Avian leukemia tetravalent epitope mRNA vaccine and application

Abstract

The invention discloses an avian leukosis tetravalent epitope mRNA vaccine and application, and belongs to the technical field of immunology and biology. According to epidemic strains of A, B, J and K subgroup avian leukosis viruses in recent 10 years, epitopes with relatively high antigenicity and conservative property in surface proteins (gp85 gene coding) are screened and connected in series, so that immune escape caused by self gene mutation of the avian leukosis viruses is avoided, the protection range is wider, and the kit has a good application prospect. And a molecular adjuvant is also added into an mRNA vaccine sequence to enhance the cellular immunity of the vaccine, so that the problem of low antigenicity of other vaccines is solved. Compared with purification investment, the vaccine has the advantages that the capital cost is saved by more than 50%, the consumption of time, manpower and material resources is greatly reduced, and the vaccine has a good application prospect.

Description

Technical Field

[0001] This invention relates to the fields of immunology and biotechnology, specifically to a tetravalent epitope mRNA vaccine for avian leukosis and its application. Background Technology

[0002] Avian leukosis (AL) is a neoplastic disease in birds caused by avian leukosis virus (ALV). ALV belongs to the family Retroviridae, subfamily Orthoretrovirinae, and genus Alpha Retrovirus. The symptoms and pathological changes in chickens after infection are complex, but can be summarized in two aspects: First, ALV infection induces typical tumor development and leads to death, with mortality rates sometimes reaching 20% ​​or even higher. Second, infected chickens develop subclinical pathological changes due to ALV, resulting in immunosuppression, slowed growth and development, and reduced egg production, affecting key production performance aspects of the flock. Furthermore, co-infection with other pathogens such as infectious bursal disease virus, Newcastle disease virus, Marek's disease virus, and reticuloendotheliosis virus is increasingly common, leading to highly complex clinical manifestations and significantly increasing the difficulty of diagnosis and treatment. When ALV works in conjunction with other immunosuppressive factors, it can cause more severe immunosuppression in chickens, clinically manifested as slow growth, anemia, emaciation, and even multiple infections of various diseases, leading to increased mortality and decreased egg production, thus posing a huge threat to the poultry industry.

[0003] Currently, both domestically and internationally, ALV is mainly controlled and eliminated through the purification of breeding flocks. However, this method is susceptible to interference from endogenous viruses, requires several generations, is time-consuming and costly, and faces limitations in batch testing technology. Achieving complete ALV eradication in China presents a significant challenge. Therefore, there is an urgent need to develop an effective, commercially viable avian leukosis vaccine with cross-protective capabilities. Because ALV, as a retrovirus, can integrate its proviral genome into the host genome and replicate along with cellular replication, the virus can remain stable in the body for a long time, making it impossible to completely eliminate and leading to exponential amplification through vertical transmission. Furthermore, given ALV's highly variable nature and immune evasion mechanisms, traditional attenuated and inactivated vaccines cannot provide effective protection. mRNA vaccines, as a novel vaccine technology, have received widespread attention in recent years. They offer significant advantages: translation begins immediately upon reaching the cytoplasm. Furthermore, due to their non-infectious and non-integrative characteristics, mRNA vaccines do not carry the risks of infection or insertional mutations like attenuated vaccines. They also overcome the possibility of virulence recovery and shedding caused by traditional vaccines. Compared to subunit vaccines, they can induce effective humoral and cellular immunity, providing more complete and targeted immune protection and creating conditions for disease control. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a tetravalent epitope mRNA vaccine for avian leukosis and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a combined peptide, the amino acid sequence of which is shown in SEQ ID NO:29.

[0006] In a second aspect, the present invention provides a nucleic acid molecule that encodes the aforementioned combined peptides.

[0007] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 28.

[0008] A third aspect of the present invention provides a recombinant vector containing the aforementioned nucleic acid molecule.

[0009] In a fourth aspect, the present invention provides an mRNA, wherein the mRNA is formed by sequentially linking a T7 promoter, a 5'UTR, the nucleic acid molecule, a 3'UTR, and a polyA from the 5' end to the 3' end; The nucleotide sequence of the T7 promoter is shown in SEQ ID NO:24, the nucleotide sequence of the 5'UTR is shown in SEQ ID NO:25, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO:26, and the nucleotide sequence of polyA is shown in SEQ ID NO:27.

