Recombinant rabbit hemorrhagic disease virus vp60 protein, vaccine and application thereof

Abstract

The application provides a recombinant rabbit hemorrhagic disease virus VP60 protein, a vaccine and application thereof, and belongs to the technical field of biotechnology.A strong immunogenic non-neutralizing epitope (monoclonal antibody A3C recognition epitope, amino acid sequence DGMDPG, aa 28-33 of VP60) in the N terminal of VP60 is replaced by a neutralizing B cell epitope (HBGAs receptor binding epitope, amino acid sequence NPISQV) to construct a VP60-TD recombinant protein.The VP60-TD recombinant protein constructed in the application can generate higher levels of antibodies of VP60 and HBGAs neutralizing epitopes, and the replaced A3C epitope can be used as a detection marker for the differentiation of rabbit hemorrhagic disease vaccine immunization and virus infection, thereby providing a new technical product for the prevention and control of rabbit hemorrhagic disease.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant rabbit hemorrhagic disease virus VP60 protein, a vaccine, and its applications. Background Technology

[0002] Rabbit hemorrhagic disease (RHD) is a highly contagious and deadly disease caused by rabbit hemorrhagic disease virus (RHDV), severely impacting the rabbit industry. Infected rabbits typically exhibit acute hepatitis, hepatocellular necrosis, apoptosis, and disseminated intravascular coagulation (DIC). There are two genotypes of RHDV: RHDV1 (GI.1) and RHDV2 (GI.2), both currently co-circulating in my country. The VP60 protein is the main structural protein of RHDV, capable of self-assembling in vitro into virus-like particles (VLPs) with a morphology similar to natural RHDV virus particles. Simultaneously, the VP60 protein can induce the production of neutralizing antibodies in animals, serving as the main immunoprotective antigen for RHDV and a primary target molecule in domestic and international research on genetically engineered subunit vaccines; however, its immunogenicity needs further improvement. Meanwhile, young rabbits infected with GI.1 type RHDV do not die and are characterized by carrying and shedding the virus, making it difficult to distinguish between vaccine-immunized rabbits and wild-type virus-infected rabbits. Existing vaccine and antibody detection methods cannot differentiate between rabbit hemorrhagic disease vaccine immunization and viral infection. Summary of the Invention

[0003] This invention provides a recombinant rabbit hemorrhagic disease virus VP60 protein, a vaccine, and its applications. The recombinant rabbit hemorrhagic disease virus VP60 protein can produce antibodies with higher levels of VP60 and HBGAs neutralizing epitopes, and its substituted A3C epitope can be used as a detection marker for the identification of rabbit hemorrhagic disease vaccine immunization and viral infection.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0005] This invention provides a recombinant rabbit hemorrhagic virus VP60 protein, which is a VP60-TD recombinant protein obtained by replacing the A3C epitope in the N-terminus of VP60 with the HBGAs epitope; the amino acid sequence of the recombinant rabbit hemorrhagic virus VP60 protein is shown in SEQ ID No. 1.

[0006] This invention provides the application of the recombinant rabbit hemorrhagic disease virus VP60 protein in the preparation of vaccines for the prevention or treatment of rabbit hemorrhagic disease.

[0007] This invention provides the application of the recombinant rabbit hemorrhagic disease virus VP60 protein in the preparation of products that distinguish between rabbit hemorrhagic disease virus infection and vaccine immunization.

[0008] This invention provides a recombinant rabbit hemorrhagic virus VP60 protein vaccine, comprising the recombinant rabbit hemorrhagic virus VP60 protein.

[0009] Preferably, it also includes a vector for which the recombinant rabbit hemorrhagic virus VP60 protein is pharmacologically acceptable.

[0010] Preferably, the content of the recombinant rabbit hemorrhagic virus VP60 protein is not less than 25 μg.

[0011] The present invention provides a gene encoding recombinant rabbit hemorrhagic virus VP60 protein, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0012] The present invention provides a recombinant expression vector, cell line or recombinant bacteria containing the gene encoding the recombinant rabbit hemorrhagic virus VP60 protein.

[0013] This invention provides the application of the recombinant expression vector, cell line, or recombinant bacteria in the preparation of products for the prevention or treatment of rabbit hemorrhage.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] This invention replaces the highly immunogenic non-neutralizing site (A3C recognition epitope, amino acid sequence DGMDPG, 28-33aa) at the N-terminus of VP60 with a neutralizing B-cell epitope (HBGAs receptor-binding epitope, amino acid sequence NPISKV) to construct the VP60-TD recombinant protein. The VP60-TD recombinant protein, based on the HBGAs receptor-binding site-substituted rabbit hemorrhagic disease epitope, can generate higher levels of antibodies against both VP60 and HBGAs neutralizing epitopes. The substituted A3C epitope can serve as a detection marker for differentiating between rabbit hemorrhagic disease vaccine immunization and viral infection, thus providing a new technological product for the prevention and control of rabbit hemorrhagic disease. Attached Figure Description

[0016] Figure 1 SDS-PAGE electrophoresis and Western Blot identification of VP60 NTA1-4 proteins (M: protein marker; 1-4: SDS-PAGE of VP60 NTA1-4; 5-8: Western Blot of VP60 NTA1-4).

