Recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein as well as preparation method and application thereof

Abstract

The invention relates to the technical field of animal vaccines, in particular to a recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein as well as a preparation method and application thereof. According to the present invention, the SpyTag sequence is inserted into the S1 gene sequence, the eukaryotic cell HEK293 is adopted to express the protein, such that the obtained recombinant S1 protein has sufficient glycosylation modification, and when the recombinant S1 protein is mixed with the recombinant transferrin containing the SpyCatcher sequence, the K31 on the SpyTag and the N117 on the SpyCatcher are subjected to spontaneous condensation to form the isomeric peptide so as to display the antigen on the transferrin 24 polymer surface; the technical system not only maintains the natural conformation of the virus antigen protein, but also has the advantage of stimulating the innate and adaptive immune response functions of the host, and has a better protection effect on the host.

Description

Technical Field

[0001] The present invention relates to the field of animal vaccine technology, and more specifically to a recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein, and a preparation method and application thereof. Background Art

[0002] Porcine Epidemic Diarrhea (PED) is a highly contagious acute intestinal disease caused by Porcine Epidemic Diarrhea Virus (PEDV). The disease develops rapidly and is accompanied by clinical symptoms such as severe diarrhea, vomiting and dehydration. PEDV can infect pigs of all ages, especially suckling piglets, causing nearly 100% mortality, causing significant economic losses to the pig industry.

[0003] PEDV is an enveloped, single-stranded, positive-strand RNA virus belonging to the genus Alphacoronavirus of the family Coronaviridae. The PEDV genome is approximately 28 kb long and encodes a variety of nonstructural replicase proteins and four major structural proteins, including spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), and auxiliary open reading frame 3 (ORF3) between S and E. The S protein of PEDV is divided into two subunits, S1 and S2. S1 is located on the surface of the virus and its main function is to recognize receptors and bind to host cell receptors and mediate the production of neutralizing antibodies. S2 is mainly responsible for the fusion of the viral envelope with the host cell membrane and the introduction of viral RNA into the host cell, thereby causing cell infection. Therefore, the correct expression and sufficient glycosylation modification of the PEDV S protein are major challenges in the development of genetically engineered subunit vaccines.

[0004] The vaccines used to control and prevent PEDV in the domestic market are basically PEDV attenuated live vaccines and inactivated vaccines. Although inactivated vaccines have many advantages, such as good safety. However, the immunogenicity of inactivated vaccines may be destroyed during the inactivation process. Attenuated live vaccines are highly immunogenic, and a single immunization is usually sufficient to induce protective immunity. However, attenuated live vaccines have the potential risk of toxic recovery, and may change from the original attenuated strain to the in vivo strain. The vaccine recipient may become ill due to vaccination, which limits its applicability. The key to developing a vaccine is to select strains with low toxicity, high titer and high immunogenicity. However, it is very difficult to isolate PEDV strains in vitro, especially for pandemic variable strains. Even if the strains are successfully isolated, they may not have high titers. In recent years, with the development of molecular biology technology, the research direction of PEDV vaccines has begun to turn to genetically engineered vaccines, which have significant advantages such as safety, high efficiency, few side effects, and industrial production. However, how to improve the effectiveness of subunit antigens is the current research direction (selection of key antigens, selection of suitable expression systems, application of virus-like particle technology, development of subunit antigen supporting immune enhancers and adjuvants, etc.). At present, the main expression systems for the preparation of S protein subunits are prokaryotic, insect baculovirus system and eukaryotic expression system, such as the invention patent with application number 201610348237.8 uses insect baculovirus to express S1 region 21-789 protein, and the patent with application number 201610256701.0 uses Escherichia coli to express the three core regions of S protein in series. The expression products of the prokaryotic expression system have the problem that the structure cannot guarantee complete folding and the glycosylation modification is imperfect, so that the immunogenicity of the obtained protein is lower than that of the virus particles themselves, resulting in insufficient protection. Although the product of the baculovirus expression system has better protein folding, its glycosylation modification is still imperfect. Therefore, eukaryotic cell expression is still the first choice.

[0005] Based on the knowledge of the structure and function of coronavirus, the basic research of diarrhea S protein has been promoted. Although S protein forms a trimer by entanglement of C-terminal sequences, it is generally believed that the N-terminal (S1) part is the key antigenic region. Therefore, the correct expression of S1 and the formation of virus-like particles (VLP) on this basis may obtain better immunogenicity.

