mg / mL, the concentration of manganese chloride is 0.03 - 0.05 mg / mL, and the concentration of peptidoglycan with nano size is 8 - 12 mg / mL.

[0015] In the present invention, in the ultra-high pressure microfluidization technology in step (2), a microfluidizer is used, and the treatment conditions are: treating at 21000 - 25000 Psi for 2 - 4 cycles.

[0016] In the present invention, in the composite peptidoglycan immune enhancer in step (3), the mass ratio of peptidoglycan particles to peptidoglycan with nano size is 4:5 - 7.

[0017] The present invention also provides the application of the composite peptidoglycan immune enhancer in the preparation of veterinary vaccines.

[0018] In the present invention, the veterinary vaccine is a porcine epidemic diarrhea vaccine.

[0019] In the present invention, after mixing the composite peptidoglycan immune enhancer with an antigen, it is mixed with ISA201 adjuvant and emulsified to obtain the veterinary vaccine.

[0020] The MPP-3 prepared by the present invention is a compound preparation of peptidoglycan particles and peptidoglycan metal nanoparticles, and has the following beneficial effects compared with the prior art: The present invention uses a high-pressure hydrothermal method to prepare peptidoglycan particles, combines the ultra-high pressure microfluidization technology and the nanoparticle technology, and adds metal ions at the same time to prepare a nanoscale metal ion peptidoglycan suspension, obtaining a composite peptidoglycan immune enhancer, which significantly enhances the promotion effect on the maturation and activation of antigen-presenting cells, can induce rapid and efficient humoral and mucosal immune responses, significantly improves the antibody level, shortens the immune window period, and is of great significance for the development of vaccines against porcine epidemic diarrhea disease and the like. Description of the Drawings

[0021] In order to more clearly illustrate the embodiments of the present invention, the drawings that appear in the embodiments will be briefly introduced below. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other more drawings can be obtained based on these drawings.

[0022] Figure 1 are the results of particle size and potential analysis of peptidoglycan particles, peptidoglycan particle lysate, and composite peptidoglycan immune enhancers MPP-0 to MPP-3 prepared by using dynamic light scattering technology and potentiometer analysis. Among them Figure 1 A is the particle size detection result, and the vertical axis represents the percentage of the intensity of particles with different particle sizes (Intensity). Figure 1 B is the potential detection result.

[0023] Figure 2 are the results of detecting the mRNA transcription levels of cytokines and chemokines produced by the in vitro activation of mouse dendritic cells (DCs) by qRT-PCR method. The vertical axis is the change in the fluorescence intensity of the mRNA of each detection index relative to the fluorescence intensity of the internal reference gene β-actin (i.e., relative mRNA fold). Among them Figure 2 A, 2B, 2C, 2D are the relative mRNA transcription levels of IL-12, TNF-α, IL-1β, and MIP-3α respectively. 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).

[0024] Figure 3It is the relative transcription levels of cytokines and chemokine mRNAs produced by the in vitro activation of porcine dendritic cells (DCs) detected by qRT-PCR method. The vertical axis is the change in the fluorescence intensity of the mRNA of each detection index relative to the fluorescence intensity of the internal reference gene β-actin (i.e., relative mRNA fold). Among them, Figure 3 A, 3B, 3C, and 3D are the relative mRNA transcription levels of IL-12, TNF-α, IL-1β, and CCL-20, respectively. 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).

[0025] Figure 4 It is the relative transcription results of cytokines IFN-γ, IL-4, and IL-17A of mouse T cell in vitro polarization detected by qRT-PCR method. Among them, Figure 4 A, 4B, and 4C represent the relative mRNA transcription levels of IL-4, IFN-γ, and IL-17A, respectively. 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).

[0026] Figure 5 It is the evaluation result of the serum antibodies of piglets immunized with the composite peptidoglycan immune enhancer MPP-3 combined with the inactivated PEDV vaccine at 14 days and 28 days. 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).

[0027] Figure 6It is the evaluation result of oral mucus antibody of piglets immunized with the compound peptidoglycan immune enhancer MPP-3 in combination with PEDV inactivated vaccine for 28 days. 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).

[0028] Figure 7 It is the detection result of serum antibody of piglets immunized with the compound peptidoglycan immune enhancer MPP-3 in combination with PEDV S protein subunit vaccine for 14 days and 28 days. 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).

[0029] Figure 8 It is the detection result of oral mucus IgA antibody of piglets immunized with the compound peptidoglycan immune enhancer MPP-3 in combination with PEDV S protein subunit vaccine for 28 days. 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).

[0030] In the present invention, the porcine epidemic diarrhea virus NJ strain is disclosed in CN201611248419.4, deposit number: CGMCC NO.13283. Specific embodiments

[0031] Example 1 Preparation and quantification of the compound peptidoglycan immune enhancer MPP-3

[0032] 1. Cultivation of Lactococcus lactis

[0033] ① Prepare a commercial GM17 liquid medium (purchased from BD Difco, USA) according to the instructions with deionized water, and autoclave at 110°C for 15 min.

[0034] ② Take out the Lactococcus lactis MG1363 strain stored at -80 °C (disclosed in CN201310065186.4), inoculate it into GM17 liquid medium, and culture it statically in a sealed sterile Erlenmeyer flask at 37 °C for 14 h under anaerobic conditions to obtain a bacterial mother liquor with a large number of proliferated bacteria.

[0035] ③ Inoculate the bacterial mother liquor into 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 under anaerobic conditions to obtain a fermentation broth.

[0036] 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: 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.

[0037] 2. Preparation of peptidoglycan particles

[0038] ① Place the Lactococcus lactis suspension obtained in step 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.

[0039] ② Wait for the high-pressure steam sterilizer to cool to 70 °C, take out the conical flask, transfer the bacterial liquid into a sterile centrifuge tube, centrifuge at 10000 rpm for 5 min, and collect the precipitate.

[0040] ③ Add 0.1 mol / L hydrochloric acid (pre-warmed in a 65 °C water bath) to the precipitate obtained in step ② to a final concentration of 25 g / L of the bacteria, vortex and shake to fully disperse, soak and wash under constant temperature at 65 °C and 120 r / min for 30 min, centrifuge at 10000 rpm for 5 min, and collect the bacterial precipitate.

