Recombinant adenovirus and application thereof in preparation of avian reovirus multivalent vaccine

Abstract

The invention discloses a recombinant adenovirus and application thereof in preparation of an avian reovirus multivalent vaccine. The recombinant adenovirus constructed by the invention contains a coding gene of a chicken reovirus type III sigma C protein, a coding gene of a chicken reovirus type IV sigma C protein and a coding gene of a duck novel reovirus sigma C protein, and contains all sequences except for ORF6 / 7 and ORF6 sequences of an E1 gene, an E3 gene and an E4 gene of an adenovirus vector AdC68; the composition contains ORF6 / 7 and ORF6 sequences of the E4 gene of the human serum type 5 adenovirus, and gene sequences of adjuvant molecules porB and CD40L can also be added. Experiments prove that when the recombinant virus is used for preparing a candidate vaccine, strong and lasting specific cell and humoral immune response can be induced and generated, and a certain protection effect on live virus attack is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant adenovirus and an application thereof in preparing a polyvalent avian reovirus vaccine. Background Art

[0002] Avian reovirus (ARV) infects a variety of poultry, including chickens, ducks, and geese, causing significant economic losses to the livestock industry. The virus's continuous mutations have weakened the protective effects of traditional vaccines, making the development of safe and effective new ARV vaccines a key approach to addressing this issue.

[0003] Avian reovirus, a member of the Reoviridae family and the genus Orthoreovirus, is a double-stranded RNA virus with no envelope, an icosahedral structure, and a double-layered capsid. The viral genome is a linear, double-stranded RNA virus divided into 10 segments, each encoding 10 structural proteins. The σC protein, encoded by the S1 gene, is an outer capsid protein involved in cell attachment and can induce the production of ARV-specific neutralizing antibodies. It is an important candidate for the development of ARV diagnostic reagents and vaccines. ARVs infect a wide range of poultry species. Infection in poultry can lead to decreased production performance, low feed conversion rates, low uniformity among infected birds, and secondary infections with other viruses or bacteria, resulting in significant economic losses for poultry farming operations. ARVs spread widely through both horizontal and vertical transmission. Due to factors such as recombination within the ARV gene, the virus has a high mutation rate, necessitating the development of new vaccines. Adenovirus-vectored vaccines are widely used due to their strong immunogenicity, lack of adjuvant requirements, broad host range, ease of operation, and amenability to large-scale production.

[0004] Currently, most research on ARV vaccines focuses on attenuated vaccines or recombinant subunit vaccines, and there is no research on viral vector vaccines. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to prepare a viral vector vaccine of avian reovirus and / or how to prepare a multivalent avian reovirus vaccine based on a chimpanzee adenovirus vector.

[0006] In order to solve the above technical problems, the present invention first provides a recombinant adenovirus, which contains the coding genes of chicken reovirus type III σC protein, chicken reovirus type IV σC protein and duck novel reovirus σC protein.

[0007] The recombinant adenovirus can be obtained by inserting the coding genes of chicken reovirus type III σC protein, chicken reovirus type IV σC protein and duck novel reovirus σC protein into an adenovirus vector and then introducing the resulting recombinant adenovirus into packaging cells.

[0008] The recombinant replication-deficient adenovirus vector may be a eukaryotic gene expression vector, which may contain elements necessary for a eukaryotic gene expression vector, such as a promoter, a transcription termination sequence, a polyA addition signal sequence, a replication initiation sequence (ori), and the like.

[0009] The adenovirus vector may be AdC68, AdC6, AdC7, AdHu5, AdC63 or AdHu26, etc.

[0010] The adenoviral vector may specifically be a recombinant replication-deficient adenoviral vector plasmid AdC68, which may contain all sequences of the adenoviral vector AdC68 except for the ORF6 / 7 and ORF6 sequences of the E1, E3, and E4 genes. The recombinant replication-deficient adenoviral vector plasmid AdC68 may also contain the ORF6 / 7 and ORF6 sequences of the E4 gene of a human serotype 5 adenovirus.

[0011] The AdC68 may be a chimpanzee adenovirus vector AdC68XY3, and the GenBank accession number of the AdC68 may be: AC_000011.1. In the above recombinant adenovirus, the recombinant replication-deficient adenovirus vector plasmid may further contain a gene sequence encoding an immune adjuvant.

[0012] The immune adjuvant may be porB and / or CD40L.

[0013] The porB is an outer membrane protein from Neisseria meningitidis and is a TLR2 ligand-based adjuvant. As a vaccine adjuvant, porB increases the expression of co-stimulatory factors and cytokines by APCs, increasing antibody production, improving antigen transport to lymph nodes, and promoting a broad range of T cell responses. The CD40L is the CD40 ligand, a glycoprotein expressed on the surface of cells such as activated T cells. This molecule belongs to the necrosis factor superfamily and has been defined as the most important co-stimulatory factor for activating APCs.

[0014] The porB may be a protein whose amino acid sequence is the outer membrane protein of Neisseria meningitidis, and the CD40L may be a protein whose amino acid sequence is the ligand of CD40.

[0015] The coding sequence of the gene encoding porB may be sequence 1; the coding sequence of the gene encoding CD40L may be sequence 2.

[0016] In the above-mentioned recombinant adenovirus, the recombinant replication-deficient adenovirus vector may further contain P2A encoding gene and T2A encoding gene sequence.

[0017] The P2A may be a self-cleaving 2A short peptide from porcine teschovirus (PTV), which may result in co-translational cleavage of the encoded polypeptide.

[0018] The T2A may be a self-cleaving 2A short peptide from Thosea asigna virus, which may result in co-translational cleavage of the encoded polypeptide.

[0019] The P2A may be a self-cleaving 2A short peptide of porcine teschovirus, and the T2A may be a self-cleaving 2A short peptide of β-tetrasomal virus of the genus Platyrrhineia.

[0020] The coding sequence of the gene encoding P2A may be nucleotides 2342-2398 of SEQ.No.1; the coding sequence of the gene encoding T2A may be nucleotides 3371-3424 of SEQ.No.1.

[0021] In the above-mentioned recombinant adenovirus, the recombinant replication-deficient adenovirus vector may contain a gene encoding a fusion protein. The fusion protein may be ARV1-P2A-ARV2-T2A-ARV3, ARV1-P2A-ARV2-T2A-ARV3-P2A-porB, or ARV1-P2A-ARV2-T2A-ARV3-P2A-CD40L. The ARV1-P2A-ARV2-T2A-ARV3 may be a protein formed by linking the chicken reovirus type III σC protein, the chicken reovirus type IV σC protein, the duck novel reovirus σC protein, the P2A, and the T2A. The ARV1-P2A-ARV2-T2A-ARV3-P2A-porB may be a protein formed by connecting the chicken reovirus type III σC protein, the chicken reovirus type IV σC protein, the duck novel reovirus σC protein, the porB, the P2A, and the T2A. The ARV1-P2A-ARV2-T2A-ARV3-P2A-CD40L may be a protein formed by connecting the chicken reovirus type III σC protein, the chicken reovirus type IV σC protein, the duck novel reovirus σC protein, the CD40L, the P2A, and the T2A.

[0022] The chicken reovirus type III σC protein may be as follows (a1) or (a2) or (a3):

[0023] (a1) the amino acid sequence is sequence 3 in the sequence listing;

[0024] (a2) a protein having the same function as (a1) after amino acid residue substitution and / or deletion and / or addition;

[0025] (a3) A protein derived from chicken reovirus type III, having 98% or greater identity with (a1) and having the same function as (a1).

[0026] The chicken reovirus type IV σC protein is as follows (b1) or (b2) or (b3):

[0027] (b1) the amino acid sequence is Sequence 4 in the sequence listing;

[0028] (b2) a protein having the same function as (b1) after amino acid residue substitution and / or deletion and / or addition;

[0029] (b3) A protein derived from chicken reovirus type IV and having 98% or greater identity with (b1) and having the same function as (b1).

[0030] The duck novel reovirus σC protein is as follows (c1) or (c2) or (c3):

[0031] (c1) the amino acid sequence is Sequence 5 in the sequence listing;

[0032] (c2) a protein having the same function as (c1) after amino acid residue substitution and / or deletion and / or addition;

[0033] (c3) A protein derived from a duck novel reovirus and having 98% or greater identity with (c1) and having the same function as (c1).

[0034] The gene encoding the σC protein may be as follows (d1) or (d2) or (d3):

[0035] (d1) the coding region sequence is nucleotides 1349-2341 of SEQ.No.1;

[0036] (d2) the coding region sequence is nucleotides 3425-4429 of SEQ.No.1;

[0037] (d3) the coding region sequence is nucleotides 2399-3370 of SEQ.No.1;

[0038] (d4) a DNA molecule that hybridizes with (d1), (d2) or (d3) under stringent conditions and encodes the protein;

[0039] (d5) A DNA molecule derived from a reovirus and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA molecule defined in (d1), (d2) or (d3) and encoding the protein.

[0040] The stringent conditions may be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 2×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 0.5×SSC and 0.1% SDS.

[0041] Alternatively, the membrane may be hybridized in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA at 50°C, and rinsed in 0.1×SSC, 0.1% SDS at 50°C. Alternatively, the membrane may be hybridized in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA at 50°C, and rinsed in 0.1×SSC, 0.1% SDS at 65°C. Alternatively, the membrane may be hybridized in a solution of 6×SSC, 0.5% SDS at 65°C, and then washed once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0042] In the above-mentioned recombinant adenovirus, the gene encoding the fusion protein can be any of the following DNA molecules:

[0043] A1) a DNA molecule whose nucleotide sequence contains positions 1349-2341, 3425-4429, and 2399-3370 of SEQ.No.1 in the sequence listing;

[0044] A2) a DNA molecule containing nucleotides 1349-2341, 3425-4429, and 2399-3370 of SEQ. No. 1 and sequence 1;

[0045] A3) A DNA molecule whose nucleotide sequence contains nucleotides 1349-2341, 3425-4429, and 2399-3370 of SEQ. No. 1 and sequence 2.

[0046] In the above-mentioned recombinant adenovirus, the recombinant replication-deficient adenovirus vector may further contain an adenovirus inverted terminal repeat (ITR) sequence.

[0047] In the above-mentioned recombinant adenovirus, the recombinant replication-deficient adenovirus vector plasmid may be pAdC68XY3-ARV123, pAdC68XY3-ARV123-CD40L or pAdC68XY3-ARV123-porB.

[0048] The pAdC68XY3-ARV123 may be a double-stranded circular DNA molecule having a nucleotide sequence of SEQ.No.1.

[0049] The pAdC68XY3-ARV123-porB can be a recombinant expression vector plasmid obtained by inserting nucleotides 2342-2398 (P2A gene sequence) of SEQ.No.1 in the sequence list and sequence 1 (porB gene sequence) in the sequence list between nucleotides 4402-4403 of SEQ.No.1, while keeping the other nucleotide sequences of the pAdC68XY3-ARV123 unchanged.

[0050] The pAdC68XY3-ARV123-CD40L can be a recombinant expression vector plasmid obtained by inserting nucleotides 2342-2398 (P2A gene) of SEQ.No.1 and sequence 2 (CD40L gene sequence) in the sequence list between nucleotides 4402-4403 of SEQ.No.1, while keeping the other nucleotide sequences of the pAdC68XY3-ARV123 unchanged.

[0051] In order to solve the above technical problems, the present invention also provides a biomaterial, which can be any of the following:

[0052] 1) the recombinant replication-defective adenoviral vector described above,

[0053] 2) a recombinant microorganism or recombinant animal cell containing the recombinant replication-defective adenovirus vector described in 1),

[0054] 3) the fusion protein described above,

[0055] 4) the nucleic acid molecule of the fusion protein described above,

[0056] 5) A recombinant vector, recombinant microorganism or recombinant animal cell containing the nucleic acid molecule described in 3).

[0057] The use of the above-mentioned recombinant adenovirus and / or the above-mentioned biological material in the preparation of a polyvalent avian reovirus vaccine also falls within the scope of protection of the present invention.

[0058] The vaccine described above may also contain a suitable carrier or excipient. Carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. These materials can be used to produce a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal formulations, buccal tablets, suppositories, lyophilized powder injections, etc. These can be conventional preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems. To prepare unit dosage forms into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate with citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To prepare the unit dosage form into a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides. To prepare unit dosage forms as injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents commonly used in the art can be used, including water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters.In addition, to prepare an isotonic injection solution, an appropriate amount of sodium chloride, glucose, or glycerol may be added to the injectable formulation. Conventional cosolvents, buffers, pH adjusters, and the like may also be added. Furthermore, colorants, preservatives, fragrances, flavoring agents, sweeteners, or other materials may be added to the pharmaceutical formulation as needed. The above dosage forms can be administered via injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; cavitary administration, such as rectal and vaginal; respiratory administration, such as nasal; and mucosal administration. Among these routes of administration, injection is preferred.

[0059] This study uses the σC proteins of three currently prevalent ARV serotypes as antigens. Co-expressing the three σC proteins alone or in combination with a molecular adjuvant, using a replication-defective chimpanzee adenovirus as a vector, creates a multivalent vaccine candidate against avian reovirus. Animal experiments are conducted to evaluate the candidate's immunogenicity and protective efficacy.

[0060] The present invention cloned the σC protein genes of three prevalent serotypes of avian reovirus type IV, type III, and duck novel reovirus into the replication-deficient recombinant chimpanzee adenovirus vector AdC68XY3 constructed by our laboratory, thereby constructing a multivalent avian reovirus candidate vaccine AdC68XY3-ARV123 based on an adenovirus vector. In addition, the present invention also added molecular adjuvants porB or CD40L to AdC68XY3-ARV123 to construct candidate vaccines AdC68XY3-ARV123-porB and AdC68XY3-ARV123-CD40L. Then, the present invention evaluated the binding antibody, neutralizing antibody, and T cell immune responses induced by the three multivalent candidate vaccines and found that all three candidate vaccines had good immunogenicity. Finally, through the infection experiment of the live ARV virus S1133, it was proved that all three candidate vaccines could protect mice from attack and infection by the live ARV virus.