[0017] Figure 23 shows a schematic diagram of the recombinants (the white solid rectangle represents the VP60 protein; the gray box represents the A3C epitope, whose amino acid sequence is DGMDPG; the black box represents the HBGAs epitope, whose amino acid sequence is NPISQV. VP60: Rabbit hemorrhagic symptom virus VP60 protein; VP60-TD: Recombinant protein obtained by replacing the A3C epitope of the rabbit hemorrhagic symptom virus VP60 protein with an HBGAs epitope; VP60-CR: Recombinant protein obtained by inserting an HBGAs epitope into the start site of the rabbit hemorrhagic symptom virus VP60 protein; VP60-QS: Recombinant protein obtained by deleting the A3C epitope of the rabbit hemorrhagic symptom virus VP60 protein).

[0018] Figure 3 Baculovirus recombinant transfer vector pFastBac TM 1-VP60-TD, pFastBac TM 1-VP60-CR, pFastBac TM Identification of 1-VP60-QS (M: DL5000 DNA Marker; 1-2: pFastBac) TM 1-VP60-TD; 3~4: pFastBac TM 1-VP60-CR; 5~6: pFastBac TM 1-VP60-QS; 7: Negative control).

[0019] Figure 4 Diagram illustrating the construction of recombinant Bacmid.

[0020] Figure 5 Identification of baculovirus recombinant shuttle vectors Bacmid-VP60-TD, Bacmid-VP60-CR, and Bacmid-VP60-QS (M: DL5000 DNA Marker; 1: Bacmid-VP60-TD; 2: Bacmid-VP60-CR; 3: Bacmid-VP60-QS; 4: Bacmid-VP60 positive control; 5: negative control).

[0021] Figure 63 shows the construction of recombinant baculovirus BAC-VP60.

[0022] Figure 7 Microscopic observation of normal Sf9 cells and transfected diseased Sf9 cells (A: normal Sf9 cells; B or C or D: diseased Sf9 cells after successful transfection with Bacmid-VP60-(TD or CR or QS)).

[0023] Figure 8RT-PCR was used to identify the recombinant virus (M: DL2000 DNA Marker; 1-3: Sf9 cells infected with three recombinant baculoviruses; 4: BAC-VP60 positive control; 5: negative control).

[0024] Figure 9 Indirect immunofluorescence detection of recombinant baculovirus protein expression: (A) Normal Sf9 insect cells; (B) Sf9 insect cells infected with recombinant baculovirus VP60-TD; (C) Sf9 insect cells infected with recombinant baculovirus VP60-CR; (D) Sf9 insect cells infected with recombinant baculovirus VP60-QS.

[0025] Figure 10 Western blot was used to identify the expression of VP60-TD, VP60-CR, and VP60-QS proteins (M: protein marker; 1-3: VP60-TD, VP60-CR, and VP60-QS protein samples; 4: normal Sf9 cell samples; 5: VP60 positive control).

[0026] Figure 11 Electron microscopic observation of RHDVVP60 protein and recombinant protein virus-like particles (A): RHDVVP60 virus-like particles; (B): Recombinant protein VP60-TD virus-like particles; (C): Recombinant protein VP60-CR virus-like particles; (D): Recombinant protein VP60-QS virus-like particles.

[0027] Figure 12 SDS-PAGE electrophoresis identification of purified protein (M: protein marker; 1: VP60-TD; 2: VP60-CR; 3: VP60-QS; 4: PBS sample after washing and elution).

[0028] Figure 13 SDS-PAGE electrophoresis of BSA standard solution and purified HBGAs and A3C epitope proteins (M: protein marker; 1: 5 μL 100 μg / mL BSA standard solution; 2: 10 μL 100 μg / mL BSA standard solution; 3: 15 μL 100 μg / mL BSA standard solution; 4: 10 μL purified HBGAs epitope protein; 5: 10 μL purified A3C epitope protein).

[0029] Figure 14 Anti-VP60 antibody OD in the serum of mice in each group 450 value.

[0030] Figure 15 Anti-HBGAs antibody OD in the serum of mice in each group 450 value.

[0031] Figure 16Anti-A3C antibody OD in the serum of mice in each group 450 value. Detailed Implementation

[0032] This invention provides a recombinant rabbit hemorrhagic disease virus (TBV) VP60 protein, which is a VP60-TD recombinant protein obtained by replacing the A3C epitope at the N-terminus of VP60 with an HBGAs epitope. The amino acid sequence of the recombinant rabbit hemorrhagic disease virus VP60 protein is: MEGKARTAPQGEAAGTATTASVPGTTTNPI (SEQ ID No. 1). The amino acid sequence of the A3C epitope in this invention is DGMD PG (SEQ ID No. 3), located at amino acid sequences 28-33 of the VP60 sequence; the amino acid sequence of the HBGAs epitope is NPISQV (SEQ ID No. 4).

[0033] This invention provides the application of the recombinant rabbit hemorrhagic disease virus VP60 protein in the preparation of vaccines for the prevention and / or treatment of rabbit hemorrhagic disease. Immunizing animals with the recombinant rabbit hemorrhagic disease virus VP60 protein of this invention can produce higher levels of antibodies against VP60 and HBGAs neutralizing epitopes, effectively preventing infection with rabbit hemorrhagic disease virus.

