Highly effective subunit vaccine for porcine epidemic diarrhea virus

Abstract

The present invention provides a highly effective subunit vaccine for porcine epidemic diarrhea virus, and relates to the field of biological vaccine manufacturing. The subunit vaccine is prepared by the following method: (1) mixing a polylysine aqueous solution with a flagellin aqueous solution, stirring for 5-10 hours, and freeze-drying to obtain a freeze-dried powder of immunopotentiator nanoparticles; the amino acid sequence of the flagellin is shown in SEQ ID NO: 1; (2) adding silicic acid to the immunopotentiator nanoparticle aqueous solution, reacting for 4-6 hours, to obtain a siliconized immunopotentiator nanoparticle solution; (3) mixing the siliconized immunopotentiator nanoparticle solution with an antigen solution carrying a HIS tag in a volume ratio of 2-4:1, centrifuging, taking the precipitate, and suspending it with a solvent to obtain the vaccine. After immunization with the vaccine of the present invention, the antibody level can be significantly increased and the antibody duration can be significantly prolonged.

Description

Technical Field

[0001] The present invention relates to the field of biological vaccine manufacturing, and in particular to a highly effective subunit vaccine of porcine epidemic diarrhea virus. Background Art

[0002] Porcine epidemic diarrhea is an acute, highly contagious gastrointestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). Since 2010, PEDV has undergone significant mutations, and existing commercial vaccines cannot provide effective protection. Traditional veterinary inactivated vaccines take a long time to develop, have poor immune effects, and have extremely limited protection against mutant strains; although attenuated vaccines have good immunogenicity, there is a risk of toxin shedding; subunit vaccines can be designed for major antigenic targets and have a short development cycle. Since subunit vaccines only contain major antigenic targets, they generally have the problem of poor immunogenicity, and usually require the addition of immunopotentiators to enhance their specific immune response. However, the existing technology lacks an efficient porcine epidemic diarrhea virus subunit vaccine. Summary of the Invention

[0003] The purpose of the present invention is to provide a highly effective subunit vaccine for porcine epidemic diarrhea virus, which can significantly increase the antibody level and significantly prolong the antibody duration after immunization.

[0004] A subunit vaccine is prepared by the following method

[0005] (1) mixing a polylysine aqueous solution and a flagellin aqueous solution, stirring for 5-10 hours, and freeze-drying to obtain a freeze-dried powder of immunopotentiator nanoparticles; the amino acid sequence of the flagellin is shown in SEQ ID NO: 1;

[0006] (2) adding silicic acid to the aqueous solution of immunopotentiator nanoparticles and reacting for 4-6 hours to obtain a silicified immunopotentiator nanoparticle solution;

[0007] (3) The siliconized immunopotentiator nanoparticle solution is mixed with the antigen solution with the HIS tag in a volume ratio of 2-4:1, centrifuged, and the precipitate is collected and suspended with a solvent to obtain the vaccine.

[0008] A highly effective subunit vaccine for porcine epidemic diarrhea virus is prepared by the following method

[0009] (1) mixing a polylysine aqueous solution and a flagellin aqueous solution, stirring for 5-10 hours, and freeze-drying to obtain a freeze-dried powder of immunopotentiator nanoparticles; the amino acid sequence of the flagellin is shown in SEQ ID NO: 1;

[0010] (2) adding silicic acid to the aqueous solution of immunopotentiator nanoparticles and reacting for 4-6 hours to obtain a silicified immunopotentiator nanoparticle solution;

[0011] (3) The siliconized immunopotentiator nanoparticle solution and the PEDV antigen solution with the HIS tag are mixed in a volume ratio of 2-4:1, centrifuged, and the precipitate is collected and suspended with a solvent to obtain the vaccine.

[0012] In the present invention, the PEDV antigen is PEDV S protein.

[0013] In the present invention, the concentration of the polylysine aqueous solution in step (1) is 0.4-0.6 mg / mL, and the concentration of the flagellin aqueous solution is 0.15-0.3 mg / mL; the volume ratio of the polylysine aqueous solution to the flagellin aqueous solution is 1:0.8-1.2.

