Fusion protein and preparation method thereof

Abstract

The invention relates to a fusion protein and a preparation method thereof. The fusion protein is a mutant SMut1 monomeric protein containing the full length of the porcine epidemic diarrhea virus, or a mutant SMut2 monomeric protein containing the full length of the porcine epidemic diarrhea virus, or a mutant SMut3 monomeric protein containing the full length of the porcine epidemic diarrhea virus. The fusion protein mutant is constructed, compared with wild type fusion protein, the immunogenicity of the mutant is remarkably improved, the Tm value is remarkably increased, and the proportion of formed trimer protein is as high as 95.2%. The CHO cell strain of the recombinant subunit fusion protein is constructed and screened, the yield of the recombinant subunit fusion protein expressed by the cell strain is up to 1.6 g / L, the recombinant subunit fusion protein is easy to purify, the purity of the target protein in cell culture supernatant can reach 85% or above, the purity of the target protein can reach 95% or above only through one-step affinity chromatography, and the recombinant subunit fusion protein can be obtained. The requirements of subunit vaccines and diagnostic reagents are greatly met, and large-scale production is easy.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and relates to a fusion protein and a preparation method thereof; in particular, to a fusion protein resistant to porcine epidemic diarrhea virus and a preparation method thereof. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is a single-stranded positive-sense RNA virus belonging to the Coronaviridae family and the genus Alphacoronavirus. Its virus particles are round or polygonal in shape, with a diameter ranging from 90 to 190 nm and an average diameter of 130 nm. PEDV virus particles are composed of four main structures, namely the outer membrane protein (M protein), envelope protein (E protein), nucleocapsid protein (N protein) and spike protein (S protein).

[0003] Porcine epidemic diarrhea virus can cause a viral disease in pigs, porcine epidemic diarrhea (PED), which causes diarrhea, vomiting, loss of appetite and other symptoms in pigs. PEDV can be transmitted through feces, respiratory secretions and other channels. The virus particles are highly stable in the environment and are easily transmitted in intensive breeding sites such as pig farms. The impact of PEDV on young pigs is particularly serious. Infected young pigs are prone to diarrhea, vomiting and other symptoms, and the mortality rate is high. At present, vaccines for PEDV have been developed, but PEDV has a large variability, and the development and application of vaccines still face certain challenges. Currently, the vaccines on the market are mainly inactivated vaccines and attenuated live vaccines. Because traditional vaccines have safety issues such as virulence reversion and incomplete inactivation, the long-term use of traditional vaccines provides favorable conditions for PEDV to continuously recombine and mutate in the field, resulting in its protective power decreasing year by year. Due to the low expression of the S protein of porcine epidemic diarrhea virus, the research and production of recombinant subunit vaccines also face great obstacles and challenges. How to develop a new type of efficient, safe and low-cost vaccine has become a highly concerned issue in the field. Summary of the invention

[0004] In order to study a subunit vaccine that can effectively prevent porcine epidemic diarrhea virus and make the vaccine have high immunogenicity and stability, the present invention proposes a fusion protein and a preparation method thereof; specifically, a subunit fusion protein rSMut2-His that can stably and efficiently express the full-length S protein of porcine epidemic diarrhea virus and a construction and expression method thereof in a CHO or 293T cell system is provided, and the proportion of the protein forming a trimer can reach 95.2%. The present invention analyzes and mutates the S fragment of porcine epidemic diarrhea virus, and screens mutants that can significantly increase protein stability, protein immunogenicity and expression, and its sequence is shown in SEQ ID NO.3. The monoclonal cell strain that can secrete and express the subunit fusion protein rSMut2-His obtained by the present invention has an expression yield of up to 1.6g / L. In previous related studies, the full-length expression of S protein was only 1g / L. By optimization, we increased the expression of S protein by 60%, from 1g / L to 1.6g / L. The neutralization titer of the screened mutant rSMut2 protein is 2.37 times that of the wild-type rS protein.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] <First aspect>

[0007] The present invention provides the following recombinant fusion protein, comprising a full-length wild-type S monomer protein of porcine epidemic diarrhea virus, the amino acid sequence of which is shown in SEQ ID NO.1;

[0008] Or, a mutant SMut1 monomer protein containing the full length of porcine epidemic diarrhea virus, whose amino acid sequence is shown in SEQ ID NO.2;

[0009] Or, a mutant SMut2 monomer protein containing the full length of porcine epidemic diarrhea virus, whose amino acid sequence is shown in SEQ ID NO.3;

[0010] Or, a mutant SMut3 monomer protein containing the full length of porcine epidemic diarrhea virus, whose amino acid sequence is shown in SEQ ID NO.4;

[0011] As one embodiment, a 6×His tag is connected to the C-terminus of SMut1, SMut2 or SMut3.

