WO2021073178A1 - 禽减蛋综合征病毒tFiber蛋白片段、其制备的疫苗组合物、制备方法和应用 - Google Patents

禽减蛋综合征病毒tFiber蛋白片段、其制备的疫苗组合物、制备方法和应用 Download PDF

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WO2021073178A1
WO2021073178A1 PCT/CN2020/101961 CN2020101961W WO2021073178A1 WO 2021073178 A1 WO2021073178 A1 WO 2021073178A1 CN 2020101961 W CN2020101961 W CN 2020101961W WO 2021073178 A1 WO2021073178 A1 WO 2021073178A1
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antigen
virus
avian
tfiber
vaccine composition
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PCT/CN2020/101961
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English (en)
French (fr)
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田克恭
王孟月
刘武杰
张许科
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普莱柯生物工程股份有限公司
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Priority to US17/754,280 priority Critical patent/US20220265811A1/en
Priority to JP2022520060A priority patent/JP7289405B2/ja
Publication of WO2021073178A1 publication Critical patent/WO2021073178A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A61K39/17—Newcastle disease virus
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/075—Adenoviridae
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10111—Atadenovirus, e.g. ovine adenovirus D
    • C12N2710/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
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    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a fowl egg drop syndrome virus tFiber protein fragment, a vaccine composition prepared therefrom, a preparation method and application thereof, and belongs to the field of biomedicine.
  • Avian egg drop syndrome virus (Egg Drop Syndrome Virus, EDSV) belongs to the avian adenovirus group III. After the virus infects poultry, there is no obvious clinical symptoms or mild diarrhea. It often occurs in the peak period of egg production and can cause laying hens to lay Soft-shelled, thin-shelled, and shell-less eggs have severely reduced the egg production rate, causing serious economic losses.
  • the EDSV has a typical adenovirus morphology, has no capsule, has red blood cell agglutinating activity, and proliferates in avian fallopian tubes.
  • the viral genome is about 33 kb linear double-stranded DNA.
  • the viral particles are composed of structural proteins.
  • the nucleocapsid has a diameter of 70-80 nm and is icosahedral symmetry. The DNA is enclosed in the capsid.
  • the nucleocapsid is composed of 252 shell particles, of which 240 are hexons (Hexon protein), which constitute the 20 faces of the icosahedron and most of the edges. These shell particles are prismatic, 7nm wide and 11nm long.
  • the other 12 are Pentons (Penton proteins), which are located on the 12 vertex corners of the icosahedron. Each Penton protein has a fiber protrusion (Fiber protein).
  • Poultry egg drop syndrome is currently one of the most important diseases that seriously endanger the development of poultry farming in the world.
  • vaccine immunization is still the most important measure.
  • the EDS inactivated vaccine commonly used in the poultry industry is a vaccine that is emulsified with a mineral oil adjuvant after the virus proliferated in duck embryos is inactivated.
  • the avian egg drop syndrome virus is difficult to obtain high-titer virus in duck embryos, the prepared vaccines are often difficult to provide the ideal immune effect; in addition, the production method of the virus antigen is completely dependent on duck breeding eggs.
  • influenza and other infectious diseases occur, the supply of duck eggs is difficult, which seriously affects the prevention and control of poultry egg drop syndrome.
  • biosafety caused by incomplete virus inactivation during the production of whole virus vaccines.
  • the subunit vaccine is a new type of genetically engineered vaccine that has been developed in recent years.
  • the research on the avian egg drop syndrome subunit vaccine takes Hexon (240/252) as the main research object.
  • Hexon 240/252
  • the immune efficiency has been low. Not developed into a product.
  • the present invention provides an avian egg drop syndrome virus immunogenic protein, a vaccine composition prepared therefrom, and a preparation method and application of the vaccine composition.
  • the vaccine composition can effectively prevent and/or Treatment of avian egg drop syndrome virus infection.
  • the present invention relates to an avian egg drop syndrome virus tFiber protein fragment, wherein the gene of the avian egg drop syndrome virus tFiber protein fragment is shown in SEQ ID NO. 2 or its degenerate sequence.
  • the present invention also relates to a vaccine composition, wherein the vaccine composition comprises an immune amount of the avian egg drop syndrome virus tFiber protein fragment or an immune amount recombinant with the avian egg drop syndrome virus tFiber protein Live carriers of fragmented genes and pharmaceutically acceptable carriers.
  • the present invention uses the avian egg drop syndrome virus tFiber protein fragment for the first time.
  • a vaccine composition is prepared, which can prevent and/or treat the avian egg drop syndrome epidemic, and contains the The protein vaccine composition can enable the animal body to rapidly produce antibodies after immunizing the animal, and has a good prevention and control effect on the single or mixed infection of the currently prevalent avian egg drop syndrome virus, and has good biological safety.
  • the vaccine composition prepared by the avian egg drop syndrome virus tFiber protein fragment of the present invention has good immunogenicity, can completely protect chickens and ducks, and can prevent and/or prevent the infection of avian egg drop syndrome virus in clinical applications. Or treatment.
  • the present invention also relates to a method for preparing the vaccine composition, wherein the method comprises: step (1) cloning the avian egg drop syndrome virus tFiber protein fragment gene, and combining the avian egg drop syndrome virus The gene of the tFiber protein fragment is recombined into the expression vector to obtain a recombinant expression vector containing the avian egg drop syndrome virus tFiber protein fragment gene; step (2) the recombination of the avian egg drop syndrome virus tFiber protein fragment gene The recombinant expression vector and the molecular chaperone expression vector are transformed into Escherichia coli together to express the avian egg drop syndrome virus tFiber protein fragment; step (3) the expressed avian egg drop syndrome virus tFiber protein fragment is used on a non-ionic surface The active agent is treated to remove endotoxin; and in step (4), the endotoxin-removed avian egg drop syndrome virus tFiber protein fragment is mixed with an adjuvant to obtain
  • the invention also relates to the application of the vaccine composition in the preparation of medicines for preventing and/or treating avian egg drop syndrome virus infection.
  • Egg Drop Syndrome Virus belongs to the avian adenovirus group III, and its genome is double-stranded DNA.
  • the clinical symptoms caused by the laying hens include soft-shelled, thin-shelled, and shell-less eggs, and the egg production rate is severely reduced.
  • the pathological changes are characterized by quiescent ovarian underdevelopment and fallopian tube atrophy.
  • the present invention relates to an avian egg drop syndrome virus tFiber protein fragment, wherein the gene of the avian egg drop syndrome virus tFiber protein fragment is shown in SEQ ID NO. 2 or its degenerate sequence.
  • the avian egg drop syndrome virus tFiber protein fragment of the present invention has good immunogenicity, can produce good immune protection with low content immunization, and its immune effect is better than that of the avian egg drop syndrome virus Fiber full-length protein.
  • the present invention also relates to a vaccine composition, wherein the vaccine composition comprises an immune amount of the avian egg drop syndrome virus tFiber protein fragment or an immune amount recombinant with the avian egg drop syndrome virus tFiber protein
  • the vaccine composition comprises an immune amount of the avian egg drop syndrome virus tFiber protein fragment or an immune amount recombinant with the avian egg drop syndrome virus tFiber protein
  • a live vector of the fragment gene and a pharmaceutically acceptable vector the gene of the avian egg drop syndrome virus tFiber protein fragment is shown in SEQ ID NO. 2 or its degenerate sequence.
  • the immunogenic component in the vaccine composition of the present invention can still ensure a good immune effect when a very low immune dose is used.
  • vaccine composition used in the present invention refers to a pharmaceutical composition containing the immunogenicity of avian egg drop syndrome virus, which can induce, stimulate or enhance the immune response of chickens and ducks against avian egg drop syndrome virus.
  • immune amount should be understood as “immune effective amount”, also known as an immune protective amount or an effective amount for generating an immune response. It is an antigen amount that can effectively induce an immune response in the recipient's body, and the amount is sufficient to prevent or ameliorate the signs of disease Or symptoms, including adverse health effects or their complications.
  • the immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of disease, including adverse health outcomes or complications caused by infections caused by pathogens. Humoral immunity or cell-mediated immunity or both can be induced.
  • the immune response of the animal to the immunogenic composition can be assessed indirectly by, for example, measuring antibody titer, lymphocyte proliferation analysis, or directly by monitoring signs or symptoms after challenge with a wild-type strain, and the vaccine provided Protective immunity can be assessed by measuring, for example, the subject's clinical signs such as mortality, reduction in morbidity, temperature values, the subject's overall physiological condition, and overall health and performance.
  • the immune response may include, but is not limited to, induction of cellular and/or humoral immunity.
  • avian egg drop syndrome virus antigen refers to any composition containing at least one avian egg drop syndrome virus antigen form, which can induce, stimulate or resist avian egg drop syndrome virus Immune response to infection.
  • the antigenic form includes, but is not limited to, inactivated, attenuated or subunit antigens.
  • the avian egg drop syndrome virus tFiber protein fragment antigen of the present invention can be a recombinantly expressed tFiber protein fragment subunit antigen, and its expression system can be a prokaryotic expression system, a eukaryotic expression system, or a synthetic peptide synthesized artificially antigen.
  • Subunit antigen refers to an antigen made by cloning the protective antigen gene of a pathogen into a prokaryotic or eukaryotic expression system by means of genetic engineering, and expressing it efficiently. It is less likely to cause side effects than whole virus antigens.
  • Synthetic peptide antigen refers to a small peptide containing only immunodeterminant components, that is, an antigen made by artificially synthesizing a protective short peptide according to the amino acid sequence of a natural protein, connecting it with a carrier and adding an adjuvant.
  • Live vector refers to non-pathogenic microorganisms that carry and express a certain antigen or antigenic determinant gene through genetic engineering methods to produce immunogenicity.
  • Non-pathogenic microorganisms can be bacteria and viruses.
  • Live viral vectors are often used as Vector viruses include vaccinia virus, fowlpox virus, turkey herpes virus, adenovirus, pseudorabies virus, retrovirus, lentivirus; live bacterial vectors can include attenuated Salmonella, BCG, and attenuated mononuclear cells Bacillus, attenuated Vibrio cholerae, attenuated Shigella, Lactococcus, Lactobacillus bacillus, Streptococcus gausii.
  • the avian egg drop syndrome virus tFiber protein fragment is the protein encoded by the nucleotide sequence shown in SEQ ID NO.2.
  • the gene of the avian egg drop syndrome virus tFiber protein fragment has the nucleotide sequence shown in SEQ ID NO. 2 or its degenerate sequence .
  • the content of the avian egg drop syndrome virus tFiber protein fragment is AGP titer ⁇ 1:8.
  • the content of the avian egg drop syndrome virus tFiber protein fragment is AGP titer 1:8-1:32.
  • AGP titer is also called “Agar Gel Precipitation (test) titer”.
  • the agar diffusion test is a precipitation reaction in which antigens and antibodies can diffuse freely around in an agar gel containing electrolytes , The two combine with each other, the precipitation line appears at the optimum ratio, and the highest dilution ratio at which the precipitation line appears is the efficacy value, which is the calibrated antigen or antibody content.
