Rabbit hemorrhagic disease virus antigen protein as well as preparation method and application thereof

Abstract

The invention relates to the field of veterinary drugs, and particularly provides rabbit hemorrhagic disease virus antigen protein as well as a preparation method and application thereof. The rabbit hemorrhagic disease virus antigen protein comprises RHDV2-VP60 protein, and the amino acid sequence of the RHDV2-VP60 protein is as shown in SEQ ID NO. 4. The antigen protein can induce generation of a relatively high HI antibody.

Description

Technical Field

[0001] The present application relates to the field of veterinary drugs, and specifically provides a rabbit hemorrhagic disease virus antigen protein, a preparation method thereof and an application thereof. Background Art

[0002] Rabbit hemorrhagic disease virus (RHDV) belongs to the genus Leporivirus of the family Caliciviridae. Rabbit hemorrhagic disease caused by this virus is characterized by high infection rate and mortality, seriously hindering the development of the rabbit industry and posing a potential threat to public health safety. RHDV is divided into two genotypes: the classical strain GI.1 (RHDV1) and the variant strain GI.2 (RHDV2). In 2010, GI.2 first broke out in France, and there is a trend that RHDV2 gradually replaces the classical RHDV strain and becomes the main epidemic strain. In 2020, RHDV2 broke out in China. Compared with RHDV1, it has stronger pathogenicity and greater harmfulness. It can infect not only domestic rabbits of different ages but also wild rabbits of different breeds, and the mortality rate can reach about 90%. The similarity between RHDV2 and RHDV1 is low and the difference is large. The vaccines of traditional strains have weak cross-protection effects on RHDV2, causing great harm to the rabbit industry. Therefore, developing a vaccine against RHDV2 is the main way to control its epidemic.

[0003] In view of this, the present application is specifically proposed. Summary of the Invention

[0004] One object of the present application is to provide a rabbit hemorrhagic disease virus antigen protein to achieve effective immune protection against rabbit hemorrhagic disease virus infection; and also provide a preparation method to achieve high expression and high assembly to obtain the antigen protein.

[0005] In order to achieve the above object, the present application specifically adopts the following technical solutions.

[0006] A rabbit hemorrhagic disease virus antigen protein, comprising an RHDV2-VP60 protein, and the amino acid sequence of the RHDV2-VP60 protein is as shown in SEQ ID NO.4.

[0007] Furthermore, the antigen protein further comprises an RHDV1-VP60 protein, and the amino acid sequence of the RHDV1-VP60 protein is as shown in SEQ ID NO.2.

[0008] A preparation method of an antigen protein, rescuing a recombinant baculovirus expressing the antigen protein under the △CC backbone, then culturing the recombinant baculovirus and collecting the supernatant to obtain the antigen protein;

[0009] The △CC backbone is a recombinant baculovirus backbone with deletions of the ChiA and V-Cath genes.

[0010] Biomaterials related to the antigen protein, the biomaterials comprising:

[0011] (1) Nucleic acid, the nucleic acid containing a nucleotide fragment encoding the antigen protein;

[0012] (2) Vector, the vector containing the nucleic acid in (1);

[0013] (3) Recombinant cell, the recombinant cell containing the nucleic acid in (1) or the vector in (2).

[0014] Further, the nucleic acid in (1) contains the nucleotide sequence encoding the RHDV2-VP60 protein as shown in SEQ ID NO.3;

[0015] And / or, the nucleic acid in (1) contains the nucleotide sequence encoding the RHDV1-VP60 protein as shown in SEQ ID NO.1.

[0016] Use of the antigen protein or the biomaterials in the preparation of related products for preventing rabbit hemorrhagic disease virus infection.

[0017] A subunit vaccine against rabbit hemorrhagic disease virus, the subunit vaccine comprising an immunogenic amount of RHDV2-VP60 protein, the amino acid sequence of the RHDV2-VP60 protein being as shown in SEQ ID NO.4.

[0018] Further, the content of the RHDV2-VP60 protein is HA titer ≥ 8log2, preferably 8log2 - 10log2.

[0019] Further, the subunit vaccine further comprises an immunogenic amount of RHDV1-VP60 protein, the amino acid sequence of the RHDV1-VP60 protein being as shown in SEQ ID NO.2;

[0020] Preferably, the content of the RHDV1-VP60 protein is HA titer ≥ 8log2, preferably 8log2 - 10log2.

[0021] Further, the subunit vaccine further contains a pharmaceutically acceptable carrier;

[0022] Preferably, the pharmaceutically acceptable carrier includes at least one of adjuvant, lyoprotectant, immunostimulant, antioxidant, surfactant, colorant, volatile oil, buffer, dispersant, propellant and preservative;

[0023] Preferably, the adjuvant includes one or more of aluminum hydroxide adjuvant, saponin, afoxolaner, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, Gel adjuvant;

[0024] Preferably, the concentration range of the adjuvant is from 5% V / V to 50% V / V, preferably 10% V / V;

[0025] Preferably, the lyoprotectant is selected from sugars, polyols, polymers, surfactants, salts, amines or amino acids;

[0026] Preferably, the immunostimulant includes alpha interferon, beta interferon, gamma interferon, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor or interleukin 2.

