Celine hepatitis virus core antigen virus-like particle as well as preparation method and application thereof

Abstract

The invention relates to a cat hepatitis virus core antigen virus-like particle as well as a preparation method and application thereof. The virus-like particle is prepared by expressing a wild type gene DCHcWT of a cat hepatitis virus core antigen or a 1-149 amino acid truncated gene DCHcN149 of an amino terminal of the wild type gene DCHcWT in an insect baculovirus expression system, and purifying and self-assembling the wild type gene DCHcWT or the 1-149 amino acid truncated gene DCHcN149 in the insect baculovirus expression system. The prepared virus-like particles are regular in structure and good in immunogenicity, and antigen characteristics of natural virus particles can be efficiently simulated. The virus-like particle can be used as a diagnosis antigen of cat hepatitis B virus infection, and is used for preparing a serum detection kit for cat hepatitis. The vaccine can also be used as an immunogen to prepare a subunit vaccine for preventing cat hepatitis B; the compound can also be used for vaccine nano-delivery carriers.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a feline hepatitis virus core antigen virus-like particle, a preparation method and application thereof. BACKGROUND

[0002] As a key pathogen of feline infection, Domestic Cat Hepadnavirus (DCH) is the main pathogen causing acute or chronic hepatitis in cats, which poses a continuous and serious threat to global pet health. The liver inflammatory disease caused by the virus not only seriously affects the quality of life of the diseased animals, but also can develop into irreversible pathological changes such as liver fibrosis, liver cirrhosis and liver failure. At present, the diagnosis method and prevention means for DCH infection in the field of veterinary medicine are still insufficient. The existing diagnosis technology mainly relies on virus nucleic acid detection or antibody screening, but these methods have obvious limitations in sensitivity, specificity and accessibility. In the field of prevention, the lack of commercial vaccines exposes the cat population to the risk of DCH infection all the time, and this situation urgently needs the emergence of a breakthrough technology.

[0003] In the structural composition of DCH virus, the core antigen (DCHcAg) as the main constituent unit of the virus capsid plays multiple key roles in the life cycle of the virus and the process of host immune response. However, the direct use of natural virus antigens faces many challenges such as high biological safety risk, complex preparation process and high cost, which seriously restricts the research and development process of related products. Virus-like particles (VLPs) are hollow nanoparticles formed by the self-assembly of one or more structural proteins of a virus. The preparation process has an important influence on the product parameters. How to realize the efficient expression of DCHcAg protein and ensure that it can correctly self-assemble to form VLPs with complete structure and good stability is the primary technical challenge currently faced. At the same time, the preparation of VLPs also has problems such as low purity and poor uniformity. When used as a diagnostic antigen, how to improve performance indicators such as sensitivity, specificity and stability also needs to be solved. Whether it can be applied in the field of nano delivery has not been reported. SUMMARY

[0004] In view of the problems such as insufficient sensitivity of the existing DCH diagnostic tools, lack of commercial preventive vaccines and blank application of VLP technology in the field, the application provides a DCHcAg VLP with clear structure, excellent immunogenicity and easy scale production, and further provides a preparation method and application thereof.

[0005] The application provides a feline hepatitis virus core antigen (DCHcAg) virus-like particle (VLP) comprising a feline hepatitis virus core antigen protein, wherein the feline hepatitis virus core antigen protein comprises an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence shown in SEQ ID NO: 2. The virus-like particle can be formed by self-assembly of the DCHcAg protein. In order to meet the needs of practical application, other sequences can be added on the basis of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, for example, a tag coding sequence can be added to facilitate subsequent detection and purification.

[0006] The application provides a preparation method of the feline hepatitis virus core antigen virus-like particle, comprising the following steps: expressing the feline hepatitis virus core antigen protein in an insect baculovirus expression system. The baculovirus expression system can be The baculovirus expression system.

[0007] Further, the method can comprise the following steps:

[0008] (1) providing a recombinant expression vector comprising a polynucleotide encoding the DCHcAg protein; (2) introducing the recombinant expression vector into an insect host cell; (3) culturing the host cell under conditions allowing expression of the DCHcAg protein; (4) isolating and purifying the DCHcAg protein from the culture and allowing the DCHcAg protein to self-assemble into a virus-like particle.

[0009] The application provides a polynucleotide encoding the nucleotide sequence of the feline hepatitis virus core antigen virus-like particle. The polynucleotide comprises a nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, or a degenerate sequence thereof.

[0010] The application provides a recombinant vector comprising the nucleotide sequence of the feline hepatitis virus core antigen virus-like particle. The recombinant vector includes but is not limited to a recombinant expression vector. The vector is preferably a donor plasmid of an insect baculovirus expression system, more preferably an optimized vector pFBDopt or a pOET1.1 vector of pFastBac Dual. Among them, the pFBDopt vector introduces an Ac-ie-01 expression cassette (the sequence is shown in SEQ ID NO: 19), which can delay the apoptosis of the host cell and prolong the protein expression period; and an optimized combination type promoter p6.9 / p10 (the sequence is shown in SEQ ID NO: 17) and a cis-activation element hr1 (the sequence is shown in SEQ ID NO: 18) are used, which enhances the transcription activity of the target gene, so that the protein expression amount can be significantly improved.

[0011] The present application provides a host cell comprising the recombinant vector described above. The host cell can be an insect cell, preferably Sf9, Sf21 or High Five, etc.

[0012] The present application provides the use of the feline hepatitis virus core antigen virus-like particle described above in the preparation of a product for preventing, treating or detecting feline hepatitis virus. The product includes but is not limited to vaccine, nanocarrier, diagnostic antigen, diagnostic reagent, kit, etc. The virus-like particle described above can be used to prepare a reagent or kit for diagnosing feline hepatitis virus infection.

[0013] The present application provides a vaccine comprising the feline hepatitis virus core antigen virus-like particle described above. The vaccine can be a vaccine for preventing feline hepatitis virus. In addition to containing an immunologically effective amount of the virus-like particle described above, the vaccine can also include a pharmaceutically acceptable adjuvant. The adjuvant can preferably be .