  • pharmaceutically acceptable carrier refers to all the ingredients in the vaccine composition of the present invention except for the avian egg drop syndrome virus antigen, a carrier or dilution that does not stimulate the body and does not hinder the biological activity and characteristics of the compound used.
  • the agent is preferably an adjuvant.
  • the vaccine composition of the present invention may further add other reagents to the composition of the present invention.
  • the pharmaceutically acceptable carrier includes drugs, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants and preservatives
  • the immunostimulants include ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and interleukin-2 (IL2).
  • GM-CSF granulocyte macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • IL2 interleukin-2
  • the immunostimulant includes ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and interleukin-2 (IL2).
  • GM-CSF granulocyte macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • IL2 interleukin-2
  • the pharmaceutically acceptable carrier is an adjuvant
  • the adjuvant includes: (1) mineral oil, aluminum gum adjuvant, saponin, and Frudine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) acrylic acid or methacrylic acid polymer, maleic anhydride and alkenyl derivatives Copolymer; and RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl One or more of dipeptide, Montanide ISA 206, Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100;
  • the content of the adjuvant is 5%-70% V/V, and the content of the adjuvant is preferably 30% to 70%, more preferably 66% V/V.
  • adjuvant may include aluminum gel adjuvant; saponin, such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); water-in-oil emulsion; Oil-in-water emulsions; water-in-oil-in-water emulsions; polymers of acrylic acid or methacrylic acid; compounds selected from copolymers of maleic anhydride and alkenyl derivatives.
  • saponin such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL)
  • water-in-oil emulsion oil-in-water emulsions
  • water-in-oil-in-water emulsions polymers of acrylic acid or methacrylic acid; compounds selected from copolymers of maleic anhydride and alkenyl derivatives.
  • emulsion may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil (isoprenoid oil) produced by the oligomerization of olefins, such as squalane or squalene oil , Especially isobutylene or decene; linear alkyl-containing esters of acids or alcohols, more especially vegetable oils, ethyl oleate, propylene glycol di-(caprylate/decanoate), glycerol tri-(caprylate/decanoic acid) Esters) or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearates.
  • light liquid paraffin oil European Pharmacopea type
  • isoprenoid oil isoprenoid oil
  • isoprenoid oil produced by the oligomerization of olefins, such as squalane or squalene oil , Especially isobutylene or
  • Emulsifiers are preferably nonionic surfactants, especially esters of sorbitan, esters of mannide (such as anhydrous mannide oleate), esters of aliphatic glycol (glycol), polyglycerol ( polyglycerol), propylene glycol and oleic acid, isostearic acid, ricinoleic acid, or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxy Ethylene block copolymers, especially Pluronic products, especially L121. See “Theory and practical application of adjuvants" (Ed.
  • acrylic acid or methacrylic acid polymer is preferably a crosslinked acrylic or methacrylic acid polymer, especially crosslinked with polyalkenyl ethers or polyols of sugar.
  • Carbomer Carbopol, trade name Carbopol
  • Carbopol trade name Carbopol
  • Those skilled in the art can also refer to US Patent No. 2,09,462, which describes this type of acrylic polymer, which is cross-linked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably no more than 8, of which at least 3 hydroxyl groups
  • the hydrogen atom of is replaced by an unsaturated aliphatic radical with at least 2 carbon atoms.
  • Preferred groups are those containing 2-4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups.
  • the unsaturated group itself may contain other substituents, such as methyl.
  • Carbopol BF Goodrich, Ohio, USA
  • Carbopol 974P, 934P and 971P can be mentioned, and Carbopol 971P is most preferably used.
  • the term "copolymer of maleic anhydride and alkenyl derivative" can also consider the copolymer of maleic anhydride and ethylene, EMA (Monsanto). These polymers are dissolved in water to produce an acidic solution. After neutralization, it is preferred Neutralize to physiological pH in order to produce an adjuvant solution into which the immunogenic, immunogenic or vaccine composition itself can be incorporated.
  • adjuvant also includes, but is not limited to, RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A (monophosphoryl lipid A) lipid A), Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin (recombinant or other), cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant, etc.
  • the adjuvant includes aluminum gum adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymer of acrylic acid or methacrylic acid, maleic anhydride and alkenyl (alkenyl) derivative copolymer, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314.
  • the pharmaceutically acceptable carrier includes an adjuvant
  • the adjuvant includes: (1) aluminum gel adjuvant, saponin, afridine, DDA; (2) water-in-oil emulsion , Oil-in-water emulsions, water-in-oil-in-water emulsions; or (3) polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives; and RIBI adjuvant system, Blockco- One or more of polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant Species
  • the saponin is Quil A, QS-21, GPI-0100;
  • the emulsion is SPT emulsion, MF59 emulsion, or the emulsion is formed by combining oil and emulsifier.
  • the emulsion may be based on light liquid paraffin oil, isoprenoid oil produced by olefin oligomerization (such as squalane or squalene).
  • esters can be ethoxylated, fatty alcohols and ethers of polyhydric alcohols (such as oleyl alcohol), Polyoxypropylene-polyoxyethylene block copolymer (especially Especially L121));
  • the acrylic or methacrylic polymer is a cross-linked acrylic or methacrylic polymer, especially the compound carbomer cross-linked with polyalkenyl ether or polyol of sugar, preferably Carbopol 974P , 934P and 971P;
  • the copolymer of maleic anhydride and alkenyl derivative is the copolymer of maleic anhydride and ethylene, EMA;
  • the adjuvant is a mineral oil adjuvant, which is used to prepare a water-in-oil emulsion;
  • the concentration of the adjuvant ranges from 5% to 70% V/V, preferably from 30% to 70%, more preferably 66% V/V.
  • White oil also known as paraffin oil, white oil, mineral oil, is a widely used adjuvant in inactivated vaccines, which can delay the retention time of immunogen in the body, make it continue to be released slowly, and enhance the ability of macrophages. Swallowing and sterilizing ability.
  • the avian egg drop syndrome virus tFiber protein fragment gene of the present invention can also be applied to the development of expression vectors, nucleic acid vaccines, diagnostic reagents, and other drugs related to the prevention and/or treatment of avian egg drop syndrome virus.
  • the present invention relates to a recombinant vector that can express the tFiber protein fragment encoded by the nucleotide sequence of the present invention, which is immunogenic and can produce an immune response.
  • the present invention relates to a transformant comprising the introduced recombinant vector expressing the tFiber protein fragment of the present invention.
  • the live vector is a recombinant attenuated Salmonella, a recombinant Newcastle disease virus, or a recombinant poxvirus.
  • live carrier vaccine composition of the present invention has the advantages of both inactivated vaccines and live vaccines, it can ensure the protection of laying poultry in terms of immune efficacy, and its immune efficacy is strong, and no adjuvant can be added.
  • the vaccine composition of the present invention further contains other pathogens or antigens used in combination to prepare a combination vaccine or a composite vaccine against various diseases including avian egg drop syndrome virus infection.
  • combined vaccine refers to a vaccine prepared by a virus mixture of the avian egg drop syndrome virus of the present invention and at least one different virus.
  • combined vaccine refers to a vaccine prepared from avian egg drop syndrome virus and bacteria of the present invention.
  • the avian egg drop syndrome virus of the present invention can be combined with Newcastle Disease Virus, Avian Infectious Bronchitis Virus, Avian Influenza Virus, Avian Infectious Bursal Disease Virus, Avian Adenovirus, Avian Reovirus and/or Escherichia coli , Avian bacillus paragallinarum, Mycoplasma synovialis, Mycoplasma gallisepticum mixed or combined.
  • the vaccine composition further comprises one or more of the following antigens: chicken Newcastle disease virus antigen, avian influenza virus antigen, chicken infectivity Bronchitis virus antigen, chicken infectious bursal disease virus antigen, avian adenovirus antigen, avian reovirus antigen, Escherichia coli antigen, avian paragallina antigen, mycoplasma synovial sac antigen, mycoplasma gallisepticum antigen, polycide Pasteurella antigen, Marek's virus antigen, avian encephalomyelitis virus antigen, chicken infectious laryngotracheitis virus antigen.
  • antigens chicken Newcastle disease virus antigen, avian influenza virus antigen, chicken infectivity Bronchitis virus antigen, chicken infectious bursal disease virus antigen, avian adenovirus antigen, avian reovirus antigen, Escherichia coli antigen, avian paragallina antigen, mycoplasma synovial sac antigen, mycoplasm
  • the vaccine composition further includes one or more of the following antigens: chicken Newcastle disease virus inactivated antigen, avian influenza virus inactivated antigen Live antigen, avian infectious bronchitis virus inactivated antigen, avian infectious bursal disease virus subunit antigen, avian adenovirus inactivated antigen or subunit antigen.
  • avian egg drop syndrome virus tFiber protein fragment of the present invention Using the highly expressed avian egg drop syndrome virus tFiber protein fragment of the present invention, a five-link vaccine was creatively prepared for the first time. After immunizing animals, the animal body can quickly produce a variety of antibodies against infections including avian egg drop syndrome virus. The immune effect of a variety of diseases has good prevention and control effects.
  • the chicken Newcastle disease virus inactivated antigen is the N7a strain inactivated antigen
  • the avian influenza virus inactivated antigen is the HF strain inactivated antigen.
  • the chicken infectious bronchitis virus inactivated antigen is the M41 strain inactivated antigen
  • the chicken infectious bursal disease virus subunit antigen is the chicken infectious bursal disease virus VP2 protein
  • the avian adenovirus subunit antigen is the avian adenovirus Penton protein or Fiber-2 protein.
  • the content of the avian egg drop syndrome virus tFiber protein fragment is AGP titer 1:8 to 1:32, and the chicken Newcastle disease virus inactivated antigen
  • the content is 10 8.0 ⁇ 10 9.0 EID 50 /0.1ml before inactivation
  • the content of the avian influenza virus inactivated antigen is 10 6.5 ⁇ 10 8.5 EID 50 /0.1ml before inactivation
  • the chicken infectious bronchitis virus is inactivated before antigen content of inactivated 10 6.0 ⁇ 10 7.0 EID 50 /0.1ml
  • the AGP titer is 1:2 to 1:16, and the protein content of the avian adenovirus Fiber-2 is AGP titer of 1:2 to 1:16.
  • the content of the avian egg drop syndrome virus tFiber protein fragment is AGP titer 1:8 to 1:32, and the chicken Newcastle disease virus is inactivated
  • the antigen content is 10 8.0 EID 50 /0.1ml before inactivation
  • the avian influenza virus inactivated antigen content is 10 8.0 EID 50 /0.1ml before inactivation
  • the chicken infectious bronchitis virus inactivated antigen content is inactivated
  • the VP2 protein content of the chicken infectious bursal disease virus is AGP titer 1:16,
  • the avian adenovirus Penton protein content is AGP titer 1:4, the avian gland
  • the protein content of the virus Fiber-2 is AGP titer 1:4.
  • the tFiber protein fragment of the present invention can be prepared by any method known in the art.
  • the tFiber protein fragment can be prepared by recombinantly expressing the tFiber protein fragment gene.
  • the expression system can use any known expression system, such as eukaryotic expression system, Prokaryotic expression system. Or directly synthesize tFiber protein fragment sequence.
