A multi-epitope fusion protein containing an intramolecular adjuvant and its application in the preparation of porcine epidemic diarrhea virus vaccine

Abstract

The present invention relates to the field of biopharmaceutical technology. The present invention provides a recombinant multi-epitope fusion protein, and a multi-epitope fusion protein containing an intramolecular adjuvant comprising the recombinant multi-epitope fusion protein. The present invention also provides the use of the multi-epitope fusion protein containing an intramolecular adjuvant in the preparation of a porcine epidemic diarrhea virus vaccine. The technical solution of the present invention uses the S protein as a target, screens the epitope region based on bioinformatics analysis, and performs different permutations and combinations through reasonable truncation, concatenation, and modification to screen out permutations and combinations with higher titers, thereby minimizing the occurrence of the ADE effect while avoiding the waste of animal immune potential; at the same time, the humoral immunity and cellular immunity are further enhanced by introducing intramolecular adjuvants to improve the immune level.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a multi-epitope fusion protein containing an intramolecular adjuvant and application thereof in the preparation of a porcine epidemic diarrhea virus vaccine. Background Art

[0002] 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 several vaccines (as shown in Table 1) have been used for more than ten years, they cannot meet the demand due to the rapid mutation of the virus, low intramuscular cellular / mucosal immunity, and potential ADE.

[0003] Table 1 Comparison of different types of vaccines

[0004]

[0005] The development of vaccines for PEDV not only needs to deal with the impact of high mutation and potential ADE effects, but also needs to consider the economic feasibility of industrial applications. Based on the current technology and status quo, subunit vaccines have become the preferred direction of research and development because they do not have biosafety issues, have clear proteins, clear structures, precise dosages, do not waste animal immune potential, and can minimize the ADE effect. This also provides a good reference for the development of PEDV vaccines.

[0006] PEDV is a single-stranded RNA enveloped virus. The PEDV S protein plays a key role in mediating virus entry into cells, inducing neutralizing antibodies and viral virulence in vivo, and is the main target for the development of live attenuated vaccines and subunit vaccines. At present, attempts to develop PED vaccines based on the S protein through intramuscular (IM) administration have not been successful; few people have paid attention to the impact of the presence of ADE effector epitopes on the S protein on the efficacy of the vaccine. In addition, studies have shown that PEDV M is relatively conserved among different PEDV strains and may contain certain neutralizing epitopes, but relevant subunit vaccines based on PEDV N and M proteins have not yet been reported or studied.

[0007] Therefore, it is very necessary to develop an effective PEDV subunit vaccine to address key issues such as virus mutation, balance of humoral and cellular immunity, enhancement of mucosal immunity, and elimination of ADE effects. Summary of the invention

[0008] The purpose of the present invention is to provide a multi-epitope fusion protein containing an intramolecular adjuvant and its application in the preparation of porcine epidemic diarrhea virus vaccine. PEDV is based on the S protein as the core, M, N antigen epitopes and intramolecular adjuvants are added through a suitable linker, and different permutations and combinations are performed through reasonable truncation, concatenation and modification to screen out permutations and combinations with higher titers, thereby minimizing the occurrence of ADE effect while avoiding the waste of animal immune potential and improving the immune level.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The invention provides a recombinant multi-epitope fusion protein, wherein the constituent fragments of the recombinant multi-epitope fusion protein include a PEDV S protein neutralizing epitope region S1D region, a core neutralizing epitope region COE region of the S1 region of the PEDV S protein, and a PEDVM protein neutralizing epitope peptide segment.

[0011] Preferably, the amino acid sequence of the S1D region of the neutralizing epitope region of the PEDV S protein is shown in SEQ ID NO: 1, the amino acid sequence of the COE region of the core neutralizing epitope region of the S1 region of the PEDV S protein is shown in SEQ ID NO: 2, and the amino acid sequence of the neutralizing epitope peptide segment of the PEDVM protein is shown in SEQ ID NO: 3.

[0012] Preferably, it also includes a T cell and B cell co-stimulatory epitope region, and the amino acid sequence of the T cell and B cell co-stimulatory epitope region is shown in SEQ ID NO:4.

