PEDV COE protein containing intramolecular adjuvant and application of PEDV COE protein in preparation of porcine epidemic diarrhea virus vaccine

Abstract

The invention relates to the technical field of biological pharmacy. The invention provides a PEDV (Porcine Epidemic Diarrhea Virus) COE region multi-epitope fusion protein, and a PEDV COE protein which comprises the PEDV COE region multi-epitope fusion protein and contains an intramolecular adjuvant. The invention also provides an application of the PEDV COE protein containing the intramolecular adjuvant in preparation of a porcine epidemic diarrhea virus vaccine. According to the invention, different neutralizing epitopes are screened for a PEDV COE region, and effective fusion is carried out through proper connection and arrangement modes, so that potential ADE effect and immune potential waste are reduced; and then a proper intramolecular adjuvant is introduced to further improve the immune level and enhance the immunogenicity.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, and in particular to a PEDV COE protein containing an intramolecular adjuvant and application thereof in preparing a porcine epidemic diarrhea virus vaccine. Background Art

[0002] Animal husbandry is related to the national economy and people's livelihood. Porcine epidemic diarrhea virus (PEDV) is the most common swine pathogen in China's pig herds and a major disease that affects the healthy and sustainable development of the pig industry. Although commercial vaccines are available, PEDV will undergo significant mutations, and existing commercial vaccines cannot provide effective protection; the existing commercial vaccines have low levels of intramuscular cellular / mucosal immunity; and existing commercial vaccines have potential ADE effect problems. Traditional veterinary inactivated vaccines take a long time to develop and have poor immune effects, and their protection against mutant strains is extremely limited; although attenuated vaccines have good immunogenicity, there is a risk of spreading the virus; subunit vaccines can be designed for major antigenic targets, with a short development cycle, but they generally have the problem of poor immunogenicity. Therefore, existing vaccines cannot meet the demand.

[0003] In summary, for the development of PEDV vaccines, it is very necessary to study an efficient porcine epidemic diarrhea virus subunit vaccine that can cope with high mutation and potential ADE effects. Summary of the invention

[0004] The purpose of the present invention is to provide a PEDV COE protein containing an internal adjuvant and application thereof in preparing a porcine epidemic diarrhea virus subunit vaccine.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a PEDV COE region multi-epitope fusion protein, and the constituent fragments of the PEDV COE region recombinant protein include COE region neutralizing epitope 1, COE region neutralizing epitope 2, COE region neutralizing epitope 3, COE region neutralizing epitope 4, COE region neutralizing epitope 5, COE region neutralizing epitope 6, COE region neutralizing epitope 7, COE region neutralizing epitope 8, COE region neutralizing epitope 9, COE region neutralizing epitope 10, COE region neutralizing epitope 11, COE region neutralizing epitope 12, COE region neutralizing epitope 13, COE region neutralizing epitope 14 and COE region neutralizing epitope 15.

[0007] Preferably, the amino acid sequence of the neutralizing epitope 1 in the COE region is as shown in SEQ ID NO:1; the amino acid sequence of the neutralizing epitope 2 in the COE region is as shown in SEQ ID NO:2; the amino acid sequence of the neutralizing epitope 3 in the COE region is as shown in SEQ ID NO:3; the amino acid sequence of the neutralizing epitope 4 in the COE region is as shown in SEQ ID NO:4; the amino acid sequence of the neutralizing epitope 5 in the COE region is as shown in SEQ ID NO:5; the amino acid sequence of the neutralizing epitope 6 in the COE region is as shown in SEQ ID NO:6; the amino acid sequence of the neutralizing epitope 7 in the COE region is as shown in SEQ ID NO:7; the amino acid sequence of the neutralizing epitope 8 in the COE region is as shown in SEQ ID NO:8; the amino acid sequence of the neutralizing epitope 9 in the COE region is as shown in SEQ ID NO:9; the amino acid sequence of the neutralizing epitope 10 in the COE region is as shown in SEQ ID NO:10; the amino acid sequence of the neutralizing epitope 11 in the COE region is as shown in SEQ ID NO:11; the amino acid sequence of the neutralizing epitope 12 in the COE region is as shown in SEQ ID NO:12; the amino acid sequence of the neutralizing epitope 13 in the COE region is shown in SEQ ID NO:13; the amino acid sequence of the neutralizing epitope 14 in the COE region is shown in SEQ ID NO:14; the amino acid sequence of the neutralizing epitope 15 in the COE region is shown in SEQ ID NO:15.