[0006] Transferrin can spontaneously form 24-mers, and display S1 protein on the surface of the 24-mers, which may improve immunogenicity. Based on this hypothesis, the present invention has conducted further research and verified its immunogenicity in animals. Summary of the invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one aspect of the present invention aims to provide a recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein, wherein the amino acid sequence of the recombinant porcine epidemic diarrhea virus S1 protein is derived from the S1 subunit of the porcine epidemic diarrhea virus S protein (the full length of the S protein is separated and sequenced in this experiment, NCBI accession number: PP083729.1, the amino acid sequence of the S1 part is 1-734, (SEQ ID No.1), signal peptide amino acid sequence (SEQ ID No.2), 6*His tag sequence (SEQID No.3), Linker sequence (SEQ IDNo.4) and SpyTag sequence (SEQ ID No.5, reference PDB number: 4MLI_B), the amino acid sequence of the recombinant porcine epidemic diarrhea virus S1 protein (S1-CS) is SEQ ID No.6; the transferrin fusion protein amino acid sequence is derived from Helicobacter pylori (SEQ ID No.8, reference UniProt: Q9ZLI1), Enterokinase Site sequence (SEQID No.9), Linker sequence (SEQ ID No.10), SpyCatcher sequence (SEQ ID No.11, refer to GeneBank accession number: QGX07219.1), and the amino acid sequence of the transferrin fusion protein is SEQ ID No.12.

[0008] Preferably, the nucleic acid encoding the recombinant porcine epidemic diarrhea virus S1 protein can be obtained according to the codon rules and can be codon optimized according to the preference of the host cell. The preferred nucleotide sequence is shown in SEQ ID No.7.

[0009] Preferably, the encoding nucleic acid of the recombinant transferrin (SpyCatcher-Ferritin) can be obtained according to the codon rules and can be codon optimized according to the preference of the host cell; the preferred nucleotide sequence is shown in SEQ ID No.13.

[0010] Another aspect of the present invention is to provide a method for preparing a recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein, wherein the specific steps of the preparation method are as follows: S1. The coding gene of the recombinant porcine epidemic diarrhea virus S1 protein was cloned into the expression vector pCDNA3.1 to obtain the recombinant expression vector pCDNA3.1-S1-CS, and the recombinant expression vector pCDNA3.1-S1-CS was transiently transferred into eukaryotic cells by chemical transfection reagent, and the cell supernatant was collected by centrifugation and purified using a Bogelon 5 mL nickel column to obtain the recombinant porcine epidemic diarrhea virus S1 protein (S1-CS); S2. The transferrin encoding nucleic acid is cloned into the expression vector pET21a to obtain the recombinant expression vector pET21a-SpyCatcher-Ferritin, and the recombinant expression vector pET21a-SpyCatcher-Ferritin is transformed into the Escherichia coli host cell to obtain the recombinant genetically engineered bacteria, and the bacterial sludge is collected by centrifugation after induction of expression, and the bacterial sludge is resuspended and ultrasonically broken, and the supernatant is centrifuged and purified using a Bogelon 5 mL nickel column to obtain the recombinant transferrin (SpyCatcher-Ferritin); S3. Prepare recombinant transferrin SpyCatcher-Ferritin and recombinant porcine epidemic diarrhea virus S1 protein (S1-CS) in a molar ratio, mix well after preparation, place at 25±2℃, and assemble for 6-8h to prepare recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein S1-F-VLP.

[0011] Preferably, the nucleic acid sequence encoding the recombinant porcine epidemic diarrhea virus S1 protein in S1 is SEQ ID No.7.

[0012] Preferably, the recombinant expression vector in S1 is pcDNA3.1, pEE17 or pks001, the transfection reagent is PEI, the eukaryotic cells are HEK293 cells or CHO cells, and the culture time is 5-16 days.

[0013] Preferably, the centrifugation conditions in S1 are first at 4°C, 1000 rpm for 10 min, and then at 4°C, 8000 rpm for 30 min.

[0014] Preferably, the encoding nucleic acid sequence of the recombinant transferrin in S2 is SEQ ID No.13; the expression vector is an Escherichia coli expression vector; more preferably, it is an Escherichia coli expression vector containing a T7 promoter; more preferably, it is a pET series expression vector; and most preferably, it is a pET21a vector.

