Preparation method and application of chicken infectious anemia virus polyepitope chimeric virus-like particle vaccine

Abstract

The application discloses a preparation method and application of a chicken infectious anemia virus multi-epitope chimeric virus-like particle vaccine, wherein the chicken infectious anemia virus chimeric multi-epitope is composed of a universal Th epitope sequence and a VP1 protein B cell epitope sequence in linear series through flexible peptides in sequence, and is inserted into an amino terminal of the chicken infectious anemia virus VP1 protein, and the gene fragment is named Epitope-vp1, as shown in SEQ ID NO. 1. First, the Epitope-vp1 gene fragment is chemically synthesized, then the gene is constructed into a baculovirus vector, a recombinant carrier baculovirus P3 generation virus is bred, and insect cells are infected respectively, and the recombinant chicken infectious anemia virus-like particle is successfully expressed in vitro, which is named as CIAV VLPs. The CIAV VLPs are used as antigens to immunize animals, can stimulate the body to produce specific antibodies against the chicken infectious anemia virus, and have a good preventive effect on the chicken infectious anemia.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a multi-epitope chimeric virus-like particle vaccine for chicken infectious anemia virus and its preparation method. Background Technology

[0002] Chicken infectious anemia (CIA) is an immunosuppressive disease caused by chicken infectious anemia virus (CIAV) of the Circovirus genus in the family Circoviridae. It primarily infects chicks under 20 days old, leading to lymphoid tissue atrophy, aplastic anemia, and often accompanied by other viral, bacterial, or fungal infections, causing significant economic losses to the poultry industry. Currently, there is no specific drug treatment for this disease; early infection control through vaccination is crucial for controlling CIAV. The low viral titer of CIAV in chicken embryos and MSB1 cells makes the production cost of inactivated CIAV vaccines very high, hindering its widespread application in the poultry industry. The use of live CAV vaccines carries the risk of vertical and horizontal transmission, limiting their widespread application. Therefore, timely molecular epidemiological surveys of CIAV, understanding the genomic characteristics of circulating strains, and developing novel genetically engineered vaccines are the main research directions at present.

[0003] Virus-like particle (VLP) vaccines have become a hot topic in modern vaccine research due to their safety, ease of production, and immunogenic properties. VLPs are hollow particles resembling viruses, assembled from one or more proteins. VLPs are structurally highly similar to viruses, do not contain viral genetic material, lack replication capacity and infectivity, and can activate both innate and acquired immune responses in the host. In particular, compared to inactivated vaccines or single viral proteins or peptides, VLP vaccines offer advantages such as economy, safety, high efficacy, and benefits for disease eradication, making them the most likely antigenic form to replace traditional inactivated vaccines. According to domestic and international research, VLPs have achieved significant results in treating various infectious diseases and tumors in humans and animals, and commercially available VLP vaccines for hepatitis B, human papillomavirus, hepatitis E, and porcine circovirus are already available. However, research on the in vitro recombinant expression of chicken infectious anemia virus-like particle vaccines is currently scarce.

[0004] Epitope vaccines generally refer to vaccines prepared by using multiple interdisciplinary fields such as bioinformatics, structural biology, and molecular immunology to screen for effective viral antigenic epitopes, tandem them with spacer sequences to mimic their spatial conformation, express them in vitro using genetic engineering techniques, or synthesize and efficiently express and purify artificial microbial antigenic epitopes before adding immunostimulants. Epitope vaccines have advantages such as ease of production, low cost, high safety, good stability, and no risk of virus shedding. VP1 is also the most important structural protein of CIAV, possessing multiple neutralizing epitopes and being crucial for CIAV replication and transmission. The presence of VP2 protein is a necessary condition for VP1 protein to form the correct conformation; only when VP1 and VP2 proteins are present simultaneously can the body induce the production of antibodies against CIAV. Currently, research on the screening and fusion expression of dominant antigenic epitopes of CIAV structural proteins to form VLPs and their immune mechanisms is still in its infancy both domestically and internationally.

