Preparation method and application of PEDV COE subunit vaccine targeting M cells

Abstract

The application discloses a preparation method of a PEDV COE subunit vaccine targeting M cells and application thereof. The application connects RGD peptide targeting M cells, a core neutralizing epitope COE region on a PEDV S protein and a fluorescent protein, expresses a recombinant protein, introduces a sulfur-containing amino acid into the protein to facilitate modification of the recombinant protein and connection of the protein to other substances in a covalent bond mode, and obtains a safe and effective mucosal subunit vaccine with green fluorescent labeling. Unlike common vaccines, the antigen of the vaccine can not only be directly taken by dendritic cells, but also be presented to dendritic cells through targeting M cells, the antigen presentation efficiency is significantly improved, the downstream immunity is more effectively started, and a series of immune responses of the body are induced, so that the mucosal immune system of the body produces a large number of PEDV specific antibodies to resist invasion of external pathogens, and the effect of specific immune protection is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing a PEDV COE subunit vaccine targeting M cells and its application. Background Technology

[0002] Porcine epidemic diarrhea (PED) is an intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). The virus invades the body through the respiratory and digestive tract mucosa, mainly manifesting as diarrhea, vomiting, and dehydration. There is currently no specific antidote for PED; vaccination is the primary means of prevention and control. Unlike the classic G1 strain, the predominantly circulating strain in my country is the G2 strain, which has higher pathogenicity. However, most currently used inactivated or attenuated vaccines, administered subcutaneously, intramuscularly, or via the Houhai acupoint, cannot directly induce a highly effective mucosal immune response to resist PEDV infection via the respiratory and digestive tract mucosa. Therefore, there is an urgent need to develop novel mucosal vaccines with good safety and immunogenicity.

[0003] There is currently no specific treatment for PED. Prevention is usually achieved through inactivated or attenuated vaccines. However, inactivated PEDV vaccines carry viral nucleic acid, posing certain safety risks. The preparation of attenuated vaccines involves a degree of randomness in selecting suitable vaccine strains and requires systematic validation, which is too costly and prone to reversion, posing a potential risk of infection. Summary of the Invention

[0004] The first aspect of the present invention is to provide a fusion protein.

[0005] A second aspect of the present invention aims to provide biomaterials related to the fusion protein described in the first aspect of the present invention.

[0006] The third aspect of this invention aims to provide the application of the fusion protein described in the first aspect of this invention or the biomaterial described in the second aspect of this invention.

[0007] The fourth aspect of this invention is to provide a porcine epidemic diarrhea virus vaccine.

[0008] The fifth aspect of this invention is to provide a method for preparing the vaccine described in the fourth aspect of this invention.

[0009] The sixth aspect of this invention aims to provide a method for preparing an antibody.

[0010] The technical solution adopted in this invention is:

[0011] In a first aspect, the present invention provides a fusion protein comprising a PEDV structural protein and an M-cell targeting peptide.

[0012] Preferably, the PEDV structural protein includes the PEDV S protein.

[0013] Preferably, the PEDV structural protein includes the COE region of the PEDV S protein.

[0014] Preferably, the amino acid sequence of the core neutralizing epitope of the PEDV S protein is shown in SEQ ID NO.1.

[0015] Preferably, the gene encoding the core neutralizing epitope of the PEDV S protein is shown in SEQ ID NO.2.

[0016] Preferably, the amino acid sequence of the M cell-targeting peptide is shown in SEQ ID NO.6.

[0017] Preferably, the fusion protein includes one or more M-cell-targeting peptides.

[0018] M cells are specialized antigen-transporting cells in the mucosal immune system, scattered among mucosal epithelial cells. M cells are also known as microfolded cells because of the numerous microfolds on their surface. These folds allow for better capture of intraluminal antigens reaching the apical surface of the M cell. Through uptake, processing, and presentation by antigen-presenting cells (APCs), the antigens are transported to downstream lymphoid tissues and induce a mucosal immune response. The basal base of the M cell forms a pocket-like depression, which is highly active in phagocytosis of antigens and transports them to dendritic cells for further processing and induction of a series of mucosal immune responses. Arginine-glycine-aspartic peptide (RGD peptide) is a peptide capable of effective cell adhesion, which can be used to locate or target specific cells. It binds to integrin β1, which is highly expressed on the surface of M cells; therefore, this property can be used to target M cells.

[0019] Preferably, the fusion protein further includes an optional tag protein sequence to assist in expression and / or purification.

[0020] Preferably, the optional tag protein sequences for assisting expression and / or purification and / or detection include, but are not limited to, (His)6, GST, MBP, FLAG, HA, Strep, GFP, and mcherry tag protein sequences.

[0021] Preferably, the Strep-tagged protein sequence is shown in SEQ ID NO.3.

[0022] Preferably, the GFP tag protein includes red fluorescent protein, green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, and orange fluorescent protein.

[0023] Preferably, the amino acid sequence of the green fluorescent protein is shown in SEQ ID NO.4.

[0024] Preferably, the tag protein sequence is a tag protein sequence containing cysteine.

[0025] Methionine (Met) and cysteine ​​(Cys) are the only two naturally occurring sulfur-containing amino acids. Sulfur atoms play crucial roles in catalysis, metal bonding, redox regulation, and other post-translational modifications. Cysteine, in particular, has a highly reactive thiol group (-SH) on its side chain. The hydrogen atom in this group can be easily replaced by free radicals and other groups, thus readily forming covalent bonds with other molecules and providing favorable prerequisites for subsequent research on protein modification and processing.