[0027] Compared with the prior art, the technical effects of this application are as follows:

[0028] In this application, type 1 and type 2 RHDV are obtained through screening. By expressing and studying the VP60 proteins of the two, it is found that they can induce the production of high levels of HI antibodies. At the same time, through the optimization of the coding nucleotides and the expression system, high expression, high assembly and reduced production costs are achieved. Detailed implementation manners

[0029] Explanation of relevant terms in this application:

[0030] The term "rabbit hemorrhagic disease virus (RHDV)" belongs to the genus Leporivirus in the family Caliciviridae. Rabbit hemorrhagic disease caused by this virus is characterized by a high infection rate and mortality, seriously hindering the development of the rabbit farming industry and posing a potential threat to public health safety. RHDV is divided into two genotypes: the classical strain GI.1 (RHDV1) and the variant strain GI.2 (RHDV2). RHDV belongs to the members of the virus strains in the family Caliciviridae, without an envelope on the surface, about 40 nm in diameter, the nucleocapsid is icosahedral symmetry, and like other virus structures, it is composed of a viral genome and 180 capsid protein subunits polymerized. The viral genome is single-stranded positive-strand RNA, the entire genome includes 7437 nucleotide bases, there is no cap structure at the 5' end of the genome, and there is a short polyadenine tail at the 3' end, encoding two open reading frames ORF1 and ORF2, encoding structural proteins and non-structural proteins respectively. Among them, the VP60 protein is the main protective antigen of the body.

[0031] The term "antigen" refers to a substance that can induce an immune response in an organism, that is, a substance that can be specifically recognized and bound by the antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activate T / B cells, cause them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to the corresponding products in vivo and in vitro.

[0032] As used herein, the term "vaccine" refers to a drug containing the protein antigen of rabbit hemorrhagic disease virus, which can induce, stimulate or enhance the immune response of rabbits against rabbit hemorrhagic disease virus.

[0033] The term "immunizing amount" should be understood as "immunologically effective amount", also known as immunoprotective amount or effective amount for generating an immune response, which is the amount of antigen that can effectively induce an immune response in a recipient, and this amount is sufficient to prevent or ameliorate the signs or symptoms of a disease, including adverse health effects or their complications. The said immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing the signs or symptoms of a disease, including the adverse health outcomes or their complications caused by an infection by a pathogen. Humoral immunity or cell-mediated immunity or both may be induced. The immune response of an animal to an immunogenic antigen can be indirectly evaluated, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly evaluated by monitoring signs or symptoms after challenge with a wild-type strain, and the protective immunity provided by the vaccine can be evaluated by measuring, for example, the clinical signs of the subject such as reduction in mortality, morbidity, temperature values, the overall physiological condition of the subject and overall health and performance. The said immune response may include, but is not limited to, inducing cellular and / or humoral immunity.

[0034] The term "pharmaceutically acceptable carrier" refers to all other components in the vaccine of the present application except the subunit antigen protein of rabbit hemorrhagic disease virus, a carrier or diluent that does not stimulate the organism and does not hinder the biological activity and properties of the compound used, and is preferably an adjuvant.

[0035] The term "adjuvant" may include aluminum hydroxide adjuvant; saponins, such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); water-in-oil emulsions; oil-in-water emulsions; water-in-oil-in-water emulsions; polymers of acrylic acid or methacrylic acid; compounds selected from copolymers of maleic anhydride and alkenyl derivatives.

[0036] The term "emulsion" may in particular be based on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils resulting from olefin oligomerization, such as squalane or squalene oil, in particular isobutene or decene; esters of linear alkyl-containing acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di-(caprylate / caprate), glyceryl tri-(caprylate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearates. The oil is used in combination with an emulsifier in order to form an emulsion. The emulsifier is preferably a non-ionic surfactant, in particular esters of sorbitan, esters of mannitol (such as anhydrous mannitol oleate), esters of aliphatic glycols, esters of polyglycerol, esters of propylene glycol and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic products, especially L121. See "The theory and practical application of adjuvants" by Hunter et al. (Ed. by DES Stewart-Tull, John Wiley and Sons, New York, 1995: 51-94) and "Vaccine" by Todd et al. (1997, 15: 564-570). For example, the SPT emulsion described on page 147 of "Vaccine design, the Subunit and adjuvant approach" by Powell M and Newman M (Plenum Press, 1995) and the MF59 emulsion described on page 183 may be used.

[0037] The term "polymer of acrylic or methacrylic acid" is preferably a cross-linked polymer of acrylic or methacrylic acid, in particular cross-linked with a polyalkenyl ether or polyol of a sugar, these compounds being known as Carbomer (trade name Carbopol) (Phameuropa, 1996, 8(2)). Those skilled in the art may also refer to US Patent US2909462, which describes such acrylic polymers cross-linked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, where at least 3 hydrogen atoms of the hydroxyl groups are replaced by an unsaturated aliphatic radical having at least 2 carbon atoms. Preferred groups are those containing 2-4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated groups themselves may contain other substituents, such as methyl. These products are sold under the name Carbopol, (BF Goodrich, Ohio, USA) being particularly suitable. They are cross-linked with allyl sucrose or with allyl pentaerythritol. Among these may be mentioned Carbopol 974P, 934P and 971P, with Carbopol 971P being most preferably used.

