A s protein mutant of porcine transmissible gastroenteritis virus, subunit vaccine and application thereof

Abstract

This invention provides a mutant S protein of porcine transmissible gastroenteritis virus (TGEV), a subunit vaccine, and its application; the amino acid sequence of the S protein mutant is shown in SEQ ID NO.3. The S protein mutant of TGEV constructed by this invention is significantly superior to the wild-type SWT protein in both humoral and cellular immunity, and its protein expression level also shows a clear advantage, demonstrating excellent prospects for subunit vaccine development and application. Specifically, in terms of immunity, after the S protein mutant is formulated into a vaccine, this invention can enhance post-immunization antibody levels, TGEV virus neutralizing antibody titers, cellular immune responses, and T lymphocyte proliferation.

Description

Technical Field

[0001] This invention belongs to the field of vaccine development for porcine transmissible gastroenteritis virus (TGEV), and particularly relates to a mutant S protein of TGEV and its subunit vaccine and its application. Background Technology

[0002] Transmissible gastroenteritis (TGE) is a viral diarrheal disease in pigs caused by the transmissible gastroenteritis virus (TGEV). Once piglets contract the disease, the mortality rate can reach 100%. TGEV mutates rapidly and easily produces recombinant strains, posing a significant potential threat to current public health. Therefore, developing novel TGEV subunit vaccines and monitoring its molecular epidemiology in real time are of paramount importance.

[0003] TGEV is an enveloped, single-stranded, positive-sense, linear RNA virus with a particle diameter of approximately 90-200 nm. The surface of the viral particle contains trimeric spikes measuring 12-25 nm. The TGEV S (Spike protein, S) protein is a type I transmembrane protein embedded on the surface of the viral particle. It is responsible for binding to specific receptors on the cell surface, mediating viral invasion. Antibodies against the S protein can effectively inhibit viral invasion and are one of the ideal antigens for current TGEV subunit vaccine development. However, when wild-type S protein is used as the active ingredient in a vaccine, its effectiveness in post-immunization antibody levels, TGEV virus neutralizing antibody titers, cellular immune responses, and T lymphocyte proliferation still needs further improvement.

[0004] In view of this, a vaccine and its application based on the S protein mutant of porcine transmissible gastroenteritis virus (TGEV) are provided to solve or at least alleviate the technical problems of how to improve the effectiveness of antibody levels after immunization, TGEV virus neutralizing antibody titers, cellular immune responses, and T lymphocyte proliferation. Summary of the Invention

[0005] The main objective of this invention is to provide a mutant S protein of porcine transmissible gastroenteritis virus (TGEV) and its subunit vaccine and application, in order to solve or at least alleviate the technical problems of how to improve the level of antibodies after immunization, the titer of neutralizing antibodies against TGEV, cellular immune response, and the proliferation of T lymphocytes.

[0006] To achieve the above objectives, the present invention provides an S protein mutant of porcine transmissible gastroenteritis virus, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] The present invention also provides a nucleic acid molecule encoding an S protein mutant of porcine transmissible gastroenteritis virus, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0008] The present invention also provides a recombinant expression vector having a nucleotide sequence encoding any of the S protein mutants described above.

[0009] The present invention also provides a recombinant expression vector having any of the nucleic acid molecules described above.

[0010] The present invention also provides an engineered cell capable of expressing any of the S protein mutants described above.

[0011] The present invention also provides a method for expressing a mutant S protein of porcine transmissible gastroenteritis virus, comprising: constructing a recombinant expression vector as described above, and expressing it through engineered cells.

[0012] The present invention also provides the application of any of the S protein mutants described above in the preparation of vaccines against porcine transmissible gastroenteritis virus.

[0013] The present invention also provides a subunit vaccine for porcine transmissible gastroenteritis virus, wherein the porcine transmissible gastroenteritis virus vaccine comprises any of the S protein mutants described above.

[0014] Furthermore, the vaccine for porcine transmissible gastroenteritis virus also includes a vaccine adjuvant.

[0015] The present invention also provides the use of any of the S protein mutants described above in one or more of the following:

[0016] A: Preparation of antibodies against porcine transmissible gastroenteritis virus;

[0017] B: Induces cellular immune response;

[0018] C: Induces T lymphocyte proliferation.