[0013] Preferably, it also includes a B cell epitope region and a T cell epitope region, the amino acid sequence of the B cell epitope region is shown in SEQ ID NO:5, and the amino acid sequence of the T cell epitope region is shown in SEQ ID NO:6.

[0014] The invention provides a multi-epitope fusion protein containing an intramolecular adjuvant, comprising the recombinant multi-epitope fusion protein and the intramolecular adjuvant.

[0015] Preferably, the intramolecular adjuvant includes any one of a p22T vector carrying Pelb and Tufisn, a p28T vector carrying PT8A and Tufisn, a p28ND vector carrying flagellin CD0 / 1 and flagellin ND0 / 1, a p28fcM vector carrying a mouse Fc fragment and Tufisn, a p28FcP vector carrying a porcine Fc fragment and Tufisn, a p28cTB vector carrying CTB and Tufisn, a p28T4 vector carrying T4-Fd and Tufisn, a p28GCN vector carrying GCN4 and Tufisn, and a p28Fe vector carrying ferritin and Tufisn.

[0016] Preferably, the amino acid sequence of Pelb is as shown in SEQ ID NO:13; the amino acid sequence of Tufisn is as shown in SEQ ID NO:14; the amino acid sequence of PT8A is as shown in SEQ ID NO:15; the amino acid sequence of flagellin CD0 / 1 is as shown in SEQ ID NO:16; the amino acid sequence of flagellin ND0 / 1 is as shown in SEQ ID NO:17; the amino acid sequence of the polymer peptide T4-Fd is as shown in SEQ ID NO:18; the amino acid sequence of the polymer peptide GCN4 is as shown in SEQ ID NO:19; the amino acid sequence of the CTB protein is as shown in SEQ ID NO:20; the amino acid sequence of ferritin is as shown in SEQ ID NO:21; the amino acid sequence of the mouse Fc fragment is as shown in SEQ ID NO:22; the amino acid sequence of the porcine Fc fragment is as shown in SEQ ID NO:23.

[0017] The present invention also provides the use of the multi-epitope fusion protein containing the intramolecular adjuvant in the preparation of porcine epidemic diarrhea virus vaccine.

[0018] The present invention also provides a porcine epidemic diarrhea virus subunit vaccine, comprising the multi-epitope fusion protein containing an intramolecular adjuvant and a pharmaceutically acceptable adjuvant.

[0019] By adopting the above technical solution, the present invention has the following beneficial effects:

[0020] The technical solution of the present invention uses S protein as a target, screens the epitope region based on bioinformatics analysis, and screens out the permutations and combinations with higher titers through reasonable truncation, concatenation, and modification, thereby minimizing the occurrence of ADE effects and avoiding the waste of animal immune potential; at the same time, the humoral immunity and cellular immunity are further strengthened by introducing intramolecular adjuvants to improve the immune level. Compared with traditional subunit vaccines, it has better immunogenicity. DETAILED DESCRIPTION

[0021] The invention provides a recombinant multi-epitope fusion protein, wherein the constituent fragments of the recombinant multi-epitope fusion protein include a PEDV S protein neutralizing epitope region S1D region, a core neutralizing epitope region COE region of the S1 region of the PEDV S protein, and a PEDVM protein neutralizing epitope peptide segment.

[0022] In the present invention, the amino acid sequence of the neutralizing epitope region S1D of the PEDV S protein is shown in SEQ ID NO: 1, and the specific sequence is TDBSFMTLDVCTKYTIYGFKGEGIITLTNSSILAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVNDDIVGVISSLSNSTFNNTRELPGFFYHSNDGSNCTEPVLVYSNIGVCKSGSIGYVPSQYGQVKIAPTVTGNISIPTNFSMSI.

[0023] In the present invention, the amino acid sequence of the COE region of the core neutralizing epitope region of the S1 region of the PEDV S protein is shown in SEQ ID NO: 2, and the specific sequence is RGSAISFVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGV.