[0005] Therefore, in order to cope with the continuous mutation of CIAV and develop a safe and effective CIA vaccine, this invention designs a method for preparing a multi-epitope chimeric virus-like particle vaccine for chicken infectious anemia virus. Specifically, multiple epitope peptide gene sequences on the VP1 protein of chicken infectious anemia, which are relatively conserved and can induce a neutralizing reaction to produce antibodies, are selected, linked together using a flexible linker, and inserted into the front of the VP1 gene expression frame. These sequences are then self-assembled into virus-like particles using a baculovirus eukaryotic expression system. Summary of the Invention

[0006] In view of this, the present invention provides a multi-epitope chimeric virus-like particle of chicken infectious anemia virus, which can stimulate the body to produce a good immune response and can be used in the preparation of vaccines, and has great development potential. To achieve the above objectives, the present invention adopts the following technical solution: A recombinant transfer vector, using pFastBac-Dual as a backbone, inserts a gene fragment encoding the fusion protein Epitope-VP1 between the EcoRI and SalI restriction sites downstream of the PH promoter, and inserts a gene fragment encoding the VP2 protein between the XhoI and NheI restriction sites downstream of the P10 promoter; the fusion protein Epitope-VP1, from the amino terminus to the carboxyl terminus, sequentially includes the universal Th epitope PADRE, B cell epitopes B1, B2, B3, B4, B5, and chicken infectious anemia virus VP1 protein, with adjacent B cell epitopes linked by flexible peptides, and B cell epitope B5 linked to the VP1 protein by the flexible peptide GGGGS, the nucleotide sequence of the gene encoding the fusion protein Epitope-VP1 is shown in SEQ ID NO:1, and the nucleotide sequence of the gene encoding the VP2 protein is shown in SEQ ID NO:10.

[0007] The gene sequence of the epitope polypeptide Epitope-vp1 is shown in SEQ ID NO: 1; the universal Th epitope is shown in SEQ ID NO: 2; the B cell epitope polypeptide B1 is shown in SEQ ID NO: 3; the B cell epitope polypeptide B2 is shown in SEQ ID NO: 4; the B cell epitope polypeptide B3 is shown in SEQ ID NO: 5; the B cell epitope polypeptide B4 is shown in SEQ ID NO: 6; and the B cell epitope polypeptide B5 is shown in SEQ ID NO: 7.

[0008] A recombinant rod-like particle is obtained by transforming the recombinant transfer vector into Escherichia coli DH10Bac competent cells, followed by blue-white screening and PCR identification, resulting in the recombinant rod-like particle rBac-Epitope-vp1-vp2.

[0009] A recombinant baculovirus is obtained by transfecting the recombinant baculovirus into sf9 insect cells for culture, and harvesting the recombinant baculovirus rEpitope-vp1-vp2 from the culture.

[0010] A method for preparing multi-epitope chimeric virus-like particles of chicken infectious anemia virus using the recombinant baculovirus involves transfecting the recombinant baculovirus into sf9 insect cells for culture, harvesting the recombinant baculovirus and continuously passaged and amplifying it, then inoculating it into High Five insect cells for expression, and finally purifying the chicken infectious anemia virus multi-epitope chimeric virus-like particles from the culture by sucrose density gradient centrifugation.

[0011] Preferably, the method includes the following steps: A universal Th epitope sequence and a VP1 protein B-cell epitope sequence obtained through bioinformatics screening are tandemly inserted into the amino terminus of the chicken infectious anemia virus VP1 protein via a flexible peptide, and the resulting gene fragment is named Epitope-vp1. The gene fragment Epitope-vp1 is then chemically synthesized, as shown in SEQ ID NO: 1. Using the vector pFastBac-Dual as a backbone, the gene fragment Epitope-vp1 is inserted between the restriction enzyme sites EcoRI and SalI, and the vp2 gene fragment is inserted between the restriction enzyme sites XhoI and NheI to obtain the correct recombinant transfer plasmid pFastBac-Dual-Epitope-vp1-vp2.

[0012] The obtained recombinant transfer plasmid pFastBac-Dual-Epitope-vp1-vp2 was transformed into *E. coli* DH10Bac competent cells to obtain recombinant rod particles rBac-Epitope-vp1-vp2. These recombinant rod particles were then transfected into sf9 insect cells, and the expressed recombinant proteins were identified using indirect immunofluorescence and Western blot.

[0013] The supernatant from sf9 insect cells transfected with recombinant baculovirus was collected to obtain the recombinant baculovirus P1 progeny. This progeny was passaged twice more in sf9 cells. Western blot and indirect immunofluorescence were used to detect the expression of the fusion protein. After purification, the formation of virus-like particles was observed under an electron microscope. CIAV multi-epitope chimeric VLPs were prepared using this method. These VLPs are formed by the co-assembly of the fusion protein Epitope-VP1 (SEQ ID NO:8) and the VP2 protein (SEQ ID NO:9).

[0014] Preferably, the amino acid sequence of the fusion protein Epitope-VP1 is shown in SEQ ID NO:8, and the amino acid sequence of the VP2 protein is shown in SEQ ID NO:9.

