CN113728011B - Recombinant protein for preventing classical swine fever virus infection and composition and cell containing the same - Google Patents

Abstract

The present disclosure relates to a recombinant protein for preventing infection with classical swine fever virus and a composition and cell containing the same. The recombinant protein comprises an antigen portion and an iron-carrying protein portion. The antigen portion is the E2 protein of classical swine fever virus. The recombinant protein disclosed herein can induce pigs to produce an immune response against infection with classical swine fever virus, which is helpful for the prevention and control of classical swine fever in the pig farming industry.

Description

Recombinant protein for preventing classical swine fever virus infection and composition and cell containing the same

Technical Field

The present disclosure relates to a composition for preventing classical swine fever virus infection, and more particularly to a subunit vaccine for preventing classical swine fever virus infection.

Background Art

Classical swine fever, also known as classical swine fever, is an infectious disease caused by the classical swine fever virus. It is highly contagious and has a high mortality rate. It can cause mass deaths of pigs and cause serious losses to the pig farming industry.

Current swine fever vaccines can be divided into three categories. 1. Traditional rabbitized swine fever vaccine: The manufacturing method of this vaccine is to inoculate weakened swine fever virus into rabbits, collect organs at specific time points, grind, filter and freeze-dry them to obtain rabbitized swine fever vaccine. 2. Tissue culture live virus vaccine: The weakened swine fever virus is used to infect pig kidney cells that are not contaminated by type 1 porcine circovirus. After virus proliferation, virus fluid collection, filtration and freeze-drying, the tissue culture live virus vaccine is obtained. 3. Subunit vaccine: The subunit vaccines currently on the market, such as Bayovac CSF-E2 Vaccine, are produced using an insect baculovirus expression system, which is to infect insect cells with a virus carrying the E2 gene, secrete and express the recombinant E2 protein, and then use the recombinant E2 protein to make a vaccine.

In view of the damage and potential threat caused by African swine fever to the pig farming industry, the industry needs more vaccines that can effectively prevent African swine fever virus infection to provide more diverse and efficient options for epidemic prevention work.

Summary of the invention

One 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 classical 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 carrying the expression cassette or expression vector, which can be used to express the recombinant protein of the present disclosure.

To meet the above objectives, the present disclosure provides a recombinant protein comprising: an antigen portion, whose amino acid sequence is SEQ ID NO: 01; and a ferritin portion.

The present disclosure further provides a composition for preventing classical 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 encodes SEQ ID NO: 01, and the second polynucleotide encodes an iron-carrying protein.

The present disclosure further provides an expression vector comprising the expression cassette of the present disclosure.

The present disclosure further provides a mammalian cell carrying 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the expression vector of Experiment 1. The tag DNA includes a C-myc tag, a Strep-tag II, and a His tag.

Figure 2 is a protein electrophoresis diagram of Experiment 1, which shows the secretion expression of recombinant proteins in C5-1, C5-4, and C5-7 cell lines. The arrows point to the recombinant proteins disclosed herein. M is a commercially available product, BenchMark ^™ Protein Ladder (Thermo Fisher Scientific).

FIG3 is a protein electrophoresis diagram of Experiment 2, which shows the secretion expression of recombinant proteins of C5-1 cell line on days 3, 4, 6, 8, 9, 10, and 11. The arrows point to the recombinant proteins disclosed herein. M is a commercially available product, BenchMark ^™ Protein Ladder (Thermo Fisher Scientific).

FIG4 is a protein electrophoresis diagram of Experiment 2, which shows the monomers and polymers of the recombinant protein disclosed herein. The arrows point to the recombinant protein disclosed herein. M is a commercial product, BenchMark ^™ Protein Ladder (Thermo Fisher Scientific). DTT: dithiothreitol. +: treated with DTT; -: not treated with DTT.

FIG. 5 is a dynamic light scattering analysis result of Experiment 2, which shows the formation of nanoparticles by the recombinant protein of the present disclosure.

Figure 6 is a transmission electron microscope image of Experiment 2, which shows the formation of nanoparticles by the recombinant protein disclosed in the present invention. Left image: scale bar 100 nm; right image: scale bar 20 nm.

FIG. 7 shows the anti-swine fever virus antibody titers of the experimental pig sera in Experiment 3.

