S protein mutants, vaccines, cell lines, antibodies, methods and applications of porcine deltacoronavirus

Abstract

The present invention provides an S protein mutant, vaccine, cell line, antibody, method and application of swine delta coronavirus; the S protein mutant has an amino acid sequence as shown in SEQ ID NO.3, and the present invention can achieve high expression of immunogens and enhance the immunogenicity of the S protein; based on the immune test of the S protein mutant, the present invention also obtains a hybridoma cell line that produces monoclonal antibodies against swine delta coronavirus, which can produce monoclonal antibodies against swine delta coronavirus.

Description

Technical Field

[0001] The present invention belongs to the field of immunization of swine delta coronavirus, and in particular relates to an S protein mutant, vaccine, cell line, antibody, method and application of the swine delta coronavirus. Background Art

[0002] Porcine deltacoronavirus (PDCoV), a recently discovered, highly contagious enteropathogenic coronavirus of pigs belonging to the genus Deltacoronavirus, can infect pigs of all ages, with piglets being the most susceptible. It causes severe clinical symptoms such as acute diarrhea, vomiting, dehydration, and even death. PDCoV is primarily transmitted via the fecal-oral route, but can also be transmitted via aerosols, primarily infecting the small intestine of pigs. Compared to PEDV and TGEV, PDCoV-infected piglets develop mild interstitial pneumonia in the lungs, and antigens can be detected in lung tissue homogenates and bronchial epithelial cells.

[0003] Chinese invention patent application publication number CN109655621A discloses an indirect ELISA method and kit for detecting antibodies against the N protein of swine deltacoronavirus. While the method uses recombinant N protein as a coating antigen and establishes a method for identifying antibodies produced by swine deltacoronavirus infection, it does not design mutants of the S protein, obtain immunogenic S protein mutants, or utilize S protein mutants to enhance immunogenicity. Furthermore, the method does not produce an antibody product against swine deltacoronavirus.

[0004] In view of this, it is necessary to provide a S protein mutant, vaccine, cell line, antibody, method and application of swine delta coronavirus to solve or at least alleviate the technical problems of how to obtain immunogenic S protein mutants, how to improve the immunogenicity of S protein, and how to obtain swine delta coronavirus monoclonal antibodies. Summary of the Invention

[0005] The main purpose of the present invention is to provide a S protein mutant, vaccine, cell line, antibody, method and application of swine delta coronavirus to solve the technical problems of how to obtain an immunogenic S protein mutant, how to improve the immunogenicity of the S protein, and how to obtain a monoclonal antibody against swine delta coronavirus.

[0006] To achieve the above objectives, the present invention provides an S protein mutant of swine delta coronavirus, wherein the S protein mutant has an amino acid sequence as shown in SEQ ID NO.3.

[0007] The present invention also provides a method for obtaining an immunogen of swine delta coronavirus, comprising: expressing any of the above-described S protein mutants through a eukaryotic expression system.

[0008] The present invention also provides a swine delta coronavirus vaccine, wherein the immunogen of the swine delta coronavirus vaccine comprises any of the S protein mutants described above.

[0009] The present invention also provides a method for preparing a swine delta coronavirus vaccine, comprising: obtaining an immunogen, and then mixing the immunogen with a vaccine adjuvant; the immunogen comprises any of the S protein mutants described above.

[0010] The present invention also provides a hybridoma cell strain that produces monoclonal antibodies to swine delta coronavirus. The hybridoma cell strain is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024107 and a deposit date of April 10, 2024.

[0011] The present invention also provides a monoclonal antibody against swine delta coronavirus, wherein the monoclonal antibody is an antibody produced by any of the hybridoma cell lines described above.

[0012] The present invention also provides a method for preparing any of the above-mentioned monoclonal antibodies, comprising: culturing the hybridoma cell line and collecting the supernatant.

[0013] The present invention also provides a swine delta coronavirus detection device, which comprises any of the monoclonal antibodies described above.

[0014] The present invention also provides a use of any of the above-described monoclonal antibodies in neutralizing swine delta coronavirus.