[0014] In the present invention, the concentration of silicic acid in step (2) is 8-10 mg / mL, the concentration of the immunopotentiator nanoparticle solution is 0.008-0.012 mg / mL, and the volume ratio of the silicic acid to the immunopotentiator nanoparticle aqueous solution is 1:0.8-1.2.

[0015] In the present invention, the PEDV S protein in step (3) carries a HIS tag, and the concentration of the PEDV S protein solution is 450-550 μg / mL.

[0016] In the present invention, the solvent in step (3) is PBS buffer or physiological saline.

[0017] In the present invention, the pH of the PBS buffer is 6.8-7.5.

[0018] In the present invention, the flagellin is obtained by inserting the gene shown in SEQ ID NO: 2 into a pET32a vector, introducing the vector into Escherichia coli, inducing expression with IPTG, lysing the resulting bacteria, and purifying the supernatant with a nickel column.

[0019] In the present invention, the PEDV S protein is obtained by inserting the PEDV S protein gene into a pET32a vector, introducing the protein into Escherichia coli, inducing expression with IPTG, lysing the resulting bacteria, and purifying the supernatant with a nickel column.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the high-efficiency subunit vaccine prepared by the present invention has the characteristics of immune enhancement, antigen purification, and immune targeting. The present invention nano-particles the immunopotentiator and, using a coating technology, coats a layer of silica on its surface. At the same time, it uses the principle of forming hydrogen bonds with the histidine (HIS) imidazole group to purify the antigen with the HIS tag, and displays the antigen on the surface of the enhancer, realizing the integration of the immunopotentiator and the antigen structure, solving the key problem of poor immunogenicity of the subunit vaccine at the source, and realizing the efficient purification of the S protein. After immunization with the high-efficiency subunit vaccine of porcine epidemic diarrhea virus of the present invention, the antibody level can be significantly improved and the antibody duration can be significantly prolonged. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is an electron microscope photo of the immune enhancer nanoparticle solution.

[0022] Figure 2 This is an electron microscope photo of siliconized immune enhancer nanoparticles.

[0023] Figure 3 This is the SDS-PAGE electrophoresis diagram of PEDV S protein purified by siliconized immunopotentiator nanoparticles, M-protein standard marker, 1-PEDV S protein solution, 2-precipitate after binding of siliconized immunopotentiator nanoparticle solution to PEDV S protein solution at a volume ratio of 1:1, 3-supernatant after binding of siliconized immunopotentiator nanoparticle solution to PEDV S protein solution at a volume ratio of 1:1, 4-precipitate after binding of siliconized immunopotentiator nanoparticle solution to PEDV S protein solution at a volume ratio of 2:1, 5-supernatant after binding of siliconized immunopotentiator nanoparticle solution to PEDV S protein solution at a volume ratio of 2:1, 6-precipitate after binding of siliconized immunopotentiator nanoparticle solution to PEDV S protein solution at a volume ratio of 3:1, 7-supernatant after binding of siliconized immunopotentiator nanoparticle solution to PEDV S protein solution at a volume ratio of 3:1.

[0024] Figure 4 It is the detection result of specific IgG antibodies in the serum of each group of immunized mice.

[0025] Figure 5 This is the result of neutralizing antibody detection in the serum of immunized mice.

[0026] Figure 6 CD8 + T cell activation detection results. DETAILED DESCRIPTION

[0027] The polylysine used in the examples of the present invention has a weight average molecular weight of 150,000 and was purchased from Sigma-Aldrich Shanghai Trading Co., Ltd. Tetramethoxysilane was purchased from Beijing J&K Technology Co., Ltd.

[0028] Example 1 Preparation of a highly effective subunit vaccine for porcine epidemic diarrhea virus

[0029] 1. Preparation of Vaccine A

[0030] (1) Preparation of immunopotentiator nanoparticles

[0031] A 0.5 mg / mL polylysine aqueous solution and a 0.25 mg / mL flagellin aqueous solution were mixed at a volume ratio of 1:1 and stirred continuously at 28°C and 1000 rpm for 8 h to obtain an immunopotentiator nanoparticle solution. The average particle size of the immunopotentiator nanoparticles was 20 nm ( Figure 1 ), good dispersibility, and uniform size. The immunopotentiator nanoparticle solution was lyophilized to obtain an immunopotentiator nanoparticle lyophilized powder. 1 mg of the immunopotentiator nanoparticle lyophilized powder was dissolved in 100 mL of deionized water to obtain a 0.01 mg / mL immunopotentiator nanoparticle solution.