  • Eukaryotic expression systems can include mammalian cell expression systems, yeast expression systems and insect expression systems.
  • the present invention also relates to a method for preparing the vaccine composition, wherein the method comprises: step (1) cloning the gene of the avian egg drop syndrome virus tFiber protein fragment, and combining the avian egg drop syndrome The gene of the viral tFiber protein fragment is recombined into the expression vector to obtain a recombinant expression vector containing the gene of the avian egg drop syndrome virus tFiber protein fragment; step (2) the recombination of the avian egg drop syndrome virus tFiber The recombinant expression vector of the gene of the protein fragment and the expression vector of the molecular chaperone are transformed into Escherichia coli together to express the avian egg drop syndrome virus tFiber protein fragment; step (3) the expressed avian egg drop syndrome virus tFiber protein fragment Treating with a nonionic surfactant to remove endotoxin; and step (4) mixing the endotoxin-removed avian egg drop syndrome virus tFiber protein fragment with an adju
  • the present invention found that the Fiber protein with very little surface content of the avian egg drop syndrome virus shell particles has good immunogenicity, but its expression level is low. Further research on the Fiber protein, select its fragment tFiber to prepare subunit antigens or recombine it. The live vector of its fragment gene can also produce good immune efficacy after immunization. Surprisingly, the expression level is greatly increased. The preparation method of the present invention can greatly improve the expression of tFiber protein fragments, and is economical and efficient.
  • the recombinant expression vector that is recombined with the avian egg drop syndrome virus tFiber protein fragment gene is a recombinant pET28a plasmid
  • the expression vector of the molecular chaperone in the step (2) is pG-Tf2
  • the Escherichia coli is E. coli BL21 (DE3)
  • the nonionic surfactant in the step (3) is Triton X- 114.
  • the invention also relates to the application of the vaccine composition in the preparation of medicines for preventing and/or treating avian egg drop syndrome.
  • the invention also relates to the application of the vaccine composition in the preparation of a medicine for preventing and/or treating avian egg drop syndrome virus infection.
  • the subject of administration of the medicament for preventing and/or treating avian egg drop syndrome virus infection of the present invention includes chickens or ducks.
  • prevention and/or treatment when referring to avian egg drop syndrome virus infection refers to inhibiting the replication of the avian egg drop syndrome virus, inhibiting the spread of the avian egg drop syndrome virus, or preventing the avian egg drop syndrome virus in its host Settle in the body and relieve the symptoms of diseases or symptoms of avian egg drop syndrome virus infection. If the viral load is reduced, the disease is reduced, and/or food intake and/or growth is increased, then the treatment can be considered to have achieved a therapeutic effect.
  • the chemical reagents used in the examples of the present invention are all analytically pure and purchased from Sinopharm Group.
  • the experimental methods of the present invention are all conventional methods; the biological materials, unless otherwise specified, can be obtained from commercial sources.
  • the viral DNA extraction kit take 0.2ml infected with avian egg drop syndrome virus HX strain (Egg Drop Syndrome Virus, Strain HX), the preservation number is CCTCC NO: V201942, and the preservation date is 2019 On June 19, 2010, the SPF duck embryo allantoic fluid stored at Wuhan ⁇ Wuhan University, China) was placed in a sterile 1.5ml centrifuge tube, 0.4ml VB was added to the sample solution, vortexed to mix, and allowed to stand at room temperature for 10 minutes. Add 0.45ml AD buffer to the sample solution and mix vigorously.
  • avian egg drop syndrome virus HX strain Egg Drop Syndrome Virus, Strain HX
  • the preservation number is CCTCC NO: V201942
  • the preservation date 2019 On June 19, 2010, the SPF duck embryo allantoic fluid stored at Wuhan ⁇ Wuhan University, China
  • the VB column into a 2ml collection tube, take 0.6ml of the mixed solution into the VB column, centrifuge at 14000g for 1 minute, add the remaining mixed solution to the VB column, centrifuge at 14000g for 1 minute, discard the 2ml collection tube, and place the VB column Put into a new 2ml collection tube, add 0.4ml W1buffer, centrifuge at 14000g for 30 seconds, add 0.6ml Wash buffer to the VB column, centrifuge at 14000g for 30 seconds, leave it for 3 minutes, put the VB column into a new sterile 1.5ml centrifuge tube Add 50 ⁇ l RNase free water to the center of the membrane, let it stand for 3 minutes, and centrifuge at 14000g for 1 minute. The centrifuged liquid is the virus DNA solution.
  • Oligonucleotide primers were synthesized based on the conserved region sequences at the 5'and 3'ends of the Fiber protein gene, and PCR was performed. The primer sequences are shown in Table 1.
  • the PCR product was sent to Invitrogen for sequencing, and the Fiber protein gene was codon optimized according to the sequencing results.
  • the optimized Fiber protein gene sequence is shown in SEQ ID NO.1.
  • the Fiber gene was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for full sequence synthesis, and the pUC57-EDS-Fiber plasmid containing the optimized sequence of the Fiber gene codon was obtained.
  • the PCR product was sent to Invitrogen for sequencing.
  • the gene sequence of the tFiber protein fragment is shown in SEQ ID NO.2.
  • the DNA gel recovery kit is used to purify the DNA fragments.
  • the DNA fragments obtained are the same after digestion with Nco I and Xho I.
  • the pET-28a(+) plasmid treated with double enzyme digestion was ligated, the ligation product was transformed into E. coli DH5 ⁇ competent cells, positive clones were screened, plasmid extraction kit was used to extract the plasmid and double enzyme digestion was performed, and the correct plasmid was identified by enzyme digestion. Sequencing analysis showed that the correctly sequenced recombinant plasmid was named pET28a-EDS-tFiber.
  • the pET28a-EDS-tFiber plasmid was transformed into Escherichia coli BL21 (DE3), and a single clone was picked and cultured overnight in an LB medium containing 100 ⁇ g/ml kanamycin to form the pET28a-EDS-tFiber expression strain.
  • IPTG Isopropyl- ⁇ -D-thiogalactopyranoside
  • Example 3 E. coli expression of tFiber protein fragments endotoxin elimination
  • Triton X-114 can eliminate the residual endotoxin in the recombinant protein, and has no effect on the immunogenicity of tFiber protein fragments.
  • Example 5 The immunogenicity test of avian egg drop syndrome virus tFiber protein fragment subunit vaccine on SPF chickens
  • the color of the eggshell becomes lighter, soft-shelled eggs, shellless eggs, deformed eggs, etc.; the egg-laying rate in the 6th week after the challenge is about 69%, which has not returned to normal levels.
  • Syndrome virus tFiber protein fragment subunit vaccine immunized chickens all produced higher HI antibody titers.
  • the tFiber protein fragment antigen of the present invention has good immunogenicity and can provide effective immune protection to chickens with low content.
  • Example 7 The immunogenicity test of avian egg drop syndrome virus tFiber protein fragment subunit vaccine on cherry valley ducks
  • the pET28a-EDS-Fiber expression strain was constructed referring to the method in Example 1, and the full-length Fiber protein was prepared according to the method in Example 2.
  • the HA titer of the Fiber protein was 1:512
  • the AGP titer was 1:64
  • the endotoxin content was 0.51 ⁇ 10 5 EU/ml.
  • the endotoxin removal was carried out according to the method in Example 3. After determination, the AGP titer of the Fiber protein reached 1:64, and the endotoxin content decreased to 0.009 ⁇ 10 5 EU/ml.
  • the purified Fiber protein was slowly added to the mineral oil adjuvant, while the motor was turned on, stirred at 17,500 r/min for 5 minutes, and 1% thimerosal solution was added before the stirring to make the final concentration 0.01%. See Table 7 for the specific ratio.
  • Example 9 Comparative test of immunity duration of avian egg drop syndrome virus subunit vaccine
  • the HI antibody titer of the 15th control group was 0 after immunization; the 13th immunization group produced higher HI antibody titer to the immunized chickens, and maintained a higher HI for 6 months after immunization.
  • Antibody titer Although the 14th immunization group also produced higher HI antibody titers for the immunized chickens, the overall level was lower than that of the 13th group. It shows that the avian egg drop syndrome virus tFiber protein fragment subunit vaccine provided by the present invention has better immunogenicity than the avian egg drop syndrome virus Fiber full-length protein subunit vaccine, which can enable chickens to maintain HI for a longer period of time.
  • the antibody titer can provide long-term and effective immune protection to chickens.
  • N7a strain Newcastle Disease Virus (genotype VII), strain N7a) (preserved at the China Type Culture Collection, the preservation number is CCTCC NO: V201545, the preservation date is October 19, 2015 , The preservation address is Wuhan ⁇ Wuhan University, China, published in Chinese Patent Application CN107281479A), inoculate susceptible chicken embryos of 10 to 11 days old with sterile normal saline (10 -4 or 10 -5), 0.1ml per embryo After inoculation, keep at 37°C and continue incubating.
  • H9 subtype avian influenza virus HF strain (Avian Influenza Virus (Subtype H9), Strain HF), the preservation number is CCTCC NO: V201941, and the preservation date is June 19, 2019 , The preservation address is Wuhan, Wuhan University, China)
  • the virus seed diluted with sterile normal saline to 10 -3 (take 0.1ml of the virus liquid and add it to 0.9ml of sterile normal saline, shake and mix, and then dilute 2 times in sequence), 10-day-old susceptible chicken embryos were inoculated through the allantoic cavity (incubated with SPF eggs purchased from Beijing Meriya Weitong Experimental Animal Technology Co., Ltd.), 0.1 ml per embryo (containing 10 5 EID 50 ).
  • the pinhole After inoculation, the pinhole is sealed, and the incubation is continued at 36 ⁇ 37°C without turning the eggs. After 96 hours, take it out, put the air chamber upright, and cool it at 2-8°C for 12-24 hours.
  • the embryo fluid is harvested from the cooled chicken embryos. The virus content was determined to be 10 8.5 EID 50 /0.1ml. Add a formaldehyde solution (v/v) with a final concentration of 0.1%, inactivate at 37°C, stir once every 4-6h during this period, inactivate for 24h, and use after the inactivation is complete.
  • IBDV virus RNA was extracted from SPF chicken bursal bursa infected with the super virulent Chengdu strain of infectious bursal disease virus, and reverse transcription was performed with random primers.
  • Oligonucleotide primers were synthesized according to the sequences of the conserved regions at the 5'and 3'ends of the VP2 gene. The sequence of the synthetic oligonucleotide primers is shown in Table 9, and PCR amplification was carried out.
  • the agarose gel recovery kit was used to recover the oligonucleotide primers. , Store at -20°C.
  • VP2-EcoR1-F CCGGAATTCATGACAAACCTGCAAGATCAAAC
  • VP2-Sal1-R ACGCGTCGACTTACCTTAGGGCCCGGATTATGT
  • the culture tank to ventilate the culture, and fill in 70% medium and peanut oil defoamer according to the volume. After sterilization, inoculate the pColdIII-VP2/E.Coli BL21(DE3) strain seed solution at 2% ⁇ 4% of the amount of the culture medium, cultivate at 37°C, and add 0.2mol/L ⁇ after the OD 600 value of the bacterial solution reaches 0.6 ⁇ 1.0 -Lactose, make the final concentration reach 0.02mol/L, and continue to incubate for 5-8h.