[0015] The present invention also provides a drug targeting swine delta coronavirus, wherein the drug comprises any of the above-mentioned monoclonal antibodies.

[0016] The beneficial effects of the present invention include at least:

[0017] The present invention provides an S protein mutant that can be highly expressed in a eukaryotic expression system to obtain an immunogenic S protein mutant, and the immunogenicity of the S protein can be enhanced. Specifically, compared to the extracellular domain of the wild-type swine deltacoronavirus S protein, the present invention obtains the S protein mutant by mutating glutamic acid at position 854 to proline, valine at position 855 to proline, and aspartic acid at position 862 to glutamine. In addition, based on immunoassays of the S protein mutant, the present invention obtains a hybridoma cell line that produces monoclonal antibodies against swine deltacoronavirus. The hybridoma cell line is capable of producing monoclonal antibodies against swine deltacoronavirus and has virus-neutralizing properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0019] Figure 1 This is the enzyme digestion verification diagram of the recombinant plasmid pcDNA3.4-PDCoV-S in Example 1;

[0020] Figure 2 This is the enzyme digestion verification diagram of pcDNA3.4-PDCoV-S-IGκSP in Example 1;

[0021] Figure 3 This is a Western Blot analysis of the recombinant protein in Example 1 (comparison with signal peptide replacement);

[0022] Figure 4 This is a Western Blot grayscale analysis of the recombinant protein in Example 1 (comparison with signal peptide replacement);

[0023] Figure 5 This is a double enzyme digestion verification diagram of the mutant recombinant plasmids pcDNA3.4-PDCoV-IGS-SE1, pcDNA3.4-PDCoV-IGS-SE 2, and pcDNA3.4-PDCoV-IGS-SE3 in Example 1;

[0024] Figure 6 This is a Western Blot analysis of the recombinant protein in Example 1 (comparison of various proteins);

[0025] Figure 7 This is a Western Blot grayscale analysis of the recombinant protein in Example 1 (comparison of various proteins);

[0026] Figure 8 Figure 1 is a diagram of the purification and identification of the recombinant protein IGS (PDCoV S protein extracellular domain) in Example 1;

[0027] Figure 9 This is a diagram of the purification and identification of the recombinant protein SE1 (mutant protein SE1) in Example 1;

[0028] Figure 10 This is a diagram of the purification and identification of the recombinant protein SE2 (mutant protein SE2) in Example 1;

[0029] Figure 11 This is a graph showing the virus neutralization experiment with serum from immune mice in Example 1;

[0030] Figure 12 This is a graph showing the antibody titer of the mouse immune serum in Example 1;

[0031] Figure 13 This is a diagram showing the Western Blot reaction results between MAb and S protein in Example 1;

[0032] Figure 14 This is a graph showing the results of the IFA reaction between MAb and PDCoV-infected ST cells in Example 1;

[0033] Figure 15 This is a graph showing the results of the neutralization test between MAb and PDCoV virus in Example 1.

[0034] The realization of the objectives, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0035] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Furthermore, the technical solutions between the various embodiments of the present invention can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. In the examples of the present invention, the PDCoV used is the PDCoV virus from China; when conducting various comparative experiments in this example, in the same comparative experiment, except for the experimental conditions that need to be compared, other experimental conditions remain consistent.

[0037] It is important to note that the spike protein (S protein) is a structural protein on the surface of PDCoV virus particles. It has both receptor binding and membrane fusion activities, playing a key role in viral attachment and invasion. This protein determines the host or tissue tropism of the virus and is also an important target for directly activating the immune response. Therefore, the S protein is an important target for the development of PDCoV antiviral drugs and vaccines.

[0038] The present invention utilizes a baculovirus expression system to efficiently express and purify mutants of the PDCoV S protein in insect cells, and prepares and screens PDCoV neutralizing antibodies by immunizing mice. This lays the foundation for exploring the protective mechanism of neutralizing antibodies and the development of vaccines, and is of great significance for the clinical diagnosis, epidemiological investigation, and epidemic prevention and control of the disease.