FIG. 8 shows the survival rate of the experimental pigs in Experiment 3 after challenge with classical swine fever virus.

DETAILED DESCRIPTION

The description herein is exemplary and explanatory only and is not intended to limit the present disclosure. Technical and scientific terms used herein should be understood as the meanings commonly understood by those of ordinary skill in the art unless explicitly defined otherwise.

Unless the context clearly indicates otherwise, the singular forms "a" or "an" herein and in the description of the claims include plural meanings. Thus, for example, "a protein" refers to one or more proteins, and "a compound" refers to one or more compounds. The use of "comprise", "comprises", "comprising", "include", "includes", "including" are interchangeable and non-limiting. It should be further understood that in the description of each specific embodiment, when the term "comprising" is used, those skilled in the art will understand that in some specific cases, the language "essentially consisting of" or "consisting of" can be used instead.

When a range of values is provided, unless the context clearly dictates otherwise, it should be understood that the integers in the numerical interval and one-tenth of each integer in the numerical interval, between the upper and lower limits of the range, and any other stated or intervening values in the range, are encompassed within the disclosure.

All publications, patents, patent applications, and other documents cited in this disclosure are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, patent application, or other document were individually denoted as being incorporated herein by reference.

definition:

The "encode/encoding" described herein refers to the process in which the polynucleotide is transcribed and/or translated to produce a polypeptide, or further forms a protein. The "first polynucleotide encodes SEQ ID NO: 01" means that the first polynucleotide is transcribed and/or translated to produce a protein, and its 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 descriptions in this article can be interpreted based on this concept. The encoding can be performed in vivo or in vitro. The encoding can be performed in homologous cells or heterologous cells.

The "prevention of classical swine fever virus infection" mentioned herein refers to the prevention of discomfort (illness) or symptoms (symdrome) caused by classical swine fever virus infection. Specifically, for example, the discomfort or symptoms caused by classical swine fever virus are not caused, or the degree of discomfort or symptoms is alleviated. It should be understood by those skilled in the art that the "prevention of classical swine fever virus infection" does not mean that the individual referred to is completely free from classical swine fever virus infection, but from the perspective of epidemic prevention, the harm of classical swine fever virus to the individual referred to is reduced.

The "sequence is SEQ ID NO" or similar descriptions described herein means that the protein or polynucleotide referred to includes the sequence referred to, but is not limited to the sequence referred to. For example, the "antigen portion, whose amino acid sequence is SEQ ID NO:01" described herein means that the amino acid sequence of the antigen portion includes SEQ ID NO:01 (in a specific embodiment, it is mainly composed of SEQ ID NO:01), but a person skilled in the art can modify the sequence referred to based on the common knowledge in the field according to their needs, so that the modified sequence includes a sequence other than SEQ ID NO:01. The present disclosure does not exclude extending 1 to several amino acids at the N-terminus or C-terminus of the protein disclosed herein. The present disclosure also does not exclude extending the sequence of other proteins at the N-terminus or C-terminus of the protein disclosed herein based on the needs of specific use. For example, various affinity tags such as His tag, Strep tag and T7 tag can be combined at the N-terminus or C-terminus of SEQ ID NO:01. Thereby, the antibodies corresponding to these affinity tags can be used to detect the expression of recombinant proteins (for example, applied to Western blot). Unless the modified sequence has lost the effect of preventing classical swine fever virus infection claimed in the present disclosure, it should still fall within the scope of the present disclosure.

As used herein, "operably linked" means that two or more polynucleotides are connected to each other by genetic engineering means, and these polynucleotides are ensured to be recognized by the host (here, the organism used to express the nucleotide sequence) and encode the desired protein. Specifically, if it is necessary to fill several nucleotides between these interconnected polynucleotides in actual operation, it is necessary to ensure that the filled nucleotides will not cause a coding deviation in the downstream polynucleotides. For example, the full length of the sequence of the filled nucleotides should be a multiple of 3, because a codon should have 3 nucleotides.