[0032] The amino acid sequence of flagellin is shown in SEQ ID NO: 1, and the sequence of its encoding gene is shown in SEQ ID NO: 2. Flagellin is obtained by inserting the gene shown in SEQ ID NO: 2 between the NdeI and XhoI restriction sites of the pET32a vector, introducing it into Escherichia coli, inducing expression with IPTG, lysing the resulting cells, and purifying the supernatant using a nickel column.

[0033] (2) Preparation of siliconized immunopotentiator nanoparticles

[0034] 0.15 g of methyl silicate (TMOS) was added to 1 mL of deionized water and hydrolyzed to obtain silicic acid, which was then diluted with deionized water to obtain 9 mg / mL of silicic acid.

[0035] 9 mg / mL of silicic acid was added to an equal volume of 0.01 mg / mL of immunopotentiator nanoparticle solution and reacted for 5 hours to obtain a silicified immunopotentiator nanoparticle solution. The average particle size of the silicified immunopotentiator nanoparticles was 70 nm. The results were as follows after electron microscopy observation: Figure 2 , and found that a layer of silica shell was wrapped on the surface of the immune enhancer nanoparticles.

[0036] (3) Purification of antigens by siliconized immunopotentiator nanoparticles

[0037] The silicified immunopotentiator nanoparticle solution obtained in step (2) was mixed with a 500 μg / mL PEDV S protein solution (the solvent was 0.01 M PBS buffer at pH 7.4) at a volume ratio of 3:1. The PEDV S protein was labeled with HIS, so the PEDV S protein was bound to the silicified immunopotentiator nanoparticles. The mixture was centrifuged, and the precipitate was taken and resuspended with an equal volume of PBS buffer (pH 7.4, concentration 0.01 M) as the PEDV S protein solution to obtain vaccine A.

[0038] Depend on Figure 3 It can be seen that when the silicified immunopotentiator nanoparticle solution and the PEDV S protein solution with a concentration of 500 μg / mL were mixed at a volume ratio of 3:1, all the PEDV S proteins were bound to the silicified immunopotentiator nanoparticles.

[0039] The amino acid sequence of the PEDV S protein is shown in SEQ ID NO:3, and the encoding gene sequence is shown in SEQ ID NO:4. The PEDV S protein was prepared using the following method: the encoding gene for the PEDV S protein was inserted between the NdeI and XhoI restriction sites of the pET32a vector, introduced into Escherichia coli, and expressed using IPTG. The resulting cells were lysed, and the supernatant was purified using a nickel column. Because the pET32a vector carries the HIS tag encoding sequence (CACCACCACCACCACCAC), the expressed and purified PEDV S protein carries a HIS tag (HHHHHH).

[0040] In vaccine A, the concentration of PEDV S protein was 500 μg / mL, and the concentration of immunopotentiator nanoparticles was 0.015 mg / mL.

[0041] 2. Preparation of other vaccines

[0042] Vaccine B: 1 mL of a 500 μg / mL aqueous solution of PEDV S protein (the solvent is 0.01 M PBS buffer at pH 7.4) was mixed with 10.38 mg / mL of commercial silicon dioxide (silicon dioxide, Beijing Dekedaojin Technology Co., Ltd., product number JL-SiO2-S30) and 15 μg of the freeze-dried powder of the immunopotentiator nanoparticles prepared in Example 1(1), and stirred uniformly to obtain Vaccine B. In Vaccine B, the concentration of silicon in the commercial silicon dioxide was the same as that in Vaccine A.

[0043] Vaccine C: 1 mL of a 500 μg / mL aqueous solution of PEDV S protein (the solvent is 0.01 M PBS buffer at pH 7.4) was mixed with 10.38 mg / mL commercial silica and stirred evenly to obtain Vaccine C.