[0026] Preferably, the amino acid sequence of the fusion protein is as follows:

[0027] a1)SEQ ID NO.8; or

[0028] a2) An amino acid sequence of SEQ ID NO. 8 with one or more amino acid substitutions and / or deletions and / or additions that have the same function as the protein shown in SEQ ID NO. 8; or

[0029] a3) An amino acid sequence that shares 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with SEQ ID NO. 8 and has the same function as the protein shown in SEQ ID NO. 8;

[0030] Preferably, the PEDV structural protein, M cell targeting peptide, and / or tag protein sequence in the fusion protein are linked by a linker peptide.

[0031] Preferably, the linker peptides include, but are not limited to, GSS, GSGGGS, GGGGS, GGGSGGSG, GGGGGGGG, GGSGGSGGSGGSGGS, GGGGSGGGGSGGGSGGGS, GSGGGSGGGGSGGGSGGGS.

[0032] A second aspect of the invention provides a biomaterial related to the fusion protein described in the first aspect of the invention, said biomaterial comprising at least one of a1) to a16):

[0033] a1) A nucleic acid molecule encoding the fusion protein described in the first aspect of the present invention;

[0034] a2) An expression cassette containing the nucleic acid molecule described in a1);

[0035] a3) A carrier containing the nucleic acid molecule described in a1);

[0036] a4) A carrier containing the expression box described in a2);

[0037] a5) Transgenic cell lines containing the nucleic acid molecules described in a1);

[0038] a6) Transgenic cell lines containing the expression cassette described in a2);

[0039] a7) A transgenic cell line containing the vector described in a3);

[0040] a8) A transgenic cell line containing the vector described in a4);

[0041] a9) Microorganisms containing the nucleic acid molecules described in a1);

[0042] a10) contains microorganisms containing the expression cassette described in a2);

[0043] a11) contains microorganisms that contain the carrier described in a3);

[0044] a12) contains microorganisms that contain the carrier described in a4);

[0045] a13) Viruses containing the nucleic acid molecules described in a1);

[0046] a14) A virus containing the expression cassette described in a2);

[0047] a15) contains a virus containing the vector described in a3);

[0048] a16) contains a virus with the vector described in a4).

[0049] Preferably, the transgenic cell line does not contain propagation material.

[0050] Preferably, the sequence of the nucleic acid molecule includes:

[0051] a1)SEQ ID NO.7; or

[0052] a2) A nucleic acid molecule that has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with SEQ ID NO.7 and encodes the fusion protein.

[0053] A third aspect of the invention provides the use of the fusion protein described in the first aspect of the invention or the biomaterial described in the second aspect of the invention in any of the following:

[0054] b1) Prepare drugs for the prevention and / or treatment of porcine epidemic diarrhea;

[0055] b2) Drugs that inhibit porcine epidemic diarrhea virus;

[0056] b3) Drugs used to induce an immune response to porcine epidemic diarrhea virus antigens.

[0057] Preferably, the drug includes a vaccine.

[0058] In a fourth aspect, the present invention provides a vaccine comprising the fusion protein described in the first aspect of the present invention or the biological material described in the second aspect of the present invention.

[0059] Preferably, the vaccine further includes an immunologically and pharmaceutically acceptable carrier or adjuvant.

[0060] Preferably, the adjuvant includes aluminum adjuvants such as aluminum hydroxide, aluminum phosphate, or squalene oil-in-water adjuvant, or liposomal adjuvants or adjuvants expressed using viral vectors, etc., and the adjuvant contains at least one agonist that induces an immune response in the body.

[0061] Preferably, the immunologically and pharmaceutically acceptable vector includes viral and non-viral vectors.

[0062] Preferably, the immunologically and pharmaceutically acceptable viral vectors include, but are not limited to, adenovirus vectors, retroviruses, lentiviruses, herpesviruses, or virus-like particles.

[0063] Preferably, the non-viral vector includes, but is not limited to, naked DNA, liposomes, nanocarriers, and exosomes.

[0064] Preferably, the nanocarrier includes, but is not limited to, mesoporous silica-chitosan derivative Chi-c composite material (Ag / MSN-Chi).

[0065] Preferably, the preparation method of Ag / MSN-Chi is as follows: mesoporous silica is mixed with chitosan derivative Chi-c and centrifuged to collect the precipitate.

[0066] Preferably, the mixing conditions are 22–26°C for 20–28 hours.

[0067] Preferably, the centrifugation conditions are: a centrifugation speed of 10,000-16,000 rpm; and / or a centrifugation time of 8-15 min.

[0068] Preferably, the preparation of the mesoporous silica includes, but is not limited to, the following method: mixing hexadecyltrimethylammonium bromide (CTAB) solution with sodium hydroxide solution, then adding tetraethyl orthosilicate (TEOS) and mixing evenly, and then refluxing with acetone to remove the CTAB micelle template.

[0069] Preferably, the preparation method of the chitosan derivative Chi-c includes, but is not limited to, the following method: adding 3,4-dihydroxyphenylpropionic acid, 1-ethyl-(3-dimethylaminopropionic acid)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) to a chitosan solution, reacting, and then dialysis.

[0070] A fifth aspect of the present invention provides a method for preparing a vaccine, comprising the following steps: mixing the fusion protein described in the first aspect of the present invention with the adjuvant to obtain a vaccine.

[0071] In a sixth aspect, the present invention provides an antibody that is induced to be produced by using the fusion protein described in the first aspect of the present invention or the biological material described in the second aspect of the present invention as an antigen.