[0061] Currently, most ARV vaccine research focuses on attenuated or recombinant subunit vaccines, with no viral vector-based vaccines being developed. This study, by analyzing prevalent avian reovirus serotypes, successfully constructed a novel multivalent avian reovirus vaccine based on a chimpanzee adenovirus 68 vector. After a single intramuscular immunization of mice, binding antibodies, neutralizing antibodies, T cell immune responses, and protective efficacy after live virus challenge were assessed. Results showed that the three recombinant chimpanzee adenovirus vaccine candidates, AdC68XY3-ARV123, AdC68XY3-ARV123-porB, and AdC68XY3-ARV123-CD40L, all induced robust and sustained specific cellular and humoral immune responses and demonstrated limited protective efficacy against live virus challenge.

[0062] In the design of the recombinant adenovirus vector vaccine, in addition to the ARVσC protein, two adjuvant molecule genes, porB and CD40L, were added to the vector to enhance the immune effect of the vaccine. Although both adjuvant molecules were successfully expressed, the induced immune response did not optimize that of the candidate vaccine AdC68XY3-ARV123 without the addition of molecular adjuvants. However, these two molecular adjuvants have been shown to enhance the immunogenicity of protein vaccines. This further demonstrates that adenovirus vectors inherently have excellent immunogenicity without the need for additional adjuvants. In addition, in subsequent studies, adjuvant molecules can also be mixed with recombinant adenovirus vaccines in the form of proteins to prepare new ARV vaccines to test whether they produce improved immune effects.

[0063] Compared with traditional attenuated vaccines and protein vaccines, the adenovirus-based vaccine candidate can simultaneously induce high levels of CD4 + and CD8 + T cell immune response. Protein vaccines induce CD8 + The ability to induce T cell immune responses is insufficient. In addition, a recombinant adenovirus vector vaccine candidate can co-express three avian reovirus antigens to form a multivalent vaccine, simultaneously inducing immune responses against multiple avian reoviruses. However, if a live attenuated vaccine, inactivated vaccine, or protein vaccine is to be prepared as a multivalent vaccine, it is necessary to express and purify multiple viruses or protein antigens. In contrast, adenovirus vaccine candidates are simpler to prepare and less expensive. In addition, traditional attenuated vaccines and protein vaccines require the addition of adjuvants, but adenovirus vaccine candidates do not. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Construction and characterization of recombinant adenovirus vaccine vectors for avian reovirus. (A) Schematic diagram of the plasmid design for the chimpanzee adenovirus vector vaccine for avian reovirus. ARV1: σC gene of chicken reovirus type IV; ARV2: σC gene of duck novel reovirus; ARV3: σC gene of chicken reovirus type III. (B) Enzyme digestion and characterization of the three recombinant adenovirus plasmids; (C) Plaque formation after packaging of the three recombinant adenoviruses; (D) Enzyme digestion and characterization of the recombinant adenovirus vaccine genome.

[0065] Figure 2 After HEK293 cells were infected with adenoviruses at different titers, the expression of the three target proteins ARV1, ARV2, and ARV3 was detected by Western blotting. ARV1: σC protein of chicken reovirus type IV; ARV2: σC protein of duck novel reovirus; ARV3: σC protein of chicken reovirus type III.

[0066] Figure 3Detection of specific binding antibodies induced by the ARV recombinant chimpanzee adenovirus vaccine. (A) Long-term specific binding antibody immune monitoring against the ARV1 protein; the vertical axis represents the endpoint titer (log10); the horizontal axis represents the number of weeks after immunization; (B) Long-term specific binding antibody immune monitoring against the ARV2 protein; the vertical axis represents the endpoint titer (log10); the horizontal axis represents the number of weeks after immunization; (C) Long-term specific binding antibody immune monitoring against the ARV3 protein; the vertical axis represents the endpoint titer (log10); the horizontal axis represents the number of weeks after immunization.

[0067] Figure 4 Neutralizing antibody testing for live virus S1133. (A) Neutralizing antibody titer against S1133 virus 4 weeks after immunization; the vertical axis represents the neutralizing antibody titer; (B) Neutralizing antibody titer against S1133 virus 8 weeks after immunization; the vertical axis represents the neutralizing antibody titer; (C) Long-term neutralizing antibody titer against S1133 virus in serum; the vertical axis represents the neutralizing antibody titer.

[0068] Figure 5 CD4 induced by recombinant adenovirus vaccine + Effector T cell immune response. (A) Two weeks after immunization, the specific CD4 T cells in the spleen cells of mice against the three ARV proteins + The percentage of Th1 cytokine IL2; the vertical axis is CD4 + T cell percentage; (B) CD4 + The percentage of Th1 cytokine IFN-γ; the vertical axis is CD4 + T cell percentage; (C) CD4 + The percentage of Th1 cytokine TNF-α; the vertical axis is CD4 + T cell percentage; (D) CD4 + The percentage of Th2 cytokine IL4; the vertical axis is CD4 + T cell percentage; (E) CD4 + The percentage of Th2 cytokine IL13; the vertical axis is CD4 + T cell percentage.

[0069] Figure 6 CD8 induced by recombinant adenovirus vaccine + Effector T cell immune response. (A) Two weeks after immunization, the specific CD8 T cells in the spleen cells of mice against the three ARV proteins + The percentage of Th1 cytokine IL2; the vertical axis is CD8 + T cell percentage; (B) CD8 + The percentage of Th1 cytokine IFN-γ; the vertical axis is CD8 +T cell percentage; (C) CD8 + The percentage of Th1 cytokine TNF-α; the vertical axis is CD8 + T cell percentage; (D) CD8 + The percentage of Th2 cytokine IL4; the vertical axis is CD8 + T cell percentage; (E) CD8 + The percentage of Th2 cytokine IL13; the vertical axis is CD8 + T cell percentage.

[0070] Figure 7 CD4 induced by recombinant adenovirus vaccine + Memory T cell immune response. (A) Specific CD4 T cells against three ARV-σC proteins in mouse spleen cells 20 weeks after immunization. + The percentage of Tm cytokine IL2; the vertical axis is CD4 + Tm cell percentage; (B) CD4 + The percentage of Tm cytokine IFN-γ; the vertical axis is CD4 + Tm cell percentage; (C) CD4 + The percentage of Tm cytokine TNF-α; the vertical axis is CD4 + Tm cell percentage; (D) CD4 + The percentage of Tm cytokine IL4; the vertical axis is CD4 + Tm cell percentage; (E) CD4 + The percentage of Tm cytokine IL13; the vertical axis is CD4 + Percentage of Tm cells.

[0071] Figure 8 CD8 induced by recombinant adenovirus vaccine + Memory T cell immune response. (A) 20 weeks after immunization, the specific CD8 T cells in the spleen cells of mice against the three ARV-σC proteins + The percentage of Tm cytokine IL2; the vertical axis is CD8 + Tm cell percentage; (B) CD8 + The percentage of Tm cytokine IFN-γ; the vertical axis is CD8 + Tm cell percentage; (C) CD8 + The percentage of Tm cytokine TNF-α; the vertical axis is CD8 + Tm cell percentage; (D) CD8 + The percentage of Tm cytokine IL4; the vertical axis is CD8 + Tm cell percentage; (E) CD8 + The percentage of Tm cytokine IL13; the vertical axis is CD8+ Percentage of Tm cells.

[0072] Figure 9 Evaluation of the protective efficacy of a recombinant adenovirus vaccine against challenge with the live virus S1133. (A) Viral load in mouse livers 5 days after challenge; the vertical axis represents the number of viral copies per 0.1 g of tissue (log10). (B) Viral load in mouse spleens 5 days after challenge; the vertical axis represents the number of viral copies per 0.1 g of tissue (log10). DETAILED DESCRIPTION

[0073] Animal viruses: The public may obtain this biological material from the applicant in accordance with relevant national biosafety regulations. This biological material may only be used to repeat the relevant experiments of the present invention and may not be used for other purposes.

[0074] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0075] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0076] The reagents, consumables and sequence information in the embodiments of the present invention are as follows:

[0077] Sequence: The σC gene (sigma C protein gene) of chicken reovirus type III and IV was optimized according to the chicken codon to obtain the optimized chicken reovirus type IV σC gene (ARV1, the nucleotide sequence is SEQ.No.1, positions 1349-2341) and chicken reovirus type III σC gene (ARV3, the nucleotide sequence is SEQ.No.1, positions 3425-4429). The optimized duck novel reovirus σC gene (ARV2, the nucleotide sequence is SEQ.No.1, positions 2399-3370) was obtained according to the duck codon optimization. The three σC genes were linked by P2A and T2A (nucleotide sequences are positions 2342-2398 of SEQ. No. 1 and positions 3371-3424 of SEQ. No. 1, respectively). The σC genes of chicken reovirus type III, chicken reovirus type IV, and duck novel reovirus (abbreviated as ARV123) were synthesized into the pshuttle vector (Clontech, K1650-1) to obtain the plasmid pshuttle-ARV123.

[0078] The plasmid vector pAdC68XY3-empty was maintained in our laboratory, and its nucleotide sequence is obtained by removing nucleotides 432-4710 of SEQ.No.1;

[0079] Competent E. coli Stabl2 was obtained from Shanghai Weidi Biotechnology Co., Ltd.;

[0080] Competent E. coli BL21 (DE3) was obtained from Shanghai Weidi Biotechnology Co., Ltd.;

[0081] Adenovirus packaging cell line HEK293 was obtained from ATCC.

[0082] Avian reovirus S1133 was a kind gift from Researcher Yu Kexiang of the Poultry Research Institute of Shandong Academy of Agricultural Sciences.

[0083] Vero cell line for amplification of avian reovirus was obtained from ATCC.

[0084] Experimental animals: Female BALB / c mice aged 6-8 weeks were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd.

[0085] Main reagents: ApaⅠ, MfeⅠ, Xhol, SrfⅠ, PacⅠ restriction endonucleases were from NEB, X-tremeGENE HP DNA transfection reagent was from Roche, 2×Phanta Max Master Mix and FastPure ViralDNA / RNA Mini Kit V2 were from Vazyme, The Gel Extraction Kit was from BIO-TEK, the Adenovirus Type 1 Hexon Monoclonal Antibody was from Invitrogen, and the DAB color development kit was from Zhongshan Jinqiao Company.

[0086] All statistical analyses in this invention were performed using GraphPad Prism 9 software. Data presented in the study graphs are mean ± standard error (mean ± SEM). Data were compared using one-way ANOVA. A P value greater than or equal to 0.05 indicated no statistical difference (ns); a P value less than 0.05 indicated a statistical difference. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0087] Example 1. Preparation and verification of recombinant adenovirus

[0088] In order to construct a candidate vaccine for avian reovirus based on a recombinant chimpanzee adenovirus vector, the present invention selected a laboratory-preserved replication-deficient chimpanzee adenovirus vector pAdC68XY3-empty. This vector deleted the E1 and E3 genes on the genome of the original chimpanzee adenovirus vector AdC68 (GenBank accession number: AC_000011.1), and modified its E4 gene: the ORF6 / 7 and ORF6 sequences of the corresponding E4 region in the human serum type 5 adenovirus AdHu5 genome replaced the ORF6 / 7 and ORF6 sequences in the E4 region of the AdC68 genome. The codon-optimized σC genes of the three ARVs were connected with 2A. Subsequently, the molecular adjuvant porB or CD40L was connected to the C-terminus of this fragment as the other two candidate vaccine designs. The relevant design pattern diagram is shown in FIG. Figure 1 Middle A.

[0089] 1. Construction of recombinant adenoviral vector plasmid

[0090] Plasmid pshuttle-ARV123 was codon-optimized and synthesized by Qingke Biotechnology. PCR primers (F: 5'-ctgaccgtaactataacggtcctaa-3' and R: 5'-aatAccatttcattacctctttctcc-3') were designed and used with pshuttle-ARV123 as a template to amplify the target gene expression cassette consisting of three σCs linked by 2A under the control of the CMV promoter. The PCR product (4353 bp) was separated and recovered by agarose gel electrophoresis. The plasmid vector pAdC68XY3-empty was double-digested with I-CeuI and PI-SceI endonucleases, and the digested vector was separated and recovered by agarose gel electrophoresis. The PCR product containing the target gene expression cassette was inserted into the E1 deletion region of pAdC68XY3-empty by homologous recombination to obtain a recombinant vector. The recombinant vector was transformed into E. coli Stabl2 competent cells to obtain a transformation product. The transformation product was spread on an ampicillin-resistant agar plate and cultured at 30°C for 20 hours. Monoclonal colonies were selected, identified by colony PCR, plasmids were extracted, and the correctness of the cloned sequence was verified by sequencing. In this way, a candidate vaccine positive recombinant plasmid (37364 bp, SEQ. No. 1) pAdC68XY3-ARV123 ( Figure 1 Middle A).

[0091] Subsequently, based on the pAdC68XY3-ARV123 plasmid, the adjuvant molecules porB (sequence 1 in the sequence list) and CD40L (sequence 2 in the sequence list) were cloned into the E1 region of the recombinant plasmid pAdC68XY3-ARV123 by homologous recombination, located at the C-terminus of the ARV123 target gene, and connected with 2A in the middle. The candidate vaccine positive recombinant plasmids pAdC68XY3-ARV123-porB and pAdC68XY3-ARV123-CD40L ( Figure 1 Middle A).

[0092] pAdC68XY3-ARV123-porB is a recombinant expression vector plasmid obtained by inserting the P2A gene (nucleotides 2342-2398 in SEQ.No.1) and the porB gene (sequence 1) into nucleotides 4402-4403 of SEQ.No.1 (pAdC68XY3-ARV123), while keeping the other nucleotide sequences of the pAdC68XY3-ARV123 unchanged.

[0093] The sequence of Sequence 1 is as follows (5'-3'):

[0094] Atgaagaagagcctgatcgccctgaccctggccgctctgcctgtggctgctatggctgatgtgaccctgtacggcaccatcaaggccggagtggagacctctaggagcgtgttccaccagaacggacaggtgaccgaagtgaccaccgccacaggaatcgtggacctgggatctaagattggctttaagggacaggaggacctgggaaatggactgaaggccatctggcaggtggagcagaaggccagtatcgccggaaccgacagcggatggggaaatagacagagctttatcgggctgaaggggggattcggcaagctgagagtgggaagactgaacagcgtgctgaaggacaccggggacatcaacccttgggacagcaagagcgactacctgggagtgaacaagatcgctgaaccagaggccagactgatctccgtgagatatgactctccagaattcgccggcctgagcggaagcgtgcagtatgctctgaacgacaacgctggcaggcacaatagcgagagctaccacgccggcttcaactacaagaacggaggattcttcgtgcagtacggcggagcttacaaaagacaccatcaggtgcaggagggcctgaacatcgagaaataccagatccacagactggtgagcggatacgacaacgacgccctgtacgcctccgtggctgtgcagcagcaggatgctaagctgaccgacgcctccaacagccacaacagccagaccgaggtggccgctacactggcttacagattcggaaacgtgacccccagagtgagctacgcccatggattcaaggggctggtggacgacgccgatatcggaaacgagtacgaccaggtggtggtgggagccgaatacgatttcagcaagagaacctctgccctggtgagcgccggatggctgcaggaaggaaaaggcgagaacaagttcgtggccaccgccggaggagtgggactgagacataagttctga。

[0095] pAdC68XY3-ARV123-CD40L is a recombinant expression vector plasmid obtained by inserting the P2A gene (nucleotides 2342-2398 in SEQ.No.1) and the CD40L gene (SEQ ID NO: 2) into nucleotides 4402-4403 of SEQ.No.1 (pAdC68XY3-ARV123), while maintaining the other nucleotide sequences of pAdC68XY3-ARV123 unchanged.

