WO2020215266A1 - Recombinant protein for preventing swine fever virus infection and composition and cell containing the same - Google Patents

Abstract

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

Description

Recombinant protein for preventing swine fever virus infection, composition and cell containing the same Technical field

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

Background technique

Classical swine fever, also known as classical swine fever, is a contagious disease caused by swine fever virus. It has the characteristics of high infectivity and high mortality, which will cause a large number of pig deaths and cause serious losses in the pig industry. .

The current swine fever vaccines can be divided into three categories. 1. Traditional Lapinized Swine Fever Vaccine: This vaccine is manufactured by inoculating the weakened swine fever seed virus into rabbits, collecting organs at a specific time point, grinding, filtering and freeze-drying to make lapinized pigs Plague vaccine. 2. Tissue culture live virus vaccine: use weakened swine fever virus to infect pig kidney cells that are not contaminated by type 1 porcine circovirus. After virus propagation, virus liquid collection, filtration and freeze drying, tissue culture live virus vaccine is obtained. 3. Subunit vaccines: The currently commercially available subunit vaccines such as Bayovac CSF-E2 Vaccine are produced by an insect baculovirus expression system, which is to infect insect cells with a virus carrying the E2 gene to secrete recombinant E2 protein Expression, and then recombinant E2 protein to prepare vaccine.

In view of the harm and potential threats caused by swine fever to the pig industry, there is a need for more vaccines that can effectively prevent swine fever virus infection in order to provide more diversified and more efficient options for epidemic prevention.

Summary of the invention

One purpose of the present disclosure is to provide a novel recombinant protein and a composition containing the same, which can induce an immune protective response and achieve the purpose of preventing swine fever virus infection. Another objective of the present disclosure is to provide an expression cassette, an expression vector containing the same, and mammalian cells carrying these expression cassettes or expression vectors, which can be used to express the recombinant protein of the present disclosure.

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

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

The present disclosure further provides an expression cassette, which comprises: an expression element including a promoter; and a first polynucleotide and a second polynucleotide operably linked to the expression element; The encoding of the first polynucleotide is SEQ ID NO: 01, and the encoding of the second polynucleotide is an iron-carrying protein.

The present disclosure also provides an expression vector, which includes the expression 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, which comprises: expressing the expression cassette of the present disclosure in a host cell.

Description of the drawings

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

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

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

Fig. 4 is a protein electrophoresis diagram of Experiment 2, which shows the monomers and multimers of the recombinant protein disclosed in the present disclosure. The arrow points to the recombinant protein disclosed. M is a commercially available product BenchMark ^™ Protein Ladder (Thermo Fisher Scientific). DTT: Dithiothreitol. +: treated with DTT; -: treated without DTT.

FIG. 5 is the analysis result of the dynamic light scattering instrument in Experiment 2, which shows that the recombinant protein of the present disclosure forms nanoparticles.

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

Figure 7 shows the anti-swine fever virus antibody titer of the pig serum in experiment three.

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

Detailed ways

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

Unless the context clearly indicates otherwise, the singular form "a or an" in the description herein and the scope of the patent application includes a majority meaning. Thus, for example, "a protein" refers to one or more (a) proteins, and "a compound" refers to one or more (a) compounds. The use of "comprise", "comprises", "comprising", "include", "includes" and "including" are interchangeable, and Not restrictive. 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 "basically composed of... .. constitute" or "consisting of" instead.

When a certain range of numerical values is provided, unless the context clearly dictates otherwise, it should be understood that the integers of the numerical range and one tenth of each integer of the numerical range are between the upper and lower limits of the range , And any other stated values or intermediate values within the stated range are covered by this disclosure.

All documents, patents, patent applications, and other documents cited in this disclosure are fully incorporated herein as reference materials, and their contents are as pointed out in each independent document, patent, patent application or other document, and are incorporated herein. 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 form a protein. The "first polynucleotide encoding 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 encoded as 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 narratives 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.