[0012] As one embodiment, the present invention provides the following recombinant (subunit) fusion proteins: rS-His, rSMut1-His, rSMut2-His, rSMut3-His, preferably rSMut2-His.

[0013] As an embodiment, the recombinant fusion protein rS-His is a protein with an amino acid sequence as shown in SEQ ID NO.5.

[0014] As an embodiment, the recombinant fusion protein rSMut1-His is a protein with an amino acid sequence as shown in SEQ ID NO.6.

[0015] As an embodiment, the recombinant fusion protein rSMut2-His is a protein with an amino acid sequence as shown in SEQ ID NO.7.

[0016] As an embodiment, the recombinant fusion protein rSMut3-His is a protein with an amino acid sequence as shown in SEQ ID NO.8.

[0017] As an embodiment, the expression system of the recombinant fusion protein includes but is not limited to mammalian cells.

[0018] As an embodiment, the mammalian cells include CHO cells and 293T cells. More preferably, the mammalian cells are CHO cells.

[0019] <Second Aspect>

[0020] The present invention provides a method for preparing a recombinant fusion protein, comprising the following steps:

[0021] S1. Cloning the gene encoding the recombinant fusion protein into a eukaryotic expression vector to obtain a recombinant plasmid containing the gene encoding the recombinant fusion protein;

[0022] For example, the gene encoding the rS-His fusion protein is cloned into a eukaryotic expression vector to obtain a recombinant plasmid containing the gene encoding the rS-His fusion protein;

[0023] Or the gene encoding the rSMut1-His fusion protein is cloned into a eukaryotic expression vector to obtain a recombinant plasmid containing the gene encoding the rSMut1-His fusion protein;

[0024] Or the gene encoding the rSMut2-His fusion protein is cloned into a eukaryotic expression vector to obtain a recombinant plasmid containing the gene encoding the rSMut2-His fusion protein;

[0025] Or the gene encoding the rSMut3-His fusion protein is cloned into a eukaryotic expression vector to obtain a recombinant plasmid containing the gene encoding the rSMut3-His fusion protein;

[0026] S2, transfecting the recombinant plasmid into a cell line;

[0027] For example, a recombinant plasmid containing the gene encoding the rS-His fusion protein is transfected into a CHO cell line;

[0028] Or transfect the recombinant plasmid containing the gene encoding the rSMut1-His fusion protein into the CHO cell line;

[0029] Or transfect the recombinant plasmid containing the gene encoding the rSMut2-His fusion protein into the CHO cell line;

[0030] Or transfect the recombinant plasmid containing the gene encoding the rSMut3-His fusion protein into the CHO cell line;

[0031] S3, fermentation, purification, and screening of recombinant fusion protein expressing cell lines;

[0032] Specifically, it includes: fermenting and culturing the transiently expressed CHO cells in step S2, purifying to obtain each recombinant fusion protein; screening out a recombinant fusion protein expressing cell strain with high expression amount and good immunogenicity;

[0033] S4, obtaining a highly expressing cell strain by culturing, screening, and acclimating the recombinant fusion protein expressing cell strain screened in step S3;

[0034] S5. Fermenting and culturing the highly expressing cell line, and obtaining the recombinant fusion protein after purification.

[0035] As an embodiment, in step S1, the gene sequence encoded by the rS-His fusion protein is shown as SEQ ID NO.9; the gene sequences encoded by the rSMut1-His, rSMut2-His, and rSMut3-His fusion proteins are shown as SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.

[0036] As an embodiment, in step S1, the eukaryotic expression vector is a mammalian cell expression vector. Preferably, the eukaryotic expression vector includes pEE6.4, pEE12.4, pGL4.13, pcDNA3.1, and more preferably, the eukaryotic expression vector is pcDNA3.1.

[0037] As an embodiment, in step S1, the cell line is a CHO cell line. The cell used to express the recombinant (subunit) fusion protein rSMut2-His is a CHO cell. Preferably, the CHO cell line includes DG44, DXB11, CHO-K1 or CHO-S cell line. More preferably, the CHO cell line is CHO-K1.