[0096] The sequence 2 is as follows (5'-3'):

[0097] Atgatcgagacctacaaccagaccagccccagaagcgccgctacaggactgcctatcagcatgaagatcttcatgtacctgctgaccgtgttcctgatcacccagatgatcggcagcgccctgttcgctgtgtacctgcatagaagactggacaagatcgaggacgagagaaacctgcacgaggactttgtgttcatgaagaccattcagagatgcaacaccggcgagagaagcctgagtctgctgaattgcgaagagatcaaatcccagttcgagggattcgtgaaggacattatgctgaacaaggaggagaccaagaaagaaaacagcttcgaaatgcagaagggagaccagaacccccagatcgccgctcatgtgatcagcgaggctagcagtaagaccacctccgtgctgcagtgggccgaaaagggatactacacaatgagcaacaacctggtgaccctggagaacggcaaacagctgaccgtgaagagacagggcctgtactacatctacgcccaggtgactttctgcagcaacagagaggccagcagccaggctccattcatcgcttctctgtgcctgaagtctcccggcagattcgagagaatcctgctgcgcgccgccaacacacattcaagtgccaagccctgcggccagcagtcaatccatctgggaggcgtgttcgagctgcagcctggagcttctgtgttcgtgaacgtgaccgacccctcccaggtgagtcacggaacaggattcacttccttcggcctgctgaagctgtga。

[0098] The sequence of SEQ.No.1 is as follows (5'-3'):

[0099]

[0100] gttctggggcgggggaggacctgcatgagggccagaataactgaaatctgtgcttttctgtgtgttgcagcagcatgagcggaagcggctcctttgagggaggggtattcagcccttatctgacgg

[0101] ggcgtctcccctcctgggcgggagtgcgtcagaatgtgatgggatccacggtggacggccggcccgtgcagcccgcgaactcttcaaccctgacctatgcaaccctgagctcttcgtcgttggac

[0102] gcagctgccgccgcagctgctgcatctgccgccagcgccgtgcgcggaatggccatgggcgccggctactacggcactctggtggccaactcgagttccaccaataatcccgccagcctgaac

[0103] gaggagaagctgttgctgctgatggcccagctcgaggccttgacccagcgcctgggcgagctgacccagcaggtggctcagctgcaggagcagacgcgggccgcggttgccacggtgaaat

[0104] ccaaataaaaaatgaatcaataaataaacggagacggttgttgattttaacacagagtctgaatctttatttgatttttcgcgcgcggtaggccctggaccaccggtctcgatcattgagcacccggtg

[0105] gatcttttccaggacccggtagaggtgggcttggatgttgaggtacatgggcatgagcccgtcccgggggtggaggtagctccattgcagggcctcgtgctcgggggtggtgttgtaaatcaccc

[0106] agtcatagcaggggcgcagggcatggtgttgcacaatatctttgaggaggagactgatggccacgggcagccctttggtgtaggtgtttacaaatctgttgagctgggagggatgcatgcggggg

[0107] gagatgaggtgcatcttggcctggatcttgagattggcgatgttaccgcccagatcccgcctggggttcatgttgtgcaggaccaccagcacggtgtatccggtgcacttggggaatttatcatgca

[0108] acttggaagggaaggcgtgaaagaatttggcgacgcctttgtgcccgcccaggttttccatgcactcatccatgatgatggcgatgggcccgtgggcggcggcctgggcaaagacgtttcgggg

[0109] gtcggacacatcatagttgtggtcctgggtgaggtcatcataggccattttaatgaatttggggcggagggtgccggactgggggacaaaggtaccctcgatcccgggggcgtagttcccctcaca

[0110] gatctgcatctcccaggctttgagctcggagggggggatcatgtccacctgcggggcgataaagaacacggtttccggggcgggggagatgagctgggccgaaagcaagttccggagcagct

[0111] gggacttgccgcagccggtggggccgtagatgaccccgatgaccggctgcaggtggtagttgagggagagacagctgccgtcctcccggaggaggggggccacctcgttcatcatctcgcgc

[0112] acgtgcatgttctcgcgcaccagttccgccaggaggcgctctccccccagggataggagctcctggagcgaggcgaagtttttcagcggcttgagtccgtcggccatgggcattttggagagggt

[0113] ttgttgcaagagttccaggcggtcccagagctcggtgatgtgctctacggcatctcgatccagcagacctcctcgtttcgcgggttgggacggctgcgggagtagggcaccagacgatgggcgtc

[0114] cagcgcagccagggtccggtccttccagggtcgcagcgtccgcgtcagggtggtctccgtcacggtgaaggggtgcgcgccgggctgggcgcttgcgagggtgcgcttcaggctcatccggc

[0115] tggtcgaaaaccgctcccgatcggcgccctgcgcgtcggccaggtagcaattgaccatgagttcgtagttgagcgcctcggccgcgtggcctttggcgcggagcttacctttggaagtctgcccg

[0116] caggcgggacagaggagggacttgagggcgtagagcttgggggcgaggaagacggactcgggggcgtaggcgtccgcgccgcagtgggcgcagacggtctcgcactccacgagccagg

[0117] tgaggtcgggctggtcggggtcaaaaaccagtttcccgccgttctttttgatgcgtttcttacctttggtctccatgagctcgtgtccccgctgggtgacaaagaggctgtccgtgtccccgtagaccg

[0118] actttatgggccggtcctcgagcggtgtgccgcggtcctcctcgtagaggaaccccgcccactccgagacgaaagcccgggtccaggccagcacgaaggaggccacgtgggacgggtagcg

[0119] gtcgttgtccaccagcgggtccaccttttccagggtatgcaaacacatgtccccctcgtccacatccaggaaggtgattggcttgtaagtgtaggccacgtgaccgggggtcccggccggggggg

[0120] tataaaagggtgcgggtccctgctcgtcctcactgtcttccggatcgctgtccaggagcgccagctgttggggtaggtattccctctcgaaggcgggcatgacctcggcactcaggttgtcagtttct

[0121] agaaacgaggaggatttgatattgacggtgccggcggagatgcctttcaagagcccctcgtccatctggtcagaaaagacgatctttttgttgtcgagcttggtggcgaaggagccgtagagggcg

[0122] ttggagaggagcttggcgatggagcgcatggtctggtttttttccttgtcggcgcgctccttggcggcgatgttgagctgcacgtactcgcgcgccacgcacttccattcggggaagacggtggtca

[0123] gctcgtcgggcacgattctgacctgccagccccgattatgcagggtgatgaggtccacactggtggccacctcgccgcgcaggggctcattagtccagcagaggcgtccgcccttgcgcgagc

[0124] agaaggggggcagggggtccagcatgacctcgtcgggggggtcggcatcgatggtgaagatgccgggcaggaggtcggggtcaaagtagctgatggaagtggccagatcgtccagggcag

[0125] cttgccattcgcgcacggccagcgcgcgctcgtagggactgaggggcgtgccccagggcatgggatgggtaagcgcggaggcgtacatgccgcagatgtcgtagacgtagaggggctcctc

[0126] gaggatgccgatgtaggtggggtagcagcgccccccgcggatgctggcgcgcacgtagtcatacagctcgtgcgagggggcgaggagccccgggcccaggttggtgcgactgggcttttcg

[0127] gcgcggtagacgatctggcggaaaatggcatgcgagttggaggagatggtgggcctttggaagatgttgaagtgggcgtggggcagtccgaccgagtcgcggatgaagtgggcgtaggagtc

[0128] ttgcagcttggcgacgagctcggcggtgactaggacgtccagagcgcagtagtcgagggtctcctggatgatgtcatacttgagctgtcccttttgtttccacagctcgcggttgagaaggaactctt

[0129] cgcggtccttccagtactcttcgagggggaacccgtcctgatctgcacggtaagagcctagcatgtagaactggttgacggccttgtaggcgcagcagcccttctccacggggagggcgtaggc

[0130] ctgggcggccttgcgcagggaggtgtgcgtgagggcgaaagtgtccctgaccatgaccttgaggaactggtgcttgaagtcgatatcgtcgcagcccccctgctcccagagctggaagtccgtg

[0131] cgcttcttgtaggcggggttgggcaaagcgaaagtaacatcgttgaagaggatcttgcccgcgcggggcataaagttgcgagtgatgcggaaaggttggggcacctcggcccggttgttgatga

[0132] cctgggcggcgagcacgatctcgtcgaagccgttgatgttgtggcccacgatgtagagttccacgaatcgcggacggcccttgacgtggggcagtttcttgagctcctcgtaggtgagctcgtcg

[0133] gggtcgctgagcccgtgctgctcgagcgcccagtcggcgagatgggggttggcgcggaggaaggaagtccagagatccacggccagggcggtttgcagacggtcccggtactgacggaact

[0134] gctgcccgacggccattttttcgggggtgacgcagtagaaggtgcgggggtccccgtgccagcgatcccatttgagctggagggcgagatcgagggcgagctcgacgagccggtcgtccccg

[0135] gagagtttcatgaccagcatgaaggggacgagctgcttgccgaaggaccccatccaggtgtaggtttccacatcgtaggtgaggaagagcctttcggtgcgaggatgcgagccgatggggaag

[0136] aactggatctcctgccaccaattggaggaatggctgttgatgtgatggaagtagaaatgccgacggcgcgccgaacactcgtgcttgtgtttatacaagcggccacagtgctcgcaacgctgcacg

[0137] ggatgcacgtgctgcacgagctgtacctgagttcctttgacgaggaatttcagtgggaagtggagtcgtggcgcctgcatctcgtgctgtactacgtcgtggtggtcggcctggccctcttctgcctc

[0138] gatggtggtcatgctgacgagcccgcgcgggaggcaggtccagacctcggcgcgagcgggtcggagagcgaggacgagggcgcgcaggccggagctgtccagggtcctgagacgctgc

[0139] ggagtcaggtcagtgggcagcggcggcgcgcggttgacttgcaggagtttttccagggcgcgcgggaggtccagatggtacttgatctccaccgcgccattggtggcgacgtcgatggcttgca

[0140] gggtcccgtgcccctggggtgtgaccaccgtcccccgtttcttcttgggcggctggggcgacgggggcggtgcctcttccatggttagaagcggcggcgaggacgcgcgccgggcggcagg

[0141] ggcggctcggggcccggaggcaggggcggcaggggcacgtcggcgccgcgcgcgggtaggttctggtactgcgcccggagaagactggcgtgagcgacgacgcgacggttgacgtcct

[0142] ggatctgacgcctctgggtgaaggccacgggacccgtgagtttgaacctgaaagagagttcgacagaatcaatctcggtatcgttgacggcggcctgccgcaggatctcttgcacgtcgcccga

[0143] gttgtcctggtaggcgatctcggtcatgaactgctcgatctcctcctcttgaaggtctccgcggccggcgcgctccacggtggccgcgaggtcgttggagatgcggcccatgagctgcgagaagg

[0144] cgttcatgcccgcctcgttccagacgcggctgtagaccacgacgccctcgggatcgcGggcgcgcatgaccacctgggcgaggttgagctccacgtggcgcgtgaagaccgcgtagttgcag

[0145] aggcgctggtagaggtagttgagcgtggtggcgatgtgctcggtgacgaagaaatacatgatccagcggcggagcggcatctcgctgacgtcgcccagcgcctccaaacgttccatggcctcgt

[0146] aaaagtccacggcgaagttgaaaaactgggagttgcgcgccgagacggtcaactcctcctccagaagacggatgagctcggcgatggtggcgcgcacctcgcgctcgaaggcccccgggag

[0147] ttcctccacttcctcttcttcctcctccactaacatctcttctacttcctcctcaggcggcagtggtggcgggggagggggcctgcgtcgccggcggcgcacgggcagacggtcgatgaagcgctc

[0148] gatggtctcgccgcgccggcgtcgcatggtctcggtgacggcgcgcccgtcctcgcggggccgcagcgtgaagacgccgccgcgcatctccaggtggccgggggggtccccgttgggcag

[0149] ggagagggcgctgacgatgcatcttatcaattgccccgtagggactccgcgcaaggacctgagcgtctcgagatccacgggatctgaaaaccgctgaacgaaggcttcgagccagtcgcagtc

[0150] gcaaggtaggctgagcacggtttcttctggcgggtcatgttggttgggagcggggcgggcgatgctgctggtgatgaagttgaaataggcggttctgagacggcggatggtggcgaggagcac

[0151] caggtctttgggcccggcttgctggatgcgcagacggtcggccatgccccaggcgtggtcctgacacctggccaggtccttgtagtagtcctgcatgagccgctccacgggcacctcctcctcgc

[0152] ccgcgcggccgtgcatgcgcgtgagcccgaagccgcgctggggctggacgagcgccaggtcggcgacgacgcgctcggcgaggatggcttgctggatctgggtgagggtggtctggaagt

[0153] catcaaagtcgacgaagcggtggtaggctccggtgttgatggtgtaggagcagttggccatgacggaccagttgacggtctggtggcccggacgcacgagctcgtggtacttgaggcgcgagt

[0154] aggcgcgcgtgtcgaagatgtagtcgttgcaggtgcgcaccaggtactggtagccgatgaggaagtgcggcggcggctggcggtagagcggccatcgctcggtggcgggggcgccgggcg

[0155] cgaggtcctcgagcatggtgcggtggtagccgtagatgtacctggacatccaggtgatgccggcggcggtggtggaggcgcgcgggaactcgcggacgcggttccagatgttgcgcagcgg