  • the cells were collected by centrifugation, resuspended, broken by ultrasound, and centrifuged to collect the supernatant. After precipitation with ammonium sulfate, the VP2 protein solution was collected.
  • Example 3 Refer to the method in Example 3 for endotoxin elimination. After determination, the endotoxin content decreased to 0.3 EU/ml.
  • the preservation number is: CCTCC NO: V201609
  • the preservation unit is the Chinese typical culture The preservation center
  • the preservation address is Wuhan ⁇ Wuhan University, China
  • the preservation time is February 29, 2016.
  • Oligonucleotide primers were synthesized according to the conserved region sequence at the 5'and 3'ends of the Fiber-2 protein gene. The synthetic oligonucleotide primer sequence is shown in Table 10, and PCR amplification was carried out and recovered by agarose gel. The kit is recovered and stored at -20°C.
  • Fiber-2-F CTCCGGGCCCCTAAAAG Fiber-2-R CGGGACGGAGGCCGC
  • the Fiber-2 protein gene was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for full sequence synthesis, and respectively ligated to the pET28a plasmid.
  • the ligated plasmid was transformed into Escherichia coli BL21 (DE3), a single clone was picked and cultured overnight in LB medium containing 100 ⁇ g kanamycin, and the plasmid was extracted and sequenced and analyzed.
  • the positive clone was the pET28a-FADV-Fiber-2 expression strain .
  • the pET28a-FADV-Fiber-2/E.Coli BL21(DE3) strain prepared in Example 1 was inoculated into LB medium containing 50-100 ⁇ g/ml kanamycin, and the inoculum amount was 1% (V/ V), shaking culture at 37°C. When the OD 600 value reaches 0.4 to 0.6, place it at 28°C for 30 minutes. Isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) was added to make the final concentration 1.0mM, and cultured with shaking at 28°C for 24 hours.
  • IPTG Isopropyl- ⁇ -D-thiogalactopyranoside
  • the avian egg drop syndrome virus tFiber protein fragment antigen purified in Example 3 was respectively compared with the chicken Newcastle disease antigen prepared in Example 10, the avian influenza antigen prepared in Example 11, and the chicken infectious bronchitis antigen prepared in Example 12.
  • the infectious bursal antigen prepared in 13 and the avian adenovirus antigen prepared in Example 14 were mixed in proportions, added to the mineral oil adjuvant, and the motor was turned on at 17500r/min for 5 minutes, and 1% thimerosal solution was added before the stirring was terminated. , Its final concentration is 0.01%. See Tables 11, 12, 13, and 14 for specific ratios.
  • Vaccine 10 Vaccine 11 Vaccine 12 Vaccine 13 tFiber protein fragment (AGP titer) 1:32 1:8 1:16 1:32 N7a strain antigen (EID 50 /0.1ml) 10 8.0 10 8.0 10 8.0 10 8.0
  • Vaccine 19 Vaccine 20 tFiber protein fragment (AGP titer) 1:16 1:16 N7a strain antigen (EID 50 /0.1ml) 10 8.0 10 8.0 HF strain antigen (EID 50 /0.1ml) 10 8.0 10 8.0 M41 strain antigen (EID 50 /0.1ml) 10 6.0 10 6.0 VP2 protein (AGP titer) 1:16 — Fiber-2 protein (AGP titer) — 1:4 Mineral oil adjuvant (V/V%) 66% 66% 66% 66%
  • the vaccine 5 to vaccine 20 immunized group produced higher HI antibody titer 21 days after immunization, which can effectively protect chickens from the occurrence of egg-laying syndrome. It shows that the tFiber protein fragment of the avian egg drop syndrome virus provided by the present invention can provide complete protection to chickens by using the oil emulsion combined vaccine prepared as an antigen.
  • a total of 130 SPF chickens aged 21 days were divided into 13 groups, each with 10 chickens.
  • Groups 33 to 44 were subcutaneously injected into the neck to immunize the vaccine prepared in Example 15 5, vaccine 10 to vaccine 20, 20 ⁇ l/bird ;
  • the 45th group was injected with 20 ⁇ l of saline subcutaneously as a challenge control. All experimental chickens were raised in isolation. 21 days after immunization, the immunized chickens from groups 33 to 44, together with the 45th challenge control chickens, were collected for blood and serum separation. Detect the Newcastle disease virus HI antibody, and at the same time use the virulent HN1101 strain of Newcastle disease virus to challenge by intramuscular injection, observe for 14 days, and record the number of morbidity, death and protection. The results are shown in Table 16.
  • HI antibody is determined as the geometric mean of immunized chicken antibodies.
  • the results show that the vaccine 5, vaccine 10-20 vaccine group can produce higher Newcastle disease antibodies 21 days after immunization, and the immunization group can completely protect against the virulent attack compared with the control. It shows that the oil emulsion combined vaccine prepared with the N7a strain of Newcastle disease virus liquid provided by the present invention as an antigen can provide complete protection to chickens.
  • Eighty 21-day-old SPF chickens were divided into 8 groups, each with 10 chickens.
  • Groups 46 to 52 were injected subcutaneously into the neck to immunize vaccine 6, vaccine 10, vaccine 14, and vaccine 17 ⁇ Vaccine 20, 0.3ml/mouse;
  • group 53 was injected subcutaneously with 0.3ml saline as a challenge control. All the experimental chickens were raised in isolation, 21 days after immunization, the 46th to 52th groups of immunized chickens, together with the 53rd group of control chickens, were blood collected and serum separated. Detect the antibody titer of H9 subtype avian influenza HI, and challenge with intravenous injection of HF strain virus liquid at the same time, 0.2ml each (containing 10 7.0 EID 50 ).
  • a cloaca swab was collected. After treatment, the allantoic cavity was inoculated with 5 10--11-day-old SPF chicken embryos, and incubated for 5 days. Both dead and live embryos should be tested for red blood cell agglutination in chicken embryos. As long as one of the 5 chicken embryos inoculated with each swab sample has an agglutination value of not less than 1:16 (micro method), it can be judged as positive for virus isolation. For samples that are negative for virus isolation, they should be blindly transmitted once before being judged. At least 9 chickens in the immunization group should be negative for virus isolation; at least 4 chickens in the control group should be positive for virus isolation. The results are shown in Table 17.
  • HI antibody is determined as the geometric mean of immunized chicken antibodies.
  • vaccine 6, vaccine 10, vaccine 14, and vaccine 17 to vaccine 20 can all produce higher avian influenza antibodies 21 days after immunization, and the immunized group can completely protect against the virulent attack compared with the control. It shows that the H9 subtype avian influenza virus liquid provided by the present invention can provide complete protection to chickens by using the oil emulsion combined vaccine prepared as an antigen.
  • the geometric mean of HI antibody titer in the second immunized serum of the immunization group was not less than 4 times the geometric mean of the HI antibody titer in the first immunized serum, and the geometric mean of the serum HI antibody titer of the non-immunized control group was not higher than 1:8 (minimum law).
  • a chicken infectious bronchitis M41 virulent toxin was used to challenge 10 3.0 EID 50 nasal drops per feather for a challenge experiment. The results are shown in Table 18.
  • each of the 62 to 67 groups was vaccinated with a 100-fold dilution of the infectious bursal disease virus BC6-85 ((CVCC AV7 strain)) purchased from China Veterinary Drug Inspection Institute) strain virus liquid 0.1ml (actual poison content ⁇ 100 BID).
  • the clinical manifestations of the chickens were observed every day, and the number of morbid and dead chickens was recorded. After 72 to 96 hours, the surviving chickens were culled, and the chickens were dissected one by one to observe the disease of the bursa of Fabricius. At least 8 immunized chickens should be normal without bursal disease; control chickens should have at least 4 chickens with disease and obvious bursal disease (such as pectoral or leg muscle strip hemorrhage, enlargement or atrophy of bursal disease, yellowing , There are more than one type of lesions such as jelly-like secretions). The results are shown in Table 19.
  • vaccine 8 vaccine 12, vaccine 15, vaccine 17, and vaccine 19 can completely protect chickens from the virulent infectious bursal disease attack 21 days after immunization.
  • mice 60 SPF chickens aged 21 days were divided into 6 groups, each with 10 chickens.
  • Groups 68 to 72 were immunized with vaccine 9, vaccine 13, vaccine 16, vaccine 18, and vaccine prepared in Example 15 by subcutaneous injection in the neck.
  • Vaccine 20, 0.3ml/mouse; group 73 was injected subcutaneously with 0.3ml saline as a challenge control. All test chickens were reared in isolation, 21 days after immunization, they were challenged by intramuscular injection with FAV-HN strain virus liquid, and observed for 14 days, and recorded the number of morbidity, death and protection. The results are shown in Table 20.
  • the avian adenovirus antigen provided by the present invention can provide complete protection to chickens by using the oil emulsion combined vaccine prepared as the antigen.
  • the avian egg drop syndrome virus combined vaccine provided by the present invention can resist the invasion of related pathogens, shows good immunogenicity, and can effectively control the prevalence of avian egg drop syndrome virus-related diseases in my country.
  • coli has made the development of polyvalent vaccines against major poultry diseases a reality; prepared poultry The subunit antigen of the egg drop syndrome virus tFiber protein fragment, and the chicken Newcastle disease antigen, and the avian influenza antigen, and the chicken infectious bronchitis antigen, and the infectious bursa subunit antigen five vaccines, and the avian egg drop syndrome virus
  • the subunit antigen of the tFiber protein fragment, the chicken Newcastle disease antigen, the avian influenza antigen, the chicken infectious bronchitis antigen, and the avian adenovirus subunit antigen quinqueous vaccine achieves the first simultaneous immunization of five antigens, omitting complicated immunization procedures .