[0039] The present invention provides an S protein mutant of swine delta coronavirus, wherein the S protein mutant has an amino acid sequence as shown in SEQ ID NO.3; as an illustration, the specific mutation method of the S protein mutant is: compared with the extracellular domain of the wild-type swine delta coronavirus S protein, the glutamic acid at position 854 is mutated to proline, the valine at position 855 is mutated to proline, and the aspartic acid at position 862 is mutated to glutamine; the amino acid sequence of the extracellular domain of the wild-type swine delta coronavirus S protein is shown in SEQ ID NO.1.

[0040] In the present invention, PDCoV S protein refers to the extracellular domain of the S protein of swine delta coronavirus, and the wild-type protein corresponding to the S protein mutant is the extracellular domain of the S protein of swine delta coronavirus; the S protein mutant is the mutant protein SE2 in the embodiment of the present invention.

[0041] In the present invention, the amino acid sequence of SEQ ID NO.3 is as follows:

[0042]

[0043] The present invention also provides a method for obtaining an immunogen of swine delta coronavirus, comprising: expressing any of the above-mentioned S protein mutants through a eukaryotic expression system; and constructing a eukaryotic expression plasmid during the expression process.

[0044] In the present invention, the method of expressing any of the above-mentioned S protein mutants through a eukaryotic expression system may include: expressing the S protein mutant in insect cells using a mammalian expression system or a baculovirus expression system.

[0045] In the present invention, the signal peptide of the target gene of the S protein mutant is IGκ, thereby further increasing the expression level of the S protein mutant. It should be noted that designing a target gene based on the S protein mutant and its expression method is a conventional technical means in the field and is therefore not described in detail.

[0046] The present invention also provides a swine delta coronavirus vaccine, characterized in that the immunogen of the swine delta coronavirus vaccine includes any of the S protein mutants described above.

[0047] Specifically, the present invention utilizes the S protein mutant for immunization against swine delta coronavirus, producing antibodies. Furthermore, the S protein mutant exhibits higher immunogenicity than the wild-type S protein extracellular domain. As is conventional in the art, a vaccine adjuvant may be incorporated into the vaccine, and the vaccine may include a mixture of the vaccine adjuvant and the immunogen.

[0048] The present invention also provides a method for preparing a swine delta coronavirus vaccine, comprising: obtaining an immunogen, and then mixing the immunogen with a vaccine adjuvant; the immunogen comprises any of the S protein mutants described above.

[0049] The present invention also provides a hybridoma cell line that produces monoclonal antibodies against swine deltacoronavirus. The hybridoma cell line is PDCoV-S-2F7, deposited with the China Center for Type Culture Collection under the deposit number CCTCC NO: C2024107 on April 10, 2024, at Wuhan University, Wuhan, China. Specifically, the hybridoma cell line corresponds to the 2F7 hybridoma cell line described in the Examples of the present invention.

[0050] The present invention also provides a monoclonal antibody against swine delta coronavirus, wherein the monoclonal antibody is an antibody produced by any of the hybridoma cell lines described above.

[0051] The present invention also provides a method for preparing any of the above-mentioned monoclonal antibodies, comprising: culturing the hybridoma cell line and collecting the supernatant; the supernatant contains the monoclonal antibody.

[0052] The present invention also provides a swine deltacoronavirus detection device, which contains any of the monoclonal antibodies described above; the swine deltacoronavirus detection device may include a detection kit, which may include an ELISA kit for detecting swine deltacoronavirus; as another embodiment, the swine deltacoronavirus detection device may include a Western blot device for detecting swine deltacoronavirus.

[0053] The present invention also provides a use of any of the above-described monoclonal antibodies in neutralizing swine delta coronavirus.

[0054] The present invention also provides a drug against swine delta coronavirus, which comprises any of the monoclonal antibodies described above.