[0015] A multi-epitope chimeric virus-like particle vaccine for chicken infectious anemia virus, comprising the virus-like particles and a vaccine adjuvant.

[0016] Preferably, the virus-like particles are virus-like particles purified by sucrose density gradient centrifugation.

[0017] More preferably, the vaccine adjuvant is a water-in-oil adjuvant composed of white oil adjuvant and Tween 80.

[0018] The application of the recombinant transfer vector, the recombinant baculovirus, the recombinant baculovirus, and the multi-epitope chimeric virus-like particles of chicken infectious anemia virus in the preparation of drugs for the prevention of chicken infectious anemia.

[0019] A further embodiment of the method for preparing the chicken infectious anemia virus multi-epitope chimeric virus-like particle vaccine includes the following steps:

[0020] (1) The recombinant baculovirus was inoculated into insect cells, and the insect cell supernatant was harvested 3 days later;

[0021] (2) The insect cell supernatant was purified to obtain relatively pure chicken infectious anemia virus-like particles;

[0022] (3) The virus-like particles are mixed with an adjuvant to obtain the CIAV virus-like particle vaccine.

[0023] In a further embodiment, the insect cells in (1) are high five cells.

[0024] In a further embodiment, the purification method in (2) is sucrose gradient density centrifugation.

[0025] In a further embodiment, the adjuvant type in (3) is a white oil adjuvant.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention first utilizes bioinformatics techniques to screen dominant antigenic epitopes of the VP1 protein of chicken infectious anemia virus (TIV). These epitopes, along with universal Th epitopes, are cleverly fused to the amino terminus of the VP1 protein coding sequence using a flexible peptide. The constructed multi-epitope chimeric VLPs combine the advantages of both VLPs and epitope vaccines, and to a certain extent, outperform conventional epitope research expression plasmids and protein forms.

[0028] Currently, there are no reported studies utilizing CIAV VLPs as a presentation platform to display epitopes of the same or different pathogens. This invention leverages the strong safety and immunogenicity of VLPs, and draws upon their ability to repeatedly express or carry antigenic epitopes and small molecules at high density on their surface. For the first time, it simultaneously displays the dominant antigenic epitopes of CIAV VP1 with VP1, constructing chimeric VLPs and studying their immune response mechanism. This provides a theoretical foundation and technical support for the future development of safe, efficient, and broad-spectrum novel multivalent vaccines.

[0029] This invention establishes superior prevention and control technologies from the perspective of the ultimate eradication of animal diseases. It develops different vaccine formulations using high-purity virus-like particles as the dominant antigen, which not only saves time, effort, and costs in production and application, but also maximizes the stimulation of animal immune responses. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings required in the description of the embodiments or the prior art will be briefly introduced below.

[0031] Figure 1 This is a schematic diagram of the fusion protein Epitope-VP1 in Example 1 of the present invention;

[0032] Figure 2 For the amplification and identification of the target gene Epitope-vp1, M: DNA marker (DL2000); 1-2: Epitope-vp1 gene amplification results.

[0033] Figure 3 For the amplification and identification of the target gene vp2, M: DNA marker (DL2000); 1-2: VP2 gene amplification results.

[0034] Figure 4To identify the target gene Epitope-vp1 in Bacmid for picking white spots, M: DNA marker (DL5000); 1-2: Epitope-vp1 gene amplification results.

[0035] Figure 5 This is a schematic diagram of morphological changes in sf9 cells after infection with the P3 generation recombinant virus rEpitope-vp1-vp2. A: sf9 cells infected with recombinant baculovirus rEpitope-vp1-vp2 for 72 hours; B: uninfected sf9 cells.

[0036] Figure 6 To identify the expression of Epitope-VP1 protein in sf9 cells infected with the P3 generation recombinant virus rEpitope-vp1-vp2 by Western blot, M: protein marker; 1: sf9 cell supernatant; 2: sf9 cell pellet.

[0037] Figure 7 To indirectly detect the expression of Epitope-VP1 protein in sf9 cells using immunofluorescence, A: sf9 cells were infected with the P3 generation recombinant virus rEpitope-vp1-vp2; B: uninfected blank sf9 cells.

