A fusion protein, rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine and application

Abstract

The application provides a fusion protein, a rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine and application, and belongs to the technical field of biological medicine. The application provides a fusion protein, which comprises a first polypeptide, a hinge region of a rabbit hemorrhagic disease virus type 2 capsid protein, a P region of a rabbit hemorrhagic disease virus capsid protein and a second polypeptide connected in sequence; the first polypeptide and the second polypeptide are polypeptides containing four cysteines respectively. The fusion protein RGD4C-HP-RGD4C of the application can self-assemble to form nanoparticles, and the particle size is 40-50 nm. The fusion protein of the application can induce domestic rabbits to produce specific antibodies against RHDV2, produce a strong humoral immune response, and provide complete protection for the domestic rabbits.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a fusion protein, a rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine and its application. Background Technology

[0002] Rabbit hemorrhagic disease (RHD) is a highly contagious and deadly disease caused by rabbit hemorrhagic disease virus (RHDV). More than 90% of infected rabbits typically die within 3 days, severely impacting the rabbit industry. The VP60 protein is the main structural protein of RHDV, capable of self-assembling in vitro into virus-like particles (VLPs) with a morphology similar to natural RHDV virus particles. Simultaneously, the VP60 protein can induce the production of neutralizing antibodies in animals, serving as an immunoprotective antigen for RHDV. The VP60 protein can be divided into three domains: NTA, S, and P, with the S and P domains connected by a hinge region (H). The NTA and S domains are located inside the virus-like particles, while the P domain is located on the outer surface, forming spike structures that are closely related to neutralizing antibody levels. The antigenicity of the P domain is only 1 / 10 to 1 / 100 that of the NTA and S domains. Therefore, genetically engineered subunit vaccines prepared using VP60 protein as an antigen suffer from low antibody levels in the P domain, resulting in less precise and efficient antibody production. Summary of the Invention

[0003] The purpose of this invention is to provide a fusion protein, a rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine, and its application.

[0004] This invention provides a fusion protein comprising, in sequence, a first polypeptide, a hinge region of a rabbit hemorrhagic virus type 2 capsid protein, a P region of a rabbit hemorrhagic virus capsid protein, and a second polypeptide; the first polypeptide and the second polypeptide are each polypeptide containing four cysteine ​​residues; the polypeptide containing four cysteine ​​residues includes RGD4C; the amino acid sequence of RGD4C is shown in SEQ ID NO.1; the amino acid sequence of the hinge region of the rabbit hemorrhagic virus type 2 capsid protein is shown in SEQ ID NO.2; the P region of the rabbit hemorrhagic virus capsid protein includes the P region of the rabbit hemorrhagic virus type 2 capsid protein; the amino acid sequence of the P region of the rabbit hemorrhagic virus type 2 capsid protein is shown in SEQ ID NO.3.

[0005] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.4.

[0006] The present invention also provides the encoding gene of the fusion protein described in the above scheme.

[0007] Preferably, the nucleotide sequence encoding the gene includes the nucleotide sequence shown in SEQ ID NO.5 or a sequence after codon optimization of the nucleotide sequence shown in SEQ ID NO.5.

[0008] Preferably, the nucleotide sequence of the codon-optimized nucleotide sequence shown in SEQ ID NO.5 is as shown in SEQ ID NO.6 or SEQ ID NO.7.

[0009] The present invention also provides a recombinant vector into which the coding gene described in the above scheme is inserted.

[0010] The present invention also provides a recombinant bacterium or recombinant cell comprising the recombinant vector described in the above-described scheme.

[0011] The present invention also provides the use of the fusion protein, the encoding gene, the recombinant vector, or the recombinant bacteria or recombinant cells described above in at least one of the following:

[0012] 1) To prepare drugs for the prevention or treatment of rabbit hemorrhagic virus type 2 infection;

[0013] 2) Application in the preparation of biological products for the prevention of rabbit hemorrhagic disease virus type 2 infection;

[0014] 3) Detection reagents or kits for preparing rabbit hemorrhagic disease virus type 2 capsid protein-specific antibodies.

[0015] The present invention also provides a rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine, comprising the fusion protein described in the above-described scheme.

[0016] The present invention also provides a detection reagent or kit for rabbit hemorrhagic disease virus type 2 capsid protein-specific antibody, using the fusion protein described in the above scheme as the coating antigen.

[0017] This invention provides a fusion protein comprising, in sequence, a first polypeptide, a hinge region of the rabbit hemorrhagic disease virus type 2 capsid protein, a P region of the rabbit hemorrhagic disease virus capsid protein, and a second polypeptide; the first polypeptide and the second polypeptide are each polypeptide containing four cysteine ​​residues. The fusion protein of this invention can self-assemble into nanoparticles with a particle size of 40-50 nm. These self-assembled nanoparticles form polymers with a larger effective surface area, and since they contain only the P region of VP60, they directly expose neutralizing epitopes, facilitating the activation of pattern recognition receptors (such as TLRs), promoting B cell activation, and reducing the waste of immune resources against non-protective epitopes (NTA region, S region). The fusion protein of this invention can induce rabbits to produce specific antibodies against RHDV2, generating a strong humoral immune response and providing complete protection for rabbits. Verification has shown that immunization of rabbits with the fusion protein of this invention results in high specific IgG antibody titers against both RHDV2 VP60 and RHDV2 VP60-P, and the antibody level of the fusion protein is higher than that of RHDV2 VP60. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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 these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the recombinant carrier structure;

[0020] Figure 2 A schematic diagram of RHDV2 VP60-P recombinant Bacmid;

[0021] Figure 3 Figure showing the expression results of recombinant pCI-RHDV2 VP60-P granule protein in HEK 293; where M is the 14~100kDa Marker; 1, pCI-RHDV2-P; 2, pCI-P-RGD4C; 3, pCI-P-CNGRC; 4, pCI-RGD4C-P; 5, pCI-CNGRC-P; 6, pCI-RGD4C-P-RGD4C; 7, pCI-CNGRC-P-CNGRC; 8, pCI-RHDV2-HP; 9, pCI-HP-RGD4C; 10, pCI-HP-CNGRC; 11, pCI-RGD4C-HP; 12, pCI-CNGRC-HP; 13, pCI-RGD4C-HP-RGD4C; 14, pCI-CNGRC-HP-CNGRC.

[0022] Figure 4 The results of the evaluation of the ability of recombinant pCI-RHDV2 VP60-P granule protein to form protein aggregates in HEK 293; where M is the 10~180kDa Marker; 1, pCI-RHDV2-P; 2, pCI-P-RGD4C; 3, pCI-P-CNGRC; 4, pCI-RGD4C-P; 5, pCI-CNGRC-P; 6, pCI-RGD4C-P-RGD4C; 7, pCI-CNGRC-P-CNGRC; 8, pCI-RHDV2-HP; 9, pCI-HP-RGD4C; 10, pCI-HP-CNGRC; 11, pCI-RGD4C-HP; 12, pCI-CNGRC-HP; 13, pCI-RGD4C-HP-RGD4C; 14, pCI-CNGRC-HP-CNGRC.

[0023] Figure 5 The image shows the results of the first blue-white screening of recombinant transfer vectors; where M is the DL5000 Marker; 1~13 RGD4C-HP was screened for the first time; 14~23 RGD4C-HP-RGD4C was screened for the first time.

[0024] Figure 6 The image shows the results of the second and third blue-white screenings of the recombinant transfer vector; where M is the DL5000 Marker; 1~5 are RGD4C-HP for the second screening; 6~10 are RGD4C-HP-RGD4C for the second screening; 11 and 12 are RGD4C-HP for the third screening; and 13 and 14 are RGD4C-HP-RGD4C for the third screening.

[0025] Figure 7 Normal Sf9 cells and Sf9 cells transfected with the disease at 24 h, 48 h, and 72 h.

[0026] Figure 8 Figure showing the results of indirect immunofluorescence assay for the expression of recombinant rBac-RHDV2 VP60-P virus in Sf9 monolayer cells; where NC: Sf9 monolayer cells without plasmid extraction and transfection; R-HP: Sf9 monolayer cells seeded with rBac-RGD4C-HP; R-HP-R: Sf9 monolayer cells seeded with rBac-RGD4C-HP-RGD4C.

[0027] Figure 9Figure 1 shows the results of Western blot analysis of recombinant RHDV2 VP60-P granular protein expressed in High Five cells; where M represents the 14–100 kDa Marker; 1, 24 h sample of recombinant RHDV2 VP60-P granular protein; 2, 48 h sample of recombinant RHDV2 VP60-P granular protein; 3, 72 h sample of recombinant RHDV2 VP60-P granular protein; 4, 96 h sample of recombinant RHDV2 VP60-P granular protein; 5, 120 h sample of recombinant RHDV2 VP60-P granular protein; 6, 144 h sample of recombinant RHDV2 VP60-P granular protein; 7, 168 h sample of recombinant RHDV2 VP60-P granular protein.

[0028] Figure 10 The purpose of this study was to detect the formation of VLPs from recombinant RHDV2 VP60-P particle antigen using transmission electron microscopy with negative staining. In this study, A represents RHDV2 VP60 protein, B represents RGD4C-HP-RGD4C protein, and C represents RGD4C-HP protein.

