Self-assembled fusion protein based on grass carp ferritin and application of self-assembled fusion protein

Abstract

The invention relates to the technical field of biology, in particular to self-assembled fusion protein based on grass carp ferritin and application of the self-assembled fusion protein. The fusion protein is obtained by connecting grass carp ferritin and II-type grass carp reovirus outer capsid protein through a connecting peptide, and the II-type grass carp reovirus outer capsid protein is II-type grass carp reovirus outer capsid protein VP4 or II-type grass carp reovirus outer capsid protein VP56. The invention further provides application of the self-assembled fusion protein in preparation of vaccines for preventing aquatic diseases. According to the invention, grass carp ferritin and II type grass carp reovirus outer capsid protein are fused through a connecting peptide, and spherical nanoparticles are formed by using the natural self-assembly characteristic of grass carp ferritin, so that high stability of ferritin is retained, high-density display of antigen is realized, and immunogenicity is significantly enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a self-assembly fusion protein based on grass carp ferritin and applications thereof. Background Art

[0002] Grass carp is one of the major freshwater fish species farmed. With the increasing intensiveness of grass carp farming, various diseases have become increasingly common, seriously impacting the high-quality development of the grass carp aquaculture industry. Among them, grass carp hemorrhagic disease, caused by grass carp reovirus (GCRV), has a mortality rate of up to 90%, causing significant economic losses to farmers after an outbreak. Based on strain isolation and sequencing results, GCRV is divided into three genotypes, of which type II GCRV (CGRV-II) is currently the predominant strain.

[0003] In recent years, ferritin has been used as a nanomaterial in many fields. Ferritin is a cage-like protein that is widely present in organisms. Its protein shell is self-assembled from 24 ferritin subunits, where each three subunits form a trimer subunit. The N-terminus of ferritin extends to the outer surface, making it easy to genetically modify and fuse protein peptides. In recent years, research on ferritin has mainly focused on: (1) Modifying the inner surface of ferritin to encapsulate specific drugs or promote the synthesis of nanomaterials; (2) Modifying the outer surface of ferritin to connect it with PEG or antibodies to expand new functions; (3) Modifying the outer surface of ferritin or the contact surface between subunits to control the self-assembly of ferritin. Summary of the Invention

[0004] The present invention aims to provide a self-assembling fusion protein that utilizes the natural self-assembly properties of grass carp ferritin to form spherical nanoparticles, which can retain the high stability of ferritin and significantly enhance the immunogenicity.

[0005] To achieve the above object, the technical solution provided by the present invention is a self-assembling fusion protein based on grass carp ferritin, wherein the self-assembling fusion protein is obtained by connecting grass carp ferritin and type II grass carp reovirus outer capsid protein through a connecting peptide;

[0006] The amino acid sequence of the grass carp ferritin is shown in SEQ ID NO: 1;

[0007] The type II grass carp reovirus outer capsid protein is type II grass carp reovirus outer capsid protein VP4 or type II grass carp reovirus outer capsid protein VP56;

[0008] The amino acid sequence of the type II grass carp reovirus outer capsid protein VP4 is shown in SEQ ID NO: 2. When the type II grass carp reovirus outer capsid protein is type II grass carp reovirus outer capsid protein VP4, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 4, and the nucleotide sequence of the encoding gene thereof is shown in SEQ ID NO: 7.

[0009] The amino acid sequence of the type II grass carp reovirus outer capsid protein VP56 is shown in SEQ ID NO: 3; when the type II grass carp reovirus outer capsid protein is type II grass carp reovirus outer capsid protein VP56, the amino acid of the fusion protein is shown in SEQ ID NO: 5, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 6.

[0010] In the above-mentioned self-assembling fusion protein based on grass carp ferritin, the connecting peptide is (G4S)3.

[0011] On the other hand, the present invention also provides the use of the self-assembling fusion protein in preparing a vaccine for preventing aquatic diseases, wherein the aquatic diseases are caused by type II grass carp reovirus.

[0012] On the other hand, the present invention also provides the above-mentioned vaccine for preventing aquatic diseases, wherein the active ingredient of the vaccine is a self-assembling fusion protein, the amino acid sequence of the self-assembling fusion protein is shown in SEQ ID NO:4, and the nucleotide sequence of the gene encoding the self-assembling fusion protein is shown in SEQ ID NO:7.