[0156] caggaagtagttcatggtgggcacggtctggcccgtgaggcgcgcgcagtcgtggatgctctatacgggcaaaaacgaaagcggtcagcggctcgactccgtggcctggaggctaagcgaac

[0157] gggttgggctgcgcgtgtaccccggttcgaatctcgaatcaggctggagccgcagctaacgtggtattggcactcccgtctcgacccaagcctgcaccaaccctccaggatacggaggcgggtc

[0158] gttttgcaacttttttttggaggccggatgagactagtaagcgcggaaagcggccgaccgcgatggctcgctgccgtagtctggagaagaatcgccagggttgcgttgcggtgtgccccggttcga

[0159] ggccggccggattccgcggctaacgagggcgtggctgccccgtcgtttccaagaccccatagccagccgacttctccagttacggagcgagcccctcttttgttttgtttgtttttgccagatgcatc

[0160] ccgtactgcggcagatgcgcccccaccaccctccaccgcaacaacagccccctccacagccggcgcttctgcccccgccccagcagcaacttccagccacgaccgccgcggccgccgtgag

[0161] cggggctggacagagttatgatcaccagctggccttggaagagggcgaggggctggcgcgcctgggggcgtcgtcgccggagcggcacccgcgcgtgcagatgaaaagggacgctcgcg

[0162] aggcctacgtgcccaagcagaacctgttcagagacaggagcggcgaggagcccgaggagatgcgcgcggcccggttccacgcggggcgggagctgcggcgcggcctggaccgaaagag

[0163] ggtgctgagggacgaggatttcgaggcggacgagctgacggggatcagccccgcgcgcgcgcacgtggccgcggccaacctggtcacggcgtacgagcagaccgtgaaggaggagagc

[0164] aacttccaaaaatccttcaacaaccacgtgcgcaccctgatcgcgcgcgaggaggtgaccctgggcctgatgcacctgtgggacctgctggaggccatcgtgcagaaccccaccagcaagccg

[0165] ctgacggcgcagctgttcctggtggtgcagcatagtcgggacaacgaagcgttcagggaggcgctgctgaatatcaccgagcccgagggccgctggctcctggacctggtgaacattctgcag

[0166] agcatcgtggtgcaggagcgcgggctgccgctgtccgagaagctggcggccatcaacttctcggtgctgagtttgggcaagtactacgctaggaagatctacaagaccccgtacgtgcccatag

[0167] acaaggaggtgaagatcgacgggttttacatgcgcatgaccctgaaagtgctgaccctgagcgacgatctgggggtgtaccgcaacgacaggatgcaccgtgcggtgagcgccagcaggcgg

[0168] cgcgagctgagcgaccaggagctgatgcatagtctgcagcgggccctgaccggggccgggaccgagggggagagctactttgacatgggcgcggacctgcactggcagcccagccgccg

[0169] ggccttggaggcggcggcaggaccctacgtagaagaggtggacgatgaggtggacgaggagggcgagtacctggaagactgatggcgcgaccgtatttttgctagatgcaacaacaacagc

[0170] cacctcctgatcccgcgatgcgggcggcgctgcagagccagccgtccggcattaactcctcggacgattggacccaggccatgcaacgcatcatggcgctgacgacccgcaaccccgaagcc

[0171] tttagacagcagccccaggccaaccggctctcggccatcctggaggccgtggtgccctcgcgctccaaccccacgcacgagaaggtcctggccatcgtgaacgcgctggtggagaacaaggc

[0172] catccgcggcgacgaggccggcctggtgtacaacgcgctgctggagcgcgtggcccgctacaacagcaccaacgtgcagaccaacctggaccgcatggtgaccgacgtgcgcgaggccgt

[0173] ggcccagcgcgagcggttccaccgcgagtccaacctgggatccatggtggcgctgaacgccttcctcagcacccagcccgccaacgtgccccggggccaggaggactacaccaacttcatca

[0174] gcgccctgcgcctgatggtgaccgaggtgccccagagcgaggtgtaccagtccgggccggactacttcttccagaccagtcgccagggcttgcagaccgtgaacctgagccaggctttcaaga

[0175] acttgcagggcctgtggggcgtgcaggccccggtcggggaccgcgcgacggtgtcgagcctgctgacgccgaactcgcgcctgctgctgctgctggtggcccccttcacggacagcggcag

[0176] catcaaccgcaactcgtacctgggctacctgattaacctgtaccgcgaggccatcggccaggcgcacgtggacgagcagacctaccaggagatcacccacgtgagccgcgccctgggccagg

[0177] acgacccgggcaacctggaagccaccctgaactttttgctgaccaaccggtcgcagaagatcccgccccagtacgcgctcagcaccgaggaggagcgcatcctgcgttacgtgcagcagagc

[0178] gtgggcctgttcctgatgcaggagggggccacccccagcgccgcgctcgacatgaccgcgcgcaacatggagcccagcatgtacgccagcaaccgcccgttcatcaataaactgatggacta

[0179] cttgcatcgggcggccgccatgaactctgactatttcaccaacgccatcctgaatccccactggctcccgccgccggggttctacacgggcgagtacgacatgcccgaccccaatgacgggttcc

[0180] tgtgggacgatgtggacagcagcgtgttctccccccgaccgggtgctaacgagcgccccttgtggaagaaggaaggcagcgaccgacgcccgtcctcggcgctgtccggccgcgagggtgct

[0181] gccgcggcggtgcccgaggccgccagtcctttcccgagcttgcccttctcgctgaacagtatccgcagcagcgagctgggcaggatcacgcgcccgcgcttgctgggcgaagaggagtacttg

[0182] aatgactcgctgttgagacccgagcgggagaagaacttccccaataacgggatagaaagcctggtggacaagatgagccgctggaagacgtatgcgcaggagcacagggacgatccccggg

[0183] cgtcgcagggggccacgagccggggcagcgccgcccgtaaacgccggtggcacgacaggcagcggggacagatgtgggacgatgaggactccgccgacgacagcagcgtgttggactt

[0184] gggtgggagtggtaacccgttcgctcacctgcgcccccgtatcgggcgcatgatgtaagagaaaccgaaaataaatgatactcaccaaggccatggcgaccagcgtgcgttcgtttcttctctgtt

[0185] gttgttgtatctagtatgatgaggcgtgcgtacccggagggtcctcctccctcgtacgagagcgtgatgcagcaggcgatggcggcggcggcgatgcagcccccgctggaggctccttacgtgc

[0186] ccccgcggtacctggcgcctacggaggggcggaacagcattcgttactcggagctggcacccttgtacgataccacccggttgtacctggtggacaacaagtcggcggacatcgcctcgctgaa

[0187] ctaccagaacgaccacagcaacttcctgaccaccgtggtgcagaacaatgacttcacccccacggaggccagcacccagaccatcaactttgacgagcgctcgcggtggggcggccagctga

[0188] aaaccatcatgcacaccaacatgcccaacgtgaacgagttcatgtacagcaacaagttcaaggcgcgggtgatggtctcccgcaagacccccaatggggtgacagtgacagaggattatgatgg

[0189] tagtcaggatgagctgaagtatgaatgggtggaatttgagctgcccgaaggcaacttctcggtgaccatgaccatcgacctgatgaacaacgccatcatcgacaattacttggcggtggggcggc

[0190] agaacggggtgctggagagcgacatcggcgtgaagttcgacactaggaacttcaggctgggctgggaccccgtgaccgagctggtcatgcccggggtgtacaccaacgaggctttccatcccg

[0191] atattgtcttgctgcccggctgcggggtggacttcaccgagagccgcctcagcaacctgctgggcattcgcaagaggcagcccttccaggaaggcttccagatcatgtacgaggatctggaggg

[0192] gggcaacatccccgcgctcctggatgtcgacgcctatgagaaaagcaaggaggatgcagcagctgaagcaactgcagccgtagctaccgcctctaccgaggtcaggggcgataattttgcaag

[0193] cgccgcagcagtggcagcggccgaggcggctgaaaccgaaagtaagatagtcattcagccggtggagaaggatagcaagaacaggagctacaacgtactaccggacaagataaacaccgc

[0194] ctaccgcagctggtacctagcctacaactatggcgaccccgagaagggcgtgcgctcctggacgctgctcaccacctcggacgtcacctgcggcgtggagcaagtctactggtcgctgcccga

[0195] catgatgcaagacccggtcaccttccgctccacgcgtcaagttagcaactacccggtggtgggcgccgagctcctgcccgtctactccaagagcttcttcaacgagcaggccgtctactcgcagc

[0196] agctgcgcgccttcacctcgcttacgcacgtcttcaaccgcttccccgagaaccagatcctcgtccgcccgcccgcgcccaccattaccaccgtcagtgaaaacgttcctgctctcacagatcacg

[0197] ggaccctgccgctgcgcagcagtatccggggagtccagcgcgtgaccgttactgacgccagacgccgcacctgcccctacgtctacaaggccctgggcatagtcgcgccgcgcgtcctctcga

[0198] gccgcaccttctaaatgtccattctcatctcgcccagtaataacaccggttggggcctgcgcgcgcccagcaagatgtacggaggcgctcgccaacgctccacgcaacaccccgtgcgcgtgcg

[0199] cgggcacttccgcgctccctggggcgccctcaagggccgcgtgcggtcgcgcaccaccgtcgacgacgtgatcgaccaggtggtggccgacgcgcgcaactacacccccgccgccgcgcc

[0200] cgtctccaccgtggacgccgtcatcgacagcgtggtggcCgacgcgcgccggtacgcccgcgccaagagccggcggcggcgcatcgcccggcggcaccggagcacccccgccatgcgc

[0201] gcggcgcgagccttgctgcgcagggccaggcgcacgggacgcagggccatgctcagggcggccagacgcgcggcttcaggcgccagcgccggcaggacccggagacgcgcggccac

[0202] ggcggcggcagcggccatcgccagcatgtcccgcccgcggcgagggaacgtgtactgggtgcgcgacgccgccaccggtgtgcgcgtgcccgtgcgcacccgcccccctcgcacttgaag

[0203] atgttcacttcgcgatgttgatgtgtcccagcggcgaggaggatgtccaagcgcaaattcaaggaagagatgctccaggtcatcgcgcctgagatctacggccctgcggtggtgaaggaggaaa

[0204] gaaagccccgcaaaatcaagcgggtcaaaaaggacaaaaaggaagaagaaagtgatgtggacggattggtggagtttgtgcgcgagttcgccccccggcggcgcgtgcagtggcgcgggc

[0205] ggaaggtgcaaccggtgctgagacccggcaccaccgtggtcttcacgcccggcgagcgctccggcaccgcttccaagcgctcctacgacgaggtgtacggggatgatgatattctggagcag

[0206] gcggccgagcgcctgggcgagtttgcttacggcaagcgcagccgttccgcaccgaaggaagaggcggtgtccatcccgctggaccacggcaaccccacgccgagcctcaagcccgtgacct

[0207] tgcagcaggtgctgccgaccgcggcgccgcgccgggggttcaagcgcgagggcgaggatctgtaccccaccatgcagctgatggtgcccaagcgccagaagctggaagacgtgctggaga

[0208] ccatgaaggtggacccggacgtgcagcccgaggtcaaggtgcggcccatcaagcaggtggccccgggcctgggcgtgcagaccgtggacatcaagattcccacggagcccatggaaacgc

[0209] agaccgagcccatgatcaagcccagcaccagcaccatggaggtgcagacggatccctggatgccatcggctcctagtcgaagaccccggcgcaagtacggcgcggccagcctgctgatgcc

[0210] caactacgcgctgcatccttccatcatccccacgccgggctaccgcggcacgcgcttctaccgcggtcataccagcagccgccgccgcaagaccaccactcgccgccgccgtcgccgcaccg

[0211] ccgctgcaaccacccctgccgccctggtgcggagagtgtaccgccgcggccgcgcacctctgaccctgccgcgcgcgcgctaccacccgagcatcgccatttaaactttcgccTgctttgcag

[0212] atcaatggccctcacatgccgccttcgcgttcccattacgggctaccgaggaagaaaaccgcgccgtagaaggctggcggggaacgggatgcgtcgccaccaccaccggcggcggcgcgcc

[0213] atcagcaagcggttggggggaggcttcctgcccgcgctgatccccatcatcgccgcggcgatcggggcgatccccggcattgcttccgtggcggtgcaggcctctcagcgccactgagacaca

[0214] cttggaaacatcttgtaataaaccAatggactctgacgctcctggtcctgtgatgtgttttcgtagacagatggaagacatcaatttttcgtccctggctccgcgacacggcacgcggccgttcatgg

[0215] gcacctggagcgacatcggcaccagccaactgaacgggggcgccttcaattggagcagtctctggagcgggcttaagaatttcgggtccacgcttaaaacctatggcagcaaggcgtggaaca

[0216] gcaccacagggcaggcgctgagggataagctgaaagagcagaacttccagcagaaggtggtcgatgggctcgcctcgggcatcaacggggtggtggacctggccaaccaggccgtgcagc

[0217] ggcagatcaacagccgcctggacccggtgccgcccgccggctccgtggagatgccgcaggtggaggaggagctgcctcccctggacaagcggggcgagaagcgaccccgccccgatgcg

[0218] gaggagacgctgctgacgcacacggacgagccgcccccgtacgaggaggcggtgaaactgggtctgcccaccacgcggcccatcgcgcccctggccaccggggtgctgaaacccgaaaa

[0219] gcccgcgaccctggacttgcctcctccccagccttcccgcccctctacagtggctaagcccctgccgccggtggccgtggcccgcgcgcgacccgggggcaccgcccgccctcatgcgaact

[0220] ggcagagcactctgaacagcatcgtgggtctgggagtgcagagtgtgaagcgccgccgctgctattaaacctaccgtagcgcttaacttgcttgtctgtgtgtgtatgtattatgtcgccgccgccgc

[0221] tgtccaccagaaggaggagtgaagaggcgcgtcgccgagttgcaagatggccaccccatcgatgctgccccagtgggcgtacatgcacatcgccggacaggacgcttcggagtacctgagtc

[0222] cgggtctggtgcagtttgcccgcgccacagacacctacttcagtctggggaacaagtttaggaaccccacggtggcgcccacgcacgatgtgaccaccgaccgcagccagcggctgacgctgc

[0223] gcttcgtgcccgtggaccgcgaggacaacacctactcgtacaaagtgcgctacacgctggccgtgggcgacaaccgcgtgctggacatggccagcacctactttgacatccgcggcgtgctgg