[0010] Furthermore, the nucleotide sequence of the mRNA is shown in SEQ ID NO:1, and the 5' end of the mRNA has a cap structure; the DNA sequence transcribed from the mRNA is shown in SEQ ID NO:2.

[0011] In a fifth aspect, the present invention provides a tetravalent epitope mRNA vaccine for avian leukosis, the avian leukosis tetravalent epitope mRNA vaccine comprising the nucleic acid molecule and / or the mRNA, and lipid nanoparticles.

[0012] Furthermore, in the lipid nanoparticles, the molar ratio of DOTAP, 1,2-DSPC, CholesUerol, and DMG-PEG2000 is DOTAP:1,2-DSPC:CholesUerol:DMG-PEG2000 = (45-55):(5-15):(35-40):(1-2).

[0013] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the mRNA and / or the avian leukosis tetravalent epitope mRNA vaccine, and a pharmaceutically acceptable carrier.

[0014] A seventh aspect of the invention provides the use of the said combined peptide, the said nucleic acid molecule, the said recombinant vector, the said mRNA, the said avian leukosis tetravalent epitope mRNA vaccine, or the said pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of avian leukosis.

[0015] The beneficial effects of this invention are: (1) This invention screened highly antigenic and conserved epitopes in the gp85 protein based on the prevalent strains of avian leukosis virus from subgroups A, B, J, and K over the past 10 years, and tandemly protected them to avoid immune escape caused by gene mutations in avian leukosis virus, thus providing broader protection. Furthermore, molecular adjuvants were added to the mRNA vaccine sequence to enhance the cellular immune response of the vaccine, without the risk of integration into the genome.

[0016] (2) Compared with purification, this invention not only saves more than 50% of the capital cost, but also greatly reduces the consumption of time, manpower and material resources. When used to prepare drugs for the prevention of avian leukosis, the sequence coding can be changed to modify and add recombinant proteins or adjuvant elements, which is very suitable for the rapid development of vaccines and avoids the complex process development and purification process. Attached Figure Description

[0017] Figure 1 The image shows the recombinant plasmid pGP-T7-RME-polyA.

[0018] Figure 2 The image shows a gel electrophoresis result of double enzyme digestion identification using a template.

[0019] Figure 3 This is an electrophoresis image after transcription capping.

[0020] Figure 4 The image shows the Western blot validation results for mRNA transfection expression.

[0021] Figure 5 The image shows the results of particle size analysis by the Malvern particle size analyzer. Figure 5 A in the diagram represents the particle size distribution of mRNA-LNPs. Figure 5 B in the diagram represents the zeta potential.

[0022] Figure 6 The figure shows the Western blot validation results for mRNA-LNPs transfection expression.

[0023] Figure 7Figure showing the weight changes in SPF chickens after immunization with mRNA-LNPs vaccine.

[0024] Figure 8 The graph shows the antibody titer results produced by SPF chickens immunized with mRNA-LNPs vaccine.

[0025] Figure 9 The graph shows the neutralizing antibody titers produced by SPF chickens immunized with mRNA-LNPs vaccine. Figure 9 A in the image represents the titer of the ALV-J neutralizing antibody. Figure 9 Figure B shows the titer of ALV-A neutralizing antibody. Figure 9 The figure in C represents the titer of the ALV-K neutralizing antibody.

[0026] Figure 10 The image shows the cytokine detection results after SPF chickens were immunized with mRNA-LNPs vaccine.

[0027] Figure 11 The graph shows the monitoring results of weight changes in SPF chickens after viral challenge.

[0028] Figure 12 A graph showing the dynamic changes in immune organ indices after SPF chickens were challenged with ALV-J.

[0029] Figure 13 A graph showing the dynamic changes in immune organ indices after SPF chickens were challenged with ALV-A. Detailed Implementation

[0030] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0031] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0032] The test materials used in the embodiments of the present invention, unless otherwise specified, are all conventional test materials in the art and can be purchased through commercial channels.

[0033] Example 1: Construction of an in vitro transcription system for mRNA vaccines Download the gp85 gene of ALV prevalent strains from subgroups A, B, J, and K over the past 10 years from NCBI GenBank (see Table 1). Screen for antigenic epitopes with high antigenicity and conservation. Epitope peptides are shown as SEQ ID NO:3-SEQ ID NO:23, and the corresponding relationships are shown in Table 2 below.