[0029] Figure 11 This is a comparison chart of immunization dosages; where M is the 14-100 kDa Marker; 1 is the RHDV2 VP60 protein; 2 is the RGD4C-HP-RGD4C protein; and 3 is the RGD4C-HP protein.

[0030] Figure 12 Image showing the purification results of the P region antigen of rabbit hemorrhagic disease virus type 2 capsid protein; where M: 14~100 kDa Marker; 1: Induced expression; 2: Ultrasonic disruption and centrifugation precipitation; 3: Ultrasonic disruption and centrifugation supernatant; 4: 8M dissolution to remove precipitate; 5: 4M dissolution to remove precipitate; 6: 0M dissolution to remove precipitate; 7: Sample after refolding; 8: 0.5 mg / ml BSA standard;

[0031] Figure 13 The results represent the immune protection outcomes; among them, CT and PBS are control groups.

[0032] Figure 14 The image shows a dead rabbit; among which, A: bleeding from the mouth and nose; B: pulmonary congestion and bleeding; C: liver hemorrhage; D: splenomegaly.

[0033] Figure 15 Figure showing the monitoring results of RHDV2 VP60 protein-specific IgG antibody levels; *: P <0.05, **: P <0.01, ***: P <0.001, ****: P <0.0001;

[0034] Figure 16 The graph shows the monitoring results of RHDV2 VP60-P protein-specific IgG antibody levels; *: P <0.05, **: P <0.01, ***: P <0.001, ****: P <0.0001. Detailed Implementation

[0035] The present invention provides a fusion protein RGD4C-HP-RGD4C, comprising a first polypeptide, a hinge region of rabbit hemorrhagic virus type 2 capsid protein, a P region of rabbit hemorrhagic virus capsid protein, and a second polypeptide connected in sequence; the first polypeptide and the second polypeptide are each polypeptides containing 4 cysteine ​​residues.

[0036] In one implementation, the sequential connection includes a sequential connection from the N end to the C end.

[0037] Cysteine ​​(abbreviated Cys or C) plays a crucial role in maintaining the higher-order structure of proteins. Two spatially close cysteine ​​residues can maintain protein stability through the formation of disulfide bonds. The amino acid sequence of RGD4C is CDCRGDCFC, containing four cysteine ​​(C) residues. The expressed protein forms disulfide bonds between these residues, potentially leading to the formation of multimeric structures, such as nanoparticles. The fusion protein RGD4C-HP-RGD4C of this invention, by linking multiple cysteine ​​residues to both ends of the P region, can form a nanoparticle structure. Furthermore, vaccines prepared with this protein exhibit excellent immunogenicity, showing higher antibody levels against the P region than the full-length VP60.

[0038] In one embodiment, the polypeptide containing four cysteine ​​residues includes RGD4C; the amino acid sequence of RGD4C is shown in SEQ ID NO.1, specifically: CDCRGDCFC.

[0039] As one embodiment, the amino acid sequence of the hinge region of the rabbit hemorrhagic disease virus type 2 capsid protein is shown in SEQ ID NO.2, specifically as follows: SSKTVDSIThe P region of the rabbit hemorrhagic virus capsid protein includes the P region of the rabbit hemorrhagic virus type 2 capsid protein; the amino acid sequence of the P region of the rabbit hemorrhagic virus type 2 capsid protein is as shown in SEQ ID. As shown in NO.3, specifically: SPADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFAAIDHDRGNASFPGSSSSNVLELWYASAGSAADNPISQIAPDGFPDMSFVPFSGITIPTAGWVGFGGIWNSSNGAPYVTTMQAYELGFATGVPSNPQPTTTTSGAQIVAKS IYGVANGINQTTAGLFVMASGVISTPNSSATTYTPQPNRIVNAPGTPAAAPIGKNTPIMFASVVRRTGDINAEAGSTNGTQYGAGSQPLPVTIGLSLNNYSSALMPGQFFVWQLNFASGFMELGLSVDGYFYAGTGASATLIDLSDLVDIRPVGPRPSTSTLVYNLGGTTNGFSYV.

[0040] In one embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO.4, specifically: CDCRGDCFC SSKTVDSI SPADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFAAIDHDRGNASFPGSSSSNVLELWYASAGSAADNPISQIAPDGFPDMSFVPFSGITIPTAGWVGFGGIWNSSNGAPYVTTMQAYELGFATGVPSNPQPTTTTSGAQIVAKSIYGVANGINQ TTAGLFVMASGVISTPNSSATTYTPQPNRIVNAPGTPAAAPIGKNTPIMFASVVRRTGDINAEAGSTNGTQYGAGSQPLPVTIGLSLNNYSSALMPGQFFVWQLNFASGFMELGLSVDGYFYAGTGASATLIDLSDLVDIRPVGPRPSTSTLVYNLGGTTNGFSYVCDCRGDCFC.

[0041] In one implementation, RGD4C, the hinge region of the rabbit hemorrhagic virus type 2 capsid protein, the P region of the rabbit hemorrhagic virus type 2 capsid protein, and RGD4C are directly connected in sequence without any linkers in between.

[0042] The present invention also provides the encoding gene of the fusion protein described in the above scheme.

[0043] In one embodiment, the nucleotide sequence encoding the gene includes the nucleotide sequence shown in SEQ ID NO. 5 or a sequence obtained by codon optimization of the nucleotide sequence shown in SEQ ID NO. 5. Specifically, the nucleotide sequence shown in SEQ ID NO. 5 is: tgcgactgccgcggcgactgcttctgc agcagcaagaccgtggacagcatcagccccgc cgacctgctgaccacccccgtgctgaccggcgtgggcaccgacaaccgctggaacggcgagatcgtgggcctgcag cccgtgcccggcggcttcagcacctgcaaccgccactggaacctgaacggcagcacctacggctggagcagccccc gcttcgccgccatcgaccacgaccgcggcaacgccagcttccccggcagcagcagcagcaacgtgctggagctgtg gtacgccagcgccggcagcgccgccgacaaccccatcagccagatcgcccccgacggcttccccgacatgagcttc gtgcccttcagcggcatcaccatccccaccgccggctgggtgggcttcggcggcatctggaacagcagcaacggcg ccccctacgtgaccaccatgcaggcctacgagctgggcttcgccaccggcgtgcccagcaacccccagcccaccac caccaccagcggcgcccagatcgtggccaagagcatctacggcgtggccaacggcatcaaccagaccaccgccggc ctgttcgtgatggccagcggcgtgatcagcacccccaacagcagcgccaccacctacaccccccagcccaaccgca tcgtgaacgcccccggcacccccgccgccgcccccatcggcaagaacacccccatcatgttcgccagcgtggtgcg ccgcaccggcgacatcaacgccgaggccggcagcaccaacggcacccagtacggcgccggcagccagcccctgccc gtgaccatcggcctgagcctgaacaactacagcagcgccctgatgcccggccagttcttcgtgtggcagctgaact tcgccagcggcttcatggagctgggcctgagcgtggacggctacttctacgccggcaccggcgccagcgccaccct gatcgacctgagcgacctggtggacatccgccccgtgggcccccgccccagcaccagcaccctggtgtacaacctg ggcggcaccaccaacggcttcagctacgtg tgcgactgccgcggcgactgcttctgc. The bolded sequence is the coding gene for RGD4C, the wavy line sequence is the coding gene for the hinge region of the rabbit hemorrhagic virus type 2 capsid protein, and the double underlined sequence is the coding gene for the P region of the rabbit hemorrhagic virus type 2 capsid protein.