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Abstract

提供了SEQ ID NO.2所示核苷酸序列或其简并序列所编码的禽减蛋综合征病毒tFiber蛋白片段，以及该蛋白片段制备的疫苗组合物，该片段具有良好的免疫原性，并能与多种抗原制备联苗，对鸡和鸭群产生完全保护。

Description

禽减蛋综合征病毒tFiber蛋白片段、其制备的疫苗组合物、制备方法和应用 技术领域
本发明涉及一种禽减蛋综合征病毒tFiber蛋白片段、其制备的疫苗组合物、制备方法和应用，属于生物医药领域。
背景技术
禽减蛋综合征病毒(Egg Drop Syndrome Virus,EDSV)属于禽腺病毒Ⅲ群，该病毒感染家禽后无明显临床症状，或可见轻微程度腹泻，常发生于产蛋高峰期，能够使蛋鸡产软壳、薄壳、无壳蛋，产蛋率严重下降，造成严重的经济损失。
EDSV具有典型的腺病毒形态，没有囊膜，具有红细胞凝集活性，在禽输卵管中增殖。病毒基因组为约33kb的线状双股DNA，病毒颗粒由结构蛋白构成，核衣壳直径70～80nm，呈二十面体对称，DNA被包于衣壳内。核衣壳由252个壳粒组成，其中240个是六邻体(Hexon蛋白)，构成二十面体的20个面以及棱的大部分。这些壳粒呈棱柱状，宽7nm，长11nm。另外12个是五邻体(Penton蛋白)，分别位于二十面体的12个顶角上。每个Penton蛋白有一条纤维突起(Fiber蛋白)。
禽减蛋综合征是当前世界范围内严重危害家禽养殖业发展的重要疾病之一。在各种防控措施中，疫苗免疫仍为最重要的措施。目前养禽业中普遍使用的EDS灭活疫苗是基于鸭胚增殖的病毒灭活后与矿物油佐剂乳化而成的疫苗。但是，由于禽减蛋综合征病毒在鸭胚上培养难以获得高滴度的病毒，导致制备的疫苗往往难以提供理想的免疫效果；此外，该病毒抗原的生产方式完全依赖于鸭种蛋，在禽流感等传染病发生时，鸭种蛋供应困难，严重影响禽减蛋综合征的防控。另外全病毒疫苗生产过程中还存在病毒灭活不完全造成的生物安全方面的风险。
亚单位疫苗是近几年发展起来的一种比较切实可行的新型基因工程疫苗，禽减蛋综合征亚单位疫苗研究以Hexon(240/252)为主要研究对象，然而一直以来，免疫效率低下，未开发成产品。现有技术中至今未有禽减蛋综合征亚单位疫苗上市。因此，临床上急需开发出免疫效果好的亚单位疫苗组合物，能够有效防止该病的流行，且能避免鸭种蛋供应波动的影响。
针对多种疫病的多联疫苗因免疫一次即可产生针对多种病原抗体，是疫苗发展的一个方向，然而实际中，多联疫苗的制备面临着多种限制：1、抗原浓缩技术，联用的抗原越多，需要浓缩的倍数越高，针对当前多数禽用疫苗采用禽胚来生产抗原，高倍浓缩技术制约了其的研制和发展；2、而使用亚单位抗原替代全菌或全病毒抗原时，表达亚单位抗原产生的内毒素，随着联用的抗原越多，叠加的内毒素越多，当疫苗中有两种或两种以上的亚单位抗原，内毒素的含量很高，已经不适合制备联苗。因此，如何克服上述困难制备禽类主要疫病的多联苗是临床实践中亟需解决的问题。
发明内容
为解决现有技术的不足，本发明提供一种禽减蛋综合征病毒免疫原性蛋白、其制备的疫苗组合物及该疫苗组合物的制备方法以及应用，该疫苗组合物能够有效预防和/或治疗禽减蛋综合征病毒的感染。
本发明涉及一种禽减蛋综合征病毒tFiber蛋白片段，其中，所述禽减蛋综合征病毒tFiber蛋白片段的基因如SEQ ID NO.2或其简并序列所示。
本发明还涉及一种疫苗组合物，其中，所述疫苗组合物包括免疫量的所述的禽减蛋综合征病毒tFiber蛋白片段或免疫量的重组有所述的禽减蛋综合征病毒tFiber蛋白片段的基因的活载体以及药学上可接受的载体。
本发明首次采用禽减蛋综合征病毒tFiber蛋白片段，选取的禽减蛋综合征病毒蛋白片段基因高效表达后，制备疫苗组合物，可预防和/或 治疗禽减蛋综合征疫情，并且含有该蛋白的疫苗组合物免疫动物后能使动物机体快速产生抗体，对当前流行的禽减蛋综合征病毒单独或混合感染有良好的预防和控制效果，生物安全性好。
本发明禽减蛋综合征病毒tFiber蛋白片段制备的疫苗组合物，其免疫原性好，能对鸡和鸭产生完全保护，在临床应用上能对禽减蛋综合征病毒的感染进行预防和/或治疗。
本发明还涉及一种制备所述疫苗组合物的方法，其中，所述方法包括：步骤(1)克隆所述禽减蛋综合征病毒tFiber蛋白片段基因，并将所述禽减蛋综合征病毒tFiber蛋白片段基因重组到表达载体以获得重组有所述禽减蛋综合征病毒tFiber蛋白片段基因的重组表达载体；步骤(2)将所述重组有所述禽减蛋综合征病毒tFiber蛋白片段基因的重组表达载体和分子伴侣的表达载体一同转化大肠杆菌，表达所述禽减蛋综合征病毒tFiber蛋白片段；步骤(3)将所述表达的禽减蛋综合征病毒tFiber蛋白片段使用非离子表面活性剂处理除去内毒素；以及步骤(4)将所述去除内毒素的禽减蛋综合征病毒tFiber蛋白片段与佐剂混匀，得到所述疫苗组合物。
本发明还涉及所述的疫苗组合物在制备预防和/或治疗禽减蛋综合征病毒感染的药物中的应用。
具体实施方式
以下，对本发明的实施方式进行说明。
术语“禽减蛋综合征病毒”(Egg Drop Syndrome Virus,EDSV)属于禽腺病毒Ⅲ群，基因组为双股DNA。所引发的临床症状包括使蛋鸡产软壳、薄壳、无壳蛋，产蛋率严重下降。病理变化以卵巢静止不发育和输卵管萎缩为特征。
本发明涉及一种禽减蛋综合征病毒tFiber蛋白片段，其中，所述禽减蛋综合征病毒tFiber蛋白片段的基因如SEQ ID NO.2或其简并序列所示。
本发明的禽减蛋综合征病毒tFiber蛋白片段具有良好的免疫原性， 以低含量免疫即能产生良好的免疫保护，其免疫效果较禽减蛋综合征病毒Fiber全长蛋白更优。
本发明还涉及一种疫苗组合物，其中，所述疫苗组合物包括免疫量的所述的禽减蛋综合征病毒tFiber蛋白片段或免疫量的重组有所述的禽减蛋综合征病毒tFiber蛋白片段的基因的活载体以及药学上可接受的载体；所述禽减蛋综合征病毒tFiber蛋白片段的基因如SEQ ID NO.2或其简并序列所示。
本发明的疫苗组合物中免疫原性成分，禽减蛋综合征病毒tFiber蛋白片段当使用很低的免疫量时，仍能保证良好的免疫效果。
本发明所用术语“疫苗组合物”指含有禽减蛋综合征病毒免疫原性的药物组合物，该药物组合物可诱发、刺激或增强鸡、鸭只针对禽减蛋综合征病毒的免疫反应。
术语“免疫量”应当理解为“免疫有效量”，又称免疫保护量或产生免疫应答的有效量，为可在接受者体内有效诱导免疫应答的抗原量，该量足以预防或改善疾病的体征或症状，包括不利的健康影响或其并发症。所述免疫应答可能足以用于诊断目的或其它试验，或可能适合用于预防疾病的征兆或症状，包括由病原体引起的感染所造成的不利的健康结果或其并发症。体液免疫力或由细胞介导的免疫力或此二者均可被诱导。动物对免疫原性组合物的免疫应答可通过例如测量抗体效价、淋巴细胞增殖分析而间接评估，或在以野生型毒株攻击后通过监测征兆或症状来直接评估，而该由疫苗提供的保护性免疫力可通过测量例如受试者的临床征兆如死亡率、发病率的减少、温度数值、受试者总体生理状况及总体健康和表现来评估。所述免疫应答可包括但不限于诱导细胞性和/或体液免疫力。
术语“禽减蛋综合征病毒抗原”是指至少含有一种禽减蛋综合征病毒抗原形式的任何组合物，所述禽减蛋综合征病毒抗原可诱导、刺激或能抵抗禽减蛋综合征病毒感染的免疫应答，所述抗原形式包括但不限于灭活的、减毒的或亚单位的抗原。
本发明所述的禽减蛋综合征病毒tFiber蛋白片段抗原可以是重组表 达的tFiber蛋白片段亚单位抗原，其表达体系可以为原核表达系统、真核表达系统，也可以是通过人工合成的合成肽抗原。
“亚单位抗原”指的是利用基因工程方法将病原体的保护性抗原基因克隆到原核或真核表达系统中，使其高效表达而制成的抗原。它比全病毒抗原引起副反应的可能性小。
“合成肽抗原”指的是一种仅含免疫决定簇组分的小肽,即用人工方法按天然蛋白质的氨基酸顺序合成保护性短肽,与载体连接后加佐剂所制成的抗原。
“活载体”指的是非致病微生物通过基因工程的方法使之携带并表达某种抗原或抗原决定簇的基因，产生免疫原性，非致病微生物可以是细菌和病毒，病毒活载体常作为载体的病毒包括痘苗病毒、禽痘病毒、火鸡疱疹病毒、腺病毒、伪狂犬病毒、反转录病毒、慢病毒；细菌活载体可以包括减毒沙门菌、卡介苗、减毒单核细胞李氏杆菌、减毒霍乱弧菌、减毒志贺氏菌、乳酸球菌、芽胚乳酸杆菌、高氏链球菌。
作为本发明的一种实施方式，在本发明的疫苗组合物中，所述的禽减蛋综合征病毒tFiber蛋白片段为SEQ ID NO.2所示的核苷酸序列编码的蛋白。
作为本发明的一种实施方式，在本发明的疫苗组合物中，所述的禽减蛋综合征病毒tFiber蛋白片段的基因具有SEQ ID NO.2所示的核苷酸序列或其简并序列。
作为本发明的一种实施方式，本发明所述疫苗组合物中，所述禽减蛋综合征病毒tFiber蛋白片段含量为AGP效价≥1:8。
作为本发明的一种优选实施方式，本发明所述疫苗组合物中，所述禽减蛋综合征病毒tFiber蛋白片段含量为AGP效价1:8～1:32。
“AGP效价”又称为“琼脂扩散试验效价(Agar Gel Precipitation(test)titer)”，琼脂扩散试验是一种沉淀反应，抗原、抗体在含有电解质的琼脂凝胶中可以向四周自由扩散，二者相互结合，在最适比例处出现沉淀线，出现沉淀线的最高稀释比例即为效价值，即为标定的抗原或抗体含量。
术语“药学上可接受的载体”是指在本发明疫苗组合物中除禽减蛋综合征病毒抗原之外的其它所有成分，不刺激机体不阻碍使用化合物的生物学活性和特性的载体或者稀释剂，优选为佐剂。
本发明疫苗组合物还可以进一步将其它的试剂加入到本发明的组合物。
作为本发明的一种实施方式，所述的药学上可接受的载体包括药物，免疫刺激剂、抗氧化剂、表面活性剂、着色剂、挥发性油、缓冲剂、分散剂、推进剂和防腐剂；所述免疫刺激剂包括α-干扰素、β-干扰素、γ-干扰素、粒细胞巨噬细胞集落刺激因子(GM-CSF)、巨噬细胞集落刺激因子(M-CSF)和白介素2(IL2)。
