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

Abstract

The invention 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 invention provides a fusion protein. The fusion protein comprises a first polypeptide, a hinge region of a rabbit hemorrhagic disease virus type 2 capsid protein, a P region of the rabbit hemorrhagic disease virus capsid protein and a second polypeptide which are connected in sequence, the first polypeptide and the second polypeptide are respectively polypeptide containing four cysteines. The fusion protein RGD4C-HP-RGD4C disclosed by the invention can be self-assembled to form nano particles, and the particle size is 40 to 50 nm. The fusion protein provided by the invention can induce a rabbit to generate a specific antibody aiming at RHDV2, generate a relatively strong humoral immune response and provide complete protection for the rabbit.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a fusion protein, a rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine and applications thereof. Background Art

[0002] Rabbit hemorrhagic disease (RHD) is a highly contagious disease with high morbidity and mortality caused by the rabbit hemorrhagic disease virus (RHDV). Over 90% of infected rabbits typically die within three days, severely impacting the development of the rabbit industry. The VP60 protein, a major structural protein of RHDV, can self-assemble into virus-like particles (VLPs) in vitro that physically resemble native RHDV virions. VP60 can also induce neutralizing antibodies in animals and serves as a protective antigen for RHDV. The VP60 protein is divided into three structural domains: NTA, S, and P. The S and P domains are connected by a hinge region (H). The NTA and S domains are located within the VLP, while the P domain is located on the outer surface, forming a spike structure that is closely associated with neutralizing antibody levels. The antigenicity of the P domain is only 1 / 10 to 1 / 100 of that of the NTA and S domains. Consequently, genetically engineered subunit vaccines using VP60 as an antigen suffer from low antibody levels against the P domain, resulting in inaccurate and ineffective antibody production. Summary of the Invention

[0003] The purpose of the present invention is to provide a fusion protein, a rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine and applications thereof.

[0004] The present invention provides a fusion protein, comprising 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 respectively polypeptides containing four cysteines; the polypeptide containing four cysteines comprises 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 disease virus type 2 capsid protein is shown in SEQ ID NO.2; the P region of the rabbit hemorrhagic disease virus capsid protein comprises the P region of the rabbit hemorrhagic disease virus type 2 capsid protein; the amino acid sequence of the P region of the rabbit hemorrhagic disease virus type 2 capsid protein is shown in SEQ ID NO.3.

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

[0006] The present invention also provides a gene encoding 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 obtained by codon-optimizing the nucleotide sequence shown in SEQ ID NO.5.

[0008] Preferably, the nucleotide sequence of the sequence after codon optimization of the nucleotide sequence shown in SEQ ID NO.5 is shown as 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 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 in the above scheme in at least one of the following: 1) Preparation of drugs for preventing or treating rabbit hemorrhagic disease virus type 2 infection; 2) Application in the preparation of biological products for preventing rabbit hemorrhagic disease virus type 2 infection; 3) Preparation of detection reagents or kits for rabbit hemorrhagic disease virus type 2 capsid protein-specific antibodies.

[0012] The present invention also provides a rabbit hemorrhagic disease virus type 2 self-assembly nanoparticle vaccine, comprising the fusion protein described in the above scheme.

[0013] The present invention also provides a detection reagent or kit for rabbit hemorrhagic disease virus type 2 capsid protein-specific antibodies, using the fusion protein described in the above scheme as a coating antigen.

[0014] The present invention provides a fusion protein comprising a first polypeptide, a hinge region of the capsid protein of rabbit hemorrhagic disease virus type 2, a P region of the capsid protein of rabbit hemorrhagic disease virus type 2, and a second polypeptide, each of which contains four cysteines. The fusion protein of the present invention can self-assemble into nanoparticles with a particle size of 40-50 nm. The self-assembled nanoparticles form multimers with a larger effective surface area. The self-assembled nanoparticles contain only the P region of VP60, directly exposing neutralizing epitopes, more conveniently activating pattern recognition receptors (such as TLRs), promoting B cell activation, and reducing the waste of immune resources targeting non-protective epitopes (NTA region, S region). The fusion protein of the present invention can induce rabbits to produce specific antibodies against RHDV2, generating a strong humoral immune response and providing complete protection. Immunization of rabbits with the fusion protein of the present invention has been shown to produce high titers of specific IgG antibodies against both RHDV2 VP60 and RHDV2 VP60-P, with the antibody levels of the fusion protein of the present invention being higher than those against RHDV2 VP60. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 Schematic diagram of the recombinant vector structure; Figure 2 Schematic diagram of the recombinant Bacmid of RHDV2 VP60-P; Figure 3 Figure 2 shows the expression results of recombinant pCI-RHDV2 VP60-P granule protein in HEK 293 cells; M, 14-100 kDa 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; Figure 4Evaluation results of the ability of recombinant pCI-RHDV2 VP60-P particle protein to form protein aggregates in HEK 293 cells; M, 10-180 kDa 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; Figure 5 The results of the first blue-white screening of the recombinant transfer vector are shown in Figure 1. M, DL5000 Marker; 1-13, RGD4C-HP, first screening; 14-23, RGD4C-HP-RGD4C, first screening; Figure 6 The results of the second and third blue-white screening of the recombinant transfer vector; M, DL5000 Marker; 1-5 RGD4C-HP, second screening; 6-10 RGD4C-HP-RGD4C, second screening; 11, 12 RGD4C-HP, third screening; 13, 14 RGD4C-HP-RGD4C, third screening; Figure 7 Normal Sf9 cells and Sf9 cells transfected with lesions for 24 h, 48 h, and 72 h; Figure 8 The results of indirect immunofluorescence detection of the expression of recombinant rBac-RHDV2 VP60-P virus in Sf9 cell monolayers; NC: Sf9 cell monolayers transfected with unextracted plasmids; R-HP: Sf9 cell monolayers inoculated with rBac-RGD4C-HP; R-HP-R: Sf9 cell monolayers inoculated with rBac-RGD4C-HP-RGD4C; Figure 9Figure 2 shows the results of Western blot analysis of recombinant RHDV2 VP60-P granule protein expressed in High Five cells; M, 14-100 kDa Marker; 1, 24 h sample of recombinant RHDV2 VP60-P granule protein; 2, 48 h sample of recombinant RHDV2 VP60-P granule protein; 3, 72 h sample of recombinant RHDV2 VP60-P granule protein; 4, 96 h sample of recombinant RHDV2 VP60-P granule protein; 5, 120 h sample of recombinant RHDV2 VP60-P granule protein; 6, 144 h sample of recombinant RHDV2 VP60-P granule protein; 7, 168 h sample of recombinant RHDV2 VP60-P granule protein; Figure 10 The negative staining method for transmission electron microscopy was used to detect the formation of VLPs by recombinant RHDV2 VP60-P particle antigen; A is RHDV2 VP60 protein, B is RGD4C-HP-RGD4C protein, and C is RGD4C-HP protein; Figure 11 The figure shows the comparison of immunization dosage; M, 14-100 kDa marker; 1, RHDV2 VP60 protein; 2, RGD4C-HP-RGD4C protein; 3, RGD4C-HP protein; Figure 12 Figure 1 shows the results of the purification of the P region antigen of the capsid protein of rabbit hemorrhagic disease virus type 2; where M is 14-100 kDa marker; 1 is induced expression; 2 is ultrasonically disrupted and centrifuged; 3 is the supernatant of ultrasonically disrupted and centrifuged; 4 is the precipitate after elution at 8 M; 5 is the precipitate after elution at 4 M; 6 is the precipitate after elution at 0 M; 7 is the sample after renaturation; 8 is 0.5 mg / ml BSA standard; Figure 13 is the immune protection result; CT, PBS control group; Figure 14 This is a picture of a dead rabbit; A: bleeding from the mouth and nose B: congestion and bleeding in the lungs C: bleeding in the liver D: enlarged spleen; Figure 15 This is the monitoring result of RHDV2 VP60 protein specific IgG antibody level; *: P <0.05,**: P <0.01,***: P <0.001, ****: P <0.0001; Figure 16 This is the monitoring result of RHDV2 VP60-P protein specific IgG antibody level; *: P <0.05,**: P<0.01,***: P <0.001, ****: P <0.0001. DETAILED DESCRIPTION

[0017] The present invention provides a fusion protein RGD4C-HP-RGD4C, comprising 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 respectively polypeptides containing four cysteines.

