TWI776048B - Recombinant protein for preventing swine fever virus infection and composition and cell comprising the same. - Google Patents

Abstract

The present disclosure is related to a recombinant protein for preventing swine fever virus infection and a composition and a cell comprising the same. The recombinant protein comprises an antigenic moiety and a moiety of ferritin. The antigenic moiety is the E2 protein of swine fever virus. The recombinant protein of the present disclosure is able to induce immune response against the infection of swine fever virus in swine; thus is useful for the epidemic prevention work in pig farming industry.

Description

Recombinant protein for preventing swine fever virus infection and compositions and cells containing the same

The present disclosure relates to a composition for preventing swine fever virus infection, especially a sub-unit vaccine for preventing swine fever virus infection.

Swine fever, also known as classic swine fever, is an infectious disease caused by swine fever virus. It has the characteristics of high infectivity and high mortality. It will cause a large number of pig deaths and cause serious losses to the pig industry. .

Current swine fever vaccines can be divided into three categories. 1. Traditional Rabbitized Swine Fever Vaccine: The production method of this vaccine is to inoculate the weakened swine fever seed virus into rabbits, collect the organs at a specific time point, grind, filter and freeze-dry, so as to prepare rabbitized pigs Plague vaccine. 2. Tissue cultured live virus vaccine: Infect pig kidney cells not contaminated by type 1 porcine circular virus with attenuated swine fever virus. After virus propagation, virus liquid collection, filtration and freeze-drying, the tissue culture live virus vaccine is obtained. 3. Sub-unit vaccines: At present, commercially available sub-unit vaccines such as Bayovac CSF-E2 Vaccine are produced by the insect baculovirus expression system, which is to infect insect cells with the virus with the E2 gene, and then secrete the recombinant E2 protein. performance, and then the recombinant E2 protein was used to prepare the vaccine.

In view of the harm and potential threat of swine fever to the pig industry, more vaccines that can effectively prevent swine fever virus infection are needed in the field to provide more diversified and efficient options for epidemic prevention.

An object of the present disclosure is to provide a novel recombinant protein and a composition containing the same, which can induce an immune protection response to prevent swine fever virus infection. Another object of the present disclosure is to provide an expression cassette, an expression vector containing the same, and a mammalian cell with the same, which can be used to express the recombinant protein of the present disclosure.

In order to meet the above purpose, the present disclosure provides a recombinant protein, which comprises: an antigenic part whose amino acid sequence is SEQ ID NO: 01; and a ferritin part.

The present disclosure further provides a composition for preventing swine fever virus infection, comprising: the recombinant protein of the present disclosure and a pharmaceutically acceptable carrier.

The present disclosure further provides an expression cassette, comprising: an expression element comprising a promoter; and a first polynucleotide and a second polynucleotide operably linked to the expression element; the first polynucleotide A polynucleotide is encoded as SEQ ID NO: 01, and the second polynucleotide is encoded as an iron-carrying protein.

The present disclosure further provides a presentation carrier comprising the presentation cassette of the present disclosure.

The present disclosure also provides a mammalian cell with the expression cassette of the present disclosure.

The present disclosure further provides a method for expressing the recombinant protein of the present disclosure, comprising: expressing the expression cassette of the present disclosure in a host cell.

The descriptions herein are exemplary and explanatory only and are not intended to limit the present disclosure. Technical and scientific terms used herein are to be understood as commonly understood by one of ordinary skill in the art, unless explicitly defined otherwise.

The singular form "a (a or an)" herein and in the description of the claimed scope includes the plural unless the context clearly dictates otherwise. Thus, for example, "a protein" is meant to include one or more (s) proteins, and "a compound" is meant to include one or more (s) of compounds. The uses of "comprise", "comprises", "comprising", "include", "includes", "including" are interchangeable, not restrictive. It should be further understood that in the description of each specific embodiment, where the term "comprising" is used, those skilled in the art will understand that in some specific cases, the language "substantially consisting of... ..consists of" or "consisting of" instead.