[0034] This invention provides the application of the recombinant rabbit hemorrhagic disease virus VP60 protein in the preparation of products that distinguish between rabbit hemorrhagic disease virus infection and vaccine immunization. This invention uses the A3C epitope substituted by the recombinant rabbit hemorrhagic disease virus VP60 protein as a detection marker for the differentiation between rabbit hemorrhagic disease vaccine immunization and viral infection.

[0035] This invention provides a recombinant rabbit hemorrhagic disease virus (TBV) VP60 protein vaccine, comprising the aforementioned recombinant TBV VP60 protein. The recombinant TBV VP60 protein vaccine of this invention also includes a pharmacodynamically acceptable carrier for the recombinant TBV VP60 protein. The carrier of this invention includes aluminum hydroxide gel adjuvant and other feasible adjuvants such as mineral oil adjuvant, propolis adjuvant, etc. The content of the recombinant TBV VP60 protein of this invention is not less than 25 μg, preferably not less than 50 μg.

[0036] The present invention provides a gene encoding recombinant rabbit hemorrhagic virus VP60 protein, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0037] This invention provides a recombinant expression vector, cell line, or recombinant bacterium containing the gene encoding the recombinant rabbit hemorrhagic disease virus VP60 protein. The recombinant expression vector is a recombinant baculovirus shuttle vector, Bacmid-VP60-TD, Bacmid-VP60-CR, or Bacmid-VP60-QS; the cell line is BAC-VP60-TD, BAC-VP60-CR, or BAC-VP60-QS.

[0038] This invention provides the use of the recombinant expression vector, cell line, or recombinant bacteria in the preparation of products for the prevention and / or treatment of rabbit hemorrhage.

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0040] Example 1: Identification of the A3C epitope site in the VP60 NTA region

[0041] 1. Construction of recombinant plasmids

[0042] A series of gene sequences of VP60 NTA region overlapping peptides were designed and ligated into the pGEX-4T-1 vector by Nanjing GenScript Biotech Co., Ltd. The gene sequences and corresponding amino acid sequences are shown in Table 1.

[0043] Table 1 Gene synthesis and corresponding amino acid sequences

[0044]

[0045] 2. Identification of the A3C recognition epitope of monoclonal antibody

[0046] The synthesized recombinant expression plasmid pGEX-4T-1-VP60 NTA1-4 was transformed into E. coli BL21(DE3) competent cells. After positive bacteria were induced to express the recombinant protein VP60 NTA1-4 by 0.5 mM IPTG, the protein was transferred to a PVDF membrane after SDS-PAGE electrophoresis and blocked with 5% skim milk. The monoclonal antibody A3C, which recognizes the VP60 NTA region, was used as the primary antibody and goat anti-mouse HRP-IgG (1:10000) as the secondary antibody. Western blotting confirmed that the precise epitope recognized by the A3C monoclonal antibody was DGMDPG.

[0047] like Figure 1 The results showed that VP60 NTA1, VP60 NTA2 and VP60 NTA4 proteins had positive bands, while VP60NTA3 had no band. Therefore, the precise amino acid site recognized by the monoclonal antibody A3C was determined to be DGMDPG, and the corresponding nucleotide sequence was GACGCATGGATCCTGGT (SEQ ID No. 13).

[0048] Example 2: Identification of Recombinant Shuttle Vectors

[0049] 1. Nucleic acid sequence analysis

[0050] Homologous sequence alignment and secondary structure analysis ultimately identified the relevant substitution, insertion, and deletion sites in the NTA region, and experiments were designed accordingly (see Table 2).

[0051] Table 2 Experimental Design Groups

[0052]

[0053] This study inserted HBGAs epitopes (amino acid sequence NPISKV) at the N-terminus of RHDVVP60, replaced the A3C epitope (amino acid sequence DGMDPG, 28–33 aa) with HBGAs epitopes (amino acid sequence NPISKV), and deleted the A3C epitope (amino acid sequence DGMDPG). Figure 2 Three recombinants were constructed as shown.

[0054] 2. Primer design

[0055] Primers were designed using Primer 5.0 software based on the RHDV Anhui strain (serial number: FJ794180) from the GenBank database, including the HBGAs epitope (base sequence: AACCCTATCTCCCAGGTT (SEQ ID No. 14)) and the A3C epitope (base sequence: GACGGCATGGATCCTGGT), and synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0056] Table 3 Primer names and sequences

[0057]

[0058] 3. Amplification of the target gene fragment

[0059] Using the recombinant plasmid pFastBac preserved in our laboratory TM Using 1-VP60 as a template, PCR reactions were performed with TD-F / R, CR-F / R, and QS-F / R primers to amplify VP60 with HBGAs recognizing epitopes replacing the A3C recognizing epitope, HBGAs recognizing epitope insertion, and A3C recognizing epitope deletion. The PCR reaction program was: 95℃ for 30 s; 95℃ for 15 s, 65℃ for 15 s, 72℃ for 7 min, 30 cycles; 72℃ for 5 min. The PCR products were identified on a 1.2% agarose gel. After electrophoresis at 120V for 30 min, an electrophoretic band of approximately 6000 bp was obtained (e.g., ...). Figure 3 PCR-positive products were recovered, and ligated and transformed using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.). The resulting positive bacterial cultures were identified using pFastBac-F / R primers and then sequenced to obtain correctly substituted, inserted, and deleted baculovirus recombinant transfer vectors, which were named pFastBac, respectively. TM 1-VP60-TD, pFastBac TM 1-VP60-CR, pFastBac TM 1-VP60-QS.