[0014] The present application provides a nanocarrier of epitope vaccine, in which a heterologous antigen epitope is inserted into the MIR region of the feline hepatitis virus core antigen described above. The virus-like particle described above can be used as a nanocarrier in the construction of a chimeric vaccine. Specifically, a heterologous antigen epitope is inserted into the MIR region (corresponding to between amino acids 78-79) of the DCHcAg, and the heterologous antigen epitope is preferably a cytotoxic T lymphocyte (CTL) epitope of the feline infectious peritonitis virus (FIPV) membrane protein (M) or nucleocapsid protein (N), the CTL epitope of the feline infectious peritonitis virus membrane protein is shown as SEQ ID NO: 20, and the CTL epitope of the nucleocapsid protein is shown as SEQ ID NO: 21, thereby constructing a recombinant chimeric VLP vaccine and .

[0015] The present application provides a diagnostic antigen for feline hepatitis virus, comprising the feline hepatitis virus core antigen virus-like particle described above. The diagnostic antigen described above can be further prepared into a reagent or kit for diagnosing feline hepatitis virus infection. The virus-like particle, in particular the DCHcWT VLP, can be used as a coating antigen for establishing an indirect ELISA detection method. The method uses DCHcVLP to coat the enzyme-labeled plate, uses the cat serum to be tested as the primary antibody, and uses the enzyme-labeled anti-cat IgG as the secondary antibody, and the presence and level of anti-DCHc antibody in the serum are determined by detecting the OD value.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] (1) High safety: the VLP prepared by the application does not contain viral genetic material and is non-infectious, and the biological safety is significantly better than that of inactivated vaccine or attenuated live vaccine; (2) Strong immunogenicity: the VLP completely simulates the conformational epitope of the natural virus, and can simultaneously stimulate strong humoral immunity and cellular immunity, and the immune effect is comprehensive; (3) Novel structure and diverse use: the application first utilizes the structural characteristics of DCHcAg, which is not only developed as a diagnostic antigen and a preventive vaccine, but also expanded as a universal nano delivery platform through molecular modification, which can be used to display protective antigens of other pathogens, and realize one platform for multiple uses; (4) Mature and stable preparation process: the detailed preparation process based on two mainstream baculovirus expression systems is provided, the steps are clear, the repeatability is good, and it is suitable for industrialized large-scale production; (5) Avoiding pre-existing immunity: due to the key sequence difference with HBcAg, the vaccine based on DCHcVLP is expected to avoid the problem of reduced vaccine efficacy caused by wide pre-existing immunity of HBcAg. In summary, the application first successfully designs, prepares and verifies the feline hepatitis virus core antigen virus-like particles, and systematically develops the application value of the feline hepatitis virus core antigen virus-like particles in diagnosis, prevention and even as a universal nano carrier platform, and provides a new and efficient technical solution for the prevention and control of feline hepatitis and other related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a schematic diagram of multiple sequence alignment of feline hepatitis virus core antigen; Figure 2 It is a schematic diagram of donor plasmid optimization in Example 2; Figure 3 It is a recombinant plasmid pFBDopt-DCHc in Example 2 WT and pFBDopt-DCHc N149 PCR verification result graph; Figure 4 It is a schematic diagram of homologous recombination of parent baculovirus plasmid and donor plasmid in Example 2; Figure 5 It is a CPE result graph of recombinant baculovirus in Example 2; Figure 6 It is a verification result graph of indirect immunofluorescence of recombinant baculovirus in Example 2; Figure 7 It is a Western Blot and SDS-PAGE verification result graph of recombinant baculovirus in Example 2; Figure 8 It is a transmission electron microscope observation result graph of recombinant virus-like particles in Example 2; Figure 9 It is a recombinant plasmid pOET1.1-DCHc in Example 3 WT and pOET1.1-DCHc N149 PCR verification result graph; Figure 10 It is a schematic diagram of homologous recombination of parent baculovirus plasmid and donor plasmid in Example 3; Figure 11 It is a CPE result graph of recombinant baculovirus in Example 3; Figure 12Figure for verification result of recombinant baculovirus indirect immunofluorescence in Example 3; Figure 13 Figure for verification result of recombinant baculovirus Western Blot and SDS-PAGE in Example 3; Figure 14 Figure for transmission electron microscope observation result of recombinant virus-like particle in Example 3; Figure 15 Figure for ELISA detection result of DCHc VLP as diagnostic antigen in Example 4; Figure 16 Figure for antigen-specific antibody titer result of DCHc VLP in Example 5; Figure 17 Figure for cellular immune response result of DCHc VLP as epitope vaccine nano-carrier produced in Example 6. DETAILED DESCRIPTION

[0019] The principles and features of the present application are described below in conjunction with the accompanying drawings, and the examples are only used to explain the present application and not to limit the scope of the present application.

[0020] The virus-like particle (VLP) of the present application is prepared by expressing the wild-type gene DCHc of feline hepatitis virus core antigen (DCHcAg) or its amino-terminal 1-149 amino acid truncated gene DCHcN WT 149 in an insect baculovirus expression system, and self-assembling after purification.The virus-like particle prepared in the present application has regular structure, good immunogenicity, and can efficiently simulate the antigenic properties of natural virus particles. The virus-like particle can be used as diagnostic antigen for feline hepatitis B virus infection, for preparing feline hepatitis serum detection kit; can also be used as immunogen, for preparing subunit vaccine for preventing feline hepatitis B; and can also be used as vaccine nano-delivery carrier.

[0021] The sequences involved in the examples are shown as follows.

[0022] The amino acid sequence of DCHc WT is shown as SEQ ID NO: 1, specifically: mhlfhlcvlfcsiptvqasklclrwlwgmdidpykefgttsqlisflpsdffpalndlvdtiqalyeeeltgrehcsphhtalrvllncweesarmatwvranvegaplqdaivayvnstvslklrqqmwfhlscltfgqhtvleflvsfgtwirtpapyrppnapilstlpehtvirargaarrparsprrrtpsprrrrsqsprrrrsqsptqsnc.

