A porcine rotavirus nanoparticle and a preparation method and application thereof

Abstract

The application belongs to the technical field of veterinary vaccines, and particularly relates to a porcine rotavirus nanoparticle, a preparation method and application thereof. The application provides a porcine rotavirus nanoparticle, which comprises a porcine rotavirus VP8 protein fragment, a dioxygen tetrahydrofolate synthetase and a linker; the porcine rotavirus VP8 protein fragment and the dioxygen tetrahydrofolate synthetase are connected through the linker; and the amino acid sequence of the porcine rotavirus VP8 protein fragment is shown as SEQ ID NO. 1. The porcine rotavirus nanoparticle can display a larger number of antigens at a time, improve the immunogenicity of a vaccine, and can induce animals to produce a higher antibody level. The results of examples show that the porcine rotavirus nanoparticle vaccine prepared by the application can cause a higher specific antibody level, and compared with a porcine rotavirus VP8 protein fragment monomer immunization group, can cause a higher neutralizing antibody level.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary vaccines, and in particular relates to porcine rotavirus nanoparticles and a preparation method and application thereof. Background Art

[0002] Porcine rotavirus (PoRV) belongs to the family Reoviridae and the genus Rotavirus. Mature, intact rotavirus particles have an icosahedral symmetry, with a dense hexagonal core at the center, a diameter of 37-40 nm, and lack an envelope. The rotavirus genome consists of 11 independent segments of positive double-stranded RNA (dsRNA), encoding six structural proteins (VP1-VP4, VP6, and VP7) and five nonstructural proteins (NSP1-NSP5). Porcine rotavirus can cause diarrhea and mortality in pigs, resulting in significant economic losses for pig farmers. Vaccination has become an important approach to reduce morbidity and mortality, and alleviate the economic burden.

[0003] Research has shown that the Porcine Rotavirus (Porcine Rotavirus) capsid spike protein, VP4, is a key anti-rotavirus target, mediating rotavirus attachment and entry into cells and effectively inducing the production of neutralizing antibodies, making it an ideal candidate for subunit vaccine development. However, VP4 protein is prone to forming inclusion bodies in prokaryotic expression systems, resulting in low solubility and increasing the difficulty of subsequent processing. Therefore, the development of a new, simple, and highly effective porcine rotavirus nanoparticle vaccine is needed. Summary of the Invention

[0004] The purpose of the present invention is to provide a porcine rotavirus nanoparticle and its preparation method and application. The porcine rotavirus nanoparticle of the present invention can display a larger number of antigens at a single time, improve the immunogenicity of the vaccine, and induce animals to produce higher antibody levels.

[0005] The present invention provides a porcine rotavirus nanoparticle, which comprises a porcine rotavirus VP8 protein fragment, a dioxopterin synthase and a linker; the porcine rotavirus VP8 protein fragment and the dioxopterin synthase are connected by the linker; the amino acid sequence of the porcine rotavirus VP8 protein fragment is shown in SEQ ID NO.1.

[0006] As a preferred embodiment, the amino acid sequence of the dioxotetrahydropterin synthase is shown in SEQ ID NO.2.

[0007] As a preferred embodiment, a purification tag is added to the C-terminus of the porcine rotavirus nanoparticles.

[0008] As a preferred embodiment, the purification tag includes a His tag.

[0009] The present invention also provides a gene encoding the porcine rotavirus nanoparticles described in the above scheme, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO.5.

[0010] The present invention also provides a recombinant expression vector containing the coding gene described in the above scheme.

[0011] The present invention also provides a method for preparing the porcine rotavirus nanoparticles described in the above scheme, comprising the following steps: transforming the recombinant expression vector described in the above scheme into a prokaryotic expression cell to obtain an engineered strain; culturing the engineered strain, and obtaining the porcine rotavirus nanoparticles through expression and purification.

[0012] As a preferred embodiment, the prokaryotic expression cells include Escherichia coli competent cells.

[0013] The present invention also provides the use of the porcine rotavirus nanoparticles described in the above scheme, or the encoding gene described in the above scheme, or the recombinant expression vector described in the above scheme, or the preparation method described in the above scheme in the preparation of porcine rotavirus nanoparticle vaccines.

[0014] The present invention also provides a porcine rotavirus nanoparticle vaccine, comprising an adjuvant and the porcine rotavirus nanoparticles described in the above scheme or the porcine rotavirus nanoparticles prepared by the preparation method described in the above scheme.

[0015] The preparation process of the porcine rotavirus nanoparticle vaccine of the present invention is as follows: Figure 1 shown.