[0055] It should be noted that PDCoV is a new type of porcine enteric coronavirus and is also one of the important intestinal pathogens that cause diarrhea and vomiting in piglets in large-scale pig farming. The widespread prevalence and spread of PDCoV have seriously restricted the development of the pig industry. Compared with other structural proteins of PDCoV, the extracellular domain of PDCoV S protein has strong immunogenicity. Since the S protein is a trimeric class I transmembrane glycoprotein, it is anchored on the viral envelope, and most of the amino acids are located in the extracellular region. The expression of the extracellular domain protein can increase its secretory expression, and the S protein is a highly glycosylated protein. Glycosylation is very important for the conformation of natural proteins. The antigen protein prepared by the prokaryotic expression system lacks glycosylation modification and does not have sufficient biological activity. The eukaryotic expression system can correctly fold the protein and perform glycosylation modification, thereby obtaining a protein similar to the natural virus structure. The present invention uses the insect-baculovirus expression system to prepare the corresponding S protein to obtain the desired target protein.

[0056] The following are specific examples of the present invention:

[0057] Example 1

[0058] 1. Construction and expression comparison of recombinant plasmids with replaced signal peptides

[0059] The original sequence signal peptide of the PDCoV S protein extracellular domain target gene was replaced with IGκ. Amplification primers were designed based on the IGκ signal peptide nucleotide sequence, and restriction sites recognized by EcoRI and HindIII were added to the forward primer and reverse primer, respectively. The amplified fragment size of PDCoV-S-IGκSP was 3438 bp. The original sequence of the PDCoV S protein extracellular domain gene in GenBank is accession number MT260149.

[0060] The recombinant plasmid pcDNA3.4-PDCoV-S was constructed using the PDCoV S protein extracellular domain target gene; the recombinant plasmid pcDNA3.4-PDCoV-S-IGκSP was constructed by replacing the signal peptide of the PDCoV S protein extracellular domain target gene with IGκ; see Figure 1 As shown in the figure, after double enzyme digestion, the recombinant plasmid pcDNA3.4-PDCoV-S was detected by agarose gel electrophoresis. The results showed that two bands of about 6011bp and 3420bp appeared after enzyme digestion, indicating that the enzyme digestion results were correct. Figure 2 As shown, the recombinant plasmid pcDNA3.4-PDCoV-S-IGκSP was digested with double enzymes and detected by agarose gel electrophoresis. The results showed that two bands of approximately 6011bp and 3438bp appeared after enzyme digestion, respectively, indicating that the enzyme digestion results were correct. The recombinant plasmids pcDNA3.4-PDCoV-S and pcDNA3.4-PDCoV-S-IGκSP were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing and alignment with the template sequence. The recombinant plasmid pcDNA3.4-PDCoV-S-IGκSP was successfully constructed.

[0061] Figure 1 In the figure, M is DL5000 Marker, 1-4 are enzyme-digested recombinant plasmid pcDNA3.4-PDCoV-S; Figure 2 In the figure, M is DL5000 Marker, and 1-4 are enzyme-digested recombinant plasmid pcDNA3.4-PDCoV-S-IGκSP.

[0062] Recombinant plasmids pcDNA3.4-PDCoV-S and pcDNA3.4-PDCoV-S-IGκSP were transfected into HEK293FT cells, and the supernatants were collected after 48 h. Figure 3-4 As shown, Western Blot verification showed that the expression level of the recombinant plasmid pcDNA3.4-PDCoV-S-IGκSP was high, indicating that the expression level was higher after replacing the original sequence signal peptide with IGκSP; therefore, the IGκSP signal peptide was used in the construction of the recombinant plasmid in subsequent experiments; to avoid redundant descriptions and because the IGκSP signal peptide was used for subsequent replacement, the expression of IGκ was omitted in the subsequent description and expressed as IGS.

[0063] Figure 3 In the figure, M is protein marker, NC is the supernatant of cells not transfected with plasmid, S is the supernatant of cells transfected with recombinant plasmid pcDNA3.4-PDCoV-S, and IGS is the supernatant of cells transfected with recombinant plasmid pcDNA3.4-PDCoV-S-IGκSP.

[0064] Figure 4In the figure, S is the supernatant of cells transfected with the recombinant plasmid pcDNA3.4-PDCoV-S, and IGS is the supernatant of cells transfected with the recombinant plasmid pcDNA3.4-PDCoV-S-IGκSP.

