Recombinant chimeric protein and application thereof in preparation of goat pox virus subunit vaccine

Abstract

The invention discloses a recombinant chimeric protein and application thereof in preparation of a goatpox virus virus vaccine, and belongs to the technical field of biological medicines. The amino acid sequence of the recombinant chimeric protein is as shown in SEQ ID NO: 1, and the recombinant chimeric protein is formed by connecting four common B cell antigen epitopes of LSDV and GTPV in series through flexible connecting peptides. The protein can be subjected to efficient soluble expression through a prokaryotic expression system, can be specifically recognized by LSDV infected serum and GTPV immune serum, and has good immunogenicity. Animal experiments show that a subunit vaccine prepared by taking the protein as an immunogen can induce a mouse to generate a neutralizing antibody aiming at LSDV and GTPV, and after challenge, visceral viral load is remarkably reduced, tissue pathological damage is relieved, and effective immune protection is provided. As a safe and efficient subunit vaccine antigen, the recombinant chimeric protein provided by the invention has a wide application prospect in the aspect of preparing broad-spectrum goat pox virus virus vaccines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a recombinant chimeric protein, and further relates to application of the protein in preparation of a broad-spectrum capripoxvirus subunit vaccine. BACKGROUND

[0002] Lumpy skin disease virus (LSDV) is a member of Capripoxvirus (CaPV) in Poxviridae, which also includes Goatpox virus (GTPV) and Sheeppox virus (SPPV). The nucleotide sequence similarity of LSDV with SPPV and GTPV is as high as 97% or more. At present, lumpy skin disease in cattle is generally prevented and controlled by using a live attenuated vaccine of goatpox, which is a "heterologous vaccine" strategy. Although this strategy has been proved to be effective, it has problems such as low cross-protection efficacy, large individual difference, short duration of immunity, high rate of adverse reactions, etc. Especially under immunological pressure or in the face of a strong virus strain, the live attenuated vaccine of goatpox may not be able to completely prevent LSDV infection or virus replication, which means that the vaccinated cattle may still be infected, shed virus and become a potential source of infection. Therefore, the most ideal choice for lumpy skin disease in cattle is to use a homologous live attenuated vaccine specifically for LSDV.

[0003] However, LSDV is a large double-stranded DNA virus with a complex genome (about 150 kb) containing a large number of non-essential genes and genes involved in host range, virulence and immune escape, making it very difficult to precisely control the degree of attenuation. Traditional attenuation methods (such as continuous passage in non-natural host cell lines or at lower temperatures) are a long and unpredictable process (which may take years or even decades). During the passage process, unexpected genetic mutations may occur in the virus, affecting not only virulence but also immunogenicity. In addition, there is a risk of residual virulence or virulence reversion in LSDV live attenuated vaccines, which may cause local reactions or even transient systemic reactions in some cattle. These reasons have resulted in few high-quality LSDV homologous vaccine products available on the market.

[0004] Compared with live attenuated vaccines, subunit vaccines have no risk of virulence reversion, virus spread and recombination, better stability and biosafety, and higher specificity (narrower antibody spectrum than live vaccines), which are the main direction of current vaccine research and development.

[0005] Based on this, the present application develops a subunit vaccine for preventing and controlling LSDV, which not only overcomes the defects of "heterologous vaccines" and live attenuated vaccines, but also has a certain cross-protection effect on members of Capripoxvirus, and can be better applied to the prevention and control of Capripoxvirus. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing vaccines in the prevention and control of goatpox virus diseases by providing a new recombinant chimeric protein and its application in the preparation of subunit vaccines.

[0007] To achieve the above objectives, the applicant used a phage display library to screen for specific peptides in sera infected with bovine nodular dermatitis and sera immunized with goatpox attenuated live vaccine. Peptides shared by both were selected as candidate antigenic epitopes. Through bioinformatics analysis, conservation analysis, and expression verification, four peptides were ultimately selected as effective antigenic epitopes. These four epitopes were then chimeric using a flexible peptide to construct the recombinant chimeric protein LSDV-B-4. Using a prokaryotic expression system, the synthesized gene sequence was cloned into the pET28a-SUMO vector to obtain the recombinant plasmid pET28a-SUMO-LSDV-B-4 linked to the target gene. Finally, the constructed recombinant plasmid was transformed into E. coli BL21(DE3) competent cells for induced expression. The SUMO tag was used to enhance soluble expression and could be precisely removed by enzyme digestion.