[0016] Beneficial effects:

[0017] The present invention provides porcine rotavirus nanoparticles, comprising a porcine rotavirus VP8 protein fragment, a dioxopterin synthase, and a linker; the porcine rotavirus VP8 protein fragment and the dioxopterin synthase are connected by the linker; the amino acid sequence of the porcine rotavirus VP8 protein fragment is shown in SEQ ID NO. 1. The porcine rotavirus VP8 protein fragment (VP8*) of the present invention contains the major antigenic epitopes of VP4 and is highly conserved among the same serotypes, can induce the production of neutralizing antibodies, and is more soluble than VP4 in an Escherichia coli expression system. Dioxopterin synthase (LS) can spontaneously form icosahedral nanoparticles, capable of displaying a larger number of antigens at a time, improving the immunogenicity of the vaccine and enhancing its immune effect. The porcine rotavirus nanoparticles of the present invention are non-whole virus antigens and, when used to prepare porcine rotavirus nanoparticle vaccines, do not pose the risk of virus spread. The production process is very safe and can achieve efficient production of genetically engineered nanoparticle vaccines. The results of the examples show that the porcine rotavirus nanoparticle vaccine prepared by the present invention can induce a higher level of specific antibodies, and can induce a higher level of neutralizing antibodies compared with the porcine rotavirus VP8 protein fragment monomer immunization group. 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 drawings required for use in the embodiments are briefly introduced below.

[0019] Figure 1 This is a technical roadmap for preparing the novel porcine rotavirus nanoparticle vaccine of the present invention;

[0020] Figure 2 This is the double enzyme digestion identification pattern of pHAT2-VP8* and pHAT2-LS-VP8* in Example 1;

[0021] Figure 3 This is a diagram showing the expression, purification, and detection of VP8* and LS-VP8* proteins in Example 1;

[0022] Figure 4 This is an SDS-PAGE detection image of LS-VP8* and LS protein in Example 1;

[0023] Figure 5 This is the particle size diagram of LS-VP8* measured by dynamic laser scattering in Example 2;

[0024] Figure 6 The transmission electron microscopy results of the purified LS-VP8* and LS nanoparticle proteins in Example 2 are shown; the scale bar in the horizontal 100 nm figure represents 100 nm, and the scale bar in the horizontal 200 nm figure represents 200 nm;

[0025] Figure 7 The results of serum IgG antibody level detection at different time points of each group of immunized BALB / c mice in Example 3 are shown in FIG.

[0026] Figure 8 The neutralizing antibody levels against the P23 strain in the serum of BALB / c mice in each group immunized at different time points in Example 3 were detected; DETAILED DESCRIPTION

[0027] The present invention provides porcine rotavirus nanoparticles, comprising a porcine rotavirus VP8 protein fragment, a dioxopterin synthase, and a linker; the porcine rotavirus VP8 protein fragment and the dioxopterin synthase are connected by the linker; the amino acid sequence of the porcine rotavirus VP8 protein fragment is shown in SEQ ID NO. 1, specifically as follows: VLDGPYQPTTFNPPV SYWILLSPSNAGVVVEGTNNSDRWLATILIEPNVTSQSRTYTLFGQQEQITVEN VSTTKWKFVDLAKTDVNGTFTQHGPLLSDTKLYGVMKFSGRLYTYNGETPN ATTSYYTTTNYDTVNMTSHCDFYIIPRSEETMCTNYINNG. The porcine rotavirus VP8 protein fragment of the present invention contains the main antigenic epitope of VP4, is highly conserved among the same serotype, can induce the production of neutralizing antibodies, and is more soluble than VP4 in an Escherichia coli expression system.

[0028] As an embodiment, the amino acid sequence of the dioxetidine synthase of the present invention is shown in SEQ ID NO. 2, specifically as follows: QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDA IVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYI ASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIE MANLFKSLR. The dioxetidine synthase (LS) of the present invention can spontaneously form icosahedral nanoparticles, which can display a larger number of antigens at a time, thereby improving the immunogenicity of the vaccine and enhancing its immune effect.

[0029] As an embodiment, the amino acid sequence of the linker of the present invention is shown in SEQ ID NO.3, specifically as follows: GGSGGS.

[0030] As an embodiment, the amino acid sequence of the porcine rotavirus nanoparticles of the present invention is shown in SEQ ID NO.4, specifically as follows: QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAID AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSVLDGPYQPTTFNPPVSYWILLSPSNAGVVVEGTNNSDRWLATILIEPNVTSQSRTYTLFGQQEQITVENVSTTKWKFVDLAKTDVNGTFTQHGPLLSDTKLYGVMKFSGRLYTYNGETPNATTSYYTTTNYDTVNMTSHCDFYIIPRSEETMCTNYINNG. The porcine rotavirus nanoparticles described in the present invention can display a larger number of antigens at a time, improve the immunogenicity of the vaccine, and can induce animals to produce higher antibody levels. Moreover, since they are non-whole virus antigens, they will not pose the risk of virus spread when used to prepare porcine rotavirus nanoparticle vaccines. The production process is very safe and can achieve efficient production of genetically engineered nanoparticle vaccines.