[0019] The beneficial effects of the present invention include at least the following:

[0020] In obtaining the S protein mutant, this invention optimized the structure of the TGEV S protein sequence by mutating glutamic acid (E) and leucine (L) at positions 1139-1140 of the TGEV S protein to two proline residues, and mutating leucine (L) at position 1091 and alanine (A) at position 1092 of the TGEV S protein to two proline residues. The porcine transmissible gastroenteritis virus (TGEV) S protein mutant constructed by this invention significantly outperforms the wild-type SWT protein in terms of protein expression level and immunogenicity (humoral and cellular immunity), showing great promise for subunit vaccine development. Specifically, this invention can enhance post-immunization antibody levels, TGEV virus neutralizing antibody titers, cellular immune responses, T lymphocyte proliferation, and increase the expression level of the target protein. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0022] Figure 1 The results of transmembrane region analysis of TGEV S protein in Example 1 of this invention (membrane: transmembrane; inside: intracellular region; outside: extracellular region);

[0023] Figure 2 This is the pFastBac 1-TGEV-SWT plasmid map in Example 1 of the present invention;

[0024] Figure 3 This is the pFastBac 1-TGEV-SM4 plasmid map in Example 1 of the present invention;

[0025] Figure 4 The Western blot results of TGEV S protein in Example 1 of this invention are shown in Figure A: Western blot results; B: relative protein expression level; 1: SWT protein; 2: SM4 protein.

[0026] Figure 5The results of SDS-PAGE and Native-PAGE of TGEV S protein in Example 1 of this invention are shown below (A: SDS-PAGE result; B: Native-PAGE result; M: molecular weight of standard protein; SWT: TGEV SWT protein; SM4: TGEV SM4 protein).

[0027] Figure 6 The results of TGEV indirect ELISA antibody level detection in Example 1 of this invention (NC: negative control group; WT: SWT immunization group; M4: SM4 immunization group);

[0028] Figure 7 The results of neutralizing antibody detection in Example 1 of this invention (NC: negative control group; WT: SWT immunization group; M4: SM4 immunization group);

[0029] Figure 8 The results of ELISpot cell immunoassay in Example 1 of this invention are as follows (NC: negative control group; WT: SWT experimental group; M4: SM4 experimental group; PMA: phorbol ester positive control; Background: background negative control).

[0030] Figure 9 The results of the T lymphocyte proliferation experiment in Example 1 of this invention (NC: negative control group; WT: SWT experimental group; M4: SM4 experimental group; Con-A: concanavalin A positive control).

[0031] The realization of the objective, functional characteristics and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0034] This invention provides a mutant S protein of porcine transmissible gastroenteritis virus, the amino acid sequence of which is shown in SEQ ID NO.3; in this invention, the S protein mutant refers to the SM4 protein (TGEVSM4 protein) in the examples; this invention is mainly designed for the extracellular region of the porcine transmissible gastroenteritis virus S protein.

[0035] The present invention also provides a nucleic acid molecule encoding an S protein mutant of porcine transmissible gastroenteritis virus as described above, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0036] This invention also provides a recombinant expression vector having a nucleotide sequence encoding any of the S protein mutants described above. Specifically, the nucleotide sequence can be the nucleotide sequence shown in SEQ ID NO.4; in this case, this invention also provides a recombinant expression vector having a nucleic acid molecule as described above. This invention can obtain the recombinant expression vector by cloning the nucleic acid molecule into the pFastBac 1 vector via XbaⅠ and KpnⅠ restriction enzyme sites, or by cloning the nucleic acid molecule into the pcDNA3.4 eukaryotic expression vector.

[0037] This invention also provides an engineered cell capable of expressing any of the S protein mutants described above; specifically, the S protein mutant can be expressed using any of the nucleic acid molecules described above. The engineered cell can be obtained by: cloning the nucleic acid molecule into the pFastBac1 vector via XbaⅠ and KpnⅠ restriction sites, and then transfecting the vector into Sf9 cells; or by: cloning the nucleic acid molecule into the pcDNA3.4 eukaryotic expression vector, and then transfecting the vector into 293FT cells.

[0038] This invention also provides a method for expressing a mutant S protein of porcine transmissible gastroenteritis virus, comprising: constructing a recombinant expression vector as described above, and expressing it using engineered cells. Specifically, this invention clones the nucleic acid molecule into the pFastBac1 vector via XbaⅠ and KpnⅠ restriction enzyme sites, and expresses the S protein using Sf9 cells; or, this invention clones the nucleic acid molecule into the pcDNA3.4 eukaryotic expression vector, and expresses the S protein using 293FT cells.

[0039] In this invention, compared with the wild-type S protein of porcine transmissible gastroenteritis virus that has not undergone targeted mutation, the expression level of the S protein mutant is significantly increased, reaching 9 times that of the wild-type S protein.

[0040] The present invention also provides the application of the S protein mutant as described above in the preparation of a vaccine for porcine transmissible gastroenteritis virus; the application process includes: expressing the S protein mutant using the expression method described above, and using the S protein mutant of porcine transmissible gastroenteritis virus as an immunogen.

[0041] The present invention also provides a subunit vaccine for porcine transmissible gastroenteritis virus, wherein the porcine transmissible gastroenteritis virus vaccine includes any of the S protein mutants described above; the vaccine is a subunit vaccine; and the porcine transmissible gastroenteritis virus vaccine further includes a vaccine adjuvant.