[0060] 4. Obtaining and identifying recombinant baculovirus shuttle vectors

[0061] (1) Construction and extraction of recombinant baculovirus shuttle vector

[0062] Recombinant transfer vector pFastBac TM 1-VP60-TD, pFastBac TM 1-VP60-CR, pFastBac TM1-VP60-QS was transformed into DH10 Bac competent cells containing the shuttle vector Bacmid. After incubation at 37°C in LB broth with shaking for 2 h, a portion was spread onto LB plates containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 20 μg / mL X-gal, and 50 μg / mL IPTG. The plates were incubated overnight at 37°C. White single colonies were picked and passaged once more on the same LB plates. White single colonies were picked again and inoculated into LB broth containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline. The plates were incubated overnight at 37°C with shaking (200 rpm). The three Bacmid plasmids were extracted using the Omega D2156-01 BAC / PAC large plasmid extraction kit (Beijing Jiehui Bogao Biotechnology Co., Ltd.). pFastBac TM The reconstruction of 1-VP60 and Bacmid is shown in [link to documentation]. Figure 4 .

[0063] (2) Identification of recombinant baculovirus shuttle vector

[0064] Using the three extracted recombinant Bacmid plasmids as templates, PCR identification was performed using M13-F / R primers. A control group of Bacmid plasmids without the target gene was also set up. The PCR reaction system was as follows: 45 μL of TSINGKE TSE101 Gold Mix (green), 2 μL of 10 μmol / L upstream primer M13-F, 2 μL of 10 μmol / L downstream primer M13-R, and 1 μL of template, for a total volume of 50 μL. The mixture was briefly centrifuged and then subjected to PCR amplification: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 20 s, 72℃ extension for 90 s, 30 cycles; and 72℃ extension for 5 min.

[0065] The results showed that the positive amplification product was approximately 4000 bp in size, while the negative control amplification product was approximately 300 bp in size, indicating successful acquisition of the recombinant baculovirus shuttle vectors Bacmid-VP60-TD, Bacmid-VP60-CR, and Bacmid-VP60-QS (e.g., ...). Figure 5 ).

[0066] 5. Transfection of Sf9 insect cells

[0067] Sf9 cells were transfected in 24-well plates using Lipofectamine 3000 transfection reagent (Invitrogen) from liposomes, following the kit instructions. The steps are as follows:

[0068] (1) Transfect cells when they reach 70%–90% confluence (adherence density of 0.5–2 × 10⁶ cells / well in 24-well tanks). 5 ).

[0069] (2) Dilute 1.5 μL / well of Lipofectamine 3000 reagent with 25 μL / well serum-free and antibiotic-free Grace complete culture medium (GIBCO) and mix thoroughly.

[0070] (3) Dilute 1 μL / well of DNA (0.5-5 μg / μL) (recombinant baculovirus shuttle vector Bacmid-VP60-TD, Bacmid-VP60-CR, or Bacmid-VP60-QS) with 25 μL / well serum-free and antibiotic-free Grace complete culture medium, mix well, and prepare DNA premix. Then add 1 μL / well of 2 μg / μL LP3000 reagent and mix thoroughly.

[0071] (4) Add diluted DNA to each tube of diluted Lipofectamine 3000 reagent at a ratio of 1:1, with 25 μL of diluted DNA and 25 μL of diluted Lipofectamine 3000 reagent per well.

[0072] (5) After centrifugation at 500-1000 r / min, incubate at room temperature for about 10 min;

[0073] (6) Add the well-mixed DNA-liposome complex into the cells under aseptic conditions;

[0074] (7) Incubate cells at 27℃ for 5–7 days and analyze transfected cells.

[0075] Cells were observed under a microscope every 24 hours after transfection until obvious pathological features such as increased cell diameter and rounded shape appeared. The cell supernatant was then collected by centrifugation and used as the recombinant baculovirus stock solution, named BAC-VP60-TD, BAC-VP60-CR, and BAC-VP60-QS, respectively, and stored at 4°C. The construction procedure is described below. Figure 6 .

[0076] Recombinant shuttle vectors Bacmid-VP60-TD, Bacmid-VP60-CR, and Bacmid-VP60-QS were transfected into Sf9 monolayer insect cells. Four days later, cell division ceased, cell diameter and intercellular spaces increased, and the refractive index of each cell increased. Normal cells, except for an increase in density, showed no significant changes, indicating successful transfection. Figure 7 ).

[0077] 6. Passaging and Preliminary Identification of Recombinant Baculoviruses

[0078] (1) Passage of recombinant baculovirus: The recombinant baculovirus stock solutions prepared above were inoculated into Sf9 cells with a density of 90% to 95% and in good condition to obtain second-generation recombinant baculoviruses BAC-VP60-TD, BAC-VP60-CR and BAC-VP60-QS.

[0079] (2) RT-PCR identification of recombinant baculovirus: Second-generation Sf9 cells inoculated with recombinant baculoviruses BAC-VP60-TD, BAC-VP60-CR and BAC-VP60-QS were collected, and total RNA was extracted from the cells using the conventional Trizol method and identified by reverse transcription-polymerase chain reaction (RT-PCR).

[0080] Reverse transcription was performed according to the instructions for the TransScript Uni One-Step gDNARemoval and cDNA Synthesis SuperMix kit.