[0015] Preferably, the molar ratio in S3 is 1-5:1-5, the temperature is 25° C., and the assembly time is 6 h.

[0016] Preferably, the recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein S1-F-VLP are used as subunit vaccines in the field of animal vaccines.

[0017] The beneficial effects of the present invention are as follows: The present invention inserts the SpyTag sequence into the S1 gene sequence and uses eukaryotic cells HEK293 to express the protein, so that the obtained recombinant S1 protein has sufficient glycosylation modification, and when it is mixed with recombinant transferrin containing the SpyCatcher sequence, K31 on the SpyTag and N117 on the SpyCatcher (based on Streptococcus pyogenes The FbaB protein sequence encoded by the transferrin protein (encoded by the FbaB protein sequence) spontaneously condenses to form isomeric peptides, thereby displaying the antigen on the surface of the transferrin 24-mer. This technical system not only maintains the natural conformation of the viral antigen protein, but also has the advantages of stimulating the host's innate and adaptive immune response functions, and has a good protective effect on the host.

[0018] Additional aspects and advantages of the invention will become apparent from the following description, or may be learned by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 Schematic diagram of the sequence distribution of PEDV-S protein in Example 1 of the present invention; Figure 2 This is a schematic diagram of the S1-CS vector map and gene sequence of Example 2 of the present invention; Figure 3 This is a schematic diagram of the SpyCatcher-Ferritin vector map and gene sequence of Example 2 of the present invention; Figure 4 This is the chromatogram and electrophoresis diagram of the S1-CS protein purification in Example 3 of the present invention; Figure 5 This is the SpyCatcher-Ferritin purification chromatogram and electrophoretogram of Example 3 of the present invention; Figure 6 This is the electrophoresis diagram of the VLP assembly process development of SpyCatcher-Ferritin and S1-CS protein in Example 4 of the present invention; Figure 7 This is the SEC-HPLC verification of Example 5 of the present invention before and after the VLP assembly of SpyCatcher-Ferritin and S1-CS protein; Figure 8 This is an electron microscopic verification image of the SpyCatcher-Ferritin and S1-CS protein before and after VLP assembly in Example 6 of the present invention; Fig. 9 This is a diagram showing the process and clinical manifestations of immunizing animals with the S1-F-VLP vaccine according to Example 7 of the present invention; Fig.10This is an anatomical diagram of the animal experiment on animals immunized with the S1-F-VLP vaccine according to Example 7 of the present invention. DETAILED DESCRIPTION

[0020] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0021] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from the description. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0022] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment. Unless otherwise specified, the materials and reagents used in the present invention are commercially available. Among them, the cell line HEK293 cells used in the present invention were purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd., and pcDNA3.1 was stored by this laboratory.

[0023] Example 1: Selection of PEDV-S1 protein gene The virus strain of the S protein of the present invention is GⅡ type, the coding gene sequence of the PEDV-S protein is sequenced based on the highly pathogenic PEDV virus isolated by the present invention and registered in GeneBank, accession number: PP083729.1, the S1 protein is 1-734aa, such as Figure 1 shown.

[0024] Example 2: Construction of S1-CS and SpyCatcher-Ferritin 2.1 A signal peptide was introduced at the N-terminus of the PEDV-S1 sequence, and a linker, SpyTag sequence and 6*His tag were introduced at the C-terminus. The codon-optimized gene was inserted into the pcDNA3.1 vector via HindIII / EcoRI by chemical synthesis. The constructed expression vector was named pcDNA3.1-S1-CS. Figure 2 As shown, the left figure is the recombinant expression vector, and the right figure is the amino acid sequence of the target protein. The recombinant expression vector was transformed into DH5α Escherichia coli to obtain cloned bacteria.

[0025] 2.2 Refer to UniProt: Q9ZLI1, select the target sequence Ferritin, add 6*His tag, Enterokinase Site, Linker, SpyCatcher sequence at the N-terminus, insert the codon-optimized gene into the pET21a expression vector through NdeI / XhoI by chemical synthesis, and the constructed expression vector is named pET21a-SpyCatcher-Ferritin, as shown in Figure 3 As shown, the left figure is the recombinant expression vector, and the right figure is the amino acid sequence of the target protein. The recombinant expression vector was transformed into Escherichia coli BL21 (DE3) to obtain the expression strain.