As used herein, "prevention of swine fever virus infection" refers to the prevention of illness or symdrome caused by swine fever virus infection. Specifically, for example, preventing the occurrence of discomfort or symptoms caused by swine fever virus, or reducing the degree of discomfort or symptoms. Those skilled in the art should understand that the "prevention of swine fever virus infection" does not mean that the designated individual is completely free from swine fever virus infection, but from the viewpoint of epidemic prevention, the harm of swine fever virus to the designated individual is reduced .

The "sequence is SEQ ID NO" or similar descriptions as used herein means that the referred protein or polynucleotide contains the referred sequence, but is not limited to the referred sequence. For example, the "antigen portion whose amino acid sequence is SEQ ID NO: 01" as 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). :01), but those skilled in the art can modify the referred sequence based on general knowledge in the field according to their needs, so that the modified sequence includes sequences other than SEQ ID NO: 01. The present disclosure does not exclude the N-terminal or C-terminal extension of the protein of the present disclosure by 1 to several amino acids. The present disclosure does not exclude the extension of sequences of other proteins at the N-terminus or C-terminus of the protein of the present disclosure based on specific usage requirements. 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. In this way, the antibodies corresponding to these affinity tags can be used to detect the expression of recombinant protein (for example, applied to the Western blot method). This modified sequence should still belong to the scope of this disclosure unless it has lost the effect of this disclosure for preventing swine fever virus infection.

As used herein, "operably linked" means that two or more segments of polynucleotides are connected to each other by genetic engineering means, and these polynucleotides are guaranteed to be able to be used by the host (referred to herein as being used for expression). The nucleotide sequence of the organism) is recognized and encoded as the desired protein. Specifically, if it is necessary to fill in several nucleotides between the connected polynucleotides in actual operation, it must be ensured that the filled-in nucleotides will not cause the downstream polynucleotides to encode的Offset. For example, the full length of the sequence of the filled-in nucleotides should be a multiple of 3, because a codon should have 3 nucleotides.

The first aspect of this disclosure is about a recombinant protein and a composition containing the same. The recombinant protein is a fusion protein and includes an antigenic moiety and an iron carrying protein moiety (ferritin). The antigen part 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 host immune response. Preferably, the amino acid sequence of the antigen part is SEQ ID NO: 01. Feasibly, the antigen part is encoded by SEQ ID NO:03. Those skilled in the art should understand that when the antigen portion is expressed in different organisms, the sequence used to encode the antigen portion may be changed to meet the codon usage bias of the organism. ).

The iron-carrying protein is as defined in the art; preferably, the iron-carrying protein used in the present disclosure is partially derived from Helicobacter pylori. As used herein, "derived from Helicobacter pylori" 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 this 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 part is encoded by SEQ ID NO:04.

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

The composition for preventing swine fever virus infection of the present disclosure includes: the recombinant protein of the present disclosure and a pharmaceutically acceptable carrier. In a feasible embodiment, 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, it is 7.5 to 15μg/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, 21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,60μg/mL.

In a feasible aspect, the pharmaceutically acceptable carrier is water, phosphate buffered saline, alcohol, glycerol, chitin, alginate, chondroitin, vitamin E, minerals, or a combination thereof. In a specific embodiment, the composition is adjusted to be solid, liquid, or colloidal, depending on the needs of the user. In another embodiment, the composition is stored in a container (for example, a glass bottle) for the convenience of users.

In a preferred embodiment, the composition further includes an adjuvant. The adjuvant can be, but is not limited to: Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum glue, surfactant, anionic polymer, peptide, oil emulsion or a combination thereof. Specifically, commercially available adjuvants can be selected, for example, but not limited to: Montanide ^™ ISA 201 VG (SEPPIC, France). The ratio of the adjuvant to the recombinant protein may be determined according to the situation: feasible, the ratio is 1:1 (w/w).

The second aspect of the present disclosure relates to an expression cassette, an expression vector containing the same, and mammalian cells with these expression cassettes or expression vectors. The expression cassette referred to in the present disclosure refers to a polynucleotide comprising an expression element, and a first polynucleotide and a second polynucleotide operably linked to the expression element: The expression element includes 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 encodes SEQ ID NO: 02. In a feasible aspect, the second polynucleotide is SEQ ID NO: 04. Preferably, the recombinant protein disclosed in the present disclosure can be obtained after the expression cassette is expressed. Feasibly, the expression cassette is SEQ ID NO: 08.