[0031] In one embodiment, a purification tag is added to the C-terminus of the porcine rotavirus nanoparticles of the present invention. In another embodiment, the purification tag of the present invention comprises a His tag. The purification tag of the present invention can simplify subsequent purification steps and improve purification efficiency. In one embodiment, the porcine rotavirus nanoparticles of the present invention and the purification tag are connected via a linker, the amino acid sequence of which is shown in SEQ ID NO. 3.

[0032] The present invention also provides a gene encoding the porcine rotavirus nanoparticles described in the above scheme, wherein the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.5所示,具体如下:5'-CAGATCTATGAGG GGAAACTGACAGCCGAGGGACTGAGGTTTGGAATCGTCGCTTCACGCTTCAATCACGCACTGGTGGACAGACTGGTGGAAGGCGCTATCGACGCAATTGTCCGGCACGGCGGGAGAGAGGAAGATATCACTCTGGTGAGAGTCCCAGGGAGCTGGGAGATTCCAGTGGCAGCTGGAGAACTGGCTCGGAAGGAGGACATCGATGCCGTGATCGCTATTGGAGTCCTGATTAGAGGCGCAACACCTCACTTCGACTACATCGCCTCAGAAGTGAGCAAGGGACTGGCTGATCTGTCCCTGGAGCTGAGGAAACCAATCACCTTTGGCGTCATTACTGCCGACACCCTGGAACAGGCAATCGAGCGCGCCGGGACAAAGCATGGCAACAAAGGGTGGGAAGCAGCCCTGTCAGCTATTGAGATGGCAAATCTGTTCAAAAGCCTGCGAGGAGGCTCCGGAGGATCTGTTCTGGATGGTCCGTATCAGCCGACCACCTTTAATCCGCCGGTTAGCTATTGGATTCTGCTGAGCCCGAGCAATGCAGGTGTTGTTGTTGAAGGTACCAATAATAGCGATCGTTGGCTGGCAACCATTCTGATTGAACCGAATGTTACCAGCCAGAGCCGTACCTATACCCTGTTTGGTCAGCAGGAACAGATTACCGTTGAAAATGTTAGCACCACCAAATGGAAATTTGTTGATCTGGCAAAAACCGATGTTAATGGTACCTTTACCCAGCATGGTCCGCTGCTGAGCGATACCAAACTGTATGGTGTTATGAAATTTAGCGGTCGTCTGTATACCTATAATGGTGAAACCCCGAATGCAACCACCAGCTATTATACCACCACCAATTATGATACCGTTAATATGACCAGCCATTGTGATTTTTATATTATTCCGCGTAGCGAAGAAACCATGTGTACCAATTATATTAATAATGGTTAA-3'。.

[0033] The present invention also provides a recombinant expression vector containing the encoding gene described in the above scheme. In one embodiment, the base vector of the recombinant expression vector of the present invention includes a pHAT2 vector; in another embodiment, the encoding gene is inserted between the Nco I and BamHI restriction sites of the base vector.

[0034] The present invention also provides a method for preparing the porcine rotavirus nanoparticles described in the above scheme, comprising the following steps: transforming the recombinant expression vector described in the above scheme into a prokaryotic expression cell to obtain an engineered strain; culturing the engineered strain, and obtaining the porcine rotavirus nanoparticles through expression and purification.

[0035] The present invention transforms the recombinant expression vector described in the above scheme into a prokaryotic expression cell to obtain an engineered strain. In one embodiment, the method for transforming the recombinant expression vector described in the present invention into the prokaryotic expression cell includes a CaCl2 heat shock method. In one embodiment, the prokaryotic expression cell described in the present invention includes an Escherichia coli competent cell. In a specific embodiment, the Escherichia coli competent cell can be an Escherichia coli DE3 competent cell.

[0036] In the present invention, after obtaining the engineered strain, the engineered strain is cultured, and the porcine rotavirus nanoparticles are obtained through expression and purification. In one embodiment, the expression comprises inducing expression using IPTG to obtain expressed bacteria. In another embodiment, the purification comprises low-temperature ultrasonic disruption of the expressed bacteria, centrifugation to obtain a supernatant, and passing the supernatant through a Ni-filled chromatography column to elute the purified fusion protein.

[0037] The present invention also provides the use of the porcine rotavirus nanoparticles described in the above scheme, or the encoding gene described in the above scheme, or the recombinant expression vector described in the above scheme, or the preparation method described in the above scheme in the preparation of porcine rotavirus nanoparticle vaccines.