[0072] The beneficial effects of this invention are:

[0073] This invention connects the RGD peptide targeting M cells, the core neutralizing epitope COE region on the S protein (one of the PEDV structural proteins), and a well-designed folded version of the GFP fluorescent protein sfGFP (superfolder GFP) to express a recombinant protein. A sulfur-containing amino acid, Cys, is introduced onto this protein to facilitate subsequent modification and covalent bonding to other substances, thus designing a safe and effective mucosal subunit vaccine with green fluorescent labeling. Unlike conventional vaccines, the antigen in this vaccine can be directly taken up by dendritic cells and also presented to dendritic cells through targeting M cells. This combination of antigen uptake significantly improves antigen presentation efficiency, more effectively initiating downstream immunity and inducing a series of immune responses. This results in the production of large amounts of PEDV-specific antibodies by the mucosal immune system to resist invading pathogens, achieving specific immune protection. Attached Figure Description

[0074] Figure 1 Simplified structural formulas of Cys-Strep-sfGFP-COE-RGD and Cys-Strep-sfGFP-COE recombinant proteins.

[0075] Figure 2 Recombinant protein particle map.

[0076] Figure 3 SDS-PAGE Coomassie Brilliant Blue gel images of recombinant proteins COER and COE ( Figure 3 A) WB chemiluminescence spectrum ( Figure 3 B).

[0077] Figure 4 Targeting of recombinant proteins COER and COE on mouse nasal mucosal M cells (immunofluorescence, IF), scale bar: 10 μm.

[0078] Figure 5 Targeting of recombinant proteins COER and COE on mouse nasal mucosal M cells (ratio of antigen to M cell area).

[0079] Figure 6 Figure showing changes in body weight in immunized mice.

[0080] Figure 7 Serum IgG antibody titer of immunized mice.

[0081] Figure 8 The titers of sIgA antibodies in vaginal mucosal lavage fluid, nasal mucosal lavage fluid, lung homogenate, and gastrointestinal mucosal lavage fluid of immunized mice.

[0082] Figure 9 The thiol group of the Cys-containing recombinant protein undergoes Michael addition with catechol, while the amino group undergoes Schiff base reaction with catechol.

[0083] Figure 10 TEM image of recombinant protein (COE) linked to antigen delivery system, scale bar: 100 nm. Detailed Implementation

[0084] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0085] Example 1: Preparation of recombinant proteins COER and COE

[0086] This embodiment provides a recombinant protein Cys-Strep-sfGFP-COE-RGD (hereinafter referred to as COER) containing the PEDV COE region and labeled with green fluorescent GFP, which can target M cells. It comprises Cys, a flexible "GS" linker that readily forms covalent bonds with other molecules, a Strep-tag for easy subsequent purification, fluorescent protein sfGFP, the PEDV COE region, two RGD peptides (GRGDS) to increase targeting binding efficiency, and a linker connecting the components. An enzyme cleavage site is introduced after the Strep-tag for subsequent tag removal. A recombinant protein Cys-Strep-sfGFP-COE (hereinafter referred to as COE) without the targeting peptide GRGDS is designed using the same method, with all other components identical. The simplified structural formulas of the two proteins are as follows: Figure 1 As shown.

[0087] The recombinant protein used in this study is the amino acid sequence of the core neutralizing antigenic epitope (COE) region of the S protein of the PEDV G2 strain:

[0088] ISFVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGV(SEQID NO.1),

[0089] The base sequence encoding the COE region obtained after codon optimization is: ATTAGCTTTGTGACCCTGCCGAGCTTTAACGATCATAGCTTTGTGAACATTACCGTGAGCGCGAGCTTTGGCGGCCATAGCGGCGCGAACCTGATTGCGAGCGATACCACCATTAACGGCTTTAGCAGCTTTTGCGTGGATACCCGCCAGTTTACCATTAGCCTGTTTTATAACGTGACCAACAGCTATGGCTATGTGAG CAAAAGCCAGGATAGCAACTGCCCGTTTACCCTGCAGAGCGTGAACGATTATCTGAGCTTTAGCAAATTTTGCGTGAGCACCAGCCTGCTGGCGAGCGCGTGCACCATTGATCTGTTTGGCTATCCGGAATTTGGCAGCGGCGTGAAATTTACCAGCTGTATTTTCAGTTTACCAAAGGCGAACTGATTACCGGCACCCCGAAACCGCTGGAAGGCGTG (SEQ ID NO.2).

[0090] To facilitate the purification of the expressed protein, the Strep-tag system, which can achieve a purity of up to 95%, was selected as the affinity chromatography tag for the purified protein. The amino acid sequence of the tag is WSHPQFEK (SEQ ID NO.3).

[0091] To facilitate subsequent experimental observation of the protein, a well-designed folded version of green fluorescent protein GFP, namely sfGFP (superfold GFP), with greater tolerance to chemical denaturants and extreme temperatures, was selected. The amino acid sequence is MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK (SEQ ID NO.4).

[0092] To avoid interactions between different functional peptides, flexible linkers were added between them. The amino acid sequences of the linkers located between Cys-Streptag and each small protein were GSS and GSGGGS (SEQ ID NO.5), respectively. The selected RGD peptide sequence was GRGDS (SEQ ID NO.6). To increase the targeting efficiency of this protein, two GRGDS peptides were linked to the protein.

[0093]

[0094] The corresponding amino acid sequence is: MCGSSWSHPQFEKSQDPMSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGP VLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGSGGGGSISFVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGFSSFCVDTRQFTISLF YNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVGSGSGSGRGDSGSGSGSGRGDS(SEQ IDNO.8).

[0095] As a control, a recombinant protein (COE) without the target peptide RGD (GRGDS) was designed using the same method. The only difference between the two recombinant proteins is whether or not they contain the RGD (GRGDS) peptide.