[0008] The amino acid sequence of the recombinant chimeric protein LSDV-B-4 is shown in SEQ ID NO:1, and the encoding polynucleotide sequence is shown in SEQ ID NO:2.

[0009] This invention successfully designed, expressed, and purified the recombinant chimeric protein LSDV-B-4 based on multiple epitope tandem. This protein exhibits good immunogenicity, specifically being recognized by serum from animals infected with or immunized with goatpoxviruses, and can induce the production of neutralizing antibodies against goatpoxviruses, demonstrating its cross-protective potential. Immunized animals showed significant protection against viral attack, effectively inhibiting viral replication, reducing pathological damage, and maintaining a healthy state.

[0010] Therefore, the recombinant chimeric protein LSDV-B-4, as an immunogen for subunit vaccines, fundamentally avoids the biosafety risks of virulence reversion, viral shedding, and recombination associated with attenuated live vaccines, and has high application prospects in the field of goatpox virus disease control.

[0011] The preparation method of the recombinant chimeric protein LSDV-B-4 includes the following steps: 1) Synthesize the polynucleotide sequence of LSDV-B-4; 2) The plasmid was ligated into the pET28a-SUMO vector by enzyme digestion to construct the pET28a-SUMO-LSDV-B-4 recombinant plasmid; 3) IPTG-induced expression of recombinant plasmids; 4) SUMO protein cleavage removes the tag.

[0012] The present invention also provides the use of the recombinant chimeric protein in the preparation of a goatpoxvirus subunit vaccine. The goatpoxvirus genus includes one or more of bovine nodular dermatovirus (LSDV), goatpoxvirus (GTPV), and sheep poxvirus (SPPV), and the drug is used to stimulate the body to produce neutralizing antibodies against the goatpoxvirus genus.

[0013] The present invention further provides a subunit vaccine comprising the recombinant chimeric protein LSDV-B-4, and a pharmaceutically acceptable adjuvant and / or carrier.

[0014] Products derived from the recombinant chimeric protein LSDV-B-4, including but not limited to polynucleotides encoding the protein, recombinant expression plasmids containing the polynucleotides, or recombinant bacteria, are all within the scope of protection of this invention.

[0015] According to a specific embodiment of the present invention, it has been confirmed that LSDV-infected serum and GTPV-immune serum can specifically react with the recombinant chimeric protein LSDV-B-4 and produce a clear target band. Therefore, the recombinant chimeric protein provided by the present invention can also be used to prepare a kit for detecting goatpoxvirus infection. Specifically, the kit can be an indirect ELLIA kit for detecting goatpoxvirus-specific antibodies.

[0016] The beneficial effects of this invention are: The recombinant chimeric protein provided by this invention contains multiple B-cell antigenic epitopes shared by LSDV and GTPV. These epitopes can be simultaneously captured and processed by the same antigen-presenting cell and presented to multiple B-cell clones, stimulating a synergistic and balanced immune response against multiple targets, thereby ensuring broad-spectrum cross-protection. By fusing multiple B-cell antigenic epitopes, this invention not only improves protein solubility and successfully achieves supernatant expression, but also enhances the protein's immunogenicity, facilitating a strong and durable immune response. The flexible linker design ensures spatial accessibility of each epitope, avoiding epitope masking. Furthermore, this invention requires only one genetic engineering fermentation and one purification process to obtain a single product containing all target epitopes, thus offering advantages such as high production efficiency and low cost. Attached Figure Description

[0017] Figure 1 It is a recombinant plasmid pET28a-SUMO-LSDV-B-4 PCR identification results; In the figure: Lane 1: DL500; 2-3: Bacterial culture sample (LSDV-B-4 nucleotide length 288bp, with enzyme sites and homologous arms added on both sides, so the PCR product size is 317bp); 4: Negative control.

[0018] Figure 2 This is the SDS-PAGE analysis result of SUMO-LSDV-B-4 protein; In the figure: Lane 1: Protein molecular weight standard; Lanes 2-9: Different bacterial culture samples after induction (37℃, 10h); Lane 10: pET28a-SUMO empty vector induction (37℃, 10h).

[0019] Figure 3 This is the SDS-PAGE analysis result of LSDV-B-4 protein after cleaving the SUMO tag; in the figure: lane 1: protein molecular weight standard; lane 2: LSDV-B-4 protein.