[0013] In another aspect, the present invention also provides a vaccine for preventing aquatic diseases, wherein the active ingredient of the vaccine is a self-assembling fusion protein, the amino acid sequence of the self-assembling fusion protein is shown in SEQ ID NO: 5, and the nucleotide sequence of the gene encoding the self-assembling fusion protein is shown in SEQ ID NO: 6.

[0014] The above-mentioned vaccine for preventing aquatic diseases, wherein the aquatic disease is grass carp hemorrhagic disease.

[0015] The above-mentioned vaccine for preventing aquatic diseases is implemented by immersion immunization.

[0016] The following are the serial numbers involved in the present invention:

[0017] Grass carp ferritin amino acid sequence (SEQ ID NO: 1)

[0018] MDSQIRQNYDRDCEALINKMVNLELYAGYTYTSMAHYFKRDDVALPGFAKFFKKNSEEEREHAEKFMEFQNKRGGRIVLQDVKKPERDEWDNGLVAMQCALQLEKNVNQALLDLHKAASEKGDPHLCDFLETHYLNEQVEAIKKLGDHITNLSKMDAGKNRMAEYLFDKHTLDGDS

[0019] Amino acid sequence of VP4-3 (SEQ ID NO:2)

[0020] MVGTTSKTTYYLSMGNSGGGDLMIDLKRLPACGLEYSLRGIPIIYDTNLTAAKLAKVTPALLMLQTAKPLSAEITAADIQAITPLVVGTDKLNTLVTTGFGNIRNITDFSMSAIWEPETVSAAGNYYLWPTVIGDASMTSDWGTISTSLANGRLRVAPLDLTHALHKGYVVESI

[0021] Amino acid sequence of VP56-2 (SEQ ID NO:3)

[0022] DATGIDAVDVPLYIYIVDDQRRLAMATGDGLFVEDRKLNGYDVRSFPPIAVAKYNDILSFSLSSAPPLDIVDGKLAVSTTSRLFITSGKLDTNSYTGSSSVDISGATAEKTVSVR

[0023] Amino acid sequence of VP4-3-Fn (SEQ ID NO:4)

[0024] MVGTTSKTTYYLSMGNSGGGDLMIDLKRLPACGLEYSLRGIPIIYDTNLTAAKLAKVTPALLMLQTAKPLSAEITAADIQAITPLVVGTDKLNTLVTTGFGNIRNITDFSMSAIWEPETVSAAGNYYLWPTVIGDASMTSDWGTISTSLANGRLRVAPLDLTHALHKGYVVESIGGGGSGGGGSGGGGSMDSQIRQNYDRDCEALINKMVNLELYAGYTYTSMAHYFKRDDVALPGFAKFFKKNSEEEREHAEKFMEFQNKRGGRIVLQDVKKPERDEWDNGLVAMQCALQLEKNVNQALLDLHKAASEKGDPHLCDFLETHYLNEQVEAIKKLGDHITNLSKMDAGKNRMAEYLFDKHTLDGDS

[0025] Amino acid sequence of VP56-2-Fn (SEQ ID NO:5)

[0026] DATGIDAVDVPLYIYIVDDQRRLAMATGDGLFVEDRKLNGYDVRSFPPIAVAKYNDILSFSLSSAPPLDIVDGKLAVSTTSRLFITSGKLDTNSYTGSSSVDISGATAEKTVSVRGGGGSGGGGSGGGGSMDSQIRQNYDRDCEALINKMVNLELYAGYTYTSMAHYFKRDDVALPGFAKFFKKNSEEEREHAEKFMEFQNKRGGRIVLQDVKKPERDEWDNGLVAMQCALQLEKNVNQALLDLHKAASEKGDPHLCDFLETHYLNEQVEAIKKLGDHITNLSKMDAGKNRMAEYLFDKHTLDGDSNucleotide sequence of VP56-2-Fn (SEQ ID NO:6)