[0044] Vaccine D: 1 mL of a 500 μg / mL aqueous solution of PEDV S protein (solvent: PBS buffer at pH 7.4, concentration: 0.01 M) was mixed with 15 μg of the lyophilized powder of the immunopotentiator nanoparticles prepared in Example 1(1), and stirred evenly to obtain Vaccine D.

[0045] Vaccine E (antigen control group): 500 μg / mL of PEDV S protein in water (the solvent is PBS buffer with a concentration of 0.01 M and a pH of 7.4).

[0046] Vaccine F (blank control group): PBS buffer with a concentration of 0.01 M and a pH of 7.4.

[0047] Example 2 Immunization Experiment and Effect of Highly Effective Subunit Vaccine of Porcine Epidemic Diarrhea Virus 1. Animal Grouping and Immunization

[0048] Sixty healthy ICR mice were randomly divided into six groups, with 10 mice in each group. Each group of mice was immunized with vaccines A, B, C, D, E, and F by subcutaneous injection at a dose of 100 μL per mouse.

[0049] 2. Specific antibody detection

[0050] Serum was collected 14, 28, 42, 70, 84, 98, 120, 150, 180, and 210 days after immunization, and the IgG antibody level in the serum was detected by indirect ELISA. PEDV S protein (Example 1) was diluted to 100 ng / mL with coating buffer (50 mM carbonate buffer at pH 9.6), 100 μL / well, added to the ELISA plate, and coated overnight at 4°C; the coating solution was discarded, and the plate was washed 3 times with PBST (0.01 M, pH 7.4 PBS buffer supplemented with 0.5% Tween-20), 5 min / time, and patted dry; PBST containing 1% BSA was added to each well. 200 μL, block at 37°C for 1h; wash the plate with PBST 3 times, 5 min / time, and pat dry; dilute the serum to be tested with PBST containing 1% BSA at a volume ratio of 1:800, 100 μL / well, incubate at 37°C for 1h; wash the plate with PBST 3 times, 5 min / time, and pat dry; HRP-labeled goat anti-mouse IgG (Biyuntian Biological Reagent, Product No. A0216) is diluted with PBST containing 1% BSA at a volume ratio of 1:5000, 100 μL / well, incubate at 37°C for 1h; wash the plate with PBST 3 times, 5 min / time, and pat dry; add TMB colorimetric solution and react at 37°C in the dark for 15min; add 2M H2SO4 aqueous solution, 100 μL / well, to terminate the reaction; place the enzyme plate in an enzyme reader and read the OD value at a wavelength of 450nm.

[0051] The results of the detection of specific IgG antibodies in the serum of immune mice are as follows Figure 4 As shown, when OD450nm ≥ 0.5 is positive, the specific IgG antibody level in the serum of mice in vaccine group A was significantly higher than that of all control groups 14 days after immunization, reaching the highest level 42 days after immunization, and then reaching a plateau 98 days after immunization, and maintained a high level until the monitoring period of 210 days. The specific IgG antibody level in the serum of mice in vaccine group B was significantly lower than that of vaccine group A throughout the duration period, reaching the highest antibody level 42 days after immunization, and then the antibody level dropped sharply. The specific IgG antibody level in the serum of mice in vaccine groups C and D reached the highest level 28 days after immunization, and then the antibody level began to show a downward trend. The specific IgG antibody level in the serum of mice in vaccine group E, the antigen control group, was still at a low level 14 days after immunization, and the antibody level increased slowly. Except for the vaccine A immunization group, the specific IgG antibody level of the other vaccine immunization groups was < 0.5 at OD450nm within 98 days and turned negative. The experimental results showed that vaccine A can significantly increase the antibody levels of mice after immunization with PEDV S protein, and the high antibody levels are maintained until 210 days after immunization, confirming that the siliconized immunopotentiator nanoparticles can effectively improve the body's humoral immune efficacy and immune duration.