[0034] Table 1. gp85 gene of ALV epidemic strains in subgroups A, B, J, and K. Table 2. Epitope peptide sequences used and their corresponding types and numbers. The above-mentioned antigenic epitope peptides were rearranged to obtain a recombinant protein, the amino acid sequence of which is shown in SEQ ID NO:29. The gene corresponding to SEQ ID NO:29 was codon-optimized to obtain the recombinant antigen-encoding gene (RME), the sequence of which is shown in SEQ ID NO:28. The RME sequence was used for subsequent mRNA vaccine sequence synthesis.

[0035] The mRNA vaccine sequence comprises the following elements: a T7 promoter, a 5' untranslated region (5'UTR), a recombinant antigen-encoding gene (RME), a 3' untranslated region (3'UTR), and polyadenylated nucleotides (polyA). A linearized restriction enzyme site (Hind III) is attached downstream of the polyA tail structure. A His tag is added at the N-terminus. The sequence of the recombinant antigen-encoding gene is then codon-optimized. The T7 promoter sequence is shown in SEQ ID NO:24, the 5'UTR sequence in SEQ ID NO:25, the 3'UTR sequence in SEQ ID NO:26, and the polyA sequence, with a length ranging from 30 to 150 Å, is 46 Å, as shown in SEQ ID NO:27. The nucleotide sequence of the finally synthesized mRNA vaccine is shown in SEQ ID NO:1, and the DNA sequence transcribed from the mRNA vaccine is shown in SEQ ID NO:2.

[0036] The mRNA vaccine sequence was synthesized by Gemma Gene and cloned into the pGP-Amp vector (the recombinant plasmid pGP-T7-RME-polyA pattern can be found in...). Figure 1 The synthesized sequence was verified by sequencing and then amplified in *E. coli* competent cells (DH5α). Positive colonies were screened, and plasmids (purchased from Tiangen Biotech Co., Ltd.) were extracted. The plasmids were then double-digested using the restriction endonucleases FastDigest Acc65I (purchased from Thermo Fisher Scientific) and FastDigest NotI (purchased from Thermo Fisher Scientific). (The gel electrophoresis image of the double-digestion identification of the sequence template is shown in...) Figure 2 The reaction system is shown in Table 3.

[0037] Table 3 Enzyme digestion reaction system The plasmid was linearized using FastDigest Hind III (Thermo Fisher Scientific) (37°C, 1 h), and then purified using a gel extraction kit (Tiangen Biotech). The linearized plasmid was then used as a template for in vitro transcription. The linearization reaction system is shown in Table 4.

[0038] Table 4 Linearized reaction system Example 2: In vitro transcription, capping, purification and validation In vitro transcription was performed using the T7 in vitro transcription kit (purchased from Beijing TransGen Biotech Co., Ltd.). Following the product instructions, all components except the T7 Enzyme Mix were briefly centrifuged before adding the T7 Enzyme Mix. The reaction was incubated at 37°C for 2 hours, followed by the addition of 2 μL of DNAI and incubation at 37°C for 30 minutes. Finally, 1 μL of 500 mM EDTA was added to terminate the reaction. The in vitro transcription reaction system is shown in Table 5.

[0039] Table 5. In vitro transcription reaction system of linearized plasmids After in vitro transcription, RNA was purified using RNA purification magnetic beads (purchased from Beijing TransGen Biotech Co., Ltd.). First, remove the magnetic beads from the kit and let them stand at room temperature for 30 minutes. Shake the magnetic beads to mix thoroughly. Add the RNA solution to a 1.5 ml centrifuge tube, then add the magnetic beads according to the RNA solution volume (magnetic bead volume = RNA volume × 1.8). Mix well and let stand for 5 minutes. Place the centrifuge tube on a magnetic rack and let it stand until the solution is clear, allowing the magnetic beads to fully adhere to the tube wall close to the rack. Discard the supernatant. Add 80% ethanol (freshly prepared using enzyme-free water) to the tube, without blowing or aspirating the magnetic beads. Let it stand at room temperature for 30 seconds and discard the supernatant. Repeat the above steps. Then, air-dry the magnetic beads at room temperature until they just begin to crack. Remove the centrifuge tube from the magnetic rack, add 20 μL of enzyme-free water to wash away the RNA, mix well, and let it stand at room temperature for 2 minutes. Place the centrifuge tube back on the magnetic rack and let it stand until the solution becomes clear, allowing the magnetic beads to fully adhere to the tube wall close to the magnetic rack. Transfer the elution buffer to a new enzyme-free centrifuge tube and store at -80°C.