[0038] The present invention also provides a porcine rotavirus nanoparticle vaccine, comprising an adjuvant and the porcine rotavirus nanoparticles described in the above scheme or the porcine rotavirus nanoparticles prepared by the preparation method described in the above scheme. In one embodiment, the adjuvant of the present invention includes Qm01E adjuvant; the Qm01E adjuvant includes ginsenosides, astragalus polysaccharides, and a divalent manganese salt; the mass ratio of the ginsenosides, astragalus polysaccharides, and divalent manganese salt is (1-10):(1-10):0.1. In a specific embodiment, the mass ratio of the ginsenosides, astragalus polysaccharides, and divalent manganese salt can be 1:1:0.1. Using the Qm01E adjuvant, the present invention can prepare the vaccine by simply mixing the Qm01E adjuvant and protein, without the need for emulsification. This simplifies the vaccine production process, reduces vaccine production costs, and enhances vaccine immunogenicity.

[0039] To further illustrate the present invention, the porcine rotavirus nanoparticles, preparation method and application thereof provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] 1. Construction of recombinant plasmids expressing VP8* and LS-VP8* proteins

[0042] (1) The LS gene (SEQ ID NO. 6) containing a 6His tag was synthesized with reference to GenBank: KX527854.1. The sequence information is as follows: 5'-CAGATCTATGAGGGGAAACTGACAGCCGAGGGACTGAGGTTTGGAATC GTCGCTTCACGCTTCAATCACGCACTGGTGGACAGACTGGTGGAAGGCGCTATCGACGCAATTGTCCGGCACGGCGGGAGAGGAAGATATCACTCTGGTGAGAGTCCCAGGGAGCTGGGAGATTCCAGTGGCAGCTGGAGAACTGGCTCGGAAGGAGGACATCGATGCCGTGATCGCTATTGGAGTCCTGATTAGAGGCGCAACACCTCACTTCGACTACATC GCCTCAGAAGTGAGCAAGGGACTGGCTGATCTGTCCCTGGAGCTGAGGAAACCAATCACCTTTGGCGTCATTACTGCCGACACCCTGGAACAGGCAATCGAGCGCGCCGGGACAAAGCATGGCAACAAAGGGTGGGAAGCAGCCCTGTCAGCTATTGAGATGGCAAATCTGTTCAAAAGCCTGCGAGGAGGCTCCGGAGGATCTCATCACCATCACCATCAC-3'.

[0043] (2) The gene of P23 type PoRV was extracted [published in “Establishment of TaqMan fluorescent quantitative RT-PCR method for porcine rotavirus and virus isolation and identification”, Lin Zhengdan, master’s thesis of Huazhong Agricultural University, 2023]. The extracted P23 type PoRV gene was used as a template to amplify the VP8* gene fragment (SEQ ID NO. 7) using the upstream and downstream primers. The primers are VP8*-F (SEQ ID NO. 8) and VP8*-R (SEQ ID NO. 9). The sequence is shown below:

[0044] VP8* gene fragment (SEQ ID NO. 7): 5'-GTTCTGGATGGTCCGTATCAGCCG ACCACCTTTAATCCGCCGGTTAGCTATTGGATTCTGCTGAGCCCGAGCAATGCAGGTGTTGTTGTTGAAGGTACCAATAATAGCGATCGTTGGCTGGCAACCATTCTGATTGAACCGAATGTTACCAGCCAGAGCCGTACCTATACCCTGTTTGGTCAGCAGGAACAGATTACCGTTGAAAATGTTAGCACCACCAAATGGAAATTTGTTGATCTGGCAAAAACCGATGT TAATGGTACCTTTACCCAGCATGGTCCGCTGCTGAGCGATACCAAACTGTATGGTGTTATGAAATTTAGCGGTCGTCTGTATAACCTATAATGGTGAAACCCCGAATGCAACCACCAGCTATTATACCACCACCAATTATGATACCGTTAATATGACCAGCCATTGTGATTTTTATATTATTCCGCGTAGCGAAGAAACCATGTGTACCAATTATATTAATAATGGT-3';

[0045] VP8*-F (SEQ ID NO.8): 5'-CCATGGATGTTCTGGATGGTCCGTATCAG CCG-3';

[0046] VP8*-R (SEQ ID NO. 9): 5'-CTAGAGGGCCCGGATCCTTAACCATTATT AATAT-3'.

[0047] (3) A 6His tag was connected to the 3' end of the VP8* gene fragment obtained in the above step (2) via a linker peptide linker (SEQ ID NO: 10) to obtain a VP8* gene fragment containing a 6His tag.

[0048] linker (SEQ ID NO: 10): 5'-GGAGGCTCCGGAGGATCT-3'.