[0081] Table 4 Reverse Transcription System

[0082]

[0083] After mixing and centrifuging the reaction complex, incubate it in a metal bath at 42°C for 30 min. The product is the recombinant baculovirus cDNA, which should be stored at -20°C.

[0084] Table 5 PCR reaction system

[0085]

[0086] The obtained cDNA was used as a template for PCR amplification: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 45 s, 55℃ annealing for 45 s, 72℃ extension for 2 min, 30 cycles; 72℃ extension for 10 min.

[0087] The results showed an electrophoretic band of 1740 bp, consistent with the size of the target gene (e.g., ...). Figure 8 The results indicate that the genes VP60-TD, VP60-CR, and VP60-QS are transcribed in Sf9 insect cells.

[0088] 7. Identification of Expression Products

[0089] (1) Indirect immunofluorescence assay (IFA)

[0090] Sf9 cells were pre-cultured in 24-well cell culture plates to a density of 85-90%, and then inoculated with second-generation recombinant baculoviruses BAC-VP60-TD, BAC-VP60-CR, and BAC-VP60-QS, respectively. 24 hours after infection, the culture medium was discarded, and the cells were washed twice with PBS. A methanol:acetone fixative solution, pre-stored at -20°C, was added, and the cells were incubated at 4°C for 1 hour. The cells were then washed three times with PBS, and a 1:50 dilution of anti-rabbit hemorrhagic disease virus monoclonal antibody 1D4 was added, and the cells were incubated at 37°C for 1 hour. After three washes with PBS, a 1:100 dilution of goat anti-mouse FITC-IgG was added, and the cells were incubated at 37°C for 1 hour. The cells were then washed three times with PBS, and the cell plates were observed and photographed under a fluorescence microscope.

[0091] The results showed that cells infected with the three recombinant baculoviruses all exhibited strong specific fluorescence (e.g., Figure 9 This indicates that VP60-TD, VP60-CR, and VP60-QS proteins are effectively expressed.

[0092] (2) Protein electrophoresis (SDS-PAGE): The purified recombinant baculoviruses of each group were inoculated into Sf9 insect cells with a growth density of 90% at a volume ratio of 1%. After observing obvious lesions in the cells under a microscope, the cells were collected by centrifugation, resuspended in sterile PBS buffer and washed 3 times, dissolved in 1 mL sterile PBS, and subjected to repeated freeze-thaw cycles. The cells were then lysed by sonication, centrifuged at 3000 r / min for 5 min at room temperature, and the supernatant was collected. 5× loading buffer was added according to the ratio, and the mixture was in a metal bath at 100℃ for 10 min. 25 μL was centrifuged and taken as the SDS-PAGE electrophoresis sample.

[0093] (3) Immunoblotting (Western Blot): Semi-dry transfer method was used. After SDS-PAGE electrophoresis, the gel was semi-dry transferred to PVDF membrane, blocked with 5% skim milk at 4°C overnight, washed 3 times with PBST, then monoclonal antibody 1D4 diluted 1:1000 was added, and reacted at 37°C for 1 h. After washing 3 times with PBST, goat anti-mouse HRP-IgG diluted 1:10000 was added, and reacted at 37°C for 1 h. After washing 3 times with PBST, color development was performed according to the ECL colorimetric kit instructions, and the image was photographed and saved.

[0094] The results showed that Sf9 cells infected with the three recombinant baculoviruses exhibited distinct protein bands at approximately 60 kDa (e.g., ...). Figure 10 The size was consistent with the expected size, indicating that VP60-TD, VP60-CR, and VP60-QS proteins were effectively expressed.

[0095] (4) Hemagglutination test (HA): Sf9 cells and their cultures inoculated with three recombinant baculoviruses were harvested, frozen and thawed three times, centrifuged at 10,000 r / min for 5 min, and 50 μL of the supernatant was taken into 50-well U-shaped hemagglutination plates. After serial dilution with sterile PBS, 50 μL of 1% human "O" type red blood cell suspension diluted with sterile PBS was added to each well. Normal cell culture supernatant and diseased rabbit liver homogenate were used as controls. After shaking and incubation at 4℃ for 1 h, the results were observed to determine the hemagglutination titers of the three expressed proteins.

[0096] The results showed that, compared with the control group where the hemagglutination titer of normal cell culture was 0, the hemagglutination titer of diseased rabbit liver homogenate was 1:256, and the hemagglutination titer of VP60 protein was 1:1024, all three recombinant proteins had high hemagglutination activity (see Table 6), indicating that the three recombinant proteins were efficiently expressed in Sf9 cells.

[0097] Table 6. Protein and hemagglutination titers for each group

[0098]

[0099] 8. Electron microscopic observation of recombinant proteins

[0100] Take an appropriate amount of recombinant protein samples identified by SDS-PAGE and Western Blot for electron microscopy observation of VLP formation. Drop the three recombinant protein samples onto a copper grid, allow to adsorb for 2 min, remove residual sample with filter paper, and fix with 2% phosphotungstic acid staining solution for 2 min. After drying at room temperature for 5 min, observe the formation of recombinant protein virus-like particles using an H-7650 transmission electron microscope.

[0101] Electron microscopy revealed that the expressed proteins could all form virus-like particles (VLPs) with a size of 35–40 nm and a morphology and structure similar to the VP60 protein of rabbit hemorrhagic disease virus, indicating that none of the three recombinant proteins affected the assembly of virus-like particles (e.g., Figure 11 ).