[0038] The term "copolymer of maleic anhydride and alkenyl derivatives" also includes the copolymer of maleic anhydride and ethylene, EMA (Monsanto), these polymers dissolving in water to produce an acidic solution which, upon neutralization, preferably to physiological pH, gives an adjuvant solution into which an immunogenic, immunizing or vaccinal agent per se can be incorporated.

[0039] The term "adjuvant" also includes, but is not limited to, the Ribi Adjuvant System (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin (recombinant or otherwise), cholera toxin, IMS1314, muramyl dipeptide, Gel adjuvant, etc.

[0040] In a preferred embodiment, the adjuvant includes one or more of mineral oil, aluminum hydroxide adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, Montanide ISA 206 or Gel adjuvant.

[0041] The term "lyoprotectant" refers to a component that, in addition to excipients, protects the efficacy of the drug active ingredient during the freeze-drying process and during storage after freeze-drying.

[0042] When referring to rabbit hemorrhagic disease virus infection, the term "prevention" means inhibiting the replication of rabbit hemorrhagic disease virus, inhibiting the transmission of rabbit hemorrhagic disease virus, or preventing rabbit hemorrhagic disease virus from colonizing in its host, as well as alleviating the symptoms of diseases or disorders caused by rabbit hemorrhagic disease virus infection.

[0043] Hereinafter, embodiments of the present application will be described.

[0044] The rabbit hemorrhagic disease virus antigen protein obtained by the applicant includes the RHDV2-VP60 protein, and the amino acid sequence of the RHDV2-VP60 protein is shown in SEQ ID NO.4. Further, the rabbit hemorrhagic disease virus antigen protein also includes the RHDV1-VP60 protein, and the amino acid sequence of the RHDV1-VP60 protein is shown in SEQ ID NO.2.

[0045] The present application also protects biomaterials related to the above antigen proteins, such as nucleotide fragments encoding the above antigen proteins, vectors containing the nucleotide fragments (such as cloning plasmids and expression plasmids, etc.), and recombinant cells containing the nucleotide fragments. These biomaterials can be directly used as biological modules for the production of the antigen proteins of the present application, and have the advantages of rapidity and high efficiency. The nucleotide fragments of the present application can be obtained by primer amplification or can also be synthesized artificially. In some embodiments, the nucleotide sequence encoding the RHDV1-VP60 protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the RHDV2-VP60 protein is shown in SEQ ID NO.3.

[0046] The present application provides a method for efficiently expressing rabbit hemorrhagic disease virus antigen protein in a baculovirus expression system. The method includes: step (1) deleting ChiA and V-Cath from the baculovirus genome to obtain a recombinant baculovirus backbone with ChiA and V-Cath genes deleted (△CC); step (2) rescuing recombinant baculoviruses of RHDV2-VP60 or RHDV1-VP60 under the △CC backbone; and step (3) culturing the recombinant baculoviruses obtained in step (2), harvesting the supernatant, and obtaining the expressed rabbit hemorrhagic disease virus antigen protein.

[0047] The antigen protein provided by the present application can be used to prepare related products for preventing rabbit hemorrhagic disease virus infection, such as vaccines for preventing infection, antibody detection reagents, and so on.

[0048] The present application provides a subunit vaccine for rabbit hemorrhagic disease virus. In addition to the active ingredient being mainly the antigen protein provided by the present application, it can also contain a pharmaceutically acceptable carrier, aiming to meet different requirements in actual applications such as production, transportation, dosage form, administration method, etc.

[0049] In some embodiments, the subunit vaccine contains an immunogenic amount of RHDV2-VP60 protein, and the content of RHDV2-VP60 protein is HA titer ≥ 8log2, preferably 8log2 - 10log2. For example, it can be but is not limited to 8.0log2, 8.1log2, 8.2log2, 8.3log2, 8.4log2, 8.5log2, 8.6log2, 8.7log2, 8.8log2, 8.9log2, 9.0log2, 9.1log2, 9.2log2, 9.3log2, 9.4log2, 9.5log2, 9.6log2, 9.7log2, 9.8log2, 9.9log2 or 10.0log2.

[0050] In some embodiments, the subunit vaccine contains an immunogenic amount of RHDV2-VP60 protein and an immunogenic amount of RHDV1-VP60 protein, and the content of RHDV1-VP60 protein is HA titer ≥ 8log2, preferably 8log2 - 10log2. For example, it can be but is not limited to 8.0log2, 8.1log2, 8.2log2, 8.3log2, 8.4log2, 8.5log2, 8.6log2, 8.7log2, 8.8log2, 8.9log2, 9.0log2, 9.1log2, 9.2log2, 9.3log2, 9.4log2, 9.5log2, 9.6log2, 9.7log2, 9.8log2, 9.9log2 or 10.0log2.

[0051] In some embodiments, the pharmaceutically acceptable carrier includes at least one of an adjuvant, a cryoprotectant, an immunostimulant, an antioxidant, a surfactant, a coloring agent, a volatile oil, a buffer, a dispersant, a propellant, and a preservative.

[0052] In some embodiments, the adjuvant includes one or more of an aluminum hydroxide adjuvant, saponin, afrividin, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and an alkenyl derivative, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, and Gel adjuvant.