[0027] GACGCGACGGGAATAGATGCAGTGGACGTACCACTATACATATATATAGTCGATGACCAGAGGCGCCTCGCAATGGCCACGGGAGACGGGTTATTTGTGGAAGATCGGAAGTTAAACGGGTACGATGTGCGTAGCTTTCCACCTATAGCCGTCGCTAAATATAACGACATATTATCGTTCTCGCTGAGCTCAGCCCCCCCACTGGACATAGTTGATGGTAAGTTGGCTGTCAGCACAACTAGCAGGCTATTCATCACCAGTGGAAAATTAGACACTAATTCATATACCGGTTCATCGAGTGTGGACATATCTGGAGCGACGGCAGAAAAGACGGTGAGCGTTAGAGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCGATGGATTCTCAGATTCGCCAGAACTACGACCGCGACTGCGAGGCTTTGATCAACAAGATGGTGAATCTGGAGCTTTATGCTGGCTACACTTACACTTCCATGGCTCACTATTTTAAACGGGATGATGTGGCTCTTCCCGGTTTTGCCAAGTTCTTCAAGAAGAACAGCGAGGAGGAGCGCGAGCATGCCGAGAAATTCATGGAGTTCCAGAACAAGAGGGGCGGACGCATTGTCCTTCAGGACGTAAAGAAACCTGAGCGCGATGAGTGGGACAATGGGCTGGTTGCTATGCAGTGCGCTCTTCAGCTGGAGAAGAACGTCAACCAGGCTCTGCTGGACCTGCATAAGGCTGCATCTGAGAAGGGAGACCCTCATCTGTGTGACTTCCTGGAGACTCACTACCTGAATGAGCAGGTTGAGGCCATCAAGAAGCTTGGTGACCACATCACCAACCTTTCCAAGATGGATGCTGGCAAAAACAGGATGGCGGAGTACTTGTTTGACAAGCACACCCTGGATGGAGACAGC

[0028] VP4-3-Fn nucleotide sequence (SEQ ID NO:7)

[0029]

[0030] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0031] 1. The present invention fuses grass carp ferritin with the outer capsid protein of type II grass carp reovirus through a connecting peptide, and utilizes the natural self-assembly properties of grass carp ferritin to form spherical nanoparticles, which not only retains the high stability of ferritin but also achieves high-density display of antigens, significantly enhancing immunogenicity.

[0032] 2. The vaccine provided by the present invention can be administered by immersion immunization, breaking through the limitations of traditional injection vaccines on aquatic animals. It can efficiently deliver antigens through the gills and body mucosa, which not only reduces the immune stress response but also saves labor costs. It is particularly suitable for large-scale breeding scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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 or the description of the prior art. 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.

[0034] Figure 1 The following are the double enzyme digestion identification images of prokaryotic expression vectors, and they are the double enzyme digestion identification results of pET-32a-VP4-3, pET-32a-VP56-2, pET-32a-Fn, pET-32a-VP4-3-Fn and pET-32a-VP56-2-Fn respectively; among them: lane M is the DL2000 plus DNA relative molecular mass standard; lane 1 is the double enzyme digestion plasmid; lane 2 is the undigested plasmid.

[0035] Figure 2 The figures are SDS-PAGE analysis charts of recombinant protein expression and purification, and are the analysis results of recombinant proteins VP4-3, VP56-2, Fn, VP4-3-Fn and VP56-2-Fn respectively; lane M is the relative molecular mass of the protein; lane 1 is the bacterial protein in the supernatant of BL21 containing an empty vector induced by IPTG; lane 2: the bacterial protein in the supernatant of BL21 containing a recombinant expression vector induced by IPTG; lane 3: the purified recombinant protein.

[0036] Figure 3 The following are Western Blot identification images of the purified recombinant proteins, which are the identification results of recombinant proteins VP4-3, VP56-2, Fn, VP4-3-Fn and VP56-2-Fn respectively; lane M is the relative molecular mass of the protein; lane 1 is the corresponding recombinant protein.

[0037] Figure 4 This is the transmission electron microscopy identification result of recombinant proteins Fn, VP4-3-Fn and VP56-2-Fn.

[0038] Figure 5 Analysis of serum antibody titers in grass carp after immunization; data are the average of three tests and expressed as mean ± standard error. Different letters indicate significant differences (p < 0.05).

[0039] Figure 6 This is the survival rate data of each group 28 days after infection. DETAILED DESCRIPTION

[0040] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental and detection methods described in each example are conventional methods unless otherwise specified. The reagents and materials described are commercially available unless otherwise specified. The percentages in the following examples are percentages by mass unless otherwise specified. The ratios in the following examples are ratios by mass unless otherwise specified.

[0041] Example 1

[0042] This embodiment provides two vaccines for preventing aquatic diseases, namely a vaccine comprising a self-assembled fusion protein of grass carp ferritin linked to the outer capsid protein VP4 of type II grass carp reovirus (VP4-3-Fn), and a vaccine comprising a self-assembled fusion protein of grass carp ferritin linked to the outer capsid protein VP56 of type II grass carp reovirus (VP56-2-Fn).