When a range of values is provided, unless the context clearly dictates otherwise, it is to be understood that integers in that range of values and tenths of each integer in the range of values are between the upper and lower limits of the range, and between the upper and lower limits of the range, and Any other stated value or intervening value within that range is encompassed within this disclosure.

All documents, patents, patent applications, and other documents cited in this disclosure are hereby incorporated by reference in their entirety, the contents of which are hereby incorporated by reference as if each individual document, patent, patent application, or other document were individually indicated. for reference purposes.

definition:

As used herein, "encode/encoding" refers to the process by which the polynucleotide is transcribed and/or translated to produce a polypeptide, or further to form a protein. The "the first polynucleotide encodes SEQ ID NO: 01" means that the first polynucleotide is transcribed and/or translated to produce a protein whose sequence is SEQ ID NO: 01. "The second polynucleotide encodes an iron-carrying protein" means that the second polynucleotide is transcribed and/or translated to produce a protein; the protein is an iron-carrying protein. Other similar statements in this article can be interpreted according to this concept. The encoding can be performed in vivo or in vitro. The encoding can be performed in syngeneic or heterologous cells.

The term "prevention of swine fever virus infection" as mentioned herein refers to the prevention of illness or symdrome caused by swine fever virus infection. Specifically, for example, the discomfort or symptoms caused by the swine fever virus are prevented from occurring, or the degree of the discomfort or symptoms is relieved. Those with ordinary knowledge in the field should understand that the "prevention of swine fever virus infection" does not mean that the referred individual is completely free from swine fever virus infection, but from the point of view of epidemic prevention, the swine fever virus can affect the referred individual. Hazard reduction.

Reference herein to "the sequence is SEQ ID NO" or similar recitations means that the referenced protein or polynucleotide comprises, but is not limited to, the referenced sequence. For example, "an antigenic moiety whose amino acid sequence is SEQ ID NO: 01" as described herein means that the amino acid sequence of the antigenic moiety comprises SEQ ID NO: 01 (in a specific embodiment, the main It consists of SEQ ID NO: 01), but those with ordinary knowledge in the field can modify the indicated sequence based on the ordinary knowledge in the field according to their needs, and make the modified sequence include sequences other than SEQ ID NO: 01. . The present disclosure does not exclude the N-terminal or C-terminal extension of the proteins of the present disclosure by 1 to several amino acids. The present disclosure also does not preclude the extension of sequences of other proteins at the N-terminus or C-terminus of the proteins of the present disclosure based on the needs of a particular use. For example, various affinity tags such as His tag, Strep tag and T7 tag can be bound to the N-terminus or C-terminus of SEQ ID NO: 01. Thereby, the expression of the recombinant protein can be detected by the antibodies corresponding to these affinity tags respectively (for example, applied to the Western blotting method). The modified sequence, unless the effect of preventing swine fever virus infection claimed in the present disclosure, should still fall within the scope of the present disclosure.

The term "operably linked" as used herein means that two or more polynucleotides are connected to each other by means of genetic engineering, and these polynucleotides are ensured to be accessible to the host (herein, the polynucleotides used to express the refers to the nucleotide sequence of an organism) recognized and encoded as the desired protein. Specifically, if several nucleotides need to be padded between the interconnected polynucleotides in practice, it must be ensured that the padded nucleotides will not cause the downstream polynucleotides to encode offset on. For example, the full length of the sequence of the padded nucleotides should be a multiple of 3, since a codon should have 3 nucleotides.

A first aspect of the present disclosure relates to a recombinant protein and a composition containing the same. The recombinant protein is a fusion protein and includes an antigenic moiety and a moiety of ferritin. The antigenic portion refers to the portion of the recombinant protein that primarily elicits an immune response in the host. The present disclosure does not exclude that other parts of the recombinant protein also have the effect of inducing host immune response. Preferably, the amino acid sequence of the antigenic moiety is SEQ ID NO: 01. Possibly, the antigenic portion is encoded by SEQ ID NO:03. It will be understood by those of ordinary skill in the art that when the antigenic portion is expressed in different organisms, the sequence used to encode the antigenic portion may vary to conform to the codon usage bias of that organism. .