[0020] Figure 4 This is the result of Western Blot analysis of LSDV-B-4 protein with LSDV-infected bovine serum, GTPV-immunized bovine serum, and FBS; In the figure: Lanes 1, 3, and 5: protein molecular weight standard; 2: LSDV-infected serum diluted 1:800 times; 4: GTPV-immunized serum diluted 1:200 times; 6: FBS diluted 1:200 times.

[0021] Figure 5 These are the results of mouse weight changes during the LSDV-B-4 immune challenge protection experiment.

[0022] Figure 6 This is the result of the viral load in mouse organs during the LSDV-B-4 immune challenge protection experiment.

[0023] Figure 7 These are the results of specific antibodies in mouse serum during the LSDV-B-4 immune challenge protection experiment.

[0024] Figure 8 These are the results of histopathological analysis of mouse organ tissues in the LSDV-B-4 immune challenge protection experiment. Detailed Implementation

[0025] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and not for limiting the scope of protection of the present invention. Various modifications or equivalent substitutions made by those skilled in the art based on the following embodiments should also be considered to fall within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions or reference books such as *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory, 2017), or according to the methods recommended in the manufacturer's operating manual. Materials in the embodiments that do not specify their source are all commonly used materials well known in the art, which can be constructed based on literature reports or obtained commercially.

[0026] Example 1: Preparation of recombinant chimeric protein 1. Design and Synthesis pET28a-SUMO-LSDV-B-4 Recombinant plasmid The applicant initially used a phage display library to screen for specific antigenic epitopes from infected serum of bovine nodular dermatitis and serum immunized with a live attenuated goatpox vaccine. This yielded three types of antigenic epitopes: those specific to LSDV infection, those specific to GTPV immunization, and those shared by both. Subsequently, the shared epitopes were selected as candidate vaccine epitopes. Combined with conservation analysis, target specificity analysis, and expression validation analysis, four peptides were ultimately selected as effective antigenic epitopes. The sequences of these four peptides and their information in the LSDV and GTPV genomes are as follows: Table 1: Four peptides and their genetic information in LSDV and GTPV

[0027] Next, the four selected epitopes were ligated using a flexible peptide and sent to the company for codon optimization synthesis. The synthesized sequence was 288 bp in length (shown in SEQ ID NO.1). GGATCC was added to the 5' end of the sequence as a BamHI restriction site, and CTCGAG was added to the 3' end of the sequence as a BamHI restriction site. Xho I restriction site, then clone the entire sequence into pET28a-SUMO The vector was used to obtain a recombinant plasmid linked with the target gene, which was named... pET28a-SUMO-LSDV-B-4 Plasmid. The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells for expression, tagged with... SUMO-LSDV-B-4 The protein is approximately 25 kDa. ， After removing the label LSDV-B-4 The protein expression size is approximately 11 kDa. PCR identification of the recombinant bacteria. pET28a-SUMO-LSDV-B-4 The results showed that the identification of the bacterial culture band size was correct. Figure 1 The samples were sent to the company for sequencing and no base mutations or frameshifts were found.

[0028] The amino acid sequence of the fusion protein LSDV-B-4 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0029] The amino acid sequence of LSDV-B-4 (SEQ ID NO.1): MDQIMQAVTNGGKIVYGTMKEGGGGGSQDLLGIESVNAGRRRNKNISQGGGGSLSFFNKSSRDSRMMRSLKNQGGGGSTTDEMMTVDAFGPENIVFKK The nucleotide sequence of LSDV-B-4 (SEQ ID NO.2): ATGGATCAGATTATGCAGGCGGTTACCAACGGTGGTAAAATTGTGTATGGTACGATGAAAGAAGGCGGTGGTGGTAGCCAGGATCTGCTGGGTATTGAAAGCGTTAACGCGGGTCGTCGTAATAAAAACATTAGCCAGGGTGGC GGTGGTAGCCTGAGCTTTCTTCAATAAAAGCAGCCGTGATAGTCGTATGATGCGTAGCCTGAAAAATCAGGGTGGTGGTGGTAGTACTACCGATGAAATGATGACCGTTGATGCATTTGGTCCGGAAAATATTGTTTTCAAGAAG 2. Small-scale preparation of plasmid DNA The method for preparing plasmid DNA in small quantities was performed according to the instructions of the Tiangen High Purity Plasmid Mini-Prep Kit. The specific steps are as follows: (1) Take 5 mL of overnight cultured bacterial solution and add it to a centrifuge tube. Centrifuge at 8000 r / min for 3 min to collect the bacterial precipitate and discard the supernatant as much as possible.

