Neuronecrosis virus polypeptide chimeric cell exosomes and vaccines and preparation methods and applications thereof

Abstract

The present invention discloses a neuronecrosis virus polypeptide chimeric cell exosome vaccine, wherein the cell exosome stably expresses a recombinant protein, wherein the recombinant protein is a fusion protein of a key antigen epitope peptide segment of a CP protein and an exosome transmembrane protein, wherein the key antigen epitope peptide segment of the CP protein is CP 105‑118 or CP 193‑211 . The present invention also discloses a preparation method of the cell exosomes, a neuronecrosis virus vaccine, a fusion protein, and the use of the cell exosomes in the preparation of a drug for preventing and treating viral neuronecrosis. The present invention anchors the key antigenic epitopes of viral proteins on cell exosomes to construct exosomes, which have the advantages of subunit vaccines and the stability of exosomes, and can be used for aquatic disease prevention and control, especially for the prevention and treatment of viral neuronecrosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a neuronecrosis virus polypeptide chimeric cell exosome and vaccine, and a preparation method and application thereof. Background Art

[0002] my country is a major aquaculture country. With the promotion of high-density, intensive, large-scale and rapid development of aquaculture models, aquatic diseases have become a key factor seriously restricting the healthy and sustainable development of aquaculture. Fish vaccination can not only prevent and control the occurrence of aquatic diseases and reduce the economic losses of the industry, but also reduce or eliminate the health hazards, ecological risks and food safety of aquatic products caused by the large-scale use of antibiotics, thereby supporting the healthy and green development of the aquaculture industry.

[0003] Fish viral nervous necrosis is a Class II animal disease caused by infection with the nervous necrosis virus NNV (nervous necrosis virus). Globally, NNV can infect more than 120 species of fish, with a mortality rate of up to 100% for fry, juveniles and young fish. In addition, NNV has the ability to spread vertically. The infected broodstock do not become ill, but NNV can infect embryos through vertical transmission, causing large-scale mortality and deformity of fry. Viral nervous necrosis causes huge economic losses to the aquaculture industry, but there are still no effective prevention and treatment measures. The main NNV strain prevalent in my country is the red-spotted nervous necrosis virus RGNNV (Red-spotted nervous necrosis virus).

[0004] At present, the research on vaccines for viral neuronecrosis mainly includes virus-like particle vaccines, subunit vaccines, DNA vaccines, etc. A study showed that the coat protein CP (coat protein) of neuronecrosis virus NNV was displayed on the surface of α-enolase of Streptococcus iberimangiosus to form virus-like particle injection vaccine, and the virus protection rates in flounder and zebrafish were 40% and 30%, respectively. In another related study, Artemia, which can express CP protein and β-defensin recombinant protein, was used as bait to vaccinate grouper orally, and the virus protection rate of this method was as high as 100%. However, CP protein has certain cytotoxicity, and there is a potential risk of damaging the tissues and organs of fish when using the complete CP protein as an immunogen; choosing the CP protein antigen epitope peptide as an immunogen can avoid the cell and tissue damage that may be caused by CP protein on the one hand, and enhance the immunogenicity of the vaccine on the other hand.

[0005] Exosomes are disc-shaped vesicles with a diameter of 30-200nm, which contain substances such as RNA, proteins and lipids. They have small particle size, high stability, strong targeting and excellent biocompatibility. As a delivery carrier for drugs or vaccines, they are widely used in cancer treatment and vaccine development. For aquatic virus infections, immersion vaccines have many advantages, such as simple operation, small stress response and the ability to cope with vertical transmission of viruses. Combined with genetic engineering technology, exosomes can be used as carriers of aquatic disease vaccines to carry key peptides, which can achieve vaccination by immersion, which is of great significance for promoting the development of aquaculture disease vaccines. Summary of the invention

[0006] One object of the present invention is to provide a chimeric exosome containing a neuronecrosis virus polypeptide with good effect, high specificity and good stability in response to the above technical problems.

[0007] Another object of the present invention is to provide a neuronecrosis virus vaccine.

[0008] Another object of the present invention is to provide a method for preparing the neuronecrosis virus polypeptide chimeric cell exosomes.