The iron-carrying protein is as defined in the art; preferably, the iron-carrying protein moiety used in the present disclosure is derived from Helicobacter pylori . As used herein, "derived from Helicobacter pylori" means that the amino acid sequence of the iron-carrying protein moiety is substantially identical to the amino acid sequence of the iron-carrying protein carried by wild-type Helicobacter pylori. This statement does not limit the iron-carrying proteins used in this disclosure must be purified or isolated from Helicobacter pylori. In a preferred embodiment, the amino acid sequence of the iron-carrying protein moiety is SEQ ID NO: 02. Possibly, the iron-carrying protein portion is encoded by SEQ ID NO:04.

In a specific embodiment, a linker is further included between the antigenic moiety and the iron-carrying protein moiety. In a possible embodiment, the amino acid sequence of the linker is SEQ ID NO: 05.

The composition for preventing swine fever virus infection of the present disclosure comprises: the recombinant protein of the present disclosure and a pharmaceutically acceptable carrier. In a possible embodiment, the concentration of the recombinant protein is 1 to 60 µg/mL, based on the total volume of the composition: preferably, 7.5 to 30 µg/mL: more preferably, it is 7.5 to 15 µg/mL. In a specific embodiment, the concentration of the recombinant protein is any one of the following concentrations or a concentration between any two concentrations: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 60 µg/mL.

In a possible embodiment, the pharmaceutically acceptable carrier is water, phosphate buffered saline, alcohol, glycerol, chitin, alginate, chondroitin, vitamin E, minerals, or a combination thereof. In one embodiment, the composition is formulated into a solid, liquid, or colloidal state, depending on the needs of the user. In yet another embodiment, the composition is stored in a container (eg, a glass bottle) for use by the user.

In a preferred embodiment, the composition further comprises an adjuvant. The adjuvant may be, but is not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum gum, surfactants, polyanions, peptides, oil emulsions, or combinations thereof. In particular, commercially available adjuvants such as, but not limited to, Montanide ^™ ISA 201 VG (SEPPIC, France) may be used. The ratio of the adjuvant to the recombinant protein is optional: practically, the ratio is 1:1 (w/w).

A second aspect of the present disclosure relates to an expression cassette, an expression vector containing the same, and a mammalian cell bearing the same. The expression cassette in the present disclosure refers to a polynucleotide comprising a expression element, a first polynucleotide and a second polynucleotide operably linked to the expression element: the expression The element comprises a promoter; the first polynucleotide encodes SEQ ID NO: 01, and the second polynucleotide encodes an iron-carrying protein.

In a preferred embodiment, the second polynucleotide is encoded as SEQ ID NO: 02. In a possible embodiment, the second polynucleotide is SEQ ID NO: 04. Preferably, the recombinant protein of the present disclosure can be obtained after the expression cassette is expressed. Aptly, the performance cassette is SEQ ID NO: 08.

The performance carrier of the present disclosure carries the performance cassette of the present disclosure. In a possible embodiment, the expression vector has sequences that are replicable in a predetermined host. In another possible embodiment, the expression vector further comprises a sequence encoding a message peptide, a tag (tag) DNA, or a combination thereof. In a preferred embodiment, the expression vector is used in a mammalian cell expression system.

The mammalian cell with the expression cassette or the expression vector in the present disclosure refers to a mammalian cell, which is transfected into the cell by the expression cassette or the expression vector through cell engineering technology. Suitably, the transfection is performed by electroporation techniques.

Preferably, the expression cassette or the expression vector will be maintained in the cell after being transfected into the cell. More preferably, after the expression cassette or the expression vector is transfected into the cell, it will replicate as the cell replicates. In a possible embodiment, the mammalian cells are Chinese hamster ovary cells (CHO cells).

The present disclosure also relates to a method of expressing the recombinant protein of the present disclosure, comprising: expressing the expression cassette in the mammalian cell of the present disclosure. Possibly, the presentation cassette is a presentation carrier present in the present disclosure. The method may further comprise a purification step to obtain the recombinant protein expressed by the mammalian cells.

Experiment 1: The construction and CHO Transfection of cells.