[0044] In one implementation, the codon optimization includes codon optimization using human cells or insect cells; the nucleotide sequence encoding the gene is optimized using human cell codons and then further linked with restriction enzyme sites, a KOZAK sequence, a start codon, and a stop codon sequence, as shown in SEQ ID NO.6, specifically: gaattcgccacc atgtgcgactgcagaggcgattgcttttgt agcagcaagaccgtggacagcatcagccccgccgatctgctgaccac acctgtgctgaccggagtgggcaccgacaacagatggaacggcgagatcgtgggcctgcagcccgtgcctggtggc ttcagcacatgcaaccgccactggaatctgaacggctccacatacggctggtcctcccctagattcgccgctatcg accacgatagaggcaacgcttctttcccaggctctagcagctccaatgtgctggaactgtggtatgccagcgccgg ctccgcggccgacaaccccatctctcagatcgcccctgatggcttccccgacatgagcttcgtgccattcagcggg attacaatccccaccgccggatgggtgggcttcggcggcatctggaacagcagcaacggcgctccttatgtgacca caatgcaggcctacgagctgggattcgccaccggcgtgcccagcaatcctcagcctaccacaaccacatctggcgc ccagatcgtggccaaaagcatctacggagtcgccaacggaatcaaccagaccaccgccggcctgttcgtcatggcc agcggcgtcatcagcaccccaaacagctctgccaccacctacaccccacaacctaatcggatcgtgaacgcccctg gcacacccgctgctgctcctattggaaagaacacccctatcatgttcgcctctgtggtgcggagaaccggcgacat caacgccgaggccggatctaccaacggaacacagtacggcgccggctcacagcctctgcctgtgaccatcggcctc agcctgaataactacagcagcgccctgatgcctggccaatttttcgtgtggcagctgaacttcgccagcggcttta tggaactgggcctgtctgttgacggctacttctacgccggcacaggcgcctcggcaaccctgatcgacctgagcga ccttgtggacatcagacccgtgggacctagacctagcacctctacactggtgtacaacctgggcggaaccacgaac ggctttagctacgtg tgtgattgtcggggcgactgcttctgctga gtcgac The nucleotide sequence encoding the gene is optimized using insect cell codons and further includes restriction enzyme sites, a KOZAK sequence, a start codon, and a stop codon sequence. The specific sequence is shown in SEQ ID NO.7, and is as follows: gaattcgccacc atgtgcgattgtaggggcgattgcttctgt t cgtctaagaccgtcgacagcatcagccctgcagacttattaacgacaccagtgcttactggagtcggaacagataa ccgttggaatggagaaatcgtcggtttgcaaccagtcccggggggtttctcaacttgtaatcgtcactggaaccta aatggctctacgtatggatggtcatccccccgattcgcggcgattgaccatgatagagggaacgcaagcttcccgg ggagttcctcctcaaatgttctcgagctatggtacgcttcggcgggctcagcggctgataatcctatatcccagat tgccccggatggttttccagatatgtcttttgtgccattctcgggaataacaattcctactgctggctgggttggt tttggtggcatttggaatagtagtaacggggctccctacgttacgacgatgcaagcgtacgagttaggttttgcaa cgggtgtaccatccaacccccaacctacgacaaccacaagcggggctcagattgtagcaaaatctatctatggggt cgcgaacggcattaaccagacgacagccgggctctttgtaatggcaagtggagttatctcgacccctaactcttcg gccaccacctatacgccgcagcccaatcggatagtgaacgcccccggtacccccgctgctgcgcctatcgggaaaa ataccccaataatgtttgccagcgtggttcgccgcacaggcgatattaacgccgaagcaggttccacaaatggaac ccaatatggggcagggtcacaaccgttgccggtaactatcggcttatctttgaacaactatagcagtgcccttatg ccaggccagttctttgtatggcagctaaatttcgccagcggattcatggagctcggactctcggtggacggctact tttacgctggtactggtgcatcggcgactctgatagacctgagtgacctggtggacataagacccgtaggccctcg gccgtccacttcaactcttgtctacaatctggggggaacgaccaatggatttagttatgtt tgcgattgtaggggcgattgcttctgttga gtcgac .

[0045] The present invention also provides a recombinant vector into which the coding gene described in the above scheme is inserted.

[0046] In one embodiment, the backbone plasmid of the recombinant vector includes pCI - neo The encoding gene is inserted into EcorI and SalI Between the restriction enzyme sites. The specific sequence is shown in SEQ ID NO.6, via... EcorI The restriction enzyme site gaattc and SalI Enzyme cleavage site gtcgac and vector pCI - neo connect.

[0047] In another embodiment, the backbone plasmid of the recombinant vector includes pFastBac1 The encoding gene is inserted into EcorI and SalI Between the restriction enzyme sites. The specific sequence is shown in SEQ ID NO.7, via... EcorI cleavage site gaattc and SalI Enzyme cleavage site gtcgac and vector pFastbac1 connect.

[0048] The present invention also provides a recombinant bacterium or recombinant cell comprising the recombinant vector described in the above-described scheme.

[0049] In one embodiment, the original cells of the recombinant bacteria or recombinant cells include DH5α competent cells, DH10Bac competent cells, HEK 293 cells, or Sf9 insect cells.

[0050] The present invention does not impose any special restrictions on the construction method of the recombinant bacteria or recombinant cells; conventional methods in the field can be used.

[0051] The present invention also provides the use of the fusion protein, the encoding gene, the recombinant vector, or the recombinant bacteria or recombinant cells described above in at least one of the following:

[0052] 1) To prepare drugs for the prevention or treatment of rabbit hemorrhagic virus type 2 infection;

[0053] 2) Application in the preparation of biological products for the prevention of rabbit hemorrhagic disease virus type 2 infection;

[0054] 3) Detection reagents or kits for preparing rabbit hemorrhagic disease virus type 2 capsid protein-specific antibodies.

[0055] The present invention also provides a rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine, comprising the fusion protein described in the above-described scheme.

[0056] As one implementation, the rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine also includes a pharmaceutically acceptable adjuvant.

[0057] As one implementation method, the rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine is in the form of an injection.

[0058] The present invention also provides a detection reagent or kit for rabbit hemorrhagic disease virus type 2 capsid protein-specific antibody, using the fusion protein described in the above scheme as the coating antigen.

[0059] In one embodiment, the kit includes an indirect ELISA detection kit. In another embodiment, the indirect ELISA detection kit further includes an enzyme-labeled plate, coating buffer, PBST, 5% skim milk, HRP-labeled goat anti-rabbit IgG, TMB chromogenic solution, H2SO4 stop solution, negative control, and positive control. In another embodiment, the working concentration of the fusion protein as the coating antigen is 1 μg / ml.

[0060] The present invention also provides a method for detecting rabbit hemorrhagic disease virus type 2 capsid protein-specific antibodies for non-diagnostic purposes, comprising the following steps: using the indirect ELISA detection kit to detect the sample to be tested.

[0061] The present invention does not impose any special restrictions on the specific detection process; conventional steps in the field can be used.

[0062] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a fusion protein, a rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine, and their applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1

[0064] Construction, expression and identification of self-assembled nanoparticles of the P region of rabbit hemorrhagic disease virus type 2 capsid protein

[0065] This embodiment analyzes the formation mechanism and antigenicity of virus-like particles (VLPs) of rabbit hemorrhagic disease virus type 2 (RHDV2) structural proteins, constructs various spike protein (P region) self-assembled nanoparticles that can improve immunogenicity and neutralizing antibody levels, thereby preparing a RHDV2 P region self-assembled nanoparticle vaccine and improving the efficacy and accuracy of vaccine immunization.

[0066] by pCI - neo As a carrier, Ecor I (GAATTC) 、Sal I (GTCGAC) is the restriction enzyme site, located upstream of the restriction enzyme site. Ecor Insert the KOZAK sequence (GCCACC) between I and the start codon ATG. Ecor I and Sal The RHDV2VP60-HP sequence was inserted between the I and P regions. RGD4C or CNGRC sequences were inserted before the hinge region (H) and after the P region of the RHDV2 VP60-HP sequence, respectively, to construct two recombinant plasmids: pCI-RGD4C-HP-RGD4C and pCI-CNGRC-HP-CNGRC. Further partial base deletion mutations were performed on the hinge region and either RGD4C or CNGRC to obtain 12 recombinant plasmids. pCI - neo The pCI-RHDV2 VP60-P recombinant plasmid is the vector. See the schematic diagram of the complete recombinant plasmid structure for details. Figure 1 The amino acid sequence of RGD4C-HP-RGD4C is shown in SEQ ID NO.4; the unoptimized nucleotide sequence of RGD4C-HP-RGD4C is shown in SEQ ID NO.5; the nucleotide sequence of RGD4C-HP-RGD4C optimized with human cell codons and with added restriction enzyme sites, KOZAK sequence, start codon, and stop codon sequence is shown in SEQ ID NO.6; the nucleotide sequence of RGD4C-HP-RGD4C optimized with insect cell codons and with added restriction enzyme sites, KOZAK sequence, start codon, and stop codon sequence is shown in SEQ ID NO.7.

[0067] The amino acid sequence of CNGRC-HP-CNGRC is shown in SEQ ID NO.8, specifically as follows: CNGRC SSKTVDS ISPADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFAAIDHDRGNASFPGSSSSNV LELWYASAGSAADNPISQIAPDGFPDMSFVPFSGITIPTAGWVGFGGIWNSSNGAPYVTTMQAYELGFATGVPSNP QPTTTTSGAQIVAKSIYGVANGINQTTAGLFVMASGVISTPNSSATTYTPQPNRIVNAPGTPAAAPIGKNTPIMFA SVVRRTGDINAEAGSTNGTQYGAGSQPLPVTIGLSLNNYSSALMPGQFFVWQLNFASGFMELGLSVDGYFYAGTGA SATLIDLSDLVDIRPVGPRPSTSTLVYNLGGTTNGFSYV CNGRC The italicized sequence is the amino acid sequence of CNGRC.