[0065] 2. Construction and expression comparison of recombinant plasmids of different mutants

[0066] Site mutations were designed on the extracellular domain of the wild-type PDCoV S protein, and three groups of mutations were selected. The S protein mutant sequences were amplified separately to obtain mutant proteins SE1, SE2, and SE3, as shown in Table 1.

[0067] Table 1 Mutation sites of mutant proteins

[0068] Mutant name mutation site SE1 E854P、V855P SE2 E854P, V855P, D862Q SE3 I457P、T412P

[0069] In this example, PDCoV S protein refers to the extracellular domain of the wild-type PDCoV S protein, GenBank accession number is MT260149, and the amino acid sequence is shown in SEQ ID NO.1.

[0070] In this embodiment, the mutation mode of the mutant protein SE1 is as follows: compared with the aforementioned wild-type PDCoV S protein extracellular domain, the 854th glutamic acid is mutated to proline, and the 855th valine is mutated to proline.

[0071] The mutation mode of the mutant protein SE2 is as follows: compared with the aforementioned wild-type PDCoV S protein extracellular domain, the 854th glutamic acid is mutated to proline, the 855th valine is mutated to proline, and the 862nd aspartic acid is mutated to glutamine.

[0072] The specific mutation mode of the mutant protein SE3 is as follows: compared with the aforementioned wild-type PDCoV S protein extracellular domain, the 457th isoleucine is mutated to proline, and the 412th threonine is mutated to proline.

[0073] In this embodiment, the amino acid sequences of mutant protein SE1, mutant protein SE2, and mutant protein SE3 are shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively.

[0074] In this example, the original sequence signal peptides of the target genes corresponding to the PDCoV S protein extracellular domain, mutant protein SE-1, mutant protein SE-2, and mutant protein SE-3 were all replaced with IGκSP.

[0075] The target genes were digested and ligated to construct mutant recombinant plasmids, resulting in pcDNA3.4-PDCoV-S-IGκSP (abbreviated as IGS), pcDNA3.4-PDCoV-IGS-SE1 (abbreviated as SE1), pcDNA3.4-PDCoV-IGS-SE2 (abbreviated as SE2), and pcDNA3.4-PDCoV-IGS-SE3 (abbreviated as SE3).

[0076] See also Figure 5 As shown in the figure, the electrophoresis results of plasmid verification showed that two bands of approximately 6011 bp and 3438 bp appeared after enzyme digestion of SE1, SE2, and SE3, respectively, and the enzyme digestion results were correct; and, after sequence alignment analysis, the sequencing results and the template sequence alignment results showed that the amino acid mutation site was correct, indicating that the mutant recombinant plasmid was successfully constructed.

[0077] Figure 5 In the figure, M is DL5000 Marker, SE1 is pcDNA3.4-PDCoV-IGS-SE1, SE2 is pcDNA3.4-PDCoV-IGS-SE2, and SE3 is pcDNA3.4-PDCoV-IGS-SE3.

[0078] Each recombinant plasmid was transfected into HEK293FT cells, and the supernatant was collected after 48 h; see Figure 6-7 As shown, Western Blot analysis and identification showed that after transfection and expression, SE1, SE2, and SE3 all showed bands at approximately 180 kDa, which was consistent with the expected size, indicating that the recombinant plasmid was successfully expressed; among them, SE1 and SE2 were expressed at higher levels.

[0079] Figure 6 In the figure, M is protein marker, NC is the supernatant of cells not transfected with plasmid, IGS is the supernatant of cells transfected with recombinant plasmid pcDNA3.4-PDCoV-S-IGκSP, SE1 is the supernatant of cells transfected with recombinant plasmid pcDNA3.4-PDCoV-IGS-SE1, SE2 is the supernatant of cells transfected with pcDNA3.4-PDCoV-IGS-SE2, and SE3 is the supernatant of cells transfected with recombinant plasmid pcDNA3.4-PDCoV-IGS-SE3.