[0041] ④ Add 0.4 times the volume of the fermentation broth corresponding to the bacterial precipitate obtained in step ③ of sterile water to resuspend the bacterial precipitate, fully disperse and wash, 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 0.1 times the volume of the fermentation broth corresponding to the bacterial precipitate of PBS buffer (0.01 mol / L, pH 7.2 - 7.5) to resuspend, fully disperse and wash, centrifuge at 10000 rpm for 5 min to remove the washing solution.

[0042] ⑤ The precipitate after washing in step ④ is the peptidoglycan particles. Disperse the particles in sterile PBS buffer (0.05 mol / L, pH 7.4 - 7.5) to obtain a peptidoglycan particle suspension, and store it at -20 - 4 °C for later use.

[0043] 3. Quantitative determination of peptidoglycan particles

[0044] ①Precisely take three samples, each with a volume of 10 mL, from the peptidoglycan particle suspension obtained in Step 2, and place them in a rotary evaporator to dry overnight to remove excess moisture.

[0045] ②Weigh the dried samples three times using 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, thereby calculating the concentration of the peptidoglycan particle suspension obtained in Step 2 and the total mass of the peptidoglycan particles.

[0046] 4. Preparation of peptidoglycan particle lysate

[0047] ①Dilute the peptidoglycan particle suspension obtained in Step 2 with sterile PBS buffer (0.05 mol / L, pH 7.4 - 7.5) to 12 mg / mL and disperse it evenly.

[0048] ②Process the peptidoglycan particle suspension in Step ① using a microfluidic homogenizer (Shanghai Nozzle, Nano-Lab type) to obtain a peptidoglycan particle lysate (denoted as MP-0). The processing conditions of the microfluidic homogenizer are as follows: control the temperature at 6°C, adjust the injection speed to 180 mL / min, first process at a high pressure of 20000 Psi for 3 cycles; then process at a high pressure of 25000 Psi for 3 cycles. The peptidoglycan in the peptidoglycan particle lysate is in nanoscale. In the following reagent preparation, the peptidoglycan derived from the peptidoglycan particle lysate is denoted as nanoscale peptidoglycan.

[0049] 5. Preparation of metal peptidoglycan nanosuspension

[0050] (1) Peptidoglycan nanosuspension containing calcium chloride and manganese chloride

[0051] The preparation method of the peptidoglycan nanosuspension containing calcium chloride and manganese chloride is as follows:

[0052] ①Under stirring, sequentially add 100 mg / mL calcium chloride aqueous solution and 100 mg / mL manganese chloride aqueous solution to the peptidoglycan particle lysate obtained in Step 4, and supplement PBS buffer (pH 7.4 - 7.5, 0.05 mol / L) to make the final concentration of calcium chloride 0.08 mg / mL, the final concentration of manganese chloride 0.04 mg / mL, and the final concentration of nanoscale peptidoglycan 10 mg / mL, and mix well.

[0053] ② Treat the above suspension with a microfluidic homogenizer (Shanghai Nozo, Nano-Lab type) to obtain a peptidoglycan nano-suspension containing calcium chloride and manganese chloride (denoted as MP-3). The treatment conditions of the microfluidic homogenizer are as follows: control the temperature at 15 °C, adjust the injection speed to 120 mL / min, treat with a high pressure of 23,000 Psi, and circulate 3 times.

[0054] (2) Peptidoglycan nano-suspension containing calcium chloride

[0055] In the peptidoglycan nano-suspension containing calcium chloride (denoted as MP-1), the concentration of calcium chloride is 0.08 mg / mL, and the concentration of nano-sized peptidoglycan is 10 mg / mL. Prepare the peptidoglycan nano-suspension containing calcium chloride according to the preparation method of the peptidoglycan nano-suspension containing calcium chloride and manganese chloride, with the difference that only calcium chloride aqueous solution is added in step ①, and no manganese chloride aqueous solution is added.

[0056] (3) Peptidoglycan nano-suspension containing manganese chloride

[0057] In the peptidoglycan nano-suspension containing manganese chloride (denoted as MP-2), the concentration of manganese chloride is 0.04 mg / mL, and the concentration of nano-sized peptidoglycan is 10 mg / mL. Prepare the peptidoglycan nano-suspension containing manganese chloride according to the preparation method of the peptidoglycan nano-suspension containing calcium chloride and manganese chloride, with the difference that only manganese chloride aqueous solution is added in step ①, and no calcium chloride aqueous solution is added.

[0058] 6. Preparation of composite peptidoglycan immune enhancer MPP-3

[0059] Under sterile conditions, dilute the peptidoglycan particle suspension obtained in step 2 with PBS buffer (0.05 mol / L, pH 7.4 - 7.5) to 10 mg / mL, and mix it with the peptidoglycan nano-suspension MP-3 containing calcium chloride and manganese chloride obtained in step 5 (the concentration of nano-sized peptidoglycan is 10 mg / mL) according to a volume ratio of 4:6, so that the final concentration of peptidoglycan particles in the solution is 4 mg / mL, and the final concentration of nano-sized peptidoglycan is 6 mg / mL. Stir well, and the resulting mixture is the composite peptidoglycan immune enhancer MPP-3, which is stored at 4 °C for standby.

[0060] According to the preparation method of the composite peptidoglycan immune enhancer MPP-3, replace the peptidoglycan nano-suspension containing calcium chloride and manganese chloride with a peptidoglycan particle lysate without metal ions (the concentration of nano-sized peptidoglycan is 10 mg / mL), and keep the others unchanged to prepare the composite peptidoglycan immune enhancer MPP-0; replace the peptidoglycan nano-suspension containing calcium chloride and manganese chloride with a peptidoglycan nano-suspension containing calcium chloride (MP-1), and keep the others unchanged to prepare the composite peptidoglycan immune enhancer MPP-1; according to the preparation method of the composite peptidoglycan immune enhancer MPP-3, replace the peptidoglycan nano-suspension containing calcium chloride and manganese chloride with a peptidoglycan nano-suspension containing manganese chloride (MP-2), and keep the others unchanged to prepare the composite peptidoglycan immune enhancer MPP-2. The concentrations of each substance in MPP-0, MPP-1, MPP-2, and MPP-3 are shown in Table 1.