[0009] Another object of the present invention is to provide the application of the neuronecrosis virus polypeptide chimeric cell exosomes.

[0010] Another object of the present invention is to provide the application of the neuronecrosis virus vaccine.

[0011] In order to achieve the above invention objectives, the present invention provides a chimeric cell exosome containing a neuronecrosis virus polypeptide, wherein the exosome stably expresses a recombinant protein, and the recombinant protein is a fusion protein of a key antigenic epitope peptide segment of a CP protein and an exosome transmembrane protein, and the key antigenic epitope peptide segment of the CP protein is CP 105-118 or CP 193-211 .

[0012] The recombinant protein has both antigenic epitope characteristics and the characteristics of anchoring in exosomes. The key antigenic epitope peptide segment is the interaction site between the RGNNV virus CP protein and the key receptor heat shock protein Hsc70 (Heatshock cognate protein 70) for viral infection obtained by molecular docking, which is the key antigenic epitope peptide segment CP of the CP protein. 105-118 or CP 193-211 .

[0013] Preferably, the exosomal transmembrane protein is CD63.

[0014] Preferably, the peptide segment CP 105-118 The amino acid sequence is shown in SEQ ID NO:1.

[0015] Preferably, the peptide segment CP 193-211 The amino acid sequence is shown in SEQ ID NO:2.

[0016] Preferably, the amino acid sequence of the recombinant protein is shown in SEQ ID NO: 4. The nucleotide sequence encoding the recombinant protein is shown in SEQ ID NO: 3.

[0017] Preferably, the amino acid sequence of the recombinant protein is shown in SEQ ID NO: 6. The nucleotide sequence encoding the recombinant protein is shown in SEQ ID NO: 5.

[0018] On the other hand, the present invention also provides a method for preparing the exosomes, which comprises the following steps: infecting cells with a lentivirus containing a nucleotide sequence encoding the recombinant protein, obtaining cells that stably express the recombinant protein through puromycin screening, and culturing and extracting the exosomes.

[0019] On the other hand, the present invention also provides a neuronecrosis virus vaccine, wherein the neuronecrosis virus vaccine comprises the exosomes as an active ingredient.

[0020] On the other hand, the present invention also provides the use of the exosomes in the preparation of a drug for preventing and treating viral neuropathy.

[0021] On the other hand, the present invention also provides the use of the neuronecrosis virus vaccine in the preparation of a drug for preventing and treating viral neuronecrosis.

[0022] On the other hand, the present invention also provides a fusion protein, wherein the fusion protein is a fusion of a key antigen epitope peptide segment of a CP protein to the second extracellular region of an exosome transmembrane protein, wherein the key antigen epitope peptide segment of the CP protein is CP 105 -118 or CP 193-211 .

[0023] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 4. Preferably, the nucleotide sequence encoding the recombinant protein is shown in SEQ ID NO: 3.

[0024] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 6. Preferably, the nucleotide sequence encoding the recombinant protein is shown in SEQ ID NO: 5.

[0025] On the other hand, the present invention also provides a nucleic acid molecule encoding the fusion protein.

[0026] On the other hand, the present invention also provides a recombinant vector comprising the nucleic acid molecule. Preferably, the recombinant vector is a lentivirus.

[0027] On the other hand, the present invention also provides a host cell, which comprises the recombinant vector. Preferably, the host cell is a 293T cell.

[0028] The present invention constructs a recombinant protein of CD63 and CP peptide segments, anchoring the key antigenic epitopes of viral proteins on cell exosomes to construct an exosome vaccine, which has both the advantages of subunit vaccines and the stability of exosomes. It can be used as an immersion vaccine for the prevention and control of aquatic diseases, especially for the prevention and treatment of viral neuronecrosis, a common disease in aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A flow chart of the preparation of exosomes according to the present invention is shown.

[0030] Figure 2 A schematic diagram of the structure of exosomes according to the present invention is shown.

[0031] Figure 3 The stable cell morphology producing exosomes according to the present invention after puromycin selection is shown.

[0032] Figure 4 The results of PCR detection of inserted genes in stable cells are shown.