[0040] Purified mRNA was capped using a capping kit (purchased from Beijing TransGen Biotech Co., Ltd.). Following the product instructions, the required SAM volume was calculated, and the 32mM stock solution was diluted with enzyme-free water to a 20mM working solution. The solution was kept on ice to prevent SAM degradation. Then, 10 μg of RNA was transferred to a PCR tube and heated in a 65°C metal bath for 10 min, followed by 5 min on ice to complete RNA denaturation. All components were thoroughly mixed and incubated at 37°C for 1 h. The capping reaction system is shown in Table 6. Electrophoresis images after transcription capping (Cap-mRME) are shown in [Table 6]. Figure 3 .

[0041] Table 6. Capped Reaction System After capping, purify the Cap-mRNA using the same steps as described above and store at -80°C.

[0042] Example 3: Validation of RME protein expression The obtained mRNA was transfected into HEK-293T cells in 12-well plates using the lipo3000 transfection kit (purchased from Thermo Fisher Scientific), with 1 μg of RNA transfected into each well. The specific steps were followed according to the transfection kit instructions: the cell concentration was adjusted to 2 x 10⁻⁶ cells the day before. 5 HEK-293T cells were seeded in 12-well plates at a concentration of / mL. The next day, when the HEK-293T cells reached 70-80% confluency, transfection was performed. 300μL of Opti-MEM and 7μL of Lipo300 were added to tube A and gently mixed by pipetting. 300μL of Opti-MEM and 7μg of mRNA were added to tube B and gently mixed by pipetting. The liquid from tube B was then added to tube A, mixed, and incubated at room temperature for 15 min. The HEK-293T cells that had reached 70-80% confluency were replaced with fresh complete culture medium, and the prepared transfection reagent and mRNA mixture was added dropwise. 48 h after transfection, the cell supernatant was discarded, and the cells were washed three times with PBS. Lysis was performed at 4°C for 1 h using RIPA lysis buffer (purchased from Xinsaimei Biotechnology Co., Ltd.) and PMSF protease inhibitor (purchased from Xinsaimei Biotechnology Co., Ltd.). Protein loading buffer was added, and the cells were incubated in a boiling water bath for 10 min.

[0043] A 10% protein gel (purchased from Xinsaimei Biotechnology Co., Ltd.) was prepared for SDS-PAGE electrophoresis under the following conditions: constant voltage 150V for 90 min, and constant current 400mA for 30 min for membrane transfer. After transfer, the membrane was blocked with 5% skim milk at 37℃ for 2 h, and washed three times with PBS for 5 min each time. His protein monoclonal antibody (purchased from Engibody) was diluted 1:5000 and incubated at 4℃ for 14 h. The membrane was washed three times with PBST for 10 min each time. Horseradish enzyme-labeled goat anti-mouse IgG (purchased from Engibody) was diluted 1:5000 and incubated at 37℃ for 1 h. The membrane was washed three times with PBST for 10 min each time. Finally, ECL chemiluminescence solution (purchased from Xinsaimei Biotechnology Co., Ltd.) was prepared according to the manufacturer's instructions, and ECL chemiluminescence was performed.

[0044] The results are as follows Figure 4 As shown, compared with the control group, a band of approximately 45 kDa appeared in the cell lysate lanes of the experimental group transfected with mRNA. The results indicate that in vitro transcribed mRNA can be expressed in HEK-293T cells.