[0008] Preferably, it further comprises a connecting peptide 1 and / or a connecting peptide 2; the amino acid sequence of the connecting peptide 1 is shown in SEQ ID NO: 16, and the amino acid sequence of the connecting peptide 2 is shown in SEQ ID NO: 17.

[0009] The present invention also provides a PEDV COE protein containing an intramolecular adjuvant, comprising the PEDV COE region multi-epitope fusion protein and the intramolecular adjuvant.

[0010] Preferably, the internal adjuvant comprises any one of a p28T vector carrying Tufisn and PT8A, a p28ND vector carrying flagellin CD0 / 1 and flagellin ND0 / 1, and a p28cTB vector carrying Tufisn and CTB.

[0011] Preferably, the amino acid sequence of Tufisn is shown in SEQ ID NO:18, the amino acid sequence of PT8A is shown in SEQ ID NO:19, the amino acid sequence of flagellin CD0 / 1 is shown in SEQ ID NO:20, the amino acid sequence of flagellin ND0 / 1 is shown in SEQ ID NO:21, and the amino acid sequence of CTB is shown in SEQ ID NO:22.

[0012] The present invention also provides the use of the PEDV COE protein containing the internal adjuvant in the preparation of porcine epidemic diarrhea virus vaccine.

[0013] The present invention also provides a porcine epidemic diarrhea virus subunit vaccine, comprising the PEDV COE protein containing an intramolecular adjuvant and a pharmaceutically acceptable adjuvant.

[0014] By adopting the above technical solution, the present invention has the following beneficial effects:

[0015] The technical solution of the present invention screens different neutralizing epitopes for the PEDV COE region, and combines them through appropriate connection and arrangement methods, thereby minimizing the occurrence of ADE effects and the waste of immune potential; then introducing appropriate intramolecular adjuvants to further improve the immune level and enhance immunogenicity. The technical solution of the present invention ensures the effective fusion of the multi-epitope fusion protein and the intramolecular adjuvant in the PEDV COE region, achieving the goal of not affecting the fragment structure and exerting the role of the intramolecular adjuvant, while trying not to form new dominant antigenic determinants, and avoiding the goal of inducing non-neutralizing antibodies to escape the immune response. DETAILED DESCRIPTION

[0016] The present invention provides a PEDV COE region multi-epitope fusion protein, and the constituent fragments of the PEDV COE region recombinant protein include COE region neutralizing epitope 1, COE region neutralizing epitope 2, COE region neutralizing epitope 3, COE region neutralizing epitope 4, COE region neutralizing epitope 5, COE region neutralizing epitope 6, COE region neutralizing epitope 7, COE region neutralizing epitope 8, COE region neutralizing epitope 9, COE region neutralizing epitope 10, COE region neutralizing epitope 11, COE region neutralizing epitope 12, COE region neutralizing epitope 13, COE region neutralizing epitope 14 and COE region neutralizing epitope 15.

[0017] In the present invention, the amino acid sequence of the neutralizing epitope 1 in the COE region is shown in SEQ ID NO: 1, and the specific sequence is NSYGYVSKSQDSNCPFTLQSVND.

[0018] In the present invention, the amino acid sequence of the neutralizing epitope 2 in the COE region is shown in SEQ ID NO: 2, and the specific sequence is GELITGTPKPLEGVTDVSFM.

[0019] In the present invention, the amino acid sequence of the neutralizing epitope 3 in the COE region is shown in SEQ ID NO: 3, and the specific sequence is DLFGYPEFGGGVKF.

[0020] In the present invention, the amino acid sequence of the neutralizing epitope 4 in the COE region is shown in SEQ ID NO: 4, and the specific sequence is NSYGYVSNSQDSNCPFTLQSVNDSLPQDVTRCKKTNFRRFFSKFNVQ.

[0021] In the present invention, the amino acid sequence of the neutralizing epitope 5 in the COE region is shown in SEQ ID NO: 5, and the specific sequence is EIGISQEPFDPSGY.