[0030] (2) Add 250µL Buffer P1 to the centrifuge tube containing the bacterial precipitate. First check to ensure that RNase A has been added. Use a 1mL pipette or vortex mixer to mix thoroughly and suspend the bacterial precipitate.

[0031] (3) Add 250µL Buffer P2 to the centrifuge tube and gently invert it 4-6 times to mix thoroughly to lyse the bacteria. At this point, the solution should become clear and viscous.

[0032] (4) Add 350µL Buffer P3 to the centrifuge tube and immediately gently invert it 4-6 times to mix thoroughly. A white flocculent precipitate should appear at this time. Centrifuge at 13000r / min for 10min.

[0033] (5) Transfer the supernatant obtained in step 4 to the adsorption column Spin Column CM with the collection tube already installed, centrifuge at 13000 r / min for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0034] (6) Add 750µL Buffer PW to the adsorption column to check that anhydrous ethanol has been added, centrifuge at 13000 r / min for 1 min, and discard the waste liquid in the collection tube.

[0035] (7) Put the adsorption column back into the collection tube, centrifuge at 13000r / min for 2min, discard the waste liquid, and place the adsorption column at room temperature for several minutes to dry it completely.

[0036] (8) Place the adsorption column in a new centrifuge tube, add 50µL of Buffer EB to the middle of the adsorption membrane, place at room temperature for 3 min, centrifuge at 13000r / min for 1 min, collect in a centrifuge tube, and store the plasmid at -20℃.

[0037] 3. Plasmid was transformed into E. coli BL21(DE3) competent cells. (1) Take 100 μL of competent cell suspension and add 1 μL of plasmid. Gently rotate to mix the contents and place on ice for 30 min (an empty vector control without plasmid DNA was set up in the experiment).

[0038] (2) Place the centrifuge tubes in a circulating water bath preheated to 42°C for 45 seconds for thermal shock.

[0039] (3) Quickly transfer the centrifuge tube to ice to cool the cells for 2 minutes.

[0040] (4) Add 700 μL of LB liquid medium to each tube. Heat the medium to 37°C in a water bath, and then transfer the centrifuge tube to a 37°C shaker for 1 hour to allow the bacteria to recover (in order to achieve effective transformation, the rotation speed should not exceed 225 r / min during recovery).

[0041] (5) Take 100 μL of transformed competent cells and transfer them to an LB agar plate containing antibiotics (Kana resistant). Use a sterile bent glass rod to spread the transformed cells evenly on the surface of the agar plate, which is considered a low concentration. Centrifuge at 5000 r / min for 3 min, collect the bacteria, discard the culture medium, and keep 100 μL to resuspend the bacteria. Spread the resuspended bacteria evenly on the surface of the agar plate, which is considered a high concentration.

[0042] (6) Incubate the petri dish upright at 37°C until the liquid is absorbed, then invert the petri dish and incubate for 10-12 hours to observe the results.

[0043] 4. Low-level expression and confirmation of expression form of LSDV-B-4 protein (1) The plasmid with correct sequencing was transformed into the BL21 expression strain and cultured overnight on a plate containing Kana resistance.

[0044] (2) Pick a single colony containing the recombinant plasmid and add it to 5 mL of LB liquid medium (50 mg / mL Kana resistance, added at a concentration of 0.1%, i.e., 5 µL added to 5 mL of medium) and incubate overnight at 37°C; at the same time, pick the corresponding bacterial cells transformed with the empty vector as the later control.

[0045] (3) Transplant the inoculum at a ratio of 1:100. Generally, add 100µL of recombinant plasmid bacterial culture and empty vector plasmid bacterial culture to two bacterial bottles containing 10mL of liquid LB medium (ensure that the LB medium is shaken thoroughly). Add Kana antibiotic and incubate at 37℃ with shaking until OD. 600 The value reaches 0.6-0.8 (it takes about 3 hours; pay attention to the turbidity of the bacterial cells, i.e., the initial turbidity of the bacterial solution).