[0023] The amino acid sequence of DCHc N149The amino acid sequence of DCHc is shown as SEQ ID NO: 2, specifically: mdidpykefgttsqlisflpsdffpalndlvdtiqalyeeeltgrehcsphhtalrvllncweesarmatwvranvegaplqdaivayvnstvslklrqqmwfhlscltfgqhtvleflvsfgtwirtpapyrppnapilstlpehtvi. N149 The amino acid sequence of DCHc is shown as SEQ ID NO: 2, specifically: mdidpykefgttsqlisflpsdffpalndlvdtiqalyeeeltgrehcsphhtalrvllncweesarmatwvranvegaplqdaivayvnstvslklrqqmwfhlscltfgqhtvleflvsfgtwirtpapyrppnapilstlpehtvi. N149 The amino acid sequence of DCHc is shown as SEQ ID NO: 2, specifically: mdidpykefgttsqlisflpsdffpalndlvdtiqalyeeeltgrehcsphhtalrvllncweesarmatwvranvegaplqdaivayvnstvslklrqqmwfhlscltfgqhtvleflvsfgtwirtpapyrppnapilstlpehtvi.

[0024] The present application provides an isolated polynucleotide encoding the above-mentioned DCHc Ag protein, comprising the following nucleotide sequence or degenerate sequence thereof.

[0025] The present application provides an isolated polynucleotide encoding the above-mentioned DCHc Ag protein, comprising the following nucleotide sequence or degenerate sequence thereof. WT The nucleotide sequence of DCHc is shown as SEQ ID NO: 3, specifically: atggacattgacccttataaagaatttggaactacatctcagcttatctcttttttgccgtctgacttttttcctgctctgaatgatctggtcgataccattcaagcgctctatgaagaggaacttacaggtagggaacactgttcccctcaccatacggcccttcgtgtgctcttgaactgttgggaggagtcagctagaatggctacatgggttagagccaatgtggaaggagccccattgcaggatgccattgtggcttatgttaattctactgtaagtttgaaattgaggcaacaaatgtggttccatctctcttgcctcacttttggacaacatactgtcttggagtttttagtatcttttggcacctggattcgcacacctgcaccgtataggccccccaatgcacccattctctccactcttccggagcatacggttatcagagcacggggagcagcgcgtagacctgctaggtcccccagaagacgtactccctctcctcgcagacgcagatctcaatctccgcgtcgcagacgttctcaatctccaactcagtcaaactgc.

[0026] The present application provides an isolated polynucleotide encoding the above-mentioned DCHc Ag protein, comprising the following nucleotide sequence or degenerate sequence thereof. N149The nucleotide sequence of the nucleotide sequence is shown as SEQ ID NO: 4, specifically: atggacattgacccttataaagaatttggaactacatctcagcttatctcttttttgccgtctgacttttttcctgctctgaatgatctggtcgataccattcaagcgctctatgaagaggaacttacaggtagggaacactgttcccctcaccatacggcccttcgtgtgctcttgaactgttgggaggagtcagctagaatggctacatgggttagagccaatgtggaaggagccccattgcaggatgccattgtggcttatgttaattctactgtaagtttgaaattgaggcaacaaatgtggttccatctctcttgcctcacttttggacaacatactgtcttggagtttttagtatcttttggcacctggattcgcacacctgcaccgtataggccccccaatgcacccattctctccactcttccggagcatacggttatc.

[0027] The present application provides a recombinant expression vector comprising the above-mentioned polynucleotide. The vector is preferably a donor plasmid of an insect baculovirus expression system, more preferably an optimized vector pFBDopt of pFastBac Dual or a pOET1.1 vector. Among them, the pFBDopt vector introduces an Ac-ie-01 expression cassette (SEQ ID NO: 19) to delay the apoptosis of host cells, prolong the protein expression period; and uses an optimized combined promoter p6.9 / p10 (SEQ ID NO: 17) and a cis-activation element hr1 (SEQ ID NO: 18) to enhance the transcriptional activity of the target gene, thereby significantly improving the protein expression amount.

[0028] The present application provides a host cell comprising the above-mentioned recombinant expression vector. The host cell is preferably an insect cell, more preferably an Sf9, Sf21 or High Five cell.

[0029] The present application provides a preparation method of the above-mentioned feline hepatitis virus core antigen virus-like particle, comprising the following steps:

[0030] (1) Provide a recombinant expression vector containing a polynucleotide encoding a DCHcAg protein; (2) Introduce the recombinant expression vector into an insect host cell; (3) Culture the host cell under conditions that allow expression of the DCHcAg protein; (4) Isolate and purify the DCHcAg protein from the culture and allow it to self-assemble into virus-like particles.

[0031] The preparation method can be implemented using the following two approaches: In the first embodiment, using... Baculovirus expression system. The gene encoding DCHcAg (SEQ ID NO:3 or SEQ ID NO:4) was cloned into the optimized donor plasmid pFBDopt to obtain a recombinant donor plasmid; this plasmid was then integrated into the baculovirus Bacmid via transposition to obtain a recombinant baculovirus plasmid; recombinant baculovirus was obtained by transfecting insect cells; the virus was used to infect insect cells for protein expression; and finally, VLPs were purified by ultracentrifugation, ultrafiltration concentration, and sucrose density gradient centrifugation. In the second embodiment, a method was adopted... Baculovirus expression system. The gene encoding DCHcAg (SEQ ID NO:3 or SEQ ID NO:4) was cloned into the donor plasmid pOET1.1 to obtain the recombinant donor plasmid; it was then combined with... Plasmids were co-transfected into insect cells, and recombinant baculovirus was obtained intracellularly via homologous recombination; subsequent protein expression and VLP purification steps were similar to those in the first embodiment. DCHc prepared by the above method... WT The diameter of the VLP is 29-31 nm (e.g., 30.2 ± 2.0 nm), DCHc N149 The diameter of the VLP is 25-27 nm (e.g., 26.0 ± 2.4 nm).

[0032] This invention provides the application of the aforementioned virus-like particles in the preparation of reagents or kits for diagnosing feline hepatitis virus (FHV) infection. The virus-like particles, particularly DCHcWT VLP, can serve as a coating antigen for establishing an indirect ELISA detection method. This method coats an ELISA plate with DCHc VLP, uses feline serum as the primary antibody, and enzyme-labeled anti-feline IgG as the secondary antibody. The presence and level of anti-DCHc antibodies in the serum are determined by detecting the OD value. This method exhibits good specificity and clinical concordance, effectively distinguishing between DCHV infection-positive and DCHV-negative sera.