[0042] In this invention, the S protein mutant and the vaccine exhibit outstanding effects in terms of post-immunization antibody levels, TGEV virus neutralizing antibody titers, cellular immune responses, and T lymphocyte proliferation; therefore, this invention also provides an application of the S protein mutant as described above in one or more of the following:

[0043] A: Preparation of antibodies against porcine transmissible gastroenteritis virus;

[0044] B: Induces cellular immune response;

[0045] C: Induces T lymphocyte proliferation.

[0046] In this invention, the S protein mutant is used as a vaccine; the application is for non-disease treatment and non-disease diagnosis purposes, and is only used for scientific research or antibody acquisition.

[0047] Specifically, the present invention also provides the use of the S protein mutant as described above in the preparation of antibodies against porcine transmissible gastroenteritis virus.

[0048] The present invention also provides the application of any of the S protein mutants described above in inducing cellular immune responses.

[0049] This invention also provides the application of any of the S protein mutants described above in inducing T lymphocyte proliferation. Specific examples of this invention are as follows:

[0050] Example 1

[0051] 1. Codon sequence optimization and recombinant plasmid construction of the TGEV S protein gene:

[0052] The TGEV S protein is a type I transmembrane glycoprotein containing extracellular, transmembrane, and intracellular regions. The transmembrane and intracellular regions were predicted using TMHMM2.0 software, and the extracellular region of the S protein was selected. The signal peptide of the S protein was predicted and analyzed using SignalP-5.0 software. The extracellular region of the S protein from a clinically isolated strain (TGEV-SHXB strain) was selected. A T4foldon trimer motif and an 8×His Tag sequence were introduced to the C-terminus of the S gene. After codon sequence optimization, the S protein gene sequence was ligated into the pFastBac1 vector using XbaI and KpnI restriction sites, named pFastBac1-TGEV-SWT. Simultaneously, the S gene was cloned into the pcDNA3.4 eukaryotic expression vector, named pcDNA3.4-TGEV-SWT.

[0053] In this embodiment, the TMHMM2.0 software prediction analysis results showed that the first 1388 aa of the TGEV S protein were located in the extracellular region, 1389-1410 aa were located in the transmembrane region, and 1411-1447 aa were located in the intracellular region. Figure 1 The SignalP-5.0 software prediction results show that 1-16aa is the signal peptide region.

[0054] In this embodiment, the TGEV S protein gene sequence was cloned into the pFastBac1 vector using XbaⅠ and KpnⅠ restriction sites. The recombinant plasmid was named pFastBac1-TGEV-SWT, and the recombinant plasmid map is shown below. Figure 2 The plasmid sequencing and alignment results showed that it was consistent with the target gene sequence, indicating that the recombinant plasmid was successfully constructed.

[0055] The amino acid sequence of the TGEV SWT protein is shown in SEQ ID NO.1. The amino acid sequence of SEQ ID NO.1 is as follows:

[0056] LA TVAKALAKVQDVVNIQGQALSHLTVQLQNNFQAISSSISDIYNRLD EL SADAQVDRLITGRLTALNAFVSQTLTRQAEVRASRQLAKDKVNECVRSQSQRFGFCGNGTHLFSLANAAPNGMIFFHTVLLPTAYETVTAWPGICASDGDRTFGLVVKDVQLTLFRNLDDKFYLTPRTMYQPRVATSSDFVQIE GCDVLFVNATVSDLPSIIPDYIDINQTVQDILENFRPNWTVPELTFDIFNATYLNLTGEIDDLEFRSEKLHNTTVELAILIDNINNTLVNLEWLNRIETYVKGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGSHHHHHHHH.

[0057] The nucleotide sequence encoding the TGEV SWT protein is shown in SEQ ID NO.2. The nucleotide sequence of SEQ ID NO.2 is as follows:

[0058] CTTGCC ACCGTTGCGAAGGCCCTTGCCAAAGTACAGGATGTAGTGAATATTCAAGGCCAAGCTCTGTCACACCTAACCGTACAATTGCAGAACAACTTTCAGGCTATTTCTAGCTCGATCTCTGATATATACAACCGGCTAGAC GAGCTTTCCGCCGATGCACAGGTCGATCGATTGATCACTGGTCGTCTAACGGCTTTAAACGCGTTCGTGTCACAGACTCTGACTCGCCAGGCTGAAGTAAGGGCGTCAAGGCAATTGGCAAAGGATAAGGTCAACGAATGCGTCCGTTCCCAATCACAGAGGTTTGGCTTTTGCGGCAACGGGACACATCTATTCTCTCTAGCCAATGCAGCCCCGAACGGCATGATCTTTTTCCACACTGTCCTGCTGCCAACCGCTTATGAGACTGTGACTGCATGGCCGGGGATCTGCGCCAGTGACGGCGATCGTACCTTTGGTCTCGTAGTGAAAGACGTGCAACTCACCCTTTTCCGCAACCTCGACGATAAGTTCTATTTAACTCCCAGAACCATGTACCAACCGCGGGTAGCCACGTCCTCCGATTTTGTTCAGATTGAAGGTTGTGATGTGCTCTTTGTCAACGCAACGGTGTCGGACCTACCATCCATCATTCCAGACTATATTGACATCAATCAGACGGTTCAGGACATTTTGGAGAACTTCCGTCCGAATTGGACAGTCCCCGAACTTACTTTTGATATTTTCAACGCGACATATCTGAATTTGACCGGGGAGATAGACGACCTTGAATTTCGTTCCGAAAAGTTGCATAACACGACCGTCGAGTTAGCGATACTGATCGATAATATAAATAACACACTCGTAAACCTTGAATGGTTGAATCGAATAGAGACCTATGTTAAGGGGGGAGGATCAGGCTATATACCCGAAGCACCCAGGGATGGACAGGCTTACGTAAGAAAAGACGGCGAATGGGTATTATTAAGTACCTTCCTCGGGAGCCACCATCACCATCACCATCATCACTAA。

[0059] 2. Design and construction of recombinant plasmid of TGEV S protein mutant:

[0060] Protein homology modeling was performed using SWISS-MODEL to predict the three-dimensional structure of the TGEV S protein. The three-dimensional structure of the S protein was analyzed using PyMOL software to screen key amino acid sites that maintain the stability of the S protein structure. A multi-site proline (P) mutation strategy was used to design the S protein with a stable conformation before fusion. Primers were designed, and amino acid sites were mutated using a rapid site-directed mutagenesis kit to construct a recombinant expression plasmid of the TGEV S protein mutant, named pFastBac1-TGEV-SM4. At the same time, the S gene was cloned into the pcDNA3.4 eukaryotic expression vector, named pcDNA3.4-TGEV-SM4.

[0061] This embodiment analyzes the three-dimensional structure of the TGEV S protein and finds that the glutamic acid (E) and leucine (L) at positions 1139-1140 of the TGEV S protein are key amino acid sites connecting the S2 subunit HR1 (Helixregion 1) and CH (Central helix). 1139 EL 1140 The mutation resulted in two proline residues. 1139 PP 1140 This can disrupt the formation of secondary structures. Using the same strategy, the leucine (L) at position 1091 and the alanine (A) at position 1092, which connect the two helical structures, are mutated into two proline residues. 1091 PP 1092 The mutant protein was named S mutant 4 (SM4). The recombinant plasmid pFastBac1-TGEV-SM4 was successfully constructed using a site-directed mutagenesis kit. The recombinant plasmid map is shown below. Figure 3 The plasmid sequencing results were consistent with the target gene sequence.

[0062] The amino acid sequence of the TGEV SM4 protein is shown in SEQ ID NO.3. The amino acid sequence of SEQ ID NO.3 is as follows:

[0063] PP TVAKALAKVQDVVNIQGQALSHLTVQLQNNFQAISSSISDIYNRLD PP SADAQVDRLITGRLTALNAFVSQTLTRQAEVRASRQLAKDKVNECVRSQSQRFGFCGNGTHLFSLANAAPNGMIFFHTVLLPTAYETVTAWPGICASDGDRTFGLVVKDVQLTLFRNLDDKFYLTPRTMYQPRVATSSDFVQIE GCDVLFVNATVSDLPSIIPDYIDINQTVQDILENFRPNWTVPELTFDIFNATYLNLTGEIDDLEFRSEKLHNTTVELAILIDNINNTLVNLEWLNRIETYVKGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGSHHHHHHHH.

[0064] The nucleotide sequence encoding the TGEV SM4 protein is shown in SEQ ID NO.4. The nucleotide sequence of SEQ ID NO.4 is as follows:

[0065] CCTCCC ACCGTTGCGAAGGCCCTTGCCAAAGTACAGGATGTAGTGAATATTCAAGGCCAAGCTCTGTCACACCTAACCGTACAATTGCAGAACAACTTTCAGGCTATTTCTAGCTCGATCTCTGATATATACAACCGGCTAGAC CCACCCTCCGCCGATGCACAGGTCGATCGATTGATCACTGGTCGTCTAACGGCTTTAAACGCGTTCGTGTCACAGACTCTGACTCGCCAGGCTGAAGTAAGGGCGTCAAGGCAATTGGCAAAGGATAAGGTCAACGAATGCGTCCGTTCCCAATCACAGAGGTTTGGCTTTTGCGGCAACGGGACACATCTATTCTCTCTAGCCAATGCAGCCCCGAACGGCATGATCTTTTTCCACACTGTCCTGCTGCCAACCGCTTATGAGACTGTGACTGCATGGCCGGGGATCTGCGCCAGTGACGGCGATCGTACCTTTGGTCTCGTAGTGAAAGACGTGCAACTCACCCTTTTCCGCAACCTCGACGATAAGTTCTATTTAACTCCCAGAACCATGTACCAACCGCGGGTAGCCACGTCCTCCGATTTTGTTCAGATTGAAGGTTGTGATGTGCTCTTTGTCAACGCAACGGTGTCGGACCTACCATCCATCATTCCAGACTATATTGACATCAATCAGACGGTTCAGGACATTTTGGAGAACTTCCGTCCGAATTGGACAGTCCCCGAACTTACTTTTGATATTTTCAACGCGACATATCTGAATTTGACCGGGGAGATAGACGACCTTGAATTTCGTTCCGAAAAGTTGCATAACACGACCGTCGAGTTAGCGATACTGATCGATAATATAAATAACACACTCGTAAACCTTGAATGGTTGAATCGAATAGAGACCTATGTTAAGGGGGGAGGATCAGGCTATATACCCGAAGCACCCAGGGATGGACAGGCTTACGTAAGAAAAGACGGCGAATGGGTATTATTAAGTACCTTCCTCGGGAGCCACCATCACCATCACCATCATCACTAA。

[0066] 3. Comparative analysis of the expression levels of TGEV S protein (TGEV SWT and SM4 proteins):

[0067] (1) TGEV SWT and SM4 proteins were expressed in 293FT cells. Before transfection, 293FT cells were seeded into 6-well plates at a cell density of 0.5 × 10⁻⁶ cells / well. 6 cells / mL.

[0068] (2) On the day of transfection, discard the culture medium in the well and add diluted expression plasmids pcDNA3.4-TGEV-SWT and pcDNA3.4-TGEV-SM4. Transfect 2 μg in each well. 6 hours after transfection, discard the culture medium and add 2 ml of DMEM medium containing 2% FBS in each well. Incubate in a constant temperature incubator at 37℃ and 5% CO2.

[0069] (3) Collect the cell culture supernatant from each well 48 h after transfection and verify the protein expression level by Western blot.

[0070] In this embodiment, pcDNA3.4-TGEV-SWT and pcDNA3.4-TGEV-SM4 plasmids were transfected into 293FT cells, and the expression level of the target protein in the cell supernatant was detected by Western blot. (See also...) Figure 4 As shown in the figure, the results indicate that the expression level of mutant SM4 protein is 9 times that of wild-type SWT protein.

[0071] 4. Expression and purification of TGEV S protein:

[0072] 4.1 Expression of TGEV S proteins (TGEV SWT and SM4 proteins):

[0073] (1) Extraction of recombinant Bacmid DNA: After transposition of the recombinant plasmids (pFastBac 1-TGEV-SWT and pFastBac 1-TGEV-SM4) into DH10 competent cells, white positive clones were picked from the plate, and the recombinant Bacmid DNA was extracted and identified by PCR.

[0074] (2) Transfection: Recombinant proteins were expressed using Sf9 cells. Approximately 9 × 10⁶ cells were added to each well of a six-well plate. 5 Each well of Sf9 cells was transfected with 2 μg of recombinant Bacmid DNA and cultured at 27°C for 4 days before harvesting P1 cells.

[0075] (3) Recombinant baculovirus amplification: On the same day as amplifying P2 generation baculovirus, the cell density was adjusted to 2×10⁻⁶. 6 Normal cells were infected with P1 generation baculovirus with an MOI of 0.05 and cultured at 27°C and 130 rpm for 48 h before harvesting P2 cells.

[0076] (4) Protein expression: The cell density was adjusted to 2×10⁻⁶.6 Sf9 cells / mL were infected with P2 generation baculovirus with MOI=1.0 and cultured at 27℃ and 130rpm for 72h before being collected.

[0077] 4.2 Purification of TGEV S protein (TGEV SWT and SM4 protein):

[0078] The S protein was purified using nickel-column affinity chromatography. Cell culture supernatant was collected 72 h post-transfection. The culture product was centrifuged at 3,000 g for 30 min to collect the supernatant, which was then filtered through a 0.45 μm filter and transferred to loading buffer (20 mM imidazole, pH 8) using 100 kDa membrane exchange buffer. Protein purification was then performed using nickel-column affinity chromatography. The supernatant was bound to Ni-NTA packing material at room temperature for 30 min. Impurities were eluted with a washbuffer (20 mM Tris-HCl, 100 mM NaCl, 20 mM Imidazole, pH 8.0), followed by elution with an elution buffer (20 mM Tris-HCl, 100 mM NaCl, 200 mM Imidazole, pH 6.0). The purified protein was then identified.