[0024] In the present invention, the amino acid sequence of the neutralizing epitope peptide of the PEDV M protein is shown in SEQ ID NO: 3, and the specific sequence is VQVSQLPNFVTVAKATTTIVYGRVGRSVNASSGTGWAFYVRSKHGDYSAVSNPSAVLTDSEKVLHLVGSGSSLPQDVTRCQSTTGSGSPDNKTLGPTVNDVTTGRGSGSTSDSGQLGSGSFDPSGYQLGSGSYIDVNKTLGSGSTLGPTVNDVTTGSGSLAESFNSAIGNITSAGSGSLDEILASLPNRTGPS.

[0025] In the present invention, the constituent fragments of the recombinant multi-epitope fusion protein also include T and B cell co-stimulatory epitope regions, and the amino acid sequence of the T and B cell co-stimulatory epitope regions is shown in SEQ ID NO: 4, and the specific sequence is NKTLGPTVNDVTTGRNCLEAAAKSLPQDVTRCQSTTNFRRFFSKFNVQAPAVVVLGGYLPSMNSSSWYEAAAKILASLPNRTGPSLPLDVFNATYEAAAKLTSDQLPDVIPDYIDVNKTLDEIEAAAKSEEALQLATISSFNGDGY.

[0026] In the present invention, the constituent fragments of the recombinant multi-epitope fusion protein also include a B cell epitope region and a T cell epitope region, the amino acid sequence of the B cell epitope region is shown in SEQ ID NO:5, and the specific sequence is TLGPTVNDVTTEAAAKLAESFNSAIGNITSAEAAAKLDEILASLPNRTGPS. The amino acid sequence of the T cell epitope region is shown in SEQ ID NO:6, and the specific sequence is SLPQDVTRCQSTTEAAAKPDNKTLGPTVNDVTTGREAAAKTSDSGQLEAAAKFDPSGYQLEAAAKYIDVNKTL.

[0027] In the present invention, the constituent fragments of the recombinant multi-epitope fusion protein are preferably connected via a connecting peptide, and the connecting peptide is preferably GGGGSGGGGS (SEQ ID NO: 7) and / or EAAAK (SEQ ID NO: 8).

[0028] In the present invention, the composition of the recombinant multi-epitope fusion protein preferably includes COE region + S1D region + M protein peptide segment + EcoRI + T, B cell co-stimulatory epitope region, M protein peptide segment + COE region + S1D region + EcoRI + T, B cell co-stimulatory epitope region, T, B cell co-stimulatory epitope region + EcoRI + COE region + S1D region + M protein peptide segment, and COE region + T cell epitope region + S1D region + M protein peptide segment + B cell epitope region.

[0029] In the present invention, the head and tail of the recombinant multi-epitope fusion protein have specific restriction sites respectively. The head specific restriction site of the recombinant multi-epitope fusion protein of the present invention is preferably BamHI, and the tail specific restriction site of the recombinant multi-epitope fusion protein is preferably HindIII.

[0030] The invention provides a multi-epitope fusion protein containing an intramolecular adjuvant, comprising the recombinant multi-epitope fusion protein and the intramolecular adjuvant.

[0031] In the present invention, the internal adjuvant includes any one of the vectors p22T, p28T, p28ND, p28FcM, p28FcP, p28cTB, p28T4, p28GCN and p28Fe.

[0032] In the present invention, the vector p22T is obtained by synthesizing Pelb and Tufisn sequences onto the PET28a vector, the amino acid sequence of Pelb is shown in SEQ ID NO: 13, and the specific sequence is MKYLLPTAAAGLLLLAAQPA; the amino acid sequence of Tufisn is shown in SEQ ID NO: 14, and the specific sequence is TKPR.

[0033] In the present invention, the vector p28T is obtained by synthesizing PT8A and Tufisn sequences onto the PET28a vector, the amino acid sequence of PT8A is shown in SEQ ID NO: 15, and the specific sequence is HIDSQKKA.

[0034] 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: 16, and the specific sequence is MAQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNA NDGISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSN; the amino acid sequence of the flagellin ND0 / 1 is shown in SEQ ID NO: 17, and the specific sequence is KSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSNMSKAQ ILQQAGTSVLAQANQVPQNVLSLL.