[0038] <Third Aspect>

[0039] The present invention provides a CHO cell line, which is deposited in China Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit time is September 3, 2024, the deposit name is Chinese Hamster Ovary Cell SLPCB07, and the deposit number is CCTCC NO: C2024299.

[0040] <Fourth Aspect>

[0041] The present invention provides the use of the above-mentioned recombinant fusion protein or CHO cell line in preparing a vaccine for preventing the viral disease porcine epidemic diarrhea.

[0042] <Fifth Aspect>

[0043] The present invention provides the use of the above-mentioned recombinant fusion protein or CHO cell line in preparing a medicine for preventing and / or treating the viral disease porcine epidemic diarrhea.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1) The present invention constructs and screens a CHO cell line that can stably and efficiently secrete a recombinant subunit fusion protein rSMut2-His that expresses the full-length S protein of porcine epidemic diarrhea virus in suspension. The cell line expresses the recombinant subunit fusion protein rSMut2-His at a yield of up to 1.6 g / L and is easy to purify. The purity of the target protein in the cell culture supernatant can reach more than 85%, and only one-step affinity chromatography is required to make the purity of the target protein reach more than 95%, which far exceeds the requirements of subunit vaccines and diagnostic reagents and is easy to mass produce.

[0046] 2) The present invention constructs a rSMut2-His fusion protein mutant, and the immunogenicity of the mutant is significantly improved compared with the wild-type fusion protein.

[0047] 3) The Tm value of the rSMut2-His fusion protein constructed by the present invention is significantly improved, and the proportion of the formed trimer protein is as high as 95.2%.

[0048] 4) In addition, the CHO cell lines used in production are highly controllable during culture, easy to control quality, produce stable protein batches, have high biosafety, are virus-free, and do not pose a risk of spreading the virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0050] Figure 1 This is the SDS-PAGE image of the purification of rS-His, rSMut1-His, rSMut2-His and rSMut3-His target proteins in Example 4;

[0051] Figure 2 TEM images of rS-His, rSMut1-His, rSMut2-His and rSMut3-His proteins in Example 5;

[0052] Figure 3 This is a schematic diagram of the pcDNA3.1-rSMut2-His plasmid map in Example 2. DETAILED DESCRIPTION

[0053] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are implemented on the premise of the technical solution of the present invention, provide detailed implementation methods and specific operation processes, and will help those skilled in the art to further understand the present invention. It should be pointed out that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made under the premise of the concept of the present invention all belong to the protection scope of the present invention.

[0054] Example 1: Optimization and synthesis of gene sequences.

[0055] Using CHO as the host cell, the present invention optimizes the codons of the base sequence encoding the recombinant subunit fusion protein rS-His (amino acid sequence as shown in SEQ ID NO.5), and the optimized base sequence is shown in SEQ ID NO.9, and the gene is marked as s-his in the embodiment; the base sequence encoding the recombinant subunit protein rSMut1-His, rSMut2-His, and rSMut3-His (amino acid sequences as shown in SEQ ID NO.6-8, respectively) is optimized by codons, and the optimized base sequence is shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, and the gene is marked as SMut1-his, SMut2-his, and SMut3-his in the embodiment. The optimized base sequence is synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0056] Example 2: Construction of recombinant expression vector.

[0057] 2.1 Construction of recombinant expression plasmids pcDNA3.1-rS-His, pcDNA3.1-rSMut1-His, pcDNA3.1-rSMut2-His, and pcDNA3.1-rSMut3-His.

[0058] (1) The s-his, SMut1-his, SMut2-his, and SMut3-his gene sequences were synthesized by GenScript Biotech, and a restriction endonuclease Nhe I site was introduced at the 5' end of the upstream sequence, wherein the sequence of the Nhe I site was GCTAGC; a restriction endonuclease Xho I site and a stop codon were introduced at the 3' end of the downstream sequence, wherein the sequence of the Xho I site was CTCGAG.