[0079] In this embodiment, TGEV SWT and SM4 proteins were expressed using a baculovirus insect cell expression system, and the proteins were purified by nickel column affinity chromatography; see [link to documentation]. Figure 5 As shown, the SDS-PAGE results indicate that the molecular weight of the purified S protein (TGEV SWT and SM4 proteins) is approximately 220 kDa, consistent with the expected protein molecular weight; see [link to relevant documentation] for further details. Figure 5 As shown, the S protein was analyzed by non-denaturing electrophoresis (NativePAGE). The results showed that the molecular weight of the S protein under non-denaturing conditions was approximately 660 kDa, indicating that the S protein (TGEVSWT and SM4 proteins) is a trimer.

[0080] 5. Immune test for piglets:

[0081] 5.1 Formulation of TGEV subunit vaccine:

[0082] The purified S proteins (TGEV SWT and SM4 proteins) obtained in section 4.2 were used as antigen proteins after their protein concentration was determined by the BCA method. The two antigen proteins and Gel O2 adjuvant were thoroughly mixed with each other at a volume ratio of 9:1 to prepare vaccines with a concentration of 100 μg / mL, which were then stored at 4°C for later use. The vaccine using TGEV SWT protein as the antigen protein was designated as the SWT vaccine, and the vaccine using TGEV SM4 protein as the antigen protein was designated as the SM4 vaccine.

[0083] 5.2 Animal Experiment Grouping:

[0084] For the vaccines described above, which use TGEV SWT protein and TGEV SM4 protein as antigen proteins respectively, 12 healthy piglets that tested negative for both TGEV antibodies and pathogens were randomly divided into 3 groups (4 piglets in each group). Group 1 was the SWT immunization group, which received 2 mL (100 μg / mL) of SWT vaccine intramuscularly in the neck on day 0; Group 2 was the SM4 immunization group, which received 2 mL (100 μg / mL) of SM4 vaccine intramuscularly in the neck on day 0; and Group 3 was the non-immunized control group. Blood samples were collected before immunization and on days 14 and 42 after immunization, and serum antibody levels were measured.

[0085] 5.3 Serum ELISA antibody detection:

[0086] Blood samples were collected at 14 and 42 days post-immunization, and serum was separated. The level of TGEV S protein-specific IgG antibody was detected by indirect ELISA.

[0087] (1) Antigen coating: Dilute the protein to 1 μg / mL with coating solution, coat 100 μL per well, place at 4℃ overnight for coating, and wash 3 times with washing solution.

[0088] (2) Incubation of serum samples to be tested: The serum samples to be tested were diluted at a ratio of 1:100. 100 μL of the diluted serum samples to be tested was added to each well, along with 100 μL of the diluted positive and negative control serums. The samples were then incubated at 37°C for 1 h.

[0089] (3) Secondary antibody incubation: Wash 3 times with washing solution, add 100 μL of enzyme-labeled secondary antibody (1:5000 dilution) to each well, and incubate at 37℃ for 30 min.

[0090] (4) TMB color development: Wash 4 times with washing solution, add 100 μL of substrate TMB solution to each well, and incubate at room temperature in the dark for 15 min.

[0091] (5) Termination and reading: Add 100 μL of termination solution to each well and read the OD value at a wavelength of 450 nm.

[0092] This embodiment uses ELISA to detect antibodies in serum on days 14 and 42 post-immunization. (See [link to documentation]) Figure 6 As shown, the results indicate that after immunization with a vaccine using TGEV SM4 protein as the antigen protein, antibody levels increased significantly on day 14 and gradually increased on day 42, indicating that the TGEV subunit vaccine using TGEV SM4 protein as the antigen protein has good immunogenicity and can induce the body to produce high levels of antibodies after immunization.

[0093] Furthermore, compared to vaccines using TGEV SWT protein as the antigen protein, vaccines using TGEV SM4 protein as the antigen protein induced higher levels of antibodies, with significantly higher antibody levels on days 14 and 42 than the SWT immunization group (p<0.01).

[0094] 5.4 Neutralizing antibody test:

[0095] (1) Cell seeding: PK-15 cells were seeded into 96-well plates one day in advance and cultured at 37°C. The experiment was carried out when the cells grew to about 80% density.

[0096] (2) Serum treatment and co-incubation with virus: The serum to be tested was inactivated at 56°C for 30 min, and serially diluted 2-fold starting from 1:4. Each dilution of serum was mixed with 200 TCID50. 50 After mixing equal volumes of TGEV virus, incubate at 37°C for 2 hours.

[0097] (3) Cell infection: Discard the cell culture medium, add 100 μL of virus-serum mixture to each well, and incubate at 37°C for 1 h.