[0061] Table 1 Final Concentrations of Each Substance in MPP-0, MPP-1, MPP-2, and MPP-3

[0062] Name Peptidoglycan particle concentration Nanoscale peptidoglycan concentration Calcium chloride Manganese chloride MPP-0 4mg / mL 6mg / mL 0 0 MPP-1 4mg / mL 6mg / mL 0.048mg / mL 0 MPP-2 4mg / mL 6mg / mL 0 0.024mg / mL MPP-3 4mg / mL 6mg / mL 0.048mg / mL 0.024mg / mL

[0063] Sample analysis: Dilute the peptidoglycan particles obtained in step 2, the peptidoglycan particle lysate obtained in step 4, and the composite peptidoglycan immune enhancers (MPP-0, MPP-1, MPP-2, MPP-3) obtained in step 6 with PBS buffer (0.01 mol / L, pH 7.2 - 7.5) to a total peptidoglycan concentration (the sum of the peptidoglycan particle and nano-sized peptidoglycan peptide concentrations) of 0.8 mg / mL, and use dynamic light scattering technology and a Zeta potential analyzer to detect and analyze the particle size and potential of the samples. The results show ( Figure 1 A), the peptidoglycan particles with a complete structure ( Figure 1 A blue broken line) have a particle size of about 1100 nm, and precipitation occurs after standing for 4 h and can be dispersed by shaking; the peptidoglycan particle lysate ( Figure 1 A red broken line) is a slightly yellow, transparent, and uniform solution visually, with a particle size of about 10 nm and is stable upon centrifugation. Therefore, the peptidoglycan particle lysate contains nano-sized peptidoglycan. The composite peptidoglycan immune enhancers MPP-0, MPP-1, MPP-2, and MPP-3 are suspensions (corresponding to Figure 1 A green broken line, Figure 1 A purple broken line, Figure 1 A orange broken line and Figure 1A black broken line), the particle size distribution mainly includes two regions. Region 1 basically coincides with the particle size distribution of peptidoglycan particles, about 1100 nm; Region 2 respectively contains the corresponding peptidoglycan nano-suspensions, with particle sizes of about 10 nm, 120 nm, 220 nm, and 150 nm. Among them, MPP-0, MPP-1, and MPP-3 are relatively stable and can be stably stored at 4 °C for 6 to 8 months. After shaking evenly to disperse, a little light brown precipitate appears in MPP-2 after being placed at 4 °C for 4 weeks. The potential differences of each sample are not obvious ( Figure 1 B), showing a negative charge in a solution with pH 7.2 - 7.5. Therefore, the peptidoglycan derived from the peptidoglycan particle lysate in the composite peptidoglycan immune enhancer MPP-0, MPP-1, MPP-2, and MPP-3 has a particle size greater than 0 and less than 400 nm, and the peptidoglycan derived from the peptidoglycan particle part has a particle size greater than 500 nm and less than 2500 nm, and the particle size is concentrated between 1000 nm - 1500 nm.

[0064] Example 2 Verification of the in vitro immune enhancement efficacy of the composite peptidoglycan immune enhancer MPP-3

[0065] To verify the immune enhancement effect of the composite peptidoglycan immune enhancer MPP-3, MPP-3 was co-cultured with mouse or porcine dendritic cells (DCs) in vitro. By analyzing the transcriptional levels of DCs activation-related cytokines IL-12, IL-1β, TNF-α, and chemokine CCL-20 (porcine) / MIP-3α (mouse) after co-culture, the effect of MPP-3 on DCs activation and its antigen presentation ability was evaluated.

[0066] 1. DCs activation in vitro

[0067] (1) Preparation of mouse bone marrow dendritic cells (DCs)

[0068] ① Disinfection: Select 5-week-old Balb / c mice, decapitate and immerse them in 75% ethanol for disinfection;

[0069] ② Sampling: Lie the mouse on its back and fix its limbs in a sterile operating table, cut open the skin of the hind limbs, aseptically remove tissues such as muscles, take out the complete hind limb femurs, and wash them twice in PBS (0.01 mol / L, pH 7.2 - 7.5).

[0070] ③ Bone marrow cell extraction: Aseptically cut open both ends of the femur, use a 5 mL syringe to suck RPMI1640 medium (Gibco, USA) and pierce it from one end, repeatedly rinse the bone marrow cavity 3 - 5 times, collect the bone marrow cells, and filter them twice through a 70 μm filter membrane to remove large particle impurities.

[0071] ④ Erythrocyte removal: Centrifuge the above cells at 1000 rpm for 5 min to collect them, add commercial erythrocyte lysate and treat for 2 - 5 min, remove the cell lysate, add PBS (0.01 mol / L, pH 7.2 - 7.5) and wash once to obtain bone marrow mononuclear cells.

[0072] ⑤ Cell culture and counting: Use a cell counting chamber to count the number of bone marrow mononuclear cells, inoculate 2×10 7 bone marrow mononuclear cells into a 10-cm diameter cell culture dish, add RPMI 1640 medium containing 10% fetal bovine serum (FBS, Gibco, USA), and add murine GM-CSF (granulocyte macrophage-stimulating factor) and IL-4 (interleukin-4) to a final concentration of 10 ng / mL each. Culture under the conditions of 37°C and 5% CO2. After 72 hours, replace half of the culture medium with fresh RPMI 1640 medium containing 10% FBS, and at the same time supplement the stimulating factors GM-CSF (ABclonal, Wuhan) and IL-4 (ABclonal, Wuhan) to a final concentration of 10 ng / mL each. After culturing for 7 days, collect the cells in the culture dish with a cell scraper, which are murine dendritic cells (DCs), and count them for standby.

[0073] (2) Preparation of porcine bone marrow dendritic cells (DCs)

[0074] ① Selection of piglets: Select healthy young pigs, collect the femurs of the hind limbs of the pigs, and rinse and disinfect them successively with 75% alcohol and sterile PBS (0.01 mol / L, pH 7.2 - 7.5).

[0075] ② Extraction of bone marrow cells: Open the femoral head in a sterile operating table, use a 50-mL syringe to aspirate an appropriate amount of medium to rinse the bone marrow cavity, collect the bone marrow cells, filter them twice through a 70-μm filter membrane, and centrifuge to collect the cells;

[0076] ③ Erythrocyte removal: Add commercial erythrocyte lysate to remove erythrocytes and collect mononuclear cells.

[0077] ④ Cell culture and counting: Resuspend the isolated mononuclear cells with RPMI 1640 medium containing 10% FBS, add porcine GM-CSF (MCE, USA) and IL-4 (MCE, USA) as stimulating factors to a concentration of 10 ng / mL each. Adjust the cell concentration and inoculate it into a cell culture dish, and culture it in an incubator at 37°C and 5% CO2. Regularly observe the cell status and replace the culture medium within 72 - 96 hours of culture, and at the same time supplement the stimulating factors. After culturing for 7 days, collect the cells, which are porcine dendritic cells (DCs), and count them for standby.