[0033] Figure 5 The results of exosome transmission electron microscopy, nanoflow cytometry and immunoblotting are shown.

[0034] Figure 6 Exosome cytotoxicity assay results are shown.

[0035] Figure 7 Real-time fluorescence quantitative PCR and immunoblotting were used to detect the antiviral function of exosomes. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. It is worth noting that for the sake of brevity, all technical operation steps are not described in detail below, but unless otherwise specified, these operations are known and mastered by those skilled in the art.

[0037] 1. Screening of key peptides of RGNNV virus CP protein

[0038] In order to obtain the key peptides of RGNNV-CP protein, the binding region and interaction mode of RGNNV-CP protein and Hsc70 protein were first analyzed, and the professional protein-protein and protein-DNA / RNA docking HDOCK program was used for docking, and the structure with the best docking score was selected as the standard result for subsequent interaction analysis. The docking score is calculated based on the iterative scoring function of ITScorePP or ITScorePR. The best result of this protein-protein docking, DockingScore, is -284.68, and Confidence Score is 0.9366. The result shows that the complex model obtained by docking has a high credibility. To further analyze the interaction between RGNNV-CP and Hsc70 protein complexes, pymol uses InterfaceResidue to analyze the interface residues of proteins, and obtains two RGNNV-CP action peptides in the interaction distribution map, as shown below:

[0039] CP 105-118 :N'-ETLEFEIQPMCPAN-C' (SEQ ID NO: 1)

[0040] CP 193-211 :N'-DVVNVSVLCRWSVRLSVPS-C' (SEQ ID NO: 2)

[0041] 2. Screening of exosome-stable cell lines

[0042] According to the molecular docking results, the designated peptide segment of the RGNNV virus CP protein was selected, and the DNA sequence of 5 copies of the designated peptide segment was seamlessly cloned into the CD63 gene sequence. The amino acid sequence and encoding nucleotide sequence of the obtained fusion protein are shown below:

[0043] SEQ ID NO: 3 (fusion protein CD63-CP 105-118 Coding nucleotide sequence):

[0044] 5’-ATGGCGGTGGAAGGAGGAATGAAATGTGTGAAGTTCTTGCTCTACGTCCTCCTG CTGGCCTTTTGCGCCTGTGCAGTGGGACTGATTGCCGTGGGTGTCGGGGCACAGCTTGTCCTGAGTCAGACCATAATCCAGGGGGCTACCCCTGGCTCTCTGTTGCCAGTGGTCATCATCGCAGTGGGTGTCTTCCTCTTCCTGGTGGCTTTTGTGGGCTGCTGCGGGGCCTGCAAGGAGAACTATTGTCTTATGATCACGTTTGCCATCTTTCTGTCTCTTATCATGTTGGTGGAGGTGGCCGCAGCCATTGCTGGCTATGTGTTTAGAGATAAGGCAGCCATT GAAACACTGGAGTTTGAAATTCAGCCAAT GTGCCCCGCAAACGAAACACTGGAGTTTGAAATTCAGCCAATGTGCCCCGCAAACGAAACACTGGAGTTTGAAATT CAGCCAATGTGCCCCGCAAACGAAACACTGGAGTTTGAAATTCAGCCAATGTGCCCCGCAAACGAAACACTGGAGT TTGAAATTCAGCCAATGTGCCCCGCAAAC GCTGGCTATGTGTTTAGAGATAAGGTGATGTCAGAGTTTAATAACAACTTCCGGCAGCAGATGGAGAATTACCCGAAAAACAACCACACTGCTTCGATCCTGGACAGGATGCAGGCAGATTTTAAGTGCTGTGGGGCTGCTAACTACACAGATTGGGAGAAAATCCCTTCCATGTCGAAGAACCGAGTCCCCGACTCCTGCTGCATTAATGTTACTGTGGGCTGTGGGATTAATTTCAACGAGAAGGCGATCCATAAGGAGGGCTGTGTGGAGAAGATTGGGGGCTGGCTGAGGAAAAATGTGCTGGTGGTAGCTGCAGCAGCCCTTGGAATTGCTTTTGTCGAGGTTTTGGGAATTGTCTTTGCCTGCTGCCTCGTGAAGAGTATCAGAAGTGGCTACGAGGTGATGTAG-3’