[0068] The unoptimized nucleotide sequence of CNGRC-HP-CNGRC is shown in SEQ ID NO.9, specifically: tgcaatgggaggt gc agcagcaagaccgtggacagcatcagccccgccgacctgctgaccacccccgtgctgaccggcgtgggcaccga caaccgctggaacggcgagatcgtgggcctgcagcccgtgcccggcggcttcagcacctgcaaccgccactggaac ctgaacggcagcacctacggctggagcagcccccgcttcgccgccatcgaccacgaccgcggcaacgccagcttcc ccggcagcagcagcagcaacgtgctggagctgtggtacgccagcgccggcagcgccgccgacaaccccatcagcca gatcgcccccgacggcttccccgacatgagcttcgtgcccttcagcggcatcaccatccccaccgccggctgggtg ggcttcggcggcatctggaacagcagcaacggcgccccctacgtgaccaccatgcaggcctacgagctgggcttcg ccaccggcgtgcccagcaacccccagcccaccaccaccaccagcggcgcccagatcgtggccaagagcatctacgg cgtggccaacggcatcaaccagaccaccgccggcctgttcgtgatggccagcggcgtgatcagcacccccaacagc agcgccaccacctacaccccccagcccaaccgcatcgtgaacgcccccggcacccccgccgccgcccccatcggca agaacacccccatcatgttcgccagcgtggtgcgccgcaccggcgacatcaacgccgaggccggcagcaccaacgg cacccagtacggcgccggcagccagcccctgcccgtgaccatcggcctgagcctgaacaactacagcagcgccctg atgcccggccagttcttcgtgtggcagctgaacttcgccagcggcttcatggagctgggcctgagcgtggacggct acttctacgccggcaccggcgccagcgccaccctgatcgacctgagcgacctggtggacatccgccccgtgggccc ccgccccagcaccagcaccctggtgtacaacctgggcggcaccaccaacggcttcagctacgtg tgcaatgggagg tgc The italicized sequence is the nucleotide sequence of the CNGRC coding gene.

[0069] The CNGRC-HP-CNGRC nucleotide sequence, optimized with human cell codons and supplemented with restriction enzyme sites, KOZAK sequence, start codon, and stop codon sequence, is shown in SEQ ID NO.10. Specifically:

[0070] gaattcgccacc atg tgcaacggcaggtgc agctcaaagaccgtggatagcatcagccctgccgacct gctgaccacccccgtgctgaccggcgttggaacagacaaccggtggaacggcgaaatcgtgggcctgcagcctgtc cctggaggctttagcacatgcaaccgccactggaatctgaatggctctacatatggctggagctctccaagattcg ccgctatcgaccacgacagaggcaacgcttcttttcccggctccagcagttctaatgtgttagagctgtggtacgc ctccgccggctctgccgcagataaccccatctcgcaaatcgcccctgatggctttcccgacatgtctttcgtgccc ttcagcggcatcaccatccccaccgccggatgggttggcttcggcggaatctggaacagcagcaacggcgctccct acgtgaccacaatgcaggcctacgagctgggcttcgccaccggcgtgccaagcaaccctcagcctacaacaaccac cagcggagcccagattgtggccaagagcatctacggcgtggccaacggcattaatcagacaaccgccggactgttc gtgatggccagcggcgtgatcagcacaccaaactccagcgccaccacctatacccctcaacctaatagaatcgtga acgcccctggcaccccagccgccgcccctatcggcaaaaacacccctatcatgttcgctagcgtggtgcggagaac cggagatatcaacgccgaggccggctctacaaacggcacccagtacggcgctggcagccagcctctgcctgtcaca atcggcctcagcctgaacaactacagcagcgccctgatgcctggccagttcttcgtgtggcagctgaacttcgcct ctggctttatggaactgggcctgagcgtggacggctacttctacgccgggaccggcgcctccgctaccctgatcga cctgagcgacctggtggacatccggcctgtgggacctagacccagcacaagcacactggtctacaacctgggtgga accaccaacggcttcagctacgtg tgtaatggaagatgt tga gtcgac .

[0071] The nucleotide sequence, amino acid sequence, human cell codon-optimized nucleotide sequence, and insect cell codon-optimized nucleotide sequence of RGD4C-HP were adjusted according to the above sequences.

[0072] 1. Materials and Methods

[0073] 1.1.1 Materials

[0074] 1.1.1.1 Plasmids and Cells

[0075] The original vectors RGD4C-HP-RGD4C and CNGRC-HP-CNGRC were synthesized by Nanjing Genscript Biotech Co., Ltd.; DH5α competent cells and DH10Bac competent cells were purchased from Beijing Qingke Biotechnology Co., Ltd.; HEK 293 cells, Sf9 and High Five insect cells were provided by the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences.

[0076] 1.1.1.2 Reagents

[0077] The Mut Express II Fast Mutagenesis Kit V2 plasmid single-point mutagenesis kit, SuperPicoECL Chemiluminssence Kit, and 2×Rapid Taq Master Mix were purchased from Nanjing Novizan Biotechnology Co., Ltd.; Ampicillin, Kanamycin sulfate, Gentamicin, and Tetracycline were purchased from Nanjing Wobo Biotechnology Co., Ltd.; Sodium chloride (NaCl), Tryptone, and Yeast Extract were purchased from Qingdao Sangon Biotech Co., Ltd.; Agar and agarose were purchased from Guangzhou Saiguo Biotechnology Co., Ltd.; the Fast Plasmid Miniprep Kit was purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.; Transfection reagents, DMEM high-glucose medium, and Grace... ’ Insect Cell Culture Medium (IB905) was purchased from Zhejiang Yishengke Biotechnology Co., Ltd.; Omega Endo-Free Bac / PAC DNA Kit was purchased from Beijing Jiehui Bogao Biotechnology Co., Ltd.; X-gal (β-galactosidase chromogenic substrate) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; 50×TAE electrophoresis buffer was purchased from Wuhan Sewell Biotechnology Co., Ltd.; GelStain fluorescent nucleic acid staining reagent was purchased from Beijing TransGen Biotechnology Co., Ltd.; Protein gel preparation kit was purchased from Shanghai Wansheng Haotian Biotechnology Co., Ltd.; 5×SDS-PAGE protein loading buffer, 5×SDS-PAGE protein loading buffer (non-denaturing and non-reducing), HRP-labeled goat anti-mouse IgG, and FITC-labeled goat anti-mouse IgG were purchased from Beijing Lanjieke Technology Co., Ltd.; Anti-rabbit hemorrhagic disease virus monoclonal antibody 1B8 was prepared by the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences.

[0078] 1.1.2 Method

[0079] 1.1.2.1 Construction of expression plasmid based on HEK 293 cells

[0080] 1.1.2.1.1 Construction of the original plasmid

[0081] The structural protein VP60 gene of rabbit hemorrhagic disease virus strain SC2020 / 0401 (GenBank ID: MT586027) was obtained from GenBank, and the hinge region and P region gene sequences (HP) of the structural protein VP60 were obtained by alignment analysis. KOZAK The sequence (GCCACC) is added to the 5′ end of the sequence to increase expression levels. RGD4C or CNGRC is inserted before the hinge region and after the P region, with ATG as the start codon and TGA as the stop codon. The modified sequence is then inserted into the [previous region / region]. pCI-neo carrier EcorI and SalI Between the restriction enzyme sites, two original expression plasmids were constructed: PCI-RGD4C-HP-RGD4C and PCI-CNGRC-HP-CNGRC.

[0082] 1.1.2.1.2 Design of recombinant pCI-RHDV2 VP60-P plasmid

[0083] Using the constructed PCI-RGD4C-HP-RGD4C and PCI-CNGRC-HP-CNGRC plasmids as templates, a series of recombinant VP60-P plasmids with different cysteine ​​residues were obtained through partial base deletion mutations using the Novizan Mut Express II Fast Mutagenesis Kit V2 plasmid single-point mutagenesis kit, such as... Figure 1 As shown.

[0084] 1.1.2.1.3 Primer Design

[0085] The primer sequences for homologous recombination and M13 blue-white screening are shown in Table 1:

[0086] Table 1 Primer names and sequences

[0087]

[0088] 1.1.2.1.4 Amplification and Identification of Recombinant pCI-RHDV2 VP60-P Vector

[0089] The plasmid amplification system is shown in Table 2; the amplification program is as follows: pre-denaturation 95℃, 30 s; denaturation 95℃, 15 s; annealing 65℃, 15 s; extension 72℃, 7 min; 30 cycles; final extension 72℃, 5 min.

[0090] Table 2 Plasmid amplification system

[0091]

[0092] Dpn1 methylation template digestion system: 1 μl Dpn1 and 40–50 μl amplification product. Digestion conditions: 37 °C, 1–2 h.

[0093] The homologous recombination reaction system is shown in Table 3. Reaction conditions: 37℃, 30 min.

[0094] Table 3 Homologous recombination reaction system

[0095]

[0096] The products obtained after homologous recombination are P-region vectors with different cysteine ​​residues, containing and without hinge regions. The recombinant pCI-RHDV2 VP60-P vector was transformed into DH5α plates, and single colonies were picked. Single colony sequencing analysis was used to identify P-region vectors of different lengths and amplified to extract plasmids.

[0097] 1.1.2.2 Expression of recombinant pCI-RHDV2 VP60-P vector in HEK 293 cells

[0098] The recombinant pCI-RHDV2 VP60-P vector plasmid, with accurate sequencing, was transiently transfected into HEK 293 cells at approximately 90% confluency (1 μg per well). After incubation at 37°C for 48 h, the cell morphology was observed under a microscope, the culture medium was discarded, and the cells were washed twice with PBS. Then, 200 μl of cell lysis buffer without denaturing and reducing agents was added to each well, and the cells were incubated at 4°C for 10 min. The lysate containing lysed HEK 293 cells was collected. The lysate was centrifuged at 12000 r / min for 10 min at 4°C, and the supernatant was the target protein.