[0022] In the present invention, the amino acid sequence of the neutralizing epitope 6 in the COE region is shown in SEQ ID NO: 6, and the specific sequence is YSNIGVCKSGSIGKKALTKVQEVVN.

[0023] In the present invention, the amino acid sequence of the neutralizing epitope 7 in the COE region is shown in SEQ ID NO: 7, and the specific sequence is SRRMFEPRKPTVSDFVKKLTRDQLPDVIPDYIDVNK.

[0024] In the present invention, the amino acid sequence of the neutralizing epitope 8 in the COE region is shown in SEQ ID NO: 8, and the specific sequence is GQHPTASGVHG.

[0025] In the present invention, the amino acid sequence of the neutralizing epitope 9 in the COE region is shown in SEQ ID NO: 9, and the specific sequence is SYQLCTANCIGY.

[0026] In the present invention, the amino acid sequence of the neutralizing epitope 10 in the COE region is shown in SEQ ID NO: 10, and the specific sequence is YVPSQSGQVKIA.

[0027] In the present invention, the amino acid sequence of the neutralizing epitope 11 in the COE region is shown in SEQ ID NO: 11, and the specific sequence is NSYGYVSNSQDSNCPFTLQSVNDSLPQDVTRCKKYSNIGVCKSGSIG.

[0028] In the present invention, the amino acid sequence of the neutralizing epitope 12 in the COE region is shown in SEQ ID NO: 12, and the specific sequence is EGFSFNNWFLLS.

[0029] In the present invention, the amino acid sequence of the neutralizing epitope 13 in the COE region is shown in SEQ ID NO: 13, and the specific sequence is PLDVFNATYLKLTGEIADLEQRSESLRNTTEELCNTLVDLEWLNRVET.

[0030] In the present invention, the amino acid sequence of the neutralizing epitope 14 in the COE region is shown in SEQ ID NO: 14, and the specific sequence is RGPRLQPYE.

[0031] In the present invention, the amino acid sequence of the neutralizing epitope 15 in the COE region is shown in SEQ ID NO: 15, and the specific sequence is SHRLPYPKKGPRPVTHHEYGAEALERAG.

[0032] In the present invention, the PEDV COE region multi-epitope fusion protein further comprises a connecting peptide 1 and / or a connecting peptide 2. The amino acid sequence of the connecting peptide 1 of the present invention is shown in SEQ ID NO: 16, and the specific sequence is KK; the amino acid sequence of the connecting peptide 2 is shown in SEQ ID NO: 17, and the specific sequence is GGGGS.

[0033] In the present invention, the composition of the PEDV COE region multi-epitope fusion protein is preferably that the COE region neutralization epitope 1 to the COE region neutralization epitope 15 are sequentially connected by a connecting peptide 1, and the head end of the PEDV COE region multi-epitope fusion protein has NcoI and BamHI specific restriction sites in sequence, and the tail end has HindIII and xhoI specific restriction sites in sequence, wherein the HindIII and xhoI specific restriction sites are connected by a connecting peptide 2. The composition of the PEDV COE region multi-epitope fusion protein of the present invention is further expressed as: NcoI+BamHI+EAAAKKNSYGYVSKSQDSNCPFTLQSVNDKKGELITGTPK PLEGVTDVSFMKKDLFGYPEFGGGVKFKKNSYGYVSNSQDSNCPFTLQSVNDSLPQDVTRCKKTNFRRFFSKFNVQKKEIGISQEPFDPSGYKKYSNIGVCKSGSIGKKALTKVQEVVNKKSRRMFEPRKPTVSDFVKKLTRDQLPDVIPDYIDVNKKKGQHPTASGVHGKKSYQLCTANCIGYK KYVPSQSGQVKIAKKNSYGYVSNSQDSNCPFTLQSVNDSLPQDVTRCKKYSNIGVCKSGSIGKKEGFSFNNWFLLSKKPLDVFNATYLKLTGEIADLEQRSESLRNTTEELCNTLVDLEWLNRVETKKRGPRLQPYEKKSHRLPYPKKGPRPVTHHEYGAEALERAG+HindIII+GGGGS+xhoI

[0034] The present invention also provides a PEDV COE protein containing an intramolecular adjuvant, comprising the PEDV COE region multi-epitope fusion protein and the intramolecular adjuvant.