[0043] 1 Materials and Methods

[0044] 1.1 Materials

[0045] 1.1.1 Plasmids and strains

[0046] The recombinant expression plasmids pET-32a-VP4-3, pET-32a-VP56-2, pET-32a-Fn, pET-32a-VP4-3-Fn and pET-32a-VP56-2-Fn and the corresponding E. coli BL-21; the prokaryotic expression strains are all preserved by the Aquatic Disease Control and Disease Resistance Biological Breeding Innovation Team of the College of Animal Science and Technology, Northwest Agriculture and Forestry University.

[0047] 1.1.2 Reagents

[0048] Sodium chloride, anhydrous ethanol, glycerol, urea, and imidazole were purchased from Sinopharm Chemical Reagent Co., Ltd.; TMB enzyme colorimetric kit, dialysis bag, and ELISA stop buffer were purchased from Beijing Solebold Technology Co., Ltd.; BCA protein concentration determination kit, 12% PAGE color gel rapid preparation kit, Tris-glycine-SDS rapid electrophoresis buffer, and rapid transfer buffer were purchased from Xi'an Aorui Jingchuang Biotechnology Co., Ltd.; mouse anti-6× histidine (Histidine, His) tag monoclonal antibody, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG monoclonal antibody, His-tag protein purification kit, EcoR I restriction enzyme, and Xho I restriction enzyme were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; protein marker was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; tryptone and yeast extract powder were purchased from Sigma; IPTG and ampicillin were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0049] 1.1.3 Test instruments

[0050] High-pressure steam autoclave FD100, Xiamen Zhiwei Instrument Co., Ltd.; electrophoresis apparatus DYY-11B, Beijing Liuyi Biotechnology Co., Ltd.; precision electronic balance ALC-1100.2, Beijing Sartorius Instrument Systems Co., Ltd.; gel imaging instrument Beijing Sensi Saizhi Technology Co., Ltd.; SCIENZ-10N freeze-vacuum dryer, Ningbo Xinzhi Biotechnology Co., Ltd.; WD-9405B decolorization shaker, Beijing Liuyi Biotechnology Co., Ltd.; YT-CJ-2ND clean bench, Beijing Yatai Kelong Instrument Technology Co., Ltd.; MyCycler PCR instrument, Bio-Rad Corporation, USA; H1650-W desktop high-speed microcentrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; ultrasonic cell disruptor, Ningbo Xinzhi Biotechnology Co., Ltd.; Thermo Multiskan MK3 microplate reader, Thermo Fisher Scientific, USA; Hitachi H-7650 transmission electron microscope, Hitachi (China) Co., Ltd.

[0051] 1.2 Test methods

[0052] 1.2.1 Construction of recombinant expression vector

[0053] The recombinant expression vectors pET-32a-VP4-3, pET-32a-VP56-2, pET-32a-Fn, pET-32a-VP4-3-Fn and pET-32a-VP56-2-Fn were commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis. After double enzyme digestion with EcoR I and Xho I, they were transformed into Escherichia coli (E. coli) BL21 competent cells. Positive clones were then picked for expansion and culture, and the plasmids were extracted and sequenced for identification.

[0054] 1.2.2 Recombinant protein expression and identification

[0055] The BL21 positive clones identified by sequencing were expanded and cultured into 3 L LB liquid medium containing ampicillin (100 μg / mL) and cultured at 37°C and 180 rpm until the bacteria grew to the logarithmic phase (OD 600 =0.4-0.6), add IPTG (1.0mM) to the culture medium to induce recombinant protein expression, and continue shaking and culturing for 4-6h. Then, centrifuge at 12000g and 4°C for 10min to collect the bacterial precipitate, add 1 / 10 volume of PBS of the culture medium to resuspend the precipitate, and use a high-pressure homogenizer to crush it until it is clear and transparent. Centrifuge at 12000g and 4°C for 10min to collect the supernatant and precipitate after crushing. Purify it using a His tag protein purification kit. After lyophilization, the purified protein solution was stored at -80°C and dissolved in sterile water when used. The expression and purification effect of the recombinant protein were analyzed by 12% SDS-PAGE.

[0056] Western Blot was used to identify whether the purified recombinant protein was of the expected size, using mouse anti-6×His tag monoclonal antibody as the primary antibody and HRP-labeled goat anti-mouse IgG monoclonal antibody as the secondary antibody.