[0038] Figure 8 Transmission electron microscopy identification results of VLPs in chicken infectious anemia. Detailed Implementation

[0039] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0040] Example 1: Screening for suitable VP1 protein epitope peptides for chicken infectious anemia

[0041] Through extensive and in-depth research, based on the sequence characteristics and structural analysis of the VP1 protein of chicken infectious anemia virus, the inventors analyzed the B-cell epitopes and physicochemical properties of the VP1 protein, and for the first time screened and fused it with the VP1 protein for expression. At the same time, they studied its immunoprotective effect and found that it can stimulate the body to produce strong cellular immunity and humoral immunity. Therefore, this invention has broad application prospects in the prevention or treatment of chicken infectious anemia. Based on this, the inventors completed this invention.

[0042] Specifically, based on the analysis of the structural information of the VP1 protein in chicken infectious anemia, this invention identifies the linear / conformal B-cell epitopes of the VP1 protein and, taking into account the structural surface characteristics of the VP1 protein, screens and determines representative B-cell epitope sequences of the VP1 protein. These epitopes are then tandemly linked with universal Th epitopes (such as PADRE sequences) to form novel tandem epitope peptides. This improves upon the problem of low immunogenicity of ordinary peptides while also taking into account the advantages of high specificity and good safety of ordinary peptides.

[0043] The whole genome sequence of the typical chicken infectious anemia virus strain JS22101784 (GenBank No. PP355008), which has been published in the GenBank database and isolated in my country, was analyzed. The dominant linear antigenic epitope of the VP1 protein was selected, linked by a flexible linker peptide, and inserted into the N-terminus of the VP1 protein coding sequence. The nucleotide sequence corresponding to the recombinant protein fragment was codon-optimized according to the expression preferences of baculoviruses, and double restriction enzyme sites were designed at both ends. The optimized nucleotides were synthesized and ligated to construct the recombinant plasmid.

[0044] The specific plan is as follows:

[0045] (1) The amino acid sequence of the VP1 protein of chicken infectious anemia virus strain JS22101784 was selected, and its B cell antigenic epitope prediction analysis was performed using the online tools ImmunomedicineGroup (http: / / imed.med.ucm.es / Tools / antigenic.pl) and SVMTriP (http: / / sysbio.un1.edu / SVMTriP / prediction.php). Based on the combined prediction results, the amino acid sequence shown in SEQ ID NO:3 in the antigenic epitope set was selected and listed as follows:

[0046] SEQ ID NO: 3 RWHHLKRLRRRYKFRHRRRQRYRRRAFRKAFHNPRPGTYSVRLPNPQS SEQ ID NO: 4 QGEYLTRRDDVKYSSDHQNRWRKGEQPMT SEQ ID NO: 5 TGKMRPDEQQYPAMPPDPPIITSTTAQGT SEQ ID NO: 6 WSFPPGQRSVSRRSFNHHKARGAGDPKGQ SEQ ID NO: 7 QLGNRQRPYPWDVNWANSTMYW

[0047] (2) The nucleotide sequences corresponding to the epitope peptides obtained above were linked by inserting flexible linker peptides in the order (general Th epitope - SEQ ID NO: 3 - SEQ ID NO: 4 - SEQ ID NO: 5 - SEQ ID NO: 6 - SEQ ID NO: 7) to construct an antigenic polypeptide with strong antigenicity, good hydrophilicity and flexibility. The specific amino acid sequence information is as follows:

[0048] AKFVAAWTLKAAAKKRWHHLKRLRRRYKFRHRRRQRYRRRAFRKAFHNPRPGTYSVRLPNPQSKKQGEYLTRRDDVKYSSDHQNRWRKGEQPMTKKTGKMRPDEQQYPAMPPDPPIITSTTAQGTKKWSFPPGQRSVSRRSFNHHKARGAGDPKGQKK QLGNRQRPYPWDVNWANSTMYW (SEQ ID NO: 8).

[0049] (3) The selected tandem epitope peptide was inserted into the amino terminus of chicken infectious anemia virus VP1 protein, and the corresponding gene fragment was named Epitope-vp1. The gene fragment Epitope-vp1 was optimized according to the insect cell preferred codon and chemically synthesized, as shown in SEQ ID NO: 1.

[0050] SEQ ID NO: 1 sequence information:

[0051]

[0052] Subsequently, the Phyre2 portal was used to model the structure of the fusion protein, and the model was scored using key parameters such as confidence, linear coverage, and homology (>90%). The results showed that the inserted exogenous cellular epitopes did not disrupt the native conformation of the VP1 protein backbone, while also contributing to the expected antigen presentation effect.