[0096] The designed base sequence was used as a template to amplify the sequence in large quantities via PCR. The target protein fragment was then ligated into an E. coli plasmid with kanamycin resistance (KanaR) for subsequent expression and purification. The plasmid structure is shown below. Figure 2 The homologous recombination vector and fragment were mixed at a molar ratio of 1:2 and transformed into *E. coli* (Top10) competent cells using a heat shock method (42°C water bath for 80 s, followed immediately by placing on ice for 2 min). The cells were then plated in a gradient onto LB agar containing 20 μg / mL kanamycin and incubated overnight at 37°C until single colonies appeared. Single colonies were picked and transferred to LB liquid agar containing 20 μg / mL kanamycin and incubated overnight at 37°C and 180 rpm for 18 h. After incubation, plasmids were extracted using a plasmid miniprep kit and sequenced to verify successful plasmid construction.

[0097] The successfully constructed plasmid was transformed into competent *E. coli* (E. coli BL21(DE3)) cells using the heat shock method described above, and cultured overnight for 18 h in LB broth containing 20 μg / mL kanamycin at 37°C and 180 rpm in a shaker. After culturing, the bacterial culture was inoculated at different gradients onto LB agar solid medium containing 20 μg / mL kanamycin and cultured at 37°C until single colonies grew. Target colonies were selected for propagation and preservation, as well as for subsequent protein expression.

[0098] The above-mentioned culture was cultured overnight for 18 h in LB liquid medium containing 20 μg / mL kanamycin at 37°C and 180 rpm in a constant temperature shaker. The bacterial culture was then inoculated at a 1:50 dilution into TSB liquid medium containing 20 μg / mL kanamycin and cultured at 37°C and 180 rpm in a constant temperature shaker until the OD value reached 0.5-0.6. Immediately after incubation, 0.5 mM isopropyl galactothioglycoside (IPTG) was added to induce protein expression. Expression was carried out for 4 h in a constant temperature shaker at 37°C and 180 rpm. After expression, the culture was centrifuged at 8000 × g for 3 min to collect all bacterial cells, and the cells were resuspended and washed twice with sterile PBS. Bacterial particles were sonicated in lysis buffer and centrifuged at 12000×g for 20 min to remove cell debris. The supernatant was collected and centrifuged again under the same conditions. The supernatant from both centrifugations was filtered through a 0.45 μm sterile filter to remove residual cell debris from the protein solution. The collected protein supernatant was purified using a Strep-Tactin affinity chromatography column, and the protein solution dissolved in elution buffer was collected.

[0099] The collected proteins were subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining to detect protein expression and purification. The resulting protein solution was dialyzed for 24 hours in a dialysis bag with a molecular weight cutoff of 3.5 kDa, with the water changed every 2 hours. The dialysate was a mixture of 5 mM KH₂PO₄ and 50 mM NaCl at pH 8. The dialyzed proteins were then lyophilized and stored protected from light.

[0100] SDS-PAGE electrophoresis and Coomassie brilliant blue staining showed that the location of the purified protein was consistent with the expected size. Figure 3 A). The lyophilized protein is a fluorescent green honeycomb powder that dissolves well in ultrapure water, PBS, and physiological saline.

[0101] Example 2: Immunogenicity test of recombinant proteins COER and COE

[0102] Experiment 1: Preparation of PEDV COE polyclonal antibody

[0103] The lyophilized protein COE was sterilized by UV irradiation and then dissolved in sterile physiological saline at a concentration of 0.25 mg / mL. Sterile Al(OH)3 aluminum glue was used as an adjuvant at a concentration of 20 mg / mL. 80 μL of the protein solution and 20 μL of the aluminum glue adjuvant (i.e., 20 μg protein and 400 μg aluminum glue adjuvant) were administered as one dose to mice via subcutaneous injection in the neck. Simultaneously, three 6-8 week old female BALB / c mice (18–22 g) were also immunized. Immunization was repeated twice, 14 days apart. Blood was collected from the mice 14 days after the second immunization, and the collected serum was the prepared COE polyclonal serum antibody. The obtained serum was aliquoted and stored at -80°C.

[0104] Experiment 2: PEDV COE polyclonal serum antibody enzyme-linked immunosorbent assay (ELISA)

[0105] The recombinant protein was analyzed for immunogenicity using an enzyme-linked immunosorbent assay (ELISA) with the polyclonal serum antibody obtained in Experiment 1 of Example 2.

[0106] The specific method is as follows:

[0107] (1) Dilute the recombinant protein COE to 50 μg / mL using bicarbonate buffer, add 100 μL / well to the microplate and coat overnight at 4°C;

[0108] (2) Discard the coating solution and wash the microplate three times with PBST washing buffer;

[0109] (3) Add 5% skim milk powder solution and seal at 37°C for 1 hour;

[0110] (4) Discard the blocking solution and wash the microplate three times with PBST;

[0111] (5) Add the COE polyclonal serum antibody obtained in Experiment 1, diluted by a certain factor (1:1000, 1:10000, 1:100000), the serum antibody dilution solution is 1% skim milk powder solution, and incubate at 37℃ for 1h.

[0112] (6) Discard the serum diluent and wash the ELISA plate three times with PBST;

[0113] (7) Add a certain proportion of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG diluted with 1% skim milk powder solution as a secondary antibody, and incubate at 37°C for 1 hour.

[0114] (8) Discard the secondary antibody and wash the microplate three times with PBST;

[0115] (9) Add TMB colorimetric solution and incubate at 37°C in the dark for 15 min.

[0116] (10) Add 2M sulfuric acid (H2SO4) solution to stop the color development, and measure the absorbance (OD value) at 450nm.

[0117] Wells with OD values ​​higher than twice that of the blank control were considered positive. The dilution factor of the lightest positive well was the titer of the target antibody in that serum sample. In Example 2, the antibody titers of the three mouse polyclonal serums obtained in Experiment 1 were all greater than 1:100,000.