The expression vector of the present disclosure is an expression cassette with the present disclosure. In a feasible aspect, the expression vector has a sequence that can be replicated in a predetermined host. In another feasible aspect, the expression vector further comprises a sequence encoding a signal peptide, 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 in the present disclosure refers to a mammalian cell, which is transfected into the cell by the cell engineering technology. Feasibly, the transfection is performed with electroporation technology.

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

The present disclosure also relates to a method for expressing the recombinant protein of the present disclosure, which comprises: expressing the expression cassette in the mammalian cell of the present disclosure. Feasibly, the expression cassette is an expression vector in the present disclosure. The method may further include 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.

1.1CHO cells and culture medium:

CHO-S cells (Thermo Fisher Scientific, USA) were used as host cells for the production of recombinant proteins. HyClone CDM4PERMAb culture medium (GE Healthcare, USA) was used for serum-free suspension culture of CHO cells, and additional Penicillin-Streptomycin (Penicillin-Streptomycin, Thermo Fisher Scientific; Penicillin final concentration was 100U/mL, Streptomycin final concentration 100μg/mL) and GlutaMAX ^TM Supplement (Thermo Fisher Scientific; final concentration is 6mM).

The semi-solid medium used to screen stable cell lines is ClonaCell ^TM -CHO ACF methylcellulose-based semi-solid medium (STEMCELL Technologies, USA), and additional hygromycin B (Hygromycin B, Thermo Fisher Scientific; final The concentration is 400μg/mL). In the subsequent process of amplifying and culturing 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:

The US GenScript company was commissioned to synthesize a polynucleotide (SEQ ID NO:07) that can encode the recombinant protein disclosed in the present disclosure according to the preferred codons of CHO cells. As shown in Table 1 below, the polynucleotide may encode a fusion protein of SEQ ID NO: 06, which includes the E2 protein of classical swine fever virus and the iron-carrying protein derived from Helicobacter pylori.

Table 1: The amino acid sequence of the fusion protein prepared in Experiment 1.

The polynucleotide is embedded in a mammalian cell expression vector. In addition to the polynucleotides used to encode the recombinant protein disclosed in the present disclosure, the expression vector used in this experiment still carries an enhancer-promoter from the immediately-early gene of human cytomegalovirus (CMV). promoter, sequence encoding mouse IgK secretory signal (immunoglobulin kappa secretory signal), and tag DNA (please refer to Figure 1). After confirming that the sequence of the expression vector is correct, DNA transfection was performed using Amaxa ^TM Cell Line Nucleofector ^TM Kit V (Lonza Bioscience, USA) transfection reagent and Nucleofector 2b Device electroporation cell transfection instrument. The number of CHO cells during transfection was 2×10 ⁶ , and the dosage of expression vector was 1 μg.

1.3 Screening of high antigen expression cell lines 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 select drug-resistant cell lines. The mini-pool selected by hygromycin B was cultured in ClonaCell ^™ -CHO ACF semi-solid medium at a concentration of about 600 cells/mL. After a single cell grows into a clump (about 7 to 9 days), use the ClonePix FL instrument to select candidate cell lines to culture in a 96-well plate. After the cells grow to nearly full coverage, move the cells to a 48-well plate and continue to culture for two days. Next, 100 μL of the cell culture supernatant was taken for sandwich enzyme-linked immunosorbent assay (ELISA) analysis to screen cell populations that can highly express the recombinant protein disclosed herein.

The capture antibody used in the ELISA method is a rabbit anti-His antibody (Rabbit anti-6-His Antibody, Bethyl Laboratories, USA); the detection antibody is a 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). Measure the absorbance of each well with an ELISA reader at 450nm. From the ELISA results, cell lines with high antigen expression were screened out.