[0053] In some embodiments, the concentration range of the adjuvant is from 5% V / V to 50% V / V, preferably 10% V / V.

[0054] In some embodiments, the cryoprotectant is selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids.

[0055] In some embodiments, the immunostimulant includes α-interferon, β-interferon, γ-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin 2.

[0056] The present application will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0057] Example 1: Modification of the baculovirus genome (deletion of ChiA and V-cath genes)

[0058] 1. Experimental materials

[0059] The pFastBacI vector, pKD46 plasmid, and DH10Bac competent cells were purchased from Thermo; the DNA polymerase was purchased from TaKaRa, and the gel extraction kit was purchased from Omega.

[0060] 2. Experimental methods

[0061] 2.1 Deletion of ChiA and V-cath genes

[0062] The ChiA and V-cath genes are adjacent in the viral genome, so they are deleted simultaneously. The gene deletion primers are as follows;

[0063] DCC-F: GTGCGTTTATCGCGTTGAGCAAGTCGCCGTTATCGGCATCAATCTCCGCCAAGTTGACCAGTGCCGTTC;

[0064] DCC-R: GGCGGCGGCGGCGCATTCTGCCTTTGCGGCGGAGCACTGCAACGCGCGTCCTGCTCCTCGGCCACGAAG;

[0065] Replace the ChiA / V-cath gene with the CmR gene using the Red recombination technique.

[0066] 2.2 Identification of ChiA and V-cath gene deletion

[0067] Design verification primers outside of ChiA and V-cath, and the verification primers are as follows:

[0068] DCC-C-F: TCGTGTCTATAATTTCTAATGGTAAAC;

[0069] DCC-C-R: GATGCCTCAATGCTACTAGTAAATC;

[0070] Identify whether the gene deletion is successful.

[0071] 3. Results (Gene deletion identification)

[0072] Pick single colonies for PCR identification, and a target band of about 1300 bp appears, indicating that the baculovirus genome with ChiA and V-cath gene deletion is obtained.

[0073] Example 2 Construction of donor plasmid

[0074] Sequence the VP60 protein genes of the isolated type 1 and type 2 RHDV strains, and optimize the codons of the sequencing results.

[0075] 1. Experimental materials

[0076] Restriction endonucleases were purchased from Thermo; Gel extraction kits were purchased from Omega; Plasmid miniprep kits were purchased from Tiangen; T4 DNA Ligase was purchased from Thermo, and Trans1-T1 competent cells were purchased from TransGen Biotech.

[0077] 2. Experimental methods

[0078] 2.1 Extraction of viral DNA and sequencing of VP60 gene

[0079] Extract the DNA from the isolated virus solution according to the instructions of the DNA extraction kit. Design sequencing primers based on the conserved region sequence of VP60, send the amplified PCR product of VP60 to Genewiz for sequencing, and then optimize the codons of the sequencing results. The sequence is as follows:

[0080] Nucleotide sequence encoding RHDV1-VP60 (SEQ ID NO.1): ATGGAGGGAAAAGCAAGGACAGCGCCACAGGGGGAGGCTGCAGGTACGGCAACCACAGCGTCGGTGCCTGGCACGACAACGGACGGC ATGGACCCTGGTGTTGTGGCAGCGACGTCAGTTGTAACCGCCGAA AATAGTTCCGCGTCTGTTGCGACCGCTGGAATTGGTGGACCACCG CAACAGGTCGACCAGCAGGAAACTTGGCGTACAAACTTCTATTAC AACGATGTATTCACCTGGAGTGTAGCTGACGCACCCGGAAGCATA TTATATACGGTCCAACACAGCCCACAAAATAATCCCTTTACGGCT GTCTTAAGCCAAATGTATGCAGGCTGGGCCGGAGGCATGCAGTTC CGTTTCATCGTTGCCGGATCAGGTGTGTTTGGCGGAAGGCTAGTC GCGGCTGTTATTCCACCAGGTATCGAAATAGGGCCGGGTCTAGAA GTTCGGCAGTTTCCTCATGTAGTTATAGACGCTCGATCGTTAGAG CCTGTGACCATAACGATGCCCGATTTGAGACCCAACATGTATCAT CCCACCGGAGACCCAGGCCTCGTACCGACTCTTGTTCTAAGTGTA TATAATAATTTAATAAACCCCTTTGGGGGTTCGACTAACGCGATA CAAGTTACCGTTGAGACAAGGCCCTCTGATGATTTCGAATTCGTC ATGATCCGAGCTCCCTCAAGTAAGACAGTCGATAGTATCAGTCCA GCCGGGCTACTCACCACACCGGTCTTGACTGGCGTAGGTAACGAT AATCGCTGGAATGGCCAGATAGTAGGACTCCAACCAGTGCCAGG CGGCTTTTCAACTTGCAACAGACACTGGAACCTGAACGGTTCGAC TTACGGCTGGAGCTCTCCCAGATTTGCAGACATCGACCACAGGCG TGGTTCTGCATCCTATTCAGGGAATAACTCCACAAACGTGCTGCA ATTTTGGTACGCAAACGCAGGTTCCGCTATTGACAATCCAATATC CCAGGTGGCCCCCGACGGGTTCCCGGACATGAGCTTCGTGCCGTT TAACTCGCCCAATATTCCGACAGCCGGGTGGGTTGGCTTCGGGGG GATCTGGAACTCCAATAACGGGGCACCTGCTGCTACGACGGTCCA AGCATACGAGCTGGGGTTTGCTACAGGGGCCCCTAACAATCTTCA GCCTACTACAAATACCTCTGGGGCACAGACAGTCGCTAAGAGCAT TTACGCCGTGGTGACGGGCACCAACCAAAATCCAACTGGACTGTT CGTGATGGCATCTGGGGTCATCTCAACACCTAATGCCTCAGCCGT TACCTACACGCCGCAACCGGACCGCATCGTAACGACTCCGGGCAC GCCTGCGGCAGCGCCTGTGGGCAAAAACACTCCGATTATGTTTGC GAGCGTAGTGCGACGCACAGGAGATGTTAATGCTGCCGCGGGGT CGACCAATGGGACTCAATATGGTACGGGATCGCAACCCCTCCCTG TCACTATAGGATTGTCCCTCAACAATTATTCGTCCGCGTTGATGCC GGGACAGTTCTTCGTCTGGCAGCTTACTTTTGCGAGCGGTTTCATG GAGATTGGTTTAAGTGTCGATGGGTATTTTTACGCTGGTACCGGA GCCAGTACCACTCTTATTGATTTGACCGAACTAATCGATGTACGG CCTGTCGGACCTCGGCCATCAAAATCTACGCTGGTTTTTAACCTTGGTGGTACTACAAATGGATTTTCTTACGTATGA。