[0046] (4) Take 1 mL of liquid as the uninduced control, and add IPTG inducer to the remainder to make the final concentration reach 1 mM (IPTG: liquid medium = 1:1000, that is, 10 mL of medium is added to 10 µL). Induce protein expression by shaking at 37℃ for 5 h.

[0047] (5) Determine that the protein can E. coli After stable expression in BL21, 10 mL of expression bacteria were induced again. Bacterial culture was collected in 2 mL centrifuge tubes, centrifuged at 12000 rpm for 2 min, washed with PBS, and the bacterial pellet was resuspended in 1 mL PBS. The bacterial cells were then disrupted using an ultrasonic homogenizer and centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant and pellet were processed separately to prepare protein electrophoresis samples for SDS-PAGE analysis. The samples were compared with an empty vector control to confirm successful expression of the prokaryotic recombinant protein. Figure 2 ).

[0048] 5. Large-scale expression and purification of LSDV-B-4 protein (1) Dilute the seed bacteria at a ratio of 1:100. Generally, add 10 mL of recombinant plasmid bacterial solution to a culture flask containing 1 L of LB medium (use a large conical flask to shake the bacteria to ensure sufficient shaking), add Kana antibiotic, and incubate at 37°C with shaking until OD. 600 The value reached 0.6-0.8, and the time was about 3 hours.

[0049] (2) Add IPTG at a ratio of 1:1000 to achieve a final concentration of 1mM, and induce culture at 37℃ for 10h with shaking.

[0050] (3) Before high-pressure disruption, collect the bacterial culture in a 50 mL centrifuge tube, centrifuge at 12000 r / min for 10 min at 4℃ to collect the bacteria, and resuspend and wash twice with PBS. Then, high-pressure disruption is used to make the bacterial cells clear and transparent, and the liquid is collected.

[0051] (4) Pre-cool the high-speed centrifuge to 4°C, centrifuge at 12000r / min for 10min and collect the supernatant.

[0052] (5) Resuspend Ni-NTA packing material. Add an appropriate amount of packing material to the chromatography column and let it stand.

[0053] (6) Equilibrate the chromatography column with 5-10 column volumes of equilibration buffer. (7) After centrifugation, filter the supernatant of the sample through a 0.45µm filter before passing it through the column. Load the sample at a uniform rate. After loading, wash the chromatography column with 5-10 column volumes of equilibration buffer and collect the eluent.

[0054] (8) Elute the target protein with different concentrations of imidazole. Prepare imidazole of different concentrations using equilibration buffer and perform gradient elution. Collect the eluent.

[0055] (9) The target protein liquid after elution was concentrated by ultrafiltration using a 10kDa ultrafiltration tube. The target protein was displaced from the high concentration of imidazole using PBS solution. The protein was centrifuged at 4℃ and 4000r / min. The ultrafiltration time was determined according to the filtration efficiency of the ultrafiltration tube. The purity of the SUMO-LSDV-B-4 fusion protein after ultrafiltration was observed by SDS-PAGE electrophoresis and ensured.

[0056] (10) The protein concentration was determined using the BCA kit, aliquoted, and stored at -80℃ for later use. The results showed that the final protein concentration determined by the BCA kit was 5 mg / mL.

[0057] (11) The SUMO label cutting system is as follows: Table 1. SUMO tag cutting system for SUMO-LSDV-B-4

[0058] Simultaneously, prepare 10 of the above-mentioned cutting systems in PCR tubes, mix them thoroughly, and place them in a 4-degree refrigerator overnight for cutting.

[0059] (12) After cutting, the sample was purified again by column chromatography. The permeate was taken and ultrafiltered using a 3KD ultrafiltration tube. The target protein was removed from the cutting system using PBS solution. SDS-PAGE electrophoresis was performed to observe and ensure the purity of the LSDV-B-4 fusion protein after ultrafiltration. Figure 3 The final protein concentration, as determined by the BCA kit, was approximately 1 mg / mL. The protein was aliquoted and stored at -80°C for later use.

[0060] Example 2: Western Blot validation of the immunogenicity of the recombinant chimeric protein Take out three prepared SDS-polyacrylamide gels, use SDS-PAGE to confirm that the protein loading amount is consistent, and use Western Blot to verify the reactivity of LSDV-B-4 protein.