[0078] (3) In vitro DC activation experiment

[0079] Inoculate the prepared mouse DCs into a 24-well cell culture plate, adjust the cell concentration to 5×10 6 cells / well, add the composite peptidoglycan immune enhancer MPP-3 and co-culture with the cells. Add 2 mL of culture medium to each well. The final concentration of peptidoglycan in the system (referring to the total final concentration of peptidoglycan, which is the sum of the concentration of peptidoglycan particles and nano-sized peptidoglycan) is 20 μg / mL, serving as the experimental group. Meanwhile, replace the composite peptidoglycan immune enhancer MPP-3 with peptidoglycan particles, peptidoglycan particle lysate, composite peptidoglycan immune enhancers MPP-0, MPP-1, and MPP-2 respectively as control groups (each sample needs to be shaken for 5 - 10 min before use to be fully dispersed). The final concentration of peptidoglycan in each control group (referring to the total final concentration of peptidoglycan, which is the sum of the concentration of peptidoglycan particles and nano-sized peptidoglycan) is 20 μg / mL; additionally, set up a blank cell control group that only adds cells and replaces the stimulator with PBS (0.05 mol / L, pH 7.4 - 7.5) buffer. The final concentrations of each stimulator in the experimental group, control groups, and blank cell control group are shown in Table 2. Set three parallel wells for each sample. After co-culturing for 12 h, collect the cells.

[0080] According to the above method for in vitro activation test of mouse DCs, conduct an in vitro activation test on porcine DCs. The only difference is that in the experimental group, add the stimulator composite peptidoglycan immune enhancer MPP-3 until the final concentration of peptidoglycan (referring to the total final concentration of peptidoglycan, which is the sum of the concentration of peptidoglycan particles and nano-sized peptidoglycan) reaches 50 μg / mL. In each of the other control groups, add the corresponding stimulator until the final concentration of peptidoglycan (referring to the total final concentration of peptidoglycan, which is the sum of the concentration of peptidoglycan particles and nano-sized peptidoglycan) reaches 50 μg / mL.

[0081] Refer to Table 2 and Table 3 for the grouping of the in vitro activation test of DCs.

[0082] Table 2 Stimulators and stimulation concentrations for mouse DCs

[0083]

[0084]

[0085] Note: In Table 2, the "final concentration of peptidoglycan" refers to the total final concentration of peptidoglycan in the system, which is the sum of the concentration of peptidoglycan particles and nano-sized peptidoglycan; "-" indicates none.

[0086] Table 3 Stimulators and stimulation concentrations for porcine DCs

[0087] Grouping Stimulus components Final concentration of peptidoglycan in the stimulation system μg / mL Control group 1 Peptidoglycan particles 50 Control group 2 Peptidoglycan particle lysate 50 Control group 3 MPP-0 50 Control group 4 MPP-1 50 Control group 5 MPP-2 50 Experimental group MPP-3 50 Blank control group PBS buffer -

[0088] Note: In Table 3, the "final concentration of peptidoglycan" refers to the total final concentration of peptidoglycan in the system, which is the sum of the concentration of peptidoglycan particles and nano-sized peptidoglycan; "-" indicates none.

[0089] (4) Relative fluorescence quantitative (qRT-PCR) detection

[0090] ① RNA extraction: Collect the cells after stimulation in item (3) of the title, and extract total RNA according to the instructions of the commercially available RNA extraction kit (Aibotech, Wuhan). Detect the A260 / A280 reading value with Nano-drop, analyze the purity and concentration of the extracted RNA, and store it at -70 °C for later use.

[0091] ② cDNA preparation: Take the same amount of RNA (0.5 μg), and perform cDNA synthesis according to the reverse transcription instructions (Aibotech, Wuhan). The synthesis system is as follows: 5 μL of 4×ABScript Neo RT Master Mix, 1 μL of 5×gDNAClean Buffer, 16 μL of RNA template. The reaction program is 42 °C for 2 min, 55 °C for 15 min, and 85 °C for 5 min.

[0092] ③ qRT-PCR: Using the housekeeping gene β-actin as the internal reference gene, use the qRT-PCR method to analyze the mRNA transcription levels of DCs activation-related cytokines IL-12, IL-1β, TNF-α, and chemokines CCL-20 (pig), MIP-3α (mouse). Set three parallel controls for each gene. The qRT-PCR reaction system and program refer to the instructions (Aibotech, Wuhan), and the relevant detection primer sequences are shown in Table 5.

[0093] ④ Relative transcription level of cytokine mRNA (relative mRNAfold): Calculate using the Livak method, and the formula is as follows: relative mRNAfold = 2 -ΔΔCt , where ΔΔCt = (Ct value of the target gene in the experimental group - Ct value of β-actin in the experimental group) - (Ct value of the target gene in the blank control group - Ct value of β-actin in the blank control group).

[0094] The detection results show that ( Figure 2 、 Figure 3) The stimulant was co-cultured with mouse DCs in vitro for 12 h. Overall, compared with the large-sized peptidoglycan particles of a single component (control group 1), the small-sized peptidoglycan of a single component with a size of about 10 nm (control group 2) could increase the transcriptional level of IL-12 in DCs cells (the relative mRNAs of DCs cytokines IL-12 in control group 1 and control group 2 were 158.4 and 203.1 respectively), but the transcriptional levels of chemokines related to mucosal immunity were significantly decreased (the relative mRNAs of chemokines in control group 1 and control group 2 were 8.67 and 0.81 respectively). Compared with the single-sized peptidoglycan component groups (control groups 1 and 2), directly mixing the two in proportion (multi-sized composite preparation without metal ions, control group 3) could slightly increase the relative transcriptional levels of each target gene. Comparing among the composite preparation groups (control groups 3, 4, 5 and the experimental group), compared with the composite peptidoglycan immune enhancer without metal ions (control group 3), the transcription of pro-inflammatory cytokines IL-1β and TNF-α in the peptidoglycan nano-suspension containing metal ions (control groups 4, 5 and the experimental group) was significantly enhanced. Compared with control groups 4 and 5, the transcriptional levels of DCs cytokines and chemokines in the experimental group with the composite preparation of the peptidoglycan nano-suspension containing double metal ions were significantly increased, indicating that the components of the double metal ion composite peptidoglycan immune enhancer MPP-3 could exert a synergistic effect. The results of in vitro activation of porcine DCs were basically the same as those of mice. Compared with the other groups, the transcriptional levels of each target gene of MPP-3 in the experimental group were significantly increased. The above results indicate that MPP-3 can efficiently promote the in vitro activation of DCs and induce the maturation of DCs through the synergistic effect among its components.