[0081]

[0082] The amino acid sequence of the optimized VP60 remains unchanged and is as follows:

[0083] RHDV1-VP60 (SEQ ID NO.2): MEGKARTAPQGEAAGTATTASVPGTTTDGMDPGVVAATSVVTAEN SSASVATAGIGGPPQQVDQQETWRTNFYYNDVFTWSVADAPGSILY TVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFGGRLVAA VIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTGDPG LVPTLVLSVYNNLINPFGGSTNAIQVTVETRPSDDFEFVMIRAPSSKT VDSISPAGLLTTPVLTGVGNDNRWNGQIVGLQPVPGGFSTCNRHWN LNGSTYGWSSPRFADIDHRRGSASYSGNNSTNVLQFWYANAGSAID NPISQVAPDGFPDMSFVPFNSPNIPTAGWVGFGGIWNSNNGAPAATT VQAYELGFATGAPNNLQPTTNTSGAQTVAKSIYAVVTGTNQNPTGL FVMASGVISTPNASAVTYTPQPDRIVTTPGTPAAAPVGKNTPIMFASV VRRTGDVNAAAGSTNGTQYGTGSQPLPVTIGLSLNNYSSALMPGQFF VWQLTFASGFMEIGLSVDGYFYAGTGASTTLIDLTELIDVRPVGPRPSKSTLVFNLGGTTNGFSYV。

[0084] RHDV2-VP60 (SEQ ID NO.4): MEGKARATPQGETAGTATTASVPGTTTDGMDPGVVATTSVVTTENASTSIATAGIGGPPQQMDQQETWRTNFYYNDVFTWSVADAPGNILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTGNPGLVPTLVLSVYNNLINPFGGSTSAIQVTVETRPSEDFEFVMIRAPSSKTVDSISPADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFAAIDHDRGNASFPGSSSSNVLELWYASAGSAADNPISQIAPDGFPDMSFVPFSGTTIPTAGWVGFGGIWNSSNGAPYVTTMQAYELGFATGVPSNPKPTTTTSGAQIVAKSIYGVANGINQTTAGLFVMASGVISTPNSSATTYTPQPNRIVNAPGTPAAAPIGKNTPIMFASVVRRTGDINAEAGSTNGTQYGAGSQPLPVTIGLSLNNYSSALMPGQFFVWQLNFASGFMELGLSVDGYFYAGTGALATLIDLSDLVDIRPVGPRPSTSTLVYNLGGTTNGFSYV。

[0085] The optimized gene was synthesized by Genewiz (Suzhou) Inc. and cloned into the PUC-57 vector respectively.

[0086] 2.2 Construction of recombinant donor plasmids

[0087] 2.2.1 Construction of RHDV2-VP60 recombinant donor plasmid

[0088] The plasmid containing the RHDV2-VP60 gene and the pFastBacI vector were double digested with BamHI and Hind III, and the target fragment and the vector were recovered. They were ligated at 22 °C for 1 h, and the ligation product was transformed into competent Trans1-T1 cells and cultured at 37 °C for 12 h. Single colonies were picked and inoculated into 3 mL of LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37 °C with shaking at 200 r / min for 12 h. The plasmid was extracted and identified by double digestion with BamH I and Hind III, and the correctly identified pFastBacI-RHDV2-VP60 plasmid was sent to Genewiz for sequencing.