[0098] (4) Result determination: After removing the mixture, wash once with serum-free medium, replace with DMEM medium containing 2% FBS, and continue to culture at 37℃ for 3-4 days. Observe the cytopathic effect (CPE). The reciprocal of the maximum serum dilution factor that completely protects PK-15 cells from CPE is used to determine the virus neutralizing antibody titer.

[0099] This embodiment measures the TGEV virus neutralizing antibody titer in serum 42 days post-immunization. (See [link to relevant documentation]). Figure 7 As shown, the results indicated that the neutralizing antibody titers in the SWT immunization group (the vaccine corresponding to TGEV SWT protein as the antigen protein) and the SM4 immunization group (the vaccine corresponding to GEV SM4 protein as the antigen protein) were 1:149 and 1:177, respectively (geometric mean). No neutralizing antibody titers were detected in the serum of the control group. These results suggest that the SM4 protein has better immunogenicity and can induce higher levels of neutralizing antibodies.

[0100] 5.5 Isolation of porcine peripheral blood lymphocytes (PBMCs):

[0101] PBMCs were isolated from pigs in the SWT immunization group, SM4 immunization group, and non-immunized control group 42 days after immunization. The PBMC isolation method is as follows:

[0102] (1) After restraining the pigs, blood is drawn from the superior vena cava into the anticoagulated blood collection tube, and the blood collection tube should be kept as horizontal as possible.

[0103] (2) Dilute the whole blood 1:1 with an equal volume of sterile PBS and gently invert to mix.

[0104] (3) Take a new sterile 15mL centrifuge tube and gently add the diluted blood sample to the upper layer of the lymphocyte separation solution.

[0105] (4) Centrifuge at 700×g for 25 min at room temperature using a horizontal rotor centrifuge.

[0106] (5) The PBMC cell separation solution used in this experiment adopts the density gradient sedimentation method. After centrifugation, the red blood cells will sink to the bottom of the tube, while the specific gravity of lymphocytes and monocytes is less than or equal to the weight of the separation solution, and they float on the surface of the separation solution, forming a silvery-white film. The silvery-white film liquid is transferred to a new sterile 15mL centrifuge tube.

[0107] (6) Add 5 mL of sterile PBS to each tube, gently invert and mix, centrifuge at 250×g for 15 min at room temperature, and repeat washing 1-2 times.

[0108] (7) Resuspend the cells with a cell viability of more than 95% for subsequent experiments related to PBMC.

[0109] 5.6 ELISpot Experiment:

[0110] (1) The secretion of IFN-γ in porcine PBMCs was detected using the commercial kit from Dakota. All reagents were brought to room temperature before use.

[0111] (2) Activation of pre-coated plate: Add 200 μL of preheated 1640 medium to each well, let stand at room temperature for 8 min and then discard the liquid.

[0112] (3) Cell plating:

[0113] SWT experimental group: PBMC cells added to the SWT immunization group (cell density 4×10⁶ cells / year). 6 (pcs / mL), 100μL per well;

[0114] SM4 experimental group: PBMCs added to the SM4 immunization group (cell density 4×10⁶) 6 (pcs / mL), 100μL per well;

[0115] Positive control: PBMC cells (cell density 1×10⁻⁶) added to the non-immunized control group. 6 (pcs / mL), 100μL per well;

[0116] Negative control: PBMC cells (cell density 1×10⁻⁶) added to the non-immunized control group. 6 (pcs / mL), 100μL per well;

[0117] Background negative control: 10% FBS1640 medium with 100 μL of cells resuspended.

[0118] (4) Add stimulants:

[0119] SWT experimental group: 10 μL (1 mg / mL) of purified TGEV SWT protein was added to each well;

[0120] SM4 experimental group: 10 μL (1 mg / mL) of purified TGEV SM4 protein was added to each well;

[0121] Positive control: Add 10 μL of PMA + Ionomycin to each well;

[0122] Negative control: Add 10 μL of 10% FBS1640 medium to each well;

[0123] Background negative control: 10 μL 10% FBS1640 medium;

[0124] Incubate at 37℃ for 36 hours.

[0125] (5) Cell lysis: Discard the culture medium, add 200 μL of pre-cooled sterile deionized water to each well, and let stand at 4℃ for 10 min for hypotonic lysis.

[0126] (6) Washing: Shake off the liquid in the wells, add 260μL of 1×Wash Buffer to each well, let stand for 1 minute and then discard. Repeat the washing 6 times, and pat dry each time.

[0127] (7) Antibody incubation test: Add 100 μL of 1×Biotinylated Antibody working solution to each well, incubate at 37°C for 1 h, and wash the plate 6 times.

[0128] (8) Enzyme-linked avidin incubation: Add 100 μL of 1×Streptavidin-HRP working solution to each well, incubate at 37℃ for 1 h, and wash the plate 6 times; after the last wash, remove the plate base, rinse the bottom of the membrane and the base with deionized water, dry and close the base.