[0080] Figure 7In the figure, IGS is the supernatant of cells transfected with the recombinant plasmid pcDNA3.4-PDCoV-S-IGκSP, SE1 is the supernatant of cells transfected with the recombinant plasmid pcDNA3.4-PDCoV-IGS-SE1, SE2 is the supernatant of cells transfected with the recombinant plasmid pcDNA3.4-PDCoV-IGS-SE2, and SE3 is the supernatant of cells transfected with the recombinant plasmid pcDNA3.4-PDCoV-IGS-SE3.

[0081] 3. Expression and purification of recombinant proteins

[0082] The target genes corresponding to the PDCoV S protein extracellular domain, mutant protein SE1, and mutant protein SE2 (the original sequence signal peptides of the target genes were replaced by IGκSP) were used to construct recombinant plasmids pFastBacⅠ-PDCoV-IGS, pFastBacⅠ-PDCoV-IGS-SE1, and pFastBacⅠ-PDCoV-IGS-SE2, respectively.

[0083] The recombinant plasmid was transformed into DH10Bac competent cells and cultured by shaking. The recombinant bacmid was extracted and identified by PCR using universal primer M13. The recombinant bacmids Bacmid-IGS, Bacmid-SE1, and Bacmid-SE2 were transfected into Sf9 cells. The P3 generation baculovirus was inoculated into Sf9 cells and cultured for 48 hours. The cells and culture supernatant were collected. The culture supernatant was filtrated by changing the medium. After changing the medium, the filtrate was purified and verified. Figure 8-10 As shown, the SDS-PAGE results showed that the eluted recombinant proteins all had bands at about 180 KDa, indicating that three recombinant proteins were successfully purified, and the purified PDCoV S protein extracellular domain and mutant protein SE1 and mutant protein SE2 were obtained.

[0084] Figure 8-10 In the figure, M is the protein marker, the stock solution is the total protein of 500 mL of expressed supernatant, the filtrate is the protein filtered out by the liquid exchange and concentration; the liquid exchange is the protein after the liquid exchange and concentration, FT is the protein not bound to the nickel column filler, W is the protein eluted by low concentration imidazole, and E1-E9 are the proteins eluted by high concentration imidazole, respectively.

[0085] 4. Immunization of mice with S protein and mutants

[0086] As shown in Table 2, Freund's complete adjuvant was mixed with antigens (purified PDCoV S protein extracellular domain, mutant protein SE1, and mutant protein SE2, respectively), emulsified, and immunized with an antigen dose of 20 μg / mouse. Two weeks later, a second immunization was performed with antigens emulsified with incomplete Freund's adjuvant (the type and dose remained unchanged); two weeks later, a third immunization was performed in the same manner using antigens emulsified with incomplete Freund's adjuvant (the type and dose remained unchanged).

[0087] Table 2 Immunization methods

[0088]

[0089]

[0090] 5. Neutralizing antibody detection in serum of mice immunized with S protein and mutants

[0091] ST cells were plated in a 48-well plate, and when the cells grew to about 80%, the mouse serum one week after the third immunization in Part 4 of this example was diluted to a corresponding ratio (for details on the dilution multiple, see Figure 11 ) were mixed with PDCoV and incubated at 37°C for 1 h. The serum and PDCoV virus (100 TCID 50 ) mixture to infect cells, the PDCoV virus group alone was set as a positive control, and the group without PDCoV virus infection was set as a negative control. After infection at 37°C for 2 h, the medium was replaced with DMEM medium containing 0.4% trypsin, and the cells were cultured in a cell culture incubator at 37°C containing 5% CO2 for 48 h. The CPE was observed and the neutralization index was calculated.

[0092] See also Figure 11 As shown, it can be seen that: under the same experimental conditions, the neutralization level of the mouse serum after SE2 protein immunization was close to 100% after dilution of 1:50, and the neutralization level of the mouse serum was still 75% after dilution of 1:200, which was 50% higher than the neutralization level of the mouse serum immunized with SE1 protein. Therefore, the neutralization level of the mouse serum immunized with SE2 protein is higher.

[0093] Figure 11 In the table, IGS is the serum of mice immunized with the extracellular domain of PDCoV S protein, SE1 is the serum of mice immunized with the mutant protein SE1 protein, SE2 is the serum of mice immunized with the mutant protein SE2 protein, and PBS is the serum of mice immunized with PBS (negative control). Figure 11 The unit of the vertical axis is %.