[0059] (2) The synthesized fragment was digested with endonuclease Nhe I and endonuclease Xho I, the gene fragment after enzyme digestion was recovered, and ligated to the pcDNA3.1 vector treated with the same endonuclease Nhe I and endonuclease Xho I. The ligation product was transformed into E. coli DH5α competent cells, spread on LB medium plates containing 100 μg / ml ampicillin, and cultured at 37°C. When the colonies on the plate were clearly visible, a single colony was picked and placed in 3 ml of liquid LB medium containing 100 μg / ml ampicillin, cultured at 37°C, and then the plasmid was extracted. The recombinant plasmids pcDNA3.1-rS-His, pcDNA3.1-rSMut1-His, and pcDNA3.1-rSMut2-His were obtained (the construction map is shown in FIG. Figure 3 As shown), pcDNA3.1-rSMut3-His, and the recombinant plasmid was confirmed to be consistent with the target sequence by sequencing.

[0060] 2.4 Preparation of plasmid for transfection.

[0061] (1) Refer to the instructions of the endotoxin-free plasmid extraction kit to extract 2 ml of plasmid pcDNA3.1-rS-His, pcDNA3.1-rSMut1-His, pcDNA3.1-rSMut2-His, and pcDNA3.1-rSMut3-His plasmids respectively.

[0062] (2) Take 1 μl of sample and perform agarose gel electrophoresis.

[0063] (3) Nanodrop was used to detect the concentration of the extracted plasmids, including 826 ng / ul of plasmid pcDNA3.1-rS-His, 834 ng / ul of pcDNA3.1-rSMut1-His, 786 ng / ul of pcDNA3.1-rSMut2-His, and 783 ng / ul of pcDNA3.1-rSMut3-His.

[0064] Example 3: Transient expression of target protein

[0065] (1) The cells used for transfection are CHO-K1 cells that have been adapted for suspension culture.

[0066] (2) One day before transfection (Day-1), CHO-K1 cells were cultured at 1×10 6 The cells were inoculated at a density of 10 viable cells / ml into a shake flask containing 30 ml of Acti CHO serum-free medium (Source Bio).

[0067] (3) On the day of transfection (Day 0), detect the viable cell density and viability. The viable cell density should reach 2×10 6 -6×10 6 The cell viability should reach 95-99%.

[0068] (4) Use 1500 μL Acti CHO serum-free medium to dilute 30 μg of each recombinant plasmid and mix thoroughly to form a dilution; immediately add 60 μL of PEI transfection reagent (1 mg / mL) to the dilution, vortex for 10 seconds, and mix thoroughly; incubate at room temperature for 15 minutes to form a DNA-PEI cationic nucleic acid transfection reagent complex.

[0069] (5) Add the complex to the cell solution and culture with shaking.

[0070] (6) On the second day after transfection (Day 2), samples were taken and analyzed by SDS-PAGE electrophoresis.

[0071] (7) On Day 6 after transfection, terminate the culture and collect the fermentation supernatant.

[0072] (8) The content of the target protein in the fermentation broth supernatant was detected by HPLC. The results are shown in Table 1.

[0073] Table 1 Expression of recombinant fusion protein

[0074]

[0075] From the above results, it can be seen that compared with the wild type, the transient expression levels of all mutant proteins are increased, and the expression level of rSMut2-His is the highest.

[0076] Example 4: Protein purification

[0077] (1) The fermentation supernatant was harvested, centrifuged at 8000 rpm for 30 min, and then filtered using a 0.45 μm filter membrane.

[0078] (2) Sample loading: Purification was performed using a 5 mL pre-packed Ni column at a flow rate of 2 mL / min.

[0079] (3) Washing: Buffer A (20 mM PB + 1 M NaCl + 30 mM imidazole), flow rate 2 mL / min.

[0080] (4) Elution: Buffer B (500 mM imidazole (pH 7.0)), flow rate 2 mL / min. Collect the elution peak.

[0081] (5) Balance: Buffer C (20 mM PB + 1 M NaCl (pH 7.2)), flow rate 2 mL / min.

[0082] (6) Storage: 20% ethanol, volume 10 mL, flow rate 2 mL / min.

[0083] (7) The collected elution peak samples were subjected to SDS-PAGE electrophoresis. Some of the results are as follows: Figure 1 As shown (Lane 1: Marker; Lane 2: rS-His protein; Lane 3: rSMut1-His protein; Lane 4: rSMut2-His protein; Lane 5: rSMut3-His protein), the protein purity was detected by grayscale analysis method, and it was found that the protein purity was higher than 95%.

[0084] (8) The trimer content in the target protein was detected by HPLC. The results showed that the content of rS-His trimer was 92.1%, the content of rSMut1-His trimer was 93.3%, the content of rSMut2-His trimer was 95.2%, and the content of rSMut3-His trimer was 62.9%.