[0089] 2.2.2 Construction of RHDV1-VP60 recombinant donor plasmid

[0090] The plasmid containing the RHDV1-VP60 gene and the pFastBacI vector were double digested with BamHI and HindIII, and the target fragment and the vector were recovered. They were ligated at 22 °C for 1 h, and the ligation product was transformed into competent Trans1-T1 cells and cultured at 37 °C for 12 h. Single colonies were picked and inoculated into 3 mL of LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37 °C with shaking at 200 r / min for 12 h. The plasmid was extracted and identified by double digestion with BamH I and Hind III. The correctly identified pFastBacI-RHDV1-VP60 plasmid was sent to Genewiz for sequencing.

[0091] Results: Identification of pFastBacI-RHDV2-VP60 recombinant donor plasmid: Specific bands of about 1800 bp were visible by digestion identification, while no target band was seen in the empty vector. Therefore, the recombinant donor plasmid containing the RHDV2-VP60 gene was successfully constructed and named pFBI-RHDV2-VP60. Identification of pFastBacI-RHDV1-VP60 recombinant donor plasmid: Specific bands of about 1800 bp were visible by digestion identification, while no target band was seen in the empty vector. Therefore, the recombinant donor plasmid containing the RHDV1-VP60 gene was successfully constructed and named pFBI-RHDV1-VP60.

[0092] Example 3 Obtaining of RHDV2-vp60 and RHDV1-VP60 recombinant Bacmid

[0093] 1. Experimental materials

[0094] The plasmid miniprep kit was purchased from Tiangen; DH10Bac competent cells were purchased from Thermo, and DH10Bac(△CC) competent cells were constructed in Example 1.

[0095] 2. Experimental methods

[0096] 2.1 Respectively transform the recombinant donor plasmids pFBI-RHDV2-VP60 and pFBI-RHDV1-VP60 constructed in Example 2 into DH10Bac (normal backbone) and DH10Bac (△CC) competent cells, culture them with shaking at 220 r / min at 37 °C for 4 h, take 100 μL of the bacterial solution and spread it on an LB solid medium containing three antibiotics, kanamycin, tetracycline, and gentamicin, as well as IPTG and X-gal;

[0097] 2.2 Incubate at 37 °C for 48 h. When the blue and white colonies are obvious, pick the white single colonies for streaking in zones and culture at 37 °C for 24 h;

[0098] 2.3 Pick the white single colonies for colony PCR identification. Transfer the correctly identified bacterial solution to an LB liquid medium containing three antibiotics, kanamycin, tetracycline, and gentamicin, at a ratio of 1:100, culture with shaking at 220 r / min at 37 °C for 16 h, and extract the recombinant Bacmid;

[0099] 3. Experimental Results

[0100] Specific target bands were found by amplification with pH primers, while no obvious target bands were observed in the negative control, indicating that the recombinant Bacmids of RHDV2-VP60 and RHDV1-VP60 under the normal backbone and gene-deleted (△CC) backbone were successfully obtained.

[0101] Example 4 Rescue of Recombinant Baculoviruses of RHDV2-VP60 and RHDV1-VP60 under the Normal Backbone

[0102] 1. Experimental Materials

[0103] Cellfectin II Reagent transfection reagent was purchased from Thermo, the DNA extraction kit was purchased from Qiagen, and the 2×Taq premix was purchased from Tiangen;

[0104] 2. Experimental Methods

[0105] 2.1 Seed cells, seed 3×10 6 cells in each T25 cell flask;

[0106] 2.2 Transfect the recombinant Bacmids of RHDV2-VP60 and RHDV1-VP60 under the normal backbone in Example 3 into the cells according to the transfection reagent instructions. At the same time, set a normal cell control, observe the cytopathic effect every day. When the cytopathic effect is obvious, collect the supernatant, which is the P1 generation;

[0107] 2.3 Inoculate the harvested P1 generation virus at an MOI = 1 into a flask containing 3×10 6In a T25 cell flask with cells, when the lesion reaches about 80%, collect the supernatant as the P2 generation;

[0108] 2.4 Extract the recombinant nucleic acid of the P2 generation and perform PCR identification using specific primers.

[0109] 3. Results

[0110] Perform PCR identification on the nucleic acid of the P2 generation and find a target band with the same size as the target band, indicating that the recombinant baculoviruses of RHDV2-VP60 and RHDV1-VP60 under the normal backbone have been successfully rescued, named rAC-RHDV2-VP60 and rAC-RHDV1-VP60.

[0111] Example 5 Rescue of recombinant baculoviruses of RHDV2-VP60 and RHDV1-VP60 under the △CC backbone

[0112] 1 Experimental materials

[0113] Cellfectin II Reagent transfection reagent was purchased from Thermo, DNA extraction kit was purchased from Qiagen, and 2×Taq premix was purchased from Tiangen;

[0114] 2 Experimental methods

[0115] 2.1 Seed cells, seed 3×10 6 cells in each T25 cell flask;

[0116] 2.2 Transfect the gene-deleted RHDV2-VP60 and RHDV1-VP60 recombinant Bacmids in Example 3 into cells according to the transfection reagent instructions, and set up a normal cell control at the same time. Observe the lesions every day. When the lesions are obvious, collect the supernatant as the P1 generation;

[0117] 2.3 Inoculate the harvested P1 generation virus into a T25 cell flask containing 3×10 6 cells at an MOI = 1. When the lesion reaches about 80%, collect the supernatant as the P2 generation;

[0118] 2.4 Extract the recombinant nucleic acid of the P2 generation and perform PCR identification using specific primers

[0119] 3. Results

[0120] Perform PCR identification on the nucleic acid of the P2 generation and find a target band with the same size as the target band, indicating that the recombinant baculoviruses of RHDV2-VP60 and RHDV1-VP60 in the △CC state have been successfully rescued.