[0018] As an embodiment, the sequential connection includes sequential connection from the N-terminus to the C-terminus.

[0019] Cysteine ​​(abbreviated as 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. RGD4C, with its amino acid sequence being CDCRGDCFC, contains four cysteines (C). The expressed protein forms disulfide bonds between these cysteine ​​residues, potentially allowing proteins to form multimeric structures through disulfide bonds, thereby forming nanoparticles. The fusion protein RGD4C-HP-RGD4C of the present invention, by linking multiple cysteines at both ends of the P region, can form a nanoparticle structure. Vaccines prepared with this fusion protein exhibit excellent immune efficacy, with higher levels of antibodies against the P region than full-length VP60.

[0020] As an embodiment, the polypeptide containing four cysteines includes RGD4C; the amino acid sequence of RGD4C is shown in SEQ ID NO.1, specifically: CDCRGDCFC.

[0021] As an 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: SSKTVDSIThe P region of the rabbit hemorrhagic disease virus capsid protein includes the P region of the rabbit hemorrhagic disease virus type 2 capsid protein; the amino acid sequence of the P region of the rabbit hemorrhagic disease virus type 2 capsid protein is as shown in SEQ ID As shown in NO.3, specifically: SPADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFAAIDHDRGNASFPGSSSSNVLELWYASAGSAADNPISQIAPDGFPDMSFVPFSGITIPTAGWVGFGGIWNSSNGAPYVTTMQAYELGFATGVPSNPQPTTTTSGAQIVAKS IYGVANGINQTTAGLFVMASGVISTPNSSATTYTPQPNRIVNAPGTPAAAPIGKNTPIMFASVVRRTGDINAEAGSTNGTQYGAGSQPLPVTIGLSLNNYSSALMPGQFFVWQLNFASGFMELGLSVDGYFYAGTGASATLIDLSDLVDIRPVGPRPSTSTLVYNLGGTTNGFSYV.

[0022] As an embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO.4, specifically: CDCRGDCFC SSKTVDSI SPADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFAAIDHDRGNASFPGSSSSNVLELWYASAGSAADNPISQIAPDGFPDMSFVPFSGITIPTAGWVGFGGIWNSSNGAPYVTTMQAYELGFATGVPSNPQPTTTTSGAQIVAKSIYGVANGINQ TTAGLFVMASGVISTPNSSATTYTPQPNRIVNAPGTPAAAPIGKNTPIMFASVVRRTGDINAEAGSTNGTQYGAGSQPLPVTIGLSLNNYSSALMPGQFFVWQLNFASGFMELGLSVDGYFYAGTGASATLIDLSDLVDIRPVGPRPSTSTLVYNLGGTTNGFSYVCDCRGDCFC.

[0023] As an embodiment, RGD4C, the hinge region of the rabbit hemorrhagic disease virus type 2 capsid protein, the P region of the rabbit hemorrhagic disease virus type 2 capsid protein and RGD4C are directly linked in sequence without any linker in between.

[0024] The present invention also provides a gene encoding the fusion protein described in the above scheme.

[0025] As an embodiment, the nucleotide sequence of the coding 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, and the nucleotide sequence shown in SEQ ID NO.5 is specifically: tgcgactgccgcggcgactgcttctgc agcagcaagaccgtggacagcatcagccccgc cgacctgctgaccacccccgtgctgaccggcgtgggcaccgacaaccgctggaacggcgagatcgtgggcctgcag cccgtgcccggcggcttcagcacctgcaaccgccactggaacctgaacggcagcacctacggctggagcagccccc gcttcgccgccatcgaccacgaccgcggcaacgccagcttccccggcagcagcagcagcaacgtgctggagctgtg gtacgccagcgccggcagcgccgccgacaaccccatcagccagatcgcccccgacggcttccccgacatgagcttc gtgcccttcagcggcatcaccatccccaccgccggctgggtgggcttcggcggcatctggaacagcagcaacggcg ccccctacgtgaccaccatgcaggcctacgagctgggcttcgccaccggcgtgcccagcaacccccagcccaccac caccaccagcggcgcccagatcgtggccaagagcatctacggcgtggccaacggcatcaaccagaccaccgccggc ctgttcgtgatggccagcggcgtgatcagcacccccaacagcagcgccaccacctacaccccccagcccaaccgca tcgtgaacgcccccggcacccccgccgccgcccccatcggcaagaacacccccatcatgttcgccagcgtggtgcg ccgcaccggcgacatcaacgccgaggccggcagcaccaacggcacccagtacggcgccggcagccagcccctgccc gtgaccatcggcctgagcctgaacaactacagcagcgccctgatgcccggccagttcttcgtgtggcagctgaact tcgccagcggcttcatggagctgggcctgagcgtggacggctacttctacgccggcaccggcgccagcgccaccct gatcgacctgagcgacctggtggacatccgccccgtgggcccccgccccagcaccagcaccctggtgtacaacctg ggcggcaccaccaacggcttcagctacgtg Among them, the bold sequence is the coding gene of RGD4C, the wavy line sequence is the coding gene of the hinge region of the capsid protein of rabbit hemorrhagic disease virus type 2, and the double-underlined sequence is the coding gene of the P region of the capsid protein of rabbit hemorrhagic disease virus type 2.

[0026] In one embodiment, the codon optimization includes human cell codon optimization or insect cell codon optimization; the nucleotide sequence of the coding gene is optimized for human cell codons and further connected to a restriction enzyme cleavage site, a KOZAK sequence, a start codon, and a stop codon sequence. The specific sequence is 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 of the coding gene is optimized for insect cell codons and is also connected to the restriction enzyme cleavage site, KOZAK sequence, start codon and stop codon sequence. The specific sequence is shown in SEQ ID NO.7, specifically: gaattcgccacc atgtgcgattgtaggggcgattgcttctgt t cgtctaagaccgtcgacagcatcagccctgcagacttattaacgacaccagtgcttactggagtcggaacagataa ccgttggaatggagaaatcgtcggtttgcaaccagtcccggggggtttctcaacttgtaatcgtcactggaaccta aatggctctacgtatggatggtcatccccccgattcgcggcgattgaccatgatagagggaacgcaagcttcccgg ggagttcctcctcaaatgttctcgagctatggtacgcttcggcgggctcagcggctgataatcctatatcccagat tgccccggatggttttccagatatgtcttttgtgccattctcgggaataacaattcctactgctggctgggttggt tttggtggcatttggaatagtagtaacggggctccctacgttacgacgatgcaagcgtacgagttaggttttgcaa cgggtgtaccatccaacccccaacctacgacaaccacaagcggggctcagattgtagcaaaatctatctatggggt cgcgaacggcattaaccagacgacagccgggctctttgtaatggcaagtggagttatctcgacccctaactcttcg gccaccacctatacgccgcagcccaatcggatagtgaacgcccccggtacccccgctgctgcgcctatcgggaaaa ataccccaataatgtttgccagcgtggttcgccgcacaggcgatattaacgccgaagcaggttccacaaatggaac ccaatatggggcagggtcacaaccgttgccggtaactatcggcttatctttgaacaactatagcagtgcccttatg ccaggccagttctttgtatggcagctaaatttcgccagcggattcatggagctcggactctcggtggacggctact tttacgctggtactggtgcatcggcgactctgatagacctgagtgacctggtggacataagacccgtaggccctcg gccgtccacttcaactcttgtctacaatctggggggaacgaccaatggatttagttatgtt tgcgattgtaggggcgattgcttctgttga gtcgac .