优选地，免疫刺激剂包括α-干扰素、β-干扰素、γ-干扰素、粒细胞巨噬细胞集落刺激因子(GM-CSF)、巨噬细胞集落刺激因子(M-CSF)和白介素2(IL2)。
为了制备这样的组合物，可以使用本领域公知的方法。
作为本发明的一种实施方式，本发明所述疫苗组合物中，所述药学上可接受的载体为佐剂，所述佐剂包括：(1)矿物油、铝胶佐剂、皂苷、阿夫立定、DDA；(2)油包水乳剂、水包油乳剂、水包油包水乳剂；或(3)丙烯酸或甲基丙烯酸的聚合物、顺丁烯二酸酐和链烯基衍生物的共聚物；以及RIBI佐剂系统、Block co-polymer、SAF-M、单磷酰脂质A、Avridine脂质-胺佐剂、大肠杆菌不耐热肠毒素、霍乱毒素、IMS 1314、胞壁酰二肽、Montanide ISA 206、Gel佐剂中的一种或几种；优选地，皂苷为Quil A、QS-21、GPI-0100；
所述佐剂含量为5％-70％V/V，所述佐剂含量优选30％到70％，更优选66％V/V。
术语“佐剂”可包括铝胶佐剂；皂苷(saponin)，如Quil A、QS-21(Cambridge Biotech Incorporation,Cambridge MA)、GPI-0100(Galenica Pharmaceuticals Incorporation，Birmingham AL)；油包水乳剂；水包油乳剂；水包油包水乳剂；丙烯酸或甲基丙烯酸的聚合物；顺丁烯二酸酐和链烯基(alkenyl)衍生物的共聚物选出的化合物。术语“乳剂”可尤其 基于轻液体石蜡油(European Pharmacopea类型)；因烯烃寡聚产生的类异戊二烯油(isoprenoid oil)，如角鲨烷(squalane)或角鲨烯油(squalene oil)，尤其异丁烯或葵烯；酸或醇的含线性烷基的酯，更尤其植物油、油酸乙酯、丙二醇二-(辛酸酯/葵酸酯)、甘油三-(辛酸酯/葵酸酯)或丙二醇二油酸酯；支链脂肪酸或醇的酯，尤其异硬脂酸酯。油与乳化剂组合使用以便形成乳剂。乳化剂优选非离子表面活性剂，尤其山梨聚糖的酯、二缩甘露醇(mannide)的酯(如无水甘露醇油酸酯)、脂肪族二元醇(glycol)的酯、聚甘油(polyglycerol)的酯、丙二醇的酯以及油酸的酯、异硬脂酸的酯、蓖麻油酸的酯或羟基硬脂酸的酯，它们任选乙氧基化，还有聚氧丙烯-聚氧乙烯嵌段共聚物，尤其Pluronic产品，特别是L121。参见Hunter等编写的《The theory and practical application of adjuvants》(Ed.by DES Stewart-Tull,John Wiley and Sons,New York,1995:51-94)和Todd等编写的《Vaccine》(1997,15:564-570)。例如，可使用Powell M和Newman M编写的《Vaccine design,the Subunit and adiuvant approach》(Plenum Press,1995)第147页描述的SPT乳剂及第183页描述的MF59乳剂。术语“丙烯酸或甲基丙烯酸的聚合物”优选为交联的丙烯酸或甲基丙烯酸聚合物，尤其是与糖(sugar)的聚链烯基醚或聚醇交联，这些化合物已知被称为卡波姆(Carbomer，商品名Carbopol)(Phameuropa,1996,8(2))。本领域技术人员还可参见美国专利US2909462，其描述了这类丙烯酸聚合物，其与聚羟基化的化合物交联，所述化合物具有至少3个羟基，优选不超过8个，其中至少3个羟基的氢原子被具有至少2个碳原子的不饱和脂烃基(aliphatic radical)取代。优选的基团是那些含有2-4个碳原子的基团，例如乙烯基、烯丙基和其它烯属不饱和基团(ethylenically unsaturated group)。所述不饱和基团自身可包含其它取代基，如甲基。这些产品以卡波普的名义出售，(BF Goodrich,Ohio,USA)特别合适。它们与烯丙基蔗糖或与烯丙基季戊四醇(allyl pentaerythritol)交联。这其中可提及卡波普974P、934P和971P，最优选使用卡波普971P。术语“顺丁烯二酸酐和链烯基衍生物的共聚物”也可考虑顺丁烯二酸酐与乙烯的共聚物EMA(Monsanto)， 这些聚合物在水中溶解产生酸性溶液，经中和，优选中和至生理pH，以便产生佐剂溶液，能向其中掺入免疫原性、致免疫性或疫苗性组合物本身。术语“佐剂”还包括，但不限于，RIBI佐剂系统(Ribi Incorporation)、Block co-polymer(CytRx,Atlanta GA)、SAF-M(Chiron,Emeryville CA)、单磷酰脂质A(monophosphoryl lipid A)、Avridine脂质-胺佐剂、大肠杆菌不耐热肠毒素(重组或其它)、霍乱毒素、IMS 1314、胞壁酰二肽、Gel佐剂等。优选地，所述佐剂包括铝胶佐剂、皂苷、油包水乳剂、水包油乳剂、水包油包水乳剂、丙烯酸或甲基丙烯酸的聚合物、顺丁烯二酸酐和链烯基(alkenyl)衍生物的共聚物、RIBI佐剂系统、Block co-polymer、SAF-M、单磷酰脂质A、Avridine脂质-胺佐剂、大肠杆菌不耐热肠毒素、霍乱毒素、IMS 1314、胞壁酰二肽或Gel佐剂中的一种或几种。
作为本发明的一种实施方式，所述药学上可接受的载体包括佐剂，所述佐剂包括：(1)铝胶佐剂、皂苷、阿夫立定、DDA；(2)油包水乳剂、水包油乳剂、水包油包水乳剂；或(3)丙烯酸或甲基丙烯酸的聚合物、顺丁烯二酸酐和链烯基衍生物的共聚物；以及RIBI佐剂系统、Block co-polymer、SAF-M、单磷酰脂质A、Avridine脂质-胺佐剂、大肠杆菌不耐热肠毒素、霍乱毒素、IMS 1314、胞壁酰二肽、Gel佐剂中的一种或几种；
优选地，皂苷为Quil A、QS-21、GPI-0100；
优选地，乳剂为SPT乳剂、MF59乳剂，或乳剂由油与乳化剂组合形成，乳剂可基于轻液体石蜡油、因烯烃寡聚产生的类异戊二烯油(如角鲨烷或角鲨烯油，烯烃，特别是异丁烯或癸烯低聚化产生的油)、酸或醇的含线性烷基的酯(更尤其植物油、油酸乙酯、丙二醇二-(辛酸酯/葵酸酯)、甘油三-(辛酸酯/葵酸酯)或丙二醇二油酸酯)、支链脂肪酸或醇的酯(尤其异硬脂酸酯)；乳化剂为非离子表面活性剂(尤其聚氧乙烯化脂肪酸(例如油酸)的酯、山梨聚糖的酯、二缩甘露醇的酯(如无水甘露醇油酸酯)、脂肪族二元醇的酯、甘油的酯、聚甘油的酯、丙二醇的酯以及油酸的酯、异硬脂酸的酯、蓖麻油酸的酯或羟基硬脂酸 的酯，上述酯可经乙氧基化、脂肪醇和多元醇(例如油醇)的醚、聚氧丙烯-聚氧乙烯嵌段共聚物(尤其
Figure PCTCN2020101961-appb-000001
特别是L121))；
优选地，丙烯酸或甲基丙烯酸的聚合物为交联的丙烯酸或甲基丙烯酸聚合物，尤其是与糖的聚链烯基醚或聚醇交联的化合物卡波姆、优选为卡波普974P、934P和971P；
优选地，顺丁烯二酸酐和链烯基衍生物的共聚物为顺丁烯二酸酐与乙烯的共聚物EMA；
优选地，所述佐剂为矿物油佐剂，其用于制备油包水乳剂；
所述佐剂的浓度范围是从5％到70％V/V，优选从30％到70％，更优选66％V/V。
白油，别名石蜡油、白色油、矿物油，白油在灭活疫苗中是使用较广的佐剂，具有延缓免疫原在机体内的留存时间，使之持续缓慢释放，增强巨噬细胞的吞噬与杀菌能力。
本发明的禽减蛋综合征病毒tFiber蛋白片段基因还可以应用于表达载体、核酸疫苗、诊断试剂开发以及其它预防和/或治疗禽减蛋综合征病毒相关药物开发。
本发明涉及一种重组载体，所述重组载体能表达本发明所述的核苷酸序列编码的tFiber蛋白片段，其具有免疫原性并能产生免疫反应。
本发明涉及一种转化子，所述转化子包含有导入的本发明所述的表达tFiber蛋白片段的重组载体。
作为本发明的一种实施方式，本发明所述疫苗组合物中，所述活载体为重组减毒沙门氏菌、重组新城疫病毒、重组痘病毒。
本发明的活载体疫苗组合物因为兼具有灭活疫苗和活疫苗的优点，在免疫效力上可以保证能够对产蛋禽进行保护，且其免疫效力强，可以不添加佐剂。
本发明的疫苗组合物进一步包含其它病原体或抗原组合使用以制备抵抗包括禽减蛋综合征病毒感染的各种疾病的联合疫苗或复合疫苗。
术语“联合疫苗”指本发明的禽减蛋综合征病毒与至少一种不同病毒的病毒混合物制备的疫苗。术语“复合疫苗”指本发明的禽减蛋综合征 病毒和细菌制备的疫苗。例如，本发明的禽减蛋综合征病毒可与鸡新城疫病毒、鸡传染性支气管炎病毒、禽流感病毒、鸡传染性法氏囊病病毒、禽腺病毒、禽呼肠孤病毒和/或大肠杆菌、副鸡禽杆菌、滑液囊支原体、鸡毒支原体混合或组合。
作为本发明的一种实施方式，本发明所述疫苗组合物中，所述疫苗组合物进一步包含由以下抗原组成的组的一种或多种：鸡新城疫病毒抗原、禽流感病毒抗原、鸡传染性支气管炎病毒抗原、鸡传染性法氏囊病病毒抗原、禽腺病毒抗原、禽呼肠孤病毒抗原、大肠杆菌抗原、副鸡禽杆菌抗原、滑液囊支原体抗原、鸡毒支原体抗原、多杀性巴氏杆菌抗原、马立克氏病毒抗原、禽脑脊髓炎病毒抗原、鸡传染性喉气管炎病毒抗原。
作为本发明的一种优选实施方式，本发明所述疫苗组合物中，所述疫苗组合物还包括由以下抗原组成的组中的一种或多种：鸡新城疫病毒灭活抗原、禽流感病毒灭活抗原、鸡传染性支气管炎病毒灭活抗原、鸡传染性法氏囊病病毒亚单位抗原、禽腺病毒灭活抗原或亚单位抗原。
采用本发明高效表达的禽减蛋综合征病毒tFiber蛋白片段，首次创造性地制备出五联疫苗，免疫动物后能使动物机体快速产生多种抗体，针对包括禽减蛋综合征病毒感染在内的多种疫病免疫效果均有良好的预防和控制效果。
作为本发明的一种更优选实施方式，本发明所述疫苗组合物中，所述鸡新城疫病毒灭活抗原为N7a株灭活抗原，所述禽流感病毒灭活抗原为HF株灭活抗原，所述鸡传染性支气管炎病毒灭活抗原为M41株灭活抗原，所述鸡传染性法氏囊病病毒亚单位抗原为鸡传染性法氏囊病病毒VP2蛋白，所述禽腺病毒灭活抗原为FAV-HN株灭活抗原，所述禽腺病毒亚单位抗原为禽腺病毒Penton蛋白或Fiber-2蛋白。
