Porcine rotavirus nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of veterinary vaccines, and particularly relates to porcine rotavirus nanoparticles as well as a preparation method and application thereof. The invention provides a porcine rotavirus nano-particle. The porcine rotavirus nano-particle comprises a porcine rotavirus VP8 protein fragment, a dioxo tetrahydropteridine synthase and a linker, wherein the linker comprises a porcine rotavirus VP8 protein fragment and a dioxo tetrahydropteridine synthase; the porcine rotavirus VP8 protein fragment is connected with the dioxo tetrahydropteridine synthase through the linker, and the linker is connected with the dioxo tetrahydropteridine synthase; the amino acid sequence of the porcine rotavirus VP8 protein fragment is as shown in SEQ ID NO. 1. The porcine rotavirus nanoparticles can display more antigens at a time, so that the immunogenicity of vaccines is improved, and animals can be induced to generate higher antibody level. Results of the embodiment show that the prepared porcine rotavirus nanoparticle vaccine can cause a higher specific antibody level, and compared with a porcine rotavirus VP8 protein fragment monomer immune group, the porcine rotavirus nanoparticle vaccine can cause a higher neutralizing antibody level.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary vaccines, and specifically 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. The mature and complete rotavirus particles have an icosahedral symmetric shape, with a dense hexagonal core in the center, a diameter of 37-40 nm, no envelope, and a double-layer capsid. The rotavirus genome consists of 11 independent fragments of double-stranded positive-strand RNA (dsRNA), encoding 6 structural proteins (VP1-VP4, VP6 and VP7) and 5 non-structural proteins (NSP1-NSP5). Porcine rotavirus can cause diarrhea in pigs and death of piglets, causing huge economic losses to farmers. Vaccine immunization has become an important way to reduce the related morbidity, mortality and economic burden.

[0003] Studies have shown that the PoRV capsid spike protein VP4 is an important anti-rotavirus target, mediating the adsorption and cell entry of rotavirus, and can effectively induce the production of neutralizing antibodies, making it an ideal candidate molecule for subunit vaccine development. However, the VP4 protein is prone to form inclusion bodies in the prokaryotic expression system, resulting in low solubility of the VP4 protein and increasing the difficulty of subsequent processing. Therefore, it is necessary to develop a new, simple and efficient porcine rotavirus nanoparticle vaccine. Summary of the invention

[0004] The purpose of the present invention is to provide a porcine rotavirus nanoparticle and a preparation method and application thereof. 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 dioxotetrahydropterin synthase and a linker; the porcine rotavirus VP8 protein fragment and the dioxotetrahydropterin synthase are connected through 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 as SEQ ID NO.2.

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

[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 wheel-shaped nanoparticles described in the above scheme or the porcine wheel-shaped 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 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. The porcine rotavirus VP8 protein fragment (VP8*) of the present invention contains the main antigenic epitope of VP4, is highly conserved between the same serotypes, can induce the production of neutralizing antibodies, and has a solubility greater than VP4 in the Escherichia coli expression system. Dioxopterin synthase (LS) can spontaneously form icosahedral nanoparticles, can display more antigens at a time, improve the immunogenicity of the vaccine, and enhance its immune effect. The porcine rotavirus nanoparticle of the present invention is a non-whole virus antigen, which is used to prepare a porcine rotavirus nanoparticle vaccine without the risk of virus spread, and the production process is very safe, and can achieve efficient production of genetic engineering 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 to be used in the embodiments are briefly introduced below.

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

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

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

[0022] Figure 4 This is the SDS-PAGE detection diagram 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 observation results of LS-VP8* and LS nanoparticle proteins after purification in Example 2; the scale bar in the horizontal 100nm figure represents 100nm, and the scale bar in the horizontal 200nm figure represents 200nm;

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

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

[0027] The invention provides a porcine rotavirus nanoparticle, which comprises a porcine rotavirus VP8 protein fragment, a dioxotetrahydropteridine synthase and a linker; the porcine rotavirus VP8 protein fragment and the dioxotetrahydropteridine synthase are connected by the linker; the amino acid sequence of the porcine rotavirus VP8 protein fragment is shown in SEQ ID NO.1, which is specifically as follows: VLDGPYQPTTFNPPV SYWILLSPSNAGVVVEGTNNSDRWLATILIEPNVTSQSRTYTLFGQQEQITVEN VSTTKWKFVDLAKTDVNGTFTQHGPLLSDTKLYGVMKFSGRLYTYNGETPN ATTSYYTTTNYDTVNMTSHCDFYIIPRSEETMCTNYINNG. The porcine rotavirus VP8 protein fragment of the invention comprises the main antigenic epitope of VP4, is highly conserved between the same serotypes, can induce the production of neutralizing antibodies, and has a higher solubility than VP4 in an Escherichia coli expression system.