[0099] 1.1.2.3 Western Blot Analysis

[0100] 1.1.2.3.1 Expression and Identification of Recombinant pCI-RHDV2 VP60-P Vector Expression Product

[0101] First, add 10 μl of 5×SDS-PAGE protein loading buffer to 40 μl of supernatant and place in a 100℃ metal bath for 10 min to completely denature the protein. After cooling the sample to room temperature, load it onto a Tris-glycine electrophoresis system for SDS-PAGE. After electrophoresis, transfer the sample to a polyvinylidene fluoride (PVDF) membrane. Block the membrane with PBST containing 5% skim milk (PBS containing 0.5% Tween 20, the same below) at room temperature for 2 h. After blocking, wash three times with PBST, 5 min each time. Incubate with 1B8 as the primary antibody (1:1000) at room temperature for 1.5 h. After membrane incubation, wash three times with PBST, 5 min each time. Incubate with goat anti-mouse IgG (HRP) (1:10000) at room temperature for 1 h. After incubation, wash three times with PBST, 15 min each time. After incubation with HRP-labeled secondary antibody, perform blot detection on a chemiluminescence image analysis system using an enhanced chemiluminescence (ECL) detection kit.

[0102] 1.1.2.3.2 Identification of the multimer-forming ability of recombinant pCI-RHDV2 VP60-P protein

[0103] Take another 40 μl of supernatant sample and add 10 μl of 5×SDS-PAGE protein loading buffer (non-denaturing and non-reducing) to the Tris-glycine (SDS-free) electrophoresis system Native-PAGE. Subsequent operations are the same as in 1.1.2.3.1 to identify the multimer-forming ability.

[0104] 1.1.2.4 Construction of recombinant transfer vector based on Sf9 insect cells

[0105] After identifying the ability of the recombinant vector to form multimers using HEK 293 cells, the insect cell codons of the recombinant pCI-RHDV2 VP60-P vector (pCI-RGD4C-HP-RGD4C), which exhibited strong multimer-forming ability, were optimized to obtain the recombinant transfer vector pFast-RGD4C-HP-RGD4C. Further partial base deletion mutations were used to obtain pFast-RGD4C-HP. PCR amplification was performed using 2×Rapid Taq Master Mix under the following conditions: pre-denaturation 95℃ for 30 s; denaturation 95℃ for 15 s, annealing 65℃ for 15 s, extension 72℃ for 7 min, 30 cycles, with a final extension at 72℃ for 5 min. The homologous recombination product was a recombinant transfer vector based on Sf9 insect cells.

[0106] 1.1.2.5 Obtaining and Identifying Recombinant Shuttle Carriers

[0107] 1.1.2.5.1 Obtaining the Recombinant Shuttle Vehicle

[0108] The recombinant transfer vectors pFast-RGD4C-HP-RGD4C and pFast-RGD4C-HP were transformed into DH10 Bac competent cells and cultured in LB liquid medium at 37°C with shaking for 2 h. After centrifugation, part of the supernatant was discarded, and the transformation products were plated onto fresh LB agar plates containing 50 µg / ml kanamycin, 7 µg / ml gentamicin, 10 µg / ml tetracycline, 100 µg / ml X-gal, and 40 µg / ml IPTG. The plates were incubated at 37°C for 48 h. White positive colonies were picked and streaked onto triple-antibody plates for purification. The process was repeated three times. White colonies that were positive in all three rounds were selected and identified by PCR using universal primers PUC M13-F / M13-R. After successful identification, positive colonies were inoculated into triple-antibody liquid LB medium and cultured overnight at 37°C with shaking at 200 rpm. The recombinant shuttle plasmid was then extracted using the Omega large fragment plasmid extraction kit. See the schematic diagram of RHDV2 VP60-P recombinant Bacmid. Figure 2 .

[0109] 1.1.2.5.2 Identification of Recombinant Shuttle Plasmids

[0110] Using positive single colonies from three screenings of the recombinant shuttle vector as templates, PCR amplification was performed using universal primers PUC M13-F / M13-R and 2×Rapid Taq Master Mix. The reaction conditions were: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 10 s, annealing at 55℃ for 20 s, extension at 72℃ for 90 s, for 35 cycles, with a final extension at 72℃ for 5 min. The amplified products were identified by agarose gel electrophoresis for bacterial PCR. Successful identification of the vector plasmids led to their extraction as recombinant shuttle plasmids (Bacmid-RGD4C-HP-RGD4C and Bacmid-RGD4C-HP).

[0111] 1.1.2.6 Obtaining the recombinant rBac-RHDV2 VP60-P virus

[0112] Recombinant shuttle plasmids Bacmid-RGD4C-HP-RGD4C and Bacmid-RGD4C-HP were transiently transfected into Sf9 insect cells using Lip 3000 transfection reagent. Cells were observed under a microscope every 24 hours post-transfection until pathological changes were observed, including cessation of cell division and growth, rounding of cell morphology, increased intercellular spaces, and some cells floating. Normal cells in the control group showed no significant changes except for increased growth density. The supernatant was collected by centrifugation, yielding the recombinant viruses rBac-RGD4C-HP-RGD4C and rBac-RGD4C-HP, which were stored at 4°C.

[0113] 1.1.2.7 Passage and Preliminary Identification of Recombinant rBac-RHDV2 VP60-P Virus

[0114] 1.1.2.7.1 Passage of recombinant rBac-RHDV2 VP60-P virus

[0115] Sf9 insect cells were observed. When the cell density reached about 90%, recombinant rBac-RHDV2 VP60-PP1 generation virus was inoculated at a 3% inoculation dose. After being placed at 26℃ for 72 h, the cells were observed under a microscope. The cells showed obvious lesions. The supernatant was collected by centrifugation to obtain recombinant rBac-RHDV2 VP60-PP2 generation virus.

[0116] 1.1.2.7.2 Preliminary IFA identification of recombinant rBac-RHDV2 VP60-P virus

[0117] Sf9 insect cells with a normal cell density of approximately 90% were infected in 24-well plates with recombinant rBac-RHDV2 VP60-P virus and incubated at 26°C for 24 h. Obvious lesions were observed under a microscope after infection. The cell supernatant was discarded, and the cells were washed twice with PBS for 3-5 min each time. 500 μl of pre-chilled fixative (ethanol:acetone 2:3) at -20°C was added to each well, and the cells were fixed at 4°C for 1 h. After fixation, 1 ml of PBST was added to each well to wash the cells three times for 5 min each time. 200 μl of 1B8 diluted with PBS was added to each well as primary antibody (1:50), and the cells were incubated at 37°C for 1 h. After incubation, the primary antibody was discarded, and 1 ml of PBST was added to each well to wash the cells three times for 5 min each time. 200 μl of goat anti-mouse IgG (FITC) was added to each well as secondary antibody (1:100), and the cells were incubated at 37°C for 1 h. After incubation, the secondary antibody was discarded, and 1 ml of PBST was added to each well. Cells were washed three times with PBST for 5 minutes each time; IFA was performed by observing the cells under a fluorescence microscope and comparing them with those of normally infected Sf9 insects.

[0118] 1.1.2.8 Obtaining and Identifying the Recombinant RHDV2 VP60-P Expression Product

[0119] 1.1.2.8.1 Obtaining the recombinant RHDV2 VP60-P expression product

[0120] Recombinant rBac-RHDV2 VP60-P virus was inoculated at a volume ratio of 2% on a growth density of 1.6 × 10⁻⁶. 6 High-Five insect cells were cultured at 26℃ with shaking at 120 r / min. Samples were taken every 24 h for microscopic observation, and samples were taken continuously for 7 days to determine the collection time.

[0121] 1.1.2.8.2 Identification of recombinant RHDV2 VP60-P expression product

[0122] Collect cells and culture medium with obvious pathological changes observed under a microscope and freeze-thaw them three times. Centrifuge at 1000 rpm for 10 min at room temperature, take 40 μl of supernatant and add 10 μl of 5×SDS-PAGE protein loading buffer. Place in a 100℃ metal bath for 10 min to completely denature the protein. Perform Western Blot identification as in 1.1.2.3.1.

[0123] 1.1.2.9 Observation using transmission electron microscopy

[0124] To determine whether recombinant RHDV2 VP60-P particle antigen could self-assemble into nanoparticles, the recombinant virus was inoculated into High Five insect cells and subjected to three freeze-thaw cycles. The supernatant was collected by centrifugation at 5000 r / min for 10 min. After removing contaminating proteins by ultrafiltration through a 100 KD tube, the recombinant RHDV2 VP60-P particle antigen was purified by high-speed centrifugation at 30000 r / min for 2 h. The presence and morphology of recombinant RHDV2 VP60-P particle antigen and VLPs were detected by negative staining under transmission electron microscopy.

[0125] 1.2 Results

[0126] 1.2.1 Identification of the expression of recombinant pCI-RHDV2 VP60-P in HEK 293 cells

[0127] To verify the successful expression of the 14 constructed recombinant pCI-RHDV2 VP60-P plasmids, HEK 293 cells were transiently transfected with 1 μg / well of plasmid and incubated at 37°C for 48 h. Western blot analysis was performed on the recombinant RHDV2 VP60-P protein. Figure 3 The results showed that the recombinant RHDV2 VP60-P protein was successfully expressed, with a molecular weight of approximately 35 kDa. The recombinant RHDV2 VP60-P protein naturally formed a dimer of 70 kDa.