[0118] The formulations of the solutions in Experiment 2 are shown below:

[0119] (1) Bicarbonate buffer: ultrapure water containing 0.05M Na2CO3 and 0.05M NaHCO3, pH adjusted to 9.6;

[0120] (2) Washing buffer PBST: PBS solution containing 0.05% Tween-20;

[0121] (3) 5% skim milk powder solution: skim milk powder with a mass fraction of 5% dissolved in PBST;

[0122] (4) 1% skim milk powder solution: skim milk powder with a mass fraction of 1% dissolved in PBST.

[0123] Experiment 3: PEDV COE polyclonal serum antibody protein immunoblotting (Western Blot)

[0124] The polyclonal serum antibody obtained in Experiment 1 of Example 2 was used to analyze the immunogenicity of the recombinant protein using Western blotting. The specific method is as follows:

[0125] (1) Dissolve the protein collected in Experiment 1 in ultrapure water and perform SDS-PAGE electrophoresis with a sample loading of 10 μg per well. The separation gel concentration is 12%, the stacking gel is 80V for 30 min, and the separating gel is 120V for 60 min.

[0126] (2) Transfer the protein in the separating gel to a PVDF membrane. The transfer conditions are 300 mA for 80 min.

[0127] (3) After the transfer was completed, the membrane was blocked with 5% bovine serum albumin (BSA) at room temperature (approximately 25°C) for 1.5 h.

[0128] (4) Dilute the serum with 1% BSA at a dilution ratio of 1:2000 and use it as the primary antibody to incubate the PVDF membrane containing the target protein overnight at 4°C.

[0129] (5) Discard the primary antibody solution and wash the membrane 6 times with TBST buffer on a shaker at 120 rpm for 5 minutes each time;

[0130] (6) Use a certain proportion of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG diluted with 1% BSA solution as a secondary antibody and incubate at room temperature for 1 h;

[0131] (7) Discard the secondary antibody solution and wash the membrane 6 times with TBST on a shaker at 120 rpm for 5 minutes each time;

[0132] (8) Add chemiluminescent substrate (ECL) colorimetric solution to develop the color of the membrane, expose it on a luminometer and acquire the image.

[0133] Images acquired by the chemiluminescence analyzer showed that the protein was located at the expected size, and that the polyclonal serum antibody specifically bound to the protein on the PVDF membrane. Figure 3 B).

[0134] The formulations of the solutions in Experiment 3 are shown below:

[0135] (1) TBST buffer: TBS solution containing 0.1% Tween-20;

[0136] (2) 5% bovine serum albumin (BSA): Dissolved in PBST in a 5% BSA solution;

[0137] (3) 1% bovine serum albumin (BSA): Dissolved in PBST with a mass fraction of 1% BSA solution.

[0138] Experiment 4: Test of the ability of PEDV COE polyclonal serum antibodies to neutralize PEDV

[0139] The polyclonal serum antibody obtained in Experiment 1 of Example 2 was tested for its ability to neutralize PEDV using a plaque reduction assay.

[0140] The virus strain used in the experiment was PEDV ZJ08.

[0141] The cells used were African green monkey kidney cells (Vero cells).

[0142] The specific operating steps are as follows:

[0143] (1) Vero cells were inoculated at 10 6 Each cell / well is seeded into a 6-well cell culture plate and cultured in a 37°C, 5% CO2 incubator for 2 days until the cells have filled the plate.

[0144] (2) Inactivate the serum antibody obtained in Experiment 1 at 56℃ for 30 min;

[0145] (3) Mix the selected PEDV ZJ08 strain with serum antibodies at different dilutions (1:4, 1:8, 1:16, 1:32) at a volume ratio of 1:1 and incubate at 37°C for 1 hour to neutralize the virus.

[0146] (4) Wash the surface of the cells in the six-well plate with sterile PBS to remove the serum in the cell culture medium, and inoculate the incubated virus solution onto the cell surface to infect the cell monolayer. Incubate at 37°C and 5% CO2 for 1.5 hours, shaking the plate continuously to ensure that the virus infects the cells evenly.

[0147] (5) After infection, the virus fluid was aspirated, and a mixture of agarose and DMEM was covered on the cell surface. After cooling at room temperature, the cells were incubated upside down in a 37°C, 5% CO2 constant temperature cell culture incubator for about 60 hours until the formation of spots could be observed.

[0148] (6) Mix 40% formaldehyde and 1% crystal violet solution in a 1:1 ratio, so that the final working concentration is 20% formaldehyde and 0.5% crystal violet. Add the mixture to the cell well plate, fix and stain in a 37°C incubator for 1.5 h, then rinse with water, observe the plaques and count them.

[0149] (7) Calculate the half-maximal neutralizing dose (ND) of the serum antibody. 50 The calculation method is based on Karber's formula: log 10 ND 50 =m-△(∑p-0.5), where m represents the logarithm of the highest dilution of serum antibody. 10 The value, △, represents the logarithmic dilution of two dilutions. 10 The interval between them is ∑p, which represents the sum of the number of plaques in all experimental groups / the number of plaques in the virus control group. The calculation results are shown in the table below: where 1, 2, and 3 are three replicate experiments.

[0150] Table 1

[0151] 1 2 3 Virus control group 469 229 294 Antibody 1:4 dilution 209 106 144 Antibody 1:8 dilution 236 125 162 Antibody 1:16 dilution 250 146 180 Antibody 1:32 dilution 303 167 204 <![CDATA[Neutralizing antibody titer ND 50 > 98.8912 117.4057 113.2613

[0152] The calculated PEDV neutralizing antibody titer ND 50 The values ​​were 98.8912, 117.4057, and 113.2613, respectively. This indicates that the serum antibodies produced by mice subcutaneously injected with recombinant protein COE have the ability to neutralize PEDV, suggesting that this subunit vaccine has the ability to induce mice to produce anti-PEDV specific antibodies.