[0102] Example 3: Preparation of RHDV Recombinant Baculovirus Vaccine

[0103] 1. Vaccination

[0104] Recombinant baculoviruses BAC-VP60-TD, BAC-VP60-CR, BAC-VP60-QS, and control BAC-VP60 were inoculated at a ratio of 1% into High Five insect cells suspended in serum-free Sf-900 II SFM medium. The cells were cultured at 26°C with shaking and observed continuously for 4–5 days. When cytopathic effects were observed to be greater than 85% under a microscope, the cell culture was harvested and stored at -20°C.

[0105] 2. Purification of recombinant protein using erythrocyte adsorption-release method

[0106] (1) Aldehydeation of human type O erythrocytes with glutaraldehyde: Human type O erythrocytes were washed three times with PBS buffer (0.1 mol / L, pH 7.0–7.2, the same below) and prepared into a 10% suspension. An equal volume of 1.25% glutaraldehyde was slowly added to the 10% erythrocyte suspension (while shaking to ensure thorough mixing), and the mixture was incubated at 37°C for 1 hour. The aldehyde-treated erythrocytes were then washed three times with PBS buffer and finally prepared into a 50% erythrocyte suspension.

[0107] (2) Aldehyde-modified erythrocytes adsorb recombinant proteins: Add 10 mL of 50% erythrocyte suspension to 100 mL of cell culture and shake in a shaker at 4°C for 2 h;

[0108] (3) Recombinant protein released from aldehyde-treated erythrocytes: Centrifuge the suspension at 8000 r / min for 5 min at 4℃, discard the supernatant, wash the precipitate three times with pre-cooled PBS buffer, add 2 mL of PBS buffer, vortex to mix, incubate at 37℃ for 1 h (shaking several times during this period), centrifuge at 8000 r / min for 10 min at 37℃ (this can be repeated multiple times to increase the release rate), and the supernatant is the purified recombinant protein, which can be stored at -20℃ for later use.

[0109] 3. Preparation of Immunogens

[0110] A small amount of purified recombinant protein was subjected to SDS-PAGE electrophoresis and Coomassie brilliant blue staining to observe the size of the purified protein and the presence of impurities. The results showed that a distinct protein band appeared at approximately 60 kDa (e.g., Figure 12 The presence of no obvious contaminants indicates that the antigen was relatively pure after purification by aldehyde-purified erythrocytes. The concentrations of the three recombinant proteins and VP60 protein were determined using a BCA kit, and the final concentrations were: VP60-TD (0.67 mg / mL), VP60-CR (2.01 mg / mL), VP60-QS (1.37 mg / mL), and VP60 (0.955 mg / mL). The samples were stored at -20℃.

[0111] 4. Vaccine preparation

[0112] The purified VP60-TD, VP60-CR, VP60-QS and VP60 antigens were diluted with PBS to 100 μg / mL, inactivated with 2‰ formaldehyde solution, and then mixed with aluminum hydroxide gel adjuvant at a mass ratio of 9:1 to prepare VP60-TD, VP60-CR, VP60-QS and VP60 inactivated vaccines.

[0113] Example 4 Preparation of coated antigen

[0114] The A3C epitope (DGMDPG) and HBGAs epitope (NPISQV) were linked into the pGEX-4T-1 vector, and the expression plasmid was synthesized by Nanjing Genscript Biotech Co., Ltd.

[0115] The synthesized recombinant expression plasmids pGEX-4T-1-A3C and pGEX-4T-1-HBGAs were transformed into E. coli BL21(DE3) competent cells. Positive bacteria were induced to express glutathione (GSH)-tagged A3C and HBGAs recombinant proteins after 0.5 mM IPTG induction. After purification with glutathione magnetic beads, the GSH-tagged coating antigen was subjected to SDS-PAGE electrophoresis with BSA standard solution and stained with Coomassie Brilliant Blue. After staining, the gray values ​​were calculated using ImageJ software, and the corresponding protein bands on the gel were relatively quantified (e.g., ...). Figure 13 The concentrations of HBGAs protein and A3C protein were determined to be 490 μg / mL and 195 μg / mL, respectively, and stored at -20℃.

[0116] Example 5: Immune response level induced by the antigen in mice

[0117] 1. Grouping and immunization of experimental mice

[0118] Immunization schedule: immunize 3 times, with an interval of 14 days between each immunization, and collect blood from the tail every 7 days until enucleation of the eyeball for blood collection on the 42nd day.

[0119] Immunization groups: The experiment was divided into 5 groups: VP60-TD, VP60-CR, VP60-QS, VP60 (prepared in Example 3) and PBS. Eight female ICR mice aged 7-8 weeks were randomly assigned to each group, and 50 μg of antigen was injected subcutaneously into the back of the neck of each mouse (see Table 7).