[0128] 1.2.2 Assessment of the ability of recombinant RHDV2 VP60-P protein to form multimers

[0129] To evaluate the ability of recombinant RHDV2 VP60-P protein to form multimers, non-denaturing, non-reducing NativePage protein blotting was used to analyze the recombinant RHDV2 VP60-P protein. Figure 4As shown, both lane 13 RGD4C-HP-RGD4C and lane 11 RGD4C-HP show protein bands with a molecular weight greater than 180 kDa, indicating that these two recombinant proteins have the ability to form multimers.

[0130] 1.2.3 PCR Identification of Recombinant Shuttle Vectors by Blue-White Screening

[0131] The recombinant transfer vectors pFast-RGD4C-HP-RGD4C and pFast-RGD4C-HP were transformed into DH10 Bac competent cells. White colonies that tested positive in three screenings were identified by colony PCR using the universal primers PUC M13-F / M13-R. The positive PCR product was approximately 4000 bp in size, and the negative PCR product was approximately 300 bp in size. PCR results indicate that the recombinant transfer vectors pFast-RGD4C-HP-RGD4C and pFast-RGD4C-HP successfully transposed, yielding the recombinant shuttle vectors Bacmid-RGD4C-HP-RGD4C and Bacmid-RGD4C-HP. (See also...) Figure 5 and Figure 6 .

[0132] 1.2.4 Obtaining and Identifying the Expression Product of Recombinant Baculovirus rBac-RHDV2 VP60-P

[0133] 1.2.4.1 Obtaining recombinant baculovirus rBac-RHDV2 VP60-P

[0134] The extracted Bacmid-RGD4C-HP-RGD4C and Bacmid-RGD4C-HP plasmids were transfected into Sf9 monolayer cells to obtain recombinant baculoviruses rBac-RGD4C-HP-RGD4C and rBac-RGD4C-HP. See also... Figure 7 .

[0135] 1.2.4.2 Identification of recombinant baculovirus rBac-RHDV2 VP60-P

[0136] Recombinant baculoviruses rBac-RGD4C-HP-RGD4C and rBac-RGD4C-HP were inoculated into Sf9 monolayer cells. After 24 h of infection, IFA was performed using 1B8 as the primary antibody. The results are as follows: Figure 8 As shown, the recombinant viruses rBac-RGD4C-HP-RGD4C and rBac-RGD4C-HP were stably expressed in Sf9 insect cells, indicating that the recombinant viruses rBac-RGD4C-HP-RGD4C and rBac-RGD4C-HP were successfully obtained.

[0137] 1.2.4.3 Determination of the optimal expression time of recombinant RHDV2 VP60-P protein

[0138] The recombinant virus rBac-RGD4C-HP-RGD4C was inoculated into High Five insect cells in suspension culture to determine the optimal expression time of the recombinant protein, such as... Figure 9 As shown, the recombinant protein exhibits distinct bands at 35 kDa and 70 kDa, with the highest expression level observed at 96 h.

[0139] 1.2.5 Electron microscopic observation results of recombinant RHDV2 VP60-P particle protein expression products

[0140] TEM results showed that both RGD4C-HP-RGD4C and RGD4C-HP could form relatively uniform nanoparticles with sizes of 40–50 nm and 80–100 nm, respectively. Figure 10 As shown.

[0141] 1.3 Discussion

[0142] This embodiment successfully constructed the original vectors pCI-RGD4C-HP-RGD4C and pCI-CNGRC-HP-CNGRC using bioinformatics and molecular biology methods. Through homologous recombination and other operations, a series of vectors based on... pCI-neo The recombinant pCI-RHDV2 VP60-P vector was transfected into HEK 293 cells, and two multimer-forming vectors, pCI-RGD4C-HP-RGD4C and pCI-RGD4C-HP, were successfully obtained by Western blotting. The designed proteins were then efficiently expressed and functionally identified using a baculovirus expression system. Transmission electron microscopy revealed that both RGD4C-HP-RGD4C and RGD4C-HP formed nanoparticles with sizes of 40–50 nm and 80–100 nm, respectively. This result provides an important technical foundation for the development of RHDV2 subunit vaccines and also offers a reference for the design of vaccines against other viruses that cannot be cultured in vitro.

[0143] pCI-RGD4C-HP-RGD4C and pCI-RGD4C-HP can form multimers in HEK 293 cells, indicating that RGD4C has a better ability to promote protein multimer formation than CNGRC. Electron microscopy showed that the particles formed by these two proteins were slightly larger than native RHDV VLPs, indicating that the P region has independent self-assembly capabilities, which provides a basis for further research on their immunogenicity and particle structure.

[0144] This embodiment successfully constructed RHDV2 P-region self-assembled nanoparticles using molecular biology and protein engineering techniques, and verified their efficient expression and assembly capabilities in insect cells. This lays an important foundation for the development of RHDV2 subunit vaccines and provides a new approach for the preparation of antigens from unculturable viruses.

[0145] 1.4 Summary

[0146] By analyzing the formation mechanism and antigenicity of RHDV2 structural protein virus-like particles (VLPs), a series of recombinant P-region plasmids containing different cysteine ​​residues were constructed. Two RHDV2 VP60-P self-assembled nanoparticle antigens (RGD4C-HP-RGD4C and RGD4C-HP) with sizes of 40-50 nm and 80-100 nm, respectively, were obtained through the Bac-to-Bac baculovirus expression system.

[0147] Example 2

[0148] Immunogenicity study of self-assembled nanoparticles of P region of rabbit hemorrhagic disease virus type 2 capsid protein

[0149] To verify the immunoprotective effect of the self-assembled RHDV2 capsid protein P region nanoparticles constructed in Example 1, 2 ml / rabbit of expressed RHDV2 VP60-P antigen (RGD4C-HP-RGD4C and RGD4C-HP) was subcutaneously immunized in the neck of 1-month-old RHDV2 antibody-negative rabbits. Immunoprotection rate and humoral immunity were evaluated, and the results were compared with the RHDV2 VP60 vaccine group and the PBS group. The results of challenge protection were as follows: 21 days post-immunization, the RGD4C-HP-RGD4C group, RGD4C-HP group, RHDV2 VP60 vaccine group, and PBS group were challenged with RHDV2. All rabbits in the RGD4C-HP-RGD4C group and the RHDV2 VP60 vaccine group survived, while all rabbits in the RGD4C-HP group and the PBS group died and exhibited typical clinical symptoms and pathological changes of RHD. Antibody level detection results showed that from day 7 to day 60 after immunization, the levels of RHDV2-VP60-specific antibodies in the serum of rabbits in the RGD4C-HP-RGD4C group and the RHDV2 VP60 vaccine group were significantly increased and maintained at a high level, which was significantly different from that in the PBS group. At the same time, the antibody levels of the RHDV2 VP60 P region in the RGD4C-HP-RGD4C group and the RHDV2 VP60 vaccine group were detected. The results showed that both the RGD4C-HP-RGD4C group and the RHDV2 VP60 vaccine group produced RHDV2-VP60 P region-specific antibodies after day 7 after immunization. At the same time point, the antibody level in the RGD4C-HP-RGD4C group was higher than that in the RHDV2 VP60 vaccine group, and maintained a high level for a period of time. The above results indicate that the self-assembled nanoparticles (RGD4C-HP-RGD4C) of the P region of the rabbit hemorrhagic disease virus type 2 capsid protein can induce rabbits to produce antibodies specific to the P region of RHDV2-VP60, generating a strong humoral immune response and providing complete protection for rabbits.

[0150] 2.1 Materials and Methods

[0151] 2.1.1 Materials

[0152] 2.1.1.1 Virus strains and laboratory animals

[0153] Rabbit hemorrhagic disease virus strain SC2020 / 0401 (RHDV2 type), recombinant RHDV2 VP60, Sf9 and High Five insect cells were provided by the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences. The recombinant RHDV2 VP60-P antigen (RGD4C-HP-RGD4C, RGD4C-HP) was constructed in this example. One-month-old RHDV-negative weaned rabbits were purchased from the Liuhe Animal Science Base of Jiangsu Academy of Agricultural Sciences.

[0154] 2.1.1.2 Main Reagents

[0155] Grace ’ Insect cell culture medium (IB 905) was purchased from Zhejiang Yishengke Biotechnology Co., Ltd.; HRP-labeled goat anti-mouse IgG and HRP-labeled goat anti-rabbit IgG were purchased from Beijing Lanjieke Technology Co., Ltd.; single-component TMB chromogenic reagent I was purchased from Huzhou Yingchuang Biotechnology Co., Ltd.; protein gel preparation kit was purchased from Shanghai Wansheng Haotian Biotechnology Co., Ltd.; BCA protein concentration assay kit was purchased from Beyotime Biotechnology Co., Ltd.; 96-well microplates were purchased from Nanjing Saiyan Biotechnology Co., Ltd.; other reagents were domestically produced analytical grade.

[0156] 2.1.2 Method

[0157] 2.1.2.1 Preparation of self-assembled nanoparticle antigens from the P region of recombinant rabbit hemorrhagic disease virus type 2 capsid protein

[0158] 2.1.2.1.1 Vaccination

[0159] Recombinant baculoviruses rBac-SC2020 VP60, rBac-RGD4C-HP-RGD4C, and rBac-RGD4C-HP were inoculated at 2% volume into High Five insect cells cultured in serum-free IB 905 medium and cultured at 26°C for 4 days. When the cytopathic effect reached more than 85% under a microscope, the cell culture was collected and stored at -20°C.