[0045] Example 4: Preparation of mRNA-LNPs vaccine The mRNA obtained in Example 2 was packaged using lipid nanoparticles to prepare an mRNA-LNPs vaccine. The LNPs consisted of four components, weighed according to a molar ratio of DOTAP:1,2-DSPC:CholesUerol:DMG-PEG(2000) (purchased from TargetMol) of 50:10:38.5:1.5, dissolved in chloroform (5 mg / ml), and added to a round-bottom flask. Evaporation was carried out at 40°C and 25 rpm until no visible liquid remained in the flask. The flask was then dried under vacuum for 1 hour to form a homogeneous lipid film. Enzyme-free water (1 mg / ml) at 60°C and pH 7.4 was added, and the flask was magnetically stirred and shaken for 1 hour to dissolve the lipid film. The hydrated lipid solution was transferred to a centrifuge tube and sonicated in an ice bath until the solution became clear. The resulting solution was filtered three times through a 0.22 μm filter membrane. The mRNA and LNPs were mixed at a molar ratio of 1:10 to obtain the mRNA-LNPs vaccine, which was stored at 4°C. The average particle size and zeta potential of the vaccine were determined using a Malvern particle size analyzer, and the results are as follows: Figure 5 As shown, the average particle size of the vaccine is 139.6 nm, the PDI is 0.301, and the zeta potential is 41.6 mV.

[0046] mRNA-LNPs were transfected into HEK-293T cells in 12-well plates using the Lipo3000 transfection kit (Thermo Fisher Scientific). 1 μg of mRNA was transfected into each well as a positive control. Finally, the expression of mRNA-LNPs was verified by Western blot, following the same procedure as in Example 3. Results are as follows: Figure 6 As shown, a band of approximately 45 kDa appeared in the cell lysate lanes of the experimental group transfected with mRNA-LNPs. These results indicate that lipid nanoparticle-encapsulated mRNA can be expressed in HEK-293T cells.

[0047] Example 5: Safety Trial of mRNA-LNPs Vaccine Five 7-day-old SPF chickens (purchased from Shandong Jinan Spafars Co., Ltd.) were intramuscularly injected with mRNA-LNPs vaccine at a dose of 5 μg. A parallel control group was established, and the chickens' mental state and feeding behavior were observed daily for 7 consecutive days. Weight changes in SPF chickens after mRNA-LNPs vaccine immunization are shown in [Figure showing weight changes after immunization]. Figure 7 .

[0048] according to Figure 7 As a result, there was no significant difference in body weight between the vaccine group and the control group, indicating that the vaccine is safe.

[0049] Example 6: Immunogenicity-related trials of mRNA-LNPs vaccines To verify the immunization effect of the mRNA vaccine, animal experiments were conducted using SPF chickens. Thirty SPF chickens were randomly divided into two groups: a vaccine group and a control group, with 15 chickens in each group. The vaccine group received an intramuscular injection of 5 μg (0.2 ml) of mRNA-LNPs vaccine on day 7 and 0.2 ml of PBS on day 21, respectively. At days 14, 28, 42, and 49, three chickens were randomly selected, and 2 ml of anticoagulated blood was collected from the subwing vein. Serum was then separated.

[0050] Recombinant antigen protein was expressed using the pet28a vector and E. coli BL21. The protein was purified using a His-tagged protein purification kit (Beyotime Biotechnology Co., Ltd.). The protein was diluted to 10 μg / mL with coating buffer (Solepro Biotechnology Co., Ltd.). 100 μL was added to each well of an ELISA plate and incubated at 4°C for 16 h. The coating buffer was then discarded, and the plate was washed three times with 1xPBST for 1 min each time, and the residual liquid was patted dry. 200 μL of 5% skim milk powder was added to each well and the plate was incubated at 37°C for 1.5 h. After incubation, the plate was washed three times with 1xPBST for 1 min each time, and the residual liquid was patted dry. Serum samples were serially diluted (starting at 200-fold and diluting to 25600-fold), and 100 μL of serum diluent was added to each well of an ELISA plate. The plate was incubated at 37°C for 1 h. After incubation, the plate was washed three times with 1xPBST for 1 min each time, and the residual liquid was patted dry. Goat anti-chicken IgG was used as the secondary antibody, diluted 5000-fold. 100 μL of the diluted secondary antibody was added to each well and incubated at 37°C for 1 h. After incubation, the sample was washed three times with 1xPBST buffer for 1 min each time, and the residual liquid was blotted dry. Under light-protected conditions, chromogenic reagents A and B were mixed thoroughly in a 1:1 ratio. 100 μL of the mixed chromogenic reagent was added to each sample well and reacted at 37°C for 15 min. After the reaction was complete, 100 μL of 2 mM H₂SO₄ stop solution was added to each well to stop the chromogenic reaction. The absorbance of each well was measured using a spectrophotometer at 450 nm. The antibody titer results of SPF chicken immunization with mRNA-LNPs vaccine are shown below. Figure 8 .