[0035] In the present invention, the vector p28T4 is obtained by synthesizing T4-Fd and Tufisn sequences onto a PET28a vector, and the amino acid sequence of the polymer peptide T4-Fd is shown in SEQ ID NO: 18, and the specific sequence is MQALQEAGYIPEAPRDGQAYVRKDGEWVLLSTFLSPA.

[0036] In the present invention, the vector p28GCN is obtained by synthesizing GCN4 and Tufisn sequences onto a PET28a vector, and the amino acid sequence of the polymer peptide GCN4 is shown in SEQ ID NO: 19, and the specific sequence is MKQIEDKIEEILSKIYHIENEIARIKKLIGEV.

[0037] In the present invention, the vector p28cTB is obtained by synthesizing CTB and Tufisn sequences onto the PET28a vector, and the amino acid sequence of the CTB protein is shown in SEQ ID NO: 20, and the specific sequence is MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN.

[0038] In the present invention, the vector p28Fe vector is obtained by synthesizing ferritin and Tufisn sequences onto a PET28a vector, the amino acid sequence of the ferritin is shown in SEQ ID NO: 21, and the specific sequence is MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS.

[0039] In the present invention, the vector p28fcM is obtained by synthesizing a mouse Fc fragment and a Tufisn sequence onto a PET28a vector, the amino acid sequence of the mouse Fc fragment is shown in SEQ ID NO: 22, and the specific sequence is VRSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0040] In the present invention, the vector p28fcP is obtained by synthesizing a porcine Fc fragment and a Tufisn sequence onto a PET28a vector. The amino acid sequence of the porcine Fc fragment is shown in SEQ ID NO: 23, and the specific sequence is MWQLLSPTALLLLVSVPGTHAEDPPKSVVILDPPWDRLLEKDSVTLKCQGAYPPRDDSTEWRWNGTLISNKASSYSITDATVGNSGEYTCKTGLSAQSDPLRLEVYKGWLLLQAPRWVVQEGESIRLRCHTWKNITIQKVQYFQNGMGKKFSHQNFEYHIPNATLKDGGSYFCRGIIKNYDLSSEPVKVTVQGSKSPSPILSFFLPWHQIIFCLVMGFLFAVDTGLYFSVRKVLRSSKEDWRNGKVTWSRDPADKGG.

[0041] The present invention also provides the use of the multi-epitope fusion protein containing the intramolecular adjuvant in the preparation of porcine epidemic diarrhea virus vaccine.

[0042] The present invention also provides a porcine epidemic diarrhea virus subunit vaccine, comprising the multi-epitope fusion protein containing an intramolecular adjuvant and a pharmaceutically acceptable adjuvant.

[0043] In the present invention, the adjuvant is preferably an oil-emulsion adjuvant, and the vaccine is preferably in the form of water-in-oil.

[0044] 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.

[0045] Example 1

[0046] (1) Identification of candidate vaccine strains

[0047] The amino acid sequences of S protein and M protein were collected from the NCBI database in the fasta file format and used as templates. The sequence conservation was analyzed using MEGA7.0 software, and the resulting comparative data set was visualized and analyzed for conservation using the WebLogo server.

[0048] (2) Immunoinformatics design

[0049] 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.

[0050] Identification of protective epitopes and assessment of antigenic epitope conservation: Dominant epitopes were assessed by the conservation analysis tool on the IEDB server.

[0051] Concatenation of candidate epitopes: Sequences collected from literature and predicted sequences are concatenated with candidate vaccines using flexible linkers to extend the duration of protein action in vivo, effectively avoiding the introduction of new binding epitopes. GGPPG is used for CTL epitopes, AAY is used for HTL epitopes, and KK is used for B lymphocyte epitopes. In order to improve the immunogenicity of candidate vaccines, intramolecular adjuvants are added and connected to the N-terminus of the construct through an EAAAK linker, and a Flynn-sensitive linker (RVRR) is used for cross-protein connection.