[0027] The present invention also provides a recombinant vector into which the coding gene described in the above scheme is inserted.

[0028] As an embodiment, the backbone plasmid of the recombinant vector includes pCI - neo The coding gene is inserted into EcorI and SalI The specific sequence is shown in SEQ ID NO.6. EcorI Restriction sites gaattc and ​ Restriction site gtcgac and vector ​ connect.

[0029] As another embodiment, the backbone plasmid of the recombinant vector includes ​ The coding gene is inserted into ​ and ​ The specific sequence is shown in SEQ ID NO.7. ​ Restriction sites gaattc and ​ Restriction site gtcgac and vector ​ connect.

[0030] The present invention also provides a recombinant bacterium or recombinant cell comprising the recombinant vector described in the above scheme.

[0031] As an 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.

[0032] The present invention has no particular limitation on the construction method of the recombinant bacteria or recombinant cells, and conventional methods in the art may be used.

[0033] 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 in the above scheme in at least one of the following: 1) Preparation of drugs for preventing or treating rabbit hemorrhagic disease virus type 2 infection; 2) Application in the preparation of biological products for preventing rabbit hemorrhagic disease virus type 2 infection; 3) Preparation of detection reagents or kits for rabbit hemorrhagic disease virus type 2 capsid protein-specific antibodies.

[0034] The present invention also provides a rabbit hemorrhagic disease virus type 2 self-assembly nanoparticle vaccine, comprising the fusion protein described in the above scheme.

[0035] As an embodiment, the rabbit hemorrhagic disease virus type 2 self-assembling nanoparticle vaccine further includes a pharmaceutically acceptable adjuvant.

[0036] As an embodiment, the dosage form of the rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine is an injection.

[0037] The present invention also provides a detection reagent or kit for rabbit hemorrhagic disease virus type 2 capsid protein-specific antibodies, using the fusion protein described in the above scheme as a coating antigen.

[0038] In one embodiment, the kit comprises an indirect ELISA detection kit. In one embodiment, the indirect ELISA detection kit further comprises an ELISA plate, a coating solution, PBST, 5% skim milk, HRP-labeled goat anti-rabbit IgG, TMB color development solution, H2SO4 stop solution, a negative control, and a positive control. In one embodiment, the working concentration of the fusion protein as a coating antigen is 1 μg / ml.

[0039] 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 a sample to be tested.

[0040] The present invention has no particular limitation on the specific process of the detection, and conventional steps in the art may be used.

[0041] To further illustrate the present invention, a fusion protein, rabbit hemorrhagic disease virus type 2 self-assembled nanoparticle vaccine and applications provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1 Construction, expression and identification of self-assembled nanoparticles containing the P region of the capsid protein of rabbit hemorrhagic disease virus type 2 In this example, the formation mechanism and antigenicity of virus-like particles (VLPs) of the structural protein of rabbit hemorrhagic disease virus type 2 (RHDV2) were analyzed to construct a variety of spike protein (P region) self-assembled nanoparticles that can improve immunogenicity and neutralizing antibody levels, thereby preparing a rabbit hemorrhagic disease virus type 2 P region self-assembled nanoparticle vaccine to improve the efficacy and accuracy of vaccine immunity.

[0043] by ​ As a carrier, ​ I (GAATTC) ​ I (GTCGAC) is the restriction site, and the upstream restriction site ​ Insert the KOZAK sequence (GCCACC) between I and the start codon ATG. ​ I and ​ I, and the RGD4C or CNGRC sequence was inserted before the hinge region (H) and after the P region of the RHDV2 VP60-HP sequence, respectively. Two recombinant plasmids, pCI-RGD4C-HP-RGD4C and pCI-CNGRC-HP-CNGRC, were constructed. Partial base deletion mutations were performed on the hinge region, RGD4C or CNGRC, to obtain 12 recombinant plasmids. ​ The pCI-RHDV2 VP60-P recombinant plasmid is used as the vector. The structural diagram of all recombinant plasmids can be found in ​ Among them, the amino acid sequence of RGD4C-HP-RGD4C is shown in SEQ ID NO.4; the nucleotide sequence of RGD4C-HP-RGD4C before optimization is shown in SEQ ID NO.5; the nucleotide sequence of RGD4C-HP-RGD4C after human cell codon optimization and the addition of a restriction enzyme cleavage site, KOZAK sequence, start codon and stop codon sequence is shown in SEQ ID NO.6; the nucleotide sequence of RGD4C-HP-RGD4C after insect cell codon optimization and the addition of a restriction enzyme cleavage site, KOZAK sequence, start codon and stop codon sequence is shown in SEQ ID NO.7.

[0044] The amino acid sequence of CNGRC-HP-CNGRC is shown in SEQ ID NO. 8, specifically: ​ ​ ​ ​ ​ ​ ​ ​ ; Among them, the italic sequence is the amino acid sequence of CNGRC.

[0045] The nucleotide sequence of CNGRC-HP-CNGRC before optimization is shown in SEQ ID NO.9, specifically:​ ​ ​ ​ ctgaacggcagcacctacggctggagcagcccccgcttcgccgccatcgaccacgaccgcggcaacgccagcttcc ccggcagcagcagcagcaacgtgctggagctgtggtacgccagcgccggcagcgccgccgacaaccccatcagcca gatcgcccccgacggcttccccgacatgagcttcgtgcccttcagcggcatcaccatccccaccgccggctgggtg ggcttcggcggcatctggaacagcagcaacggcgccccctacgtgaccaccatgcaggcctacgagctgggcttcg ccaccggcgtgcccagcaacccccagcccaccaccaccaccagcggcgcccagatcgtggccaagagcatctacgg cgtggccaacggcatcaaccagaccaccgccggcctgttcgtgatggccagcggcgtgatcagcacccccaacagc agcgccaccacctacaccccccagcccaaccgcatcgtgaacgcccccggcacccccgccgccgcccccatcggca agaacacccccatcatgttcgccagcgtggtgcgccgcaccggcgacatcaacgccgaggccggcagcaccaacgg cacccagtacggcgccggcagccagcccctgcccgtgaccatcggcctgagcctgaacaactacagcagcgccctg atgcccggccagttcttcgtgtggcagctgaacttcgccagcggcttcatggagctgggcctgagcgtggacggct acttctacgccggcaccggcgccagcgccaccctgatcgacctgagcgacctggtggacatccgccccgtgggccc ccgccccagcaccagcaccctggtgtacaacctgggcggcaccaccaacggcttcagctacgtg tgcaatgggagg tgc ; Among them, the italic sequence is the nucleotide sequence of the coding gene of CNGRC.

