Serum type 4, type 8 and type 11 fowl adenovirus subunit vaccine and application thereof

Abstract

The invention discloses a subunit vaccine for serum type 4, type 8 and type 11 fowl adenoviruses and application of the subunit vaccine. The immunogenic composition comprises three antigen proteins with amino acid sequences shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The invention also discloses a subunit vaccine which contains the three antigen proteins and is used for preventing diseases caused by avian adenovirus infection. Experiments show that after the subunit vaccine is used for immunizing chicken, the chicken can be protected from infection or attack of the fowl adenovirus, the immune response of the body can be effectively activated, complete immune challenge protection is generated, and the protection rate reaches 100%. The strain can completely resist the attack of virulent virus without diseases, and has good practical application value. The subunit vaccine provided by the invention has the characteristics of high antigen expression quantity, high antigen stability, high purity, good safety and strong immunogenicity, and lays a solid foundation for industrial production of avian adenovirus multivalent subunit vaccines and diagnostic reagents.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biological products, and specifically relates to a serum type 4, type 8, type 11 avian adenovirus subunit vaccine and an application thereof. Background Art

[0002] Fowl Adenovirus (FAdV) is an important virus that affects the health of poultry and is known to be divided into multiple serotypes. Serotypes 4, 8, and 11 of avian adenovirus have a particularly serious impact on the poultry industry, especially the diseases they cause, such as inclusion body hepatitis and pericardial effusion, which lead to high morbidity and mortality in chickens and cause huge economic losses to the breeding industry. At present, the global poultry breeding industry is plagued by multiple subtypes of avian adenovirus, especially frequent cases of infection with serotypes 4, 8, and 11. It is difficult for farms to effectively control the spread of these viruses.

[0003] There are also some commercialized inactivated vaccines for avian adenovirus on the market, which may have poor protection against some serotypes of avian adenovirus, especially difficult to achieve simultaneous protection against serotypes 4, 8, and 11 of avian adenovirus, and cannot provide broad-spectrum protection against multiple serotypes at the same time. Therefore, the development of a subunit vaccine that can protect against these three serotypes at the same time has important research and application value.

[0004] With the development of genetic engineering technology, recombinant protein technology has provided a new approach for the development of vaccines. Subunit vaccines prepared by recombinant protein technology have gradually become a research hotspot due to their high safety and strong immunogenicity. Genetically engineered subunit vaccines are vaccines prepared by combining protein antigens expressed by genetic engineering with corresponding adjuvants. Compared with traditional vaccines, genetically engineered subunit vaccines have large production capacity, high antigen purity, and good economic benefits. At present, the types and quantities of vaccines on the market are still dominated by traditional vaccines. Genetically engineered subunit vaccines are still in their infancy, but have shown strong competitiveness. Strengthening vaccine research and development and continuously developing high-quality vaccines have become the key to ensuring the sustainable and healthy development of the poultry industry. The present invention aims to develop antigen proteins of serum types 4, 8, and 11 through genetic engineering technology, and prepare safe and efficient subunit vaccines to improve the immune effect. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to effectively prevent avian adenovirus infection or diseases caused by avian adenovirus infection against multiple serotypes. The technical problem to be solved is not limited to the technical subject matter described, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0006] In order to solve the above technical problems, the present invention first provides an immunogenic composition, which comprises three antigenic proteins shown in A1), A2) and A3): A1) The amino acid sequence of the antigenic protein may be as shown in SEQ ID NO: 1, or an antigenic protein having the same function obtained by connecting a signal peptide to the N-terminus of SEQ ID NO: 1; A2) The amino acid sequence of the antigenic protein may be as shown in SEQ ID NO: 2, or an antigenic protein having the same function obtained by connecting a signal peptide to the N-terminus of SEQ ID NO: 2; A3) The amino acid sequence of the antigenic protein may be as shown in SEQ ID NO: 3, or an antigenic protein having the same function obtained by connecting a signal peptide to the N-terminus of SEQ ID NO: 3.

[0007] The antigenic protein whose amino acid sequence is shown in SEQ ID NO:1 may be serotype 4 fowl adenovirus Fiber-2 protein. The antigenic protein whose amino acid sequence is shown in SEQ ID NO:2 may be serotype 8 fowl adenovirus Fiber protein. The antigenic protein whose amino acid sequence is shown in SEQ ID NO:3 may be serotype 11 fowl adenovirus Fiber protein.

[0008] The signal peptide is used to guide the recombinant protein to cross the cell membrane for secretory expression, which does not affect the immunogenicity of the antigenic protein. Those skilled in the art can select a suitable signal peptide, such as Melittin signal peptide suitable for insect cells, GP64 signal peptide, gp67 signal peptide, hemolin signal peptide, HIV-ENV signal peptide, etc., but are not limited thereto.

[0009] The signal peptide may be Melittin signal peptide (SEQ ID NO: 7).

[0010] The present invention also provides a nucleic acid molecule composition, which can be any of the following: B1) the nucleic acid molecule composition comprises nucleic acid molecules encoding the three antigenic proteins in the immunogenic composition respectively; B2) The nucleic acid molecule composition includes a DNA molecule whose nucleotide sequence can be shown as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0011] The DNA molecule shown in SEQ ID NO:4 can be a gene encoding serotype 4 fowl adenovirus Fiber-2 protein obtained by optimizing the nucleotide sequence of serotype 4 fowl adenovirus Fiber-2 protein (SEQ ID NO:1) for insect cell preference.

[0012] The DNA molecule shown in SEQ ID NO:5 can be a gene encoding the serotype 8 fowl adenovirus Fiber protein obtained by optimizing the nucleotide sequence of the serotype 8 fowl adenovirus Fiber protein (SEQ ID NO:2) for insect cell preference.

[0013] The DNA molecule shown in SEQ ID NO:6 can be a gene encoding the serotype 11 fowl adenovirus Fiber protein obtained by optimizing the nucleotide sequence of the serotype 11 fowl adenovirus Fiber protein (SEQ ID NO:3) for insect cell preference.

[0014] The present invention also provides a biomaterial, which may be any of the following: C1) an expression cassette containing the nucleic acid molecule composition; C2) a recombinant vector containing the nucleic acid molecule composition; C3) a recombinant microorganism containing the nucleic acid molecule composition; C4) A recombinant host cell containing the nucleic acid molecule composition.