[0085] Example 5: Stability test of recombinant fusion protein

[0086] 5.1DSF determination of Tm value of recombinant fusion protein

[0087] (1) Dilute 5000× Orange dye to 100×: add 2 μL of dye to 98 μL PBS and mix well;

[0088] (2) Dilute the protein to be tested: dilute the protein to 10 μM with PBS;

[0089] (3) Take 67.4 μL of diluted protein, add 2.6 μL of 100× dye, mix well and add to a 96-well plate, 20 μL per well, and repeat three times. Take the PBS group as the blank control. The results are shown in Table 2.

[0090] Table 2 Mean Tm of recombinant fusion protein

[0091] Target protein rS-His rSMut1-His rSMut2-His rSMut3-His Tm mean (℃) 52.3 52.8 53.4 51.7

[0092] The experimental results show that the thermal stability of rSMut1-His and rSMut2-His mutant recombinant fusion proteins is higher than that of the wild-type protein, among which rSMut2-His is more significant, and the thermal stability of rSMut3-His is reduced.

[0093] 5.2 Transmission electron microscopy observation of S protein trimer structure

[0094] The protein was diluted to 40 μg / mL with pure water, 10 μL was added to the copper mesh, adsorbed for 90 seconds, excess sample was removed with clean filter paper, and after drying, 10 μL of uranyl acetate was added for staining for 90 seconds, excess stain was removed with clean filter paper, and the sample was dried before observation. Figure 2 As shown (1, rS-His protein; 2, rSMut1-His protein; 3, rSMut2-His protein; 4, rSMut3-His protein), it can be seen that rSMut2-His forms an obvious trimer structure.

[0095] Example 6. Analysis of immunogenicity of recombinant fusion protein.

[0096] 6.1 Immunoassay sample preparation.

[0097] The purified recombinant fusion proteins rS-His, rSMut1-His, rSMut2-His and rSMut3-His were diluted to 200 μg / ml with PBS, and the diluted recombinant fusion proteins were emulsified with sterilized Montanide ISA 50V adjuvant at a volume ratio of 1:1 to prepare the samples required for immunization.

[0098] 6.2 Pig immunization experiment.

[0099] The prepared samples rS-His, rSMut1-His, rSMut2-His, rSMut3-His and PBS were divided into 5 groups for pig immunization experiments, with 5 pigs in each group. The immunization and blood collection process included: 1) about 5 ml of blood was collected before immunization; 2) Day 1: first immunization: 2 ml per pig; 3) day 21 booster immunization: 2 ml per pig; 4) day 35, 10 ml of blood was collected from each pig to detect neutralization titer.

[0100] 6.3 Neutralization titer detection

[0101] 6.3.1 Analysis of immunogenicity of rS-His, rSMut1-His, rSMut2-His, and rSMut3-His groups

[0102] This test compared the immunogenicity differences between the wild-type full-length S protein and the mutant S protein. The neutralizing antibody titer of the immune serum was tested using the virus neutralization test method, and the results are shown in Table 3.

[0103] Table 3 Analysis of immunogenicity of rS-His, rSMut1-His, rSMut2-His and rSMut3-His

[0104]

[0105]

[0106] The results show that on the 35th day of immunization, the immunogenicity of rSMut2-His was higher than that of rSMut1-His and higher than that of rS-His group, and the immunogenicity of rSMut3-His group protein was the lowest. This indicates that the immunogenicity of the S protein is uncertain after mutation. After screening, the recombinant fusion protein of the rSMut2-His mutant type was selected, and its neutralization titer was 2.37 times that of the wild-type protein group.

[0107] Example 7. Establishment of recombinant plasmid transfection into CHO-K1 cells and monoclonal screening.

[0108] 7.1 CHO-K1 cell transfection

[0109] (1) The cells used for transfection are CHO-K1 cells that have been adapted for suspension culture.

[0110] (2) One day before transfection (Day-1), CHO-K1 cells were cultured at 1×10 6 The cells were inoculated at a density of 10 viable cells / ml into a shake flask containing 30 ml of Acti CHO serum-free medium.

[0111] (3) On the day of transfection (Day 0), detect the viable cell density and viability. The viable cell density should reach 2×10 6 -6×10 6 The cell viability should reach 95-99%.