[0052] By adjusting the number of repeated copies and the construction position, the neutralizing epitopes selected from known literature were designed to enhance antigenicity.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Example 2

[0057] (I) Synthesis, expression and purification of recombinant multi-epitope fusion proteins

[0058] According to the above screening, the dominant sequences were obtained, and then four recombinant multi-epitope fusion protein combinations were designed, including COE region + S1D region + M protein peptide + EcoRI + T, B cell costimulatory epitope region, M protein peptide + COE region + S1D region + EcoRI + T, B cell costimulatory epitope region, T, B cell costimulatory epitope region + EcoRI + COE region + S1D region + M protein peptide, and COE region + T cell epitope region + S1D region + M protein peptide + B cell epitope region. See Table 2 for details.

[0059] Table 2 Recombinant multi-epitope fusion protein

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] The designed sequence was sent to BGI for synthesis, and BGI connected the synthesized sequence to pET28a to prepare the corresponding recombinant plasmid (dry powder). The recombinant plasmid was dissolved in sterile water and transferred to BL21 competent medium for coating and culture for 12 hours to obtain recombinant bacteria.

[0067] The recombinant bacteria obtained above were induced to express, and 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 expression of recombinant proteins. The results are shown in Table 3.

[0068] 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.

[0069] 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 is shown in Table 3, and the purified protein was collected and stored at -20°C.

[0070] Table 3 Conditions for inducing expression of different recombinant multi-epitope fusion proteins

[0071]

[0072]

[0073] (II) Identification and stability study of recombinant multi-epitope fusion proteins

[0074] The purified protein was extracted with TritonX-114 to remove endotoxin content. The initial protein endotoxin content was higher than 100EU / mL, and was lower than 10EU / mL after two extractions.

[0075] The Western-blot method was used to verify whether it was a recombinant protein, and the Dot-ELISA method was used to identify the immunogenicity of the protein by reacting the antigen with PEDV positive serum. The results showed that both had positive reactions.

[0076] 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.

[0077] (III) Dialysis refolding of recombinant multi-epitope fusion protein

[0078] 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.

[0079] Take 50 mL of the filtered inclusion body protein solution and add refolding solution (20 mmol / L Tris-HCl, 500 mmol / L NaCl, 45 mL / L glycerol, 1 g / L arginine) and dimercaptoethanol respectively, and put it into the pretreated dialysis bag for refolding, the refolding solution added volume is 10% of the volume of the inclusion body protein solution, and the dimercaptoethanol added volume 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 dialyzed external solution with a magnetic stirrer.

[0080] The renaturation procedure is:

[0081] 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;

[0082] 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);

[0083] 3) Change the volume of external solution C by 100 times and dialyze for 4 hours (the external solution C is 10 mM pH 9.5 CB, and the CB solution is obtained by diluting the volume to 1 L with 0.318 g of sodium carbonate and 0.586 g of sodium bicarbonate).

[0084] 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.

[0085] The results showed that the target bands existed and were of the same size after renaturation.

[0086] Example 3

[0087] 1. Vaccine preparation and mouse immunization

[0088] 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 over two months 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 received a total of 200μL, including 100μL in the neck and 50μL in the inner side of the hind legs. 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.

[0089] (II) Humoral Immunity Testing

[0090] 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. The results are shown in Table 4.

[0091] Table 4 The corresponding titers of different recombinant multi-epitope fusion proteins

[0092]

[0093] The results showed that after immunization of the above mice, the optimal serum titer was 256,000, and the protein with the best immune effect was the recombinant multi-epitope fusion protein corresponding to the titer of 256,000.

[0094] (III) Evaluation of cellular immune effects

[0095] The mice were killed by dislocating the neck after eyeball bleeding, and spleen tissues were obtained by dissection. Splenic single cell suspensions were prepared and lysed with red blood cell lysis buffer for cell stimulation and culture (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.

[0096] The results showed that CD3, CD4 and CD8 all increased.

[0097] (IV) Safety evaluation

[0098] 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.

[0099] 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. The pathological changes were observed with the naked eye, and then 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 infiltration of inflammatory cells in the tissues was observed under an optical microscope.