[0053] Example 2: Construction of recombinant baculovirus expressing VP1 and VP2 genes

[0054] Start codons were added to the amino terminus of the Epitope-VP1 and VP2 protein gene sequences, and a 6×His tag and a stop codon were added to the carboxyl terminus of the Epitope-VP1 protein gene sequence to obtain the optimized and modified chicken infectious anemia virus Epitope-VP1 and VP2 protein genes.

[0055] The insect cell-baculovirus expression vector pFastBac-Dual has two promoters, PH and P10, which can be used to construct vectors containing two target gene fragments. The optimized chicken infectious anemia Epitope-VP1 and VP2 protein gene fragments were sent to Nanjing Genscript Biotech Co., Ltd. for synthesis. The Epitope-VP1 protein gene fragment was inserted downstream of the PH promoter, between the EcoRI and SalI restriction sites, and the VP2 protein gene fragment was inserted downstream of the P10 promoter, between the XhoI and NheI restriction sites.

[0056] The above-mentioned positive bacterial cultures, which have been identified as correct, were used to extract plasmids using a small-volume plasmid extraction kit to obtain the corresponding recombinant transposon vectors. The vectors were then transformed into competent DH10Bac Escherichia coli cells. The specific steps are as follows: (1) Add 8 μl of the recombinant transposon vector plasmid to 100 μl of DH10Bac competent cells, mix gently, and place on ice for 30 min; (2) Immediately place the mixture in a 42℃ constant temperature water bath for 45 s heat shock; (3) Continue to place the mixture on ice for 2 min; (4) Add 800 μl of SOC medium solution and mix by inversion; (5) Culture on a shaker at 37℃ and 225 r / min for 5 h; (6) Take 50 μl of the above bacterial culture and spread it on LB plates with three antibiotics (kanamycin + gentamicin + tetracycline), and incubate at 37℃ for 48 h. Pick white colonies with a sterilized pipette tip and gently pipette them into 500 μl of liquid LB medium. Perform PCR identification using the M13 universal vector primers. The M13 primer sequences refer to the primers that have been validated and preserved in this invention; M13F: 5'-AGCGGATAACAATTTCACACAGGA-3'; M13R: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3';

[0057] The expected amplified fragment is 4300bp.

[0058] The reaction system is as follows:

[0059] 2×Taq PCR Master Mix 12.5 μl, M13 universal forward / reverse primers 1 μl each, RNase-free water 8.5 μl

[0060] 2 μl of bacterial solution

[0061] Total volume 25μl

[0062] The PCR reaction conditions are as follows:

[0063] After the recombinant rod-shaped PCR reaction was completed, 10 μl of the PCR product was loaded for nucleic acid electrophoresis analysis. The identification results are as follows: Figure 4 As shown. Positive bacteria were selected and streaked again on LB solid medium, then incubated at 37°C for 48 hours. White single colonies were picked and inoculated into 500 μl of LB liquid medium (containing kanamycin, gentamicin, and tetracycline resistance), and the culture was streaked three times in the same manner for purification. High-purity Bacmid recombinant band granule DNA of the above positive clones was obtained using the standard alkaline lysis method.

[0064] Example 3: Generation and Identification of Recombinant Baculovirus

[0065] Sf9 insect cells were revived. Before transfection, the Sf9 cells needed to be in the logarithmic growth phase and have a cell viability of over 90%. Transfection was performed using the prepared high-purity Bacmid recombinant band-granule DNA: rBac-Epitope-vp1-vp2. The specific steps are as follows:

[0066] (1) Add 1 μg rBac-Epitope-vp1-vp2 to 100 μl of serum-free Grace medium, mix thoroughly, and name it Solution A; add 8 μl of liposomes to 100 μl of serum-free Grace medium, name it Solution B, and let it stand at room temperature for 5 min; (2) Add Solution A to B and mix well, let it stand at room temperature for 10-15 min; then remove the monolayer of sf9 cells that has grown to about 90% in the six-well plate from the cell culture medium, wash gently twice with serum-free Grace medium, and add 2 ml of serum-free Grace medium; (3) Add the mixture of Solution A and B to the sf9 cell culture dish and let it stand at 27℃ for 5 h; (4) Discard the cell culture supernatant, add 2 ml of Grace medium containing 10% fetal bovine serum, and let it stand at 27℃ for 3-6 days; (5) Observe the cytopathic effect. After 3 days, obvious cytopathic effects were observed in the cells (see attached). Figure 5As shown in the figure, collect the cell culture medium, centrifuge at 2000 rpm for 5 min to remove cell debris, collect the supernatant to obtain the recombinant baculovirus P1 substitute, and store in a separate device at -80℃.