[0121] Example 6 Expression and Identification of RHDV2-VP60 and RHDV1-VP60 Proteins under Normal Skeleton

[0122] 1.1 Identification of Recombinant Proteins After harvesting the P2 generation of recombinant baculoviruses under normal skeleton, SDS-PAGE identification was performed on the extracellular supernatant, cell lysate supernatant, and cell lysate precipitate.

[0123] 1.2 Expression of Recombinant Proteins The harvested P2 generation of recombinant baculoviruses was used to infect sf9 cells at an MOI of 1. When the cell viability reached about 80%, the cell supernatant was harvested as the P3 generation of virus. The P3 generation of virus was inoculated into 100 mL of 2.3×10 6 cells. The extracellular supernatant and cells were collected, the mixture was frozen and thawed once, centrifuged at 5000 rpm for 10 min, the supernatant was collected to measure HA, observed under an electron microscope and identified by SDS-PAGE, and the supernatant was used for vaccine preparation;

[0124] 2 Results: Identification of the P2 generation found that protein expression could be observed in the cell culture supernatant, cell lysate supernatant, and precipitate; the HA of the proteins expressed in the P3 generation was measured. The titer of RHDV2-VP60 could reach 13 log2, and the titer of RHDV1-VP60 was 13 log2. Uniform virus-like particles could be observed under an electron microscope, and specific protein bands could be observed in the supernatant after centrifugation by SDS-PAGE.

[0125] Example 7 Expression and Identification of RHDV2-VP60 and RHDV1-VP60 Proteins under △CC Skeleton

[0126] 1.1 Identification of Recombinant Proteins After harvesting the P2 generation of recombinant baculoviruses under △CC skeleton, SDS-PAGE identification was performed on the extracellular supernatant, cell lysate supernatant, and cell lysate precipitate.

[0127] 1.2 Expression of Recombinant Proteins The harvested P2 generation of recombinant baculoviruses was used to infect sf9 cells at an MOI of 1. When the cell viability reached about 80%, the cell supernatant was harvested as the P3 generation of virus. The P3 generation of virus was inoculated into 100 mL of 2.3×10 6 cells. The extracellular supernatant and cells were collected, the mixture was frozen and thawed once, centrifuged at 5000 rpm for 10 min, the supernatant was collected to measure HA, observed under an electron microscope and identified by SDS-PAGE, and the supernatant was used for vaccine preparation;

[0128] 2 Results: Identification of the P2 generation found that protein expression could be observed in the cell culture supernatant, cell lysate supernatant, and precipitate; for the protein expressed by the P3 generation, the HA and RHDV2-VP60 titers could reach 16 log2, which was 3 titers higher than that of the normal backbone, and the RHDV1-VP60 titer could reach 16 log2, which was 3 titers higher than that of the normal backbone. Uniform virus-like particles could be observed under the electron microscope, and specific protein bands could be observed in the supernatant after centrifugation by SDS-PAGE.

[0129] Example 8 Preparation of a Inactivated Vaccine of Rabbit Hemorrhagic Disease Virus Type 2 Baculovirus Vector

[0130] The inactivated antigen of RHDV2-VP60 protein of rabbit hemorrhagic disease virus type 2 prepared in Example 7 was slowly added to the adjuvant, and stirred continuously during the addition process to mix evenly. The adjuvant applicable to this application can be the adjuvants well-known to those skilled in the art. In this application, the selected adjuvant is aluminum hydroxide gel adjuvant. The specific vaccine formulation is shown in Table 1.

[0131] Table 1 Ratio of Inactivated Vaccine of Rabbit Hemorrhagic Disease Virus Type 2 Baculovirus Vector

[0132] Example 9 Immunogenicity Evaluation of Inactivated Vaccine of Rabbit Hemorrhagic Disease Virus Type 2 Baculovirus Vector

[0133] Twenty healthy and susceptible antibody-negative rabbits weighing 1.5 - 3.0 kg were randomly divided into 4 groups, with 5 rabbits in each group. Among them, the first to third groups were subcutaneously inoculated with 1 ml / rabbit of inactivated vaccines 1, 2, and 3 respectively, and the fourth group was subcutaneously injected with 1 ml of normal saline as a blank control. Twenty-eight days after immunization, blood was collected respectively to detect the serum HI antibody titer. The specific results are shown in Table 2.

[0134] Table 2 Results of Immunogenicity Test of Inactivated Vaccine of Rabbit Hemorrhagic Disease Virus Type 2 Baculovirus Vector Group Serum RHDV2 HI antibody titer 1 1:16、1:64、1:16、1:32、1:16 2 1:64、1:32、1:128、1:32、1:64 3 1:256、1:64、1:128、1:64、1:128 4 ＜1:4、＜1:4、＜1:4、＜1:4、＜1:4

[0135] The results showed that the inactivated vaccine of rabbit hemorrhagic disease virus type 2 baculovirus vector had a good immune effect and could induce the production of serum HI antibodies.