Then, after culturing in a cell shake flask (125 mL) to confirm that the high antigen-expressing cell strains screened out above are not prone to clumping in suspension culture, the E2 protein-specific sandwich ELISA and protein electrophoresis analysis were used for further analysis. Screening of high antigen expressing cell lines.

The replenishing antibody used in ELISA specific to E2 protein is WH303 monoclonal antibody (APHA, UK); the labeled detection antibody is rabbit anti-c-myc antibody; the coloring agent used is TMB substrate solution. Measure the absorbance of each well with an ELISA reader at 450nm. Based on the results of ELISA and protein electrophoresis, cell lines with high antigen expression were screened out.

Afterwards, the selected cell lines were cultured in ClonaCell ^™ -CHO ACF semi-solid medium, and the above-mentioned screening steps were repeated 5 times.

The high antigen-expressing cell strains obtained after screening were mixed with CELLBANKER 2 (Nippon Zenyaku Kogyo, Japan) serum-free cell freezing solution and then frozen.

2. Experimental results.

The experimental results are shown in Figure 2. The C5-1 cell line has the best expression and the cell growth is stable. Therefore, in this experiment, the C5-1 cell line was selected as the seed cell for subsequent production of the recombinant protein disclosed herein, and the seed cell bank was established with it.

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

1. Materials and methods.

CHO cells from the seed cell bank were used for shaking flask culture with 5L medium. After culturing for 11 days, the cell culture solution 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. Recombinant protein was purified using immobilized metal ion affinity resin Ni Sepharose excel (GE Healthcare, Sweden). The dynamic light scattering instrument ZetaSizer ZEN 3600 (Malvern, USA) and the transmission electron microscope JEM-2100F (JEOL, Japan) were used to analyze the ability of recombinant proteins to form nanoparticles.

2. Experimental results.

Protein electrophoresis results showed that the C5-1 cell line can stably secrete and express recombinant proteins (Figure 3). In addition, extracellular recombinant proteins can be purified using immobilized metal ion affinity resin. The purified recombinant protein is treated with dithiothreitol (DTT), which can break the disulfide bond between protein molecules. The molecular weight of the monomer protein is about 70kDa; without DTT treatment, the purified recombinant protein will have intermolecular interactions. Form multimers (Figure 4).

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

Experiment 3: Vaccine preparation and pig immune challenge test.

1. Materials and methods.

1.1 Vaccine preparation:

The recombinant protein solution purified in experiment two was adjusted to a specific concentration and mixed with Montanide ^TM ISA 201VG adjuvant (SEPPIC, France) at a ratio of 1:1 (w/w) to prepare V-1311, V-1331, There are 5 vaccines, V-1332, V-1333 and V-1334 (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 the vaccine in a refrigerator at 4°C 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 Drugs Inspection Branch of the Animal Health Laboratory, Agricultural Committee of the Executive Yuan. A total of 20 specific pathogen-free (SPF) pigs at 9 weeks of age that tested negative for swine fever virus antibody were selected and randomly divided into groups A to F. The number of pigs in each group is 2 to 4; groups A to E are experimental groups, and group F is a control group. Pigs were immunized by intramuscular injection once at 9 weeks of age, with a dose of 2 mL. The pig test groups are as follows in Table 2.

Table 2: Vaccine and challenge trial design:

Group	Number of pigs	Composition of this experiment	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

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), 3-5 mL of blood was collected from the neck vein to prepare defibrillation Blood, and store in -80 ℃ refrigerator for later use. At 12 weeks of age (three weeks after immunization), the pigs in each group were injected intramuscularly with the highly virulent swine fever virus strain ALD (2 mL) into the experimental pigs for the challenge test. After the challenge, the pigs' clinical symptoms, temperature changes and survival rate were calculated daily. At 14 weeks of age (2 weeks after challenge), all pigs were sacrificed and anatomical pathological examination was performed.