[0153] Example 3: Assay on the ability of recombinant proteins COER and COE to target M cells

[0154] To investigate whether recombinant protein COER has a targeting effect on mucosal M cells, immunofluorescence (IF) was used to observe whether there was a difference in the adhesion of recombinant protein to mouse nasal mucosal M cells after intranasal administration of recombinant protein COER and COE. All mice used were 6-8 week old female BALB / c mice, weighing approximately 18-22g; each group consisted of 3 mice.

[0155] The specific operating steps are as follows:

[0156] (1) Mice were treated with COER and COE recombinant protein intranasally for 20 min, respectively;

[0157] (2) The mice were euthanized, and the entire skull of the mice was removed and fixed with 4% paraformaldehyde (PFA) for 24 hours.

[0158] (3) Soak in 10% EDTA decalcification solution for 4 days, changing the decalcification solution once a day;

[0159] (4) Mouse skulls were embedded in OCT (optimal cutting temperature compound) frozen section embedding agent and sectioned using a cryostat with a section thickness of 8-10 μm. The sectioned samples were then attached to positively charged adhesive slides.

[0160] (5) Fix the glass slide containing the sample with 4% paraformaldehyde (PFA) for 15 min;

[0161] (6) Wash three times with PBS buffer, 5 min each time;

[0162] (7) Clear the airway with 0.1% Triton X-100 for 15 minutes;

[0163] (8) Wash three times with PBS buffer, 5 min each time;

[0164] (9) 3% BSA sealed at room temperature for 1 hour;

[0165] (10) After blocking, antibody incubation was performed, and Ulex Europaeus Agglutinin I (UEA I) labeled with red fluorescent Rhodamine was used for immunofluorescence staining. The mixture was incubated at room temperature for 1 hour.

[0166] (11) Wash 3 times with PBS buffer, 5 min each time;

[0167] (12) Stain with the nuclear dye DAPI at room temperature for 10 min;

[0168] (13) Wash 3 times with PBS buffer, 5 min each time;

[0169] (14) After mixing glycerol and PBS in a 1:1 ratio, mount the slide;

[0170] (15) Observe using a fluorescence microscope.

[0171] Fluorescence microscopy revealed that recombinant protein COER containing the targeting peptide had a higher adsorption probability for nasal mucosal M cells than recombinant protein COE without the targeting peptide. Furthermore, calculations showed that the adsorption rate of recombinant protein COER containing the targeting peptide for nasal mucosal M cells was approximately four times that of recombinant protein COE without the targeting peptide. Figure 4 and Figure 5 ).

[0172] Example 4: Mouse Immunization Experiment with Recombinant Proteins COER and COE

[0173] Experiment 1: Feeding and Immunization Program for Mice

[0174] To investigate whether recombinant protein COER containing the targeting peptide RGD could induce higher antibody titers in mice than recombinant protein COE without the targeting peptide, mice were immunized with nasal mucosal drops.

[0175] Sixteen 6-8 week old SPF-grade female BALB / c mice, weighing 18-22g, were randomly divided into three groups: a blank control group (n=4 per group), a COER group (n=6 per group), and a COE group (n=6 per group). The mice were acclimatized to their environment one week prior to the experiment, with the animal room maintaining a 12-hour light-12-hour dark environment at a temperature of approximately 22±2℃.

[0176] Mice were first immunized on day 1, and then immunized a second and third time on days 15 and 29, respectively, with each immunization lasting two days, for a total experimental period of 42 days. Mice were immunized intranasally between 8:00 and 9:00 AM, with the immunized group receiving 10 μL of a protein solution containing 50 μg of antigen in each nostril, while the control group received the same volume of PBS solution. Mice were weighed and their weight recorded daily; water bottles were washed every two days; and bedding was changed daily. During the rearing process, each mouse was given 4-6g of feed and provided with ample clean drinking water daily.

[0177] During the 42 days of feeding, the mice showed stable weight gain, indicating that the immunization process had no significant impact on their growth. Figure 6 ).

[0178] Experiment 2: Collection and processing of mouse samples

[0179] On day 43, two weeks after the third immunization, samples were collected from the mice. Blood, vaginal lavage fluid, nasal lavage fluid, lung homogenate, and gastrointestinal lavage fluid were collected.

[0180] Vaginal lavage solution: Hold the mouse by the back of its neck with your left hand and fix its tail to your palm. Use a micropipette to draw 50 μL of sterile PBS solution and irrigate the mouse's vagina 10 times. Centrifuge at 3500 rpm at 4°C for 15 min, collect the supernatant, aliquot and store at -80°C.

[0181] Blood collection from the eye: Grasp the mouse's neck skin with your left hand and gently press it onto the lab table. Position the mouse in a lateral recumbent position. Use your left index finger to press the skin around the mouse's eye towards the back of the neck to make the eyeball protrude. Quickly remove the eyeball with ophthalmic forceps. Invert the mouse and collect the flowing blood in a 1.5 mL EP tube. Collect approximately 0.6–1 mL of blood each time and store at 4°C overnight. Centrifuge at 3500 rpm at 4°C for 15 min, collect the supernatant serum, aliquot it, and store it at -80°C.

[0182] Nasal lavage fluid: Open the lower jaw from the corner of the mouse's mouth to the sides to find the nasopharyngeal opening, insert a syringe and inject 300 μL of sterile PBS solution into it, collect it in the nostrils with a sterile EP tube, centrifuge at 4000 rpm at 4℃ for 10 min, take the supernatant, aliquot and store in a -80℃ freezer.