[0057] 1.2.3 Transmission electron microscopy analysis of nanoparticle vaccines

[0058] Recombinant Fn, VP4-3-Fn, and VP56-2-Fn proteins were diluted with purified water to a concentration of 20 μg / mL. Prior to testing, the samples were centrifuged at 10,000 rpm for 4 minutes to remove possible aggregates. Protein samples were dropped onto a copper grid, incubated for 5 minutes, rinsed three times with distilled water, and stained with phosphotungstic acid for 15-20 minutes. After drying, the samples were examined under a transmission electron microscope.

[0059] 2 Results and Analysis

[0060] The constructed recombinant plasmids pET-32a-VP4-3, pET-32a-VP56-2, pET-32a-Fn, pET-32a-VP4-3-Fn and pET-32a-VP56-2-Fn were identified by double enzyme digestion. Figure 1 As shown in Figure 1, the target bands appeared after double enzyme digestion of the recombinant expression vector, indicating that the construction was successful. The results of 12% SDS-PAGE analysis are shown in Figure 1. Figure 2 The five recombinant proteins were successfully expressed and the impurity protein content was reduced after purification. The WB identification results of the purified recombinant proteins are shown in Figure 3 The expected molecular weights of the purified VP4-3, VP56-2, Fn, VP4-3-Fn, and VP56-2-Fn proteins were approximately 39, 33, 42, 60, and 54 kDa, respectively. Figure 3 As shown, both VP4-3-Fn and VP56-2-Fn self-assembled to form nanoparticles as expected.

[0061] Example 2

[0062] This example is intended to illustrate the immune effects of VP4-3-Fn and VP56-2-Fn.

[0063] 1.1 Materials

[0064] 1.1.1 Experimental animals and viruses

[0065] Healthy grass carp (4.0 ± 0.4 cm) were purchased from the Menghu Aquaculture Farm in Guangdong Province. The water temperature was 26–28°C, and they were fed regularly at 8:00 AM and 5:00 PM daily. Any remaining bait and feces were removed from the tank bottom daily. The fish were kept for 14 days before testing. All fish were tested for GCRV-II infection before the formal experiment. GCRV-II tissue suspensions were prepared and maintained by the Aquatic Disease Prevention and Control and Disease Resistance Breeding Innovation Team of the College of Animal Science and Technology, Northwest Agriculture and Forestry University.

[0066] 1.1.2 Reagents

[0067] Recombinant proteins VP4-3, VP56-2, Fn, VP4-3-Fn, and VP56-2-Fn were prepared by the Aquatic Disease Control and Disease Resistance Breeding Innovation Team of the College of Animal Science and Technology, Northwest Agriculture and Forestry University, according to the method in Example 1. Other reagents were prepared according to 1.1.2 in Example 1.

[0068] 1.1.3 Instruments

[0069] The instruments used are as in 1.1.3 of Example 1.

[0070] 1.2 Test methods

[0071] 1.2.1 Grass carp immersion immunization

[0072] Grass carp were divided into four groups (Control, Fn, Pep, and Pep-Fn), with 60 fish per group, and three replicates per group. The prepared vaccine was added to a pre-prepared 5L small plastic aquarium to a concentration of 20 mg / L. The grass carp were then transferred to a beaker containing the vaccine solution and allowed to soak for 6 hours. After immunization, the grass carp were returned to the culture device.

[0073] 1.2.2 Serum antibody titer determination

[0074] Serum samples were collected from each group of grass carp on days 7, 14, 21, and 28 after immunization, with five fish sampled at each time. The grass carp were anesthetized, their necks cut with surgical scissors, and the spilled blood was quickly aspirated using a 10μL pipette. The blood was then incubated at 4°C overnight, and the next day, the supernatant serum was separated by centrifugation. Serum antibody titers were determined using an enzyme-linked immunosorbent assay (ELISA) using the corresponding recombinant protein as the antigen, a mouse anti-6×His tag monoclonal antibody as the primary antibody, and an HRP-conjugated goat anti-mouse IgG monoclonal antibody as the secondary antibody. After terminating the color reaction, the absorbance of each sample was measured at a wavelength of 450 nm.