1. Materials and methods.

1.1 CHO cells and culture medium:

CHO-S cells (Thermo Fisher Scientific, USA) were used as host cells for recombinant protein production. Serum-free suspension culture of CHO cells was performed using HyClone CDM4PERMAb medium (GE Healthcare, USA), and additionally added penicillin-streptomycin (Penicillin-Streptomycin, Thermo Fisher Scientific; the final concentration of Penicillin was 100 U/mL, and the final concentration of Streptomycin was 100 U/mL). 100 μg/mL) with GlutaMAX™ Supplement (Thermo Fisher Scientific; final concentration of 6 mM).

The semi-solid medium used to screen stable cell lines is ClonaCell™-CHO ACF methylcellulose-based semi-solid medium (STEMCELL Technologies, USA), additionally added hygromycin B (Hygromycin B, Thermo Fisher Scientific; final concentration) during the screening process 400 μg/mL). During the subsequent scale-up culturing of CHO cells, additional cell culture additives (HyClone Cell Boost Kit, GE Healthcare) may be added depending on the situation.

1.2 Construction of expression vector and transfection of CHO cells:

U.S. GenScript Company was commissioned to synthesize polynucleotides (SEQ ID NO: 07) encoding the recombinant proteins of the present disclosure according to the preferred codons of CHO cells. As shown in Table 1 below, the polynucleotide can encode a fusion protein with a sequence of SEQ ID NO: 06, which comprises the E2 protein of swine fever virus and the iron-carrying protein derived from Helicobacter pylori.

Table 1: Amino acid sequences of fusion proteins prepared in experiment 1. SEQ ID NO: 06 VKVLRGQIVQGVIWLLLVTGAQGRLACKEDYRYAISSTDEIGLLGAGGLTTTWKEYTHDLQLNDGTVKATCVAGSFKVTALNVVSRRYLASLHKKALPTSVTFELLFDGTNPSTEEMGDDFGFGLCPFDTRPVVKGKYNATLVNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFRRDKPFPHRMNCVTTTVENEDLFYCKLGGNWTCVKGEPVVYTGGLVKQCRWCGFDFNEPDGLPHYPIGKCILANETSYRVVDSTDCNRDGVVISTEGSHECLIGNTTVKVHASDERLGPMPCRPKEIVSSAGPAMKTSCTFNYAKTLKNRYYEPRDSYFQQYMLKGEYQYWFDLDATDRHSDYFAEF CPGGS DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS n Bold type is the antigenic portion of the recombinant protein of the present disclosure, SEQ ID NO: 01. n Italic type is the iron-carrying protein portion of the recombinant protein of the present disclosure, SEQ ID NO: 02. The n box is the linker of the disclosed recombinant protein, SEQ ID NO: 05.

The polynucleotide is embedded in a mammalian cell expression vector. In addition to the above-mentioned polynucleotides for encoding the recombinant proteins of the present disclosure, the expression vector used in this experiment still carries the human cytomegalovirus early gene promoter (enhancer-promoter from the immediately-early gene of human cytomegalovirus, CMV) promoter), a sequence encoding the mouse IgK secretory signal (immunoglobulin kappa secretory signal), and tag DNA (see Figure 1). After the sequence of the expression vector was confirmed to be correct, DNA transfection was performed using Amaxa™ Cell Line Nucleofector™ Kit V (Lonza Bioscience, USA) transfection reagent and Nucleofector 2b Device electroporation cell transfection instrument. The number of CHO cells at the time of transfection was 2×10 ⁶ , and the amount of expression vector was 1 μg.

1.3 Screening of cell lines with high antigen expression and establishment of seed cell bank:

After the transfected CHO cells were cultured in HyClone CDM4PERMAb medium for two days, hygromycin B was added to screen for drug-resistant cell lines. Hygromycin B-selected mini-pools were cultured in ClonaCell™-CHO ACF semi-solid medium at a concentration of about 600 cells/mL. After the single cells grow into clusters (about 7 to 9 days), the candidate cell lines are selected and cultured in 96-well plates using the ClonePix FL instrument. After the cells had grown to nearly full coverage, the cells were transferred to a 48-well plate and cultured for two days. Next, 100 μL of the cell culture supernatant was taken for sandwich enzyme-linked immunosorbent assay (ELISA) analysis, so as to screen cell populations that can highly express the recombinant proteins of the present disclosure.