[0035] In the present invention, the internal adjuvant includes any one of a p28T vector carrying Tufisn and PT8A, a p28ND vector carrying flagellin CD0 / 1 and flagellin ND0 / 1, and a p28cTB vector carrying Tufisn and CTB.

[0036] In the present invention, the vector p28T is obtained by synthesizing PT8A and Tufisn sequences onto the PET28a vector, the amino acid sequence of Tufisn is shown in SEQ ID NO: 18, and the specific sequence is TKPR; the amino acid sequence of PT8A is shown in SEQ ID NO: 19, and the specific sequence is HIDSQKKA.

[0037] In the present invention, the vector p28ND is obtained by synthesizing flagellin CD0 / 1 and flagellin ND0 / 1 sequences onto a PET28a vector, the amino acid sequence of the flagellin CD0 / 1 is shown in SEQ ID NO: 20, and the specific sequence is MAQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIA NRFTSNIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELSVQATNG TNSDSDLKSIQDEIQQRLEEIDRVSN; the amino acid sequence of the flagellin ND0 / 1 is shown in SEQ ID NO: 21, and the specific sequence is KSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLL.

[0038] In the present invention, the vector p28cTB is obtained by synthesizing the Tufisn sequence and CTB onto the PET28a vector. The amino acid sequence of the CTB protein is shown in SEQ ID NO: 22, and the specific sequence is MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN.

[0039] The present invention also provides the use of the PEDV COE protein containing the internal adjuvant in the preparation of porcine epidemic diarrhea virus vaccine.

[0040] The present invention also provides a porcine epidemic diarrhea virus subunit vaccine, comprising the PEDV COE protein containing an intramolecular adjuvant and a pharmaceutically acceptable adjuvant.

[0041] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example 1

[0043] (1) Identification of candidate vaccine strains

[0044] The amino acid sequences of COE proteins were collected from the NCBI database in the fasta file format and used as templates. The sequence conservation was analyzed using MEGA 7.0 software, and the resulting comparative datasets were then visualized using the WebLogo server to analyze conservation.

[0045] (2) Immunoinformatics design

[0046] Epitope prediction and screening: ABCpred server was used to predict candidate linear B lymphocyte epitopes of proteins. MHC-II epitopes were predicted using NetMHCIIpan-4.0 server. The potential for epitope combination with MHC-II was evaluated by EpiTOP server. Prediction was performed by IFN epitope server and IL4pred server. MHC-I was combined with NetMHCpan4.1 server and IEDB server for CTL epitope prediction. All predicted epitopes were determined by IEDB server. Non-toxic immunogenicity test was performed by ToxinPred server.

[0047] Identification of protective epitopes and assessment of antigenic epitope conservation: Dominant epitopes were assessed by the conservation analysis tool on the IEDB server.

[0048] By adjusting the number of repeated copies and the construction position, the neutralizing epitope selected from the existing COE region is designed to enhance the antigenicity.

[0049] Construction of candidate vaccines and prediction of antigenicity, sensitization, solubility and physicochemical properties: The final antigenicity analysis of the candidate vaccine was performed using the ANTIGENpro server and the VaxiJen v2.0 server; the AllerTOPv2.0 server was used for the final allergenicity analysis of the candidate vaccine; and the SOLpro server was used for the final solubility analysis of the candidate vaccine. The physicochemical properties of the candidate vaccine were analyzed using the ExPASy server, and several candidate combinations were selected.

[0050] Secondary and tertiary structure modeling, refinement and disulfide engineering of candidate vaccines: The secondary structure prediction of the final candidate vaccine was completed using PSIPRED4.0 server and SOPMA server. The tertiary structure prediction of the final candidate vaccine was completed using trRosetta server. The structure was optimized using Galaxy-WEB server. Ramachandran plots were generated by EBI-PDBSum server to evaluate the quality of the model before and after optimization. Disulfide engineering of the candidate vaccine was performed using DisulfidebyDesign32.2 server. Global detection of the model was performed using ProSA-Web server.