[0045] SEQ ID NO: 4 (Fusion protein CD63-CP 105-118 Amino acid sequence):

[0046] NH2-MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLP VVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKAAI ETLEFEIQPMCPANETLEFEIQPMCP ANETLEFEIQPMCPANETLEFEIQPMCPANETLEFEIQPMCPAN AGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM-COOH

[0047] SEQ ID NO: 5 (Fusion protein CD63-CP 193-211 Coding nucleotide sequence):

[0048] 5’-ATGGCGGTGGAAGGAGGAATGAAATGTGTGAAGTTCTTGCTCTACGTCCTCCTG CTGGCCTTTTGCGCCTGTGCAGTGGGACTGATTGCCGTGGGTGTCGGGGCACAGCTTGTCCTGAGTCAGACCATAATCCAGGGGGCTACCCCTGGCTCTCTGTTGCCAGTGGTCATCATCGCAGTGGGTGTCTTCCTCTTCCTGGTGGCTTTTGTGGGCTGCTGCGGGGCCTGCAAGGAGAACTATTGTCTTATGATCACGTTTGCCATCTTTCTGTCTCTTATCATGTTGGTGGAGGTGGCCGCAGCCATTGCTGGCTATGTGTTTAGAGATAAGGCAGCCATT GATGTGGTCAACGTGTCAGTGCTGTGTCG CTGGAGTGTTCGATTGAGCGTTCCATCTGATGTGGTCAACGTGTCAGTGCTGTGTCGCTGGAGTGTTCGATTGAGC GTTCCATCTGATGTGGTCAACGTGTCAGTGCTGTGTCGCTGGAGTGTTCGATTGAGCGTTCCATCTGATGTGGTCA ACGTGTCAGTGCTGTGTCGCTGGAGTGTTCGATTGAGCGTTCCATCTGATGTGGTCAACGTGTCAGTGCTGTGTCG CTGGAGTGTTCGATTGAGCGTTCCATCTGCTGGCTATGTGTTTAGAGATAAGGTGATGTCAGAGTTTAATAACAACTTCCGGCAGCAGATGGAGAATTACCCGAAAAACAACCACACTGCTTCGATCCTGGACAGGATGCAGGCAGATTTTAAGTGCTGTGGGGCTGCTAACTACACAGATTGGGAGAAAATCCCTTCCATGTCGAAGAACCGAGTCCCCGACTCCTGCTGCATT AATGTTACTGTGGGCTGTGGGATTAATTTCAACGAGAAGGCGATCCATAAGGAGGGCTGTGTGGAGAAGATTGGGGGCTGGCTGAGGAAAAATGTGCTGGTGGTAGCTGCAGCAGCCCTTGGAATTGCTTTTGTCGAGGTTTTGGGAATTGTCTTTGCCTGCTGCCTCGTGAAGAGTATCAGAAGTGGCTACGAGGTGATGTAG-3'

[0049] SEQ ID NO: 6 (fusion protein CD63-CP 193-211 Amino Acid Sequence):

[0050] NH2-MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFSLIMLVEVAAAIAGYVFRDKAAI DVVNVSVLCRWSVRLSVPSDVVNVSVL CRWSVRLSVPSDVVNVSVLCRWSVRLSVPSDVVNVSVLCRWSVRLSVPSDVVNVSVLCRWSVRLSVPS AGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM-COOH

[0051] The underlined part is the key peptide sequence of CP.

[0052] Figure 1 A flow chart of the preparation of exosomes according to the present invention is shown. Figure 2 The schematic diagram of the structure of the exosome according to the present invention is shown. As shown in the figure, the CP key peptide segment is inserted between the second extracellular structure and the fourth transmembrane structure of CD63, while retaining the key recognition site of the CD63 antibody.