[0067] Preparation of P2 generation virus strain:

[0068] To 2×10 6 Add 200 μl of P1 generation virus strain to each well of SF9 cells / cells, culture at 27°C for 72 h, collect 2 ml of virus-containing culture medium from each well, centrifuge at 2000 rpm for 5 min to discard cells and debris; this is the P2 generation virus, aliquoted and stored at -80°C. Preparation of P3 generation virus strain: Add 2 × 10⁻⁶ cells / well of SF9 cells to each well of SF9 cells / well of SF9 cells. 6 Add 200 μl of P2 generation virus strain to each well of sf9 cells and culture the cells at 27°C for 72 h. Collect 2 ml of virus-containing culture medium from each well, centrifuge at 2000 rpm for 5 min and discard the cells and debris. This is the P3 generation virus, which is used to infect insect cell lines as seed virus for preparing chicken infectious anemia virus-like particles. After aliquoting, store at -80°C.

[0069] The specific steps for indirect immunofluorescence (IFA) identification are as follows:

[0070] (1) Inoculate sf9 cells into a 24-well cell plate and culture at 27°C. When the cells cover 80% of the bottom of the well, inoculate the cells with P3 generation recombinant baculovirus and continue to culture for 72 hours until the cells show obvious lesions. Discard the supernatant and wash each well twice with PBST.

[0071] (2) Fix cells with anhydrous ethanol for 15 min, then wash twice with PBST;

[0072] (3) Add 5% skim milk and block at 37°C for 30 min, then wash twice with PBST;

[0073] (4) Add mouse-derived his-tag monoclonal antibody and incubate (1:500 dilution) at 37°C for 1 h, then wash twice with PBST;

[0074] (5) Add FITC-labeled goat anti-mouse IgG (1:500 dilution), incubate at 37°C for 1 h, wash twice with PBST, and observe the fluorescence under an inverted fluorescence microscope.

[0075] IFA identification results showed that 72 hours after infection of sf9 cells with recombinant baculovirus, extremely strong green specific fluorescence appeared in the cytoplasm of the recombinant protein Epitope-VP1, indicating that the recombinant protein Epitope-VP1 was expressed (see attached). Figure 7 (As shown).

[0076] Western blot identification, the specific steps are as follows:

[0077] (1) Prepare SDS-PAGE gel, with a sample loading volume of 20 μl per well, and transfer it to a PVDF membrane after electrophoresis;

[0078] (2) Place the PVDF membrane in a 5% skim milk solution and seal it overnight at 4°C;

[0079] (3) Primary antibody incubation: Wash the membrane three times with PBST for 10 min each time, and continue to incubate with mouse his-tag monoclonal antibody (1:1000 dilution) at 37°C for 1 h;

[0080] (4) Wash the membrane three times with PBST for 10 min each time, and incubate with HRP-labeled goat anti-mouse IgG (1:10000 dilution) at 37℃ for 1 h.

[0081] (5) Wash the membrane three times with PBST for 10 minutes each time, develop the color using Beyotime Biotech ECL colorimetric kit, and take a picture for record.

[0082] Western blot analysis showed that the recombinant protein Epitope-VP1 produced a specific protein band of approximately 70 kDa, consistent with the expected molecular weight, while no band was detected in sf9 cells infected with the empty vector (see attached image). Figure 6 (As shown).

[0083] The purification of CIAV VLPs is carried out by the following steps: Recombinant baculovirus rEpitope-VP1 is used to infect high five cells at a ratio of 1%, and the cells are cultured at 27°C for 72 hours. The cell culture is then harvested.

[0084] Centrifuge the cell culture at 2000 rpm for 10 min to remove the supernatant, harvest the cell pellet, wash it again with PBS, add cell lysis buffer at 1 / 5 volume of the original solution to the pellet, and resuspend the pellet with a shaker.

[0085] After precipitating the precipitate in an ice bath for 20 minutes, shake for 15 seconds, continue in an ice bath for 10 minutes, centrifuge at 4°C and 12,000 rpm for 15 minutes, and collect the supernatant.

[0086] Sucrose density gradient centrifugation was performed at three gradients: 20%, 40%, and 60%. The centrifugation was carried out at 160,000g for 3 hours. The bands between 20% and 40% were collected. PBS was added, and the centrifugation was continued at 160,000g for 2 hours to remove sugar. The precipitate was collected, which is the purified CIAV-VLPs. The precipitates were aliquoted and stored at -80℃ for later use.