[0051] Tertiary epitope prediction and protein docking analysis: The tertiary structure of the vaccine protein was predicted by ElliPro server. The interaction between TLR3 as receptor and protein vaccine as ligand was predicted using PyDockWEB server. LigPLot+ was used to determine the amino acid residues with hydrogen bonds and hydrophobic interactions after docking.

[0052] Example 2

[0053] (I) Synthesis, expression and purification of PEDV COE region multi-epitope fusion protein

[0054] According to the above screening, the neutralizing epitopes 1 to 15 in the dominant COE region were obtained, and then the PEDV COE region multi-epitope fusion protein was designed, with the composition of NcoI+BamHI+EAAAKKNSYGYVSKSQDSNCPFTLQSVNDKKGELITGTPK PLEGVTDVSFMKKDLFGYPEFGGGVKFKKNSYGYVSNSQDSNCPFTLQSVNDSLPQDVTRCKKTNFRRFFSKFNVQKKEIGISQEPFDPSGYKKYSNIGVCKSGSIGKKALTKVQEVVNKKSRRMFEPRKPTVSDFVKKLTRDQLPDVIPDYIDVNKKKGQHPTASGVHGKKSYQLCTANCIGYKK YVPSQSGQVKIAKKNSYGYVSNSQDSNCPFTLQSVNDSLPQDVTRCKKYSNIGVCKSGSIGKKEGFSFNNWFLLSKKPLDVFNATYLKLTGEIADLEQRSESLRNTTEELCNTLVDLEWLNRVETKKRGPRLQPYEKKSHRLPYPKKGPRPVTHHEYGAEALERAG+HindIII+GGGGS+xhoI.

[0055] The designed sequence was sent to BGI for synthesis, and BGI connected the synthesized sequence to pET28a to obtain the corresponding recombinant plasmid (dry powder). The recombinant plasmid was dissolved in sterile water and transferred to BL21 (DE3) competent cells for coating and culture for 12 hours to obtain recombinant bacteria.

[0056] The next day, the recombinant bacteria prepared above were picked and inoculated into 5 mL of LB liquid medium containing kanamycin to verify its expression. Different gradients of induction temperature (16°C, 28°C, 37°C), different gradients of induction time (4h, 6h, 8h, 10h), and different gradients of inducer addition (0.2mmol / L, 0.4mmol / L, 0.6mmol / L, 0.8mmol / L, 1mmol / L) were set to explore the conditions for inducing the expression of recombinant proteins.

[0057] The results showed that the optimal induction temperature was 37℃, the optimal induction time was 6h, and the optimal inducer concentration was 0.4mmol / L.

[0058] The induced protein solution is taken, and the precipitate is collected by centrifugation, and then resuspended and broken. After centrifugation, the precipitate is retained and washed with inclusion body washing solution to remove impurities to obtain inclusion body protein solution.

[0059] The inclusion body protein solution was centrifuged at 8000r / min for 10min, and the supernatant protein was filtered using a 0.22μm filter membrane to remove significant precipitate and prevent clogging of the filter. The filtered inclusion body protein solution was collected and then purified with Ni-NTA resin at 4°C. After binding, gradient elution was performed with eluents containing different concentrations of imidazole (100, 300, 500mmol / L). Among them, the optimal elution concentration was 300mmol / L imidazole, and the purified protein was collected and stored at -20°C.

[0060] (II) Dialysis refolding of PEDV COE region multi-epitope fusion protein

[0061] Cut the dialysis bag into a suitable size, put it into a beaker, add appropriate amount of pure water, boil it in a boiling water bath for 10 minutes, and take it out to cool.

[0062] Take 50mL of the above filtered inclusion body protein solution and add refolding solution (20mmol / LTris-HCl, 500mmol / LNaCl, 45mL / L glycerol, 1g / L arginine) and dithiothioethanol respectively, and put it into the pretreated dialysis bag for refolding. The volume of the refolding solution added is 10% of the volume of the inclusion body protein solution, and the volume of dithiothiothioethanol added is 0.5% of the volume of the inclusion body protein solution. After confirming that there is no leakage in the seal, place it at 4°C for refolding procedure, and stir the dialyzate with a magnetic stirrer. The refolding procedure is:

[0063] 1) Dialyze with 10 times the volume of external solution A for 1 hour (the external solution A is 8M urea diluted 100 times with 2 times the volume of dialysate), and then add 3 times the volume of external solution water for 1 hour;

[0064] 2) Change the volume of external solution B by 100 times and dialyze for 4 hours (the external solution B is the refolding solution diluted 1000 times);

[0065] 3) The external solution C was replaced with 100 times the volume and dialyzed for 4 h (the external solution C was a 10 mM CB solution with a pH of 9.5, and the CB solution was prepared by diluting the volume to 1 L with 0.318 g of sodium carbonate and 0.586 g of sodium bicarbonate).