[0049] (4) Using the LS gene sequence containing a 6His tag and the VP8* gene fragment containing a 6His tag obtained in steps (1) and (3) above as templates, LS-VP8* (SEQ ID NO: 11) with a 6His tag was amplified by overlap PCR through preferred codon optimization of the Escherichia coli prokaryotic expression system; the primers for the overlap PCR were LS-VP8*-F1 (SEQ ID NO. 12), LS-VP8*-F2 (SEQ ID NO. 13), LS-VP8*-R1 (SEQ ID NO. 14) and VP8*-R (SEQ ID NO. 9), and the sequence information is as follows:

[0050] LS-VP8 with 6His tag* (SEQ ID NO: 11): 5'-CAGATCTATGAGGGGAAACTGACAGCCGAGGGACTGAGGTTTGGAATCGTCGCTTCACGCTTCAATCACGCACTGGTGGACAGACTGGTGGAAGGCGCTATCGACGCAATTGTCCGGCACGGCGGGAGAGAGGAAGATATCACTCTGGTGAGAGTCCCAGGGAGCTGGGAGATTCCAGTGGCAGCTGGAGAACTGGCTCGGAAGGAGGACATCGATGCCGTGATCGCTATTGGAGTCCTGATTAGAGGCGCAACACCTCACTTCGACTACATCGCCTCAGAAGTGAGCAAGGGACTGGCTGATCTGTCCCTGGAGCTGAGGAAACCAATCACCTTTGGCGTCATTACTGCCGACACCCTGGAACAGGCAATCGAGCGCGCCGGGACAAAGCATGGCAACAAAGGGTGGGAAGCAGCCCTGTCAGCTATTGAGATGGCAAATCTGTTCAAAAGCCTGCGAGGAGGCTCCGGAGGATCTGTTCTGGATGGTCCGTATCAGCCGACCACCTTTAATCCGCCGGTTAGCTATTGGATTCTGCTGAGCCCGAGCAATGCAGGTGTTGTTGTTGAAGGTACCAATAATAGCGATCGTTGGCTGGCAACCATTCTGATTGAACCGAATGTTACCAGCCAGAGCCGTACCTATACCCTGTTTGGTCAGCAGGAACAGATTACCGTTGAAAATGTTAGCACCACCAAATGGAAATTTGTTGATCTGGCAAAAACCGATGTTAATGGTACCTTTACCCAGCATGGTCCGCTGCTGAGCGATACCAAACTGTATGGTGTTATGAAATTTAGCGGTCGTCTGTATACCTATAATGGTGAAACCCCGAATGCAACCACCAGCTATTATACCACCACCAATTATGATACCGTTAATATGACCAGCCATTGTGATTTTTATATTATTCCGCGTAGCGAAGAAACCATGTGTACCAATTATATTAATAATGGTGGAGGCTCCGGAGGATCTCATCACCATCACCATCAC-3';

[0051] LS-VP8*-F1 (SEQ ID NO:12): 5'-TCACCATCACTCCATGGATCAGATC TATGAGGGG-3';

[0052] LS-VP8*-F2 (SEQ ID NO:13): 5'-TTCAAAAGCCTGCGAGGAGGCTCCGGAGGATCTTGTTCTGGATGGTCCG-3';

[0053] Ls-VP8*-R1 (SEQ ID NO: 14): 5'-CGGACCATCCAGAACAGATCCTCCGGAGCCTCCTCGCAGGCTTTTGAA-3'.

[0054] The amplification reaction system consisted of 25 μL of 2× PhantaMax Buffer, 1 μL of dNTPMix, 2 μL of a 10 μM forward primer, 2 μL of a 10 μM reverse primer, 100 ng of template, and PhantaMax Super-Fidelity DNA Polymerase (1 U / μL). The system was made up to 50 μL with ddH2O. The amplification reaction protocol was 95°C for 5 min, followed by 30 cycles of (95°C for 15 s, 65°C for 15 s, and 72°C for 1 min), and 72°C for 10 min.

[0055] (5) Using the LS-VP8* obtained in step (4) as a template, primers VP8*-F (SEQ ID NO. 8) and VP8*-R (SEQ ID NO. 9) were used to amplify the VP8* gene fragment with a 6His tag.

[0056] The amplification reaction system consisted of 25 μL of 2× PhantaMax Buffer, 1 μL of dNTPMix, 2 μL of a 10 μM forward primer, 2 μL of a 10 μM reverse primer, 100 ng of template, and PhantaMax Super-Fidelity DNA Polymerase (1 U / μL). The system was made up to 50 μL with ddH2O. The amplification reaction protocol was 95°C for 5 min, followed by 30 cycles of (95°C for 15 s, 65°C for 15 s, and 72°C for 1 min), and 72°C for 10 min.

[0057] (6) The VP8* and LS-VP8* gene fragments obtained in the above steps (4) and (5) were respectively cloned into the Nco I and BamHI restriction sites of the prokaryotic expression vector pHAT2 to construct the recombinant expression vectors pHAT2-VP8* and pHAT2-LS-VP8*.