[0046] The nucleotide sequence of CNGRC-HP-CNGRC is optimized for human cell codons and is supplemented with restriction enzyme sites, KOZAK sequence, start codon, and stop codon sequences as shown in SEQ ID NO. 10. Specifically, it is: gaattcgccacc atg tgcaacggcaggtgc agctcaaagaccgtggatagcatcagccctgccgacct gctgaccacccccgtgctgaccggcgttggaacagacaaccggtggaacggcgaaatcgtgggcctgcagcctgtc cctggaggctttagcacatgcaaccgccactggaatctgaatggctctacatatggctggagctctccaagattcg ccgctatcgaccacgacagaggcaacgcttcttttcccggctccagcagttctaatgtgttagagctgtggtacgc ctccgccggctctgccgcagataaccccatctcgcaaatcgcccctgatggctttcccgacatgtctttcgtgccc ttcagcggcatcaccatccccaccgccggatgggttggcttcggcggaatctggaacagcagcaacggcgctccct acgtgaccacaatgcaggcctacgagctgggcttcgccaccggcgtgccaagcaaccctcagcctacaacaaccac cagcggagcccagattgtggccaagagcatctacggcgtggccaacggcattaatcagacaaccgccggactgttc gtgatggccagcggcgtgatcagcacaccaaactccagcgccaccacctatacccctcaacctaatagaatcgtga acgcccctggcaccccagccgccgcccctatcggcaaaaacacccctatcatgttcgctagcgtggtgcggagaac cggagatatcaacgccgaggccggctctacaaacggcacccagtacggcgctggcagccagcctctgcctgtcaca atcggcctcagcctgaacaactacagcagcgccctgatgcctggccagttcttcgtgtggcagctgaacttcgcct ctggctttatggaactgggcctgagcgtggacggctacttctacgccgggaccggcgcctccgctaccctgatcga cctgagcgacctggtggacatccggcctgtgggacctagacccagcacaagcacactggtctacaacctgggtgga accaccaacggcttcagctacgtg tgtaatggaagatgt tga gtcgac .

[0047] The nucleotide sequence, amino acid sequence, human cell codon-optimized nucleotide sequence, and insect cell codon-optimized nucleotide sequence of RGD4C-HP were adjusted with reference to the above sequences.

[0048] 1. Materials and Methods 1.1.1 Materials 1.1.1.1 Plasmids and cells The RGD4C-HP-RGD4C and CNGRC-HP-CNGRC original vectors were synthesized by Nanjing GenScript Biotechnology 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.

[0049] 1.1.1.2 Reagents Mut Express II Fast Mutagenesis Kit V2 plasmid single-point mutagenesis kit, SuperPicoECL Chemiluminsence Kit, and 2× Rapid Taq Master Mix were purchased from Nanjing Novozymes 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 Shengong Biotechnology Co., Ltd.; agar and agarose were purchased from Guangzhou Saiguo Biotechnology Co., Ltd.; Fast Plasmid Miniprep Kit was purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.; transfection reagent, DMEM high-glucose medium, Grace ’ Insect Cell Culture Medium and IB905 insect cell culture medium were 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 Solaibao 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 Quanshijin 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 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.

[0050] 1.1.2 Methods 1.1.2.1 Construction of HEK 293 cell-based expression plasmid 1.1.2.1.1 Construction of the original plasmid The structural protein VP60 gene of rabbit hemorrhagic disease virus SC2020 / 0401 strain (GenBank No.: MT586027) was obtained from GenBank, and the hinge region and P region gene sequences (HP) of the structural protein VP60 were obtained by comparison analysis. KOZAKThe sequence (GCCACC) is added to the 5′ end of the sequence to increase the expression level. 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 inserted into pCI-neo Carrier EcoRI and SalI Between the restriction enzyme cutting sites, two original expression plasmids were constructed: PCI-RGD4C-HP-RGD4C and PCI-CNGRC-HP-CNGRC.

[0051] 1.1.2.1.2 Design of recombinant pCI-RHDV2 VP60-P plasmid 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 by partial base deletion mutagenesis using the Novozymes Mut Express II Fast Mutagenesis Kit V2 plasmid single point mutagenesis kit, such as Figure 1 shown.

[0052] 1.1.2.1.3 Primer design The sequences of primers for homologous recombination and M13 blue-white spot screening and identification are shown in Table 1: Table 1 Primer names and sequences

[0053] 1.1.2.1.4 Amplification and identification of the recombinant pCI-RHDV2 VP60-P vector The plasmid amplification system is shown in Table 2; the amplification program was as follows: initial denaturation at 95°C for 30 s; denaturation at 95°C for 15 s, annealing at 65°C for 15 s, extension at 72°C for 7 min, 30 cycles, and final extension at 72°C for 5 min.

[0054] Table 2 Plasmid amplification system

[0055] Dpn1 methylation template digestion system: Dpn1 1 μl and amplification product 40-50 μl. Digestion conditions: 37°C, 1-2 hours.

[0056] The homologous recombination reaction system is shown in Table 3. Reaction conditions: 37°C, 30 min.

[0057] Table 3 Homologous recombination reaction system

[0058] The products obtained after homologous recombination are P region vectors with different cysteine ​​residues, including those without hinge regions. The recombinant pCI-RHDV2 VP60-P vector was transformed into DH5α plate and single colonies were picked. Single colony sequencing analysis was performed to determine the P region vectors with different lengths and amplify and extract the plasmids.

[0059] 1.1.2.2 Expression of recombinant pCI-RHDV2 VP60-P vector in HEK 293 cells HEK 293 cells at a cell density of approximately 90% were transiently transfected with the sequenced recombinant pCI-RHDV2 VP60-P vector plasmid (1 μg per well). After incubation at 37°C for 48 hours, cells were observed under a microscope, the medium was discarded, and the cells were washed twice with PBS. Then, 200 μl of cell lysis buffer without a denaturing reducing agent was added to each well. The cells were incubated at 4°C for 10 minutes, and the lysate containing the lysed HEK 293 cells was collected. The lysate was centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was the target protein.

[0060] 1.1.2.3 Western Blot Analysis 1.1.2.3.1 Expression and identification of the recombinant pCI-RHDV2 VP60-P vector expression product First, 10 μl of 5× SDS-PAGE protein loading buffer was added to 40 μl of supernatant, and the sample was placed in a 100°C metal bath for 10 min to completely denature the protein. After cooling to room temperature, the sample was loaded on a Tris-glycine electrophoresis system for SDS-PAGE. After electrophoresis, the sample was transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk in PBST (PBS containing 0.5% Tween 20, the same below) for 2 h at room temperature and washed three times with PBST for 5 min each. 1B8 was used as the primary antibody (1:1000) for 1.5 h at room temperature. After incubation, the membrane was washed three times with PBST for 5 min each. Goat anti-mouse IgG (HRP) (1:10,000) was incubated for 1 h at room temperature and washed three times with PBST for 15 min each. After incubation with the HRP-conjugated secondary antibody, the blot was detected using an enhanced chemiluminescence (ECL) detection kit on a chemiluminescence image analysis system.

[0061] 1.1.2.3.2 Evaluation of the Multimer-forming Ability of Recombinant pCI-RHDV2 VP60-P Protein Take another 40 μl supernatant sample and add 10 μl 5× SDS-PAGE protein loading buffer (non-denaturing, non-reducing) to the Tris-glycine (SDS-free) electrophoresis system Native-PAGE. Subsequent operations are the same as 1.1.2.3.1 to identify the polymer formation ability.