The first aspect of the present disclosure is about a recombinant protein and a composition containing the same. The recombinant protein is a fusion protein and comprises an antigenic moiety and an iron-carrying protein moiety. The antigenic moiety refers to the part of the recombinant protein that mainly induces the host immune response. The present disclosure does not exclude that other parts of the recombinant protein also have the effect of inducing the host immune response. Preferably, the amino acid sequence of the antigenic moiety is SEQ ID NO:01. Optionally, the antigenic moiety is encoded by SEQ ID NO:03. Those skilled in the art should understand that when the antigenic moiety is expressed in different organisms, the sequence used to encode the antigenic moiety may change to conform to the codon usage bias of the organism.

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

In one embodiment, a linker is further included between the antigen portion and the iron-carrying protein portion. In one feasible embodiment, the amino acid sequence of the linker is SEQ ID NO: 05.

The composition disclosed herein for preventing infection with classical swine fever virus comprises: the recombinant protein disclosed herein and a pharmaceutically acceptable carrier. In a feasible implementation, the concentration of the recombinant protein is 1 to 60 μg/mL, which is based on the total volume of the composition: preferably, 7.5 to 30 μg/mL: more preferably, 7.5 to 15 μg/mL. In a specific implementation, the concentration of the recombinant protein is any 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 one possible implementation, the pharmaceutically acceptable carrier is water, phosphate buffered saline, alcohol, glycerol, chitosan, alginate, chondroitin, vitamin E, minerals, or a combination thereof. In one specific implementation, the composition is adjusted to a solid, liquid, or colloidal state, depending on the needs of the user. In another specific implementation, the composition is stored in a container (e.g., a glass bottle) for the convenience of the user.

In a preferred embodiment, the composition further comprises an adjuvant. The adjuvant may be, but not limited to: Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum gel, surfactant, anionic polymer, peptide, oil emulsion or a combination thereof. Specifically, commercially available adjuvants may be used, such as, but not limited to: Montanide ^TM ISA 201VG (SEPPIC, France). The ratio of the adjuvant to the recombinant protein may be determined as appropriate: preferably, the ratio is 1:1 (w/w).

The second aspect of the present disclosure is an expression cassette, an expression vector containing the same, and a mammalian cell carrying the expression cassette or the expression vector. The expression cassette referred to in the present disclosure refers to a polynucleotide, which comprises an expression element, and a first polynucleotide and a second polynucleotide operably linked to the expression element: the expression 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 by SEQ ID NO: 02. In a feasible embodiment, the second polynucleotide is SEQ ID NO: 04. Preferably, the expression cassette can obtain the recombinant protein of the present disclosure after expression. Feasibly, the expression cassette is SEQ ID NO: 08.

The expression vector disclosed herein is an expression cassette disclosed herein. In one feasible embodiment, the expression vector has a sequence that can be replicated in a predetermined host. In another feasible embodiment, the expression vector further comprises a sequence encoding a signal peptide, a 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 expression vector disclosed herein refers to a mammalian cell into which the expression cassette or expression vector is transfected by cell engineering technology. Preferably, the transfection is performed by electroporation technology.

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

The present disclosure further relates to a method for expressing the recombinant protein of the present disclosure, comprising: expressing the expression cassette in the mammalian cell of the present disclosure. Preferably, the expression cassette is present in the expression vector of the present disclosure. The method may further comprise a purification step to obtain the recombinant protein expressed by the mammalian cell.

Experiment 1: Construction of expression vector and transfection of CHO 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 the production of recombinant proteins. HyClone CDM4PERMAb medium (GE Healthcare, USA) was used for serum-free suspension culture of CHO cells, and penicillin-streptomycin (Penicillin-Streptomycin, Thermo Fisher Scientific; the final concentration of Penicillin was 100 U/mL, the final concentration of Streptomycin was 100 μg/mL) and GlutaMAX ^™ Supplement (Thermo Fisher Scientific; the final concentration was 6 mM) were additionally added.

The semi-solid medium used to screen stable cell lines was ClonaCell ^TM -CHO ACF methylcellulose-based semi-solid medium (STEMCELL Technologies, USA). Hygromycin B (Thermo Fisher Scientific; final concentration was 400 μg/mL) was required during the screening process. In the subsequent amplification of CHO cells, cell culture additives (HyClone Cell Boost Kit, GE Healthcare) were added according to the manufacturer's recommendations.