[0224] atcggggccctagcttcaaaccctactccggcaccgcctacaacagtctggcccccaagggagcacccaacacttgtcagtggacatataaagccgatggtgaaactgccacagaaaaaaccta

[0225] tacatatggaaatgcacccgtgcagggcattaacatcacaaaagatggtattcaacttggaactgacaccgatgatcagccaatctacgcagataaaacctatcagcctgaacctcaagtgggtgat

[0226] gctgaatggcatgacatcactggtactgatgaaaagtatggaggcagagctcttaagcctgataccaaaatgaagccttgttatggttcttttgccaagcctactaataaagaaggaggtcaggcaaa

[0227] tgtgaaaacaggaacaggcactactaaagaatatgacatagacatggctttctttgacaacagaagtgcggctgctgctggcctagctccagaaattgttttgtatactgaaaatgtggatttggaaac

[0228] tccagatacccatattgtatacaaagcaggcacagatgacagcagctcttctattaatttgggtcagcaagccatgcccaacagacctaactacattggtttcagagacaactttatcgggctcatgta

[0229] ctacaacagcactggcaatatgggggtgctggccggtcaggcttctcagctgaatgctgtggttgacttgcaagacagaaacaccgagctgtcctaccagctcttgcttgactctctgggtgacaga

[0230] acccggtatttcagtatgtggaatcaggcggtggacagctatgatcctgatgtgcgcattattgaaaatcatggtgtggaggatgaacttcccaactattgtttccctctggatgctgttggcagaacag

[0231] atacttatcagggaattaaggctaatggaactgatcaaaccacatggaccaaagatgacagtgtcaatgatgctaatgagataggcaagggtaatccattcgccatggaaatcaacatccaagcca

[0232] acctgtggaggaacttcctctacgccaacgtggccctgtacctgcccgactcttacaagtacacgccggccaatgttaccctgcccaccaacaccaacacctacgattacatgaacggccgggtg

[0233] gtggcgccctcgctggtggactcctacatcaacatcggggcgcgctggtcgctggatcccatggacaacgtgaaccccttcaaccaccaccgcaatgcggggctgcgctaccgctccatgctcc

[0234] tgggcaacgggcgctacgtgcccttccacatccaggtgccccagaaatttttcgccatcaagagcctcctgctcctgcccgggtcctacacctacgagtggaacttccgcaaggacgtcaacatga

[0235] tcctgcagagctccctcggcaacgacctgcgcacggacggggcctccatctccttcaccagcatcaacctctacgccaccttcttccccatggcgcacaacacggcctccacgctcgaggccatg

[0236] ctgcgcaacgacaccaacgaccagtccttcaacgactacctctcggcggccaacatgctctaccccatcccggccaacgccaccaacgtgcccatctccatcccctcgcgcaactgggccgcct

[0237] tccgcggctggtccttcacgcgtctcaagaccaaggagacgccctcgctgggctccgggttcgacccctacttcgtctactcgggctccatcccctacctcgacggcaccttctacctcaaccaca

[0238] ccttcaagaaggtctccatcaccttcgactcctccgtcagctggcccggcaacgaccggctcctgacgcccaacgagttcgaaatcaagcgcaccgtcgacggcgagggctacaacgtggccc

[0239] agtgcaacatgaccaaggactggttcctggtccagatgctggcccactacaacatcggctaccagggcttctacgtgcccgagggctacaaggaccgcatgtactccttcttccgcaacttccagc

[0240] ccatgagccgccaggtggtggacgaggtcaactacaaggactaccaggccgtcaccctggcctaccagcacaacaactcgggcttcgtcggctacctcgcgcccaccatgcgccagggccag

[0241] ccctaccccgccaactacccctacccgctcatcggcaagagcgccgtcaccagcgtcacccagaaaaagttcctctgcgacagggtcatgtggcgcatccccttctccagcaacttcatgtccatg

[0242] ggcgcgctcaccgacctcggccagaacatgctctatgccaactccgcccacgcgctagacatgaatttcgaagtcgaccccatggatgagtccacccttctctatgttgtcttcgaagtcttcgacgt

[0243] cgtccgagtgcaccagccccaccgcggcgtcatcgaggccgtctacctgcgcacccccttctcggccggtaacgccaccacctaagctcttgcttcttgcaagccatggccgcgggctccggcg

[0244] agcaggagctcagggccatcatccgcgacctgggctgcgggccctacttcctgggcaccttcgataagcgcttcccgggattcatggccccgcacaagctggcctgcgccatcgtcaacacgg

[0245] ccggccgcgagaccgggggcgagcactggctggccttcgcctggaacccgcgctcgaacacctgctacctcttcgaccccttcgggttctcggacgagcgcctcaagcagatctaccagttcg

[0246] agtacgagggcctgctgcgccgcagcgccctggccaccgaggaccgctgcgtcaccctggaaaagtccacccagaccgtgcagggtccgcgctcggccgcctgcgggctcttctgctgcatg

[0247] ttcctgcacgccttcgtgcactggcccgaccgccccatggacaagaaccccaccatgaacttgctgacgggggtgcccaacggcatgctccagtcgccccaggtggaacccaccctgcgccgc

[0248] aaccaggaggcgctctaccgcttcctcaactcccactccgcctactttcgctcccaccgcgcgcgcatcgagaaggccaccgccttcgaccgcatgaatcaagacatgtaaaccgtgtgtgtatgt

[0249] taaatgtctttaataaacagcactttcatgttacacatgcatctgagatgatttatttagaaatcgaaagggttctgccgggtctcggcatggcccgcgggcagggacacgttgcggaactggtacttg

[0250] gccagccacttgaactcggggatcagcagtttgggcagcggggtgtcggggaaggagtcggtccacagcttccgcgtcagttgcagggcgcccagcaggtcgggcgcggagatcttgaaatc

[0251] gcagttgggacccgcgttctgcgcgcgggagttgcggtacacggggttgcagcactggaacaccatcagggccgggtgcttcacgctcgccagcaccgtcgcgtcggtgatgctctccacgtc

[0252] gaggtcctcggcgttggccatcccgaagggggtcatcttgcaggtctgccttcccatggtgggcacgcacccgggcttgtggttgcaatcgcagtgcagggggatcagcatcatctgggcctggt

[0253] cggcgttcatccccgggtacatggccttcatgaaagcctccaattgcctgaacgcctgctgggccttggctccctcggtgaagaagaccccgcaggacttgctagagaactggttggtggcgcac

[0254] ccggcgtcgtgcacgcagcagcgcgcgtcgttgttggccagctgcaccacgctgcgcccccagcggttctgggtgatcttggcccggtcggggttctccttcagcgcgcgctgcccgttctcgct

[0255] cgccacatccatctcgatcatgtgctccttctggatcatggtggtcccgtgcaggcaccgcagcttgccctcggcctcggtgcacccgtgcagccacagcgcgcacccggtgcactcccagttctt

[0256] gtgggcgatctgggaatgcgcgtgcacgaagccctgcaggaagcggcccatcatggtggtcagggtcttgttgctagtgaaggtcagcggaatgccgcggtgctcctcgttgatgtacaggtgg

[0257] cagatgcggcggtacacctcgccctgctcgggcatcagctggaagttggctttcaggtcggtctccacgcggtagcggtccatcagcatagtcatgatttccatacccttctcccaggccgagacg

[0258] atgggcaggctcatagggttcttcaccatcatcttagcgctagcagccgcggccagggggtcgctctcgtccagggtctcaaagctccgcttgccgtccttctcggtgatccgcaccggggggtag

[0259] ctgaagcccacggccgccagctcctcctcggcctgtctttcgtcctcgctgtcctggctgacgtcctgcaggaccacatgcttggtcttgcggggtttcttcttgggcggcagcggcggcggagatg

[0260] ttggagatggcgagggggagcgcgagttctcgctcaccactactatctcttcctcttcttggtccgaggccacgcggcggtaggtatgtctcttcgggggcagaggcggaggcgacgggctctcg

[0261] ccgccgcgacttggcggatggctggcagagccccttccgcgttcgggggtgcgctcccggcggcgctctgactgacttcctccgcggccggccattgtgttctcctagggaggaacaacaagc

[0262] atggagactcagccatcgccaacctcgccatctgcccccaccgccgacgagaagcagcagcagcagaatgaaagcttaaccgccccgccgcccagccccgccacctccgacgcggccgtcc

[0263] cagacatgcaagagatggaggaatccatcgagattgacctgggctatgtgacgcccgcggagcacgaggaggagctggcagtgcgcttttcacaagaagagatacaccaagaacagccagag

[0264] caggaagcagagaatgagcagagtcaggctgggctcgagcatgacggcgactacctccacctgagcgggggggaggacgcgctcatcaagcatctggcccggcaggccaccatcgtcaag

[0265] gatgcgctgctcgaccgcaccgaggtgcccctcagcgtggaggagctcagccgcgcctacgagttgaacctcttctcgccgcgcgtgccccccaagcgccagcccaatggcacctgcgagcc

[0266] caacccgcgcctcaacttctacccggtcttcgcggtgcccgaggccctggccacctaccacatctttttcaagaaccaaaagatccccgtctcctgccgcgccaaccgcacccgcgccgacgccc

[0267] ttttcaacctgggtcccggcgcccgcctacctgatatcgcctccttggaagaggttcccaagatcttcgagggtctgggcagcgacgagactcgggccgcgaacgctctgcaaggagaaggagg

[0268] agagcatgagcaccacagcgccctggtcgagttggaaggcgacaacgcgcggctggcggtgctcaaacgcacggtcgagctgacccatttcgcctacccggctctgaacctgccccccaaag

[0269] tcatgagcgcggtcatggaccaggtgctcatcaagcgcgcgtcgcccatctccgaggacgagggcatgcaagactccgaggagggcaagcccgtggtcagcgacgagcagctggcccggtg

[0270] gctgggtcctaatgctagtccccagagtttggaagagcggcgcaaactcatgatggccgtggtcctggtgaccgtggagctggagtgcctgcgccgcttcttcgccgacgcggagaccctgcgc

[0271] aaggtcgaggagaacctgcactacctcttcaggcacgggttcgtgcgccaggcctgcaagatctccaacgtggagctgaccaacctggtctcctacatgggcatcttgcacgagaaccgcctgg

[0272] ggcagaacgtgctgcacaccaccctgcgcggggaggcccggcgcgactacatccgcgactgcgtctacctctacctctgccacacctggcagacgggcatgggcgtgtggcagcagtgtctg

[0273] gaggagcagaacctgaaagagctctgcaagctcctgcagaagaacctcaagggtctgtggaccgggttcgacgagcgcaccaccgcctcggacctggccgacctcattttccccgagcgcctc

[0274] aggctgacgctgcgcaacggcctgcccgactttatgagccaaagcatgttgcaaaactttcgctctttcatcctcgaacgctccggaatcctgcccgccacctgctccgcgctgccctcggacttcg

[0275] tgccgctgaccttccgcgagtgccccccgccgctgtggagccactgctacctgctgcgcctggccaactacctggcctaccactcggacgtgatcgaggacgtcagcggcgagggcctgctcg

[0276] agtgccactgccgctgcaacctctgcacgccgcaccgctccctggcctgcaacccccagctgctgagcgagacccagatcatcggcaccttcgagttgcaagggcccagcgaaggcgagggt

[0277] tcagccgccaaggggggtctgaaactcaccccggggctgtggacctcggcctacttgcgcaagttcgtgcccgaggactaccatcccttcgagatcaggttctacgaggaccaatcccatccgc

[0278] ccaaggccgagctgtcggcctgcgtcatcacccagggggcgatcctggcccaattgcaagccatccagaaatcccgccaagaattcttgctgaaaaagggccgcggggtctacctcgaccccc

[0279] agaccggtgaggagctcaaccccggcttcccccaggatgccccgaggaaacaagaagctgaaagtggagctgccgcccgtggaggatttggaggaagactgggagaacagcagtcaggca

[0280] gaggaggaggagatggaggaagactgggacagcactcaggcagaggaggacagcctgcaagacagtctggaggaagacgaggaggaggcagaggaggaggtggaagaagcagccgc

[0281] cgccagaccgtcgtcctcggcgggggagaaagcaagcagcacggataccatctccgctccgggtcggggtcccgctcgaccacacagtagatgggacgagaccggacgattcccgaacccc

[0282] accacccagaccggtaagaaggagcggcagggatacaagtcctggcgggggcacaaaaacgccatcgtctcctgcttgcaggcctgcgggggcaacatctccttcacccggcgctacctgct

[0283] cttccaccgcggggtgaactttccccgcaacatcttgcattactaccgtcacctccacagcccctactacttccaagaagaggcagcagcagcagaaaaagaccagcagaaaaccagcagctag

[0284] aaaatccacagcggcggcagcaggtggactgaggatcgcggcgaacgagccggcgcaaacccgggagctgaggaaccggatctttcccaccctctatgccatcttccagcagagtcggggg

[0285] caggagcaggaactgaaagtcaagaaccgttctctgcgctcgctcacccgcagttgtctgtatcacaagagcgaagaccaacttcagcgcactctcgaggacgccgaggctctcttcaacaagta

[0286] ctgcgcgctcactcttaaagagtagcccgcgcccgcccagtcgcagaaaaaggcgggaattacgtcacctgtgcccttcgccctagccgcctccacccatcatcatgagcaaagagattcccac

[0287] gccttacatgtggagctaccagccccagatgggcctggccgccggtgccgcccaggactactccacccgcatgaattggctcagcgccgggcccgcgatgatctcacgggtgaatgacatccg

[0288] cgcccaccgaaaccagatactcctagaacagtcagcgctcaccgccacgccccgcaatcacctcaatccgcgtaattggcccgccgccctggtgtaccaggaaattccccagcccacgaccgt

[0289] actacttccgcgagacgcccaggccgaagtccagctgactaactcaggtgtccagctggcgggcggcgccaccctgtgtcgtcaccgccccgctcagggtataaagcggctggtgatccgggg

[0290] cagaggcacacagctcaacgacgaggtggtgagctcttcgctgggtctgcgacctgacggagtcttccaactcgccggatcggggagatcttccttcacgcctcgtcaggccgtcctgactttgg

[0291] agagttcgtcctcgcagccccgctcgggtggcatcggcactctccagttcgtggaggagttcactccctcggtctacttcaaccccttctccggctcccccggccactacccggacgagttcatccc

[0292] gaacttcgacgccatcagcgagtcggtggacggctacgattgaatgtcccatggtggcgcagctgacctagctcggcttcgacacctggaccactgccgccgcttccgctgcttcgctcgggatct