[0058] (7) The above recombinant expression vectors pHAT2-VP8* and pHAT2-LS-VP8* were transformed into DH5α competent cells, cultured at 37°C overnight, and positive clones were obtained. The plasmids were extracted, and after double enzyme digestion, the plasmids with correct enzyme digestion were selected for sequencing. The plasmids with successful sequencing were used for the expression of antigen proteins. The enzyme digestion test results were Figure 2 As shown, lanes 1 to 4 in the left figure are the detection results of 4 different pHAT2-VP8* positive plasmids; lanes 1 to 4 in the right figure are the detection results of 4 different pHAT2-LS-VP8* positive plasmids.

[0059] 2. Construction of recombinant expression strain

[0060] The successfully constructed recombinant plasmids pHAT2-VP8*, pHAT2-LS-VP8* and recombinant plasmid pET28a-LS [the preparation method of the recombinant plasmid pET28a-LS is disclosed in Chinese patent CN114874338A] were respectively transformed into the prokaryotic expression system BL21 (DE3) expression strain, cultured overnight at 37°C, and single clone strains were selected to obtain recombinant expression strains. The bacterial solution was preserved with 20% glycerol and stored at -80°C.

[0061] 3. Expression and purification of fusion proteins VP8*, LS-VP8*, and LS

[0062] (1) Take the bacterial solution stored in the above steps and inoculate it into fresh liquid LB medium at a ratio of 1:1000 (V / V) overnight, then take the bacterial solution cultured overnight and inoculate it into 1L liquid LB medium at a ratio of 1:100 (V / V), and culture it on a shaker at 37°C and 180 rpm for 3 hours; the liquid LB medium contains ampicillin with a final concentration of 100 μg / mL. 600 When the value reached 0.6-0.8, IPTG with a final concentration of 0.4 mmoL / L was added for induction, and the expression was induced at 16°C and 160 rpm for 18 h. The induced bacterial solution was then centrifuged at 7000 rpm for 10 min to collect the bacteria.

[0063] (2) The bacterial cells collected in step (1) were resuspended in Tris-HCl, pH 8.0 buffer at a ratio of 1:10 (V / V) and disrupted at 4°C and 1000 bar to obtain a disrupted bacterial solution.

[0064] (3) The disrupted bacterial solution described in step (2) was centrifuged at 13,000 rpm and 4°C for 30 min, the supernatant was collected, and then filtered with a 0.22 μm filter membrane to remove cell debris. The filtered supernatant was allowed to bind to the Ni filler through a peristaltic pump (the His tag at the end of the target protein can bind to Ni). After the binding was completed, the target protein was automatically eluted using a 0-500 mM gradient concentration imidazole buffer by a protein purification elution instrument at a uniform speed within 1.5 h. The imidazole buffer concentration gradient range was determined according to the peak position of the target protein, and the eluate within the range was collected using a collection tube, with 2 mL of eluate collected in each collection tube. Among them, the target protein VP8* collected 9 tubes of eluate within the gradient concentration range of 80-130mM, numbered 1-9 in sequence, the target protein LS-VP8* collected 12 tubes of eluate within the gradient concentration range of 80-150mM, numbered 1-12 in sequence, and the target protein LS collected 12 tubes of eluate within the gradient concentration range of 80-150mM, numbered 1-12 in sequence. Subsequently, the purification effect of the target protein in different eluates was detected by SDS-PAGE. The results are shown in Figure 3 , where A is the detection result of the target protein VP8*, and lanes 1 to 9 are the detection results of the target protein VP8* eluted from different collection tubes; B is the detection result of the target protein LS-VP8*, and lanes 1 to 12 are the detection results of the target protein LS-VP8* eluted from different collection tubes; C is the detection result of the target protein LS, and lanes 1 to 12 are the detection results of the target protein LS-VP8* eluted from different collection tubes. According to Figure 3 It can be seen that the target proteins VP8*, LS-VP8* and LS with the expected molecular weight were prepared.

[0065] 4. Fusion protein LS-VP8*, LS gel chromatography purification (SEC)

[0066] The fusion proteins LS-VP8* and LS were purified by SEC using a Superdex 200Increase 10 / 300GL gel filtration column. The purification steps were referred to [Purification of gap junction protein 26 by anti-GFP nanoantibody as affinity ligand [J], Tong Ling et al., Biotechnology, 2023]. The eluent was eluted at a constant rate of 1 mL / min to remove non-target proteins or unsuccessfully folded impurities. The peak position and collection tube position were determined according to UV absorbance, and the target protein was collected. LS-VP8* eluted at about 70 mL after sample loading. 14 collection tubes were used to collect the eluate at 65 mL of elution. Each collection tube contained 2 mL of eluate and was numbered 1 to 14. LS eluted at about 80 mL after sample loading. 14 collection tubes were used to collect the eluate at 75 mL of elution. Each collection tube contained 2 mL of eluate and was numbered 1 to 14. The proteins collected after purification were detected by SDS-PAGE, and the results are as follows. Figure 4 As shown, lanes 1 to 14 in the left figure represent the LS protein detection results of different collection tubes after SEC purification; lanes 1 to 14 in the right figure represent the LS-VP8* protein detection results of different collection tubes after SEC purification. Figure 4 It can be seen that relatively pure LS-VP8* and LS proteins were prepared.