[0183] Lung homogenate: Mouse lungs were collected by opening the thoracic cavity and immersed in a 2 mL homogenate tube containing 400 μL of sterile PBS solution. Homogenizing was then carried out with homogenate beads. The homogenate was centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatant was collected, aliquoted, and stored at -80 °C.

[0184] Gastrointestinal mucosal lavage fluid: The small intestine, including the duodenum, jejunum, and ileum, was collected from the mouse peritoneum. The intestinal contents were gently squeezed out with forceps. Sterile PBS solution was injected into one end of the intestine using a syringe, and the lavage fluid was collected from the other end. This lavage step was repeated twice, and the fluid was collected in sterile EP tubes. The solution was centrifuged at 13,000 rpm at 4°C for 10 min, and the supernatant was collected, aliquoted, and stored at -80°C.

[0185] Experiment 3: Detection of serum IgG antibody titer using enzyme-linked immunosorbent assay (ELISA)

[0186] The serum obtained in Experiment 2 of Example 4 was used to detect the antibody titer of the recombinant protein using an enzyme-linked immunosorbent assay (ELISA).

[0187] The specific method is as follows:

[0188] (1) Dilute the recombinant protein COE to 50 μg / mL using bicarbonate buffer, add 100 μL / well to the microplate and coat overnight at 4°C;

[0189] (2) Discard the coating solution and wash the microplate three times with PBST washing buffer;

[0190] (3) Add 5% skim milk powder solution and seal at 37°C for 1 hour;

[0191] (4) Discard the blocking solution and wash the microplate three times with PBST;

[0192] (5) Add serum diluted by a certain factor. The serum dilution solution is a 1% skim milk powder solution. Incubate at 37°C for 1 hour.

[0193] (6) Discard the serum diluent and wash the ELISA plate three times with PBST;

[0194] (7) Add a certain proportion of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG diluted with 1% skim milk powder solution as a secondary antibody, and incubate at 37°C for 1 hour.

[0195] (8) Discard the secondary antibody and wash the microplate three times with PBST;

[0196] (9) Add TMB colorimetric solution and incubate at 37°C in the dark for 15 min.

[0197] (10) Add 2M sulfuric acid (H2SO4) solution to stop the color development, and measure the absorbance (OD value) at 450nm.

[0198] Wells with OD values ​​more than twice that of the blank control are considered positive. The dilution factor of the lightest positive well is the titer of the target antibody in the serum sample.

[0199] The results showed that the COER group containing the targeting peptide had nearly twice the IgG antibody titer of the COE group without the targeting peptide. Figure 7 ).

[0200] The formulations of the solutions are shown below:

[0201] (1) Bicarbonate buffer: ultrapure water containing 0.05M Na2CO3 and 0.05M NaHCO3, pH adjusted to 9.6;

[0202] (2) Washing buffer PBST: PBS solution containing 0.05% Tween-20;

[0203] (3) 5% skim milk powder solution: skim milk powder with a mass fraction of 5% dissolved in PBST;

[0204] (4) 1% skim milk powder solution: skim milk powder with a mass fraction of 1% dissolved in PBST.

[0205] Experiment 4: Enzyme-linked immunosorbent assay (ELISA) to detect mucosal sIgA antibody titer

[0206] The recombinant protein was tested for antibody titer using enzyme-linked immunosorbent assay (ELISA) with the vaginal lavage fluid, nasal lavage fluid, lung homogenate, and gastrointestinal lavage fluid obtained in Experiment 2 of Example 4. The specific method is as follows:

[0207] (1) Dilute the recombinant protein COE to 50 μg / mL using bicarbonate buffer, add 100 μL / well to the microplate and coat overnight at 4°C;

[0208] (2) Discard the coating solution and wash the microplate three times with PBST washing buffer;

[0209] (3) Add 5% skim milk powder solution and seal at 37°C for 1 hour;

[0210] (4) Discard the blocking solution and wash the microplate three times with PBST;

[0211] (5) Add vaginal lavage solution, nasal lavage solution, lung homogenate and digestive tract lavage solution diluted by a certain multiple respectively. The diluent is 1% skim milk powder solution. Incubate at 37°C for 1 hour.

[0212] (6) Discard the diluted solutions of vaginal lavage fluid, nasal lavage fluid, lung homogenate, and gastrointestinal lavage fluid respectively, and wash the ELISA plate three times with PBST.

[0213] (7) Add a certain proportion of horseradish peroxidase (HRP)-labeled goat anti-mouse IgA diluted with 1% skim milk powder solution as a secondary antibody, and incubate at 37°C for 1 hour.

[0214] (8) Discard the secondary antibody and wash the microplate three times with PBST;

[0215] (9) Add TMB colorimetric solution and incubate at 37°C in the dark for 15 min.

[0216] (10) Add 2M sulfuric acid (H2SO4) solution to stop the color development, and measure the absorbance (OD value) at 450nm.

[0217] Wells with OD values ​​higher than twice that of the blank control are considered positive. The dilution factor of the positive well with the lightest color is the potency ratio of the sIgA antibody in that sample.

[0218] The results showed that the COER group containing the targeting peptide had nearly one-fifth higher sIgA antibody titer than the COE group without the targeting peptide. Figure 8).

[0219] The formulations of the solutions are shown below:

[0220] (1) Bicarbonate buffer: ultrapure water containing 0.05M Na2CO3 and 0.05M NaHCO3, pH adjusted to 9.6;

[0221] (2) Washing buffer PBST: PBS solution containing 0.05% Tween-20;

[0222] (3) 5% skim milk powder solution: skim milk powder with a mass fraction of 5% dissolved in PBST;

[0223] (4) 1% skim milk powder solution: skim milk powder with a mass fraction of 1% dissolved in PBST.