[0095] Table 5 qRT-PCR primer sequences

[0096]

[0097]

[0098] (5) In vitro polarization experiment of T cells

[0099] Preparation of mouse spleen cells:

[0100] ① Mouse selection: Select 5-week-old Balb / c mice, decapitate and sacrifice them, and immerse them in 75% ethanol for disinfection;

[0101] ② Specimen collection: Lie the mouse flat and fix its four limbs in a sterile workbench, successively cut open the abdominal skin and muscles, open the abdominal cavity, aseptically collect the spleen, and place it in a sterile 6-well cell culture plate;

[0102] ③ Spleen cell extraction: Gently grind the spleen in RPMI 1640 cell culture medium, put the grinding solution into a 70 μm cell sieve, filter it twice to remove impurities and tissue fragments;

[0103] ④Red blood cell removal: Collect the cells, add red blood cell lysis buffer to remove red blood cells, obtain splenic lymphocytes, and perform cell counting analysis.

[0104] Analysis of CD4+ T lymphocytes:

[0105] ① Take 2×10 4 of the above-mentioned splenic lymphocytes and resuspend them with PBS (0.01 mol / L, pH 7.2 - 7.5) containing 1% fetal bovine serum;

[0106] ② Add anti-CD4 FITC fluorescent antibody (Abcam, UK) and stain at 4°C for 1 - 2 h;

[0107] ③ Add PBS buffer (0.01 mol / L, pH 7.2 - 7.5) to wash once, and analyze the number of CD4 + T lymphocytes using a flow cytometer.

[0108] After analysis, the proportion of CD4 + T lymphocytes in the prepared splenic lymphocytes was 32%.

[0109] In vitro T cell polarization experiment:

[0110] To verify the effect of peptidoglycan samples as immune enhancers on the antigen-presenting ability of dendritic cells (DCs) to T cells after activating DCs in vitro, an in vitro T cell polarization experiment was conducted. The specific operations are as follows:

[0111] ① Collect mouse DCs stimulated with GM-CSF, and at the same time add inactivated antigen of porcine epidemic diarrhea virus NJ strain and stimulants (peptidoglycan particles, peptidoglycan particle lysate, or composite peptidoglycan immune enhancer MPP-0 - MPP-3) for co-culture. The addition amount of PEDV antigen is 10 5 TCID 50 , and each stimulant is added to a final concentration of peptidoglycan (referring to the total final concentration of peptidoglycan, which is the sum of the concentrations of peptidoglycan particles and peptidoglycan of nanometer size) of 20 μg / mL. For specific grouping, see Table 4.

[0112] ② After co-culture for 12 h, collect the DCs, resuspend them with PBS (0.01 mol / L, pH 7.2 - 7.5) buffer containing 1% FBS, add anti-CD11b PE-Cy7 and anti-CD11c FITC fluorescent antibodies (Abcam, UK), stain at 4°C for 1 - 2 h, then add PBS to wash once, and analyze the number of CD11c+ and CD11b+ double-positive DCs using a flow cytometer. After analysis, the proportion of double-positive cells of CD11b + , CD11c + was 35%.

[0113] ③According to the proportion of positive cells analyzed by flow cytometry, DCs were added to freshly prepared CD4+ T lymphocytes at a ratio of 1:10 (positive cell ratio) and cultured in an incubator at 37°C and 5% CO2 for 12 h.

[0114] ④Suspended T lymphocytes were collected, and total RNA was extracted using an RNA extraction kit (Aibotech, Wuhan). Using the housekeeping gene β-actin as a reference, the transcriptional levels of antigen-presenting related cytokines IFN-γ, IL-17A, and IL-4 were analyzed by qRT-PCR. The specific method was the same as above.

[0115] CD4 + Results of in vitro polarization experiment of CD4 Figure 4 T cells showed that ( + ), after different peptidoglycan immune enhancers were co-stimulated and activated by DCs together with PEDV antigen and interacted with CD4 + T cells, generally speaking, the relative transcriptional levels of IFN-γ, IL-17A, and IL-4 in CD4 + T cells in the co-stimulation groups containing immune enhancers (control groups 1, 2, 3, 4, 5 and experimental group) were higher than those in the antigen control group; compared with the large-sized peptidoglycan particles of single component (control group 1), the relative transcriptional level of IFN-γ mRNA in CD4 + T cells of small-sized peptidoglycan of single component (control group 2) slightly increased, but the relative transcriptional level of IL-17A significantly decreased, while the relative transcriptional levels of IFN-γ, IL-17A, and IL-4 mRNAs in CD4 + T cells in the composite preparation groups (control groups 3, 4, 5 and experimental group) were all higher than those in the single component groups (control groups 1, 2). Comparing among the composite preparation groups, the transcriptional levels of IFN-γ, IL-17A, and IL-4 in CD4

[0116] Table 4 Grouping of mouse T cell polarization test

[0117] Grouping Stimulus components 1 Final concentration of peptidoglycan (μg / mL) Stimulus components 2 Control group 1 Peptidoglycan particles 20 PEDV Control group 2 Peptidoglycan particle lysate 20 PEDV Control group 3 MPP-0 20 PEDV Control group 4 MPP-1 20 PEDV Control group 5 MPP-2 20 PEDV Experimental group MPP-3 20 PEDV Antigen control group - - PEDV Blank control group PBS buffer - PBS

[0118] Note: 1. "-" in Table 4 indicates none. 2. "Final concentration of peptidoglycan" in the table refers to the final total concentration of peptidoglycan in the system, which is the sum of the concentrations of peptidoglycan particles and peptidoglycan of nanosize.