[0033] This invention provides the use of the aforementioned virus-like particles in the preparation of a vaccine for the prevention of feline hepatitis virus. The vaccine comprises an immunologically effective amount of the aforementioned virus-like particles and a pharmaceutically acceptable adjuvant. In the embodiments, the adjuvant is preferably... Immunogenicity evaluation results showed that, regardless of DCHc WTVLP is also DCHc N149 VLP, DCHc, and DCHc 6 The above is significantly higher than the PBS control group, which proves that it is an excellent vaccine immunogen.

[0034] The application provides application of the virus-like particle as a nanocarrier in construction of a chimeric vaccine. Specifically, a heterologous antigen epitope is inserted into an MIR region (corresponding to between 78th and 79th amino acids) of the DCHcAg, and the heterologous antigen epitope is preferably a cytotoxic T lymphocyte (CTL) epitope of a feline infectious peritonitis virus (FIPV) membrane protein (M) or nucleocapsid protein (N), so that a recombinant chimeric VLP vaccine DCHc WTM DCHc WTN The ELISpot results show that the two chimeric VLPs can induce strong antigen-specific cellular immune responses in domestic cats, produce high levels of IFN-γ, and confirm the feasibility of the DCHc VLP as a multivalent vaccine platform.

[0035] In the examples, the primer sequences are shown in Table 1. If not specifically stated, the primers in the examples are synthesized by Nanjing Kingsway.

[0036] Table 1 Primer sequences

[0037]

[0038] If not specifically stated, the specific techniques or conditions in the examples are all conventional methods or techniques or conditions described in the literature in the art, or are performed according to the product instructions. If not specified, the reagents used are all conventional products that can be purchased through regular channels or prepared according to conventional methods in the art. If not specified, the instruments used are all conventional products that can be purchased through regular channels.

[0039] The following will be described through specific examples.

[0040] Example 1

[0041] The application discloses a DCHcAg as a VLP platform for the first time from the structural biology level, and reveals the theoretical basis and unique advantages of the DCHcAg as a VLP platform. Figure 1 As shown in the figure, the amino acid sequence of the DCHcAg provided by the application is subjected to multiple sequence alignment with a hepatitis B virus core antigen (HBV) and several other hepatitis virus core antigens (DDHV, BHBV, and WHBV, respectively). The alignment result clearly shows that:

[0042] (1) Structural conservation: DCHcAg shares high structural conservation with HBcAg in the secondary structure, with the spatial positions and arrangements of its α-helices (annotated as α1 to α5 in the figure) and β-sheets (annotated as η1 and η2 in the figure) being basically identical. This high degree of structural homology indicates that DCHcAg inherits the inherent and strong self-assembly ability of HBcAg, and can spontaneously form a well-structured icosahedral virus-like particle. This is the theoretical premise for the successful preparation of DCHc VLPs in the present application.

[0043] (2) Key region difference: Although the overall structure is conserved, DCHcAg exhibits significant sequence difference in the key region that determines immunogenicity. In particular, in the major immunodominant region, corresponding to the MIR region between amino acids 78-79, DCHcAg has a unique amino acid sequence (region marked by black letters in the figure). This enables the DCHc VLP-based vaccine to effectively avoid interference from pre-existing immunity against HBcAg in the vaccinated individual, thereby improving vaccine efficacy. On the other hand, this specific sequence may have stronger immunogenicity in felines, achieving species-specific optimization.

[0044] (3) Modification site is clear: The alignment map clearly indicates the modification sites of DCHcAg protein. For example, the MIR region is clearly defined as a flexible region that can accommodate the insertion of exogenous sequences without disrupting the overall structure. This finding provides direct sequence-level design basis for further development of DCHc VLPs as a general nanodelivery carrier in the present application, making it possible to precisely insert heterologous antigen epitopes (such as CTL epitopes of FIPV).

[0045] Example 2 Preparation of DCHcAg VLPs using a baculovirus expression system

[0046] (1) Construction of recombinant baculovirus expression plasmid

[0047] The full-length gene of domestic cat hepatitis virus core antigen (DCHc) (DCHc WT ) and the gene fragment encoding the N-terminal 149 amino acids of DCHc (DCHc N149 ) were obtained by gene synthesis technology (Shenguo Bioengineering). An 8×His tag coding sequence was introduced at the 3' end of the synthetic gene to facilitate subsequent detection and purification. The primers P1-F (SEQ ID NO: 5) and P1-R (SEQ ID NO: 6) for PCR amplification of DCHc WT were designed and synthesized. The primers P2-F (SEQ ID NO: 7) and P2-R (SEQ ID NO: 8) for PCR amplification of DCHc N149 ​The primers P2-F (SEQ ID NO: 7) and P2-R (SEQ ID NO: 8) were introduced with Xho I and Kpn I enzyme sites in the upstream and downstream primers, respectively (see Table 1 for primer sequences). The synthesized gene fragments were used as templates, and PrimeSTAR HS high-fidelity enzyme was used for PCR amplification. The PCR amplification reaction system included: PrimeSTAR HS DNA Polymerase 10 μL, DNA Templete 1 μL, Forward Primer 0.8 μL, Reverse Primer 0.8 μL, and ddH2O supplemented to 20 μL. The PCR amplification reaction program included: 98℃ pre-denaturation for 2 min; followed by 98℃, 10 s, 55℃, 15 s, 72℃, 1 min, for a total of 30 cycles; and finally 72℃ extension for 5 min. After the reaction was completed, the PCR products were verified by 1% agarose gel electrophoresis, and the target band was recovered using a DNA purification kit. As shown in Figure 2 As shown in the following, the donor plasmid pFastBac Dual (purchased from Invitrogen) was optimized by gene synthesis technology (Shenguo Bioengineering), and the pFastBac Dual was modified based on the pFastBac Dual. An optimized combination promoter p6.9 / p10 and a cis-activating element hr1 were used, and an Ac-ie-01 expression cassette was introduced, named pFBDopt.

[0048] The sequence of the p6.9 / 10 combination promoter is: gtcgagtgattgtaaataaaatgtaatttacagtatagtattttaattaatatacaaatgatttgataataattcttatttaactataatatattgtgttgggttgaattaaaggtccgtatgtttaaattgtgtaatttatgtagctgtaatttttaccttattaatattttttacgctttgcattcgacgactgaactcccaaatatatgtttaactcgt (SEQ ID NO: 17).