[0120] Table 7. Grouping of mice

[0121]

[0122]

[0123] 2. Detection of VP60-specific antibodies in the serum of immunized mice

[0124] The levels of VP60-specific antibodies in the serum of mice immunized in five groups (VP60-TD, VP60-CR, VP60-QS, VP60, and PBS) were detected using an indirect ELISA method coated with VP60 protein. The specific steps of the method are as follows:

[0125] (1) VP60 protein expressed in insect cells and purified from glutaraldehyde-treated erythrocytes was used as the coating antigen. The antigen was diluted to a final concentration of 0.5775 μg / mL with 0.05 M coating buffer (pH = 9.6). 100 μL of the antigen was added to each well to coat a 96-well ELISA plate, and the plate was incubated overnight at 4°C. Each well was washed three times with 300 μL of PBST for 5 min each time. 200 μL of 5% skim milk was added to each well as blocking buffer, and the plate was incubated at 37°C for 2 h. Each well was washed three times with 300 μL of PBST for 5 min each time.

[0126] (2) Dilute the serum of each group of mice with PBST at a ratio of 1:100. Add 100 μL of diluted mouse serum to each well and incubate at 37°C for 1 h. Wash each well three times with 300 μL of PBST for 5 min each time. Add 100 μL of goat anti-mouse HRP-IgG diluted 1:10000 with PBST to each well and incubate at 37°C in the dark for 1.5 h. Wash each well three times with 300 μL of PBST for 5 min each time. Add 100 μL of TMB chromogenic solution to each well and develop the color at room temperature in the dark for 10 min. Add 50 μL of 2M H2SO4 stop solution to each well and detect the absorbance at a wavelength of 450 nm.

[0127] Depend on Figure 14 It can be seen that the antibody levels in the experimental group mice increased after the first, second, and third immunizations. After the third immunization, the P / N values ​​of the three recombinant proteins were all ≥2.1, indicating that the antibodies were all positive, suggesting that all three recombinant proteins could induce a humoral immune response in mice. The ability to induce antibody levels after immunization was TD > CR > QS > VP60. Significant difference analysis of the data in each group revealed that the antibody level in the TD group was significantly higher than that in the CR and QS groups (P < 0.05), while there was no significant difference between the CR and QS groups. These results indicate that the TD group was superior to the CR and QS groups and could induce a greater degree of VP60 antibody secretion.

[0128] 3. Detection of HBGAs-specific antibodies in the serum of immunized mice

[0129] The levels of HBGAs-specific antibodies against neutralizing epitopes in the serum of five groups of immunized mice were detected using an indirect ELISA method with HBGAs protein-coated plates. The method steps are as follows:

[0130] (1) The HBGAs epitope protein purified from glutathione magnetic beads was used as the coating antigen. The coating antigen was diluted to a final concentration of 2 μg / mL with 0.05 M coating buffer (pH = 9.6). 100 μL of the antigen was added to each well to coat a 96-well ELISA plate and incubated overnight at 4°C. Each well was washed three times with 300 μL of PBST for 5 min each time. 200 μL of 5% skim milk was added to each well as blocking buffer and incubated at 37°C for 2 h. Each well was washed three times with 300 μL of PBST for 5 min each time.

[0131] (2) Same as step (2) in step 2.

[0132] Depend on Figure 15 It can be seen that the levels of anti-HBGAs antibodies in B cells of TD group mice showed a significant increasing trend after the first and second immunizations, and the antibodies remained stable at a high level after the third immunization, with a P / N value ≥2.1, indicating a positive result. The CR, QS, and VP60 groups showed a slight increase after three immunizations. After the third immunization, some mice in the CR group had a P / N ≥2.1, and the HBGAs antibody level was weakly positive. The HBGAs antibody levels in the QS and VP60 groups were negative after the third immunization. Significant difference analysis of the data for each group revealed that the HBGAs antibody level in the TD group was significantly different from that in the CR and QS groups (P < 0.01); the difference between the CR group and the QS group was significant (P < 0.05). These results indicate that the VP60-TD recombinant protein can induce high levels of anti-HBGAs epitope antibodies in mice. The VP60-CR recombinant protein induces lower levels of anti-HBGAs epitope antibodies in mice.

[0133] 4. Detection of non-neutralizing epitope A3C-specific antibodies in the serum of immunized mice

[0134] The levels of A3C-specific antibodies against non-neutralizing epitopes in the serum of five groups of immunized mice were detected using an indirect ELISA method with an A3C protein-coated plate. The method steps are as follows:

[0135] (1) The A3C epitope protein purified from glutathione magnetic beads was used as the coating antigen. The coating antigen was diluted to a final concentration of 1.155 μg / mL with 0.05 M coating buffer (pH = 9.6). 100 μL of the antigen was added to each well to coat a 96-well ELISA plate and incubated overnight at 4°C. Each well was washed three times with 300 μL of PBST for 5 min each time. 200 μL of 5% skim milk was added to each well as blocking buffer and incubated at 37°C for 2 h. Each well was washed three times with 300 μL of PBST for 5 min each time.

[0136] (2) Same as step (2) in step 2.

[0137] Depend on Figure 16It can be seen that the levels of B-cell non-neutralizing epitope A3C antibody in most mice in the CR group increased significantly after the third immunization, with a P / N value ≥ 2.1, indicating a positive A3C antibody level, but there were significant individual differences between groups. The TD and QS groups showed negative A3C antibody levels after the third immunization, while the VP60 group showed a weakly positive A3C antibody level with a P / N value ≥ 2.1. Significant difference analysis of the data from each group after the second immunization revealed a significant difference between the CR and VP60 groups (P < 0.05). This indicates that the VP60-CR recombinant protein can induce high levels of anti-A3C epitope antibodies in mice, while VP60-TD and VP60-QS do not cause changes in A3C antibody levels in mice.