[0119] Verification of the Immune Efficacy of the Composite Peptidoglycan Immunoenhancer MPP-3 in Compatibility with the Whole Viral Antigen of PEDV in Example 3

[0120] 1. Antigen Preparation

[0121] The porcine epidemic diarrhea virus (PEDV) NJ strain (accession number: CGMCC NO. 13283) was selected as the model antigen. The virus was cultured and propagated on Vero cells. The culture method was as follows: When the Vero cells grew to a monolayer, the culture medium was discarded, and the cells were washed once with serum-free DMEM. The virus was inoculated at a ratio of 1%, and the cells were placed in an incubator at 37°C and 5% CO2 for 2 h for adsorption. The virus solution was removed, and the cells were washed once with serum-free DMEM. Then, a DMEM maintenance solution containing 10 μg / mL trypsin, 2% fetal bovine serum, and 1% penicillin-streptomycin double antibody was added, and the cells were cultured in an incubator at 37°C and 5% CO2 for 48 - 72 h. The cells and the supernatant were collected. The titer of the cultured virus was 10 7.5 TCID 50 / mL. Formaldehyde with a final concentration (volume percentage concentration) of 0.2% was added, and the mixture was inactivated at 37°C for 12 h. The inactivated PEDV virus solution was inoculated onto Vero cells for inactivation detection. The completely inactivated virus was stored at -80°C for standby.

[0122] 2. Vaccine Preparation and Immunization

[0123] The peptidoglycan particle suspension, peptidoglycan particle lysate, and composite peptidoglycan immunoenhancers MPP-0 - MPP-3 with a peptidoglycan concentration (referring to the total peptidoglycan concentration, which is the sum of the peptidoglycan particle and nano-sized peptidoglycan concentrations) of 10 mg / mL were respectively added to the above inactivated PEDV virus solution. Sterile PBS (0.01 mol / L, pH 7.2 - 7.5) buffer was supplemented to make the final peptidoglycan concentration (referring to the total final peptidoglycan concentration, which is the sum of the peptidoglycan particle and nano-sized peptidoglycan concentrations) 54.5 μg / mL, and the final PEDV titer was 1.1×10 7.0 TCID 50 / mL. Then, this mixture was mixed with the ISA201 adjuvant from the French company SEPPIC at a volume ratio of 46:54 and emulsified to prepare each vaccine. The numbers of each vaccine and the contained immunoenhancers are shown in Table 6. A PBS (0.01 mol / L, pH 7.2 - 7.5) buffer with the same volume was used to replace the composite peptidoglycan immunoenhancer and added to the inactivated PEDV virus solution. This mixture was mixed with the ISA201 adjuvant from the French company SEPPIC at a volume ratio of 46:54 and emulsified to obtain the control vaccine 7, where the PEDV concentration was the same as that of the above vaccines. PBS (0.01 mol / L, pH 7.2 - 7.5) was used to replace the PEDV virus, and it was mixed with the ISA201 adjuvant from the French company SEPPIC at a volume ratio of 46:54 and emulsified to obtain the blank control vaccine.

[0124] Forty-eight 35-day-old PEDV antigen- and antibody-negative piglets (about 20 kg per head) were randomly divided into 8 groups: Control Group 1, Control Group 2, Control Group 3, Control Group 4, Control Group 5, Experimental Group, Vaccine Control Group, and Blank Control Group, and were immunized with the corresponding vaccines. The immunization grouping is shown in Table 6. The above vaccines were given a second immunization 14 days after the first immunization. The immunization method was: intramuscular injection in the neck. The immunization dose each time was: the antigen content in each dose was 10 7.0 TCID 50 , the peptidoglycan content (referring to the total peptidoglycan content, including peptidoglycan particles and peptidoglycan of nanometer size) was 50 μg, and the immunization volume each time was 2 mL per head. Serum samples were collected 14 days and 28 days after the first immunization, and oral mucus samples were collected 28 days after the first immunization. The serum IgG and oral IgA secretion levels were analyzed by ELISA method.

[0125] 3. Serum IgG detection

[0126] The inactivated virus culture solution of the porcine epidemic diarrhea virus NJ strain was purified by sucrose density gradient centrifugation and then coated on an ELISA plate. The specific operation was as follows:

[0127] ① The purified virus was diluted to 2 μg / mL with the coating solution (sodium carbonate-sodium bicarbonate buffer, pH 9.6), and added to a blank ELISA plate (Shanghai Bioengineering Co., Ltd.), 100 μL was added to each well, and it was coated overnight at 4°C (12 - 16 h);

[0128] ② The solution was discarded, and washed three times with PBST buffer (PBS buffer with 0.01 mol / L, pH 7.2 - 7.5 containing 0.5% Tween 20). 200 μL of blocking solution (PBST buffer containing 3% skim milk powder) was added to each well, and blocked at 37°C for 1 h;

[0129] ③ Washed three times, air-dried and stored at 4°C for later use.

[0130] ④ The piglet serum was diluted at a dilution ratio of 1:5000 with PBS buffer (0.01 mol / L, pH 7.2 - 7.5), added to the coated well plate, and reacted at 37°C for 1 h;

[0131] ⑤ Then washed 5 times with PBST, and then added sheep anti-pig HRP-IgG (product number: ab6915, Abcam) diluted at a dilution ratio of 1:5000 with PBST buffer, 100 μL per well, and reacted at 37°C for 45 min;

[0132] ⑥Subsequently, wash 5 times with PBST, add 100 μL / well of TMB chromogenic solution, develop color at room temperature (20 °C) for 10 min, then add 100 μL / well of 2% sulfuric acid aqueous solution to terminate the reaction, and detect the OD reading at 450 nm wavelength with an enzyme-linked immunosorbent assay (ELISA) reader.

[0133] 4. Oral IgA detection

[0134] ①Twenty-eight days after the first immunization, collect porcine oral mucus, centrifuge at 12,000 rpm for 5 min to collect the supernatant, and dilute it with PBS buffer (0.01 mol / L, pH 7.2 - 7.5) at a dilution ratio of 1:50.

[0135] ②Add it to the ELISA plate coated with the porcine epidemic diarrhea virus NJ strain in Title 3 of this example, 100 μL / well, and react at 37 °C for 1 h.

[0136] ③Subsequently, wash 5 times with PBST, and then add goat anti-pig HRP-IgA (product number: ab112746, Abcam) diluted with PBST buffer at a dilution ratio of 1:5000, and react at 37 °C for 1 h.

[0137] ④Subsequently, 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 450 nm wavelength with an ELISA reader.