[0053] The recombinant sequence was constructed by homologous recombination and seamlessly cloned into the second-generation lentiviral backbone plasmid pCDH-CMV-MCS-EF1-copGFP-T2A-Puro (System Biosciences, Cat.#CD511B-1), and the plasmid was co-transfected with the lentiviral packaging plasmid pCMV-VSV-G (Addgene, Cat.#8454) and psPAX2 (Addgene, Cat.#12260) into 293T cells for lentiviral packaging. Specifically, 293T cells were inoculated in a 10 cm culture dish and cultured in a 37-well, 5% CO2 incubator. When the cell density reached 70% to 80%, it could be used for transfection. Add 10 μg of lentiviral backbone plasmid, 5 μg of pCMV-VSV-G and 5 μg of psPAX2 to 0.5 mL of Opti-MEM (Gibco, Cat. #31985070) culture medium, and incubate at room temperature for 5 min; add 50 μL of transfection reagent Lipofectamine 2000 (ThermoFisher, Cat. #11668-019) to 0.45 mL of Opti-MEM culture medium in another sterile centrifuge tube, and incubate at room temperature for 5 min; mix the diluted plasmid with the diluted transfection reagent and let stand at room temperature for 10 min; add the above transfection mixture dropwise to the 293T cell culture medium, shake well, and culture in a CO2 cell culture incubator at 37 culture medium; replace fresh culture medium 6 h after transfection. 48 hours after transfection, the cell culture supernatant was collected, filtered through a 0.22 μm filter membrane (Millipore, Cat. # GSWP04700), and the lentivirus was aliquoted and stored at -80 ° C for later use. Different lentiviruses were used to infect 293T cells, and the lentivirus-infected cells were screened by puromycin (MCE, Cat. # HY-B1743A). After multiple screenings, a stable cell line that can express a specific protein was obtained. The state of the stable cell line was observed under a fluorescence microscope, and a green fluorescent signal was visible, indicating that the lentivirus infected the cells.

[0054] The results are as follows Figure 3 , showing the morphology of stable 293T cells producing exosomes according to the present invention after puromycin selection.

[0055] Stable 293T cells were collected, genomic DNA was extracted, and PCR detection was performed using the following primer pairs:

[0056] Seq-PF:CACGTTTTGCCATCTTTCTGT (SEQ ID NO: 7);

[0057] Seq-PR: TCGTAGCCACTTCTGATACTCTT (SEQ ID NO: 8).

[0058] The PCR product was detected by nucleic acid gel, and the size of the amplified product was in line with expectations. The PCR product was sequenced by the second generation to further verify the correctness of the inserted sequence.

[0059] The results are as follows Figure 4 As shown, the stable cell genomic DNA was extracted after puromycin selection, and PCR was used to detect the target gene fragment CD63-CP inserted into the genome. 105-118 and CD63-CP 193-211 .

[0060] 3. Extraction of Exosome Vaccine

[0061] After culturing the stable 293T cells in DMEM medium (Corning, Cat. #10-090-CM) containing 10% (w / v) exosome-free fetal bovine serum (VIVACELL, Cat. #C3801-0100) for 2-5 days, the cell culture supernatant was collected, centrifuged at 300×g to remove suspended cells, and centrifuged at 3000×g to remove cell debris. The centrifuged supernatant was filtered through a 0.22μm filter membrane, and then the exosome extraction reagent (ThermoFisher, Cat. #4478359) was added and allowed to stand at 4°C for 12h. The culture medium and exosome extraction reagent mixture was centrifuged at 4°C and 10000×g after standing, and the supernatant was removed and resuspended with an appropriate amount of PBS to obtain the exosome vaccine.

[0062] The exosome vaccine was characterized using transmission electron microscopy, nanoflow cytometry and immunoblotting.

[0063] The results are as follows Figure 5 As shown, the characterization of CD63-CP related exosomes is shown, where A, C, and E are control exosomes, CD63-CP 105-118 and CD63-CP 193-211 Transmission electron microscopy observation results of exosomes showed no significant differences among the three groups; Figures B, D, and F were the results of nanoflow cytometry, and the average particle sizes of the three groups were 81.1±2.17nm, 80.7±1.44nm, and 81.2±1.98nm, respectively. The results showed that there was no significant difference in the morphology and size of exosomes in different groups. Figure H was an immunoblotting experiment to detect exosome marker proteins ALIX and CD63, that is, anti-ALIX (Proteintech, Cat.#12422-1-AP) and anti-CD63 (Proteintech, Cat.#25682-1-AP) antibodies were used as primary antibodies, and goat anti-rabbit antibodies (Proteintech, Cat.#RGAR001) were used as secondary antibodies to detect exosome-specific proteins by immunoblotting.