[0015] The biological materials can all express the nucleic acid molecule composition.

[0016] In the above biological materials, the recombinant vector in C2) may be a recombinant baculovirus vector; the recombinant microorganism in C3) may be a recombinant baculovirus.

[0017] The recombinant baculovirus vector can be a recombinant vector (recombinant plasmid) obtained by cloning a nucleic acid molecule encoding a serotype 4 avian adenovirus fiber-2 protein (e.g., SEQ ID NO: 4), a nucleic acid molecule encoding a serotype 8 avian adenovirus fiber protein (e.g., SEQ ID NO: 5), and a nucleic acid molecule encoding a serotype 11 avian adenovirus fiber protein (e.g., SEQ ID NO: 6) into a baculovirus expression vector. Although the baculovirus expression vector used in the embodiments provided by the present invention is a pFastBac1 vector, the present invention is not limited to this specific vector. Those skilled in the art can use other known suitable baculovirus expression vectors, such as other vectors of the pFastBac series (e.g., pFastBacDual), pAc series vectors (e.g., pAC-k-Fc, pAC-L-Fc, etc.), and pVL series vectors (e.g., pVL1393, pVL1392, etc.). As long as the baculovirus expression vector can express the above-mentioned nucleic acid molecules (i.e., the nucleic acid molecule composition of the present invention), it can be used.

[0018] The recombinant baculovirus vector may also be a recombinant expression vector (recombinant bacmid) obtained by cloning a nucleic acid molecule encoding serotype 4 fowl adenovirus Fiber-2 protein (e.g., SEQ ID NO: 4), a nucleic acid molecule encoding serotype 8 fowl adenovirus Fiber protein (e.g., SEQ ID NO: 5), and a nucleic acid molecule encoding serotype 11 fowl adenovirus Fiber protein (e.g., SEQ ID NO: 6) into a baculovirus expression vector and subjecting the clones to transposition.

[0019] The method of constructing a recombinant bacmid is known to those skilled in the art, and may include, for example, the following steps: (1) Clone the target gene between two Tn7 transposition sites in a baculovirus expression vector (containing two Escherichia coli Tn7 transposition sites) to obtain a recombinant plasmid; (2) The recombinant plasmid is transformed into a host bacmid (e.g., E. coli containing a Bacmid shuttle vector and a helper plasmid that can express Tn7 transposase), and the transposase mediates the transposition of the region between the two Tn7 transposition sites on the vector to the mini-attTn7 site on the shuttle vector to complete the bacmid transposition, and the recombinant bacmid is extracted.

[0020] Furthermore, the baculovirus expression vector may be a pFastBac1 vector.

[0021] Furthermore, the recombinant baculovirus vector may be a pFB-4+8+11 vector. The pFB-4+8+11 vector is a recombinant vector (recombinant plasmid) obtained by cloning the serotype 4 fowl adenovirus fiber-2 gene (SEQ ID NO: 4), the serotype 8 fowl adenovirus fiber gene (SEQ ID NO: 5) and the serotype 11 fowl adenovirus fiber gene (SEQ ID NO: 6) into the pFastBac1 vector.

[0022] Furthermore, the recombinant baculovirus vector may also be a Bacmid-4+8+11 vector. The Bacmid-4+8+11 vector is a recombinant vector (recombinant bacmid) obtained by transforming the pFB-4+8+11 vector into a host bacterium (e.g., competent cell DH10Bac) containing a Bacmid shuttle vector and a helper plasmid capable of expressing Tn7 transposase.

[0023] The recombinant baculovirus described herein contains the gene encoding the serotype 4 fowl adenovirus fiber-2 protein (SEQ ID NO: 1), the gene encoding the serotype 8 fowl adenovirus fiber protein (SEQ ID NO: 2), and the gene encoding the serotype 11 fowl adenovirus fiber protein (SEQ ID NO: 3).

[0024] Furthermore, the recombinant baculovirus contains the DNA molecules shown by SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0025] The recombinant baculovirus can be obtained by transfecting insect cells with the recombinant baculovirus vector (such as recombinant bacmid) described herein using the Bac-to-Bac baculovirus expression system.

[0026] The recombinant baculovirus can also be obtained by introducing the linearized baculovirus genomic DNA carrying the nucleic acid molecule composition of the present invention into animal cells and then rescuing the virus.

[0027] In the above biological materials, the host cell in C4) may be an insect cell.

[0028] The recombinant host cell may be a recombinant host cell obtained by introducing the recombinant baculovirus vector described herein into an insect cell.

[0029] Furthermore, the insect cells include but are not limited to: Sf9 cells, Sf21 cells, High Five (Hi-5) cells and Tn-368 cells.

[0030] Furthermore, the insect cell may be a Sf9 cell or a High Five cell.

[0031] Furthermore, the recombinant host cell may be a recombinant host cell obtained by introducing the Bacmid-4+8+11 vector described herein into an insect cell (such as a Sf9 cell).

[0032] Furthermore, the introduction includes introduction into in vitro animal cells (transfection) by chemical transfection (such as calcium phosphate co-precipitation method), cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation (i.e. electrotransfection method), microinjection, gene gun method or virus-mediated method (such as retrovirus infection method, adenovirus infection method, lentivirus infection method) and the like.

[0033] Furthermore, the introduction can be carried out by chemical transfection.

[0034] The present invention also provides the use of the immunogenic composition, the nucleic acid molecule composition or the biological material in any of the following: D1) Use in the preparation of products for preventing and / or treating avian adenovirus infection or diseases caused by avian adenovirus infection; D2) Use in the preparation of a reagent for inducing an immune response against an avian adenovirus antigen; D3) Use in the preparation of vaccines for preventing avian adenovirus infection or diseases caused by avian adenovirus infection.

[0035] The avian adenovirus described herein includes avian adenovirus serotype 4, avian adenovirus serotype 8 and / or avian adenovirus serotype 11.

[0036] The product described herein can be a reagent, a kit (such as a therapeutic kit), a formulation, a medicament or a pharmaceutical composition.

[0037] The diseases caused by avian adenovirus infection described herein include hydropericardial hepatitis syndrome (HHS), inclusion body hepatitis and gizzard erosion, but are not limited thereto.

[0038] The present invention also provides a vaccine composition comprising the immunogenic composition described herein.