1.2 Construction of expression vector and transfection of CHO cells:

GenScript, Inc. of the United States was commissioned to synthesize a polynucleotide (SEQ ID NO: 07) encoding the recombinant protein disclosed herein 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 includes the E2 protein of classical swine fever virus and an iron-carrying protein derived from Helicobacter pylori.

Table 1: Amino acid sequences of fusion proteins prepared in Experiment 1.

The polynucleotide is inserted into a mammalian cell expression vector. In addition to the polynucleotide encoding the recombinant protein disclosed herein, the expression vector used in this experiment also carries an enhancer-promoter from the immediately-early gene of human cytomegalovirus (CMV promoter), a sequence encoding a mouse immunoglobulin kappa secretory signal, and a tag DNA (see FIG1 ). After sequencing to confirm that the sequence of the expression vector is correct, DNA transfection is performed using the Amaxa ^TM CellLine Nucleofector ^TM Kit V (Lonza Bioscience, USA) transfection reagent in combination with the Nucleofector 2bDevice electroporation cell transfection instrument. The number of CHO cells during transfection is 2×10 ⁶ , and the amount of the expression vector used is 1 μg.

1.3 Screening of high antigen-expressing cell lines and establishment of seed cell banks:

After culturing the transfected CHO cells in HyClone CDM4PERMAb culture medium for two days, hygromycin B was added to screen for drug-resistant cell lines. The mini-pool screened with hygromycin B was cultured in ClonaCell ^TM -CHO ACF semi-solid culture medium at a concentration of about 600 cells/mL. After the single cells grew into clusters (about 7 to 9 days), the candidate cell lines were selected and cultured in a 96-well plate using the ClonePix FL instrument. After the cells grew to nearly full coverage, the cells were moved 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 to screen for cell groups that can highly express the recombinant protein disclosed herein.

The capture antibody used in the ELISA method is rabbit anti-6-His antibody (Bethyl Laboratories, USA); the detection antibody is rabbit anti-c-myc antibody (Rabbit anti-c-myc Antibody HRP Conjugated, Bethyl Laboratories, USA); the coloring agent used is TMB substrate solution (United States Biological, USA). The absorbance of each well is measured at 450nm using an ELISA reader. Based on the ELISA results, cell lines with high antigen expression are screened.

Next, the cells were cultured in shake flasks (125 mL) to confirm that the cell lines with high antigen expression screened above were not easy to clump in suspension culture, and then sandwich ELISA and protein electrophoresis analysis specific for E2 protein were used to further screen the cell lines with high antigen expression.

The capture antibody used in the ELISA specific for E2 protein was WH303 monoclonal antibody (APHA, UK); the labeled detection antibody was rabbit anti-c-myc antibody; the color developing agent used was TMB substrate solution. The absorbance value of each well was measured at 450nm using an ELISA reader. Cell lines with high antigen expression were screened based on the results of ELISA and protein electrophoresis.

The screened cell lines were then cultured in ClonaCell ^™ -CHO ACF semi-solid medium, and the above screening steps were repeated 5 times in total.

The high antigen-expressing cell lines obtained through screening were mixed with CELLBANKER 2 (Nippon Zenyaku Kogyo, Japan) serum-free cell freezing medium and then cryopreserved.

2. Experimental results.

The experimental results are shown in Figure 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 the subsequent production of the recombinant protein disclosed in the present invention and used to establish the seed cell bank.

Experiment 2: Purification of recombinant antigens and analysis of nanoparticle structure.

1. Materials and methods.

CHO cells from the seed cell bank were cultured in a 5 L shake flask. After 11 days of culture, the cell culture fluid was centrifuged at 20,000 × g for 2 hours and the supernatant was collected. The supernatant was filtered with a 0.22 μm filter membrane. The recombinant protein was purified using an immobilized metal ion affinity resin Ni Sepharose excel (GE Healthcare, Sweden). The ability of the recombinant protein to form nanoparticles was analyzed using a dynamic light scattering instrument ZetaSizer ZEN 3600 instrument (Malvern, USA) and a 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, the extracellular recombinant proteins could be purified using immobilized metal ion affinity resins. The purified recombinant proteins were treated with dithiothreitol (DTT) to destroy the disulfide bonds between protein molecules. The molecular weight of the monomer protein was about 70 kDa. Without DTT treatment, the purified recombinant protein molecules would form polymers (Figure 4).