[0100] The results showed that there were no pathological changes such as edema and congestion under naked eye observation, and no tissue inflammatory cell infiltration was observed under an optical microscope.

[0101] (V) In vitro neutralization test evaluation

[0102] 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.

[0103] 1. Material Preparation

[0104] 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.

[0105] Reagents: DMEM cell culture medium, fetal bovine serum (56°C, 30 min inactivation) and 0.125% trypsin solution

[0106] Cells, viruses and serum: Vero cells, PEDV virus and PEDV virus-related serum samples to be tested (inactivated at 56°C for 30 minutes).

[0107] 2. Operation method

[0108] (1) Cell recovery and monolayer preparation. One day in advance, two frozen primary Vero cells (purchased from Pleco) were taken out from the liquid hydrogen tank, recovered, counted, and inoculated into 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.

[0109] (2) Serum inactivation and dilution. The serum to be tested was inactivated in a 56°C water bath for 30 min and then diluted 2-fold using DMEM cell culture medium containing 5% fetal bovine serum. 2 , 2 3 , 2 4 , 2 5 , 26 .

[0110] (3) Virus neutralization. A virus solution with a known virus content (purchased from Pleco) was diluted with DMEM cell culture medium supplemented with 4% fetal bovine serum to a concentration of 2×10 3 TC1D50 / ml, in a 1.5ml EP tube, take 500μl of the diluted virus solution and mix it with 500μl of the diluted serum, set up virus control and normal cell control at the same time, and incubate at 37℃ for 1 hour.

[0111] (4) Sample addition. Take a 96-well Vero cell culture plate grown into a monolayer, discard the cell culture medium, add the neutralization sample in step (3) to each well, and repeat 4 wells for each neutralization sample. Set up normal cell control wells and virus photo wells.

[0112] (5) Replacement of medium: After adding the sample in step (4) to the well for 2 hours, all the medium is discarded and replaced with DMEM cell culture medium containing 4% fetal bovine serum, and the cells are placed in an incubator at 37°C and 5% CO2 for further culture.

[0113] (6) Cultivate and observe. Cultivate for 3-4 days and observe the situation.

[0114] Table 5 Neutralization titers of different recombinant multi-epitope fusion proteins in cell experiments

[0115]

[0116] The results are shown in Table 5. It can be seen that the best is the recombinant multi-epitope fusion protein of COE region + S1D region + M protein peptide + EcoRI + T, B cell co-stimulatory epitope region combination, followed by the recombinant multi-epitope fusion protein of M protein peptide + COE region + S1D region + EcoRI + T, B cell co-stimulatory epitope region combination.

[0117] Example 4

[0118] 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, and the corresponding new vectors obtained were used as intramolecular adjuvants.

[0119] Table 6 Intramolecular adjuvant

[0120]

[0121]

[0122] Table 7 Sequences of the main dominant components in the intramolecular adjuvant

[0123]

[0124]

[0125] The two restriction sites of BamHI and HindIII were selected on each of the above-mentioned intramolecular adjuvants for subcloning and synthesis of the optimal recombinant multi-epitope fusion protein sequence screened above (completed by BGI). After obtaining the multi-epitope fusion protein containing the intramolecular adjuvant, the same vaccine preparation and mouse immunization were carried out, and the in vitro cell virus neutralization experiment was evaluated.

[0126] Table 8 Neutralization titer of multi-epitope fusion proteins containing different intramolecular adjuvants in cell experiments

[0127] Multi-epitope fusion protein containing the following built-in adjuvants Potency Cell assay neutralization titer p2T - - p28 128000 1：10 p28ND 128000 1：16 p28FcM 128000 1：12 p28FcP 128000 1：12 p28cB 128000 1：8 p28T4 256000 1：24 p28GCN 256000 1：24 p28Fe 256000 1：36

[0128] The results showed that when the internal adjuvants were p28T4, p28GCN and p28Fe, the neutralization titer in the cell experiment was relatively high, indicating that the immunogenicity of the multi-epitope fusion protein containing the three internal adjuvants p28T4, p28GCN and p28Fe was relatively good, among which the multi-epitope fusion protein containing the internal adjuvant p28Fe had the best immunogenicity.