[0062] 1.1.2.4 Construction of recombinant transfer vector based on Sf9 insect cells After identifying the multimer-forming ability of the recombinant vector using HEK 293 cells, the recombinant pCI-RHDV2 VP60-P vector (pCI-RGD4C-HP-RGD4C), which exhibited strong multimer-forming ability, was codon-optimized in insect cells to generate the recombinant transfer vector pFast-RGD4C-HP-RGD4C. A partial base deletion mutagenesis was then performed to obtain pFast-RGD4C-HP. PCR amplification was performed using 2× Rapid Taq Master Mix. The reaction conditions were: initial denaturation at 95°C for 30 s; denaturation at 95°C for 15 s; annealing at 65°C for 15 s; and extension at 72°C for 7 min for 30 cycles, with a final extension at 72°C for 5 min. The homologous recombination product was a recombinant transfer vector for Sf9 insect cells.

[0063] 1.1.2.5 Acquisition and identification of recombinant shuttle vectors 1.1.2.5.1 Obtaining recombinant shuttle vector 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 with shaking at 37°C for 2 hours. After centrifugation, the supernatant was discarded and the transformation product was 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 hours. White positive colonies were streaked onto triple-antibody plates for purification. This was repeated three times. White colonies that were positive in all three rounds were identified by PCR using the universal primers PUC M13-F / M13-R. After positive 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. Schematic diagram of RHDV2 VP60-P recombinant bacmid Figure 2 .

[0064] 1.1.2.5.2 Identification of recombinant shuttle plasmids PCR amplification was performed using single colonies screened three times for the recombinant shuttle vector as templates using universal primers PUC M13-F / M13-R and 2× Rapid Taq Master Mix. The reaction conditions were: initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 20 s, and extension at 72°C for 90 s, with a final extension at 72°C for 5 min. Amplified products were identified by agarose gel electrophoresis and bacterial culture PCR. Successful vector plasmid extraction confirmed the recombinant shuttle plasmids (Bacmid-RGD4C-HP-RGD4C and Bacmid-RGD4C-HP).

[0065] 1.1.2.6 Obtaining the recombinant rBac-RHDV2 VP60-P virus The recombinant shuttle plasmids, Bacmid-RGD4C-HP-RGD4C and Bacmid-RGD4C-HP, were transiently transfected into Sf9 insect cells using Lip 3000 as a transfection reagent. Cells were observed under a microscope every 24 hours after transfection until signs of cell division and growth ceased, with rounded cells, increased intercellular spaces, and some floating cells. Normal cells in the control group showed no significant changes except for an increase in growth density. The supernatant, representing the recombinant viruses rBac-RGD4C-HP-RGD4C and rBac-RGD4C-HP, was collected by centrifugation and stored at 4°C.

[0066] 1.1.2.7 Passaging and Preliminary Identification of the Recombinant rBac-RHDV2 VP60-P Virus 1.1.2.7.1 Passaging of recombinant rBac-RHDV2 VP60-P virus Observe Sf9 insect cells. When the cell density grows to about 90%, inoculate the recombinant rBac-RHDV2 VP60-P P1 virus at a 3% inoculum. After incubation at 26°C for 72 h, observe under a microscope. If obvious cell lesions appear, centrifuge and collect the supernatant to obtain the recombinant rBac-RHDV2 VP60-P P2 virus.

[0067] 1.1.2.7.2 Preliminary IFA Identification of Recombinant rBac-RHDV2 VP60-P Virus Sf9 insect cells grown at a density of approximately 90% of normal cells in a 24-well plate were infected with the 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 in the 24-well plate was aspirated and the cells were washed twice with PBS for 3-5 min each time. 500 μl of -20°C pre-cooled fixative (ethanol:acetone 2:3) was added to each well and fixed at 4°C for 1 h. After fixation, the cells were washed three times with 1 ml of PBST per well for 5 min each time. 200 μl of 1B8 diluted in PBS was added to each well as the primary antibody (1:50) and incubated at 37°C for 1 h. After incubation, the primary antibody was aspirated and the cells were washed three times with 1 ml of PBST per well for 5 min each time. 200 μl of goat anti-mouse IgG (FITC) secondary antibody (1:100) was added to each well and incubated at 37°C for 1 h. After incubation, the secondary antibody was aspirated and 1 ml of PBST was added to each well. The cells were washed with PBST three times, 5 min each time, and observed under a fluorescence microscope and compared with the infected normal Sf9 insect cells for IFA identification.

[0068] 1.1.2.8 Acquisition and identification of recombinant RHDV2 VP60-P expression product 1.1.2.8.1 Obtaining the recombinant RHDV2 VP60-P expression product The recombinant rBac-RHDV2 VP60-P virus was inoculated at a volume ratio of 2% in a culture medium with a growth density of 1.6×10 6 HighFive insect cells were cultured at 26°C with shaking at 120 rpm. Samples were taken every 24 hours for observation under a microscope. Samples were taken continuously for 7 days to determine the sampling time.

[0069] 1.1.2.8.2 Identification of recombinant RHDV2 VP60-P expression products Collect cells and culture medium with obvious pathological changes observed under the microscope and freeze-thaw repeatedly three times; centrifuge at 1000 rpm for 10 min at room temperature, take 40 μl of supernatant, add 10 μl 5× SDS-PAGE protein loading buffer, and place in a 100℃ metal bath for 10 min to completely denature the protein for Western Blot identification. The operation is the same as 1.1.2.3.1.

[0070] 1.1.2.9 Transmission electron microscopy To determine whether recombinant RHDV2 VP60-P particle antigen can self-assemble into nanoparticles, the recombinant virus was inoculated into High Five insect cells and then repeatedly frozen and thawed three times. The supernatant was collected by centrifugation at 5000 r / min for 10 min, and the supernatant was passed through a 100KD ultrafiltration tube to remove impurities and then centrifuged at 30000 r / min for 2 h to purify the recombinant RHDV2 VP60-P particle antigen. The presence and morphology of recombinant RHDV2 VP60-P particle antigen and VLPs were detected by transmission electron microscopy negative staining.

[0071] 1.2 Results 1.2.1 Expression and identification of recombinant pCI-RHDV2 VP60-P in HEK 293 cells To verify the successful expression of the 14 recombinant pCI-RHDV2 VP60-P plasmids, HEK 293 cells were transiently transfected with 1 μg / well of the plasmids and incubated at 37°C for 48 h for expression. The recombinant RHDV2 VP60-P protein was analyzed by Western blot. Figure 3 The results showed that the recombinant RHDV2 VP60-P proteins were successfully expressed with a molecular weight of approximately 35 kDa. The recombinant RHDV2 VP60-P proteins naturally formed a dimer of 70 kDa.

[0072] 1.2.2 Evaluation of the ability of recombinant RHDV2 VP60-P protein to form multimers To evaluate the ability of recombinant RHDV2 VP60-P protein to form multimers, the recombinant RHDV2 VP60-P protein was analyzed by non-denaturing, non-reducing Nativepage Western blotting. Figure 4 As shown, lane 13 RGD4C-HP-RGD4C and lane 11 RGD4C-HP both showed protein bands with a molecular weight greater than 180 kDa, indicating that these two recombinant proteins have the ability to form multimers.

[0073] 1.2.3 PCR Identification of Recombinant Shuttle Vectors by Blue-White Screening The recombinant transfer vectors pFast-RGD4C-HP-RGD4C and pFast-RGD4C-HP were transformed into DH10 Bac competent cells. White colonies that were positive in three screenings were identified by PCR using 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. The PCR results showed that the recombinant transfer vectors pFast-RGD4C-HP-RGD4C and pFast-RGD4C-HP were successfully transposed, and the recombinant shuttle vectors Bacmid-RGD4C-HP-RGD4C and Bacmid-RGD4C-HP were obtained. Figure 5 and Figure 6 .