[0129] (9) Color development: Add 100 μL of freshly prepared AEC color development solution to each well, develop the color at 37℃ in the dark for 15-20 min, and observe the formation of spots.

[0130] (10) Terminate color development: Discard the color development solution, remove the base, rinse the front and back with deionized water 3-5 times, let it air dry naturally, close the base and mail it to Dakowei Company for reading.

[0131] In this embodiment, the enzyme-linked spot assay (ELISpot) was used to detect the IFN-γ secretion level of peripheral blood lymphocytes in immunized pigs after stimulation with recombinant S protein, in order to assess the vaccine-induced cellular immune response. The detection results are as follows: Figure 8 As shown, in the ELISpot assay using recombinant S protein as the stimulating antigen, the SM4 experimental group exhibited a higher level of IFN-γ than the SWT experimental group. The number of positive cells per million cells in the SWT experimental group was 334, while the number of positive cells per million cells in the SM4 experimental group was 554. Statistical analysis showed a significant difference (p<0.0001). The number of spots in the NC control group and the background control group was close to zero, indicating good detection specificity. These results indicate that the SM4 protein can induce a stronger specific cellular immune response compared to the SWT protein.

[0132] 5.7 Lymphocyte proliferation experiment:

[0133] (1) Cell proliferation experiments were performed using the Solarbio commercial CCK8 kit.

[0134] (2) Prepare 100 μL of SWT or SM4 immunized PBMC cell suspension (total cell count of 5000 cells) in a 96-well plate and incubate at 37°C and 5% CO2 for 24 h.

[0135] (3) In each well containing the SWT immunized PBMC cell suspension, 10 μL (1 mg / mL) of purified TGEV SM4 protein was added to stimulate porcine PBMC cells (referred to as the SWT experimental group); in each well containing the SM4 immunized PBMC cell suspension, 10 μL (1 mg / mL) of purified TGEV SM4 protein was added to stimulate porcine PBMC cells (referred to as the SM4 experimental group); and incubated for 72 h.

[0136] (4) Change the medium and wash the cells twice with culture medium, and add 10 μL of CCK-8 solution to each well.

[0137] (5) Incubate the culture plate in the incubator for 3 hours.

[0138] (6) The absorbance at 450 nm was measured using an ELISA reader. Stimulation index (SI) = (OD immune group - OD Blank) / (OD blank group - OD Blank).

[0139] In this embodiment, the CCK8 assay was used to detect the proliferative response of PBMCs to recombinant S protein stimulation after immunization, in order to evaluate the vaccine-induced T lymphocyte proliferative activity. Figure 9The results showed that both the SWT and SM4 groups exhibited a certain degree of PBMC proliferation response. The SM4 experimental group had a Stimulation Index (SI) of 1.89, significantly higher than the SWT experimental group (SI = 1.33) (p < 0.0001), indicating that SM4 protein can more effectively activate T lymphocyte proliferation. In this embodiment, a negative control group (NC) and a positive control group were set up during the experiment. The SI value of the NC group was close to 1, and Con-A, as the positive control group, showed a high stimulation index, verifying the reliability of the experimental system.

[0140] The above technical solutions of the present invention are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made under the technical concept of the present invention using the contents of the present invention specification and drawings, or direct / indirect applications 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 porcine transmissible gastroenteritis virus, characterized in that, The amino acid sequence of the S protein mutant is shown in SEQ ID NO.

3.

2. A nucleic acid molecule encoding an S protein mutant of porcine transmissible gastroenteritis virus, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

3. A recombinant expression vector, characterized in that, The recombinant expression vector has a nucleotide sequence encoding the S protein mutant as described in claim 1.

4. A recombinant expression vector, characterized in that, The recombinant expression vector has the nucleic acid molecule as described in claim 2.

5. An engineered cell, characterized in that, The engineered cells are capable of expressing the S protein mutant as described in claim 1.

6. A method for expressing an S protein mutant of porcine transmissible gastroenteritis virus, characterized in that, include: Construct the recombinant expression vector as described in claim 3 or 4, and express it using engineered cells.

7. The use of the S protein mutant as described in claim 1 in the preparation of a vaccine for porcine transmissible gastroenteritis virus.

8. A subunit vaccine against porcine transmissible gastroenteritis virus, characterized in that, The vaccine against porcine transmissible gastroenteritis virus includes the S protein mutant as described in claim 1.

9. The subunit vaccine for porcine transmissible gastroenteritis virus according to claim 8, characterized in that, The vaccine against porcine transmissible gastroenteritis virus includes a vaccine adjuvant.

10. The use of the S protein mutant as described in claim 1 in the preparation of antibodies against porcine transmissible gastroenteritis virus.

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