[0039] The vaccine composition can be used to prevent avian adenovirus infection or diseases caused by avian adenovirus infection.

[0040] Furthermore, the vaccine composition may also include an adjuvant.

[0041] The adjuvant may be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response to the antigen inoculated with it. The adjuvant described herein may be known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic components, quinine, saponin, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharide, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokines and nucleic acid adjuvants (such as monocyte colony stimulating factor, leukocyte factor IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid vectors, etc.), emulsion adjuvants (such as Freund's adjuvant, white oil adjuvant). The adjuvant may be a pharmaceutically acceptable adjuvant.

[0042] Furthermore, the adjuvant may be a white oil adjuvant.

[0043] Furthermore, the vaccine composition may also include one or more pharmaceutically acceptable carriers.

[0044] The pharmaceutically acceptable carrier is selected from excipients, preservatives, protective agents, cosolvents, diluents (such as water, saline, PBS (phosphate buffer), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc.), wetting agents, disintegrants (such as dry starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, etc.), lubricants (such as sorbitan trioleate, soybean lecithin, lecithin, oleic acid, magnesium stearate, sodium lauryl sulfate, etc.), fillers (such as starch, dextrin, etc.), adhesives (such as gelatin, pectin, gum arabic, hydroxypropyl cellulose (CP), PVP, CMC-Na, etc.), penetration enhancers (such as Brij-78), pH regulator, stabilizer (such as sodium sulfite, citric acid, tartaric acid, EDTA, etc.), surfactant (such as Tween, Span, eucalyptus oil, polysorbate-80, sodium lauryl sulfate, soybean lecithin, sodium cholate, sodium deoxycholate, etc.), absorption promoter (such as chitosan), thickener (such as sodium hyaluronate, sodium carboxymethyl cellulose, polyvinyl alcohol, etc.), antioxidant (such as sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium thiosulfate, vitamin C, etc.), plasticizer (such as glycerol, sorbitol, phthalate, etc.), propellant (such as hydrofluoroalkane, dimethyl ether, etc.), aerosolizer, suspending agent, dispersant, colorant (such as TiO2, pigment, etc.) and flavoring agent. Those skilled in the art know that a carrier usually has multiple functions, for example, starch can be used as both a disintegrant and a binder. Those skilled in the art can make conventional selections of the above carriers according to the properties of the vaccine and the route of administration.

[0045] Excipients include, but are not limited to: (1) Excipients for injections: such as solvent water, alcohols, ethers, amides, sulfoxides, esters, etc.; (2) Excipients for powders for injection: such as sucrose, lactose, mannitol, etc.; (3) Excipients for sprays: such as soybean lecithin, propylene glycol, borneol, ethanol, phenol, etc.; (4) Excipients for tablets: such as starch, sucrose, dextrin, methyl cellulose, gelatin, polyethylene glycol, tartaric acid, boric acid, etc.; (5) Excipients for eye drops: such as sodium hyaluronate, disodium ethylenediaminetetraacetic acid (EDTA-Na2), etc.; (6) Excipients for suppositories: such as cocoa butter, semi-synthetic or fully synthetic fatty acid glycerides, glycerol gelatin, polyethylene glycol, etc.; (7) Excipients for granules: such as corn flour, bentonite, zeolite powder, etc.; (8) Excipients for capsules: such as gelatin, etc.; (9) Excipients for gels: such as gelatin, pectin, gum arabic, etc.; (10) Excipients for ointments: such as vaseline, paraffin, liquid paraffin, lanolin, lanolin alcohol, beeswax, lard, vegetable oil, silicone oil, silicone, soaps, higher fatty alcohols, fatty alcohol sulfates, polyols, polyethylene glycol, FAPG, etc.; (11) Excipients for patches: such as ethylene-vinyl acetate copolymer (EVA), pressure-sensitive adhesive (PSA), etc.; (12) Excipients for films: such as gelatin, shellac, gum arabic, polyvinyl alcohol compounds, acrylic acid copolymers, etc.

[0046] Vaccines are easily contaminated by exogenous microorganisms during production, transportation and storage, which may affect the quality of the drug. Therefore, preservatives are usually added to inhibit the growth and reproduction of microorganisms. The preservatives include but are not limited to thimerosal, formaldehyde, phenol and m-cresol.

[0047] Protective agents generally refer to any substance that can protect the activity of drugs, including lyoprotectants. Lyoprotectants can be used to change the physical and chemical environment of biological samples during freeze-drying, maintain the original biological activity, improve the stability of antigenic proteins, etc. The lyoprotectants include but are not limited to sugars / polyols (such as sucrose, trehalose, lactose, glucose, maltose, maltodextrin, mannitol, sorbitol, etc.), polymers (such as HES, PVP, PEG, dextran, albumin, etc.), anhydrous solvents (such as polyethylene glycol, ethylene glycol, glycerol, DMSO, DMF, etc.), surfactants (such as Tween-80, etc.), amino acids (such as L-serine, sodium glutamate, alanine, glycine, sarcosine, acetyltryptophan, etc.), and salts and amines (such as phosphates, acetates, citrates, etc.).

[0048] Furthermore, the vaccine composition may further include a vaccine delivery system.

[0049] The vaccine delivery system can be a type of material that can carry antigenic substances to the body's immune system, and store and exert its antigenic effect therein for a long time. The vaccine delivery system can be a saline gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system or a nano adjuvant vaccine delivery system. It is known to those skilled in the art that adjuvants and delivery systems may overlap in structure and function, and can be routinely selected according to factors such as the specific type of vaccine, inoculation route, etc.

[0050] It is well known to those skilled in the art that in order to enhance the immunogenicity of antigenic proteins, in addition to adding compounds with immunopotentiating effects as adjuvants, the gene combination can be adjusted to allow them to be expressed in a granular structure; or they can be aggregated in vitro and encapsulated in liposomes or capsule microspheres.

[0051] The dosage forms of the vaccine composition include, but are not limited to, injections (including injection solutions and powders for injection), gels, eye drops (including eye drops and intraocular injection solutions), oral solutions, suppositories, effervescent tablets, capsules, ointments, creams, sprays, aerosols, external solutions, tablets, powders, pills, powders, scratches, granules, drops, coatings, patches, and long-acting sustained-release preparations. Those skilled in the art know that the above-mentioned various dosage forms can be prepared by using active components (such as the three antigen proteins of the present invention) in combination with suitable pharmaceutically acceptable carriers according to conventional preparation processes for preparations.