[0026] Example 3: Expression and purification of S1-CS and SpyCatcher-Ferritin 3.1 Expression and purification of S1-CS 3.1.1 Plasmid mass extraction The pcDNA3.1-S1-CS strain was selected and inoculated into 150 mL LB liquid medium containing 100 μg / mL ampicillin sodium, and cultured overnight at 250 rpm and 37 °C in a shaking incubator. All bacterial liquid was used for plasmid extraction, and the plasmid extraction steps were carried out according to the instructions of the Tiangen Endotoxin-Free Plasmid Extraction Kit (Enhanced). The recovered plasmid was measured for concentration using Nano drop, and will be used for transient transfection of HEK293 cells later.

[0027] 3.1.2 Cell passaging HEK293 cells were cultured in a 125 mL shake flask using Wayne293 medium at a cell density of 0.7 × 10 6 cells / mL for subculture (temperature: 37°C, CO2 concentration: 5%, rotation speed: 125rpm), and can be used for transfection with transfection reagent PEI after one day of culture.

[0028] 3.1.3 Transient cell transfection When the cell density of HEK293 cells reached 1.3×10 6 ~1.7×10 6When the concentration of cells / mL was 100 μg, the plasmid was transiently transfected. 1 bottle of PEI transfected pcDNA3.1-S1-CS plasmid, 30 mL per bottle, prepared transfection solution (Solution A: 30 μg plasmid + 900 μL 1×PBS solution, mixed and let stand for 5 min; Solution B: 120 μL PEI + 900 μL 1×PBS solution, mixed and let stand for 5 min), added Solution B to Solution A, mixed, and let stand for 10 min. Add the mixed solution dropwise to the cell solution, shake gently, and add 600 μL of 200 mM glutamine solution. After transfection, the cells were cultured in a carbon dioxide shaking incubator (temperature: 37°C, CO2 concentration: 5%, speed: 125 rpm). The cells were counted on the 1st, 3rd, 4th and 5th days of culture. On the 3rd day, 200 mM glutamine was added. When the viability was lower than 70%, the cells were centrifuged at 4°C, 1000 rpm for 10 min to remove the cells and the supernatant was collected. The supernatant was centrifuged at 4°C, 8000 rpm for 30 min to remove the cell debris and the supernatant was collected again. The supernatant was filtered with a 0.45 μm filter membrane and set aside.

[0029] 3.1.4 Protein purification Use a 5 mL empty column tube from Boglund and pack the column with Boglund Ni Bestarose FF filler.

[0030] Column equilibration: After equilibration for 3 column volumes with purified water at a flow rate of 5 mL / min, equilibrate again for 2 to 3 column volumes with Buffer A (20 mM PB + 150 mM NaCl, pH = 7.4).

[0031] Sample loading: Load the sample at a flow rate of 5 mL / min, with a sample volume of 400 mL, and collect the flow-through liquid at the same time.

[0032] Washing: Wash the column with 5% Buffer B (20mM PB + 150mM NaCl + 0.5M imidazole, pH = 7.4) at a flow rate of 5mL / min to remove unbound proteins and weakly bound proteins until OD 280nm until the baseline is stable.

[0033] Elution: flow rate 5 mL / min, time 20 min, use 5%-100% Buffer B (20 mM PB + 150 mM NaCl + 0.5 M imidazole, pH = 7.4) buffer to linearly elute the target protein for 20 column volumes, and collect the eluate at 15 mL / tube.

[0034] Filler washing: 5 mL / min flow rate, 5 column volumes, 100% Buffer B (20 mM PB + 150 mM NaCl + 0.5 M imidazole, pH = 7.4) to wash the filler.

[0035] Desalting and replacing buffer: The target protein solution containing imidazole collected above was desalted to pH=7.4, 1× PBS buffer through G25 dextran gel particles. Figure 4 As shown, the left picture is the purification chromatogram, and the process is loading (impurities will flow through at the same time as loading), washing (using low concentration imidazole to further remove impurities), elution (the target protein appears at this stage), the target protein is located between the two black dotted lines, the blue solid line is the protein spectrum absorption value, and the green dotted line is the percentage of imidazole buffer. The right picture is the electrophoresis diagram, in which the 1st lane is the protein Marker (ThermoFisher #26616), the 2nd lane is the stock solution, the 3rd lane FT is the flow-through solution, the 4th lane is a 5% imidazole buffer for washing, and the 5-8th lanes are the elution collection solution, which is the required target protein S1-CS. The S1-CS protein was successfully purified.