[0293] cgccgagtttgcctactttgagctgcccgaggagcaccctcagggcccggcccacggagtgcggatcgtcgtcgaagggggcctcgactcccacctgcttcggatcttcagccagcgtccgatc

[0294] ctggtcgagcgcgagcaaggacagacccttctgactctgtactgcatctgcaaccaccccggcctgcatgaaagtctttgttgtctgctgtgtactgagtataataaaagctgagatcagcgactact

[0295] ccggacttccgtgtgttcctgaatccatcaaccagtctttgttcttcaccgggaacgagaccgagctccagctccagtgtaagccccacaagaagtacctcacctggctgttccagggctccccgat

[0296] cgccgttgtcaaccactgcgacaacgactatttaaatccacaatacatgcccatattagactatgaggccgagccacagcgacccatgctccccgctattagttacttcaatctaaccggcggagatg

[0297] actgacccactggccaacaacaacgtcaacgaccttctcctggacatggacggccgcgcctcggagcagcgactcgcccaacttcgcattcgccagcagcaggagagagccgtcaaggagct

[0298] gcaggatgcggtggccatccaccagtgcaagagaggcatcttctgcctggtgaaacaggccaagatctcctacgaggtcactccaaacgaccatcgcctctcctacgagctcctgcagcagcgc

[0299] cagaagttcacctgcctggtcggagtcaaccccatcgtcatcacccagcagtctggcgataccaaggggtgcatccactgctcctgcgactcccccgactgcgtccacactctgatcaagaccctc

[0300] tgcggcctccgcgacctcctccccatgaactaatcacccccttatccagtgaaataaagatcatattgatgatgattttacagaaataaaaaataatcatttgatttgaaataaagatacaatcatattgat

[0301] gatttgagtttaacaaaaaaataaagaatcacttacttgaaatctgataccaggtctctgtccatgttttctgccaacaccacttcactcccctcttcccagctctggtactgcaggccccggcgggctgc

[0302] aaacttcctccacacgctgaaggggatgtcaaattcctcctgtccctcaatcttcattttatcttctatcagatgtccaaaaagcgcgtccgggtggatgatgacttcgaccccgtctacccctacgatgc

[0303] agacaacgcaccgaccgtgcccttcatcaacccccccttcgtctcttcagatggattccaagagaagcccctgggggtgttgtccctgcgactggccgaccccgtcaccaccaagaacggggaa

[0304] atcaccctcaagctgggagagggggtggacctcgattcctcgggaaaactcatctccaacacggccaccaaggccgccgcccctctcagtttttccaacaacaccatttcccttaacatggatcac

[0305] cccttttacactaaagatggaaaattatccttacaagtttctccaccattaaatatactgagaacaagcattctaaacacactagctttaggttttggatcaggtttaggactccgtggctctgccttggcag

[0306] tacagttagtctctccacttacatttgatactgatggaaacataaagcttaccttagacagaggtttgcatgttacaacaggagatgcaattgaaagcaacataagctgggctaaaggtttaaaatttgaa

[0307] gatggagccatagcaaccaacattggaaatgggttagagtttggaagcagtagtacagaaacaggtgttgatgatgcttacccaatccaagttaaacttggatctggccttagctttgacagtacagg

[0308] agccataatggctggtaacaaagaagacgataaactcactttgtggacaacacctgatccatcaccaaactgtcaaatactcgcagaaaatgatgcaaaactaacactttgcttgactaaatgtggta

[0309] gtcaaatactggccactgtgtcagtcttagttgtaggaagtggaaacctaaaccccattactggcaccgtaagcagtgctcaggtgtttctacgttttgatgcaaacggtgttcttttaacagaacattct

[0310] acactaaaaaaatactgggggtataggcagggagatagcatagatggcactccatataccaatgctgtaggattcatgcccaatttaaaagcttatccaaagtcacaaagttctactactaaaaataat

[0311] atagtagggcaagtatacatgaatggagatgtttcaaaacctatgcttctcactataaccctcaatggtactgatgacagcaacagtacatattcaatgtcattttcatacacctggactaatggaagcta

[0312] tgttggagcaacatttggggctaactcttataccttctcatacatcgcccaagaatgaacactgtatcccaccctgcatgccaacccttcccaccccactctgtggaacaaactctgaaacacaaaata

[0313] aaataaagttcaagtgttttattgattcaacagtttcacagaaccctagtattcaacctgccacctccctcccaacacacagagtacacagtcctttctccccggctggccttaaaaagcatcatatcatg

[0314] ggtaacagacatattcttaggtgttatattccacacggtttcctgtcgagccaaacgctcatcagtgatattaataaactccccgggcagctcacttaagttcatgtcgctgtccagctgctgagccaca

[0315] ggctgctgtccaacttgcggttgcttaacgggcggcgaaggagaagtccacgcctacatgggggtagagtcataatcgtgcatcaggatagggcggtggtgctgcagcagcgcgcgaataaact

[0316] gctgccgccgccgctccgtcctgcaggaatacaacatggcagtggtctcctcagcgatgattcgcaccgcccgcagcataaggcgccttgtcctccgggcacagcagcgcaccctgatctcactt

[0317] aaatcagcacagtaactgcagcacagcaccacaatattgttcaaaatcccacagtgcaaggcgctgtatccaaagctcatggcggggaccacagaacccacgtggccatcataccacaagcgca

[0318] ggtagattaagtggcgacccctcataaacacgctggacataaacattacctcttttggcatgttgtaattcaccacctcccggtaccatataaacctctgattaaacatggcgccatccaccaccatcct

[0319] aaaccagctggccaaaacctgcccgccggctatacactgcagggaaccgggactggaacaatgacagtggagagcccaggactcgtaaccatggatcatcatgctcgtcatgatatcaatgttg

[0320] gcacaacacaggcacacgtgcatacacttcctcaggattacaagctcctcccgcgttagaaccatatcccagggaacaacccattcctgaatcagcgtaaatcccacactgcagggaagacctcg

[0321] cacgtaactcacgttgtgcattgtcaaagtgttacattcgggcagcagcggatgatcctccagtatggtagcgcgggtttctgtctcaaaaggaggtagacgatccctactgtacggagtgcgccga

[0322] gacaaccgagatcgtgttggtcgtagtgtcatgccaaatggaacgccggacgtagtcattttcgtacttgctgtagcagaacctggtccgggcgctgcacaccgatcgccggcggcggtctcggc

[0323] gcttggaacgctcggtgttgaaattgtaaaacagccactctctcagaccgtgcagcagatctagggcctcaggagtgatgaagatcccatcatgcctgatggctctgatcacatcgaccaccgtgg

[0324] aatgggccagacccagccagatgatgcaattttgttgggtttcggtgacggcgggggagggaagaacaggaagaaccatgattaacttttaatccaaacggtctcggagtacttcaaaatgaagat

[0325] cgcggagatggcacctctcgcccccgctgtgttggtggaaaataacagccaggtcaaaggtgatacggttctcgagatgttccacggtggcttccagcaaagcctccacgcgcacatccagaaa

[0326] caagacaatagcgaaagcgggagggttctctaattcctcaatcatcatgttacactcctgcaccatccccagataattttcatttttccagccttgaatgattcgaactagttcCtgaggtaaatccaagc

[0327] cagccatgataaagagctcgcgcagagcgccctccaccggcattcttaagcacaccctcataattccaagatattctgctcctggttcacctgcagcagattgacaagcggaatatcaaaatctctgc

[0328] cgcgatccctgagctcctccctcagcaataactgtaagtactctttcatatcctctccgaaatttttagccataggaccaccaggaataagattaggggcaagccacagtacagataaaccgaagtcctc

[0329] cccagtgagcattgccaaatgcaagactgctataagcatgctggctagacccggtgatatcttccagataactggacagaaaatcgcccaggcaatttttaagaaaatcaacaaaagaaaaatcctc

[0330] caggtggacgtttagagcctcgggaacaacgatgaagtaaatgcaagcggtgcgttccagcatggttagttagctgatctgtagaaaaaacaaaaatgaacattaaaccatgctagcctggcgaac

[0331] aggtgggtaaatcgttctctccagcaccaggcaggccacggggtctccggcgcgaccctcgtaaaaattgtcgctatgattgaaaaccatcacagagagacgttcccggtggccggcgtgaatg

[0332] attcgacaagatgaatacaccccgggaacattggcgtccgcgagtgaaaaaaaagcgcccgaggaagcaataaggcactacaatgctcagtctcaagtccagcaaagcgatgccatgcggatga

[0333] agcacaaaattctcaggtacaaaatgtaattactcccctcctgcacaggcagcaaagccccccgatccctccaggtacacatacaaagcctcagcgtccatagcttaccgagcagcagcacacaac

[0334] aggcgcaagagtcagagaaaggctgagctctaacctgtccacccgctctctgctcaatatatagcccagatctacactgacgtaaaggccaaagtctaaaaatacccgccaaataatcacacacg

[0335] cccagcacacgcccagaaaccggtgacacactcaaaaaatacgcgcacttctccaaacgcccaaaactgccgtcatttccgggttcccacgctacgtcatcaaaacaccgactttcaaattccgtc

[0336] gaccgttaaaaacgtcacccgccccgcccctaacggtcgccccgtctctcagccaatcagcgccccgcatccccaaattcaaacacctcatttgcatattaacgcgcacaaaaagtttgaggtatatt

[0337] attgatgatggttaattaagGGaattcactggccgtcgttttacaacgtcgtgactgggaaaacctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcg

[0338] aagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtatttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtaca

[0339] atctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgccgccctgacgggcttgtctgctccccggcatccgcttacagacaagctgtgaccgtctccgggagctg

[0340] catgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcgggga

[0341] aatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgt

[0342] cgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcg

[0343] gtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacact

[0344] attctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctga

[0345] caacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccac

[0346] gatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcg

[0347] gcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacgggga

[0348] gtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaa

[0349] aaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgta

[0350] atctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttcta

[0351] gtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaag

[0352] acgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctAtgagaaagc

[0353] gccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtt

[0354] tcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttc

[0355] ctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaat

[0356] acgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcac

[0357] cccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttgcatgcctgcaggtttaaacCCttaattaaccatcttcaataatatacctcaaactttttgtgcgcgttaatatgcaaatgaggcgtttgaatttggggaggaagggcggtgattg。

[0358] Subsequently, three recombinant adenovirus plasmids (pAdC68XY3-ARV123, pAdC68XY3-ARV123-porB, and pAdC68XY3-ARV123-CD40L) were digested with three restriction endonucleases, ApaⅠ / MfeⅠ / XhoⅠ, at 25°C (ApaⅠ) or 37°C (MfeⅠ / XhoⅠ) for 2 h, and the correctness of the band size and position was determined by 0.8% agarose gel electrophoresis. Figure 1 As shown in B, by comparison with the theoretical bands, it was found that the plasmid enzyme digestion bands were correct and the three recombinant adenovirus plasmids were successfully constructed.

[0359] 2. Preparation and Identification of Recombinant Adenovirus

[0360] The three recombinant adenovirus plasmids prepared in step 1 were linearized using PacⅠ endonuclease and inactivated at 65℃ for 20 minutes to obtain three linearized candidate vaccine plasmids. HEK293 cells were transfected in a six-well plate using the transfection reagent X-treme GENE HP. Typical plaques ( Figure 1 (C) Successfully packaged three recombinant chimpanzee adenoviruses. Then, selecting the appropriate ratio, repeated freeze-thaw cycles were used to amplify the packaged viruses in large quantities, ultimately accumulating them into 30 15cm culture dishes.

[0361] Specifically, on the tenth day after transfection, when cells showed obvious pathological changes (becoming round and suspended) and more than 60% of them showed plaques (( Figure 1 (C) Collect cells and supernatant. Repeat freeze-thaw three times, centrifuge, and remove supernatant. Infect new HEK293 cells. Repeat the above steps until 30 adenovirus-infected 15 cm culture dishes of cells have been collected. Use cesium chloride density gradient centrifugation to concentrate and purify the recombinant adenovirus solution. Store the recombinant adenovirus solution at -80°C.

[0362] The physical titer of the virus was calculated by measuring the absorbance A260: Detect the OD of the purified recombinant adenovirus solution 260nm The physical titers of the three viruses were calculated: the titer of AdC68XY3-ARV123 was 4×10 12 vp / mL, and the titer of AdC68XY3-ARV123-proB was 4.3×10 12 vp / mL, and the titer of AdC68XY3-ARV123-CD40L was 3.1×10 12 vp / mL.

[0363] Titer of virus solution = OD 260nm × dilution factor × 1.1 × 10 12 .

[0364] The unit of the titer of the virus solution is the number of virus particles / ml (vp / mL).

[0365] The IFU infection titer of the virus was detected by immunohistochemistry. Two dilution gradients (10 -6 and 10 -7 ) virus infected HEK293 cells, and Adenovirus Type 1 Hexon Monoclonal Antibody (Invitrogen, MA1-82982) was used as the primary antibody and IgG-HRP (Abcam, ab6789) as the secondary antibody. After DAB color development, the virus infection titer was calculated based on the number of positive cells. The titer of AdC68XY3-ARV123 was 3.2×10 10 IFU / mL, and the titer of AdC68XY3-ARV123-proB was 9.1×10 9 IFU / mL, and the titer of AdC68XY3-ARV123-CD40L was 1.04×10 9 IFU / mL.

[0366] The genomic DNA of the three recombinant adenovirus fluids was extracted and digested with ApaⅠ / MfeⅠ / XhoⅠ restriction enzymes at 25℃ (ApaⅠ) or 37℃ (MfeⅠ / XhoⅠ) for 2 hours. The integrity of the recombinant adenovirus genome was identified by 0.8% agarose gel electrophoresis. Figure 1 As shown in D. By comparing with the theoretical fragment, it was verified that the entire genome was complete and correct, indicating that it had not been recombined or lost during the amplification process.