[0052] The vaccine composition may be in the form of an injection (including injection solution and powder for injection).

[0053] Injections generally refer to solutions and emulsions for injection into the body made after extraction and purification of the original drug, as well as sterile powders prepared into solutions before clinical use, including injection solutions and powder injections for injection. The preparation method of injections is well known to those skilled in the art, for example, powder injections for injection can be prepared by vacuum freeze drying technology, spray drying technology, spray freeze drying technology. The original drug can also be prepared into a solution by a concentrated preparation method or a diluted preparation method, and then the injection solution is prepared by filtering (such as surface filtration and / or deep filtration), potting, sterilization and other steps.

[0054] In order to prepare the vaccine composition into an injection preparation, such as a solution, emulsion, lyophilized powder injection and suspension, the injection solution can be prepared using a commonly used diluent in the art, such as water, physiological saline, PBS (phosphate buffered saline), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc. as a solvent. In addition, in order to prepare an isotonic injection solution, an appropriate amount of a carrier such as sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, conventional cosolvents, buffers, pH adjusters and other carriers can also be added.

[0055] The administration methods of the vaccine composition include, but are not limited to, injection (such as administration in the form of injection), mucosal administration (such as administration in the form of sprays, aerosols, tablets, eye drops, suppositories, granules, capsules, etc.) and transdermal administration (such as administration in the form of gels, ointments, patches, films, etc.).

[0056] The administration methods of the vaccine composition include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, transdermal injection, intravenous injection, arterial injection, intraperitoneal injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, skin application, oral and nasal spraying, atomization inhalation, in vivo implantation, and in vitro device administration. Preferably, it can be intramuscular injection, subcutaneous injection, oral administration, oral and nasal spraying, atomization inhalation, etc.

[0057] The active ingredient of the vaccine composition may be any of the immunogenic compositions described herein (the three antigenic proteins described herein).

[0058] The present invention also provides a method for preparing a vaccine composition, which comprises mixing the immunogenic composition described herein with an adjuvant to obtain the vaccine composition.

[0059] Furthermore, the method may include the following steps: (1) cloning the gene encoding the fiber-2 protein of serotype 4 fowl adenovirus (SEQ ID NO: 1), the gene encoding the fiber protein of serotype 8 fowl adenovirus (SEQ ID NO: 2), and the gene encoding the fiber protein of serotype 11 fowl adenovirus (SEQ ID NO: 3) into a baculovirus expression vector to obtain a recombinant plasmid; (2) transforming the recombinant plasmid into a host bacterium containing a transposase for transposition, and extracting the recombinant bacmid; (3) transfecting insect cells with the recombinant bacmid to obtain a recombinant baculovirus; (4) culturing the recombinant baculovirus to prepare a composition of the three antigenic proteins described herein; (5) The combination of the three antigen proteins is used as an active ingredient of the vaccine and mixed with an adjuvant to prepare a vaccine composition.

[0060] Furthermore, in the above method, the nucleotide sequence of the gene encoding the Fiber-2 protein of the serotype 4 fowl adenovirus may be SEQ ID NO: 4, the nucleotide sequence of the gene encoding the Fiber protein of the serotype 8 fowl adenovirus may be SEQ ID NO: 5, and the nucleotide sequence of the gene encoding the Fiber protein of the serotype 11 fowl adenovirus may be SEQ ID NO: 6. SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 are all codon-optimized sequences.

[0061] In the above method, the cloning into the baculovirus expression vector in step (1) may be cloning between two Escherichia coli Tn7 transposition sites in the baculovirus expression vector.

[0062] In the above method, the genes encoding the three antigenic proteins can be placed under the control of PH promoter, P10 promoter and / or IE0 promoter for transcription.

[0063] The baculovirus expression vector may be a pFastBac series vector (such as pFastBac1, pFastBacDual), a pAc series vector (such as pAC-k-Fc, pAC-L-Fc) or a pVL series vector (such as pVL1393, pVL1392), and specifically may be a pFastBac1 vector.

[0064] The host bacteria in step (2) may be Escherichia coli, specifically Escherichia coli DH10Bac.

[0065] The insect cells in step (3) may be Sf9 cells, Sf21 cells, High Five cells or Tn-368 cells, and specifically may be Sf9 cells or High Five cells.

[0066] The adjuvant in step (5) may be an emulsion adjuvant, specifically a white oil adjuvant.

[0067] The composition of the three antigen proteins obtained in step (4) can be an antigen protein liquid. The antigen protein liquid is mixed with the white oil adjuvant in a volume ratio of 1:2 to obtain the vaccine composition.

[0068] The present invention discloses a serotype 4, serotype 8, and 11 fowl adenovirus subunit vaccine and its application. The present invention utilizes a baculovirus expression system to efficiently express serotype 4 fiber-2 fowl adenovirus protein, serotype 8 fowl adenovirus fiber protein, and serotype 11 fowl adenovirus fiber protein, and can realize secretory expression of the three antigens with an expression titer of not less than 1:128, and the expressed antigen protein maintains good immunogenicity. After chickens are immunized with the subunit vaccine of the present invention, they can be protected from infection or attack of fowl adenovirus, can effectively activate the body's immune response, produce complete immune protection against toxicity, and the protection rate reaches 100%. It can completely resist the attack of strong toxins without becoming ill, and has good practical application value.

[0069] The present invention uses insect cells to express serotype 4, serotype 8, and type 11 avian adenovirus fiber proteins to prepare a trivalent subunit vaccine, which has the characteristics of high antigen expression, high antigen stability, high purity, good safety, and strong immunogenicity, and lays a solid foundation for the industrial production of avian adenovirus multivalent subunit vaccines and diagnostic reagents.

[0070] Definition of terms In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0071] The term "expression cassette" generally refers to a nucleic acid construct containing nucleic acid elements sufficient to express a target gene. A typical expression cassette contains a promoter, an MCS (multiple cloning site) and a terminator. An expression cassette may also include a target gene, a marker gene (such as TK gene, DHFR gene, CAT gene and NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly (A) signal sequence and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette may be directly connected or indirectly connected through a linker.