[0136] Example 10 Preparation of a Bivalent Inactivated Vaccine of Rabbit Hemorrhagic Disease Virus Baculovirus Vector

[0137] The inactivated antigens of RHDV1-VP60 of rabbit hemorrhagic disease virus type 1 and RHDV2-VP60 of rabbit hemorrhagic disease virus type 2 prepared in Example 7 were slowly added to the adjuvant, and stirred continuously during the addition process to mix evenly. The adjuvant applicable to this application can be the adjuvants well-known to those skilled in the art. In this application, the selected adjuvant is aluminum hydroxide gel adjuvant. The specific vaccine formulation is shown in Table 3.

[0138] Table 3 Ratio of the Bivalent Inactivated Vaccine of Rabbit Hemorrhagic Disease Virus Recombinant Baculovirus Vector

[0139] Example 11 Immunogenicity Evaluation of the Bivalent Inactivated Vaccine of Rabbit Hemorrhagic Disease Virus Recombinant Baculovirus Vector

[0140] Twenty healthy and susceptible rabbits with negative antibodies and a body weight of 1.5 - 3.0 kg were randomly divided into 4 groups, with 5 rabbits in each group. Among them, groups 5 - 7 were subcutaneously inoculated with 1 ml / rabbit of inactivated vaccines 4, 5, and 6 respectively, and group 8 was subcutaneously injected with 1 ml of normal saline as a blank control. Twenty-eight days after immunization, blood samples were collected to detect the serum HI antibody titer. The specific results are shown in Table 4.

[0141] Table 4 Results of the Immunogenicity Test of the Bivalent Inactivated Vaccine of Rabbit Hemorrhagic Disease Virus Recombinant Baculovirus Vector

[0142] The results showed that the bivalent inactivated vaccine of rabbit hemorrhagic disease virus recombinant baculovirus vector had good immunization effects and could induce the production of serum HI antibodies against RHDV1 and RHDV2.

[0143] Unless otherwise defined, all technical and scientific terms used throughout this application have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. In case of inconsistency, the meaning stated in the full text of this application or the meaning derived from the content recorded in the full text of this application shall prevail. In addition, the terms used in this specification are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

Claims

1. A rabbit hemorrhagic disease virus antigen protein, characterized in that, It includes the RHDV2-VP60 protein, and the amino acid sequence of the RHDV2-VP60 protein is as shown in SEQ ID NO.

4.

2. The antigen protein according to claim 1, characterized in that, It also includes the RHDV1-VP60 protein, and the amino acid sequence of the RHDV1-VP60 protein is as shown in SEQ ID NO.

2.

3. A method for preparing the antigen protein according to claim 1 or 2, characterized in that, Rescue the recombinant baculovirus expressing the antigen protein under the △CC backbone, then culture the recombinant baculovirus and collect the supernatant to obtain the antigen protein; The △CC backbone is a recombinant baculovirus backbone with deletions of the ChiA and V-Cath genes.

4. A biological material related to the antigen protein according to claim 1 or 2, characterized in that, The biological material includes: (1) Nucleic acid, which contains a nucleotide fragment encoding the antigen protein; (2) Vector, which contains the nucleic acid in (1); (3) Recombinant cell, which contains the nucleic acid in (1) or the vector in (2).

5. The biological material according to claim 4, characterized in that, The nucleic acid in (1) contains the nucleotide sequence encoding the RHDV2-VP60 protein as shown in SEQ ID NO.3; and / or, the nucleotide sequence encoding the RHDV1-VP60 protein as shown in SEQ ID NO.

1.

6. Use of the antigen protein according to claim 1 or 2 or the biological material according to claim 4 or 5 in the preparation of related products for preventing rabbit hemorrhagic disease virus infection.

7. A rabbit hemorrhagic disease virus subunit vaccine, characterized in that, The subunit vaccine includes an immunogenic amount of the RHDV2-VP60 protein, and the amino acid sequence of the RHDV2-VP60 protein is as shown in SEQ ID NO.

4.

8. The subunit vaccine according to claim 7, characterized in that, The content of the RHDV2-VP60 protein is such that the HA titer ≥ 8log2, preferably 8log2 - 10log2.

9. The subunit vaccine according to claim 7, characterized in that, The subunit vaccine also includes an immunogenic amount of the RHDV1-VP60 protein, and the amino acid sequence of the RHDV1-VP60 protein is as shown in SEQ ID NO.2; Preferably, the content of the RHDV1-VP60 protein is such that the HA titer ≥ 8log2, preferably 8log2 - 10log2.

10. The subunit vaccine according to any one of claims 7 to 9, characterized in that, The subunit vaccine also contains a pharmaceutically acceptable carrier; Preferably, the pharmaceutically acceptable carrier includes at least one of adjuvants, lyoprotectants, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives; Preferably, the adjuvant includes one or more of aluminum hydroxide adjuvant, saponin, afloxacillin, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, Gel adjuvant; Preferably, the concentration range of the adjuvant is from 5% V / V to 50% V / V, preferably 10% V / V; Preferably, the lyoprotectant is selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids; Preferably, the immunostimulant includes α-interferon, β-interferon, γ-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin 2.

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