[0129] In summary, it can be seen that in the technical solution of the present invention, the S protein is used as the core of PEDV, and M, N antigen epitopes and intramolecular adjuvants are added through suitable linkers. Through reasonable truncation, concatenation and modification, different arrangements and combinations are performed to screen out multi-epitope fusion proteins containing intramolecular adjuvants with relatively superior potency. The vaccine prepared by the fusion protein minimizes the occurrence of ADE effect while avoiding the waste of animal immune potential and improving the immune level.

[0130] 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 recombinant multi-epitope fusion protein, characterized in that: The constituent fragments of the recombinant multi-epitope fusion protein include the S1D region of the neutralizing epitope region of the PEDV S protein, the COE region of the core neutralizing epitope region of the S1 region of the PEDV S protein, and the PEDVM protein neutralizing epitope peptide segment; The amino acid sequence of the S1D region of the neutralizing epitope region of the PEDV S protein is shown in SEQ ID NO: 1, the amino acid sequence of the COE region of the core neutralizing epitope region of the S1 region of the PEDV S protein is shown in SEQ ID NO: 2, and the amino acid sequence of the neutralizing epitope peptide segment of the PEDV M protein is shown in SEQ ID NO:

3.

2. The recombinant multi-epitope fusion protein according to claim 1, characterized in that: It also includes T and B cell co-stimulatory epitope regions, and the amino acid sequence of the T and B cell co-stimulatory epitope regions is shown in SEQ ID NO:

4.

3. The recombinant multi-epitope fusion protein according to claim 1, characterized in that: It also includes a B cell epitope region and a T cell epitope region. The amino acid sequence of the B cell epitope region is shown in SEQ ID NO:5, and the amino acid sequence of the T cell epitope region is shown in SEQ ID NO:

6.

4. A multi-epitope fusion protein containing an internal adjuvant, characterized in that: The invention comprises the recombinant multi-epitope fusion protein according to any one of claims 1 to 3 and an intramolecular adjuvant.

5. The multi-epitope fusion protein containing an internal adjuvant according to claim 4, characterized in that: The intramolecular adjuvant includes any one of a p22T vector carrying Pelb and Tufisn, a p28T vector carrying PT8A and Tufisn, a p28ND vector carrying flagellin CD0 / 1 and flagellin ND0 / 1, a p28fcM vector carrying a mouse Fc fragment and Tufisn, a p28FcP vector carrying a porcine Fc fragment and Tufisn, a p28cTB vector carrying CTB and Tufisn, a p28T4 vector carrying T4-Fd and Tufisn, a p28GCN vector carrying GCN4 and Tufisn, and a p28Fe vector carrying ferritin and Tufisn.

6. The multi-epitope fusion protein containing an internal adjuvant according to claim 5, characterized in that: The amino acid sequence of the Pelb is shown in SEQ ID NO:13; the amino acid sequence of the Tufisn is shown in SEQ ID NO:14; the amino acid sequence of the PT8A is shown in SEQ ID NO:15; the amino acid sequence of the flagellin CD0 / 1 is shown in SEQ ID NO:16; the amino acid sequence of the flagellin ND0 / 1 is shown in SEQ ID NO:17; the amino acid sequence of the polymer peptide T4-Fd is shown in SEQ ID NO:18; the amino acid sequence of the polymer peptide GCN4 is shown in SEQ ID NO:19; the amino acid sequence of the CTB protein is shown in SEQ ID NO:20; the amino acid sequence of the ferritin is shown in SEQ ID NO:21; the amino acid sequence of the mouse Fc fragment is shown in SEQ ID NO:22; the amino acid sequence of the porcine Fc fragment is shown in SEQID NO:

23.

7. Use of the multi-epitope fusion protein containing an intramolecular adjuvant according to claim 4 in the preparation of a porcine epidemic diarrhea virus vaccine.

8. A porcine epidemic diarrhea virus subunit vaccine, characterized in that: It comprises the multi-epitope fusion protein containing an intramolecular adjuvant as claimed in claim 4 and a pharmaceutically acceptable adjuvant.

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