[0087] Transmission electron microscopy examination

[0088] A 20 μl solution of purified CIAV VLPs protein was dropped onto a copper grid, stained, and excess liquid was removed. The morphology and size of the CIAV VLPs were then observed under a transmission electron microscope. The results showed that the protein solution contained a large number of spherical virus-like particles with a diameter of approximately 20-30 nm (see attached image). Figure 8 As shown in the image, the morphology of VP1 is similar to that of the natural particles of chicken infectious anemia virus (CAV), indicating that VP1 is expressed. Since VP2 is a scaffold protein of the virus, also known as a helper protein, it is the most conserved protein in CAV and mainly plays a role in viral particle replication and assembly, ensuring that VP1 can fold correctly and assisting it in producing virus-neutralizing antibodies in the host. Figure 8 The confirmation of VP1 expression indicates that VP2 was also expressed.

[0089] Example 4: Evaluation of the Immunization Effect of CIAV VLPs

[0090] 4.1 Vaccine preparation

[0091] After determining the protein concentration of CIAV-VLPs collected by sucrose density gradient centrifugation, the aqueous phase was prepared by mixing purified protein and Tween 80 at a volume ratio of 94:6. The aqueous phase and oil phase were mixed and emulsified at a volume ratio of 1:2 to prepare a subunit vaccine. The Epitope-VP1 protein content per milliliter of vaccine was adjusted to be 0.4 mg for subsequent animal immunization.

[0092] 4.2 Detection of vaccine components and properties

[0093] Dosage form: Water-in-oil (W / O)

[0094] Adjuvant composition: white mineral oil

[0095] Vaccine adjuvant: Tween 80

[0096] Vaccine emulsification: The aqueous phase is prepared by mixing purified protein and Tween 80 at a volume ratio of 94:6. The aqueous phase and oil phase are mixed and emulsified at a volume ratio of 1:2 to prepare a subunit vaccine.

[0097] Vaccine characteristics test: Take a clean pipette, draw a small amount of the finished vaccine and slowly drip it into cold water. Except for the first drop, the rest should not spread. The test is qualified.

[0098] Sterility test: Spread a small amount of vaccine onto a TSA plate and incubate at 37°C for 18 hours until no colonies grow.

[0099] Stability test: Add 10ml of vaccine to a centrifuge tube, centrifuge at 3000rpm for 15min. If the amount of aqueous phase separated at the bottom of the tube does not exceed 0.5ml, the test is qualified.

[0100] Vaccine safety trials

[0101] Five-day-old SPF chicks were subjected to single-dose vaccination, repeated single-dose vaccination, and single overdose vaccination trials. Results showed that all chicks were in good spirits after vaccination, with no significant abnormalities in feed and water intake. Absorption at the injection site was good, with no redness, swelling, or scab formation. No systemic adverse reactions were observed, and body temperature did not rise significantly. These results indicate that the subunit vaccine prepared in this invention has good safety performance.

[0102] 4.3 Vaccine efficacy testing

[0103] Experimental plan:

[0104] Laboratory animals: SPF chicks

[0105] Experimental groups: SPF chicks were divided into 3 groups: immunization group 1 (also known as Epitope-VP1 protein group, immunized by injection of the subunit vaccine prepared in Example 4.1), immunization group 2 (also known as VP1 protein group, immunized by injection of CIAV-VLPs vaccine prepared in Example 3 of CN121592720A), and control group, as shown in Table 1.

[0106] Table 1 SPF Chick Immunization Groups

[0107] Immunization group 10 200 Immunization group 2: 10, 200; Control group: 10 /

[0108] Immunization method: intramuscular injection in the leg, the immunization dose is 200μg / animal in the immunization group, once every 2 weeks, for a total of 2 times, and the control group is immunized with the same dose of lysed supernatant of healthy sf9 cells.

[0109] Two weeks after the second immunization, blood was collected from the subpterygoid vein for serum-specific antibody detection. After agglutination, the collected blood was centrifuged at 6000 rpm / min to separate the serum, which was then tested using the IDEXX avian anemia antibody detection kit. Results showed that the average S / N ratio in immunization group 1 was 0.166, with 10 / 10 positive; the average S / N ratio in immunization group 2 was 0.185, with 10 / 10 positive; and the average S / N ratio in the negative control group was 0.945, with all groups being negative. An S / N value ≤ 0.6 was defined as positive. This demonstrates that subunit vaccines can stimulate a good immune response in the body.