[0160] 2.1.2.1.2 Detection of Erythrocyte Agglutination Titer

[0161] Harvested cell cultures were subjected to three freeze-thaw cycles, and erythrocyte agglutination titers were determined using a 50-well plate method. 50 μl of the frozen-thawed cell culture was added to a 50-well plate; 50 μl of PBS was added and mixed thoroughly by pipetting, followed by a two-fold serial dilution; after mixing thoroughly by pipetting, 50 μl of 1% human type B erythrocyte suspension was added and mixed by tapping; the plate was incubated at 2–8°C for 60 min, and erythrocyte agglutination was observed.

[0162] 2.1.2.1.3 Determination of Immunization Dosage for Immunogens

[0163] The collected cell cultures were repeatedly frozen and thawed three times to lyse the cells and release the recombinant antigen. A full-length recombinant RHDV2 VP60 protein with a hemagglutination titer of 1:256 was used as a control. Western blotting was performed to preliminarily determine the immunization dose as 2 ml / animal.

[0164] 2.1.2.2 Purification of P region antigen of rabbit hemorrhagic disease virus type 2 capsid protein

[0165] Comparative analysis selected the P region gene of rabbit hemorrhagic disease virus strain SC2020 / 0401 (MT586027) as the basis for comparison. pCold We constructed a vector to express the P region protein of recombinant rabbit hemorrhagic disease virus type 2 based on Escherichia coli expression.

[0166] 2.1.2.2.1 Antigen-induced expression of P region of rabbit hemorrhagic disease virus type 2 capsid protein

[0167] (1) Transform the constructed recombinant P-region plasmid into BL-21(DE3) competent cells, and pick single colonies from the plate;

[0168] (2) Inoculate a single colony into 5 ml of Amp+ resistant LB and incubate overnight at 37°C with shaking at 200 r / min;

[0169] (3) The recombinant Escherichia coli cultured by shaking was inoculated into 100 ml of Amp+ LB at a 1% inoculation rate and cultured at 37℃ and 200 r / min with shaking.

[0170] (4) Every 30 min after inoculation, 200 μl of Escherichia coli was sampled into a 96-well microplate and the OD value of the bacterial solution was read at OD600 absorbance. When the OD value at OD600 absorbance was 0.3~0.5, IPTG with a final concentration of 0.1 μM was added and cultured at 37℃ with shaking at 200 r / min for 4 h to induce expression. After induction, the sample was retained for subsequent identification.

[0171] 2.1.2.2.2 Purification of P region antigen of rabbit hemorrhagic disease virus type 2 capsid protein

[0172] (1) After induction, E. coli were centrifuged at 12000 r / min at 4℃ for 20 min, the supernatant was discarded, and the bacterial pellet was resuspended in an appropriate amount of PBS. The pellet was then centrifuged at 12000 r / min at 4℃ for 10 min to wash the pellet. This process was repeated 3 times.

[0173] (2) After washing, the bacterial precipitate was resuspended in 20 ml of PBS, sonicated at 60% power for 10 min for 5 s, and the supernatant and precipitate were centrifuged separately for expression verification.

[0174] (3) Dissolve the bacterial precipitate after ultrasonic disruption with denaturing solution containing 8M urea, invert at 4°C to completely dissolve the bacterial precipitate, and retain the sample for identification.

[0175] (4) Add an equal amount of denaturing solution containing 6M urea to the completely dissolved Escherichia coli 8M denaturing mixture containing the P region protein of the recombinant rabbit hemorrhagic disease virus type 2 capsid protein, vortex mix, add to a 10000 pore size cellulose dialysis bag, place in denaturing solution containing 4M urea, and dialyze overnight at 4°C with magnetic stirring.

[0176] (5) Place the cellulose dialysis bag that has been dialyzed overnight in a denaturing solution containing 2M urea and dialyze it under magnetic stirring at 4°C for 8 hours.

[0177] (6) Place the cellulose dialysis bag that has been dialyzed overnight in a denaturing solution that does not contain urea, stir magnetically at 4°C for 24 hours, and change the denaturing solution that does not contain urea every 6 hours for dialysis refolding.

[0178] 2.1.2.2.3 Identification of the P region protein of the capsid protein of rabbit hemorrhagic disease virus type 2

[0179] The expression of the induced expression, the supernatant of ultrasonic disruption and centrifugation, the precipitation of ultrasonic disruption and centrifugation, the precipitation of each process after denaturation, and the refolded sample were verified by SDS-PAGE, and the concentration of purified protein was roughly estimated by grayscale calculation using ImageJ software.

[0180] 2.1.2.3 Virus challenge protection experiment

[0181] Twenty-four one-month-old RHDV-negative weaned rabbits were randomly divided into four groups. The immunization groups were immunized with RGD4C-HP protein, RGD4C-HP-RGD4C protein, and RHDV2 VP60 protein, respectively; the control group was injected with PBS, 2 ml per rabbit (Table 4). Twenty-one days after immunization, all immunized rabbits were challenged with 1 ml / rabbit of rabbit hemorrhagic disease virus SC strain (SC2020 / 0401 strain (MT586027)) (1000 LD50). 50 The rabbits were observed for 7 consecutive days. Daily mortality was recorded, and the immunization protection rate was calculated.

[0182] Immunization protection rate (%) = (mortality rate in the control group - mortality rate in the immunized group) / mortality rate in the control group × 100%

[0183] Table 4. Grouping of Infected Individuals

[0184]

[0185] 2.1.2.4 Grouping and Immunization

[0186] Eighteen one-month-old RHDV-negative weaned rabbits were randomly divided into three groups. The immunization groups were immunized with RGD4C-HP-RGD4C protein and RHDV2 VP60 protein, respectively; the control group was injected with PBS, 2 ml per rabbit (Table 5). Blood samples were collected on days 0, 7, 14, 21, 28, 35, 42, and 60 post-immunization, and serum was separated and stored for later use.

[0187] Table 5. Immunization Grouping

[0188]

[0189] 2.1.2.5 Detection of RHDV2 VP60 specific antibodies

[0190] The indirect ELISA method established in our laboratory was used to detect serum RHDV2 VP60-specific IgG antibody levels at different time points after immunization, and to monitor antibody response patterns. The specific procedure was as follows:

[0191] (1) The recombinant RHDV2 VP60 protein expressed by the baculovirus expression system was used as the coating antigen (the HA titer of the recombinant RHDV2 VP60 protein was 1:256 as determined by HA detection). The coating antigen was diluted 1:200 with coating buffer (pH=9.6) (concentration approximately 0.5 μg) and coated onto 96-well microplates, 100 μl per well. The plates were sealed in a resealable bag and incubated overnight at 4°C. After coating, each well was washed 3 times with 300 μl of PBST for 5 min each time. Each well was then blocked by adding 200 μl of 5% skim milk and incubating at 37°C for 2 h. After blocking, each well was washed 3 times with 300 μl of PBST for 5 min each time.

[0192] (2) Dilute the serum at different time points with 5% skim milk at a ratio of 1:100. Add 100 μl of the diluted immunized rabbit serum to each well and incubate at 37°C for 1 h. Wash each well with 300 μl of PBST 3 times, 5 min each time. Add 100 μl of 5% skim milk to each well and dilute HRP-labeled goat anti-rabbit IgG at a ratio of 1:10000. Incubate at 37°C for 1 h. Wash each well with 300 μl of PBST 3 times, 5 min each time. Add 100 μl of TMB chromogenic solution to each well and incubate at room temperature in the dark for 5-10 minutes. When the negative sample wells show slight color development, add 50 μl of 2M H2SO4 stop solution to each well and detect the absorbance at 450 nm.

[0193] 2.1.2.6 Detection of RHDV2 VP60-P specific antibody

[0194] The established indirect ELISA method was used to detect serum RHDV2 VP60-P specific IgG antibody levels at different time points after immunization, and to monitor antibody response patterns. The specific procedures were as follows:

[0195] The recombinant RHDV2 VP60-P protein expressed and purified from E. coli was used as the coating antigen. The coating antigen was diluted with coating buffer (pH=9.6) to a final concentration of 1 μg / ml. 100 μl of the antigen was coated onto each well of a 96-well ELISA plate. The plate was sealed in a self-sealing bag and incubated overnight at 4°C. Subsequent ELISA procedures were the same as in 2.1.2.5.

[0196] 2.2 Results

[0197] 2.2.1 Antigen Preparation

[0198] Recombinant baculoviruses rBac-RGD4C-HP and rBac-RGD4C-HP-RGD4C were inoculated into HighFive insect cells at a volume of 2%, cultured at 26°C for 4 days, and the cell culture was harvested. After repeated freeze-thaw cycles 3 times, the hemagglutination titer was tested using the 50-well plate method. The results showed that the recombinant protein could not agglutinate red blood cells, and the hemagglutination titer was 0.

[0199] 2.2.2 Comparison of Immunogen Dosage

[0200] The collected recombinant RGD4C-HP-RGD4C and RGD4C-HP cell cultures, along with recombinant RHDV2 VP60 protein with a hemagglutination titer of 1:256, were used as controls. Western blot analysis was performed to preliminarily determine the immunization dose as 2 ml / animal. Figure 11 ).