On the other hand, the results of dynamic light scattering analysis showed that the recombinant protein in this experiment can indeed self-assemble to form nanoparticles, and the average hydrated particle size is about 37nm (Figure 5). Further observation of the morphology of nanoparticles using a transmission electron microscope showed that the recombinant protein in this experiment can form nanoparticles with a particle size of about 20 to 50nm (Figure 6).

Experiment 3: Vaccine preparation and pig immunization 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 ^™ ISA201VG adjuvant (SEPPIC, France) at a ratio of 1:1 (w/w) to prepare 5 vaccines, namely V-1311, V-1331, V-1332, V-1333 and V-1334 (Table 2). A control group V-1335 containing no antigen was prepared by mixing physiological saline containing 0.01% Thiomersal (w/v) with ISA201VG adjuvant. The vaccine was stored in a refrigerator at 4°C for later use.

1.2 Pig immune challenge test:

This experiment was conducted in the genetically modified organism (GMO) animal house of the Animal Drug Testing Branch of the Livestock Hygiene Research Institute in Taiwan. A total of 20 9-week-old specific pathogen free (SPF) pigs with negative swine fever virus antibody tests were selected and randomly divided into groups A to F. The number of pigs in each group was 2 to 4; groups A to E were experimental groups, and group F was the control group. The pigs were immunized once intramuscularly at 9 weeks of age, with an immunization dose of 2 mL. The experimental grouping of pigs is shown in Table 2 below.

Table 2: Vaccine and challenge test design:

Group Number of pigs This experimental composition E2 antigen amount (μg)/dose (2mL) 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

3-5 mL of blood was collected from the jugular vein before immunization (9 weeks of age), 1 week after immunization (10 weeks of age), 2 weeks after immunization (11 weeks of age), and 3 weeks after immunization (12 weeks of age) to prepare defibrinated blood, which was stored in a -80°C refrigerator for use. At 12 weeks of age (three weeks after immunization), each group of pigs was injected intramuscularly with the highly toxic classical swine fever virus strain ALD (2 mL) for a challenge test. After the challenge, the clinical symptoms and body temperature changes of the pigs were observed daily, and the survival rate was calculated. All pigs were sacrificed at 14 weeks of age (2 weeks after the challenge) and subjected to anatomical pathological examinations.

2. Experimental results.

In this experiment, no adverse reactions such as redness, swelling or ulceration occurred at the injection site of each group of pigs, and the animals' spirit, activity and appetite were normal, indicating that the vaccine has good safety. The serum of the experimental pigs was analyzed with a commercial swine fever ELISA antibody detection kit (BioChek, UK). The results showed that the anti-swine fever virus antibodies of each group of pigs before immunization (9 weeks of age) were negative, indicating that the experimental pigs had never been infected before the experiment. The anti-swine fever virus antibodies of the pigs administered with the disclosed composition were observed to increase in the serum collected three weeks after immunization (12 weeks of age); among them, the results of the groups with E2 antigen immunization doses of 60, 30 and 15 μg/dose were better (Figure 7).

The survival rate of experimental pigs (Figure 8) shows that the pigs administered with the disclosed composition have an improved survival rate, especially in the groups with E2 antigen immunization doses of 60, 30 and 15 μg/dose, all pigs survived after the virus attack. This experimental result should not be interpreted as 7.5 and 3.75 μg/dose are ineffective against classical swine fever virus, because this experiment was conducted using the highly toxic classical swine fever virus strain ALD, and only one immunization injection was performed. In addition, individual differences are still inevitable in the experiment. Therefore, this experimental result should be interpreted from a comprehensive perspective, that is, the disclosed composition has shown an anti-classic swine fever virus effect at all experimental doses. The above experimental results show that the disclosed composition has good safety, and the immunization dose of more than 15 μg/dose only needs to be administered once to provide pigs with the effect of resisting classical swine fever virus infection.