[0110] 4.4 Immunoprotective efficacy test of recombinant subunit vaccine against SPF chicks

[0111] Experimental protocol

[0112] Laboratory animals: SPF chicks

[0113] Challenge virus: Chicken infectious anemia virus challenge dose: 5×10 4 EID 50

[0114] Thirty 2-week-old SPF chickens were randomly divided into three groups of 10 each. Group 1 (also known as the Epitope-VP1 protein group) was immunized with the subunit vaccine prepared in Example 4.1 via intramuscular injection in the leg, at a dose of 200 μg / chicken, administered twice every two weeks. Group 2 (also known as the VP1 protein group) was immunized with the CIAV-VLPs vaccine prepared in Example 3 (CN121592720A), using the same method and dosage as Group 1. The control group received the same dose of lysed supernatant from healthy sf9 cells. Fourteen days after the second immunization, both groups of chickens were challenged with a virulent strain preserved in our laboratory, and morbidity and mortality were observed over 14 days. The results showed that 9 out of 10 chickens in immunized group 1 were protected, with a protection rate of 90%, and 8 out of 10 chickens in immunized group 2 were protected. In the non-immunized challenge control group, 0 chickens were protected, with a protection rate of 0%. The results indicate that a 200 μg immunization dose of Epitope-VP1 protein can induce a specific immune response in the experimental chickens and protect 90% of the experimental chickens from chicken infectious anemia virus. Compared with immunization with VP1 protein, the immune protection effect is significantly improved.

[0115] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A recombinant transfer vector, characterized in that, Using pFastBac-Dual as a backbone, a gene fragment encoding the fusion protein Epitope-VP1 was inserted between the EcoRI and SalI restriction sites downstream of the PH promoter, and a gene fragment encoding the VP2 protein was inserted between the XhoI and NheI restriction sites downstream of the P10 promoter. The fusion protein Epitope-VP1 contains, from the amino terminus to the carboxyl terminus, the universal Th epitope PADRE, B cell epitopes B1, B2, B3, B4, B5, and chicken infectious anemia virus VP1 protein. Adjacent B cell epitopes are linked by flexible peptides, and B cell epitope B5 is linked to the VP1 protein by the flexible peptide GGGGS. The nucleotide sequence of the gene encoding the fusion protein Epitope-VP1 is shown in SEQ ID NO:1, and the nucleotide sequence of the gene encoding the VP2 protein is shown in SEQ ID NO:

10.

2. A recombinant rod-like particle, characterized in that, The recombinant rod-like particles rBac-Epitope-vp1-vp2 were obtained by transforming the recombinant transfer vector described in claim 1 into Escherichia coli DH10Bac competent cells, followed by blue-white screening and PCR identification.

3. A recombinant baculovirus, characterized in that, The recombinant baculovirus rEpitope-vp1-vp2 described in claim 2 is obtained by transfecting the recombinant baculovirus into sf9 insect cells and culturing them.

4. A method for preparing multi-epitope chimeric virus-like particles of chicken infectious anemia virus using the recombinant rod particles described in claim 2, characterized in that, The recombinant baculovirus was transfected into sf9 insect cells for culture. After harvesting and continuously passaged for amplification, it was inoculated into High Five insect cells for expression. Chicken infectious anemia virus multi-epitope chimeric virus-like particles were obtained from the culture by sucrose density gradient centrifugation.

5. A multi-epitope chimeric virus-like particle of chicken infectious anemia virus, characterized in that, It is prepared by the method described in claim 4 and is formed by co-assembly of the fusion protein Epitope-VP1 shown in SEQ ID NO:8 and the VP2 protein shown in SEQ ID NO:

9.

6. The virus-like particle according to claim 5, characterized in that, The amino acid sequence of the fusion protein Epitope-VP1 is shown in SEQ ID NO:8, and the amino acid sequence of the VP2 protein is shown in SEQ ID NO:

9.

7. A multi-epitope chimeric virus-like particle vaccine for chicken infectious anemia virus, characterized in that, Includes the virus-like particles and vaccine adjuvants as described in claim 5 or 6.

8. The vaccine according to claim 7, characterized in that, The virus-like particles were purified by sucrose density gradient centrifugation.

9. The vaccine according to any one of claims 7 to 9, characterized in that, The vaccine adjuvant is a water-in-oil adjuvant composed of white oil adjuvant and Tween 80.

10. The use of the recombinant transfer vector of claim 1, the recombinant baculovirus of claim 2, the recombinant baculovirus of claim 3, and the multi-epitope chimeric virus-like particle of chicken infectious anemia virus of claim 5 in the preparation of a drug for preventing chicken infectious anemia.

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