[0074] 1.2.4 Obtaining the recombinant baculovirus rBac-RHDV2 VP60-P and identifying the expression product 1.2.4.1 Obtaining recombinant baculovirus rBac-RHDV2 VP60-P The extracted Bacmid-RGD4C-HP-RGD4C and Bacmid-RGD4C-HP plasmids were transfected into Sf9 cell monolayers to obtain recombinant baculovirus rBac-RGD4C-HP-RGD4C and rBac-RGD4C-HP. Figure 7 .

[0075] 1.2.4.2 Identification of recombinant baculovirus rBac-RHDV2 VP60-P The recombinant baculoviruses rBac-RGD4C-HP-RGD4C and rBac-RGD4C-HP were inoculated into Sf9 cell monolayers. After 24 h of infection, IFA detection was performed using 1B8 as the primary antibody. The results are shown in Figure 2. Figure 8 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.

[0076] 1.2.4.3 Determination of the optimal expression time of recombinant RHDV2 VP60-P protein The recombinant virus rBac-RGD4C-HP-RGD4C was inoculated into suspension cultured High Five insect cells to determine the optimal expression time of the recombinant protein, e.g. Figure 9 The recombinant protein showed distinct bands at 35 kDa and 70 kDa, and the expression level was highest at 96 h.

[0077] 1.2.5 Electron microscopic observation of the recombinant RHDV2 VP60-P granule protein expression product TEM results show that RGD4C-HP-RGD4C and RGD4C-HP can form relatively uniform nanoparticles with sizes of 40~50 nm and 80~100 nm, respectively. Figure 10 shown.

[0078] 1.3 Discussion In this example, the original vectors pCI-RGD4C-HP-RGD4C and pCI-CNGRC-HP-CNGRC were successfully constructed by bioinformatics and molecular biology. After 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 identified by Western blot as two multimer-forming vectors, pCI-RGD4C-HP-RGD4C and pCI-RGD4C-HP. The designed proteins were then efficiently expressed and functionally characterized using a baculovirus expression system. Transmission electron microscopy revealed that both RGD4C-HP-RGD4C and RGD4C-HP formed nanoparticles measuring 40-50 nm and 80-100 nm, respectively. These results provide an important technical foundation for the development of RHDV2 subunit vaccines and offer a reference for vaccine design for other viruses that cannot be cultured in vitro.

[0079] pCI-RGD4C-HP-RGD4C and pCI-RGD4C-HP were able to form multimers in HEK 293 cells, indicating that RGD4C has a better ability to promote protein multimer formation than CNGRC. Electron microscopy revealed that the particles formed by these two proteins were slightly larger than native RHDV VLPs, indicating that the P region has the ability to independently self-assemble, which provides a basis for subsequent studies of their immunogenicity and particle structure.

[0080] This example successfully constructed RHDV2 P region self-assembling nanoparticles through molecular biology and protein engineering technology, and verified their efficient expression and assembly capabilities in insect cells, laying an important foundation for the development of RHDV2 subunit vaccines and providing new ideas for the preparation of antigens for unculturable viruses.

[0081] 1.4 Summary 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) that can form protein polymers were obtained through the Bac-to-Bac baculovirus expression system. The sizes of these two antigens were 40-50 nm and 80-100 nm, respectively.

[0082] Example 2 Study on the immunogenicity of self-assembled nanoparticles containing the P region of the capsid protein of rabbit hemorrhagic disease virus type 2 To verify the immune protection effect of the RHDV2 capsid protein P region self-assembled nanoparticles constructed in Example 1, 2 ml of the expressed RHDV2 VP60-P antigen (RGD4C-HP-RGD4C and RGD4C-HP) was administered subcutaneously to the neck of one-month-old RHDV2 antibody-negative rabbits. The immune efficacy was evaluated in terms of immune protection rate and humoral immunity, and compared with the RHDV2 VP60 vaccine group and the PBS group. The challenge protection results showed that 21 days after immunization, the RGD4C-HP-RGD4C group, the RGD4C-HP group, the RHDV2 VP60 vaccine group, and the PBS group were challenged with RHDV2. All the rabbits in the RGD4C-HP-RGD4C group and the RHDV2 VP60 vaccine group survived, while all the rabbits in the RGD4C-HP group and the PBS group died with typical RHD clinical symptoms and pathological changes. The results of antibody level detection showed that from the 7th day to the 60th day 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 increased significantly and maintained at a high level, which was significantly different from that in the PBS group. At the same time, the levels of RHDV2 VP60 P region antibodies were detected in the RGD4C-HP-RGD4C group and the RHDV2 VP60 vaccine group. The results showed that both the RGD4C-HP-RGD4C group and the RHDV2 VP60 vaccine group produced RHDV2-VP60 P region-specific antibodies after 7 days of immunization. At the same time point, the antibody level of the RGD4C-HP-RGD4C group was higher than that of the RHDV2 VP60 vaccine group and maintained at a high level for a period of time. The above results show that rabbit hemorrhagic disease virus type 2 capsid protein P region self-assembled nanoparticles (RGD4C-HP-RGD4C) can induce rabbits to produce antibodies specific to the RHDV2-VP60 P region, produce a strong humoral immune response, and provide complete protection for rabbits.

[0083] 2.1 Materials and Methods 2.1.1 Materials 2.1.1.1 Virus strains and experimental animals Rabbit hemorrhagic disease virus SC2020 / 0401 strain (RHDV2 type), recombinant RHDV2 VP60, Sf9, and High Five insect cells were provided by the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences. 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.

[0084] 2.1.1.2 Main reagents Grace ’ Insect Cell Culture Medium and IB 905 insect cell culture medium were 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.; one-component TMB colorimetric solution 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 determination kit was purchased from Beyotime Biotechnology Co., Ltd.; 96-well ELISA plate was purchased from Nanjing Saiyan Biotechnology Co., Ltd.; the remaining reagents were domestic analytical grade.

[0085] 2.1.2 Methods 2.1.2.1 Preparation of self-assembled nanoparticle antigens containing the P region of the recombinant rabbit hemorrhagic disease virus type 2 capsid protein 2.1.2.1.1 Vaccination 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 medium IB 905 and cultured at 26°C for 4 days. When the cytopathic effect reached more than 85% under a microscope, the cell culture was harvested and stored at -20°C.

[0086] 2.1.2.1.2 Hemagglutination titer test Harvested cell cultures were frozen and thawed three times, and red blood cell agglutination titers were determined using a 50-well plate assay. 50 μl of the frozen-thawed cell culture was added to a 50-well plate. 50 μl of PBS was added and pipetted to mix thoroughly, resulting in a two-fold dilution. After pipetting to mix thoroughly, 50 μl of a 1% human "B" red blood cell suspension was added and tapped to mix thoroughly. The cells were allowed to stand at 2–8°C for 60 minutes, and then red blood cell agglutination was observed.

[0087] 2.1.2.1.3 Determination of immune antigen dose The harvested cell culture was frozen and thawed three times to lyse the cells and release the recombinant antigen. A recombinant RHDV2 VP60 full-length protein with a hemagglutination titer of 1:256 was used as a control. Western Blot analysis showed that the initial immunoassay volume was 2 ml per animal.

[0088] 2.1.2.2 Purification of Rabbit Hemorrhagic Disease Virus Type 2 Capsid Protein P Region Antigen The P region gene of rabbit hemorrhagic disease virus SC2020 / 0401 strain (MT586027) was selected for comparison analysis. pCold This paper constructs a vector to express the recombinant rabbit hemorrhagic disease virus type 2 P region protein based on Escherichia coli expression.