[0367] 3. Verification of recombinant adenovirus antigen protein expression level

[0368] 3.1 Obtaining mouse polyclonal antibodies to ARVσC protein

[0369] 3.1.1 Prokaryotic expression and purification of ARVσC protein

[0370] The expression cassettes of ARV1, ARV2, and ARV3 were amplified by PCR using plasmid pshuttle-ARV123 (Qingke Biotechnology) as template. The PCR primers were (ARV1 F: 5′-ATGGGTCGCGGATCCGAATTCGCCACCATGGAGGGGCTG-3′, ARV1R: 5′-GTGGTGGTGGTGGTGCTCGAGGGTGTCAATGCCGGTTCTCA-3′; ARV2 F: 5′-ATGGGTCGCGGATCCGAATTCATGGACCGCAACGAGGTGA-3′, ARV2 R: 5′-GTGGTGGTGGTGGTGCTCGAGGCCGGTGGCCACGGTGAA-3′; ARV3 F: 5′-ATGGGTCGCGGATCCGAATTCATGGAGGGGCTGACGCCG-3′, ARV3 R: 5'-GTGGTGGTGGTGGTGCTCGAGGGTGTCAATGCCGGTTCTCA-3'). PCR products (ARV1 984 bp; ARV2 963 bp; ARV3 978 bp) were separated and recovered by agarose gel electrophoresis. The prokaryotic expression plasmid vector pET-28a (Merck, 69864) was digested with EcoRI and XhoI endonucleases, and the digested vectors were separated and recovered by agarose gel electrophoresis. The PCR product containing the target gene was inserted into pET-28a using homologous recombination to generate a recombinant plasmid. The recombinant vector was transformed into E. coli BL21(DE3) competent cells to generate a transformation product. The transformation product was plated on kanamycin-resistant agar plates and incubated at 37°C for 14-16 hours. Single colonies were selected, identified by colony PCR, and plasmids were extracted. The correctness of the cloned sequence was verified by sequencing. In this way, three prokaryotic expression recombinant plasmids pET-28a-ARV1, pET-28a-ARV2 and pET-28a-ARV3 were constructed.

[0371] The three prokaryotic expression recombinant plasmids were transformed using E. coli BL21 (DE3) competent cells. The transformation products were spread on kanamycin-resistant agar plates and cultured at 37°C for 14-16 hours. Monoclonal colonies were selected and placed in kanamycin-resistant LB culture medium. 600 mL of the culture medium was amplified on a shaker at 37°C and 220 rpm. The OD of the culture medium was 0.05. 560nmThe pH value was between 0.6 and 0.8. Add 20 μL of 1 mM IPTG (Solybol) solution per 100 mL of bacterial culture and induce protein expression at 16°C at a low speed of 180 rpm for 12-14 hours. Centrifuge at 4°C at 4000 rpm for 20 minutes, remove the supernatant, and harvest the cells. Add lysis buffer (50 mM Tris, 50 mM NaCl, 1 mM PMSF) and disrupt the cells 4-5 times at 1200 Pa in a low-temperature, ultrahigh-pressure continuous flow cell disruptor. The lysate was then affinity chromatographed (Beyotime, FCL12) using His-tag Purification Resin (Beyotime, P2233-10ml), and the sample was eluted using a high-concentration imidazole eluent (50mM Tris, 50mM NaCl, 5% glycerol, 1M imidazole). Finally, the imidazole and impurity proteins in the eluent were removed using a dialysate (50mMTris, 50mM NaCl, 10% glycerol, 0.5mM EDTA) to obtain the purified ARVσC proteins expressed in the three prokaryotic systems.

[0372] 3.1.2 Immunization strategy for preparation of mouse polyclonal antibodies

[0373] BALB / c mice were divided into 3 groups, 5 mice in each group, and immunized with three purified ARVσC proteins, ARV1 (amino acid sequence is sequence 3 in the sequence list), ARV2 (amino acid sequence is sequence 4 in the sequence list), and ARV3 (amino acid sequence is sequence 5 in the sequence list) expressed by the prokaryotic system obtained in step 3.1.1. Blood was collected from the mandibular vein one day before immunization, which was the serum of the negative control group. The second day was recorded as 0W, and 50μL of Quick Antibody-Mouse 3W (Boaolong, KX0210042) and purified protein mixture were injected into the left and right hind leg muscles of the mice, for a total of 100μL. At 2W, booster immunization was performed, and 50μL of Quick Antibody-Mouse3W+purified protein mixture were injected into the left and right hind legs of the mice, for a total of 100μL. At 4W, booster immunization was performed, and the mice were subcutaneously injected 2% adjuvant (InVivoGen, vac-alu-v) + 50 μL of purified protein mixture, totaling 100 μL. Blood was collected from the mandibular vein at 5 and 6 weeks and from the eyeball at 7 weeks, and then the mice were sacrificed by spinal dislocation. A titer of at least 1:10 was obtained. 5 Mouse polyclonal antibodies were used to detect the expression of recombinant adenovirus antigen proteins.

[0374] Sequence 3 in the sequence table is as follows:

[0375] ATMEGLTQSQRREVVGLILSLTSNVTINPGDLTELRERVSALESANASLNEIIKGVLDQLVDLAQKLGNAAGAVVDLRGELNSLTASVQTIQSSLGSLTDSISDLSSQVTTNASSPTNLRSMVAGLIADVTNLKRDVSNQGLQMTSLEQRVTSLESGTGSIPTFAAPLKLDGGIVSLDLDPYFCSVDHNLTSYSASALLMNFQWLVRGEGGSSDSFDMNVTAHSHGQRTDFMMSTTQSLTVTGNSVTLVFDLNALISPPSDYSRLIPCHGFQQATFPVDLSFKRDDVTHSYQVYGSYTTPRIFKITFSPGNPVPAVIRFITVRTGIDTGSG。

[0376] The sequence 4 in the sequence list is as follows:

[0377] MDRNEVIRLILSLLPYQSSDVDHLTTQIKSLQSAVDSLKESQVVVLRRLTTITSTVADLQSTTELLTSQVAGLSSRVASVTDEVVRVDSVIGSTITNLDNVRSELSSLSSQVSSQTSTLTNLTSTVSSQSLAISDLQRRVTVLERSGGAPTQFEAPLQLQNGVVSLQASPSFCSLSPILSGPADAAVFKVGEWLGTVISGQSQSSAIMNVRIHSFGQRTMLLMSSQNVFTIPPGSGASLQLDVTRITTPAIDVAMVTPSAAFASASFMADIAFKDSKTGEVHALHTTGSFRSPSFSIAWVPVASETRNYQIMALRFTVATGGSG。

[0378] The sequence 5 in the sequence list is as follows:

[0379] MEGLTPLQRREVVGLILSLTSSVSISSGDLIPLYERLSAIEKMCTTVNDSLGHLTSSVSEISARIDDLAETVRNTVTDLNNVQIRVTALQSSFDSLSSNVTTLSSSSVSNQESQLATVSTSVNALSTNVSNLQSDVSSTALTVTSLGQRVEALESGAGSALTFISPLKV DGKSVLLLDMDPYFCSERANLTSYSASAQLLQFQWFVRSEGGSSDSIDMNVVAHCHGRRTDYLMSTHDSLTVTGNSVTLVFNMDYITTQGVDYARLVPCHGFQQATFPVDISFTKDDATHSYQVYGAFAGPRIFKVTFSPGETSATNVRFLTVRTGIDTARASLSLNR.

[0380] 3.2 Protein extraction from recombinant adenovirus-infected cells

[0381] The AdC68XY3-empty vector virus solution was diluted to 1×10 10 vp / 100 μL, dilute the three recombinant adenovirus solutions of AdC68XY3-ARV123, AdC68XY3-ARV123-porB, and AdC68XY3-ARV123-CD40L with DMEM complete medium to 1×10 8 vp / 100μL, 1×10 9 vp / 100μL, 1×10 10 vp / 100 μL was used to infect HEK293A cells seeded in 6-well plates one day in advance. 24 hours after infection, the cells in the 6-well plates were pipetted to mix thoroughly and transferred to a 1.5 ml EP tube. Centrifuge at 800 × g for 5 minutes, remove the supernatant, and add 200 μL of RIPA cell lysis buffer to the cell pellet. After pipetting, lyse the pellet and place on ice for 10 minutes. After lysis, add 50 μL of 5× loading buffer to the sample and boil in a water bath for 5 minutes. Protein samples were stored at -20°C.

[0382] 3.3 Western blot detection of protein expression levels

[0383] Western blot experiments were used to detect the expression of three target proteins, σC of chicken reovirus type IV (ARV1), σC of duck novel reovirus (ARV2), and σC of chicken reovirus type III (ARV3), after cells were infected with viruses at different titers.

[0384] After SDS-PAGE electrophoresis, transfer the proteins on the gel to a PVDF membrane; after transfer, block with 5% skim milk; wash the membrane three times with TBST, dilute the three polyclonal antibodies mouse anti-ARV1, ARV2, and ARV3 prepared in step 3.1.2 at 1:200, and use the internal reference antibody β-tubulin at 1:1000, and incubate overnight at 4°C; wash the membrane with TBST, add 1:10000 dilution of Goat Anti-mouse IgG (H+L)-HRP Conjugate and incubate for 1 hour; wash the membrane with TBST, develop the color with ECL luminescent working solution, and image using a chemiluminescence imager.

[0385] like Figure 2 As shown, 1×10 10 vp / 100μL of AdC68XY3-empty virus infected cells did not express the three σC target proteins ( Figure 2 Empty in the figure). And 1×10 8 vp / 100μL, 1×10 9 vp / 100 μL and 1×10 10 vp / 100μL, three recombinant adenoviruses, AdC68XY3-ARV123, AdC68XY3-ARV123-porB and AdC68XY3-ARV123-CD40L, successfully expressed the target proteins. Moreover, with the increase of virus titer, the expression levels of the three σC target proteins (ARV1, ARV2 or ARV3) also increased.

[0386] 4. Recombinant Adenovirus Functional Testing

[0387] 4.1 Mouse immunization strategy and serum preparation

[0388] Take the purified three recombinant adenovirus candidate vaccine virus solutions (AdC68XY3-ARV123, AdC68XY3-ARV123-porB and AdC68XY3-ARV123-CD40L) and AdC68XY3-empty empty vector virus solution prepared in step 2 and stored at -80°C, and dilute them to 1×10 8 After 100 μL of IFU, BALB / c mice were immunized in groups, and 50 μL was injected into the left and right hind leg muscles of BALB / c mice:

[0389] BALB / c mice aged 6-8 weeks were divided into four groups, with 6 mice in each group.

[0390] Control group: 1×10 8IFU / 100uLAdC68XY3-empty empty vector virus solution;

[0391] Experimental group: Each mouse was intramuscularly immunized with 1×10 8 IFU / 100uL of three recombinant adenovirus solutions.

[0392] Blood was collected from the mandibular vein of mice at multiple time points after immunization (including 2, 4, 6, 8, 10, 12, 16, 18 and 20 weeks), placed at 37°C for 1 hour or at 4°C overnight, centrifuged at 3000×g for 10 minutes, and the supernatant was collected and inactivated at 56°C for 30 minutes to obtain the mouse immune serum of the four recombinant adenoviruses to be tested.

[0393] 4.2 ELISA detection of serum antibody titer

[0394] The three antigen proteins (ARV1, ARV2, and ARV3) obtained in step 3.1.1 were diluted to the corresponding concentrations (ARV1 and ARV2 were diluted to 200 ng / well, and ARV3 was diluted to 300 ng / well) with the coating solution, with a total volume of 100 μl / well. Incubate at 4°C overnight; wash the plate three times with 0.05% PBST, 200 μl / well; block the plate with 5% skim milk at 37°C for 1 hour; wash the plate three times with 0.05% PBST, 200 μl / well. Serum was diluted with PBS containing 1% BSA, starting at 1:100, and serially diluted at 100 μl / well. Incubate at 37°C for 2 h. Plates were washed three times with 200 μl / well of 0.05% PBST. Anti-mouse IgG-HRP secondary antibody (1:100,000) was diluted with PBS containing 1% BSA, at 100 μl / well, incubate at 37°C for 2 h. Plates were washed four times with 200 μl / well of 0.05% PBST. TMB (50 μl / well) was added, color was developed in the dark for 10 min, and the plate was terminated with 50 μl / well of 2 M hydrochloric acid. OD450 and OD630 values ​​were read on a microplate reader. Endpoint titer was calculated.

[0395] Use 1×10 8The virus dose was 100uL / 100uL. The three purified recombinant adenovirus liquids stored at -80°C prepared in step 2 and the control adenovirus empty vector AdC68XY3-empty were injected intramuscularly to immunize 6-8W BALB / C mice with six mice in each group. Then, the serum of the immunized mice was collected through the mandibular vein at 2, 4, 6, 8, 10, 12, 16, 18 and 20 weeks after immunization. The specific binding antibodies to the three proteins ARV1, ARV2 and ARV3 in the serum after immunization at each time point, that is, the total IgG antibody titer, were detected by ELISA experiments. The results are shown in the figure. Figure 3 As shown, starting from 2 weeks after immunization, the three candidate recombinant adenovirus vaccines all induced mice to produce antibodies against ARV1 ( Figure 3 ARV2( Figure 3 Middle B) and ARV3( Figure 3 Middle C) Total IgG levels of these three σC proteins, representing the specific binding antibodies induced by the mice. Furthermore, IgG levels were significantly elevated at week 4 compared to week 2, and this high level of binding antibodies persisted until the endpoint of the assay, week 20. Meanwhile, AdC68XY3-empty control mice did not produce specific binding antibodies.

[0396] 4.3 Microneutralization assay to detect neutralizing antibody titers

[0397] Vero cells (ATCC, CCL-81) were added to a 96-well plate at a ratio of 2000 cells / 100 μL per well and cultured overnight at 5% CO2 and 37°C. Avian reovirus S1133 (kindly donated by Yu Kexiang, Researcher of the Poultry Research Institute of Shandong Academy of Agricultural Sciences, related literature: Yu Kexiang, Liu Cunxia, ​​Gong Xiao, et al., Isolation and identification of a broiler reovirus variant. Journal of Animal Husbandry and Veterinary Medicine 2019.50(5)1039-1047) was diluted with DMEM medium containing 10% FBS to 100 TCID50 / 50 μL. The mouse immunization sera of the four recombinant adenoviruses prepared in step 4.1 were diluted with 10% DMEM medium, with an initial dilution of 1:10, and two-fold dilutions were used for a total of 10 dilution gradients, with 50 μL per well. Only 50 μL of 10% DMEM medium was added to the first and last columns, serving as the blank control group and the virus infection positive group. After the serum is diluted, add 50 μL of S1133 diluted virus suspension to each well. Add 50 μL of DMEM complete culture medium to the blank control group, and add 50 μL of S1133 diluted virus suspension to the positive control well. Mix the virus-serum mixture thoroughly and incubate it in a 37°C, 5% CO2 incubator for 1 hour. Remove the culture medium in the 96-well plate inoculated with Vero cells one day in advance, add 100 μL of the virus-serum mixture in the previous step to each well and mix it thoroughly. Place the well plate in a 37°C, 5% CO2 incubator and culture for 5 days. Observe the degree of cell pathology. When all the virus-infected positive control wells show lesions, while the cells in the blank control group are in good condition and the neutralizing antibody titer containing serum is within the dilution range, the neutralizing antibody titer is the highest serum dilution that completely inhibits the lesions.