The capture antibody used in the ELISA method was a rabbit anti-His antibody (Rabbit anti-6-His Antibody, Bethyl Laboratories, USA); the detection antibody was a rabbit anti-c-myc antibody (Rabbit anti-6-His Antibody, Bethyl Laboratories, USA). anti-c-myc Antibody HRP Conjugated, Bethyl Laboratories, USA); the color reagent used was TMB substrate solution (United States Biological, USA). The absorbance of each well was measured at 450 nm with an ELISA reader. Cell lines with high antigen expression were screened from ELISA results.

Next, the cells were cultured in a cell shaker flask (125 mL) to confirm that the previously screened cell lines with high antigen expression were not easy to form clumps during suspension culture, and were further analyzed by sandwich ELISA and protein electrophoresis with specificity for E2 protein. Screening of high antigen-expressing cell lines.

WH303 monoclonal antibody (APHA, UK) was used as the acquisition antibody in the ELISA specific for E2 protein; the labeled detection antibody was rabbit anti-c-myc antibody; and the colorant used was TMB substrate solution. The absorbance of each well was measured at 450 nm with an ELISA reader. The cell lines with high antigenic expression were screened out by ELISA and protein electrophoresis results.

Afterwards, the screened cell lines were re-cultured in ClonaCell™-CHO ACF semi-solid medium, and the screening steps from paragraphs 0051 to 0054 were repeated for a total of 5 times.

The screened high antigen-expressing cell lines were mixed with CELLBANKER 2 (Nippon Zenyaku Kogyo, Japan) serum-free cell cryopreservation solution for cryopreservation.

2. Experimental results.

The experimental results are shown in FIG. 2 . The C5-1 cell line has the best expression level, and the cell growth state is stable. Therefore, in this experiment, the C5-1 cell line was selected as the seed cell for subsequent production of the recombinant protein of the present disclosure, and was used to establish the seed cell bank.

Experiment 2: Purification of recombinant antigens and structural analysis of nanoparticles.

1. Materials and methods.

Shake flask culture in 5 L medium was performed using CHO cells from the seed cell bank. After 11 days of culture, the cell culture broth was centrifuged at 20,000 xg for 2 hours and the supernatant was collected. The supernatant was filtered with a 0.22 μm filter. Recombinant proteins were purified using immobilized metal ion affinity resin Ni Sepharose excel (GE Healthcare, Sweden). The ability of recombinant proteins to form nanoparticles was analyzed by dynamic light scattering instrument ZetaSizer ZEN 3600 instrument (Malvern, USA) and transmission electron microscope JEM-2100F (JEOL, Japan).

2. Experimental results.

The results of protein electrophoresis showed that the C5-1 cell line could stably secrete and express recombinant proteins (Figure 3). In addition, extracellular recombinant proteins can be purified using immobilized metal ion affinity resins. The purified recombinant protein is treated with dithiothreitol (DTT), which can destroy the disulfide bonds between the protein molecules, and the molecular weight of the monomer protein is about 70 kDa; without DTT treatment, the purified recombinant protein intermolecular Multimers are formed (Figure 4).

On the other hand, the results of dynamic light scattering analysis showed that the recombinant proteins in this experiment could indeed self-assemble to form nanoparticles with an average hydrated particle size of about 37 nm (Fig. 5). The morphology of the nanoparticles was further observed by transmission electron microscopy, and it was shown that the recombinant protein in this experiment could form nanoparticles with a particle size between 20 and 50 nm (Fig. 6).

Experiment 3: vaccine preparation and pig immunization challenge test.

1. Materials and methods.

1.1 Vaccine preparation:

The recombinant protein solution purified in experiment 2 was adjusted to a specific concentration and mixed with Montanide ^TM ISA 201 VG adjuvant (SEPPIC, France) at a ratio of 1:1 (w/w) to prepare V-1311, V-1331 , V-1332, V-1333 and V-1334, a total of 5 vaccines (Table 2). In addition, normal saline containing 0.01% Thiomersal (w/v) was mixed with ISA 201 VG adjuvant to prepare a control group V-1335 without antigen. Store vaccines in a 4°C refrigerator for later use.