[0089] 2.1.2.2.1 Inducible expression of rabbit hemorrhagic disease virus type 2 capsid protein P region antigen (1) Transform the constructed recombinant P region plasmid into BL-21 (DE3) competent cells, spread the plate and pick out a single colony; (2) Inoculate a single colony into 5 ml of Amp+ resistant LB and culture overnight at 37°C with shaking at 200 rpm. (3) Inoculate the shaken recombinant E. coli into 100 ml of Amp + LB at a 1% inoculum volume and shake at 37°C and 200 rpm; (4) After inoculation, 200 μl of the E. coli was sampled every 30 minutes and placed in a 96-well ELISA plate. The OD value of the bacterial solution at OD600 absorbance was read. When the OD value at OD600 absorbance was 0.3-0.5, IPTG with a final concentration of 0.1 μM was added and shaken at 37°C and 200 r / min for 4 hours to induce expression. After induction, samples were retained for subsequent identification.

[0090] 2.1.2.2.2 Purification of Rabbit Hemorrhagic Disease Virus Type 2 Capsid Protein P Region Antigen (1) After induction, the E. coli was centrifuged at 4°C and 12,000 r / min for 20 min, the supernatant was discarded, and an appropriate amount of PBS was added to resuspend the bacterial pellet. The pellet was then centrifuged at 4°C and 12,000 r / min for 10 min and the wash was repeated three times. (2) After washing, the bacterial pellet was resuspended in 20 ml PBS, ultrasonically disrupted for 10 min at 60% power with a 5 s pause and a 5 s supernatant, and centrifuged to obtain the supernatant and pellet for expression verification; (3) Dissolve the bacterial precipitate after ultrasonic disruption with a denaturing solution containing 8 M urea, invert at 4°C to completely dissolve the bacterial precipitate, and retain a sample for identification; (4) Add an equal amount of denaturing solution containing 6 M urea to the thoroughly dissolved 8 M denaturing mixture of Escherichia coli containing the P region protein of the recombinant rabbit hemorrhagic disease virus type 2 capsid protein, vortex mix, add to a 10,000 pore size cellulose dialysis bag, place in the denaturing solution containing 4 M urea, and dialyze and renature overnight at 4°C with magnetic stirring; (5) Place the cellulose dialysis bag that has been dialyzed overnight in a denaturing solution containing 2 M urea and perform renaturation under magnetic stirring at 4°C for 8 h. (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 replace the denaturing solution that does not contain urea every 6 hours for dialysis refolding; 2.1.2.2.3 Identification of the P region protein of the capsid protein of rabbit hemorrhagic disease virus type 2 The expression of induced expression, ultrasonic crushing centrifugal supernatant, ultrasonic crushing centrifugal precipitate, precipitate in each process after denaturation, and renaturation samples were subjected to SDS-PAGE to verify the expression, and the grayscale calculation of Image J software was used to roughly estimate the concentration of purified protein.

[0091] 2.1.2.3 Challenge and protection experiments 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 2 ml of PBS per rabbit (Table 4). Twenty-one days after immunization, all immunized rabbits were challenged with 1 ml / rabbit hemorrhagic disease virus SC strain (SC2020 / 0401 (MT586027)) (1000 LD). 50 The rabbits were observed for 7 consecutive days. The mortality of the immunized rabbits was recorded every day, and the immune protection rate was calculated.

[0092] Immune protection rate (%) = (mortality rate of control group - mortality rate of immunized group) / mortality rate of control group × 100% Table 4: Grouping of virus attack

[0093] 2.1.2.4 Grouping and Immunization 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 2 ml of PBS per rabbit (Table 5). Blood was collected on days 0, 7, 14, 21, 28, 35, 42, and 60 after immunization, and serum was separated and stored for future use.

[0094] Table 5 Immunization grouping

[0095] 2.1.2.5 RHDV2 VP60-specific antibody detection The indirect ELISA method established in our laboratory was used to detect the serum RHDV2 VP60-specific IgG antibody levels at different time points after immunization to monitor the antibody response pattern. The specific procedures are as follows: (1) The recombinant RHDV2 VP60 protein expressed in the baculovirus expression system was used as the coating antigen (the HA titer of the recombinant RHDV2 VP60 protein was 1:256 after HA detection). The coating antigen was diluted 1:200 with coating solution (pH = 9.6) (concentration was approximately 0.5 μg) and coated on a 96-well ELISA plate. 100 μl was added to each well and sealed in a ziplock bag. The plate was coated overnight at 4°C. After coating, 300 μl of PBST was added to each well and washed three times for 5 min each time. 200 μl of 5% skim milk was added to each well and incubated at 37°C for 2 h for blocking. After blocking, 300 μl of PBST was added to each well and washed three times for 5 min each time.

[0096] (2) Serum at different time points was diluted 1:100 with 5% skim milk, 100 μl of diluted immune rabbit serum was added to each well, incubated at 37°C for 1 h, and washed 3 times with 300 μl PBST per well, 5 min / time; 100 μl of 5% skim milk 1:10000 diluted HRP-labeled goat anti-rabbit IgG was added to each well, incubated at 37°C for 1 h, and washed 3 times with 300 μl PBST per well, 5 min / time; 100 μl TMB color development solution was added to each well, and color was developed at room temperature in the dark for 5-10 minutes. When the negative sample wells were slightly colored, 50 μl 2M H2SO4 stop solution was added to each well, and the absorbance was detected at 450 nm.

[0097] 2.1.2.6 RHDV2 VP60-P specific antibody detection The established indirect ELISA method was used to detect serum RHDV2 VP60-P specific IgG antibody levels at different time points after immunization to monitor the antibody response pattern. The specific procedures were as follows: Use the purified recombinant RHDV2 VP60-P protein expressed in E. coli as the coating antigen. Dilute the coating antigen with coating solution (pH = 9.6) to a final concentration of 1 μg / ml. Coat a 96-well ELISA plate with 100 μl per well. Place the plate in a sealed ziplock bag and incubate overnight at 4°C. Subsequent ELISA procedures are the same as in 2.1.2.5.

[0098] 2.2 Results 2.2.1 Antigen preparation The 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 and frozen and thawed three times. The hemagglutination titer was detected using a 50-well plate method. The results showed that the recombinant proteins could not agglutinate erythrocytes, and the hemagglutination titer was 0.

[0099] 2.2.2 Comparison of immune antigen dosage The collected recombinant RGD4C-HP-RGD4C and RGD4C-HP cell cultures were treated with recombinant RHDV2 VP60 protein with a hemagglutination titer of 1:256 as a control. Western Blot was performed to preliminarily determine the immunoassay dosage to be 2 ml / cell ( Figure 11 ).

[0100] 2.2.3 Purification of Rabbit Hemorrhagic Disease Virus Type 2 Capsid Protein P Region Antigen The recombinant RHDV2 VP60-P protein expressed in E. coli was induced for expression, the supernatant was ultrasonically disrupted, the precipitate was ultrasonically disrupted, the precipitate was precipitated during denaturation, and the sample after renaturation was subjected to SDS-PAGE to verify the expression. The grayscale calculation of Image J software was used to roughly estimate the purified protein concentration to be approximately 0.6 mg / ml ( Figure 12 ).