作为本发明的一种优选实施方式，本发明所述疫苗组合物中，所述禽减蛋综合征病毒tFiber蛋白片段含量为AGP效价1:8～1:32，所述鸡新城疫病毒灭活抗原含量为灭活前10 8.0～10 9.0EID 50/0.1ml，所述禽流感病毒灭活抗原含量为灭活前10 6.5～10 8.5EID 50/0.1ml，所述鸡传染性支气管炎病毒灭活抗原含量为灭活前10 6.0～10 7.0EID 50/0.1ml，所述鸡传染性 法氏囊病病毒VP2蛋白含量为AGP效价1:16～1:128，所述禽腺病毒Penton蛋白含量为AGP效价1:2～1:16，所述禽腺病毒Fiber-2蛋白含量为AGP效价1:2～1:16。
作为本发明的一种更优选实施方式，本发明所述疫苗组合物中，所述禽减蛋综合征病毒tFiber蛋白片段含量为AGP效价1:8～1:32，所述鸡新城疫病毒灭活抗原含量为灭活前10 8.0EID 50/0.1ml，所述禽流感病毒灭活抗原含量为灭活前10 8.0EID 50/0.1ml，所述鸡传染性支气管炎病毒灭活抗原含量为灭活前10 6.0EID 50/0.1ml，所述鸡传染性法氏囊病病毒VP2蛋白含量为AGP效价1:16，所述禽腺病毒Penton蛋白含量为AGP效价1:4，所述禽腺病毒Fiber-2蛋白含量为AGP效价1:4。
本发明的tFiber蛋白片段可以通过本领域内任何已知的方法制备，例如可以通过重组表达tFiber蛋白片段基因制备tFiber蛋白片段，表达系统可以使用任何已知的表达系统，例如：真核表达系统、原核表达系统。或者直接合成tFiber蛋白片段序列。真核表达系统可以包括哺乳动物细胞表达系统，酵母表达系统和昆虫表达系统。
本发明还涉及一种制备所述疫苗组合物的方法，其中，所述方法包括：步骤(1)克隆所述禽减蛋综合征病毒tFiber蛋白片段的基因，并将所述禽减蛋综合征病毒tFiber蛋白片段的基因重组到表达载体以获得重组有所述禽减蛋综合征病毒tFiber蛋白片段的基因的重组表达载体；步骤(2)将所述重组有所述禽减蛋综合征病毒tFiber蛋白片段的基因的重组表达载体和分子伴侣的表达载体一同转化大肠杆菌，表达所述禽减蛋综合征病毒tFiber蛋白片段；步骤(3)将所述表达的禽减蛋综合征病毒tFiber蛋白片段使用非离子表面活性剂处理除去内毒素；以及步骤(4)将所述去除内毒素的禽减蛋综合征病毒tFiber蛋白片段与佐剂混匀，得到所述疫苗组合物。
本发明在发现禽减蛋综合征病毒壳粒表面含量极少的Fiber蛋白具有良好的免疫原性，但其表达量较低，对Fiber蛋白进一步研究，选取其片段tFiber制备亚单位抗原或重组有其片段基因的活载体也能在免疫后产生良好的免疫效力，令人意外的是表达量大幅提升。本发明的制备 方法，能大幅度提高tFiber蛋白片段的表达，经济高效。
作为本发明的一种实施方式，在所述的制备所述疫苗组合物的方法中，所述步骤(1)中所述重组有禽减蛋综合征病毒tFiber蛋白片段基因的重组表达载体为重组pET28a质粒，所述步骤(2)中所述分子伴侣的表达载体为pG-Tf2，所述大肠杆菌为大肠杆菌BL21(DE3)；所述步骤(3)中非离子表面活性剂为Triton X-114。
本发明还涉及所述的疫苗组合物在制备预防和/或治疗禽减蛋综合征的药物中的应用。
本发明还涉及所述疫苗组合物在制备预防和/或治疗禽减蛋综合征病毒感染的药物中的应用。
本发明所述的制备预防和/或治疗禽减蛋综合征病毒感染的药物的施用对象包括鸡或鸭。
术语“预防和/或治疗”在涉及禽减蛋综合征病毒感染时是指抑制禽减蛋综合征病毒的复制、抑制禽减蛋综合征病毒的传播或防止禽减蛋综合征病毒在其宿主体内定居，以及减轻禽减蛋综合征病毒感染的疾病或病症的症状。若病毒荷载量减少、病症减轻和/或摄食量和/或生长增加，那么就可以认为所述治疗达到了治疗效果。
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
本发明实施例中所用到的化学试剂均为分析纯，购自国药集团。本发明所述的实验方法，若无特殊说明，均为常规方法；所述的生物材料，若无特殊说明，均可从商业途径获得。
实施例1 pET28a-EDSV-tFiber表达载体的构建
1.1 EDSV病毒DNA的提取
按照病毒DNA提取试剂盒说明书，取0.2ml感染禽减蛋综合征病毒HX株(减蛋综合征病毒HX株(Egg Drop Syndrome Virus，Strain HX)，保藏号为CCTCC NO：V201942，保藏日期为2019年06月19日，保藏地址为中国武汉·武汉大学)的SPF鸭胚尿囊液于无菌1.5ml离心管中，加0.4ml VB于样品液中，旋涡混匀，室温静置10分钟。加0.45ml AD buffer于样品液中，用力混匀。将VB柱放入2ml收集管中，取0.6ml混合液加入VB柱中，14000g离心1分钟，将剩余的混合液加入VB柱中，14000g离心1分钟，弃掉2ml收集管，将VB柱放入新的2ml收集管中，加入0.4ml W1buffer，14000g离心30秒，加0.6ml Wash buffer于VB柱，14000g离心30秒，空离3分钟，将VB柱放入新的无菌1.5ml离心管中，加入50μl RNase free water置膜中央，静置3分钟，14000g离心1分钟，离心的液体即为病毒DNA溶液。
1.2 Fiber蛋白基因扩增
根据Fiber蛋白基因5’和3’末端的保守区序列合成寡聚核苷酸引物，进行PCR。引物序列见表1。
表1 Fiber蛋白基因扩增引物
Fiber-F ATGAAGCGACTACGGTTGGA
Fiber-R CTACTGTGCTCCAACATATG
将PCR产物送Invitrogen公司测序，根据测序结果对Fiber蛋白基因进行密码子优化，优化后的Fiber蛋白基因序列如SEQ ID NO.1所示。
1.3表达载体构建
优化后Fiber基因送苏州金唯智生物科技有限公司进行全序列合成，获得包含Fiber基因密码子优化序列的pUC57-EDS-Fiber质粒。
根据优化后的Fiber序列设计合成寡聚核苷酸引物，扩增tFiber基因，引物序列见表2。
表2 tFiber蛋白片段基因扩增引物
tFiber-F CATGCCATGGGCCCGCTGTCTATCACCTCT
tFiber-R CCGCTCGAGTTACTGAGCACCAACGTA
PCR产物送Invitrogen公司测序，tFiber蛋白片段基因序列如SEQ  ID NO.2所示，经电泳后使用DNA胶回收试剂盒进行纯化，所获得的DNA片段利用Nco I和Xho I双酶切后与相同双酶切处理的pET-28a(+)质粒进行连接，连接产物转化大肠杆菌DH5α感受态细胞，筛选阳性克隆，使用质粒提取试剂盒提取质粒并进行双酶切鉴定，酶切鉴定正确的质粒进行测序分析，测序正确的重组质粒命名为pET28a-EDS-tFiber。
pET28a-EDS-tFiber质粒转化大肠杆菌BL21(DE3)，挑取单克隆在含有100μg/ml卡那霉素的LB培养基中培养过夜，即为pET28a-EDS-tFiber表达菌株。
实施例2 tFiber蛋白片段的制备
将含有实施例1中制备的pET28a-EDS-tFiber表达菌株接种到含50-100μg/ml卡那霉素的LB培养基中，接种量为1％(V/V)，37℃振荡培养。当OD 600＝0.4～0.6时，于28℃放置30分钟。加入异丙基-β-D-硫代吡喃半乳糖苷(IPTG)，使其终浓度为0.1～1.0mM，28℃振荡培养24小时。培养结束后，收集菌体，并用PBS(氯化钠8g，氯化钾0.2g，磷酸氢二钠1.44g，磷酸二氢钾0.24g，调节pH 7.4，定容至1L)重悬菌体，超声破碎，离心取上清。表达产物中可溶性目的蛋白含量较高，即可溶性tFiber蛋白片段含量较高，tFiber蛋白片段的AGP效价达到1：512，内毒素含量为0.46×10 5EU/ml，无HA活性。
实施例3大肠杆菌表达tFiber蛋白片段内毒素清除
在1.5ml离心管中加入0.5ml待处理溶液和终浓度为1％(v/v)的曲拉通X-114(Triton X-114)5μl，涡旋振荡。将样品在冰上放置5分钟。涡旋振荡冷却的样品后，将离心管立即放入37℃水浴中温浴5min，使得产生新的两相。然后，把样品在37℃离心60s。离心过后，目的蛋白将留在上层，而含有内毒素的洗涤剂将会以油滴的形状残留在离心管底部。整个去除内毒素的操作循环3次。经测定，tFiber蛋白片段的AGP效价达到1：512，内毒素含量下降为0.009×10 5EU/ml。
结果表明Triton X-114能够清除重组蛋白中残留的内毒素，且对 tFiber蛋白片段免疫原性没有影响。
实施例4禽减蛋综合征病毒tFiber蛋白片段亚单位疫苗的制备
将按实施例3的方法纯化后的tFiber蛋白片段缓缓加入到矿物油佐剂中，同时开动电机，17500r/min搅拌5min，在终止搅拌前加入1％硫柳汞溶液，使其终浓度为0.01％。具体配比见表3。
表3禽减蛋综合征病毒tFiber蛋白片段亚单位疫苗配比
组分 疫苗1 疫苗2 疫苗3
tFiber蛋白片段(AGP效价) 1:8 1:16 1:32
矿物油佐剂(V/V％) 66％ 66％ 66％
实施例5禽减蛋综合征病毒tFiber蛋白片段亚单位疫苗对SPF鸡免疫原性试验
取21日龄的SPF鸡40只，分成4组，每组10只，第1组～第3组分别经颈部皮下注射免疫实施例4制备的疫苗1～疫苗3，免疫剂量为0.5ml，第4组颈部皮下注射0.5ml生理盐水，作为空白对照。所有试验鸡均隔离饲养，免疫前及免疫后21日，每只鸡分别采血，分离血清，测定血清中禽减蛋综合征HI抗体效价。结果见表4。
表4禽减蛋综合征病毒tFiber蛋白片段亚单位疫苗对SPF鸡免疫原性试验结果
Figure PCTCN2020101961-appb-000002
结果显示，第4组对照组免后21日HI抗体效价为0，而第1组～第3组免疫组对免疫鸡均产生了较高的HI抗体效价。表明以AGP效价不低于1:8的禽减蛋综合征病毒tFiber蛋白片段亚单位疫苗，即可使鸡 群产生较高HI抗体效价，可实现对鸡群提供有效的免疫保护。