[0072] The term "vector" generally refers to a vector that can carry exogenous DNA or target gene into host cells for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into the host cell by transformation, transduction or transfection, so that the genetic material elements it carries are amplified and / or expressed in the host cell. Those skilled in the art can select a suitable vector according to the purpose of genetic engineering and the properties of the recipient cell. The vector includes, but is not limited to, plasmid, phage (such as lambda phage or M13 phage), cosmid (i.e., Cosmid), phagemid, shuttle vector (such as yeast expression vector), Ti plasmid, artificial chromosome (such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1 artificial chromosome (PAC) or Ti plasmid artificial chromosome (TAC)), viral vector (such as baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papillomavirus (such as SV40), herpes virus (such as herpes simplex virus)). A vector may contain a variety of elements for controlling expression, including but not limited to promoter sequence, transcription start sequence, enhancer sequence, selection element and reporter gene. In addition, the vector may also contain a replication start site.

[0073] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, etc. For example, the bacteria may be from the genus Corynebacterium ( Corynebacterium sp. ) (such as Corynebacterium glutamicum, Corynebacterium pekinensis, Corynebacterium blunt-toothed, etc.), Brevibacterium ( Brevibacterium sp. ) (such as lactic acid fermentation Brevibacterium, yellow Brevibacterium, ammonia-phagic Brevibacterium, etc.), Escherichia coli ( Escherichia sp. ) (such as Escherichia coli), Erwinia spp. ( Erwinia sp. ), Agrobacterium ( Agrobacterium sp. ) (such as Agrobacterium tumefaciens), Flavobacterium ( Flavobacterium sp. )、Alcaligenes spp.Alcaligenes sp. ), Pseudomonas ( Pseudomonas sp. ) and Bacillus ( Bacillus sp. ) (such as Bacillus), etc. The virus may include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (such as SV40) and herpes virus (such as herpes simplex virus), etc. The fungus may be from the genus Saccharomyces ( Saccharomyces sp. ) (such as Saccharomyces cerevisiae, Candida, Methanol yeast, Pichia pastoris), Fusarium ( Fusarium sp. ), Rhizoctonia spp. Rhizoctonia sp. ), Verticillium ( Verticillium sp. )、Penicillium( Penicillium sp. )、Aspergillus( Aspergillus sp. ) and Cephalosporium ( Cephalosporium sp. ) etc. The actinomycetes may be from the genus Streptomyces ( Streptomyces sp. ) (such as Streptomyces). The algae may be from Cyanophyta (such as cyanobacteria), Fucus ( Fucus sp. ), the genus A. Achnanthes sp. ), Cocoon algae ( Amphiprora sp. ), Diplocoriaceae ( Amphora sp. ), Fibrocystis spp. Ankistrodesmus sp. ), Astrophytum ( Asteromonas sp. ) and Chromophytes ( Boekelovia sp. )wait.

[0074] The term "host cell" is also referred to as a recipient cell, and generally refers to any type of cell that can be used to introduce a vector, such as a plant cell and an animal cell. The host cell can be understood to refer not only to a specific recipient cell, but also to the offspring of such a cell, and due to natural, accidental or intentional mutations and / or changes, the offspring may not necessarily be completely identical to the original parent cell, but is still included in the scope of the host cell. Suitable host cells are known in the art, among which: the plant cell can be Arabidopsis thaliana ( Arabidopsis thaliana ),tobacco( Nicotiana tabacum ),corn( Zea mays )、Rice( Oryza sativa ),wheat( Triticum aestivum) and other plant cells, but not limited thereto; the animal cells may be mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell substrain (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., African clawed frogs ( Xenopus laevis ) cells or giant salamander ( Andrias davidianus ) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells, Sf-9 cells or Hi-5), etc. but are not limited thereto.

[0075] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0076] The term "recombinant microorganism" generally refers to a recombinant microorganism with changed functions obtained by manipulating and modifying the genes of a target microorganism, such as introducing an exogenous target gene or a recombinant vector into the target microorganism, or directly editing the endogenous genes of the target microorganism.

[0077] The term "recombinant host cell" generally refers to a recombinant host cell whose function has been changed by manipulating and modifying the genes of the host cell, such as introducing an exogenous target gene or a recombinant vector into the host cell, or directly editing the endogenous genes of the host cell.

[0078] The term "signal peptide" generally refers to a short peptide chain that guides the transfer of newly synthesized proteins to the secretory pathway. The antigen protein of the present invention may further comprise a signal peptide, thereby facilitating the secretory expression of the antigen protein of the present invention in host cells.

[0079] The term "introduction" generally refers to the transfer of exogenous genes into recipient cells such as eukaryotic recipient cells or prokaryotic recipient cells. The method of introduction is not particularly limited, and any known transformation method can be used as long as it can transfer the target gene (such as the DNA molecule of the present invention) into the recipient cell. The introduced DNA molecule can be a single copy or multiple copies. The introduction can be the integration of the exogenous gene into the host chromosome, or it can be expressed extrachromosomally by a plasmid. The introduction method may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria by chemical transformation (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation, etc.) or physical transformation (such as electroporation transformation). (2) transducing the target gene into the host bacteria by phage transduction. (3) The target gene is transferred into plant receptor cells by physical or chemical methods, such as gene gun method (also known as microprojectile bombardment method or biological missile method), chemical stimulation method, electric shock method, liposome-mediated method, microinjection method, laser microbeam method, pollen tube channel method, ultrasound method, air gun method and vortex method, etc. (4) The target gene is transferred into plant receptor cells by vector, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system) mediated method (Agrobacterium-mediated method), plant virus vector-mediated transformation method, etc. (5) The target gene is introduced into in vitro animal cells (transfection) by calcium phosphate coprecipitation method, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation method (i.e. electrofection method), microinjection, gene gun method or virus-mediated method (such as retrovirus infection method, adenovirus infection method, lentivirus infection method), etc. (6) Introducing the target gene into in vivo animal cells through microinjection, retroviral vector method, somatic cell nuclear transfer method, sperm vector method or embryonic stem cell method, and further preparing transgenic animals.

[0080] The term "codon optimization" generally refers to a technique that improves protein expression levels in organisms by increasing the translation efficiency of target genes. Codon optimization usually involves redesigning genes to improve translation efficiency and thus protein expression levels by avoiding rare codons, using preferred codons, simplifying the secondary structure of mRNA, optimizing repetitive sequences, eliminating restriction enzyme sites, and adjusting GC content.