[0067] Example 2

[0068] 1. Determination of Nanoparticle Size by Dynamic Laser Scattering of Fusion Protein LS-VP8*

[0069] The LS-VP8* fusion protein prepared in Example 1 was ultrafiltered and concentrated using 10 kDa and 30 kDa ultrafiltration tubes at 3500 rpm at 4°C. The concentrated solution was then replaced with PBS to approximately 2 mL. The concentrated LS-VP8* protein solution was filtered using a 0.22 μm filter and diluted with pure water to 0.1 mg / mL. The particle size of the LS-VP8* fusion protein was measured and analyzed using a dynamic laser scattering instrument. The results are shown in Figure 5 .according to Figure 5 It can be seen that the particle size of the LS-VP8* fusion protein is concentrated in the range of 30-60 nm, indicating that the LS-VP8* fusion protein has high purity and good uniformity.

[0070] 2. Transmission electron microscopy observation of fusion protein LS-VP8* and LS

[0071] The LS-VP8* and LS protein samples prepared in Example 1 were adsorbed on a copper mesh, stained with a 2% (w / v) sodium phosphotungstate solution for 30 seconds, and after the copper mesh was completely dried, a transmission electron microscope was used to observe whether nanoparticles were formed. Figure 6 As shown, LS-VP8 (P23) in the picture is LS-VP8*. Figure 6 It can be seen that the fusion proteins LS-VP8* and LS prepared in the present invention can both form nanoparticles.

[0072] Example 3

[0073] Mouse immunization experiments

[0074] The LS protein, VP8* protein, and LS-VP8* fusion protein obtained in Example 1 were each diluted to 400 μg / mL with sterile PBS and mixed with an equal volume of Qm01E adjuvant to prepare a porcine rotavirus nanoparticle vaccine, achieving a final antigen concentration of 200 μg / mL. The Qm01E adjuvant consisted of ginsenosides, astragalus polysaccharides, and manganous chloride in a mass ratio of 1:1:0.1.

[0075] (1) Immunogenicity experiment of new porcine rotavirus nanoparticle vaccine

[0076] Twenty six-week-old female BALB / c mice were purchased from the Experimental Animal Center of Huazhong Agricultural University and randomly divided into four groups of five mice: LS, P23-VP8*, P23-LS-VP8*, and PBS (negative control). Immunization was performed via intramuscular injection of the hind leg. Each mouse received a dose of 40 μg of the vaccine, on days 0 and 14, respectively. The injection schedule is shown in Table 1. This experiment was repeated three times.

[0077] Table 1 Immunization methods for mice

[0078]

[0079] Blood was collected from the retro-orbital vein on days 0, 14, 28, and 42, respectively. The collected blood was allowed to stand for 30 minutes to allow the serum to precipitate, and then centrifuged at 3000 rpm and 4°C for 10 minutes to collect the serum for the detection of specific IgG levels and neutralizing antibody levels.

[0080] (2) Indirect ELISA method to detect specific IgG antibody levels in mouse serum

[0081] The P23 PoRV virus was centrifuged at 30,000 rpm for 3 h, the supernatant was discarded, and PBS was added to the virus pellet for resuspending to obtain concentrated P23 PoRV virus particles, wherein the volume ratio of the concentrated pre-virus solution to PBS was 500:1; the concentrated P23 PoRV virus particles were used to coat the enzyme label plate, 0.5 μg per well, and coated at 4°C for 14 h; after coating, the coating solution was discarded, 100 μL PBS solution was added to each well, and the plate was blocked at 37°C for 2 h, wherein the PBS solution contained BSA with a final concentration of 2% (w / v); after blocking, the coating solution was discarded, and the plate was washed 4 times with PBST, 5 min / time; the mouse serum immunized in step (1) was diluted 1:1600 as the primary antibody, 100 μL primary antibody was added to each well, and the plate was incubated at 37°C for 1.5 h; after incubation, the primary antibody was discarded, and the plate was washed 4 times with PBST, 5 min / time; goat anti-mouse HRP was diluted 1:5000 IgG was used as the secondary antibody, 100 μL of secondary antibody was added to each well and incubated at 37°C for 1 hour; after incubation, the secondary antibody was discarded and the cells were washed 4 times with PBST, 5 minutes each time; 100 μL of ELISA colorimetric solution was added to each well and the cells were reacted in the dark for 10 minutes; after the reaction was completed in the dark, 50 μL of ELISA stop solution was added and the OD value of each well was read using a microplate reader. 450 The results are shown in Table 2 and Figure 7 The results are the average of three repeated experiments. Figure 7** indicates that the data have significant differences, p < 0.01, *** indicates that the data have significant differences, p < 0.001.