[0064] 4. Exosome Function Verification

[0065] Grouper kidney (GK) cells are RGNNV susceptible cell lines, and this cell line was used to verify the function of exosome vaccines in inhibiting viruses. First, GK cells were cultured in Leibovitz L-15 medium (Basal Media, Cat. #L620KJ) containing 10% (w / v) fetal bovine serum (Excell, Cat. #FSP500) at 28°C. For the cytotoxicity analysis of exosomes, GK cells were inoculated in 96-well plates and incubated with exosome vaccines at a final concentration of 20 μg / mL. The cytotoxicity of exosome vaccines was detected by cck-8 (Selleck, Cat. #B34302) after 24 and 48 hours, respectively. 10 μl of active detection reagent was added to every 100 μL of culture medium, and the culture was continued for 1-4 hours until the color turned orange, and the 450nm light absorption value was read with an enzyme reader.

[0066] The results are as follows Figure 6 As shown, CCK-8 assay showed that CD63-CP at a final concentration of 20 μg / mL 105-118 and CD63-CP 193-211 The exosomes had no cytotoxicity to GK cells.

[0067] GK cells were then incubated with exosomes at a final concentration of 20 μg / mL and infected with RGNNV (multiplicity of infection was 1). The progeny virus production was detected by real-time fluorescence quantitative PCR and immunoblotting experiments to evaluate the virus-inhibiting function of the exosome vaccine.

[0068] The results are as follows Figure 7 As shown, CD63-CP 105-118 and CD63-CP 193-211 Exosomes can significantly inhibit the replication of RGNNV virus. Among them, A shows that exosomes were incubated with RGNNV and detected by real-time fluorescence quantitative PCR, CD63-CP 105-118 and CD63-CP 193-211 Exosomes can significantly inhibit the transcription of RGNNV-CP protein; B shows the results of immunoblotting, indicating that CD63-CP 105-118 and CD63-CP 193-211 Exosomes can inhibit the expression of RGNNV-CP protein. In summary, exosomes at a concentration of 20 μg / mL have no obvious toxicity to GK cells; exosomes can inhibit the infection and replication of RGNNV virus after incubation. It is speculated that exosomes can competitively bind to the receptor protein Hsc70, reduce the infection efficiency of RGNNV virus, and thus inhibit the infection and replication of the virus.

[0069] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope of the present invention.

Claims

1. A chimeric exosome containing a neuronecrosis virus polypeptide, wherein the exosome stably expresses a recombinant protein, characterized in that: The recombinant protein is a fusion protein of the key antigenic epitope peptide segment of the CP protein and the exosome transmembrane protein. The key antigenic epitope peptide segment of the CP protein is CP 105-118 or CP 193-211 , the amino acid sequence of the recombinant protein is shown in SEQ ID NO: 4 or SEQ ID NO:

6.

2. The method for preparing exosomes according to claim 1, comprising the following steps: The cells are infected with a lentivirus containing a nucleotide sequence encoding the recombinant protein, and the cells stably expressing the recombinant protein are obtained after puromycin screening, and the exosomes are extracted after culturing.

3. A neuronecrosis virus vaccine, wherein: The neuronecrosis virus vaccine comprises the exosomes according to claim 1 as an active ingredient.

4. Use of the exosomes according to claim 1 in the preparation of a medicament for preventing and treating viral neuropathy.

5. A fusion protein, characterized in that: The fusion protein is a key antigen epitope peptide segment of the exosome transmembrane protein fused to the CP protein. The key antigen epitope peptide segment of the CP protein is CP 105-118 or CP 193-211 The amino acid sequence of the fusion protein is shown in SEQ ID NO: 4 or SEQ ID NO:

6. A nucleic acid molecule encoding the fusion protein according to claim 5 . A recombinant vector comprising the nucleic acid molecule according to claim 6 . A host cell comprising the recombinant vector according to claim 7.

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