[0066] The renatured protein was centrifuged at 5000 r / min for 10 min, and the supernatant was retained. Then, the protein before and after renaturation was subjected to 12% SDS-PAGE electrophoresis to determine its renaturation status.

[0067] The results showed that the target band existed after renaturation, and its size was consistent with that before renaturation, and no other impurity bands were generated.

[0068] (III) Identification and stability study of PEDV COE region multi-epitope fusion protein

[0069] The purified protein was tested for endotoxin content using Triton X-114 and horseshoe crab reagent to evaluate the endotoxin removal effect. The initial protein endotoxin content was higher than 100 EU / mL, and was lower than 10 EU / mL after two extractions.

[0070] Western-blot method was used to verify whether it was a recombinant protein, and Dot-ELISA method was used to identify the immunogenicity of the protein by reacting the antigen with PRRSV positive serum. The results showed that both had positive reactions.

[0071] Long-term stability experiments were conducted to investigate the storage stability of recombinant antigens at different temperatures (-80°C, -20°C, 4°C or 37°C for accelerated destruction). The results confirmed that the protein stability was highest at -80°C.

[0072] Example 3

[0073] 1. Vaccine preparation and mouse immunization

[0074] The renatured protein was quantitatively set to 200ug / mL, and mixed with 201 adjuvant at a ratio of 1:1. After mixing, a homogenizer was used to emulsify for 5 minutes. After centrifugation, no stratification occurred, and the vaccine preparation was obtained. Balb / c mice (purchased from Henan Sikebeisi Biotechnology Co., Ltd.) were selected for immunization. The mice were selected to be 6-8 weeks old. The mice were immunized subcutaneously. Three points were selected for immunization, including subcutaneous injection in the neck and subcutaneous injection in the hind legs. Each mouse was injected with a total of 200μL, of which the injection volume in the neck was 100μL, and the injection volume in the inner side of the hind legs was 50μL on each side. The immunization was performed three times, with an interval of 14 days each time. Blood was collected from the tail vein one week after each immunization, and blood was collected from the orbits two weeks after the three immunizations. The corresponding immunization times when the titer was 1:100,000 were the common immunization endpoints. If the titer did not meet the requirements, enhanced immunization was performed. Blood was collected from the tail vein one week after immunization. The operation was the same as the previous three times. Enhanced immunization was performed until the titer was reached, and a maximum of 5 immunizations were performed.

[0075] (II) Humoral Immunity Testing

[0076] The blood of the immunized mice was collected, and the serum obtained was separated and tested for specific antibody IgM and IgG indicators according to the ELISA method.

[0077] The results showed that after the above mice were immunized, the serum titer was measured to be 128,000.

[0078] (III) Evaluation of cellular immune effects

[0079] After blood was drawn from the eyeball, the mice were killed by cervical dislocation, and the spleen tissue of the mice was dissected to prepare spleen single cell suspension, which was lysed with red blood cell lysis buffer and then stimulated and cultured (1×10 6 cells / mL), cell surface staining, fixation, membrane permeabilization, cell activation stimulation, extracellular factor staining after blocking the antibody Fc-terminal receptor, fixation, membrane permeabilization and washing, resuspended and then flow cytometry detection, detection of CD3, CD4, CD8 indicators.

[0080] The results showed that CD3, CD4 and CD8 all increased.

[0081] (IV) Safety evaluation

[0082] The mice were observed after immunization, and their appearance, behavior, spirit, and food intake were observed every day during the immunization period. No abnormalities were found.

[0083] Three days after the last immunization, a histopathological examination was performed: the mice were killed by cervical dislocation, and the lungs, hearts, spleens, kidneys, etc. were taken out on a sterile operating table. No pathological changes such as edema and congestion were observed with the naked eye.