[0138] The results show (see Figure 5 ), 14 days after the first immunization, compared with the single-component large-sized peptidoglycan control group 1 (antibody mean 0.60) and the single-component small-sized peptidoglycan control group 2 (antibody mean 0.76), the antibody is significantly higher, indicating that small-sized peptidoglycan and its composite preparations can promote the rapid action of the immune-enhancing material and shorten the antibody production period. Compared with the single-component peptidoglycan preparation control groups 1 and 2, the serum antibodies of the multi-sized peptidoglycan composite preparation control groups 3, 4, 5 and the experimental group are significantly higher, indicating that its composite preparation can play a synergistic effect and improve the immune response level. In addition, among the various composite peptidoglycan preparation groups, compared with the metal-ion-free composite peptidoglycan preparation control group 3 (antibody mean 0.82), the single calcium-ion composite peptidoglycan preparation control group 4 and the single manganese-ion composite preparation control group 5 (antibody means are both 0.90), it can be seen that the antibody level of the double-metal-ion composite peptidoglycan preparation experimental group (antibody mean 1.03) is significantly higher than that of the control groups 3 (p < 0.01), 4 (p < 0.05), and 5 (p < 0.05).

[0139] Twenty-eight days after the first immunization, the IgG antibody level of the experimental group (MPP-3) (antibody mean 2.42) was much higher than that of the other groups: compared with the vaccine control group (antibody mean 1.53), the IgG antibody level of the experimental group increased by about 0.6 times (p < 0.0001); compared with the single large-sized peptidoglycan preparation control group 1 (antibody mean 1.74) and the single small-sized peptidoglycan preparation control group 2 (antibody mean 1.85), the IgG antibody levels of the experimental group increased by about 0.4 (p < 0.0001) and 0.3 times (p < 0.0001), respectively. In addition, among the composite peptidoglycan preparation groups, compared with the metal ion-free composite peptidoglycan preparation control group 3 (antibody mean 1.92), the single calcium ion composite peptidoglycan preparation control group 4 (antibody mean 2.05), and the single manganese ion composite peptidoglycan preparation control group 5 (antibody mean 1.97), it can be seen that the antibody level of the double metal ion composite peptidoglycan preparation experimental group was significantly higher than that of control groups 3 (p < 0.0001), 4 (p < 0.0001), and 5 (p < 0.0001).

[0140] Twenty-eight days after the first immunization, the oral mucus IgA level of the experimental group (MPP-3) (antibody mean 1.19) was much higher than that of the other groups: compared with the vaccine control group (antibody mean 0.35), the IgA antibody level increased by about 2.4 times (p < 0.0001); compared with the single large-sized peptidoglycan preparation control group 1 (antibody mean 0.72), the IgA antibody level of the experimental group increased by about 0.7 times (p < 0.0001); compared with the single small-sized peptidoglycan preparation control group 2 (antibody mean 0.39), the IgA antibody level of the experimental group increased by about 2 times (p < 0.0001). In addition, when comparing among the composite preparation groups, the metal ion-free composite peptidoglycan preparation control group 3 (antibody mean 0.82), the single calcium ion composite peptidoglycan preparation control group 4 (antibody mean 0.92), and the single manganese ion composite peptidoglycan preparation control group 5 (antibody mean 0.96), it can be seen that the IgA antibody level of the double metal ion composite peptidoglycan preparation experimental group was significantly higher than that of control groups 3 (p < 0.0001), 4 (p < 0.0001), and 5 (p < 0.01), as shown in Figure 6 . Based on the above results, it shows that the composite peptidoglycan immune enhancer MPP-3 can assist the rapid humoral immune response 14 days after immunization with PEDV antigen, and effectively improve the humoral and mucosal immune response levels 28 days after vaccination.

[0141] Table 6 Piglet Immunization Grouping and Vaccine Ingredients

[0142]

[0143] Example 4 Verification of the Immune Efficacy of the Composite Peptidoglycan Immune Enhancer MPP-3 in Compatibility with PEDV S Subunit Antigen

[0144] 1. Preparation of PEDV S antigen (subunit antigen)

[0145] The S protein was expressed by the laboratory itself, and the specific method is as follows: The S gene of the porcine epidemic diarrhea virus NJ strain was codon-optimized, and the sequence is shown in SEQ ID NO:1. The amino acid sequence of the S protein encoded by it is shown in SEQ ID NO:2. The sequence shown in SEQ ID NO:1 was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis and inserted between the restriction enzyme sites BamHⅠ and NotⅠ of the pcDNA3.1 vector (CatNo.V790-20, Thermo Fisher Scientific) to obtain the recombinant eukaryotic expression plasmid pcDNA-S. By using the conventional method, pcDNA-S was transfected into CHO cells with the transfection reagent Lipofectamine TM 2000 to express the S protein. The purified S protein was obtained by nickel ion affinity chromatography technology. After quantification with the BCA protein quantification kit, the protein was stored at -80°C for later use.

[0146] 2. Vaccine preparation

[0147] The compound immune enhancer MPP-3 was added to the purified S protein, and sterile PBS buffer (0.01 mol / L, pH 7.2 - 7.5) was supplemented to make the final concentration of peptidoglycan (referring to the total final concentration of peptidoglycan, which is the sum of the concentrations of peptidoglycan particles and peptidoglycan of nanoscale size) 54.5 μg / mL and the final concentration of S protein 110 μg / mL, and they were mixed evenly. Then this mixture was mixed with the ISA201 adjuvant from SEPPIC Company, France, at a volume ratio of 46:54 and emulsified to obtain the compound peptidoglycan PEDV S antigen subunit vaccine. According to the preparation method of the compound peptidoglycan PEDV S antigen subunit vaccine, only peptidoglycan particles were used to replace MPP-3 to obtain Control Vaccine 1, with other conditions unchanged. According to the preparation method of the compound peptidoglycan PEDV S antigen subunit vaccine, only the compound peptidoglycan immune enhancer MPP-0 was used to replace MPP-3 to obtain Control Vaccine 2, with other conditions unchanged. According to the preparation method of the compound peptidoglycan PEDV S antigen subunit vaccine, only the compound peptidoglycan immune enhancer MPP-1 was used to replace MPP-3 to obtain Control Vaccine 3, with other conditions unchanged. According to the preparation method of the compound peptidoglycan PEDV S antigen subunit vaccine, only the compound peptidoglycan immune enhancer MPP-2 was used to replace MPP-3 to obtain Control Vaccine 4, with other conditions unchanged. Meanwhile, the purified S protein was mixed with the ISA201 adjuvant from SEPPIC Company, France, at a volume ratio of 46:54 and emulsified to obtain Control Vaccine 5, with the final concentration of S protein 110 μg / mL. PBS (0.01 mol / L, pH 7.2 - 7.5) was mixed with the ISA201 adjuvant from SEPPIC Company, France, at a volume ratio of 46:54 and emulsified to obtain Control Vaccine 6. The vaccine components and grouping are shown in Table 7.