[0061] (1) Preparation of separating gel: Select an appropriate volume of separating gel, refer to the formula table, add the separating gel to the appropriate amount, add isopropanol and press the gel, after the separating gel solidifies, pour out the isopropanol, rinse lightly with pure water and then absorb with absorbent paper.

[0062] (2) Preparation of concentrated glue: Select an appropriate volume of concentrated glue, refer to the formula table, add concentrated glue until full, insert the comb, and after the concentrated glue solidifies, pull out the comb.

[0063] (3) Sample preparation: Mix the sample with SDS-PAGE loading buffer, treat at 100℃ for 10 min, immediately in an ice bath for 2 min, centrifuge at 10000 r / min for 30 s, and use 1×glycine buffer as electrophoresis buffer.

[0064] (4) Sample loading: Under normal circumstances, 10 µL of sample is loaded into each well. In this experiment, the concentration of LSDV-B-4 protein was first determined using the BCA kit to adjust the specific sample loading amount. At the same time, 5 µL of marker was loaded (mixed with 5 µL of 1× Loading Buffer).

[0065] (5) Electrophoresis: Keep the constant voltage at 80V until the sample reaches the boundary between the stacking gel and the separating gel. Then adjust to 120V to ensure that the bromophenol blue electrophoresis reaches the bottom position.

[0066] (6) After the protein gel electrophoresis, directly place it in Coomassie brilliant blue staining solution for 4-6 hours, and then transfer it to destaining solution (10% glacial acetic acid, 5% ethanol) for destaining.

[0067] (7) Based on the size of the gel, cut one PVDF membrane and three filter papers slightly larger than the gel. Soak the gel and paper in the transfer buffer. Soak the membrane in methanol for 5 minutes and then transfer it to the transfer buffer.

[0068] (8) Transfer: On the transfer plate, starting from the white layer, lay filter paper, membrane, gel, and filter paper in sequence. Cover each layer with sufficient transfer buffer and remove air bubbles. Insert into the transfer tank and set a constant voltage of 70V for 1 hour for transfer.

[0069] (9) Washing the membrane: Rinse the membrane 3 times with TBST buffer, every 5 min.

[0070] (10) Blocking: Incubate with 5% (w / v) skim milk powder blocking buffer at room temperature for 2-3 hours.

[0071] (11) Same as (9).

[0072] (12) Primary antibody incubation: 1×TBST diluted serum (based on the specific titer ratio of infected serum and immune serum determined according to LSDV-B-4 protein, to ensure that the specific antibody titer in serum remains consistent, therefore the dilution of infected serum is determined to be 1:800, the dilution of immune serum is 1:200, and FBS is also 1:200), and incubated overnight on a horizontal shaker at 4°C.

[0073] (13) Same as (9).

[0074] (14) Secondary antibody incubation: Dilute horseradish peroxidase-labeled rabbit anti-bovine IgG (H+L) with 1×TBST, with a dilution of 1:5000, and incubate at room temperature on a shaker for no more than 2 hours.

[0075] (15) Same as (9).

[0076] (16) Color development: Equal volumes of chemiluminescent color development solutions A and B are mixed on the membrane for development. The purified LSDV-B-4 protein is approximately 11 kDa in size. Incubation with LSDV-infected serum and GTPV-immunized serum revealed a clear target band for the protein. Figure 4 ), while FBS incubation is stripless.

[0077] Example 3: Virus serum neutralization test after immunization of mice with recombinant chimeric protein For the first immunization, administer 100 μg of LSDV-B-4 per mouse, emulsified with Freund's complete adjuvant at a 1:1 ratio. Disinfect the skin on the mouse's back and inject the emulsified vaccine at multiple sites, 30-50 μL per site. Two weeks later, administer a second immunization, using 50 μg of LSDV-B-4 per mouse, emulsified with Freund's incomplete adjuvant at a 1:1 ratio. Disinfect the skin on the mouse's back and inject the emulsified vaccine at multiple sites, 30-50 μL per site. Two weeks after the second immunization, collect mouse serum, aliquot and store at -20°C for later use.

[0078] (1) Serum inactivation: sterile test serum was inactivated by water bath at 56°C for 30 min and used directly for the test.

[0079] (2) Serum serial dilution: In a 1.5 mL sterile EP tube, the serum to be tested was serially diluted with DMEM cell growth medium from 1:2 to 1:256. Each dilution was replicated in 3 wells, with 50 μL added to each well and then added to a 96-well plate.