[0201] 2.2.3 Purification of P region antigen of rabbit hemorrhagic disease virus type 2 capsid protein

[0202] The recombinant RHDV2 VP60-P protein expressed in *E. coli* was induced to express, and the supernatant was subjected to ultrasonic disruption and centrifugation. The supernatant was then subjected to ultrasonic disruption and centrifugation to precipitate, followed by denaturation and refolding. SDS-PAGE was then performed to verify the expression of the precipitate. The purified protein concentration was approximately estimated to be 0.6 mg / ml using ImageJ software grayscale calculation. Figure 12 ).

[0203] 2.2.4 Virus Challenge Protection Experiment

[0204] Twenty-one days after immunization, all immunized rabbits were challenged with 1 ml / rabbit of rabbit hemorrhagic disease virus SC strain (SC2020 / 0401 strain (MT586027)) (1000 LD50). 50 The patients were observed for 7 consecutive days. Results showed that all patients in the RGD4C-HP-RGD4C group and the RHDV2 VP60 group survived, with a protection rate of 100% (Table 6), exhibiting no clinical symptoms of RHD and showing no lesions upon necropsy; all patients in the RGD4C-HP group and the PBS group died 48 hours after challenge. Figure 13 The dead rabbits exhibited obvious clinical symptoms, and the necropsy revealed typical lesions. Figure 14 ).

[0205] Table 6 Immunoprotection rate

[0206]

[0207] 2.2.5 Detection of RHDV2 VP60 specific antibodies

[0208] Serum samples taken at 0, 7, 14, 21, 28, 35, 42, and 60 days post-immunization were analyzed using indirect ELISA to detect RHDV2 VP60-specific IgG antibodies. Results showed that antibody titers in the RHDV2-VP60 and RGD4C-HP-RGD4C groups gradually increased starting 7 days post-immunization, significantly exceeding those in the PBS group (P<0.05). Antibody titers in the PBS group showed no significant change during the immunization cycle. Both RHDV2-VP60 and RGD4C-HP-RGD4C immunizations induced the production of antibodies against RHDV2 VP60 in rabbits. Figure 15 ).

[0209] 2.2.6 Detection of RHDV2 VP60-P specific antibody

[0210] Serum samples from 0, 7, 14, 21, 28, 35, 42, and 60 days post-immunization were analyzed using indirect ELISA to detect RHDV2 VP60-P specific IgG antibodies. Results showed that the antibody levels in the P region of the RGD4C-HP-RGD4C and RHDV2 VP60 groups were significantly higher than those in the PBS group starting from day 7 post-immunization (P<0.05), showing an increasing trend with increasing immunization time and remaining at a high level for a certain period. At the same time point, the antibody levels in the RGD4C-HP-RGD4C group were higher than those in the RHDV2 VP60 vaccine group. Figure 16 ).

[0211] 2.3 Discussion

[0212] This embodiment systematically evaluates the immunogenicity of recombinant proteins RGD4C-HP-RGD4C and RGD4C-HP through animal experiments, revealing their significant potential in combating RHDV2 infection. Regarding immunoprotective efficacy, recombinant protein RGD4C-HP-RGD4C provided good protection to immunized rabbits, while recombinant protein RGD4C-HP failed to offer adequate protection. This provides a basis for studying the relationship between different nanoparticle structures and immunoprotection and the ability to generate neutralizing antibodies, suggesting that the bilateral insertion of RGD4C may be crucial for maintaining particle stability or antigenic epitope exposure. This result demonstrates that even minor changes in antigenic structure can significantly affect immunogenicity, and the formation of complete nanoparticles with morphological similarities to virus-like particles may be an important factor in providing good protective effects. Immunizing rabbits with recombinant proteins RGD4C-HP-RGD4C and RHDV2 VP60 resulted in high specific IgG antibody titers against both RHDV2 VP60 and RHDV2 VP60-P, with RGD4C-HP-RGD4C showing higher antibody levels than RHDV2 VP60. This may be related to the more efficient antigen presentation of nanoparticles. Because nanoparticles form polymers with a larger effective surface area, and the recombinant nanoparticles contain only the P region of VP60, they directly expose neutralizing epitopes, more easily activating pattern recognition receptors (such as TLRs), promoting B cell activation, and reducing the waste of immune resources against non-protective epitopes (NTA and S regions).

[0213] This embodiment successfully demonstrates that the RGD4C-HP-RGD4C nanoparticle antigen possesses excellent immunoprotective efficacy and can elicit antibody responses against RHDV2 VP60 and RHDV2 VP60-P. This embodiment confirms that self-assembled nanoparticles (RGD4C-HP-RGD4C) of the RHDV2 capsid protein P region can induce a potent humoral immune response and provide complete protection for rabbits. This lays an important foundation for the development of novel RHDV2 subunit vaccines and provides new ideas for the design of vaccines against other viruses that cannot be cultured in vitro.

[0214] 2.4 Summary

[0215] Two types of nanoparticles, RGD4C-HP-RGD4C and RGD4C-HP, were used to immunize RHDV2-negative rabbits, and the results were compared with those of the RHDV2 VP60 vaccine group and the PBS group. Immunization challenge and antibody detection results showed that the self-assembled RHDV2 capsid protein P region nanoparticles RGD4C-HP-RGD4C induced higher levels of antibodies in rabbits than the RHDV2 VP60 vaccine, providing complete protection for the rabbits.

[0216] In summary, this invention fuses short peptides RGD4C and CNGRC, containing different cysteine ​​residues, at one or both ends of the amino acid sequence of the P region of the rabbit hemorrhagic disease virus capsid protein VP60, into HEK293 cells for expression, screening out two recombinant proteins, RGD4C-HP-RGD4C and RGD4C-HP, which can form nanoparticles. To conduct subsequent immunization experiments by expressing the recombinant proteins RGD4C-HP-RGD4C and RGD4C-HP in large quantities, a recombinant baculovirus was constructed in Sf9 insect cells using the Bac-to-Bac system, and the recombinant proteins RGD4C-HP-RGD4C and RGD4C-HP were expressed in High Five insect cells. Through immunoprotection assays and antibody level comparisons, the recombinant protein RGD4C-HP-RGD4C constructed in this invention can produce higher levels of antibodies against the P region, providing complete protection against rabbit hemorrhagic disease virus attack after immunization, thus providing a new technological product for the prevention and control of rabbit hemorrhagic disease. Furthermore, a comparison of antibody levels between the RGD4C-HP-RGD4C protein and the entire VP60 protein showed that all individuals in both the RGD4C-HP-RGD4C and VP60 protein immunization groups survived after challenge with RHDV2, indicating that both provide complete protection. Both the RGD4C-HP-RGD4C and VP60 vaccine groups produced specific antibodies against the VP60 P region 7 days after immunization. At the same time point, the antibody levels in the RGD4C-HP-RGD4C group were higher than those in the VP60 group and remained at a higher level for a period of time. This indicates that, regarding the P region antibody level, which plays a crucial role in vaccine-induced immune protection, the antibody level of the RGD4C-HP-RGD4C protein was significantly higher than that of the VP60 protein at all time points, demonstrating that the RGD4C-HP-RGD4C protein produces higher antibody levels and has a better immunization effect.

[0217] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that, It includes a first polypeptide, a hinge region of rabbit hemorrhagic virus type 2 capsid protein, a P region of rabbit hemorrhagic virus capsid protein, and a second polypeptide connected in sequence; the first polypeptide and the second polypeptide are each polypeptides containing 4 cysteine ​​residues. The polypeptide containing four cysteine ​​residues is RGD4C; the amino acid sequence of RGD4C is shown in SEQ ID NO.1; the amino acid sequence of the hinge region of the rabbit hemorrhagic virus type 2 capsid protein is shown in SEQ ID NO.2; the P region of the rabbit hemorrhagic virus capsid protein is the P region of the rabbit hemorrhagic virus type 2 capsid protein; the amino acid sequence of the P region of the rabbit hemorrhagic virus type 2 capsid protein is shown in SEQ ID NO.

3.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO.

4.

3. The gene encoding the fusion protein of claim 1 or 2.

4. The encoding gene according to claim 3, characterized in that, The nucleotide sequence encoding the gene includes the nucleotide sequence shown in SEQ ID NO.5 or a sequence after codon optimization of the nucleotide sequence shown in SEQ ID NO.

5.

5. The encoding gene according to claim 4, characterized in that, The nucleotide sequence of the codon-optimized sequence of the nucleotide sequence shown in SEQ ID NO.5 is shown in SEQ ID NO.6 or SEQ ID NO.

7.

6. A recombinant vector, characterized in that, The gene encoding as described in any one of claims 3 to 5 is inserted.

7. A recombinant bacterium or recombinant cell, characterized in that, It includes the recombinant vector as described in claim 6.

8. The use of the fusion protein of claim 1 or 2, the encoding gene of any one of claims 3 to 5, the recombinant vector of claim 6, or the recombinant bacteria or recombinant cells of claim 7 in the preparation of a medicament for the prevention or treatment of rabbit hemorrhagic virus type 2 infection.

9. A rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine, characterized in that, Includes the fusion protein described in claim 1 or 2.

Images