[0051] according to Figure 8 As a result, antibody levels peaked one week after the booster immunization and remained at a high level for one month.

[0052] Neutralizing antibody titer assay: ALV-J, ALV-A, and ALV-K (Genbank accession number for ALV-J: MH379647, for ALV-A: HM452339, and for ALV-K: KY773912) were diluted to 200 TCID using antibody-free and serum-free DMEM. 50 / 0.1mL, serially dilute the serum to be tested with an equal volume, and add 100 TCID to each well. 50 The antigen was neutralized in a cell culture incubator for 2 hours, the neutralization solution was discarded, and the cells were washed twice with sterile PBS. DMEM maintenance medium containing 1% FBS was added, and the cells were cultured for 5 days. The supernatant from each well was collected and the results were determined using an avian leukosis-specific antigen detection kit (purchased from Tianzhitai Biotechnology Co., Ltd.). The results are shown in the attached table. Figure 9 .

[0053] according to Figure 9 As a result, the highest ratio of neutralizing antibody against ALV-J produced after immunization was 1:160, the highest ratio of neutralizing antibody against ALV-A was 1:320, and the highest ratio of neutralizing antibody against ALV-K was 1:280.

[0054] Cytokine detection: Serum cytokine levels were detected using a chicken interferon-gamma (IFN-γ) ELISA kit (purchased from ELISA Biotechnology Co., Ltd.) and a chicken interleukin-4 (IL-4) ELISA kit (purchased from ELISA Biotechnology Co., Ltd.), following the manufacturer's instructions. Standards were diluted to five concentration gradients: 120 pg / mL, 60 pg / mL, 30 pg / mL, 15 pg / mL, and 7.5 pg / mL. For sample loading, blank wells (without sample or enzyme-labeled reagents), standard wells, and serum wells were prepared. 50 μL of standard was added to the ELISA plate. For the serum wells, 40 μL of sample diluent was added first, followed by 10 μL of serum. The plate was gently shaken to mix, sealed with a sealing plate, and incubated at 37°C for 30 min. Dilute the 20x concentrated wash buffer 20x with distilled water. Carefully remove the sealing film, discard the liquid, and shake dry. Fill each well with the wash buffer, let stand for 30 seconds, then discard. Repeat 5 times, and pat dry. Add 50 μL of enzyme-labeled reagent to each well, except for the blank wells. Seal the plate with a sealing film and incubate at 37°C for 30 min, then wash 5 times. First add 50 μL of chromogenic reagent A, then add 50 μL of chromogenic reagent B, gently shake to mix, and develop at 37°C in the dark for 10 min. Add the stop solution to terminate the reaction, and measure the absorbance of each well at 450 nm using a spectrophotometer. The cytokine detection results after SPF chicken immunization with mRNA-LNPs vaccine are shown below. Figure 10 .

[0055] according to Figure 10 As a result, the vaccine was able to induce good humoral and cellular immunity in the flock, and this immunity was maintained at a high level for one month.

[0056] Example 7: Immunoprotective efficacy trial of mRNA-LNPs vaccine Among the four subgroups (A, B, J, and K) of prevalent ALV strains, ALV-A was chosen as the representative because the lesions caused by ALV-A and ALV-B are essentially the same. ALV-K, however, has weak pathogenicity and does not produce obvious lesions after inoculation; therefore, ALV-J and ALV-A were selected as the test items in this experiment. The Genbank accession number for ALV-J is MH379647, and the Genbank accession number for ALV-A is HM452339.