[0082] Table 2 OD of IgG after immunization of mice in each group 450 value

[0083] Group 0d 14d 28d 42d P23-VP8* 0.161 0.376 0.443 1.216 P23-Ls-VP8* 0.167 0.324 0.945 1.200 Ls 0.164 0.179 0.306 0.178 PBS 0.167 0.226 0.169 0.304

[0084] According to Table 2 and Figure 7 High levels of specific antibodies were observed in the serum of mice in all experimental groups 28 and 42 days after immunization. These levels increased over time, reaching their peak at 42 days. Furthermore, the P23-LS-VP8* group had higher levels of specific antibodies than the P23-VP8* monomer group. Neither the LS nor the PBS groups produced significant specific antibodies from 0 to 42 days after immunization.

[0085] (3) Neutralization test

[0086] The inactivated mouse serum was diluted to 2 10 50 μL of mouse serum and 50 μL of 200 TCID 50 The P23 porcine rotavirus solution was mixed and incubated at 37°C for 1 hour. The serum-virus mixture was then added to a 96-well plate of MA104 cells at a density of 90% (the culture medium contained trypsin at a final concentration of 4 μg / mL). The cells were cultured at 37°C for 3-5 days, the cytopathic effect was observed, and the neutralization titer was calculated. The results are shown in Table 3 (the table values ​​are log2 values) and Figure 8 As shown, Figure 8 *** on the middle bar indicates that the data are significantly different, p < 0.001, and the results are the average of three repeated experiments.

[0087] Table 3 Neutralizing antibody levels in mice in each group after immunization

[0088] Group 0d 14d 28d 42d P23-VP8* 0 1 2 1 P23-Ls-VP8* 0 2.3 6.7 7.3 Ls 0 0 0 0 PBS 0 0 0 0

[0089] According to Table 3 and Figure 8 It can be seen that from 0d to 42d after immunization, neither the LS group nor the PBS group produced neutralizing antibodies. On 28d and 42d after immunization, both the P23-VP8* and P23-LS-VP8* groups produced neutralizing antibodies. Moreover, on 42d after immunization, the neutralizing antibody titer (1:157.6) of the P23-LS-VP8* group was significantly higher than that of the control group and the P23-VP8* group. The neutralizing antibody titer was calculated using the following formula: N = 2 n , where N represents the neutralizing antibody titer and n represents the number of 2-fold dilutions, i.e., the neutralizing antibody level values ​​in Table 3. The results show that the nanoparticle vaccine prepared by the present invention can induce the production of neutralizing antibodies and the effect is higher than that of P23-VP8* monomer.

[0090] In summary, the porcine rotavirus nanoparticles of the present invention can display a larger number of antigens at a single time, improve the immunogenicity of the vaccine, and enhance its immune effect; the vaccine prepared using the porcine rotavirus nanoparticles can induce higher specific antibody levels and higher neutralizing antibody levels.

[0091] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A porcine rotavirus nanoparticle, characterized in that: The porcine rotavirus nanoparticles include a porcine rotavirus VP8 protein fragment, a dioxopterin synthase, and a linker; the porcine rotavirus VP8 protein fragment and the dioxopterin synthase are connected via the linker; the amino acid sequence of the porcine rotavirus nanoparticles is shown in SEQ ID NO.

4.

2. Porcine rotavirus nanoparticles with a purification tag added to the C-terminus of the nanoparticles according to claim 1.

3. The porcine rotavirus nanoparticles according to claim 2, characterized in that The purification tag includes a His tag.

4. The coding gene of the porcine rotavirus nanoparticles according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

5.

5. A recombinant expression vector containing the coding gene according to claim 4.

6. The method for preparing porcine rotavirus nanoparticles according to claim 1, characterized in that: The method comprises the following steps: transforming the recombinant expression vector according to claim 5 into a prokaryotic expression cell to obtain an engineered strain; culturing the engineered strain, and obtaining the porcine rotavirus nanoparticles through expression and purification.

7. The preparation method according to claim 6, characterized in that The prokaryotic expression cells include Escherichia coli competent cells.

8. Use of the porcine rotavirus nanoparticles according to claim 1, the encoding gene according to claim 4, the recombinant expression vector according to claim 5, or the preparation method according to claim 6 or 7 in the preparation of porcine rotavirus nanoparticle vaccines.

9. A porcine rotavirus nanoparticle vaccine, characterized in that: The invention comprises an adjuvant and the porcine rotavirus nanoparticles according to any one of claims 1 to 3 or the porcine rotavirus nanoparticles prepared by the preparation method according to claim 6 or 7.

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