[0138] Example 6: Rabbit Protection Experiment

[0139] Immunization and challenge procedure: Immunize once, and on day 14, all experimental rabbits were challenged with 1 mL of RHDVWF strain (HA titer 1:256). The rabbits were observed for 7 consecutive days, and the clinical changes and mortality of the challenged rabbits were recorded.

[0140] Immunization groups: The experiment was divided into 5 categories: VP60-TD inactivated vaccine, VP60-CR inactivated vaccine, VP60-QS inactivated vaccine, VP60 inactivated vaccine and PBS. Among them, VP60-TD, VP60-CR, VP60-QS and VP60 inactivated vaccines were further divided into three immunization dose groups: low, medium and high. Five susceptible two-month-old rabbits were randomly assigned to each group and subcutaneously injected with 25 μg / rabbit (0.25 mL / rabbit), 50 μg / rabbit (0.5 mL / rabbit) and 100 μg / rabbit (1 mL / rabbit) of the vaccine (prepared in Example 3) (see Table 8).

[0141] Table 8. Grouping of Experimental Rabbits

[0142]

[0143] Two-month-old susceptible New Zealand rabbits were immunized with vaccines prepared from three recombinant proteins, with the prepared VP60 vaccine used as a control. After 14 days, the rabbits were challenged to test the immunoprotective effect of the vaccines prepared from the three recombinant proteins on the rabbits.

[0144] The results showed that rabbits in the VP60-TD, VP60-CR, and VP60 immunization groups (low, medium, and high doses) achieved 100% protection, while rabbits in the VP60-QS immunization group (low dose) did not achieve 100% protection. All rabbits in the control group injected with PBS died within 36 hours (see Table 9). The deceased rabbits exhibited typical clinical symptoms, including hepatomegaly and positive hemagglutination results (see Table 10). RT-PCR detection of the livers of the deceased rabbits was performed using specific primers RHDV1-VP60 (upstream primer: TATTCTGGGAACAACTCCAC (SEQ ID No. 27); downstream primer: AACAGTCCGGTTGGATTTTG (SEQ ID No. 28) and RHDV2-VP60 (upstream primer: CCCTGGAAGCAGTTCGTCAAAC (SEQ ID No. 29); downstream primer: GATTGTCAACAAGGTCTGACAG (SEQ ID No. 30)). The results showed that the target band was present at a size of 347 bp in both PBS electrophoresis samples, indicating positive RHDV1 nucleic acid (see Table 10). These results indicate that the expressed VP60-TD and VP60-CR recombinant proteins possess good immunogenicity.

[0145] Table 9 Results of the Virus Challenge Protection Experiment

[0146]

[0147] Table 10 Results of Detection of Dead Rabbits

[0148]

[0149] In summary, this invention constructed three recombinant proteins. After purification and inactivation, these three recombinant proteins were added with adjuvants to prepare vaccines. The results showed that vaccines prepared with VP60-TD antigen and VP60-CR antigen, administered at low, medium, and high doses, could resist lethal doses of virulent virus in New Zealand rabbits. Regarding the immune response level induced by the vaccine antigen, the recombinant VP60-TD antigen, with the rabbit hemorrhagic epitope replacing the HBGAs receptor binding site, produced higher levels of antibodies against VP60 and HBGAs neutralizing epitopes. The replaced A3C epitope can also serve as a detection marker for differentiating between rabbit hemorrhagic disease vaccine immunization and viral infection, thus providing a new technological product for the prevention and control of rabbit hemorrhagic disease.

[0150] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant rabbit hemorrhagic disease virus VP60 protein, characterized in that, The recombinant rabbit hemorrhagic virus VP60 protein is the VP60-TD recombinant protein obtained by replacing the monoclonal antibody A3C recognition epitope in the N-terminus of VP60 with the HBGAs receptor binding epitope. The amino acid sequence of the recombinant rabbit hemorrhagic virus VP60 protein is shown in SEQ ID No.

1.

2. The use of the recombinant rabbit hemorrhagic disease virus VP60 protein as described in claim 1 in the preparation of a vaccine to prevent rabbit hemorrhagic disease.

3. A recombinant rabbit hemorrhagic disease virus VP60 protein vaccine, characterized in that, Includes the recombinant rabbit hemorrhagic virus VP60 protein as described in claim 1.

4. The recombinant rabbit hemorrhagic disease virus VP60 protein vaccine as described in claim 3, characterized in that, The vaccine also includes a vector for the recombinant rabbit hemorrhagic virus VP60 protein that is pharmacologically acceptable.

5. The recombinant rabbit hemorrhagic disease virus VP60 protein vaccine as described in claim 3, characterized in that, The content of the recombinant rabbit hemorrhagic virus VP60 protein is not less than 25 μg.

6. A gene encoding the VP60 protein of recombinant rabbit hemorrhagic disease virus, characterized in that, The nucleotide sequence of the gene encoding the recombinant rabbit hemorrhagic virus VP60 protein is shown in SEQ ID No.

2.

7. A recombinant expression vector, cell line, or recombinant bacterium containing the gene encoding the recombinant rabbit hemorrhagic virus VP60 protein as described in claim 6.

8. The use of the recombinant expression vector, cell line or recombinant bacteria as described in claim 7 in the preparation of a vaccine to prevent rabbit hemorrhagic disease.

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