[0081] The term "comprising" is not intended to be limiting, but is intended to be inclusive and means that there may be other elements besides the listed elements, which can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of". The terms "comprising" and "including" are used interchangeably herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1This is a map of the transfer vector constructed in Example 1 of the present invention.

[0083] Figure 2 The results of indirect immunofluorescence detection of SF9 cells infected with recombinant baculovirus. Figure 2 A and D in the figure are the results of primary antibody detection using serum positive for serotype 4 fowl adenovirus, where A is the inoculated recombinant baculovirus and D is the normal Sf9 cell; Figure 2 B and E in the figure are the results of the primary antibody test using serum positive for fowl adenovirus type 8, where B is the inoculated recombinant baculovirus and E is the normal Sf9 cell; Figure 2 C and F are the results of primary antibody detection using serum type 11 avian adenovirus positive serum, where C is the inoculated recombinant baculovirus and F is the normal Sf9 cell.

[0084] Figure 3 It is the result of antigen agar amplification detection in the present invention. Figure 3 A, B, and C are agar amplification reactions of fowl adenovirus type 4, fowl adenovirus type 8, and fowl adenovirus type 11, respectively; dilutions 1 to 5 are 1:2, 1:4, 1:8, 1:16, and 1:32, respectively; and 6 is a negative control. DETAILED DESCRIPTION

[0085] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0086] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0087] The amino acid sequence of the melittin signal peptide in the following example is: MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 7). The nucleotide sequence of the melittin signal peptide encoding gene is 5'-ATGAAGTTCCTGGTGAACGTGGCTCTGGTGTTCATGGTGGTGTACATCTCCTACATCTACGCT-3' (SEQ ID NO: 8).

[0088] The pFastBac1 vector (insect baculovirus expression vector) in the following examples was purchased from Wuhan Miaoling Biotechnology Co., Ltd., item number P0291.

[0089] The pIEx / Bac-3 vector in the following examples was provided by Jiangsu Saisuofei Biotechnology Co., Ltd.

[0090] Example 1. Construction of recombinant virus expressing protein antigen In this example, recombinant baculovirus expressing serotype 4 fowl adenovirus Fiber-2 protein, serotype 8 fowl adenovirus Fiber protein, and serotype 11 fowl adenovirus Fiber protein were constructed, as follows: 1. Optimization and acquisition of Fiber-2 gene of serotype 4 fowl adenovirus The Fiber-2 protein in the Ax25 strain of serotype 4 avian adenovirus epidemic strain was selected, and the amino acid sequence was shown in SEQ ID NO: 1. In order to increase the expression amount of the target protein in insect cells, the gene encoding the Fiber-2 protein was optimized for insect cell-preferred codons. The nucleotide sequence of the optimized Fiber-2 protein encoding gene is shown in SEQ ID NO: 4. At the same time, the Melittin signal peptide encoding gene was added to the N-terminus of SEQ ID NO: 4 for artificial synthesis. The synthesized gene was connected to the pFastBac1 vector, and the obtained recombinant vector was named pFastBac1-4.

[0091] 2. Optimization and acquisition of serotype 8 avian adenovirus fiber gene The fiber protein in the Ab26 strain of the serotype 8 fowl adenovirus epidemic strain was selected, and the amino acid sequence was shown in SEQ ID NO:2. In order to increase the expression amount of the target protein in insect cells, the gene encoding the fiber protein was optimized for insect cell-preferred codons. The nucleotide sequence of the optimized fiber protein encoding gene was shown in SEQ ID NO:5. At the same time, the melittin signal peptide encoding gene was added to the N-terminus of SEQ ID NO:5 for artificial synthesis. The synthesized gene was connected to the pFastBac1 vector, and the obtained recombinant vector was named pFastBac1-8.

[0092] 3. Optimization and acquisition of serotype 11 avian adenovirus fiber gene The fiber protein in the serotype 11 fowl adenovirus epidemic strain 1102 was selected, and the amino acid sequence was shown in SEQ ID NO:3. In order to increase the expression amount of the target protein in insect cells, the gene encoding the fiber protein was codon optimized for insect cell preference. The nucleotide sequence of the optimized fiber protein encoding gene is shown in SEQ ID NO:6. At the same time, the melittin signal peptide encoding gene was added to the N-terminus of SEQ ID NO:6 for artificial synthesis. The synthesized gene was connected to the pIEx / Bac-3 vector, and the obtained recombinant vector was named pIEx / Bac-11.

[0093] 4. Construction of transfer vectors containing serotype 4 fowl adenovirus fiber-2 gene, serotype 8 fowl adenovirus fiber gene, and serotype 11 fowl adenovirus fiber gene 4.1 Primer design and fragment preparation Primers for the pFastBac1 linearized vector containing the serotype 4 fowl adenovirus Fiber-2 gene, primers containing the serotype 8 fowl adenovirus Fiber gene expression cassette, and primers containing the serotype 11 fowl adenovirus Fiber gene expression cassette were designed to prepare PCR products for recombination reaction and purify them. The primer sequences are shown in Table 1.

[0094]

[0095] The pFastBac1 linearized vector containing the Fiber-2 gene of serotype 4 fowl adenovirus is a DNA fragment of about 6200 bp 1 obtained by amplifying the recombinant vector pFastBac1-4 as a template and using 4P1 and 4P2 as primers.

[0096] The DNA fragment 2 of about 2100 bp containing the expression cassette of the fiber gene of serotype 8 fowl adenovirus is amplified using the recombinant vector pFastBac1-8 as a template and 8P1 and 8P2 as primers.

[0097] The DNA fragment 3 of about 3400 bp containing the fiber gene expression cassette of fowl adenovirus serotype 11 was amplified using the recombinant vector pIEx / Bac-11 as a template and 11P1 and 11P2 as primers.

[0098] 4.2 Recombination reaction Use a commercial seamless cloning kit (Nanjing Novozyme Biotech Co., Ltd., product number C115) to prepare the reaction system according to Table 2, mix gently, perform recombination reaction at 50°C for 30 min to obtain the ligation product, and place on ice after the reaction.