1.2 Pig immune challenge test:

This experiment was carried out in the genetically modified organism (GMO) animal house of the Animal Health Laboratory, Animal Health Laboratory, Executive Yuan Agriculture Committee. A total of 20 9-week-old specific pathogen free (SPF) pigs with negative swine fever virus antibodies were selected and randomly divided into groups A to F. The number of pigs in each group was 2-4; groups A-E were the experimental groups, and group F was the control group. Pigs were immunized by intramuscular injection once at 9 weeks of age, and the immunization dose was 2 mL. The pigs were grouped in the following Table 2.

Table 2: Vaccine and challenge trial design: group Number of pigs The composition of this experiment Amount of E2 antigen (μg)/dose (2 mL) A 2 V-1311 60 B 4 V-1331 30 C 4 V-1332 15 D 4 V-1333 7.5 E 3 V-1334 3.75 F 3 V-1335 0

Before immunization (9 weeks old), 1 week after immunization (10 weeks old), 2 weeks after immunization (11 weeks old), and 3 weeks after immunization (12 weeks old) Fibrosis, and stored in -80 ℃ refrigerator for later use. At the age of 12 weeks (three weeks after immunization), the highly virulent swine fever virus strain ALD (2 mL) was injected into the experimental pigs by intramuscular injection for the challenge test. After challenge, pigs were observed daily for clinical symptoms, body temperature changes, and survival rate was calculated. All pigs were sacrificed at 14 weeks of age (2 weeks post-challenge) and subjected to anatomical pathology.

2. Experimental results.

In this experiment, there was no adverse reaction of redness, swelling or festering at the injection site of the pigs in each group, and the spirit, activity and appetite of the animals were normal, indicating that the vaccine has good safety. The sera of the experimental pigs were analyzed with a commercial swine fever ELISA antibody detection kit (BioChek, UK). Only before the experiment was really uninfected. Anti-swine fever virus antibody can be observed to increase in serum collected from pigs administered the composition of the present disclosure at three weeks (12 weeks of age) after immunization. Results were better in the μg/dose group (Figure 7).

The survival rate of the experimental pigs was recorded (Fig. 8), showing that the pigs administered the composition of the present disclosure had improved survival rate, especially in the groups immunized with E2 antigen at 60, 30 and 15 μg/dose , all pigs survived the challenge. The results of this experiment should not be interpreted as 7.5 and 3.75 μg/dose are ineffective against swine fever virus, because this experiment was conducted with the highly virulent swine fever virus strain ALD, and only one immunization injection was performed. In addition, individual differences are still inevitable in experiments. Therefore, the experimental results should be interpreted from a comprehensive perspective, which means that the disclosed composition exhibits anti-swine fever virus effects at all experimental doses. Based on the above test results, the composition of the present disclosure has good safety, and the immunization dose above 15 μg/dose only needs to be administered once, which can provide pigs with the effect of resisting swine fever virus infection.

none

FIG. 1 is a schematic diagram of the expression vector of experiment one. Tag DNA includes C-myc tag, Strep-tag II, and His tag.

FIG. 2 is a protein electrophoresis chart of experiment 1, which shows the expression levels of recombinant protein secretion of C5-1, C5-4, and C5-7 cell lines. The arrows point to the recombinant proteins of the present disclosure. M is the commercially available product BenchMark ^™ Protein Ladder (Thermo Fisher Scientific).

Figure 3 is the protein electrophoresis chart of experiment 2, which shows the expression level of recombinant protein secretion of C5-1 cell line on days 3, 4, 6, 8, 9, 10, and 11. The arrows point to the recombinant proteins of the present disclosure. M is the commercially available product BenchMark ^™ Protein Ladder (Thermo Fisher Scientific).