[0094] 6. Determination of serum titer in mice immunized with recombinant protein and preparation of MAb

[0095] Freund's complete adjuvant was mixed with the purified mutant protein SE2 (antigen), emulsified, and immunized BALB / c mice at an antigen dose of 20 μg / mouse. Two weeks later, a second immunization was performed with the antigen emulsified with Freund's incomplete adjuvant (the type and dose remained unchanged). Two weeks later, a third immunization was performed in the same manner using the antigen emulsified with incomplete Freund's adjuvant (the type and dose remained unchanged). One week after the third immunization, blood was collected to detect serum titer. One day before the cell fusion experiment, no adjuvant was added, and the purified mutant protein SE2 was used for shock immunization at an antigen dose of 50 ug / mouse.

[0096] See also Figure 12 As shown, the ELISA method was used to determine the serum antibody titer of mice after the third immunization. The results showed that the serum titer of 6 mice reached 1:64000, OD 450nm The titers were greater than 0.8, with mice No. 86 and No. 83 having higher titers. In this example, mouse No. 83 was used for the cell fusion experiment.

[0097] Figure 12 Among them, numbers 81-86 refer to mice No. 81-86, respectively.

[0098] Mouse spleen cells were fused with SP2 / 0 cells and cultured in 6 96-well plates with 10% HAT medium. The plates were cultured in a cell culture incubator at 37°C and 5% CO2. On the 5th day of culture, half of the medium was replaced in each well. On the 9th day, the medium was replaced with 10% HT medium.

[0099] 7. Screening of positive clones by ELISA

[0100] The purified mutant protein SE2 was used as the coating antigen, the supernatant of the fused cells was used as the primary antibody, and the goat anti-mouse IgG-HRP antibody (1:5000) was used as the secondary antibody for ELISA detection. The culture medium was used as the negative control, and the immune mouse serum was used as the positive control. The standard for determining the positive hybridoma cell wells was: the OD value of the clone well 450nm Value / OD of negative control well 450nm If the value is >2, it is determined to be a positive hybridoma cell. The positive hybridoma cells are subcloned and purified three times, expanded and cultured, and then frozen.

[0101] In this example, the supernatants of the hybridoma cells after fusion were subjected to ELISA testing, with a P / N value > 2 being considered positive and a value < 2 being considered negative. The results showed that 18 positive hybridoma cell supernatants were screened with P / N values > 2 (e.g., MAbs 2E5, 4D5, 5D10, 2F7, and 5A9). Among them, the P / N value of MAb 2F7 was 36.4.

[0102] 8. Western Blot test

[0103] The purified mutant protein SE2 was added to 5× Loading Buffer and boiled. The proteins were separated by 8% SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% skim milk powder for 3 hours. The hybridoma cell supernatant and serum of mice immunized with S protein (mutant protein SE2) (1:3000) were incubated at room temperature for 1 hour, washed three times with PBST, and incubated with HRP-labeled rabbit anti-mouse IgG at 37°C for 45 minutes. The membranes were washed three times with PBST, and visualized using an ultra-sensitive ECL chemiluminescence kit.

[0104] See also Figure 13 As shown, MAb 2E5, 4D5, 5D10, 2F7 and 5A9 and mutant protein SE2 immune serum were respectively subjected to Western blot detection with purified mutant protein SE2 and Sf9 cell protein. The results showed that MAb 2E5, 4D5, 5D10, 2F7 and 5A9 monoclonal antibodies were able to recognize S protein (mutant protein SE2), indicating that the prepared mouse anti-PDCoVS protein monoclonal antibody can be used for Western blot detection.

[0105] Figure 13 In the table, 2E5, 4D5, 5D10, 2F7, and 5A9 are hybridoma cell supernatants, serum is the corresponding immune mouse serum, M is a protein marker, NC is an Sf9 cell protein, and S is an S protein (mutant protein SE2).