[0075] 1.2.3 Challenge test

[0076] 28 days after immunization, 30 fish from each group were randomly selected and placed in new tanks for a challenge test. Each fish was injected intraperitoneally with 20 μL of GCRV-II virus suspension. During the challenge test, the water temperature was maintained at 26 ± 2°C, and other culture conditions remained unchanged. The fish were observed regularly daily, and any morbidity or mortality was recorded promptly. The relative protection rate (RPR) was calculated using the formula: RPR = (1 - mortality rate in the immunized group / mortality rate in the control group) × 100%.

[0077] 2 Results and Analysis

[0078] 2.1 Serum immune antibody titer

[0079] The results of serum antibody level determination in each group (Control, Fn, VP4-3, VP56-2, VP4-3-Fn and VP56-2-Fn) are shown in Figure 5 Serum-specific antibody levels in the VP4-3, VP56-2, VP4-3-Fn, and VP56-2-Fn groups gradually increased after immunization, reaching a peak on day 21. Serum-specific antibody levels in the VP4-3 and VP56-2 groups were significantly higher than those in the Fn and control groups from day 14 to day 28 after immunization. Furthermore, serum-specific antibody levels in the VP4-3-Fn and VP56-2-Fn groups were significantly higher than those in the VP4-3 and VP56-2 groups.

[0080] 2.2 Relative immune protection rate

[0081] The survival rate of each profession after the attack is as follows Figure 6 As shown. Grass carp mortality occurred in the control and Fn groups 2 days after challenge, whereas no grass carp mortality occurred in any of the groups 10 to 28 days after challenge. 28 days after challenge, the mortality rates of the control, Fn, VP4-3, VP56-2, VP4-3-Fn, and VP56-2-Fn groups were 83.87%, 74.19%, 61.29%, 64.61%, 38.71%, and 41.94%, respectively. The relative protection rates of each group after challenge are shown in Table 1. Compared with the VP4-3 / VP56-2 group, the RPS of the VP4-3-Fn / VP56-2-Fn group increased by 26.92% and 23.08%, respectively. For details, see Table 1.

[0082] Table 1: Relative immune protection rate of each group after immunization and challenge

[0083]

[0084]

[0085] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

Claims

1. A self-assembling fusion protein based on grass carp ferritin, characterized in that: The self-assembling fusion protein is obtained by connecting grass carp ferritin and type II grass carp reovirus outer capsid protein through a connecting peptide; The amino acid sequence of the grass carp ferritin is shown in SEQ ID NO: 1; The type II grass carp reovirus outer capsid protein is type II grass carp reovirus outer capsid protein VP4 or type II grass carp reovirus outer capsid protein VP56; The amino acid sequence of the type II grass carp reovirus outer capsid protein VP4 is shown in SEQ ID NO:

2. When the type II grass carp reovirus outer capsid protein is type II grass carp reovirus outer capsid protein VP4, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 4, and the nucleotide sequence of the encoding gene thereof is shown in SEQ ID NO:

7. The amino acid sequence of the type II grass carp reovirus outer capsid protein VP56 is shown in SEQ ID NO: 3; when the type II grass carp reovirus outer capsid protein is type II grass carp reovirus outer capsid protein VP56, the amino acid of the fusion protein is shown in SEQ ID NO: 5, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:

6.

2. The self-assembling fusion protein based on grass carp ferritin according to claim 1, characterized in that: The connecting peptide is (G4S)3.

3. Use of the self-assembling fusion protein according to claim 1 in preparing vaccines for preventing aquatic diseases, characterized in that: The aquatic disease is caused by type II grass carp reovirus.

4. A vaccine for preventing aquatic diseases, characterized in that: The active ingredient of the vaccine is a self-assembling fusion protein, the amino acid sequence of the self-assembling fusion protein is shown in SEQ ID NO: 4, and the nucleotide sequence of the gene encoding the self-assembling fusion protein is shown in SEQ ID NO:

7.

5. A vaccine for preventing aquatic diseases, characterized in that: The active ingredient of the vaccine is a self-assembling fusion protein, the amino acid sequence of the self-assembling fusion protein is shown in SEQ ID NO: 5, and the nucleotide sequence of the gene encoding the self-assembling fusion protein is shown in SEQ ID NO:

6.

6. A vaccine for preventing aquatic diseases according to claim 4 or 5, characterized in that: The aquatic disease is grass carp hemorrhagic disease.

7. A vaccine for preventing aquatic diseases according to claim 4 or 5, characterized in that: The vaccine is implemented by immersion immunization.

Images