[0049] The sequence of the cis-activating element hr1 is: gctttacgagtagaattctacttgtaacgcacgattaattatgactcataagctgatgtcatgttttgcacacggctcataaccgaactcgctttacgagtagaattctacgcgtaaaacacaatcaagtatgagtcataagctgatgtcatgttttgcacacggctcataaccgaactggctttacgagtagaattctacttgtaacgcacgatcagtggatgatgtcataaacttatctcatgttttgcacacggctcataacttgagtcataagctgatatcagaagattattaacgtgtttaggtatgactcatttgttttcaaaactgaactcgctttacggatagaattctacttgtaaaacacaatcaggggatgatgtcattatacaaatgatgtcatttgtttttcaaaactaaactcgctttacgggtagaattctacttgtaaaac (SEQ ID NO: 18).

[0050]

[0051] The recovered DCHc WT and DCHc N149 PCR products and double enzyme digestion with plasmid pFBDopt, the enzyme digestion products were verified by 1% agarose gel electrophoresis, and the target band was recovered using a DNA purification kit. The target fragment was ligated with the linearized pFBDopt vector under the action of T4 DNA ligase, transformed into E. coli DH5α competent cells, and plated on LB plates containing ampicillin (100 μg / mL) and incubated at 37°C overnight. Single colonies were picked for amplification, plasmids were extracted, PCR was performed using primers P3-F (SEQ ID NO: 9) and P3-R (SEQ ID NO: 10), and sequencing was performed. The PCR reaction system was as described in Example 2 (1). The PCR amplification reaction program was: pre-denaturation at 98°C for 2 min; followed by 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, for a total of 30 cycles; and finally 72°C for 5 min. The correct recombinant plasmids were named pFBDopt-DCHc WT and pFBDopt-DCHc N149 , respectively. The recombinant plasmids pFBDopt-DCHc WT and pFBDopt-DCHc N149 were verified by PCR, and bands of approximately 615 bp and 492 bp were obtained Figure 3 ).

[0052] (2) Rescue of recombinant baculovirus

[0053] As shown in Figure 4 , the target gene was integrated into the baculovirus genome by homologous recombination with the help of the helper plasmid. The recombinant plasmids pFBDopt-DCHc WT and pFBDopt-DCHc N149 were transformed into E. coli DH10Bac competent cells, respectively, and selected by blue-white spot screening on LB plates containing tri-antibiotics (7 μg / mL Gen, 10 μg / mL Tet, 50 μg / mL Kan) and 100 μg / mL X-gal and 40 μg / mL IPTG, and incubated at 37°C for 36-48 h. White colonies were picked for amplification and recombinant baculovirus plasmids were extracted, and PCR was performed using pUC-M13 universal primers. The correct recombinant baculovirus plasmids were named rBac-optDCHc WT and rBac-optDCHc N149. The correct recombinant baculovirus plasmid was extracted using the Endo-Free Plasmid Maxi Kit. Using Cellfectin® II reagent, 2 μg of the recombinant plasmid was transfected into Sf9 adherent cells with a confluence of more than 80% in a 6-well cell culture plate, and cultured in a 27°C incubator for 96-120 h until a clear cytopathic effect was observed. The cell culture supernatant was collected and centrifuged at 3500 rpm for 20 min at 4°C to remove cell debris, and the supernatant was collected to obtain the P0 generation of recombinant baculovirus stock, which was recorded as rBac-DCHc WT (i.e. rBV-DCHc WT , hereinafter the same) and rBac-DCHc N149 (i.e. rBV-DCHc N149 , hereinafter the same). As shown in Figure 5 , a clear cytopathic effect was observed in Sf9 cells transfected with the recombinant baculovirus plasmid about 120 h, with cell swelling, increased intracellular granules and lysis. The P0 generation of recombinant baculovirus rBac-DCHc WT and rBac-DCHc N149 was inoculated into Sf9 adherent cells with a confluence of more than 80% at a volume ratio of 1:1000, and cultured in a 27°C incubator for 72-96 h until a clear cytopathic effect was observed, and the culture supernatant was collected to obtain the P1 generation of recombinant baculovirus. The P1 generation of recombinant baculovirus was inoculated into Sf9 suspension cells with a viable cell density of 2 x 10 6 cells / mL at a volume ratio of 1:1000, and cultured at 27°C until the cell viability was less than 70%, and the culture supernatant was collected to obtain the P2 generation of recombinant baculovirus. The P2 generation of recombinant baculovirus was subjected to indirect immunofluorescence analysis, using recombinant anti-6 x His tag antibody as the primary antibody, and FITC-labeled goat anti-rabbit IgG H&L as the secondary antibody. As shown in Figure 6 , strong specific green fluorescent signals were observed in Sf9 cells infected with rBV-DCHc WT and rBV-DCHc N149 , while no signals were observed in the uninfected control group (Neg. Ctrl.), confirming the successful expression and localization of the target protein in the cells. The P2 generation of recombinant baculovirus was subjected to SDS-PAGE and Western Blot verification, using HRP Anti-6 x His tag antibody as the detection antibody. As shown in Figure 7 , the rBac-DCHc WT monomer was about 24 kDa, and the rBac-DCHc N149 monomer was about 19 kDa. Bands corresponding to the monomer and dimer were detected at the molecular weights of the monomers and dimers, proving the correct expression of the protein and its ability to form a dimeric structure.