[0101] 2.2.4 Challenge and protection experiments 21 days after immunization, all immunized rabbits were challenged with 1 ml / rabbit hemorrhagic disease virus SC strain (SC2020 / 0401 strain (MT586027)) (1000 LD 50 ), and were observed for 7 consecutive days. The results showed that all the mice in the RGD4C-HP-RGD4C group and the RHDV2 VP60 group survived, with a protection rate of 100% (Table 6), had no clinical symptoms of RHD, and no lesions were found in the autopsy. All the mice in the RGD4C-HP group and the PBS group died 48 hours after the challenge ( Figure 13 ), the dead rabbits had obvious clinical symptoms and typical lesions were found in the autopsy ( Figure 14 ).

[0102] Table 6 Immune protection rate

[0103] 2.2.5 RHDV2 VP60-specific antibody detection Indirect ELISA was used to detect RHDV2 VP60-specific IgG antibodies in sera at 0, 7, 14, 21, 28, 35, 42, and 60 days after immunization. The results showed that the antibody titers of the RHDV2-VP60 group and the RGD4C-HP-RGD4C group gradually increased from 7 days after immunization and were significantly higher than those of the PBS group (P<0.05). The antibody titers of the PBS group did not change significantly during the immunization cycle. Both RHDV2-VP60 and RGD4C-HP-RGD4C immunizations induced rabbits to produce antibodies against RHDV2 VP60 ( Figure 15 ).

[0104] 2.2.6 RHDV2 VP60-P specific antibody detection Indirect ELISA was used to detect RHDV2 VP60-P-specific IgG antibodies in sera collected on days 0, 7, 14, 21, 28, 35, 42, and 60 after immunization. The results showed that the antibody levels in the RGD4C-HP-RGD4C and RHDV2 VP60 groups were significantly higher than those in the PBS group starting from day 7 after immunization (P<0.05), and increased with the immunization time and maintained at a high level for a certain period of time. At the same time point, the antibody level in the RGD4C-HP-RGD4C group was higher than that in the RHDV2 VP60 vaccine group ( Figure 16 ).

[0105] 2.3 Discussion This example systematically evaluates the immunogenicity of recombinant proteins RGD4C-HP-RGD4C and RGD4C-HP by means of animal experiments, revealing their significant potential in anti-RHDV2 infection. In terms of immune protection effect, the recombinant protein RGD4C-HP-RGD4C can produce a good protective effect on immunized rabbits, while the recombinant protein RGD4C-HP cannot provide good protection, providing a basis for studying the relationship between the differences in nanoparticle structure and immune protection and the ability to produce neutralizing antibodies, suggesting that the bilateral insertion of RGD4C may be crucial for maintaining particle stability or antigen epitope exposure. This result shows that slight changes in the antigen structure can significantly affect the immune efficacy, and the formation of complete nanoparticles similar to the morphology of virus-like particles may be an important factor in providing good protection. Immunization of rabbits with the recombinant proteins RGD4C-HP-RGD4C and RHDV2 VP60 resulted in high titers of specific IgG antibodies against both RHDV2 VP60 and RHDV2 VP60-P, with RGD4C-HP-RGD4C antibody levels higher than those for RHDV2 VP60. This may be due to the more efficient antigen presentation of the nanoparticles. Because the nanoparticles form multimers with a larger effective surface area, and because the recombinant nanoparticles contain only the P region of VP60, neutralizing epitopes are directly exposed, more readily activating pattern recognition receptors (such as TLRs), promoting B cell activation, and reducing the waste of immune resources targeting non-protective epitopes (NTA and S regions).

[0106] This example successfully demonstrates that the RGD4C-HP-RGD4C nanoparticle antigen has a strong immune protection effect and can elicit antibody responses against RHDV2 VP60 and RHDV2 VP60-P. This example confirms that RHDV2 capsid protein P region self-assembled nanoparticles (RGD4C-HP-RGD4C) can induce a potent humoral immune response and provide complete protection in rabbits. This lays an important foundation for the development of new RHDV2 subunit vaccines and provides new ideas for the design of vaccines for other viruses that cannot be cultured in vitro.

[0107] 2.4 Summary The two nanoparticles, RGD4C-HP-RGD4C and RGD4C-HP, were used to immunize RHDV2-negative rabbits and compared with the RHDV2 VP60 vaccine and PBS groups. Immune challenge and antibody detection results showed that the RHDV2 capsid protein P region self-assembled nanoparticles, RGD4C-HP-RGD4C, induced higher levels of antibodies in rabbits than the RHDV2 VP60 vaccine, providing complete protection.

[0108] In summary, the present invention fuses one or both ends of the P region amino acid sequence of the rabbit hemorrhagic disease virus capsid protein VP60 with short peptides RGD4C and CNGRC containing different cysteine ​​residues in HEK293 cells, and screens out two recombinant proteins RGD4C-HP-RGD4C and RGD4C-HP that can form nanoparticles. In order to express the recombinant proteins RGD4C-HP-RGD4C and RGD4C-HP in large quantities for subsequent immune test studies, 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 immune protection tests and antibody level comparisons, the recombinant protein RGD4C-HP-RGD4C constructed by the present invention can produce higher levels of antibodies against the P region, and after immunizing animals, it can form complete protection against the attack of rabbit hemorrhagic disease virus, thereby providing a new technical product for the prevention and control of rabbit hemorrhagic disease. Moreover, by comparing the antibody levels of RGD4C-HP-RGD4C protein and the entire VP60 protein, it was shown that all the groups immunized with RGD4C-HP-RGD4C protein and VP60 protein survived the challenge with RHDV2, indicating that both can provide complete protection; the RGD4C-HP-RGD4C group and the VP60 vaccine group both produced specific antibodies against the VP60 P region 7 days after immunization. At the same time point, the antibody level of the RGD4C-HP-RGD4C group was higher than that of the VP60 group, and maintained a high level for a period of time, indicating that in terms of the P region antibody level, which plays a key role in vaccine immune protection, the RGD4C-HP-RGD4C protein antibody level was significantly higher than that of the VP60 protein at all time points, reflecting that the antibody level produced by the RGD4C-HP-RGD4C protein is higher and the immune effect is better.

[0109] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that The invention 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 polypeptide containing four cysteines 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 disease virus type 2 capsid protein is shown in SEQ ID NO.2; the P region of the rabbit hemorrhagic disease virus capsid protein includes the P region of the rabbit hemorrhagic disease virus type 2 capsid protein; the amino acid sequence of the P region of the rabbit hemorrhagic disease 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 according to claim 1 or 2.

4. The coding 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 obtained by codon-optimizing the nucleotide sequence shown in SEQ ID NO.

5.

5. The coding gene according to claim 4, characterized in that The nucleotide sequence of the sequence obtained by codon optimization 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 coding gene according to any one of claims 3 to 5 is inserted.

7. A recombinant bacterium or recombinant cell, characterized in that: Comprising the recombinant vector according to claim 6.

8. Use of the fusion protein according to claim 1 or 2, the encoding gene according to any one of claims 3 to 5, the recombinant vector according to claim 6, or the recombinant bacterium or recombinant cell according to claim 7 in at least one of the following: 1) Preparation of drugs for preventing or treating rabbit hemorrhagic disease virus type 2 infection; 2) Application in the preparation of biological products for preventing rabbit hemorrhagic disease virus type 2 infection; 3) Preparation of detection reagents or kits for rabbit hemorrhagic disease virus type 2 capsid protein-specific antibodies.

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

10. A detection reagent or kit for rabbit hemorrhagic disease virus type 2 capsid protein-specific antibodies, characterized in that: The fusion protein according to claim 1 or 2 is used as the coating antigen.

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