[0398] Detection of neutralizing antibodies against the live ARV virus S1133 strain in serum revealed that the neutralizing antibody titers of the three candidate adenovirus vaccines increased significantly at 4 weeks and 8 weeks after immunization compared with the control group ( Figure 4 A and B). Subsequently, a 20-week long-term immune effect test showed that the serum titer of the control group remained at 1:10 ( Figure 4 However, the neutralizing antibody titers of the three candidate adenovirus vaccines (AdC68XY3-ARV123, AdC68XY3-ARV123-porB and AdC68XY3-ARV123-CD40L) gradually increased from 2 to 10 weeks after immunization, reaching the highest level at 10 weeks, which was around 1:1280 ( Figure 4 (C) After 10 weeks, neutralizing antibody titers gradually declined, but at the final stage of long-term monitoring, at 20 weeks, neutralizing antibodies were still present at a titer of approximately 1:320. This demonstrates that all three candidate recombinant adenovirus vaccines prepared in this invention can induce neutralizing antibodies against the live S1133 virus, and that these antibodies persist for a long time.

[0399] 4.4 Flow cytometry detection of T cell responses

[0400] Mouse splenocyte extraction: According to the immunization strategy in step 4.1, at 2 weeks after immunization (effector T cell flow cytometry) or 20 weeks after immunization, mice immunized with three recombinant adenovirus vaccine candidate solutions (AdC68XY3-ARV123, AdC68XY3-ARV123-porB, and AdC68XY3-ARV123-CD40L) and AdC68XY3-empty empty vector virus solution) were dissected and the spleens were removed and placed in a 24-well plate. 1 ml of 10% 1640 culture medium and two layers of 70-mesh cell sieves were placed in the well plate in advance; the spleens were ground with a syringe pusher; centrifuged at 4°C, 500g, 5 min, and the supernatant was removed; 3 ml of red blood cell lysis buffer was added, and the red blood cell lysis was carried out at room temperature for 10 min, and then 3 ml of red blood cell lysis buffer was added. 10% 1640 culture medium was added to stop lysis; centrifuged at 4°C, 500×g for 5 minutes, and the supernatant was removed; 1 ml of 10% 1640 culture medium was added, mixed by pipetting, and the mouse spleen cells were obtained by filtering with a 70-mesh cell sieve; centrifuged at 4°C, 500×g for 5 minutes, and the supernatant was removed; 0.5 ml of 10% 1640 culture medium was added to resuspend the cells to obtain spleen cell resuspension, and the cells were counted, and 1×10 cells were taken per well. 6 Mouse spleen cells were inoculated into 96-well plates at 100 μL / well.

[0401] Mouse splenocyte culture stimulation: Splenocytes were plated into three wells per mouse in a 96-well plate. 0.2 μL of each of the three peptide libraries (synthesized by Nanjing GenScript) was added: ARV1 (SEQ ID 3 in the sequence list), ARV2 (SEQ ID 4), and ARV3 (SEQ ID 5). Each σC peptide library consisted of 15 amino acids in length, with an 11-amino acid overlap. The peptide libraries were used at a concentration of 2 μg / mL. Antibodies against CD28 (BD, 553295) and CD49 (BD, 553154) were co-stimulated, at a concentration of 0.5 μL per well. The culture medium was made up to 100 μL. The cells were incubated at 37°C in a 5% CO2 incubator for 4-5 hours. Golgi blocker (BD Biosciences) was then added, and the culture medium was made up to 200 μL. Stimulation was continued overnight for 6-8 hours. A negative control group was also established, containing only DMSO. A positive control group was treated with cell stimulation cocktail (a mixture of PMA and ionomycin, Thermo Fisher Scientific).

[0402] Flow cytometry staining: Add 1 ml of MACS buffer to the flow cytometry tube, centrifuge at 500 × g at 4°C for 5 min, and remove the supernatant. Dilute anti-CD16 / CD32 antibody (Thermo Scientific, 14-0161-85, 1:250) and dead dye (1:250) in MACS buffer and add to the cells. The cells were mixed by vortexing and incubated at 4°C in the dark for 30 min; 1 ml of MACS buffer was added, and the cells were centrifuged at 4°C, 500 × g, and the supernatant was removed; surface marker staining: surface factor antibodies were diluted 1:250 in MACS buffer: percp / cy5.5 anti-CD3 (BioLegend, 100328), FITC anti-CD4 (BioLegend, 100510), AF700 anti-CD8 (BioLegend, 100730), PE-eFlour 610 anti-CD44 (Thermo Scientific, 61-0441-82), elour450 anti-CD62L (Thermo Scientific, 48-0621-80), the cells were mixed by vortexing and incubated at 4°C in the dark for 30 min; 1 ml of MACS was added, and the cells were centrifuged at 500 × g for 5 min, and the supernatant was removed; The cells were centrifuged at 500 × g for 5 min at 4°C and the supernatant was discarded. The cells were resuspended in 120 μl of fixation and permeabilization buffer, mixed by vortexing, and incubated at 4°C in the dark for 20 min. 400 μl of fixation and permeabilization wash buffer was added and the cells were centrifuged at 500 × g for 8 min at 4°C and the supernatant was discarded. For intracellular marker staining, the intracellular factor antibodies were diluted 1:250 in fixation and permeabilization wash buffer: BV421 anti-IL2 (BioLegend, 503826), PE-cy7 anti-IL4 (BioLegend, 504117), PE anti-IL13 (BioLegend, 159403), APC anti-IFN-γ (BioLegend, 505810), and BV650 anti-TNF-α (BioLegend, 506333). Vortex to mix, incubate at 4°C in the dark for 30 min; wash: add 400 μl fixation and permeabilization wash buffer, centrifuge at 4°C, 500g, 8 min, and remove the supernatant; fix with 1% paraformaldehyde for 30 min and store at 4°C in the dark; use a high-end analytical flow cytometer Fortessa for detection and Flow jo 10 software for analysis to count the proportion of intracellular cytokine-positive cells.

[0403] 4.4.1CD4 + Effector T cell immune response

[0404] Two weeks after immunization, spleen cells were collected from mice and the effector T cell immune response specific to the three σC proteins of ARV was detected by flow cytometry. Figure 5 As shown in FIG, the three recombinant chimpanzee adenovirus candidate vaccines prepared by the present invention can induce strong CD4 specificity against the three ARV-σC proteins. + T cell immune response, mainly inducing the production of Th1 cytokine IL2 in mice.

[0405] 4.4.2CD8 + Effector T cell immune response

[0406] Two weeks after immunization, the three recombinant chimpanzee adenovirus candidate vaccines were able to induce stronger CD8 specificity against the three ARV-σC proteins compared with the control group. + T cell immune response, and CD8 + The production of Th1 cytokine IFN-γ is dominant, such as Figure 6 shown.

[0407] 4.4.3CD4 + Memory T cell immune response

[0408] 20 weeks after immunization, spleen cells were collected from mice and flow cytometry was used to detect the memory T cell immune response specific to the three ARV σC proteins. Compared with the control vaccine, the three recombinant chimpanzee adenovirus candidate vaccines can induce stronger CD4 specific to the three ARV-σC proteins. + Memory T cell immune response, and mainly the production of cytokine TNF-α, such as Figure 7 shown.

[0409] 4.4.4CD8 + Memory T cell immune response

[0410] 20 weeks after immunization, the three recombinant chimpanzee adenovirus candidate vaccines can induce stronger CD8 specificity against the three ARV-σC proteins compared with the control group. + Memory T cell immune response, and mainly the production of cytokine IFN-γ, such as Figure 8 shown.

[0411] 4.5ARV live virus S1133 challenge experiment

[0412] The virus solution obtained in step 4.1 was concentrated by S1133 and washed with PBS at 1×10 5 Each immunized mouse was injected intramuscularly with 200 μl of the virus solution diluted to a concentration of TCID50 / 100 μl. Five days after virus injection, the mice were sacrificed by spinal dislocation, and their livers and spleens were removed and frozen for further tissue viral copy number analysis. Total RNA was extracted using the FastPureViral DNA / RNAMini Kit V2, and then reverse transcribed into cDNA for RT-qPCR analysis to quantify viral copy number in tissues.

[0413] Live virus S1133 was injected into four groups of mice 10 weeks after immunization, and then on the 5th day after the challenge, the liver and spleen of the mice were taken for the detection of tissue viral load using RT-qPCR. Figure 9 As shown, the control group mice ( Figure 9 Higher viral copy numbers can be detected in the liver and spleen of Adc68-empty mice. Compared with the control group vaccine, the three recombinant chimpanzee adenovirus candidate vaccines prepared by the present invention have a higher viral copy number in the liver and spleen of mice ( Figure 9 Middle A) and spleen ( Figure 9 The detectable viral copy number in (B) was significantly decreased (p < 0.0001), and many were even below the minimum detection range of RT-qPCR, indicating that the recombinant adenovirus candidate vaccines against the three ARV viruses prepared by the present invention have obvious protective effects and can resist the attack of live viruses.

[0414] In summary, the present invention prepares a multivalent avian reovirus preventive vaccine based on a replication-defective chimpanzee adenovirus vector, which is expected to produce a vaccine product that is simple to prepare, low-cost, safe, does not require the addition of additional adjuvants, and has broad application prospects.

[0415] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A recombinant adenovirus, characterized in that: The recombinant adenovirus contains the coding gene of chicken reovirus type III σC protein, the coding gene of chicken reovirus type IV σC protein and the coding gene of duck novel reovirus σC protein.

2. The recombinant adenovirus according to claim 1, characterized in that: The recombinant adenovirus is obtained by inserting the coding gene of chicken reovirus type III σC protein, chicken reovirus type IV σC protein and duck novel reovirus σC protein into an adenovirus vector and then introducing it into packaging cells.

3. The recombinant adenovirus according to claim 2, characterized in that: The adenovirus vector is a recombinant replication-deficient adenovirus vector plasmid AdC68, which contains all sequences of the adenovirus vector AdC68 except the ORF6 / 7 and ORF6 sequences of the E1 gene, the E3 gene and the E4 gene; the recombinant replication-deficient adenovirus vector plasmid AdC68 also contains the ORF6 / 7 and ORF6 sequences of the E4 gene of human serum type 5 adenovirus.

4. The recombinant adenovirus according to claim 3, characterized in that: The recombinant replication-deficient adenovirus vector plasmid also contains a coding gene sequence of an immune adjuvant.

5. The recombinant adenovirus according to any one of claims 2 to 4, characterized in that: The recombinant replication-deficient adenovirus vector also contains the coding gene sequences of P2A and T2A.

6. The recombinant adenovirus according to any one of claims 2 to 5, characterized in that: The recombinant replication-deficient adenovirus vector contains a gene encoding a fusion protein, wherein the fusion protein is ARV1-P2A-ARV2-T2A-ARV3, ARV1-P2A-ARV2-T2A-ARV3-P2A-porB or ARV1-P2A-ARV2-T2A-ARV3-P2A-CD40L, wherein the ARV1-P2A-ARV2-T2A-ARV3 is a protein formed by connecting the chicken reovirus type III σC protein, the chicken reovirus type IV σC protein, the duck novel reovirus σC protein, the P2A and the T2A, and wherein ARV1-P2A-ARV2-T2A-ARV3-P2A-porB is a protein formed by connecting the chicken reovirus type III σC protein, the chicken reovirus type IV σC protein, the duck novel reovirus σC protein, the porB, the P2A and the T2A, and the ARV1-P2A-ARV2-T2A-ARV3-P2A-CD40L is a protein formed by connecting the chicken reovirus type III σC protein, the chicken reovirus type IV σC protein, the duck novel reovirus σC protein, the CD40L, the P2A and the T2A.

7. The recombinant adenovirus according to any one of claims 2 to 5, characterized in that: The coding gene of the fusion protein is any of the following DNA molecules: A1) a DNA molecule whose nucleotide sequence contains positions 1349-2341, 3425-4429 and 2399-3370 of SEQ.No.1; A2) a DNA molecule having a nucleotide sequence containing nucleotides 1349-2341, 3425-4429, and 2399-3370 of SEQ.No.1 and containing sequence 1; A3) A DNA molecule whose nucleotide sequence contains nucleotides 1349-2341, 3425-4429, and 2399-3370 of SEQ.No.1 and sequence 2.

8. The recombinant adenovirus according to any one of claims 2 to 5, characterized in that: The recombinant replication-deficient adenovirus vector plasmid is pAdC68XY3-ARV123, pAdC68XY3-ARV123-CD40L or pAdC68XY3-ARV123-porB, The pAdC68XY3-ARV123 is a double-stranded circular DNA molecule having a nucleotide sequence of SEQ.No.1, The pAdC68XY3-ARV123-porB is a recombinant expression vector plasmid obtained by inserting nucleotides 2342-2398 of SEQ.No.1 and sequence 1 in the sequence list between nucleotides 4402-4403 of SEQ.No.1, while keeping the other nucleotide sequences of the pAdC68XY3-ARV123 unchanged; The pAdC68XY3-ARV123-CD40L is a recombinant expression vector plasmid obtained by inserting nucleotides 2342-2398 of SEQ.No.1 and sequence 2 in the sequence list between nucleotides 4402-4403 of SEQ.No.1, while keeping the other nucleotide sequences of the pAdC68XY3-ARV123 unchanged.

9. Biological material, any of the following: 1) The recombinant replication-deficient adenoviral vector according to any one of claims 2 to 8, 2) a recombinant microorganism or a recombinant animal cell containing the recombinant replication-defective adenovirus vector described in 1), 3) The fusion protein according to claim 6, 4) a nucleic acid molecule encoding the fusion protein described in 3), 5) A recombinant vector, recombinant microorganism or recombinant animal cell containing the nucleic acid molecule described in 3).

10. Use of the recombinant adenovirus according to any one of claims 1 to 8 and / or the biological material according to claim 9 in the preparation of a polyvalent vaccine of avian reovirus.

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