[0057] To verify the protective effect of the mRNA-LNPs vaccine on chickens, 60 SPF chickens were randomly divided into 5 groups: ALV-J challenge group, ALV-A challenge group, vaccine + ALV-J challenge group, vaccine + ALV-A challenge group, and control group, with 12 chickens in each group. On day 14 (35) after the second immunization, all chickens except the control group were intraperitoneally injected with 100 TCID50. 50 Two types of viruses were used. After challenge, cells were weighed weekly (42d, 49d, 56d), and blood was collected from the subwing vein to separate plasma. 100 µL of plasma was dropped into a 24-well plate containing 400 µL of antibiotic-free and serum-free DMEM, and cultured in a monolayer of DF-1 cells. After 2 hours of plasma adsorption, the plate was replaced with DMEM maintenance medium containing 1% FBS, and the cells were cultured for 7 days. Cell supernatant was collected, and viral symptoms were detected using an avian leukosis-specific antigen detection kit (purchased from Tianzhitai Biotechnology Co., Ltd.). RT-PCR was used to detect the viral load in the blood. Anal swabs were collected, and viral shedding was detected using the avian leukosis-specific antigen detection kit. The protection rate of the vaccine was calculated using the above three methods, using the following formula: Protection rate = (Incidence rate in control group - Incidence rate in experimental group) / Incidence rate in control group × 100%.

[0058] The results are shown in Table 7.

[0059] Table 7. Disease incidence in chickens of the control group and experimental groups after viral challenge. According to the results in Table 7, after two immunizations with the vaccine, the proportion of viremia and RT-PCR positivity in the flock was significantly reduced, and the protection rate reached 83%, indicating that the vaccine was effective.

[0060] Three chickens were euthanized in each group, and organs such as liver, spleen, bone marrow, bursa of Fabricius, and thymus were collected. The lesions were observed, and the chickens were weighed and their immune organ indices were calculated. The monitoring results of weight changes in SPF chickens after challenge are shown below. Figure 11 The results of dynamic changes in immune organ indices in SPF chickens after challenge with ALV-J are shown in [the table below]. Figure 12 The dynamic changes in immune organ indices in SPF chickens after ALV-A challenge are shown in the figure below. Figure 13 .

[0061] according to Figure 11-13 The results showed no significant difference in body weight between the control group and the vaccine plus challenge group, but the immune organ index of the vaccine plus challenge group was significantly higher than that of the challenge group, indicating that the vaccine was effective.

[0062] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A combination peptide segment, characterized in that, The amino acid sequence of the combined peptide segment is shown as SEQ ID NO:

29.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the combined peptide segment of claim 1.

3. The nucleic acid molecule of claim 2, wherein, The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO:

28.

4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule of claim 2 or 3.

5. An mRNA, characterized in that The mRNA is sequentially linked from 5' end to 3' end by a T7 promoter, a 5' UTR, the nucleic acid molecule of claim 3, a 3' UTR and a polyA. The nucleotide sequence of the T7 promoter is shown as SEQ ID NO: 24, the nucleotide sequence of the 5' UTR is shown as SEQ ID NO: 25, the nucleotide sequence of the 3' UTR is shown as SEQ ID NO: 26, and the nucleotide sequence of the polyA is shown as SEQ ID NO:

27.

6. The mRNA according to claim 5, characterized in that, The nucleotide sequence of the mRNA is shown as SEQ ID NO: 1, and the 5' end of the mRNA has a cap structure; the DNA sequence transcribed to the mRNA is shown as SEQ ID NO:

2.

7. A fowl leukosis tetravalent epitope mRNA vaccine, characterized in that, The avian leukemia tetravalent epitope mRNA vaccine comprises the nucleic acid molecule of claim 2 or 3 and / or the mRNA of claim 5 or 6, and a lipid nanoparticle.

8. The avian leukosis tetravalent epitope mRNA vaccine according to claim 7, characterized in that, In the lipid nanoparticle, the molar ratio of DOTAP, 1,2-DSPC, CholesUerol and DMG-PEG2000 is DOTAP: 1,2-DSPC: CholesUerol: DMG-PEG2000 = (45-55): (5-15): (35-40): (1-2).

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the mRNA of claim 5 or 6 and / or the avian leukemia tetravalent epitope mRNA vaccine of claim 7 or 8, and a pharmaceutically acceptable carrier.

10. Use of the combined peptide segment of claim 1, the nucleic acid molecule of claim 2 or 3, the recombinant vector of claim 4, the mRNA of claim 5 or 6, the avian leukemia tetravalent epitope mRNA vaccine of claim 7 or 8, or the pharmaceutical composition of claim 9 in the preparation of a medicament for preventing and / or treating avian leukemia.

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