[0099]

[0100] 4.3 Conversion Take 100 µL of competent cells DH5α and place them in an ice bath, add 10 µL of the ligation product, mix thoroughly, and place them in an ice bath for 30 min; heat shock at 42°C for 90 s, and place them in an ice bath for 1 min; add 890 µL of LB culture medium preheated at 37°C and shake at 37°C for 45 min to revive resistance; centrifuge at 4000 rpm for 5 min, discard part of the supernatant, and evenly spread the remaining 20 µL on the LB plate containing Amp antibiotic (50 µg / mL), and invert and culture at 37°C for 12 to 18 h. Aseptically pick a single white colony and place it in 4 mL of LB (Amp+) liquid culture and shake and culture at 37°C for 10 to 14 h.

[0101] 4.4. PCR Identification of Bacterial Fluid The culture solution was used as a template for PCR identification, and the positive clones were initially screened and sent for sequencing. The PCR identification primers were: Tn7L: 5'-GCGTCGTAAGCTAATACGAAAATTAAAAATGAC-3', TN7R: 5'-GCCAAGGGCATGGTAAAGAC-3'.

[0102] 4.5 Plasmid extraction After overnight culture of the bacterial solution with correct sequencing, plasmid was extracted according to the instructions of the commercial plasmid extraction kit (QIAGEN product, catalog number 27104), and named pFB-4+8+11. The recombinant vector (transfer vector) pFB-4+8+11 contains the serotype 4 fowl adenovirus fiber-2 gene (SEQ ID NO: 4), the serotype 8 fowl adenovirus fiber gene (SEQ ID NO: 5), and the serotype 11 fowl adenovirus fiber gene (SEQ ID NO: 6). The structural diagram is shown in Figure 1 shown.

[0103] 5. Construction and identification of recombinant bacmids expressing serotype 4 fowl adenovirus fiber-2 protein, serotype 8 fowl adenovirus fiber protein, and serotype 11 fowl adenovirus fiber protein 5.1 Conversion Take 100 µL of competent cells DH10Bac and place it on ice, add 10 µL of pFB-4+8+11 plasmid, mix thoroughly, and place it on ice for 30 min; heat shock at 42°C for 90 s, and place it on ice for 1 min; add 890 µL of LB culture medium preheated at 37°C and shake and culture at 37°C for 4 h; take 100 µL of bacterial solution and dilute it 10 times with LB medium (10 -1 ~10 -3), 100 µL of each dilution of bacterial solution was evenly spread on LB plates containing X-Gal, IPTG, kanamycin (50 µg / ml), gentamicin (7 µg / ml), and tetracycline (10 µg / ml), and inverted at 37°C for 48 h. Complete bacmid transposition.

[0104] 5.2 PCR identification of recombinants Pick a single clone on the culture plate and inoculate it into LB liquid medium containing antibiotics (kanamycin, gentamicin, tetracycline), shake culture at 37℃ for 10-14 hours, and use the culture solution as a template for PCR identification of the culture solution. The PCR identification primers are: M13 / pUC-F: 5'-CCCAGTCACGACGTTGTAAAACG-3'; M13 / pUC-R: 5'-AGCGGATAACAATTTCACACAGG-3'.

[0105] 5.3. Preparation of recombinant bacmid After overnight culture of the bacterial solution identified as positive by PCR, the plasmid was extracted according to the method of the endotoxin-free plasmid extraction kit (product of Promega, catalog number A2495), and the obtained recombinant bacmid was named Bacmid-4+8+11.

[0106] 6. Construction of recombinant baculovirus expressing serotype 4 fowl adenovirus fiber-2 protein, serotype 8 fowl adenovirus fiber protein, and serotype 11 fowl adenovirus fiber protein 6.1. Transfection of recombinant bacmid into Sf9 insect cells 24 h before transfection, 0.5×10 6 Sf9 cells were inoculated into 6-well plates at a density of 1:1 / well; the cells were cultured to the logarithmic phase and transfected. After culturing at 27-28°C for 72 h, the culture supernatant was centrifuged at 1500 rpm for 5 min. The recombinant baculovirus was named Baculo-4+8+11 virus and stored at -80°C to obtain the P1 generation virus.

[0107] 6.2. Recombinant baculovirus amplification The P1 generation recombinant baculovirus was inoculated at 1% and then placed in a constant temperature cell culture incubator for culture for about 72 to 96 hours. When the cells showed obvious pathological changes and most of the cells fell off, the cell culture supernatant was collected as the P2 generation virus seed.

[0108] Example 2. Expression and identification of protein antigens The expression and identification of serotype 4 avian adenovirus fiber-2 protein, serotype 8 avian adenovirus fiber protein, and serotype 11 avian adenovirus fiber protein in Sf9 insect cells are as follows: 1. Indirect immunofluorescence assay (IFA) Sf9 cells were cultured in 6-well plates for 24 h, and insect cells SF9 were infected with the second-generation recombinant baculovirus and cultured at 27°C for 72 h. At the same time, normal SF9 cells were used as a negative control group, in which wells A, B, and C were inoculated with recombinant baculovirus, and wells D, E, and F were used as negative controls. After the cells were cultured for 72 h, the supernatant was aspirated and washed with PBS. 1 mL of pre-cooled 4% paraformaldehyde solution was added to each well for fixation for 10-15 min, and washed 3 times with PBS. 500 μL of 0.1% Triton X-100 was added to each well, and the cells were allowed to stand at room temperature for 15 min, and washed 3 times with PBS. A / D wells were treated with serum positive serum of serotype 4 avian adenovirus as the primary antibody, B / E wells were treated with serum positive serum of serotype 8 avian adenovirus as the primary antibody, and C / F wells were treated with serum positive serum of serotype 11 avian adenovirus as the primary antibody. 500 μL of each well was incubated at 37°C for 1 h, the primary antibody was discarded, and the cells were washed 3 times with PBS. FITC-labeled goat anti-chicken secondary antibody (purchased from SeraCare) was added. (KPL, catalog number 5230-0325), 500 μL per well, 37℃ for 1 h, discard the secondary antibody, wash with PBS 3 times; leave 500 μL PBS in each well, observe and take pictures under an inverted fluorescence microscope. The test results are as follows Figure 2 As shown, the results showed that the Fiber-2 protein of serotype 4 avian adenovirus, the Fiber protein of serotype 8 avian adenovirus and the Fiber protein of serotype 11 avian adenovirus were all well expressed.