[0052] 3. Neutralizing antibody detection

[0053] The serum was collected 28 days after immunization and inactivated in a 56°C water bath for 30 min. The serum was diluted with DMEM medium in a 2-fold gradient. The diluted serum was mixed with 200 TCID 50 PEDV-NJ virus solution (disclosed in ZL201611248419.4) and DMEM medium were mixed in a 1:1 volume ratio and incubated at 37°C for 1 hour. The cell culture medium in a 96-well plate filled with VERO cells was discarded, and 100 μL of the serum-virus mixture was added to each well. A negative control of DMEM medium and a positive control of virus solution were established. After 72 hours, the serum neutralization titer was calculated using the Reed-Muench method.

[0054] The results of neutralizing antibody test in immunized mice serum were as follows Figure 5 As shown, the neutralizing antibody level in the serum of mice in vaccine group A was 1:415, in vaccine group B it was 1:34, in vaccine group C it was 1:9, in vaccine group D it was 1:16, in vaccine group E it was 1:2, and in vaccine F, the blank control group, it was 0. The neutralizing antibody level in vaccine group A was significantly higher than in the other vaccine control groups. These experimental results demonstrate that the serum of mice vaccinated with vaccines containing siliconized immunopotentiator nanoparticles has a strong neutralizing effect on PEDV-infected VERO cells.

[0055] 4. CD8 + T cell detection

[0056] Seven days after immunization of each group of mice, inguinal lymph nodes were isolated and processed to prepare lymphocyte suspensions. CD8 + T cell activation status.

[0057] Immunization of mice with CD8 + T cell activation test results Figure 6 As shown, the lymph node CD8 + The T cell activation rate was significantly higher than that of other vaccine control groups. The test results showed that vaccines containing siliconized immune enhancer nanoparticles can effectively activate the CTL response of mice, help the body to quickly exert cellular immune response and achieve anti-viral infection effect.

Claims

1. A highly effective subunit vaccine for porcine epidemic diarrhea virus, characterized in that Prepared by the following method (1) Mixing a polylysine aqueous solution and a flagellin aqueous solution, stirring for 5-10 hours, and freeze-drying to obtain a freeze-dried powder of immunopotentiator nanoparticles; the amino acid sequence of the flagellin is shown in SEQ ID NO: 1; (2) adding silicic acid to the aqueous solution of immunopotentiator nanoparticles and reacting for 4-6 hours to obtain a silicified immunopotentiator nanoparticle solution; (3) The siliconized immunopotentiator nanoparticle solution and the HIS-tagged PEDV antigen solution are mixed in a volume ratio of 2-4:1, centrifuged, and the precipitate is collected and suspended with a solvent to obtain the vaccine; the PEDV antigen is the PEDV S protein; In step (1), the concentration of the polylysine aqueous solution is 0.4-0.6 mg / mL, and the concentration of the flagellin aqueous solution is 0.15-0.3 mg / mL; the volume ratio of the polylysine aqueous solution to the flagellin aqueous solution is 1:0.8-1.2; in step (2), the concentration of silicic acid is 8-10 mg / mL, the concentration of the immunopotentiator nanoparticle solution is 0.008-0.012 mg / mL, and the volume ratio of the silicic acid to the immunopotentiator nanoparticle aqueous solution is 1:0.8-1.2; in step (3), the PEDV S protein carries a HIS tag, and the concentration of the PEDV S protein solution is 450-550 μg / mL.

2. The vaccine according to claim 1, characterized in that The solvent in step (3) is PBS buffer or physiological saline.

3. The vaccine according to claim 2, characterized in that The pH of the PBS buffer is 6.8-7.

5.

4. The vaccine according to claim 3, characterized in that The flagellin is obtained by inserting the gene shown in SEQ ID NO: 2 into a pET32a vector, introducing the gene into Escherichia coli, inducing expression with IPTG, lysing the resulting bacteria, and purifying the supernatant with a nickel column.

5. The vaccine according to claim 4, characterized in that The PEDV S protein is obtained by inserting the PEDV S protein gene into a pET32a vector, introducing the protein into Escherichia coli, inducing expression with IPTG, lysing the resulting bacteria, and purifying the supernatant with a nickel column.

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