[0224] Example 5: Covalent linking of recombinant proteins COER and COE

[0225] Experiment 1: Ligation of recombinant proteins COER and COE with antigen delivery systems

[0226] Our research group has previously successfully constructed an antigen delivery system using mesoporous silica (MSN) and the catechol-containing chitosan derivative Chi-c. The recombinant proteins COER and COE, containing thiol groups, readily undergo Michael addition with the catechol groups in the chitosan derivative Chi-c. Figure 9 ).

[0227] The preparation method of the antigen delivery system of mesoporous silica (MSN) and chitosan derivative Chi-c containing catechol is as follows:

[0228] 1. Synthesis of mesoporous silica MSN

[0229] (1) Weigh 0.25g of cetyltrimethylammonium bromide (CTAB) as a surfactant and stir it in 120mL of ultrapure water for about 1 hour until it is completely dissolved;

[0230] (2) Add 1.75 mL of 1 mol / L sodium hydroxide solution and stir in an oil bath at 80 °C for 1 h;

[0231] (3) 5 mL of tetraethyl orthosilicate (TEOS) was added as a silicon source and the mixture was stirred at a constant temperature of 80 °C for 12 h.

[0232] (4) Collect the materials and wash them repeatedly with anhydrous ethanol 5 times;

[0233] (5) The cleaned material was refluxed with 100 mL of acetone at 50 °C for more than 12 hours to remove the CTAB micelle template.

[0234] (6) After the condensation and reflux are completed, collect the material, wash it three times with anhydrous ethanol, and then wash it three times with ultrapure water.

[0235] (7) After drying at 80℃, store for later use.

[0236] 2. Synthesis of Chi-c

[0237] (1) Dissolve 50 mg of chitosan in 10 mL of ultrapure water with pH = 4, and stir overnight in a round-bottom flask until dissolved, while keeping the pH of the solution in the range of 4 to 5 during the process;

[0238] (2) After dissolving, add 120 mg of 3,4-dihydroxyphenylpropionic acid (HCA);

[0239] (3) Then add 120 mg of 1-ethyl-(3-dimethylaminopropionic acid) carbodiimide (EDC) and 74 mg of N-hydroxysuccinimide (NHS) and stir for 48 h;

[0240] (4) After the reaction was completed, the dialysis was performed in ultrapure water at pH 4 for 48 hours using a dialysis bag with a molecular weight cutoff of 3.5 kDa, and the dialysis solution was changed every 2 hours during the process.

[0241] (5) After lyophilizing the dialyzed Chi-c, store it for later use.

[0242] 3. Synthesis of MC nanoparticle delivery system

[0243] (1) Dissolve MSN at a concentration of 5 mg / mL in 5 mL of PB solution (pH = 7.75) and stir evenly in a round-bottom flask until the solution turns milky white;

[0244] (2) Take an equal volume of Chi-c with a concentration of 2.5 mg / mL and slowly add it dropwise, and stir the reaction at room temperature for 24 h;

[0245] (3) After the reaction was completed, the synthesized material MC was washed three times with ultrapure water, centrifuged at 13000 rpm for 10 min to collect the precipitate, freeze-dried and stored for later use.

[0246] The preparation method of the recombinant protein antigen delivery system is as follows:

[0247] (1) Dissolve the MSN-Chic antigen delivery system in PB solution (pH=7.75) at a concentration of 1 mg / mL, stir evenly with a magnetic stirrer, and then add a certain amount of recombinant protein solution to the reaction system for reaction.

[0248] (2) After stirring at room temperature for 24 hours, the supernatant was collected by centrifugation, and the residual protein content in the supernatant was detected by BCA kit. The amount of protein adhering was calculated, and the morphology of the antigen delivery system could be changed according to the amount of protein adhering.

[0249] (3) The antigen delivery system containing the recombinant protein was observed using transmission electron microscopy (TEM), and the protein was successfully attached to the antigen delivery system. Figure 10 ).

[0250] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A fusion protein, wherein the amino acid sequence of the fusion protein is SEQ ID NO.

8.

2. A biomaterial relating to the fusion protein of claim 1, said biomaterial comprising at least one of a1) to a16): a1) A nucleic acid molecule encoding the fusion protein of claim 1; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A carrier containing the nucleic acid molecules described in a1); a4) A carrier containing the expression box described in a2); a5) Transgenic cell lines containing the nucleic acid molecules described in a1); a6) Transgenic cell lines containing the expression cassette described in a2); a7) A transgenic cell line containing the vector described in a3); a8) A transgenic cell line containing the vector described in a4); a9) Microorganisms containing the nucleic acid molecules described in a1); a10) contains microorganisms containing the expression cassette described in a2); a11) contains microorganisms that contain the carrier described in a3); a12) contains microorganisms that contain the carrier described in a4); a13) Viruses containing the nucleic acid molecules described in a1); a14) A virus containing the expression cassette described in a2); a15) contains a virus containing the vector described in a3); a16) contains a virus with the vector described in a4).

3. The biomaterial according to claim 2, characterized in that, The sequence of the nucleic acid molecule is SEQ ID NO.

7.

4. The use of the fusion protein of claim 1 or the biomaterial of claim 2 in the preparation of a vaccine for inducing an immune response to porcine epidemic diarrhea virus antigen.

5. A vaccine comprising the fusion protein of claim 1.

6. The vaccine according to claim 5, characterized in that, The vaccine also includes immunologically and pharmaceutically acceptable carriers or adjuvants.

7. A method for preparing a vaccine, comprising the following steps: mixing the fusion protein of claim 1 and the adjuvant of claim 6 to obtain a vaccine.

Images