[0036] 3.2 Expression and purification of SpyCatcher-Ferritin 3.2.1 Bacterial activation Activate the BL21 expression strain of pET21a-SpyCatcher-Ferritin. Take the stored glycerol stock and inoculate it in a 125 mL shake flask at a ratio of 1:1000. Culture it in LB medium (final concentration of 50 μg / mL Kan + ), culture volume 25mL, placed in a shaker at 37℃, 220rpm overnight culture.

[0037] 3.2.2 Protein expression Take the bacterial solution from 3.2.1 and inoculate it into a 3L shake flask at a ratio of 0.1% and culture it in LB medium (final concentration of 50 μg / mL Kan + The culture volume was 1 L and the culture was shaken at 37°C and 220 rpm for 4 h until OD 600nm =0.6-0.8, add inducer IPTG (final concentration is 1mM / L) and induce for 6-8 hours.

[0038] 3.2.3 Protein purification The overnight induced culture was centrifuged at 4°C, 8000 rpm for 30 min to collect the bacterial sludge. The bacterial sludge was re-dissolved with Buffer A (20 mM Tris, 150 mM NaCl, pH = 7.4) at a ratio of 1:10, and ultrasonicated (power: 220 W, working 5 s, interval 3 s) for 30 min, then centrifuged at 4°C, 8000 rpm for 30 min to obtain the supernatant, which was then filtered through a 0.45 μm filter membrane for purification.

[0039] The column was packed using a Boglund 5 mL column tube and Boglund Ni Bestarose FF filler.

[0040] Column equilibration: Equilibrate with purified water for 3 column volumes at a flow rate of 8 mL / min, and then equilibrate with Buffer A (20 mM Tris, 150 mM NaCl, pH = 7.4) for 2 to 3 column volumes.

[0041] Sample loading: Load the sample at a flow rate of 5 mL / min, with a sample volume of 400 mL, and collect the flow-through.

[0042] Washing: Wash the column with 5% Buffer B (20 mM PB + 150 mM NaCl + 0.5 M imidazole, pH = 7.4) at a flow rate of 8 mL / min until OD 280nm until the baseline is stable.

[0043] Elution: linearly elute the target protein with 5%-100% Buffer B (20mM PB+150mM NaCl+0.5M imidazole, pH=7.4) buffer for 20 column volumes at a flow rate of 8mL / min for 12.5min, and collect the eluate at 15mL / tube.

[0044] Washing: The flow rate was 8 mL / min, and 100% Buffer B (20 mM PB + 150 mM NaCl + 0.5 M imidazole, pH = 7.4) was used to wash 5 column volumes to clean the filler.

[0045] Desalting and replacing buffer: The above target protein solution containing imidazole was desalted through G25 dextran gel particles into 1× PBS buffer with a pH of 7.4. Figure 5 As shown, the left picture is the purification chromatogram, the process is loading (impurities will flow through at the same time of loading), washing (using low concentration imidazole to further remove impurities), elution (the target protein appears at this stage), the target protein is located between the two black dotted lines, the blue solid line is the protein spectrum absorption value, the green dotted line is the percentage of imidazole buffer, and the right picture is the electrophoresis diagram, among which, lane 1 is the stock solution, lane 2 FT is the flow-through solution, lane 3 is the protein Marker (Thermo Fisher #26616), lane 4 is a 5% imidazole buffer for washing, and lanes 5-7 are elution collection solutions, which are the desired target protein SpyCatcher-Ferritin, and the SpyCatcher-Ferritin protein is successfully purified.

[0046] Example 4: Assembly of S1-CS and SpyCatcher-Ferritin SpyCatcher-Ferritin: S1-CS were prepared in molar ratios of 1:1, 1:2, 1:3, 1:5, 2:1, and 5:1. After preparation, the mixture was mixed upside down and placed at 25°C for 6-8 hours. The results were as follows: Figure 6 As shown, the first lane is a protein marker (Thermo The 2nd lane is SpyCatcher-ferritin, the 3rd lane is S1-CS, and the 4th to 9th lanes are SpyCatcher-ferritin: S1-CS molar ratios of 1:1, 1:2, 1:3, 1:5, 2:1, and 5:1, respectively. Among them, the SpyCatcher-ferritin: S1-CS molar ratios of 1:2 and 1:1 have the highest assembly rate compared with other groups of data. It can be seen from the electrophoresis bands that the size of the assembled VLP band is slightly larger than that of S1-CS itself, because the size of the assembled VLP is equal to the sum of the size of S1-CS and the size of SpyCatcher-ferritin. At the same time, the consumption of S1-CS and SpyCatcher-ferritin proteins should also be observed. The ideal state is that both are completely consumed and assembled into VLPs, and the assembly rate is 100%. The SpyCatcher-ferritin: S1-CS molar ratios of 1:2 and 1:1 are closest to the ideal state.