[0028] As an embodiment, the amino acid sequence of the tetrahydroxeteridine synthase of the present invention is shown in SEQ ID NO.2, which is as follows: QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDA IVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYI ASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIE MANLFKSLR. The tetrahydroxeteridine synthase (LS) of the present invention can spontaneously form icosahedral nanoparticles, which can display more antigens at a single time, improve the immunogenicity of the vaccine, and enhance 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 of 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 a 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] As an embodiment, a purification tag is added to the C-terminus of the porcine rotavirus nanoparticles of the present invention. As another embodiment, the purification tag of the present invention includes a His tag. The purification tag of the present invention can simplify the subsequent purification steps and improve the purification efficiency. In one embodiment, the porcine rotavirus nanoparticles of the present invention and the purification tag are connected by a linker, and the amino acid sequence of the linker 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 gene encoding is as shown in SEQ ID NO.As shown in 5, specifically as follows: 5'-CAGATCTATGAGG GGAAACTGACAGCCGAGGGACTGAGGTTTGGAATCGTCGCTTCACGCTTCAATCACGCACTGGTGGACAGACTGGTGGAAGGCGCTATCGACGCAATTGTCCGGCACGGCGGGAGAGAGGAAGATATCACTCTGGTGAGAGTCCCAGGGAGCTGGGAGATTCCAGTGGCAGCTGGAGAACTGGCTCGGAAGGAGGACATCGATGCCGTGATCGCTATTGGAGTCCTGATTAGAGGCGCAACACCTCACTTCGACTACATCGCCTCAGAAGTGAGCAAGGGACTGGCTGATCTGTCCCTGGAGCTGAGGAAACCAATCACCTTTGGCGTCATTACTGCCGACACCCTGGAACAGGCAATCGAGCGCGCCGGGACAAAGCATGGCAACAAAGGGTGGGAAGCAGCCCTGTCAGCTATTGAGATGGCAAATCTGTTCAAAAGCCTGCGAGGAGGCTCCGGAGGATCTGTTCTGGATGGTCCGTATCAGCCGACCACCTTTAATCCGCCGGTTAGCTATTGGATTCTGCTGAGCCCGAGCAATGCAGGTGTTGTTGTTGAAGGTACCAATAATAGCGATCGTTGGCTGGCAACCATTCTGATTGAACCGAATGTTACCAGCCAGAGCCGTACCTATACCCTGTTTGGTCAGCAGGAACAGATTACCGTTGAAAATGTTAGCACCACCAAATGGAAATTTGTTGATCTGGCAAAAACCGATGTTAATGGTACCTTTACCCAGCATGGTCCGCTGCTGAGCGATACCAAACTGTATGGTGTTATGAAATTTAGCGGTCGTCTGTATACCTATAATGGTGAAACCCCGAATGCAACCACCAGCTATTATACCACCACCAATTATGATACCGTTAATATGACCAGCCATTGTGATTTTTATATTATTCCGCGTAGCGAAGAAACCATGTGTACCAATTATATTAATAATGGTTAA-3'.

[0033] The present invention also provides a recombinant expression vector containing the coding gene of the above scheme. As one embodiment, the basic vector of the recombinant expression vector of the present invention includes pHAT2 vector; as another embodiment, the present invention inserts the coding gene between the Nco I and BamHI restriction sites of the basic 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. As an embodiment, the method for transforming the recombinant expression vector described in the present invention into a prokaryotic expression cell includes a CaCl2 heat shock method. As an embodiment, the prokaryotic expression cell described in the present invention includes an Escherichia coli competent cell, and in a specific embodiment, the Escherichia coli competent cell can be an Escherichia coli competent DE3 cell.

[0036] In the present invention, after obtaining the engineered strain, the present invention cultured the engineered strain, expressed and purified to obtain the porcine rotavirus nanoparticles. As an embodiment, the expression of the present invention includes inducing expression using IPTG to obtain expressed bacteria. As an embodiment, the purification of the present invention includes low-temperature ultrasonic crushing of the expressed bacteria, centrifugation to obtain a supernatant; passing the supernatant through a Ni-filled chromatography column to elute to obtain a 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 wheel-shaped nanoparticles described in the above scheme or the porcine wheel-shaped nanoparticles prepared by the preparation method described in the above scheme. As an embodiment, the adjuvant of the present invention comprises Qm01E adjuvant; the Qm01E adjuvant comprises ginsenoside, astragalus polysaccharide and divalent manganese salt; the mass ratio of the ginsenoside, astragalus polysaccharide and divalent manganese salt is (1-10):(1-10):0.1. In a specific embodiment, the mass ratio of the ginsenoside, astragalus polysaccharide and divalent manganese salt can be 1:1:0.1. The present invention utilizes the Qm01E adjuvant, and the vaccine can be prepared by simply mixing the Qm01E adjuvant and the protein evenly without emulsification, which simplifies the vaccine production procedure, can reduce the vaccine production cost, and enhance the vaccine immunogenicity.