[0080] (3) Take out the GTPV / LSDV virus stored at -80℃, and dilute the virus solution with the measured TCID50 to 200 TCID using DMEM cell growth medium. 50 / 0.1mL, add an equal volume of 200TCID to the 96-well plate after adding the diluted serum. 50 Mix 0.1 mL of virus suspension thoroughly and incubate at 37°C for 1 hour in a 5% CO2 incubator. (4) The completed virus and serum mixture was inoculated into confluent Vero / LT cells. The cells were incubated in a 37°C incubator containing 5% CO2. The experiment was performed in triplicate, and each plate contained a serum toxicity control, a virus control, and a normal cell control.

[0081] (5) Observe and record the cytopathic effects daily until the results stabilize. Calculate the GTPV / LSDV neutralizing antibody titers in the serum to be tested using the Reed and Muench methods. After two immunizations of mice with LSDV-B-4, the neutralizing titer against GTPV was approximately 1:6, and the neutralizing titer against LSDV was approximately 1:10.

[0082] Example 4: Immunoprotective assay of recombinant chimeric protein Following the above immunization protocol, a vaccine was prepared using LSDV-B-4 with Freund's adjuvant. C57 mice were immunized twice, once every two weeks. Two weeks after the second immunization, the mice were divided into groups. The challenge group received 100 μL of the vaccine per mouse. 4.5 C57BL / 6 female mice were infected nasally with TCID50 / 1mL GTPV. Two groups were established: a non-immunized single-challenge group and a non-challenge single-immunization group, with 12 biological replicates in each group. Nine mice in each group were included in the experiment for 12 days. Clinical observation was conducted from 0 to 12 days post-challenge, with daily recording of mouse weight and mental status. Heart, liver, spleen, lung, and kidney samples were collected from the mice. Half of each organ sample was fixed in 4% paraformaldehyde for 48 hours, then embedded in paraffin, and the tissue sections were stained with hematoxylin and eosin (HE) for histopathological evaluation. The remaining half of each organ sample was ground and nucleic acid was extracted. Real-time quantitative PCR (qPCR) was used to analyze GTPV load. The remaining three mice in each group had their serum antibody levels assessed for 28 days post-immunization, with blood samples collected weekly.

[0083] Body weight changes during the challenge period showed that mice immunized with LSDV-B-4 twice continued to gain weight after challenge, with no significant difference in weight between the two groups at 12 days, while the weight of the single-challenge group continued to decrease. Figure 5 ).

[0084] The viral load in organs during the challenge period showed that the viral load in the organs of the immunized challenge group was significantly lower than that of the challenge group. Figure 6 ).

[0085] Serum-specific antibody test results during the challenge period showed that the serum-specific antibodies in the immunized challenge group were at a relatively high level and could be maintained for a relatively long time. Figure 7 ).

[0086] Histopathological analysis of mouse organs showed that the lungs of the immune challenge group had no visible lesions, while the lungs of the challenge group showed obvious inflammatory cell infiltration and alveolar wall thickening. Figure 8 ).

Claims

1. A recombinant chimeric protein having the amino acid sequence shown in SEQ ID NO:

1.

2. A polynucleotide encoding the recombinant chimeric protein as described in claim 1.

3. The polynucleotide as described in claim 2, characterized in that: The nucleotide sequence of the polynucleotide is shown in SEQ ID NO:

2.

4. A recombinant expression plasmid comprising the polynucleotide as described in claim 2 or 3.

5. The recombinant expression plasmid as described in claim 4, characterized in that: The vector for the recombinant expression plasmid is pET28a-SUMO.

6. A host cell comprising the recombinant expression plasmid as described in claim 4.

7. The use of the recombinant chimeric protein of claim 1 in the preparation of a goatpox virus subunit vaccine.

8. The application as described in claim 7, characterized in that, The goatpoxvirus genus includes one or more of bovine nodular dermatovirus (LSDV), goatpoxvirus (GTPV), and sheep poxvirus (SPPV).

9. The application as described in claim 7, characterized in that, The subunit vaccine is used to stimulate the body to produce neutralizing antibodies against goatpoxviruses.

10. A goatpox virus subunit vaccine, characterized in that, It comprises the recombinant chimeric protein as described in claim 1, and a pharmaceutically acceptable adjuvant and / or carrier.

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