FIG. 4 is a protein electropherogram of experiment 2, which shows monomers and multimers of the recombinant proteins of the present disclosure. The arrows point to the recombinant proteins of the present disclosure. M is the commercially available product BenchMark ^™ Protein Ladder (Thermo Fisher Scientific). DTT: Dithiothreitol. +: treated with DTT; -: not treated with DTT.

FIG. 5 is the dynamic light scattering analysis result of experiment 2, which shows that the recombinant proteins of the present disclosure form nanoparticles.

FIG. 6 is a transmission electron microscope image of Experiment 2, which shows that the recombinant proteins of the present disclosure form nanoparticles. Left panel: scale bar 100 nm; right panel: scale bar 20 nm.

Figure 7 shows the anti-swine fever virus antibody titers of the experimental pig sera in experiment three.

FIG. 8 shows the survival rate of experimental pigs after swine fever virus challenge in experiment three.

none

<110> Agricultural Science and Technology Research Institute

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Claims (28)

A recombinant protein comprising: an antigenic part, the amino acid sequence of which is SEQ ID NO: 01; a ferritin part; and a linker, which links the antigenic part and the ferritin part, wherein The amino acid sequence of this linker is SEQ ID NO:05. The recombinant protein of claim 1, wherein the antigenic portion is encoded by SEQ ID NO:03. The recombinant protein of claim 1, wherein the iron-carrying protein moiety is derived from Helicobacter pylori. The recombinant protein of claim 1, wherein the amino acid sequence of the iron-carrying protein portion is SEQ ID NO: 02. The recombinant protein of claim 4, wherein the iron-carrying protein portion is encoded by SEQ ID NO:04. The recombinant protein of claim 1, wherein the amino acid sequence of the recombinant protein is SEQ ID NO:06. The recombinant protein of claim 1, which is encoded by SEQ ID NO:07. A composition for preventing swine fever virus infection, comprising: the recombinant protein according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier. The composition of claim 8, wherein the concentration of the recombinant protein is 1 to 60 μg/mL, based on the total volume of the composition. The composition of claim 9, wherein the concentration of the recombinant protein is 7.5 to 30 μg/mL, based on the total volume of the composition. The composition of claim 9, wherein the concentration of the recombinant protein is 7.5 to 15 μg/mL, based on the total volume of the composition. The composition of claim 8, which further comprises an adjuvant. The composition of claim 12, wherein the adjuvant comprises: complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum gum, surfactants, polyanions, peptides, oil emulsions, or combinations thereof. The composition of claim 8, wherein the pharmaceutically acceptable carrier is water, phosphate buffered saline, alcohol, glycerol, chitin, alginate, chondroitin, vitamin E, minerals, or a combination thereof. An expression cassette comprising: an expression element comprising a promoter; a first polynucleotide and a second polynucleotide operably linked to the expression element; the first polynucleotide The code is SEQ ID NO: 01, and the second polynucleotide is encoded as an iron-carrying protein; and a linker, which links the first polynucleotide and the second polynucleotide, wherein the linker Coded as SEQ ID NO:05. The performance cassette of claim 15, wherein the second polynucleotide is encoded as SEQ ID NO:02. The performance cassette of claim 15, wherein the first polynucleotide is SEQ ID NO:03, and wherein the second polynucleotide is SEQ ID NO:04. The expression cassette of claim 15, which expresses the recombinant protein of any one of claims 1 to 7. The expression cassette of claim 18, wherein the amino acid sequence of the recombinant protein is SEQ ID NO:06. The performance cassette of claim 15, which is SEQ ID NO:08. A presentation carrier comprising the presentation cassette of any one of claims 15-20. The expression vector of claim 21, which is used in a mammalian cell expression system. A mammalian cell with the expression cassette of any one of claims 15-20. The mammalian cell of claim 23, which carries the expression vector of any one of claims 21 to 22. The mammalian cell of claim 23, which is a Chinese hamster ovary cell. A method of expressing the recombinant protein of any one of claims 1 to 7, comprising: expressing the expression cassette of any one of claims 15 to 20 in a host cell. The method of claim 26, wherein the host cell is a mammalian cell as claimed in any one of claims 23 to 25. The method of claim 26, wherein after expressing the expression cassette, further comprising purifying the recombinant protein.

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