[0106] 9. Indirect immunofluorescence assay

[0107] PDCoV (MOI = 0.01) was inoculated into a monolayer of ST cells, and uninfected cells were set as a negative control. After 2 hours of infection, cell culture medium containing 0.4% trypsin and fetal bovine serum was added. After 48 hours, the sample was collected, the supernatant was discarded, and the cells were fixed with 4% paraformaldehyde. IFA detection was performed using hybridoma cell culture supernatant as the primary antibody and rabbit anti-mouse IgG-FITC antibody as the secondary antibody.

[0108] See also Figure 14 As shown, ST cells were infected with PDCoV, and the hybridoma cell supernatant was used as the primary antibody for IFA detection. The serum of mice immunized with S protein (mutant protein SE2) was used as the primary antibody for detection as the positive control group; the results showed that compared with the group not infected with PDCoV, the cells in the PDCoV-infected group were able to react with MAbs 2E5, 5D10, 4D5, 5A9, 2F7 and immune serum, and were localized around the cell nucleus, which was consistent with the positive control group, indicating that these 5 MAbs can be used for IFA detection of PDCoV strains.

[0109] Figure 14In the figure, 2E5, 4D5, 5D10, 2F7 and 5A9 are hybridoma cell supernatants, PC is a positive control, NC is a negative control, DAPI is stained cell nuclei (blue), PDCoV is infected with PDCoV (red), and Merge is the combination of the first two.

[0110] 10. Virus neutralization experiment

[0111] ST cells were plated in 48-well plates, and when the cells grew to about 80%, the hybridoma cell culture supernatant was mixed with PDCoV (100 TCID 50 ) and incubated at 37°C for 1 h. The supernatant and virus mixture was used to infect cells. The virus-infected group alone was set as the positive control, and the virus-uninfected group was set as the negative control. After infection at 37°C for 2 h, the medium was replaced with DMEM culture medium containing 0.4% trypsin, and the cells were cultured in a cell culture incubator at 37°C containing 5% CO2 for 48 h. The supernatant was discarded, and the cells were fixed with 4% paraformaldehyde. IFA detection was performed using the hybridoma cell culture supernatant as the primary antibody and rabbit anti-mouse IgG-FITC antibody as the secondary antibody.

[0112] See also Figure 15 As shown, PDCoV was mixed with hybridoma cell supernatant and then infected ST cells. N protein polyclonal antibody was used as the primary antibody for IFA detection and CPE determination. The results showed that compared with the PDCoV-infected group, after adding 2E5, 4D5, 5D10, 2F7 and 5A9 hybridoma cell supernatant, the specific fluorescence number was significantly lower than that of the infection group, which was consistent with the fluorescence number statistical results, and there was no obvious CPE phenomenon. The fluorescence number of the experimental group was reduced by more than 5 times compared with the control group (PDCoV-infected group); among them, the fluorescence number of the 2F7 group was the lowest in the experimental group. Compared with the control group (PDCoV-infected group), the virus neutralization degree reached 97.3%, and the neutralization effect was the best, indicating that 2F7 has an excellent neutralizing effect.

[0113] The 2F7 hybridoma cell line was expanded and cultured to obtain hybridoma cell 2F7 that can stably secrete antibodies. The monoclonal antibody subtype identification results showed that 2F7 was IgG1 type.

[0114] Figure 15 Among them, 2E5, 4D5, 5D10, 2F7 and 5A9 are hybridoma cell supernatants, and PC is the group infected with PDCoV alone.

[0115] The above technical solutions of the present invention are only preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made by using the contents of the present invention's description and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A mutant of the S protein of swine delta coronavirus, characterized in that: The amino acid sequence of the S protein mutant is shown in SEQ ID NO.

3.

2. A method for obtaining an immunogen of swine delta coronavirus, characterized in that: include: The S protein mutant according to claim 1 is expressed by a eukaryotic expression system.

3. A vaccine for swine delta coronavirus, characterized in that: The immunogen of the swine delta coronavirus vaccine includes the S protein mutant as described in claim 1.

4. A method for preparing a vaccine for swine delta coronavirus, characterized in that: include: Obtain an immunogen, and then mix the immunogen with a vaccine adjuvant; the immunogen includes the S protein mutant as described in claim 1.

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