Sequence Listing

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Gly Thr Val Lys Ala Thr Cys Val Ala Gly Ser Phe Lys Val Thr Ala

65 70 75 80

Leu Asn Val Val Ser Arg Arg Tyr Leu Ala Ser Leu His Lys Lys Ala

85 90 95

Leu Pro Thr Ser Val Thr Phe Glu Leu Leu Phe Asp Gly Thr Asn Pro

100 105 110

Ser Thr Glu Glu Met Gly Asp Asp Phe Gly Phe Gly Leu Cys Pro Phe

115 120 125

Asp Thr Arg Pro Val Val Lys Gly Lys Tyr Asn Ala Thr Leu Val Asn

130 135 140

Gly Ser Ala Phe Tyr Leu Val Cys Pro Ile Gly Trp Thr Gly Val Ile

145 150 155 160

Glu Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val Val Lys

165 170 175

Thr Phe Arg Arg Asp Lys Pro Phe Pro His Arg Met Asn Cys Val Thr

180 185 190

Thr Thr Val Glu Asn Glu Asp Leu Phe Tyr Cys Lys Leu Gly Gly Asn

195 200 205

Trp Thr Cys Val Lys Gly Glu Pro Val Val Tyr Thr Gly Gly Leu Val

210 215 220

Lys Gln Cys Arg Trp Cys Gly Phe Asp Phe Asn Glu Pro Asp Gly Leu

225 230 235 240

Pro His Tyr Pro Ile Gly Lys Cys Ile Leu Ala Asn Glu Thr Ser Tyr

245 250 255

Arg Val Val Asp Ser Thr Asp Cys Asn Arg Asp Gly Val Val Ile Ser

260 265 270

Thr Glu Gly Ser His Glu Cys Leu Ile Gly Asn Thr Thr Val Lys Val

275 280 285

His Ala Ser Asp Glu Arg Leu Gly Pro Met Pro Cys Arg Pro Lys Glu

290 295 300

Ile Val Ser Ser Ala Gly Pro Ala Met Lys Thr Ser Cys Thr Phe Asn

305 310 315 320

Tyr Ala Lys Thr Leu Lys Asn Arg Tyr Tyr Glu Pro Arg Asp Ser Tyr

325 330 335

Phe Gln Gln Tyr Met Leu Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu

340 345 350

Asp Ala Thr Asp Arg His Ser Asp Tyr Phe Ala Glu Phe Cys Pro Gly

355 360 365

Gly Ser Asp Ile Ile Lys Leu Leu Asn Glu Gln Val Asn Lys Glu Met

370 375 380

Gln Ser Ser Asn Leu Tyr Met Ser Met Ser Ser Trp Cys Tyr Thr His

385 390 395 400

Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp His Ala Ala Glu Glu

405 410 415

Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu Asn Glu Asn Asn Val

420 425 430

Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu His Lys Phe Glu Gly

435 440 445

Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His Glu Gln His Ile Ser

450 455 460

Glu Ser Ile Asn Asn Ile Val Asp His Ala Ile Lys Ser Lys Asp His

465 470 475 480

Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala Glu Gln His Glu Glu

485 490 495

Glu Val Leu Phe Lys Asp Ile Leu Asp Lys Ile Glu Leu Ile Gly Asn

500 505 510

Glu Asn His Gly Leu Tyr Leu Ala Asp Gln Tyr Val Lys Gly Ile Ala

515 520 525

Lys Ser Arg Lys Ser Gly Ser

530 535

<210> 7

<211> 1605

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding recombinant protein

<400> 7

gtgaaagtgc tgcgtggtca gattgtccag ggggtcattt ggctgctgct ggtgactggc 60

gctcagggaa gactggcatg caaggaggac taccgctatg caatctccag cacagatgaa 120

attggactgc tgggtgcagg aggactgacc acaacttgga aggagtacac acacgacctg 180

cagctgaatg atggaaccgt caaggcaaca tgcgtggccg ggtctttcaa agtgacagct 240

ctgaacgtgg tcagtaggcg gtatctggcc tcactgcata agaaagctct gcctacttct 300

gtgaccttcg agctgctgtt tgacggcacc aatccaagta cagaggaaat gggcgacgat 360

ttcggctttg gactgtgccc ctttgatacc aggcctgtgg tcaagggtaa atacaacgcc 420

acactggtga atggctccgc cttctatctg gtgtgcccca tcggctggac cggagtgatt 480

gagtgtacag cagtgtctcc tactaccctg agaactgaag tggtcaagac cttcagacgc 540

gacaaaccctttcctcaccg catgaactgc gtcacaacta ccgtggagaa cgaagacctg 600

ttttactgca agctgggggg taattggaca tgcgtgaaag gcgagccagt ggtctatact 660

ggcggactgg tcaagcagtg cagatggtgt ggattcgact ttaatgagcc cgatggtctg 720

cctcattacc caatcggaaa atgtattctg gccaacgaaa cttcctatcg agtggtggac 780