[0148] 3. Immunization of Piglets and Efficacy Evaluation

[0149] Forty-two 35-day-old piglets with negative antigen and antibody (about 20 Kg per head) were randomly divided into 7 groups: Control Group 1, Control Group 2, Control Group 3, Control Group 4, Experimental Group, Vaccine Control Group, and Blank Control Group, and they were immunized with the corresponding vaccines respectively. The immunization grouping is shown in Table 7. The above vaccines were given a second immunization 14 days after the first immunization. The immunization method was: intramuscular injection in the neck, and the immunization dose was: the S protein antigen content in each dose was 100 μg, the peptidoglycan content (referring to the total peptidoglycan content, including peptidoglycan particles and peptidoglycan of nanoscale size) was 50 μg, and the immunization volume was 2 mL per head. Serum samples were collected 14 days and 28 days after the first immunization, and oral mucus samples were collected 28 days after the first immunization. The serum IgG and oral IgA secretion levels were analyzed by ELISA method. The specific method was the same as that in Example 3, with the serum dilution ratio of 1:2000 and the oral fluid dilution ratio of 1:50.

[0150] The results showed ( Figure 7) On the 14th day after the first immunization, the serum IgG of control group 1 containing a single-component large-sized peptidoglycan (antibody mean 0.48) was slightly lower than that of the vaccine control group (antibody mean 0.56), while the serum IgG of the immunized groups containing multi-sized composite peptidoglycan (control groups 2, 3, 4 and the experimental group) was significantly higher than that of control group 1 and the vaccine control group. Among the immunized groups with composite peptidoglycan preparations, compared with the control group 2 of the composite peptidoglycan preparation without metal ions (antibody mean 0.63), the control group 3 of the single calcium ion composite peptidoglycan preparation (antibody mean 0.69) and the control group 4 of the single manganese ion composite peptidoglycan preparation (antibody mean 0.78), it can be seen that the IgG antibody level of the experimental group of the double metal ion composite peptidoglycan preparation (antibody mean 0.95) was significantly higher than that of control 2 (p<0.0001), 3 (p<0.0001) and 4 (p<0.0001).

[0151] On the 28th day after immunization, the antibodies in the experimental group continued to rise to 2.16, which was significantly higher than that of the vaccine control group (antibody mean 1.08, p<0.0001), control group 1 containing a single peptidoglycan particle component (antibody mean 1.52, p<0.0001), and the other composite peptidoglycan immune enhancer group control groups 2 (antibody mean 1.75, p<0.0001), 3 (antibody mean 1.88, p<0.0001), 4 (antibody mean 1.77, p<0.0001).

[0152] On the 28th day after immunization, the level of oral mucus IgA antibodies in the experimental group was also significantly higher than that of the other groups. Compared with the vaccine control group (antibody mean 0.29), the experimental group (antibody mean 1.16) increased by 3 times (p<0.0001); compared with the single-component peptidoglycan control group 1 (antibody mean 0.79), the antibody level of the experimental group increased by nearly 0.5 times (p<0.0001); and compared with the composite peptidoglycan preparation control groups 2 (antibody mean 0.78), 3 (antibody mean 0.86), 4 (antibody mean 0.89), it can be seen that the IgA antibody level of the experimental group was also significantly higher than that of control 2 (p<0.0001), 3 (p<0.0001), 4 (p<0.0001), as shown in Figure 8 . The above results indicate that the composite peptidoglycan immune enhancer prepared by the present invention has a significant immune enhancing effect on the PEDV S subunit vaccine.

[0153] Table 7 Immunization grouping of PEDV S subunit vaccine

[0154]

[0155]

Claims

1. A composite peptidoglycan immunopotentiator, characterized in that The method is prepared by the following steps: (1) using ultra-high pressure microfluidization technology to treat peptidoglycan particles prepared from Lactococcus lactis to obtain a peptidoglycan particle lysate; (2) adding calcium chloride and manganese chloride to the peptidoglycan particle lysate, and treating the solution with ultrahigh pressure microfluidization technology to obtain a peptidoglycan nanosuspension; (3) The peptidoglycan particles are mixed with the peptidoglycan nanosuspension to obtain a composite peptidoglycan immunopotentiator.

2. The composite peptidoglycan immunopotentiator according to claim 1, characterized in that: Lactococcus lactis was treated by high pressure wet heat method and then soaked in acid to obtain peptidoglycan particles.

3. The composite peptidoglycan immunopotentiator according to claim 1 or 2, characterized in that: The ultra-high pressure microfluidization technology in step (1) uses a microfluidizer, and the treatment conditions are: treatment at 18000-22000Psi, and circulation for 2-4 times; then treatment at 23000-27000Psi, and circulation for 2-4 times.

4. The composite peptidoglycan immunopotentiator according to claim 3, characterized in that: The concentration of calcium chloride in the peptidoglycan nanosuspension is 0.06-1.0 mg / mL, the concentration of manganese chloride is 0.03-0.05 mg / mL, and the concentration of nano-sized peptidoglycan is 8-12 mg / mL.

5. The composite peptidoglycan immunopotentiator according to claim 4, characterized in that: The ultra-high pressure microfluidization technique in step (2) uses a microfluidizer, and the treatment conditions are: treatment at 21000-25000Psi, and circulation for 2-4 times.

6. The composite peptidoglycan immunopotentiator according to claim 5, characterized in that: The mass ratio of peptidoglycan particles to nano-sized peptidoglycan in the composite peptidoglycan immunoenhancer in step (3) is 4:5-7.

7. Use of the composite peptidoglycan immunoenhancer according to any one of claims 1 to 6 in the preparation of veterinary vaccines.

8. The use according to claim 7, characterized in that: The veterinary vaccine is a porcine epidemic diarrhea vaccine.

9. The use according to claim 8, characterized in that The composite peptidoglycan immunoenhancer is mixed with an antigen, and then mixed with an ISA201 adjuvant, and then emulsified to obtain the veterinary vaccine.

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