[0084] Then, the above tissues were rinsed twice with PBS and fixed in 10% formalin solution. The fixed tissues were paraffin-embedded for 24 hours, cut into 4 μm slices under a sliding microtome, and dehydrated step by step with different concentrations of ethanol. The obtained slices were stained with hematoxylin-eosin (HE), and the inflammatory cell infiltration of the tissues was observed under an optical microscope. The results showed that no inflammatory cell infiltration was observed in any tissue.

[0085] (V) In vitro neutralization test evaluation

[0086] The serum was placed in a 56°C water bath and inactivated for 30 min; the in vitro cell virus neutralization assay was evaluated according to the following experimental scheme.

[0087] 1. Material Preparation

[0088] Equipment: carbon dioxide incubator, clean workbench, biological safety cabinet, ordinary refrigerator, inverted biological microscope, cell counting plate, constant temperature water bath, centrifuge tube, cell culture flask, cell culture dish, 96-well cell plate, centrifuge tube rack, single-channel and eight-channel pipettes, 200μl and 1000μl tips.

[0089] Reagents: DMEM cell culture medium, fetal bovine serum (56°C, 30 min inactivation) and 0.125% trypsin solution.

[0090] Cells, viruses and serum: Vero cells, PEDV virus (both purchased from Pleco) and serum samples to be tested related to PEDV virus (inactivated at 56°C for 30 min).

[0091] 2. Operation method

[0092] (1) Cell recovery and monolayer preparation

[0093] One day in advance, take out two frozen primary Vero cells from the liquid hydrogen tank, resuscitate and count the cells and inoculate them in a 96-well plate, with 2×10 cells per well. 5 cells / well, the culture medium is DMEM cell culture medium containing 10% fetal bovine serum, and the cells are cultured in a 37°C, 5% CO2 incubator for 24 hours to grow into a monolayer.

[0094] (2) Serum inactivation and dilution

[0095] After the serum to be tested was inactivated in a 56°C water bath for 30 min, it was diluted 2-fold in a DMEM cell culture medium containing 5% fetal bovine serum. 2 , 2 3 , 2 4 , 2 5 , 2 6 .

[0096] (3) Virus Neutralization

[0097] The virus solution with known virus content was diluted with DMEM cell culture medium with 4% fetal bovine serum to a half tissue cell infection dose of 2×10 3 TC1D50 / ml, in a 1.5mIEP tube, take 500μl of diluted virus solution and mix with 500μl of diluted serum, set up virus control and normal cell control at the same time, and incubate at 37℃ for 1 hour.

[0098] (4) Sample addition

[0099] Take a 96-well Vero cell culture plate grown into a single layer, discard the cell culture medium, add the neutralization sample of 3 to each well, and repeat 4 wells for each neutralization sample. At the same time, set up normal cell control wells and virus photo wells.

[0100] (5) Fluid exchange

[0101] After adding the sample in step (4) to the well for 2 hours, all of it was discarded and replaced with DMEM cell culture medium containing 4% fetal bovine serum, and the cells were placed in a 37°C, 5% CO2 incubator for continued culture.

[0102] (6) Cultivate and observe: Cultivate for 3 to 4 days and observe the results.

[0103] The results showed that after mice were immunized with the PEDV COE region multi-epitope fusion protein, the neutralization titer in the cell test was 1:6.

[0104] Example 4

[0105] The sequences of the dominant components screened out in Example 1 were sent to BGI, which synthesized the sequences of the dominant components into PET28a vectors, respectively, and the corresponding new vectors obtained were used as intramolecular adjuvants, as shown in Table 1.

[0106] Table 1 Intramolecular adjuvants

[0107]

[0108] Table 2 Sequences of the main dominant components in intramolecular adjuvants

[0109]

[0110]

[0111] The two restriction sites of BamHI and HindIII were selected on the above-mentioned intramolecular adjuvants to subclone and synthesize the multi-epitope fusion protein of the PEDV COE region to obtain the multi-epitope fusion protein containing the intramolecular adjuvant. Then the same vaccine preparation and mouse immunization were carried out, and the in vitro cell virus neutralization experiment was evaluated.