[0112] (4) Use 1500 μL Acti CHO serum-free medium to dilute 30 μg of the recombinant plasmid pcDNA3.1-rSMut2-His and mix thoroughly to form a diluent; immediately add 60 μL of PEI transfection reagent (1 mg / mL) to the diluent, vortex for 10 seconds, and mix thoroughly; incubate at room temperature for 15 minutes to form a DNA-PEI cationic nucleic acid transfection reagent complex.

[0113] (5) Add the complex to the cell solution and culture with shaking.

[0114] (6) On the second day after transfection (Day 2), samples were taken and analyzed by SDS-PAGE electrophoresis.

[0115] 7.2 Cell line screening

[0116] (1) On the second day after transfection (Day 2), add G418 solution with a final concentration of 500 μg / ml.

[0117] (2) On Day 7 after transfection, the viable cell density and viability were measured according to 1×10 6 The cells were inoculated into shake flasks containing Acti CHO serum-free medium at a density of 100 viable cells / ml.

[0118] (3) Subculture every 3-4 days until cell viability recovers to more than 90% and the viable cell density reaches 1×10 6 viable cells / ml.

[0119] (4) The monoclonal cell line was screened by the limiting dilution method to obtain the subclone with the highest expression level of the recombinant fusion protein in the supernatant.

[0120] (5) Three cell lines with high rSMut2-His expression were screened, namely 2F6, 3D6, and 8A3. The expression level of the screened 2F6 cell line was increased to 1.6 g / L, and that of the 3D6 and 8A3 cells was increased to 1.2 g / L and 1.51 g / L, respectively. The purity of the target protein in the culture supernatant of the three cell lines was 85%, 86%, and 86%, respectively.

[0121] (6) The screened cell lines are cryopreserved under liquid nitrogen conditions.

[0122] (7) The 2F6 cell line (2F6-rSMut2-His) has been submitted to the China Center for Type Culture Collection for preservation at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C2024299.

[0123] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A recombinant fusion protein, characterized in that: Contains any of the following monomeric proteins: A full-length mutant SMut1 monomer protein of porcine epidemic diarrhea virus, the amino acid sequence of which is shown in SEQ ID NO.2; A full-length mutant SMut2 monomer protein of porcine epidemic diarrhea virus, the amino acid sequence of which is shown in SEQ ID NO.3; The full-length mutant SMut3 monomer protein of porcine epidemic diarrhea virus, the amino acid sequence of which is shown in SEQ ID NO.

4.

2. The recombinant fusion protein according to claim 1, characterized in that The C-terminus of the SMut1, SMut2 or SMut3 is connected to a 6×His tag to obtain recombinant fusion proteins rSMut1-His, rSMut2-His and rSMut3-His, respectively.

3. The recombinant fusion protein according to claim 2, characterized in that The amino acid sequences of the recombinant fusion proteins rSMut1-His, rSMut2-His and rSMut3-His are shown in SEQ ID NO.6 to SEQ ID NO.8 respectively.

4. A method for preparing the recombinant fusion protein according to any one of claims 1 to 3, characterized in that: The steps include: S1. Cloning the gene encoding the recombinant fusion protein into a eukaryotic expression vector to obtain a recombinant plasmid containing the gene encoding the recombinant fusion protein; S2, transfecting the recombinant plasmid into a cell line; S3, fermentation, purification, and screening of recombinant fusion protein expressing cell lines; S4, obtaining a highly expressing cell strain by culturing, screening, and acclimating the recombinant fusion protein expressing cell strain screened in step S3; S5. Fermenting and culturing the highly expressing cell line, and obtaining the recombinant fusion protein after purification.

5. The preparation method according to claim 4, characterized in that: In step S1, the gene sequence encoded by the recombinant fusion protein is shown in any sequence of SEQ ID NO.10-SEQ ID NO.

12.

6. The preparation method according to claim 4, characterized in that: In step S1, the eukaryotic expression vector is a mammalian cell expression vector.

7. The preparation method according to claim 4, characterized in that: In step S2, the cell line shown is a CHO cell line.

8. A CHO cell line, which is Chinese hamster ovary cell SLPCB07, with the deposit number being CCTCCNO: C2024299.

9. Use of the recombinant fusion protein according to any one of claims 1 to 3 or the CHO cell line according to claim 8 in the preparation of a vaccine for preventing the viral disease porcine epidemic diarrhea.

10. Use of the recombinant fusion protein according to any one of claims 1 to 3 or the CHO cell line according to claim 8 in the preparation of a drug for preventing and / or treating the viral disease porcine epidemic diarrhea.

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