2. Experimental results.

In this experiment, the injection sites of pigs in each group had no adverse reactions such as redness, swelling or ulceration, and the animals' spirit, mobility and appetite were normal, indicating that the vaccine has good safety. The commercial swine fever ELISA antibody detection kit (BioChek, UK) was used to analyze the serum of experimental pigs. The results showed that the anti-swine fever virus antibodies of each group of pigs before immunization (9 weeks old) were all negative, indicating that the experimental pigs Only before the experiment was really not infected. Anti-swine fever virus antibodies can be seen to rise in the sera collected three weeks after immunization (12 weeks old) of pigs administered the composition of the present disclosure; among them, the immunization amount of E2 antigen is 60, 30 and 15 μg The results of the /dose group were better (Figure 7).

Recording the survival rate of experimental pigs (Figure 8) shows that pigs administered with the composition of the present disclosure have improved survival rates, especially in the groups with E2 antigen immunity of 60, 30 and 15 μg/dose. All pigs survived the challenge. The results of this experiment should not be interpreted as 7.5 and 3.75μg/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, there are still individual differences in experiments. Therefore, the experimental results should be interpreted from a comprehensive perspective, which means that the composition of the present disclosure exhibits anti-swine fever virus effects at all experimental doses. Based on the above test results, it is shown that the composition of the present disclosure has good safety and only needs to be administered once with an immune amount above 15 μg/dose, which can provide pigs with the effect of resisting swine fever virus infection.

Claims (29)

1. A recombinant protein comprising:

   The antigen part, whose amino acid sequence is SEQ ID NO: 01; and

   Iron carrying protein (ferritin) part.
2. The recombinant protein of claim 1, wherein the antigen 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 according to claim 1, which comprises a linker to link the antigen part and the non-hematinous protein part; wherein the amino acid sequence of the linker is SEQ ID NO: 05.
7. The recombinant protein of claim 1, wherein the amino acid sequence of the recombinant protein is SEQ ID NO: 06.
8. The recombinant protein of claim 1, which is encoded by SEQ ID NO:07.
9. A composition for preventing swine fever virus infection, comprising: the recombinant protein according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.
10. The composition of claim 9, wherein the concentration of the recombinant protein is 1 to 60 μg/mL, which is based on the total volume of the composition.
11. The composition of claim 10, wherein the concentration of the recombinant protein is 7.5 to 30 μg/mL, which is based on the total volume of the composition.
12. The composition of claim 10, wherein the concentration of the recombinant protein is 7.5 to 15 μg/mL, which is based on the total volume of the composition.
13. The composition of claim 9, which further comprises an adjuvant.
14. The composition according to claim 13, wherein the adjuvant comprises: Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum glue, surfactant, anionic polymer, peptide, oil emulsion or a combination thereof.
15. The composition of claim 9, wherein the pharmaceutically acceptable carrier is water, phosphate buffered saline, alcohol, glycerin, chitin, alginate, chondroitin, vitamin E, minerals, or a combination thereof.
16. An expression cassette, which comprises:

    An expression element, which includes 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 Encoded as iron carrying protein.
17. The expression cassette of claim 16, wherein the second polynucleotide encodes SEQ ID NO: 02.
18. The expression cassette of claim 16, wherein the first polynucleotide is SEQ ID NO: 03, and the second polynucleotide is SEQ ID NO: 04.
19. The expression cassette according to claim 16, which expresses the recombinant protein according to any one of claims 1 to 8.
20. The expression cassette of claim 19, wherein the amino acid sequence of the recombinant protein is SEQ ID NO: 06.
21. The expression cassette of claim 16, which is SEQ ID NO: 08.
22. An expression vector comprising the expression cassette according to any one of claims 16-21.
23. The expression vector of claim 22, which is used in a mammalian cell expression system.
24. A mammalian cell with the expression cassette according to any one of claims 16 to 21.
25. The mammalian cell according to claim 24, which carries the expression vector according to any one of claims 22 to 23.
26. The mammalian cell of claim 24, which is a Chinese hamster ovary cell.
27. A method for expressing the recombinant protein according to any one of claims 1 to 8, comprising: expressing the expression cassette according to any one of claims 16 to 21 in a host cell.
28. The method according to claim 27, wherein the host cell is a mammalian cell according to any one of claims 24 to 26.
29. The method of claim 27, wherein after expressing the expression cassette, it further comprises a process of purifying the recombinant protein.

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