[0039] In order to further illustrate the present invention, a porcine rotavirus nanoparticle and a preparation method and application thereof provided by the present invention are described in detail below in conjunction with 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 containing a 6His tag was synthesized with reference to GenBank: KX527854.1 sequence (SEQ ID NO. 6). The sequence information is as follows: 5'-CAGATCTATGAGGGGAAACTGACAGCCGAGGGACTGAGGTTTGGAATC GTCGCTTCACGCTTCAATCACGCACTGGTGGACAGACTGGTGGAAGGCGCTATCGACGCAATTGTCCGGCACGGCGGGAGAGGAAGATATCACTCTGGTGAGAGTCCCAGGGAGCTGGGAGATTCCAGTGGCAGCTGGAGAACTGGCTCGGAAGGAGGACATCGATGCCGTGATCGCTATTGGAGTCCTGATTAGAGGCGCAACACCTCACTTCGACTACATC GCCTCAGAAGTGAGCAAGGGACTGGCTGATCTGTCCCTGGAGCTGAGGAAACCAATCACCTTTGGCGTCATTACTGCCGACACCCTGGAACAGGCAATCGAGCGCGCCGGGACAAAGCATGGCAACAAAGGGTGGGAAGCAGCCCTGTCAGCTATTGAGATGGCAAATCTGTTCAAAAGCCTGCGAGGAGGCTCCGGAGGATCTCATCACCATCACCATCAC-3'.

[0043] (2) Extract the gene of P23 PoRV [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], use the extracted P23 PoRV gene as a template, and amplify the VP8* gene fragment (SEQ ID NO.7) with upstream and downstream primers. The primers are VP8*-F (SEQ ID NO.8) and VP8*-R (SEQ ID NO.9), and the sequence is as follows:

[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 (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 the above steps (1) and (3) as templates, the preferred codons of the Escherichia coli prokaryotic expression system were optimized, and overlap PCR was used to amplify LS-VP8* (SEQ ID NO: 11) with a 6His tag; the primers of 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 was 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 is 2×PhantaMax Buffer 25μL, dNTPMix 1μL, 10μM forward primer 2μL, 10μM reverse primer 2μL, template 100ng, PhantaRMax Super-Fidelity DNA Polymeras (1U / μL), and ddH2O is used to make up the system to 50μL. The amplification reaction program is 95℃5min; (95℃15s, 65℃15s, 72℃1min) 30 cycles; 72℃10min.

[0055] (5) Using the LS-VP8* obtained in the above 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 is 2×PhantaMax Buffer 25μL, dNTPMix 1μL, 10μM forward primer 2μL, 10μM reverse primer 2μL, template 100ng, PhantaRMax Super-Fidelity DNA Polymeras (1U / μL), and ddH2O is used to make up the system to 50μL. The amplification reaction program is 95℃5min; (95℃15s, 65℃15s, 72℃1min) 30 cycles; 72℃10min.

[0057] (6) The VP8* and LS-VP8* gene fragments obtained in the above steps (4) and (5) are respectively cloned between 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 the plasmids with correct digestion were selected for sequencing after double restriction digestion. The plasmids with successful sequencing were used for the expression of antigen proteins, and the digestion results were detected. 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 the above 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 broken bacterial solution described in the above step (2) was centrifuged at 13000rpm and 4℃ for 30min, the supernatant was collected, and then filtered with a 0.22μm filter membrane to remove cell debris. The filtered supernatant was allowed to bind to the Ni filler (the His tag at the end of the target protein can bind to Ni) through a peristaltic pump. After the binding was completed, the target protein was automatically eluted with a 0-500mM gradient concentration imidazole buffer at a uniform speed within 1.5h through a protein purification elution instrument. According to the peak position of the target protein, the concentration gradient range of the imidazole buffer was determined, and the eluate within the range was collected using a collection tube, and 2mL of eluate was collected in each collection tube. Among them, the target protein VP8* collected 9 tubes of eluate within the gradient concentration of 80-130mM, numbered 1-9, the target protein LS-VP8* collected 12 tubes of eluate within the gradient concentration of 80-150mM, numbered 1-12, and the target protein LS collected 12 tubes of eluate within the gradient concentration of 80-150mM, numbered 1-12. Subsequently, SDS-PAGE was used to detect the purification effect of the target protein in different eluates. 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* in different eluted 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* in different eluted 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* in different eluted 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 [Anti-GFP nanoantibodies as affinity ligands to purify gap junction protein 26 [J], Tong Ling et al., Biotechnology, 2023]. The eluent was eluted at a uniform 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 the UV absorbance to collect the target protein. LS-VP8* elutes at about 70 mL after loading. 14 collection tubes were used to collect the eluate at 65 mL of elution. Each collection tube had 2 mL of eluate, which were numbered 1 to 14 in sequence. LS elutes at about 80 mL after loading. 14 collection tubes were used to collect the eluate at 75 mL of elution. Each collection tube had 2 mL of eluate, which were numbered 1 to 14 in sequence. The proteins collected after purification were detected by SDS-PAGE, and the results were 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 10kDa and 30kDa ultrafiltration tubes at 3500rpm at 4°C, and concentrated to about 2mL and replaced with PBS. The concentrated LS-VP8* protein solution was filtered with a 0.22μm filter, diluted with pure water to 0.1mg / mL, and the particle size of the LS-VP8* fusion protein was measured and analyzed by 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 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, respectively, and stained with a 2% (w / v) sodium phosphotungstate solution for 30 seconds. After the copper mesh was completely dried, a transmission electron microscope was used to observe whether nanoparticles were formed. The results are as follows: 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 experiment