[0054] (3) Purification and observation of DCHc virus-like particles (VLPs)

[0055] P2 generation recombinant baculovirus was inoculated at a volume ratio of 1:10 into live cells at a density of 2×10⁶ cells / year. 6 Sf9 suspension cells (cells / mL) were cultured at 27°C until cell viability dropped below 70%. Cell debris was removed by centrifugation at 3500 rpm, 5000 rpm, and 8000 rpm for 30 min at 4°C, and the supernatant was collected. The viral supernatant was concentrated by centrifugation at 5000 g at 4°C using a 30 KD ultrafiltration tube. VLP was purified using a sucrose density gradient centrifugation method. Sucrose solutions of 20%, 30%, 40%, 50%, and 60% (w / v) were prepared and subjected to discontinuous density gradient centrifugation. The concentrated samples were plated on top of the gradient sucrose solutions and ultracentrifuged at 200,000 × g at 4°C for 4 h. Sucrose solutions at the 20%-30%, 30%-40%, 40%-50%, and 50%-60% interfaces were collected, and 10 mL of PBS was added to each. The samples were then centrifuged at 200,000 × g at 4°C for 4 h to remove the sucrose. The supernatant was discarded, and the precipitate was resuspended in 500 μL of PBS to obtain the purified VLP samples. Protein purity was verified by SDS-PAGE and Western blotting, and the morphology and size of the VLPs were observed by transmission electron microscopy. Results Figure 8 As shown, DCHc WT VLP and DCHc N149 VLPs all exhibited typical virus-like particle structures, with intact morphology and near-spherical shape, demonstrating that the recombinant protein could correctly self-assemble within the cell. Particle size statistical analysis showed that DCHc WT The average hydrodynamic diameter of the VLP is 29.7 ± 2.1 nm, DCHc WT The average diameter of the VLP is 26.1 ± 1.5 nm.

[0056] Example 3 Preparation of DCHcAg VLP using Baculovirus Expression System

[0057] (1) Construction of recombinant baculovirus expression plasmid

[0058] The full-length gene of feline hepatitis virus core antigen (DCHc) was obtained through gene synthesis technology (Sangon Biotech). WT ) and the gene segment encoding its N-terminal 149th amino acid (DCHc N149 An 8×His tag coding sequence was introduced at the 3' end of the synthetic gene to facilitate subsequent detection and purification. A gene for DCHc was designed and synthesized. WTPrimers P4-F (SEQ ID NO:11) and P4-R (SEQ ID NO:12) for PCR amplification were used for DCHc N149 Primers P5-F (SEQ ID NO:13) and P5-R (SEQ ID NO:14) for PCR amplification were introduced into the upstream and downstream primers, respectively. Bam HI and Eco RI restriction sites (primer sequences are shown in Table 1). Using the synthesized gene fragment as a template, PCR amplification was performed using PrimeStar HS high-fidelity enzyme. The PCR reaction system was as described in Example 2 (1). The PCR amplification program was: 98℃ pre-denaturation for 2 min; followed by 30 cycles of 98℃ for 10 s, 55℃ for 15 s, and 72℃ for 1 min; and a final extension at 72℃ for 5 min. After the reaction, the PCR products were verified by 1% agarose gel electrophoresis, and the target band was recovered using a DNA purification kit. [The remaining text appears to be incomplete and requires further context.] Bam HI and Eco RI for recovered DCHc WT and DCHc N149 The PCR product and the pOET1.1 baculovirus vector plasmid were double-digested with enzymes. The digested products were verified by 1% agarose gel electrophoresis, and the target band was recovered using a DNA purification kit. The target fragment was ligated to the linearized pOET1.1 vector using T4 DNA ligase, transformed into E. coli DH5α competent cells, and plated on LB plates containing ampicillin (100 μg / mL) and incubated overnight at 37°C. Single colonies were picked for amplification, plasmids were extracted, and PCR identification was performed using primers P6-F (SEQ ID NO:15) and P6-R (SEQ ID NO:16), followed by sequencing verification. The PCR reaction system was as described in Example 2 (1). The PCR amplification reaction program was as follows: 98°C pre-denaturation for 2 min; followed by 30 cycles of 98°C, 10 s, 55°C, 15 s, 72°C, 1 min; and finally, extension at 72°C for 5 min. The correctly identified recombinant plasmids were named pOET1.1-DCHc. WT and pOET1.1-DCHc N149 The recombinant plasmid pOET1.1-DCHc WT and pOET1.1-DCHc N149 PCR verification yielded bands of approximately 613 bp and 490 bp in size, respectively. Figure 9 ).

[0059] (2) Rescue of recombinant baculovirus

[0060] like Figure 10As shown, the target gene was integrated into the baculovirus genome in Sf9 cells via homologous recombination. Correctly identified pOET1.1-DCHc was extracted using an endotoxin-free plasmid large-scale extraction kit. WT and pOET1.1-DCHc N149 Plasmid. Reference According to the instructions for transfection reagent II, mix 2 μg of recombinant plasmid with... The plasmid was co-transfected into Sf9 adherent cells with a confluence of more than 80% in 6-well cell culture plates and cultured at 27°C for 72-96 h until obvious cytopathic effects appeared. Cell culture supernatant was collected, centrifuged at 3500 rpm for 20 min at 4°C to remove cell debris, and the supernatant was collected to obtain the P0 generation recombinant baculovirus stock solution, designated as rBac-DCHc. WT (i.e., rBV-DCHc) WT (hereinafter the same) and rBac-DCHc N149 (i.e. rBV-DCHc) N149 (The same applies below). From Figure 11 It can be seen that after approximately 96 hours of transfection of Sf9 cells with the recombinant plasmid, a significant cytopathic effect was observed, with cell swelling, intracellular vacuolation, and lysis. The P0 generation recombinant baculovirus rBac-DCHc... WT and rBac-DCHc N149 Inoculate Sf9 adherent cells with confluence of 80% or higher at a volume ratio of 1:1000 and incubate at 27°C for 72-96 h until obvious cytopathic effects appear. Collect the culture supernatant to obtain P1 generation recombinant baculovirus. Inoculate the P1 generation recombinant baculovirus at a volume ratio of 1:1000 into viable cell density of 2×10⁶ cells / year. 6 Sf9 suspension cells (cells / mL) were cultured at 27°C until cell viability fell below 70%. The culture supernatant was collected to obtain P2 generation recombinant baculovirus. The P2 generation recombinant baculovirus was analyzed by indirect immunofluorescence using recombinant anti-6×His-tagged antibody as the primary antibody and FITC-labeled goat anti-rabbit IgG H&L as the secondary antibody. Results are as follows: Figure 12 As shown, in the infection of rBV-DCHc WT With rBV-DCHc N149 A strong, specific green fluorescent signal was observed in the infected Sf9 cells, while no such signal was observed in the uninfected control group (Neg. Ctrl.), confirming the successful expression and localization of the target protein within the cells. P2 generation recombinant baculovirus was used for SDS-PAGE and Western blotting verification, with HRP Anti-6×His tag antibody used as the detection antibody. Results are as follows... Figure 13 As shown, rBac-DCHc WTThe monomer is approximately 24 kDa, rBac-DCHc N149 The monomer is approximately 19 kDa. The target band was detected at the molecular weights of both the monomer and the dimer, proving that the protein was correctly expressed and had the ability to form a dimer structure.