agtaccgatt gcaatcgtga cggggtggtc atctcaaccg agggttccca cgaatgtctg 840

attggcaaca caactgtcaa ggtgcatgct tccgatgaga ggctgggacc aatgccctgc 900

cggccaaagg aaatcgtgtc ctccgccggc cccgctatga aaacatcatg tactttcaac 960

tacgctaaga cactgaaaaa tcgatactat gagccccgtg actcctactt ccagcagtat 1020

atgctgaagg gcgaatacca gtattggttt gacctggatg caaccgaccg acactccgat 1080

tacttcgccg agttttgtcc aggggggtct gatattatca agctgctgaa cgaacaggtg 1140

aacaaggaga tgcagagctc caacctgtac atgagtatgt ctagttggtg ctatactcac 1200

tccctggacg gcgctggact gttcctgttt gatcacgccg ctgaggaata cgaacatgca 1260

aagaaactga tcattttcct gaatgagaac aatgtgcccg tccagctgac ctcaatcagc 1320

gcccctgaac ataagttcga gggactgaca cagatctttc agaaggccta cgaacacgag 1380

cagcatattt ccgagtctat caacaatatt gtcgaccacg caatcaagag caaagatcat 1440

gccaccttca acttcctcca gtggtacgtg gccgagcagc acgaggaaga ggtcctgttt 1500

aaggacattc tggataaaat cgaactgatt gggaacgaga atcatggcct gtacctggca 1560

gatcagtatg tgaaaggcat cgcaaagtcc cgaaaaagcg gctcc 1605

<210> 8

<211> 2348

<212> DNA

<213> Artificial Sequence

<220>

<223> expression cassette

<400> 8

agatctcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata 60

gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact 120

tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat 180

gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggacta 240

tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc 300

tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg 360

ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg 420

gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct 480

ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa 540

atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt 600

ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat 660

gctagcatgg agacagacac actcctgcta tgggtactgc tgctctgggt tccaggttcc 720

actggtgacg gcgcgccgtg aaagtgctgc gtggtcagat tgtccagggg gtcatttggc 780

tgctgctggt gactggcgct cagggaagac tggcatgcaa ggaggactac cgctatgcaa 840

tctccagcac agatgaaatt ggactgctgg gtgcaggagg actgaccaca acttggaagg 900

agtacacaca cgacctgcag ctgaatgatg gaaccgtcaa ggcaacatgc gtggccgggt 960

ctttcaaagt gacagctctg aacgtggtca gtaggcggta tctggcctca ctgcataaga 1020

aagctctgcc tacttctgtg accttcgagc tgctgtttga cggcaccaat ccaagtacag 1080

aggaaatggg cgacgatttc ggctttggac tgtgcccctt tgataccagg cctgtggtca 1140

agggtaaata caacgccaca ctggtgaatg gctccgcctt ctatctggtg tgccccatcg 1200

gctggaccgg agtgattgag tgtacagcag tgtctcctac taccctgaga actgaagtgg 1260

tcaagacctt cagacgcgac aaaccctttc ctcaccgcat gaactgcgtc acaactaccg 1320

tggagaacga agacctgttt tactgcaagc tggggggtaa ttggacatgc gtgaaaggcg 1380

agccagtggt ctatactggc ggactggtca agcagtgcag atggtgtgga ttcgacttta 1440

atgagcccga tggtctgcct cattacccaa tcggaaaatg tattctggcc aacgaaactt 1500

cctatcgagt ggtggacagt accgattgca atcgtgacgg ggtggtcatc tcaaccgagg 1560

gttcccacga atgtctgatt ggcaacacaa ctgtcaaggt gcatgcttcc gatgagaggc 1620

tgggaccaat gccctgccgg ccaaaggaaa tcgtgtcctc cgccggcccc gctatgaaaa 1680

catcatgtac tttcaactac gctaagacac tgaaaaatcg atactatgag ccccgtgact 1740

cctacttcca gcagtatatg ctgaagggcg aataccagta ttggtttgac ctggatgcaa 1800

ccgaccgaca ctccgattac ttcgccgagt tttgtccagg ggggtctgat attatcaagc 1860

tgctgaacga acaggtgaac aaggagatgc agagctccaa cctgtacatg agtatgtcta 1920

gttggtgcta tactcactcc ctggacggcg ctggactgtt cctgtttgat cacgccgctg 1980

aggaatacga acatgcaaag aaactgatca ttttcctgaa tgagaacaat gtgcccgtcc 2040

agctgacctc aatcagcgcc cctgaacata agttcgaggg actgacacag atctttcaga 2100