[0109] 2. Bidirectional agar diffusion test The second generation recombinant baculovirus was used to infect the suspension cultured Sf9 cells to prepare the antigen protein solution. Three more agar double diffusion plates were prepared with plum blossom wells, including the first plate of serum type 4 avian adenovirus antigen detection plate: serum type 4 avian adenovirus positive serum was added to the center well, serum type 4 avian adenovirus agar antigen was added to the positive control wells, and 2-fold diluted protein solution was added to the test wells; the second plate of serum type 8 avian adenovirus antigen detection plate: serum type 8 avian adenovirus positive serum was added to the center well, serum type 8 avian adenovirus agar antigen was added to the positive control wells, and 2-fold diluted protein solution was added to the test wells; the third plate of serum type 11 avian adenovirus antigen detection plate: serum type 11 avian adenovirus positive serum was added to the center well, serum type 11 avian adenovirus agar antigen was added to the positive control wells, and 2-fold diluted protein solution was added to the test wells, and incubated in a 37°C incubator humid box for 48 to 72 hours. The results are as follows Figure 3 As shown, the titers of the three antigens were not less than 1:16. The results showed that the three protein antigens had good specificity and immunogenicity.

[0110] Example 3. Preparation of protein antigen The serotype 4 fowl adenovirus fiber-2 protein, serotype 8 fowl adenovirus fiber protein, and serotype 11 fowl adenovirus fiber protein antigens were prepared as follows: The third generation recombinant baculovirus stock solution was inoculated into High Five cells at a volume ratio of 0.5%, harvested about 72 to 96 hours after inoculation, frozen and thawed three times at -20°C, and centrifuged to collect the supernatant, which was a mixture of serotype 4 fowl adenovirus fiber-2 protein, serotype 8 fowl adenovirus fiber protein, and serotype 11 fowl adenovirus fiber protein antigens. The agar titer of serotype 4 fowl adenovirus fiber-2 protein, serotype 8 fowl adenovirus fiber protein, and serotype 11 fowl adenovirus fiber antigen was determined to be 1:128.

[0111] Example 4. Preparation and immunogenicity test of subunit vaccine 1. Vaccine preparation The three antigens expressed, namely, serotype 4 fowl adenovirus Fiber-2 protein, serotype 8 fowl adenovirus Fiber protein, and serotype 11 fowl adenovirus Fiber protein, were prepared into an antigen mixture in an equal mass ratio, and the agar titer of the three antigens in the antigen mixture was 1:42; the antigen mixture was then mixed with white oil adjuvant in a volume ratio of 1:2 to prepare a subunit vaccine.

[0112] 2. Immunogenicity test 45 SPF chickens aged 21 days were divided into two groups, including 30 in the immunization group and 15 in the control group. The vaccine immunization group was subcutaneously injected with the subunit vaccine prepared in step 1 above, with an immunization dose of 0.2 ml / chicken, and the control group was subcutaneously injected with 0.2 ml of normal saline. Serum was collected 21 to 28 days after immunization, and the antibody level was determined by two-way agar diffusion test. At the same time, the 10 6.0 TCID 50 / dose intramuscular injection of serotype 4 fowl adenovirus Ax25 strain, according to 10 6.5 TCID 50 / dose intravenous injection of serum type 8 avian adenovirus Ab26 strain, according to 10 6.5 TCID 50 The mice were intravenously injected with serum type 11 avian adenovirus strain 1102 at a dose of 1:1 / head, and observation and autopsy were performed 5 to 14 days after the challenge. The experimental results are shown in Table 3.

[0113]

[0114] The results show that the subunit vaccine of the present invention can effectively activate the body's immune response and produce complete immune protection against viruses, with a protection rate of 100%.

[0115] 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 the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An immunogenic composition, characterized in that The immunogenic composition comprises three antigenic proteins shown in A1), A2) and A3): A1) an antigenic protein having an amino acid sequence as shown in SEQ ID NO: 1, or an antigenic protein having the same function obtained by connecting a signal peptide to the N-terminus of SEQ ID NO: 1; A2) an antigenic protein having an amino acid sequence as shown in SEQ ID NO: 2, or an antigenic protein having the same function obtained by connecting a signal peptide to the N-terminus of SEQ ID NO: 2; A3) An antigenic protein having an amino acid sequence as shown in SEQ ID NO: 3, or an antigenic protein having the same function obtained by connecting a signal peptide to the N-terminus of SEQ ID NO:

3.

2. A nucleic acid molecule composition, characterized in that: The nucleic acid molecule composition is any of the following: B1) the nucleic acid molecule composition comprises nucleic acid molecules encoding the three antigenic proteins in the immunogenic composition of claim 1 respectively; B2) The nucleic acid molecule composition includes DNA molecules with nucleotide sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:

6.

3. Biomaterial, characterized in that The biological material is any of the following: C1) an expression cassette containing the nucleic acid molecule composition according to claim 2; C2) a recombinant vector containing the nucleic acid molecule composition according to claim 2; C3) a recombinant microorganism containing the nucleic acid molecule composition according to claim 2; C4) A recombinant host cell containing the nucleic acid molecule composition according to claim 2.

4. The biomaterial according to claim 3, characterized in that The recombinant vector in C2) is a recombinant baculovirus vector; the recombinant microorganism in C3) is a recombinant baculovirus.

5. Use of the immunogenic composition according to claim 1, the nucleic acid molecule composition according to claim 2, or the biological material according to claim 3 or 4 in any of the following: D1) Use in the preparation of products for preventing and / or treating avian adenovirus infection or diseases caused by avian adenovirus infection; D2) Use in the preparation of a reagent for inducing an immune response against an avian adenovirus antigen; D3) Use in the preparation of vaccines for preventing avian adenovirus infection or diseases caused by avian adenovirus infection.

6. The use according to claim 5, characterized in that: The diseases caused by the avian adenovirus infection include pericardial effusion-hepatitis syndrome, inclusion body hepatitis and gizzard erosion.

7. A vaccine composition, characterized in that The vaccine composition comprises the immunogenic composition of claim 1.

8. The vaccine composition according to claim 8, characterized in that The vaccine composition also includes an adjuvant.

9. The vaccine composition according to claim 8, characterized in that The adjuvant is a white oil adjuvant.

10. A method for preparing a vaccine composition, characterized in that: The method comprises mixing the immunogenic composition of claim 1 with an adjuvant to prepare the vaccine composition.

Images