[0061] (3) Purification and observation of DCHc virus-like particles (VLPs)

[0062] P2 generation recombinant baculovirus was inoculated at a volume ratio of 1:10 into live cells at a density of 2×10⁶ cells / year. 6 Sf9 suspension cells (cells / mL) were cultured at 27°C until cell viability dropped below 70%. Cell debris was removed by centrifugation at 3500 rpm, 5000 rpm, and 8000 rpm for 30 min at 4°C, and the supernatant was collected. The viral supernatant was concentrated by centrifugation at 5000×g using a 30 KD ultrafiltration tube. VLP was purified using a sucrose density gradient centrifugation method. Sucrose solutions of 20%, 30%, 40%, 50%, and 60% (w / v) were prepared and subjected to discontinuous density gradient centrifugation. The concentrated samples were plated on top of the gradient sucrose solutions and ultracentrifuged at 200000×g for 4 h at 4°C. Sucrose solutions at the 20%-30%, 30%-40%, 40%-50%, and 50%-60% interfaces were collected, and 10 mL of PBS was added to each. The samples were then centrifuged at 200000×g for 4 h at 4°C to remove the sucrose. Discard the supernatant and resuspend the precipitate in 500 μL of PBS to obtain the purified VLP sample. Protein purity was verified by SDS-PAGE and Western blotting, and the morphology and size of the VLPs were observed by transmission electron microscopy. Figure 14 As shown, DCHc WT VLP and DCHc N149 VLPs all exhibited typical virus-like particle structures, with intact morphology and near-spherical shape, demonstrating that the recombinant protein could correctly self-assemble within the cell. Particle size statistical analysis showed that DCHc WT The average hydrodynamic diameter of the VLP is 30.2 ± 2.0 nm, DCHc WT The average diameter of the VLP is 26.0 ± 2.4 nm.

[0063] Example 4: Application of DCHc VLP as a diagnostic antigen

[0064] Applying DCHc in Example 1 or Example 2 WTVLP was used as the diagnostic antigen and coated onto 96-well microplates at a concentration of 5 μg / mL to establish an indirect ELISA detection method. The test samples were 48 clinical cat serum samples collected from Yantai and Weifang, Shandong Province (Table 2), with SPF cat serum used as a negative control. All serum samples were diluted 1:100 (v / v), and HRP-labeled goat anti-cat IgG (H+L) was used as the secondary antibody for detection. The positive criterion was set as: OD of the test sample... 450 The value should be ≥ the negative control mean + 3 × standard deviation. The specific method is as follows: Use PBS buffer to prepare DCHc... WT Dilute VLP to 5 μg / mL and add 100 μL to each well of a 96-well ELISA plate. Coat overnight at 4°C. Discard the coating solution and wash three times with PBST for 3 min each time. Add 200 μL of casein blocking solution to each well and block at 37°C for 1 h. Discard the blocking solution and wash three times with PBST for 3 min each time. Dilute the test serum and negative control serum 1:100 (v / v) and add 100 μL to each well. Incubate at 37°C for 1 h. Include a blank control (add diluent only). Discard the serum diluent and wash three times with PBST for 3 min each time. Add 100 μL of HRP-labeled goat anti-cat IgG (H+L) diluted 1:5000 as a secondary antibody to each well and incubate at 37°C for 1 h. Discard the secondary antibody diluent and wash three times with PBST for 3 min each time. Add 100 μL of TMB substrate solution to each well and develop at room temperature in the dark for 15 min. Add 100 μL of ELISA stop solution to each well to terminate the reaction, and immediately measure the absorbance (OD value) of each well at 450 nm using a microplate reader to reflect the IgG content in the separated serum sample. Figure 15 As shown, the mean OD of the negative control serum 450 The values ​​were at low levels, and some clinical serum samples showed strong positive reactions, indicating that these samples contained high levels of anti-DCHc specific antibodies. The results of clinical sample testing are summarized in Table 2. Of the total 48 serum samples, 3 were positive, originating from Yantai (numbers Y09 and Y15) and Weifang (number W08), respectively, with a positive detection rate of approximately 6.25%. This result is consistent with the epidemiological survey expectations, demonstrating that this diagnostic method has good specificity and clinical concordance.

[0065] Table 2 Basic Information of Cat Serum Samples to be Tested

[0066]

[0067] Example 5 Immunogenicity evaluation of DCHc VLP

[0068] Two- to three-month-old negative experimental cats were randomly divided into three groups (DCHc) WT VLP group, DCHcN149 VLP group, PBS control group), 3 cats per group. The purified VLPs were mixed with an equal volume of ISA 206 adjuvant to prepare immunogens, 100 μg / mL. Each cat in the immunized group was subcutaneously injected with 1 mL of the immunogen as the primary immunization, and each cat in the control group was injected with an equal volume of the mixture of PBS and adjuvant; 14 days after the primary immunization, each cat was subcutaneously injected with 1 mL of the immunogen as the booster immunization, and each cat in the control group was injected with an equal volume of the mixture of PBS and adjuvant. Blood was collected from the cephalic vein of the forelimb 14 days after the booster immunization, and the serum was separated and stored at -20°C for testing. The level of specific total IgG antibody against DCHc in the serum was detected by indirect ELISA according to the method described in Example 4, and the highest serum dilution fold corresponding to a P / N value ≥ 3 was defined as the antibody end-point titer. The results are shown in Table 1. Figure 16 As shown in Table 1, the DCHc WT The VLP immunized group induced the production of high levels of specific IgG antibodies, and the antibody titer reached about 10 6.7 The DCHc N149 The VLP immunized group also induced a strong humoral immune response, and the antibody titer reached about 10 6.3 Statistical analysis showed that the antibody titers of the two VLP immunized groups were extremely significantly higher than those of the PBS control group, and there was no statistical difference between the two groups (marked as “ns”), which proved that the DCHc N149 The truncated expression did not weaken its ability to induce a high level of humoral immune response.