[0047] Example 5: SEC-HPLC detection of S1-F-VLP The results are as follows Figure 7As shown in the results of Example 4, the electrophoretic band assembly rates of the molar ratios of SpyCatcher-Ferritin: S1-CS of 1:2 and 1:1 are higher than those of other groups, so S1-F-VLPs with molar ratios of 1:2 and 1:1 were selected for SEC-HPLC detection. From the results, it can be seen that SpyCatcher-Ferritin: S1-CS = 1:1 is compared with SpyCatcher-Ferritin: S1-CS = 1:2, SpyCatcher-Ferritin: S1-CS = 1:1 has basically only one main peak , there are no other impurities, the retention time of the main peak is 10.535, which is close to the retention time of SpyCatcher-Ferritin 10.853, SpyCatcher-Ferritin: S1-CS = 1:2 has two peaks, the retention time of the first main peak is 10.468, which is close to the retention time of SpyCatcher-Ferritin 10.853, and the retention time of the second impurity peak (the machine does not mark the retention time of the second impurity peak, just look at the position) is close to the retention time of S1-CS 14.459, indicating that there is residual S1-CS. Therefore, at the SEC-HPLC level, it can also be explained that SpyCatcher-Ferritin: S1-CS = 1:1 has a high assembly rate and the two are consumed relatively cleanly.

[0048] The HPLC detection method is briefly described as follows: Sample volume: 800 μL, buffer: 50 mM Tris, 300 mM NaCl, pH = 8.0; chromatographic column: Saifen SRT-C SEC-300 molecular sieve 5 μm, 7.8×300 mm, CV = 14.33 mL; column temperature: 25°C; 0.5 mL / min, sample loading time: 35 min.

[0049] Example 6: Scanning electron microscopy (TEM) detection of S1-F-VLP The results are as follows Figure 8 As shown, according to the results of Example 5, the assembly rate of SpyCatcher-Ferritin: S1-CS = 1:1 is the highest, and the residual amount of both is small, so the assembly of this ratio can achieve the best assembly effect. The sample was taken for electron microscopy observation, and it can be seen at both 200nm and 100nm that the diameter of S1-F-VLP is larger than SpyCatcher-Ferritin, indicating that S1-CS protein has been carried around SpyCatcher-Ferritin, so its successful assembly can also be verified at the electron microscopy level.

[0050] 6.1 Sample staining Take 10 μL of S1-F-VLP (SpyCatcher-Ferritin: S1-CS is 1:1) sample and drop it on the copper grid. Stain the sample with 1% phosphotungstic acid, wash it, and dry it at room temperature.

[0051] 6.2 Instrument Debugging Install the sample and sample rod, open the valve, set the concentric height, and adjust the optical core to the appropriate level.

[0052] 6.3 Sample photography and end of operation Adjust the magnification, select the observation area, perform the focusing operation, take multiple photos at different magnifications, end the photo shoot, reduce the magnification, diffuse the light, close the valve, and return the sample rod to its original position.

[0053] Example 7: Animal Experiment Verification of S1-F-VLP 7.1 Immunization of pregnant sows Nine sows 30-40 days before delivery with negative PEDV antigen and antibody were selected and divided into three groups: experimental vaccine group, blank control group, and challenge control group. Three sows in each group were injected for immunization.

[0054] Experimental vaccine group: S1-F-VLP protein 50 μg / head, adjuvant 1313+GEL, vaccine preparation method was hand-shaking emulsification.

[0055] The challenge control group: no immunization, the challenge group.

[0056] Blank control group: no immunization, blank control group Immunization method: intramuscular injection, one vaccination 30 days before delivery, and the second vaccination 14 days after the first vaccination. Fig. 9 shown.