aggcctacga acacgagcag catatttccg agtctatcaa caatattgtc gaccacgcaa 2160

tcaagagcaa agatcatgcc accttcaact tcctccagtg gtacgtggcc gagcagcacg 2220

aggaagaggt cctgtttaag gacattctgg ataaaatcga actgattggg aacgagaatc 2280

atggcctgta cctggcagat cagtatgtga aaggcatcgc aaagtcccga aaaagcggct 2340

ccggatcc 2348

Claims (26)

1. A recombinant protein comprising: An antigen portion, the amino acid sequence of which is SEQ ID NO: 01; and Iron-carrying protein (ferritin) part; The amino acid sequence of the recombinant protein is SEQ ID NO: 06. 2. The recombinant protein of claim 1, wherein the antigenic portion is encoded by SEQ ID NO: 03. 3. The recombinant protein of claim 1, wherein the iron-carrying protein portion is derived from Helicobacter pylori. 4. The recombinant protein of claim 1, wherein the amino acid sequence of the iron-carrying protein portion is SEQ ID NO: 02. 5. The recombinant protein of claim 4, wherein the iron-carrying protein portion is encoded by SEQ ID NO:04. 6. The recombinant protein of claim 1, comprising a linker to link the antigen portion and the non-hematinic protein portion; wherein the amino acid sequence of the linker is SEQ ID NO: 05. 7. The recombinant protein of claim 1, which is encoded by SEQ ID NO: 07. 8. A composition for preventing classical swine fever virus infection, comprising: the recombinant protein according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier. 9. 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. 10. 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. 11. 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. 12. The composition of claim 8, further comprising an adjuvant. 13. The composition of claim 12, wherein the adjuvant comprises: Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum gel, a surfactant, anionic polymer, a peptide, an oil emulsion or a combination thereof. 14. The composition of claim 8, wherein the pharmaceutically acceptable carrier is water, phosphate buffered saline, alcohol, glycerol, chitosan, alginate, chondroitin, vitamin E, minerals, or combinations thereof. 15. 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 encodes SEQ ID NO: 01, and the second polynucleotide encodes an iron-carrying protein; The expression cassette expresses the recombinant protein according to any one of claims 1 to 7. 16. The expression cassette of claim 15, wherein the second polynucleotide encodes SEQ ID NO: 02. 17. The expression cassette of claim 15, wherein the first polynucleotide is SEQ ID NO: 03, and wherein the second polynucleotide is SEQ ID NO: 04. 18. The expression cassette of claim 15, which is SEQ ID NO: 08. 19. An expression vector comprising the expression cassette according to any one of claims 15 to 18. 20. The expression vector of claim 19, which is used in a mammalian cell expression system. 21. A mammalian cell carrying the expression cassette of any one of claims 15 to 18. 22. The mammalian cell of claim 21, which carries the expression vector of any one of claims 19 to 20. 23. The mammalian cell of claim 21, which is a Chinese hamster ovary cell. 24. A method for expressing the recombinant protein of any one of claims 1 to 7, comprising: expressing the expression cassette of any one of claims 15 to 18 in a host cell. 25. The method of claim 24, wherein the host cell is a mammalian cell as described in any one of claims 21 to 23. 26. The method of claim 24, further comprising a process of purifying the recombinant protein after expressing the expression cassette.