[0112] Table 3 Potency of PEDV COE protein containing different internal adjuvants

[0113]

[0114] The results showed that when the intramolecular adjuvant was p28ND, the neutralization titer in the cell experiment was relatively the highest, which means that the immunogenicity of the PEDV COE protein containing the intramolecular adjuvant p28ND was relatively the best.

[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A PEDV COE region multi-epitope fusion protein, characterized in that: The constituent fragments of the PEDV COE region recombinant protein include COE region neutralizing epitope 1, COE region neutralizing epitope 2, COE region neutralizing epitope 3, COE region neutralizing epitope 4, COE region neutralizing epitope 5, COE region neutralizing epitope 6, COE region neutralizing epitope 7, COE region neutralizing epitope 8, COE region neutralizing epitope 9, COE region neutralizing epitope 10, COE region neutralizing epitope 11, COE region neutralizing epitope 12, COE region neutralizing epitope 13, COE region neutralizing epitope 14 and COE region neutralizing epitope 15; The amino acid sequence of the neutralizing epitope 1 in the COE region is shown in SEQ ID NO: 1; The amino acid sequence of the neutralizing epitope 2 in the COE region is shown in SEQ ID NO: 2; The amino acid sequence of the neutralizing epitope 3 in the COE region is shown in SEQ ID NO: 3; The amino acid sequence of the neutralizing epitope 4 in the COE region is shown in SEQ ID NO: 4; The amino acid sequence of the neutralizing epitope 5 in the COE region is shown in SEQ ID NO: 5; The amino acid sequence of the neutralizing epitope 6 in the COE region is shown in SEQ ID NO: 6; The amino acid sequence of the neutralizing epitope 7 in the COE region is shown in SEQ ID NO: 7; The amino acid sequence of the neutralizing epitope 8 in the COE region is shown in SEQ ID NO: 8; The amino acid sequence of the neutralizing epitope 9 in the COE region is shown in SEQ ID NO:9; The amino acid sequence of the neutralizing epitope 10 in the COE region is shown in SEQ ID NO: 10; The amino acid sequence of the neutralizing epitope 11 in the COE region is shown in SEQ ID NO: 11; The amino acid sequence of the neutralizing epitope 12 in the COE region is shown in SEQ ID NO: 12; The amino acid sequence of the neutralizing epitope 13 in the COE region is shown in SEQ ID NO: 13; The amino acid sequence of the neutralizing epitope 14 in the COE region is shown in SEQ ID NO: 14; The amino acid sequence of the COE region neutralizing epitope 15 is shown in SEQ ID NO:

15.

2. The PEDV COE region multi-epitope fusion protein according to claim 1, characterized in that: Also includes connecting peptide 1 and / or connecting peptide 2; The amino acid sequence of the connecting peptide 1 is shown in SEQ ID NO:16, and the amino acid sequence of the connecting peptide 2 is shown in SEQ ID NO:

17.

3. A PEDV COE protein containing an internal adjuvant, characterized in that: It comprises the PEDVCOE region multi-epitope fusion protein as described in claim 1 or 2 and an intramolecular adjuvant.

4. The PEDV COE protein containing a built-in adjuvant according to claim 3, characterized in that: The internal adjuvant includes any one of a p28T vector carrying Tufisn and PT8A, a p28ND vector carrying flagellin CD0 / 1 and flagellin ND0 / 1, and a p28cTB vector carrying Tufisn and CTB.

5. The PEDV COE protein containing a built-in adjuvant according to claim 4, characterized in that: The amino acid sequence of Tufisn is shown in SEQ ID NO: 18, the amino acid sequence of PT8A is shown in SEQ ID NO: 19, the amino acid sequence of flagellin CD0 / 1 is shown in SEQ ID NO: 20, the amino acid sequence of flagellin ND0 / 1 is shown in SEQ ID NO: 21, and the amino acid sequence of CTB is shown in SEQ ID NO:

22.

6. Use of the PEDV COE protein containing an internal adjuvant according to claim 3 in the preparation of a porcine epidemic diarrhea virus vaccine.

7. A porcine epidemic diarrhea virus subunit vaccine, characterized in that: The invention comprises the PEDV COE protein containing the built-in adjuvant according to claim 3 and a pharmaceutically acceptable adjuvant.

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