[0057] 7.2 Piglet challenge protection verification The piglets were breastfed for 5-7 days, and 2-4 piglets were randomly selected from each sow for infection. The virus strain was PEDV-0221 (stored in this laboratory), 2 mL / pig, and oral administration. The growth of the piglets was observed for 10-14 days after infection. Anal swabs were collected from the sixth day after infection for follow-up research. The process results are as follows Fig. 9 As shown, records were made every day. The results are shown in Table 1 below. The experimental vaccine group became ill but had no deaths; the challenge control group became ill and died; the blank control group did not become ill or die. One of the animals in the blank control group died unexpectedly due to objective factors and was not included in the overall experimental data. It can be seen from the above experimental data that the vaccine can achieve good protection against PEDV virus.

[0058] After the whole experiment was completed, the piglets were dissected to observe the pathological changes of their internal organs. Fig.10As shown, visual observation of the intestinal organs of the experimental vaccine group and the blank control group was basically the same, which was normal, while the intestines of the challenge control group were yellow, even congested and black, which are typical symptoms of PEDV infection. Therefore, the conclusion that the experimental vaccine group can protect against PEDV can also be drawn from the level of organ lesions.

[0059] Combined with the growth and survival of piglets and anatomical organ lesions, the S1-F-VLP obtained in the present invention can achieve complete protection against PEDV challenge.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may also have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein, characterized in that: The amino acid sequence of the recombinant porcine epidemic diarrhea virus S1 protein is derived from the S1 subunit, signal peptide amino acid sequence, 6*His tag sequence, Linker sequence and SpyTag sequence of the porcine epidemic diarrhea virus S protein, and the amino acid sequence of the recombinant porcine epidemic diarrhea virus S1 protein is SEQ ID No.6; the amino acid sequence of the transferrin fusion protein is derived from Helicobacter pylori, Enterokinase Site sequence, SpyCatcher sequence, and the amino acid sequence of the transferrin fusion protein is SEQ ID No.

12.

2. The method for preparing a recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein according to claim 1, characterized in that: The specific steps of the preparation method are as follows: S1. The coding gene of the recombinant porcine epidemic diarrhea virus S1 protein was cloned into the expression vector pCDNA3.1 to obtain the recombinant expression vector pCDNA3.1-S1-CS, and the recombinant expression vector pCDNA3.1-S1-CS was transiently transferred into eukaryotic cells by chemical transfection reagent, and the cell supernatant was collected by centrifugation and purified by using a Bogelon 5 mL nickel column to obtain the recombinant porcine epidemic diarrhea virus S1 protein; S2. The transferrin encoding nucleic acid is cloned into the expression vector pET21a to obtain the recombinant expression vector pET21a-SpyCatcher-Ferritin, and the recombinant expression vector pET21a-SpyCatcher-Ferritin is transformed into the Escherichia coli host cell to obtain the recombinant genetically engineered bacteria, and the bacterial sludge is collected by centrifugation after inducing expression, and the bacterial sludge is resuspended and ultrasonically broken, and the supernatant is centrifuged and purified using a Bogelon 5mL nickel column to obtain the recombinant transferrin; S3. Prepare recombinant transferrin and recombinant porcine epidemic diarrhea virus S1 protein in a molar ratio, mix well after preparation, place at 25±2℃, and assemble for 6-8h to prepare recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein.

3. The method for preparing a recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein according to claim 2, characterized in that: The nucleic acid sequence encoding the recombinant porcine epidemic diarrhea virus S1 protein in the S1 is SEQ ID No.

7.

4. The method for preparing a recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein according to claim 2, characterized in that: The recombinant expression vector in S1 is pcDNA3.1, pEE17 or pks001, the transfection reagent is PEI, the eukaryotic cells are HEK293 cells or CHO cells, and the culture time is 5-16 days.

5. The method for preparing a recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein according to claim 2, characterized in that: The centrifugation conditions in S1 are first 4° C., 1000 rpm for 10 min, and then 4° C., 8000 rpm for 30 min.

6. The method for preparing a recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein according to claim 2, characterized in that: The encoding nucleic acid sequence of the recombinant transferrin in S2 is SEQ ID No. 13; and the expression vector is an Escherichia coli expression vector.

7. The method for preparing a recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein according to claim 2, characterized in that: The molar ratio in S3 is 1-5:1-5, the temperature is 25° C., and the assembly time is 6 hours.

8. The use of a recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein according to claim 1, characterized in that: The recombinant porcine epidemic diarrhea virus S1 protein and transferrin fusion protein are used as subunit vaccines in the field of animal vaccines.

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