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

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

[0076] Twenty 6-week-old female BALB / c mice were ordered from the Experimental Animal Center of Huazhong Agricultural University and randomly divided into 4 groups, namely LS group, P23-VP8* group, P23-LS-VP8* group and PBS group (negative control group), with 5 mice in each group, and the immunization route was hind leg muscle injection. The immunization dose for each mouse was 40 μg / mouse, and each mouse received two vaccine immunizations on day 0 and day 14, respectively. The injection method is shown in Table 1, and the above experiment was repeated 3 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 min to allow serum to precipitate, and then centrifuged at 3000 rpm and 4°C for 10 min to collect serum for detection of specific IgG levels and neutralizing antibody levels in serum.

[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 precipitate 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 ELISA 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 the secondary antibody was added to each well, and the cells were incubated at 37°C for 1 h. After the incubation, the secondary antibody was discarded and the cells were washed 4 times with PBST, 5 min / times. 100 μL of ELISA colorimetric solution was added to each well, and the cells were reacted in the dark for 10 min. After the reaction was completed in the dark, 50 μL of ELISA stop solution was added, and the OD of each well was read using an enzyme reader. 450 The results are shown in Table 2 and Figure 7 The results are shown as 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 in each group of mice after immunization 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 It can be seen that at 28 days and 42 days after immunization, the serum of mice in each experimental group produced high specific antibody levels, which increased over time and reached the highest value at 42 days. In addition, the specific antibody level of the P23-LS-VP8* experimental group was higher than that of the P23-VP8* monomer experimental group on average. From 0 days to 42 days after immunization, no obvious specific antibodies were produced in the LS group and the PBS group.

[0085] (3) Neutralization test

[0086] The inactivated mouse serum was diluted to 2 10 , take 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, and then the serum virus mixture was added to a 96-well plate of MA104 cells with a density of 90% (the culture medium contained trypsin with a final concentration of 4 μg / mL). After culturing at 37°C for 3 to 5 days, the cell lesions were 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 each group of mice 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. At 28d and 42d after immunization, both the P23-VP8* and P23-LS-VP8* groups produced neutralizing antibodies. At 42d after immunization, the neutralizing antibody titer (1:157.6) of the P23-LS-VP8* immunization 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 a higher level of specific antibodies and a higher level of neutralizing antibodies.

[0091] Although the above embodiment describes the present invention in detail, 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 protection scope 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 dioxotetrahydropterin synthase and a linker; the porcine rotavirus VP8 protein fragment and the dioxotetrahydropterin synthase are connected via the linker; the amino acid sequence of the porcine rotavirus VP8 protein fragment is shown in SEQ ID NO.

1.

2. The porcine rotavirus nanoparticle according to claim 1, characterized in that: The amino acid sequence of the dioxotetrahydropterin synthase is shown in SEQ ID NO.

2.

3. The porcine rotavirus nanoparticle according to claim 1 or 2, characterized in that: A purification tag is added to the C-terminus of the porcine rotavirus nanoparticles.

4. The porcine rotavirus nanoparticle according to claim 3, characterized in that: The purification tag includes a His tag.

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

5.

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

7. The method for preparing porcine rotavirus nanoparticles according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: transforming the recombinant expression vector according to claim 6 into a prokaryotic expression cell to obtain an engineered strain; culturing the engineered strain, and obtaining the porcine rotavirus nanoparticles through expression and purification.

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

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

10. A porcine rotavirus nanoparticle vaccine, characterized in that: The invention comprises an adjuvant and the pig wheel-shaped nanoparticles according to any one of claims 1 to 5 or the pig wheel-shaped nanoparticles prepared by the preparation method according to claim 7 or 8.

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