[0069] Example 6 Application of DCHc VLP as a feline infectious peritonitis CTL epitope vaccine nanocarrier

[0070] By comparing the amino acid sequences of DCHc Ag and HBcAg, the immunodominant CTL epitope of the membrane protein (M) and the CTL epitope of the nucleocapsid protein (N) of the feline infectious peritonitis virus (FIPV) were introduced into the MIR region of DCHc (corresponding to between amino acids 78-79), the amino acid sequence of FIPV M is shown as SEQ ID NO: 20, specifically: vygikmlimwllwpivlalt, and the amino acid sequence of FIPV N is shown as SEQ ID NO: 21, specifically: eplrfdgkippqfqlevnrs. The recombinant virus DCHc WTM and DCHc WTN The vaccine was prepared according to the method described in Example 5. 2-3-month-old negative experimental cats were randomly divided into 3 groups (DCHc WTM VLP group, DCHc WTN(VLP group and PBS control group), with 3 cats in each group. Each cat in the immunization group received a subcutaneous injection of 50 μg / 1 mL of immunogen as a basic immunization, while the control group received an equal volume of a mixture of PBS and adjuvant. Fourteen days after the basic immunization, each cat received a subcutaneous injection of 50 μg / 1 mL of immunogen as a booster immunization, while the control group received an equal volume of the mixture of PBS and adjuvant. Anticoagulated whole blood was collected on day 14 after the second immunization. Peripheral blood mononuclear cells (PBMCs) were isolated using a feline peripheral blood lymphocyte separation kit via density gradient centrifugation. After cell counting, the cells were resuspended in complete RPMI-1640 medium and the cell concentration was adjusted to 2.5 × 10⁻⁶. 6 cells / mL.

[0071] ELISpot Plus: The Cat IFN-γ (HRP) kit was purchased from Mabtech. The experimental procedure was performed according to the manufacturer's instructions with appropriate optimizations. A brief summary of the steps is as follows: Add 100 μL of sterile PBS to each well of the ELISpot plate and incubate at room temperature for 10 min, then discard the PBS. Add 100 μL of anti-cat IFN-γ monoclonal antibody capture antibody diluted in sterile PBS to each well and coat overnight at 4°C. Discard the coating solution and wash the plate three times with sterile PBS. Add 200 μL of complete culture medium to each well and block at room temperature for 2 h. Discard the blocking solution. Add 100 μL of cell suspension (2.5 × 10⁻⁶) to each well. 5 (cells), and add the following stimulants in sequence, and set up 3 duplicate wells simultaneously.

[0072] Table 3 Experimental Groups

[0073]

[0074] Incubate the plate at 37°C in a 5% CO2 incubator for 40–48 h. After incubation, discard the cells and culture medium. Wash three times with PBST, then add 100 μL of biotinylated anti-cat IFN-γ detection antibody to each well and incubate at 37°C for 1 h. After washing, add 100 μL of HRP-labeled streptavidin (diluted 1:1000) to each well and incubate at 37°C for 1 h. Wash three times with PBST, then add 100 μL of AEC substrate chromogenic solution to each well and react at room temperature in the dark until the spots are fully developed but the background is unstained. Rinse with deionized water to stop the reaction. Count the spots using an automated ELISpot spot analyzer. Record the spot-forming units (SFU) formed in each well. Results are as follows: Figure 17 As shown, DCHc WTM Peripheral blood mononuclear cells in the VLP immunization group could be induced to produce a large number of IFN-γ T cells under the stimulation of the corresponding antigen, and their speckle-forming units (SFU) were significantly higher than those in the PBS control group; DCHc WTNVLP immunization groups also elicited strong antigen-specific T cell immune responses, SFU levels were comparable between DCHcAg VLP and DCHc groups, both were significantly higher than background values; PBS control group only presented low background response under antigen stimulation, indicating good specificity of the detection system; phytohemagglutinin (PHA) as a positive control could induce strong IFN-γ secretion, confirming the reliability of cell activity and experimental system. WTM

[0075] In summary, the present application develops an efficient and stable DCHcAg VLP preparation technology, and systematically evaluates its application potential in multiple fields such as diagnosis, prevention and treatment, provides a new solution for the prevention and control of DCH infection, promotes the progress of related technologies in the field of veterinary medicine, and makes a substantial contribution to the health protection of felines.

[0076] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A feline hepatitis virus core antigen virus-like particle, characterized in that, The protein includes a feline hepatitis virus core antigen protein, wherein the feline hepatitis virus core antigen protein comprises the amino acid sequence shown in SEQ ID NO:1, or wherein the feline hepatitis virus core antigen protein comprises the amino acid sequence shown in SEQ ID NO:

2.

2. The method for preparing feline hepatitis virus core antigen virus-like particles according to claim 1, characterized in that, Includes the following steps: The core antigen protein of feline hepatitis virus was expressed in an insect baculovirus expression system.

3. A polynucleotide, characterized in that, The nucleotide sequence encoding the feline hepatitis virus core antigen virus-like particle of claim 1.

4. A recombinant vector, characterized in that, Includes the nucleotide sequence of the feline hepatitis virus core antigen virus-like particle as described in claim 1.

5. The recombinant vector according to claim 4, characterized in that, It also includes one or more of the Ac-ie-01 expression cassette, the combined promoter p6.9 / p10, and the cis-activating element hr1.

6. A host cell, characterized in that, Includes the recombinant vector as described in claim 4 or 5.

7. A vaccine, characterized in that, Includes the feline hepatitis virus core antigen virus-like particles as described in claim 1.

8. A nanocarrier for an epitope vaccine, characterized in that, A heterologous antigen epitope is inserted into the MIR region of the feline hepatitis virus core antigen as described in claim 1.

9. A diagnostic antigen for feline hepatitis virus, characterized in that, Includes the feline hepatitis virus core antigen virus-like particles as described in claim 1.

10. The use of the feline hepatitis virus core antigen virus-like particles of claim 1 in the preparation of products for the prevention, treatment or detection of feline hepatitis virus.

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