实施例6禽减蛋综合征病毒tFiber蛋白片段亚单位疫苗对蛋鸡免疫原性试验
取115日龄的海兰褐商品蛋鸡40只，分成4组，每组10只，第5组～第7组分别经颈部皮下注射免疫实施例4制备的疫苗1～疫苗3，免疫剂量为0.5ml，第8组颈部皮下注射0.5ml生理盐水，作为空白对照。免疫前及免疫后21日，每只鸡分别采血，分离血清，测定血清禽减蛋综合征HI抗体效价。当产蛋率达90％左右时(免疫后6周)，所有4组试验鸡均用HX株强毒攻击，每只口服10倍稀释毒1ml，病毒含量为10 6.5EID 50，攻毒后观察6周，观察鸡群采食、精神、粪便等状况，记录产蛋数量，计算产蛋率。结果见表5。
表5禽减蛋综合征病毒tFiber蛋白片段亚单位疫苗对蛋鸡的免疫原性试验结果
Figure PCTCN2020101961-appb-000003
结果显示，第8组对照组免后21日HI抗体效价为0，对照组试验鸡在攻毒后产蛋开始下降，攻毒后第3周产蛋率从原来的90％左右下降到46％左右，同时蛋壳颜色变淡，产软壳蛋、无壳蛋、畸形蛋等；攻毒后第6周产蛋率为69％左右，仍未恢复到正常水平。而第5组～第7组免疫组对免疫鸡均产生了较高的HI抗体效价，且攻毒后产蛋率几乎没有变化，表明以AGP效价不低于1:8的禽减蛋综合征病毒tFiber蛋白片段亚单位疫苗，免疫鸡均产生了较高的HI抗体效价，本发明的tFiber 蛋白片段抗原免疫原性好，以低含量即可实现对鸡群提供有效的免疫保护。
实施例7禽减蛋综合征病毒tFiber蛋白片段亚单位疫苗对樱桃谷鸭免疫原性试验
取42日龄的樱桃谷鸭40只，分成4组，每组10只，第9组～第11组分别经颈部皮下注射免疫实施例4制备的疫苗1～疫苗3，免疫剂量为0.5ml，第12组颈部皮下注射0.5ml生理盐水，作为空白对照。所有试验鸭均隔离饲养，免疫前及免疫后21日，每只鸭分别采血，分离血清，测定血清中禽减蛋综合征HI抗体效价。测定结果见表6。
表6禽减蛋综合征病毒tFiber蛋白片段亚单位疫苗对樱桃谷鸭免疫原性试验结果
Figure PCTCN2020101961-appb-000004
结果显示，第12组对照组鸭免后21日HI抗体效价为0，而第9组～第11组免疫组鸭均产生了较高的HI抗体效价，免疫效果良好。表明以AGP效价不低于1:8的禽减蛋综合征病毒tFiber蛋白片段亚单位疫苗，即可使免疫鸭产生较高HI抗体效价，本发明的tFiber蛋白片段抗原免疫原性好，以低含量即可对鸭群提供有效的免疫保护。
实施例8禽减蛋综合征病毒Fiber蛋白亚单位疫苗的制备
参照实施例1方法构建pET28a-EDS-Fiber表达菌株，按照实施例2方法制备Fiber全长蛋白，Fiber蛋白的HA效价为1：512，AGP效价为1：64，内毒素含量为0.51×10 5EU/ml。按照实施例3方法进行内毒素清除，经测定，Fiber蛋白的AGP效价达到1：64，内毒素含量下降 为0.009×10 5EU/ml。
将上述纯化后的Fiber蛋白缓缓加入到矿物油佐剂中，同时开动电机，17500r/min搅拌5min，在终止搅拌前加入1％硫柳汞溶液，使其终浓度为0.01％。具体配比见表7。
表7禽减蛋综合征病毒Fiber蛋白亚单位疫苗配比
组分 疫苗4
Fiber蛋白(AGP效价) 1:16
矿物油佐剂(V/V％) 66％
实施例9禽减蛋综合征病毒亚单位疫苗免疫持续期对比试验
取21日龄SPF鸡30只，分成3组，每组10只，第13组、第14组分别经颈部皮下注射免疫实施例4制备的疫苗2、实施例8制备的疫苗4，免疫剂量为0.5ml，第15组颈部皮下注射0.5ml生理盐水，作为空白对照。所有试验鸡均隔离饲养，免疫前及免疫后21日，每只鸡分别采血，分离血清，测定血清禽减蛋综合征HI抗体效价。结果见表8。
表8免疫持续期对比试验结果
Figure PCTCN2020101961-appb-000005
结果显示，第15组对照组免后HI抗体效价为0；第13组免疫组 对免疫鸡均产生了较高的HI抗体效价，在免疫后6个月内均能维持较高的HI抗体效价；第14组免疫组虽然对免疫鸡也产生了较高的HI抗体效价，但整体均低于第13组的抗体水平。表明本发明提供的禽减蛋综合征病毒tFiber蛋白片段亚单位疫苗较禽减蛋综合征病毒Fiber全长蛋白亚单位疫苗具有更好的免疫原性，即可使鸡群维持更长时间的HI抗体效价，可实现对鸡群提供长期、有效的免疫保护。
实施例10鸡新城疫抗原的制备
取新城疫病毒(基因Ⅶ型)N7a株(Newcastle Disease Virus(genotype Ⅶ),strain N7a)(保藏于中国典型培养物保藏中心，保藏号为CCTCC NO：V201545，保藏日期为2015年10月19日，保藏地址为中国武汉·武汉大学，公开于中国专利申请CN107281479A)，用灭菌生理盐水作适当稀释(10 -4或10 -5)接种10～11日龄易感鸡胚，每胚0.1ml，接种后置37℃继续孵育。选接种后48～120小时死亡和存活鸡胚，收获尿囊液，测定病毒含量，为10 8.0EID 50/0.1ml。加入终浓度为0.1％的甲醛溶液(v/v)，放37℃灭活，其间每隔4～6h搅拌一次，灭活16h，灭活完全后备用。
实施例11禽流感抗原的制备
取H9亚型禽流感病毒HF株(禽流感病毒(H9亚型)HF株(Avian Influenza Virus(Subtype H9)，Strain HF)，保藏号为CCTCC NO：V201941，保藏日期为2019年06月19日，保藏地址为中国武汉·武汉大学)毒种，用无菌生理盐水稀释至10 -3(取病毒液0.1ml加入到0.9ml无菌生理盐水中，震荡混匀后依次再稀释2次)，经尿囊腔接种10日龄易感鸡胚(使用购自北京梅里亚维通实验动物技术有限公司的SPF种蛋自行孵化)，每胚0.1ml(含10 5EID 50)。接种后密封针孔，置36～37℃继续孵育，不必翻蛋。至96小时，取出，气室向上直立，置于2～8℃冷却12～24小时。将冷却后的鸡胚收获胚液。测定病毒含量，为10 8.5EID 50/0.1ml。加入终浓度为0.1％的甲醛溶液(v/v)，放37℃灭活， 其间每隔4～6h搅拌一次，灭活24h，灭活完全后备用。
实施例12鸡传染性支气管炎抗原的制备
取鸡传染性支气管炎病毒M41株(购自中国兽医药品监察所)，用灭菌生理盐水作适当稀释(10 -2或10 -3)接种10～11日龄易感鸡胚，每胚0.1ml，接种后置36～37℃继续孵育。选接种后24～48小时死亡和存活鸡胚，收获尿囊液，测定病毒含量，为10 6.0EID 50/0.1ml。加入终浓度为0.1％的甲醛溶液(v/v)，放置于37℃灭活，其间每隔4～6h搅拌一次，灭活16h，灭活完全后备用。
实施例13法氏囊抗原的制备
13.1 VP2 cDNA制备
按病毒RNA提取试剂盒操作从感染鸡传染性法氏囊病病毒超强毒成都株的SPF鸡法氏囊中提取IBDV病毒RNA，并用随机引物进行逆转录。根据VP2基因5’和3’末端的保守区序列合成寡聚核苷酸引物，合成寡聚核苷酸引物序列见表9，并进行PCR扩增，并利用琼脂糖凝胶胶回收试剂盒回收，-20℃保存。
表9法氏囊病毒VP2基因扩增引物
VP2-EcoR1-F CCGGAATTCATGACAAACCTGCAAGATCAAAC
VP2-Sal1-R ACGCGTCGACTTACCTTAGGGCCCGGATTATGT
13.2 pColdⅢ-VP2/E.Coli BL21(DE3)菌株的构建
取上述制备的VP2 cDNA，进行双酶切，并将酶切后的片段连接到pColdⅢ载体上；连接产物直接转化大肠杆菌BL21(DE3)，并涂布在含有100μg氨苄青霉素LB固体培养基中培养过夜，长出的菌落即为pColdⅢ-VP2/E.Coli BL21(DE3)菌株。
13.3鸡传染性法氏囊病病毒VP2蛋白的制备
用培养罐通气培养，按容积装入70％培养基及花生油消泡剂。灭菌后按培养基量的2％～4％接种pColdⅢ-VP2/E.Coli BL21(DE3)菌株种子液，37℃培养，待菌液的OD 600值达到0.6～1.0，加入0.2mol/Lα-乳糖， 使终浓度达到0.02mol/L，再继续培养5～8h。
培养结束后，离心收集菌体，重悬，超声波破碎，离心收集上清液。经硫酸铵沉淀后，收集VP2蛋白液。
参照实施例3方法进行内毒素清除，经测定，内毒素含量下降为0.3EU/ml。
实施例14禽腺病毒抗原的制备
14.1 Fiber-2 cDNA制备
按病毒RNA提取试剂盒操作从感染禽腺病毒FAV-HN株(禽腺病毒，FAV-HN株(Fowl aviadenovirus,strain FAV-HN)，保藏号为：CCTCC NO：V201609，保藏单位为中国典型培养物保藏中心，保藏地址为中国武汉·武汉大学，保藏时间为2016年2月29日，公开于中国专利申请CN107523556A)中提取FADV病毒DNA。根据Fiber-2蛋白基因5’和3’末端的保守区序列合成寡聚核苷酸引物，合成寡聚核苷酸引物序列见表10，并进行PCR扩增，并利用琼脂糖凝胶胶回收试剂盒回收，-20℃保存。
表10禽腺病毒Fiber2基因扩增引物
Fiber-2-F CTCCGGGCCCCTAAAAG
Fiber-2-R CGGGACGGAGGCCGC
14.2表达载体构建
优化后Fiber-2蛋白基因送由苏州金唯智生物科技有限公司进行全序列合成，并分别连接到pET28a质粒上。连接后的质粒转化大肠杆菌BL21(DE3)，挑取单克隆在含有100μg卡那霉素的LB培养基中培养过夜，提取质粒后测序分析，阳性克隆即为pET28a-FADV-Fiber-2表达菌株。
14.3 Fiber-2蛋白的制备
将含有实施例1中制备的pET28a-FADV-Fiber-2/E.Coli BL21(DE3)菌株接种到含50-100μg/ml卡那霉素的LB培养基中，接种量为1％(V/V)，37℃振荡培养。当OD 600值达到0.4～0.6时，于28℃放置30 分钟。加入异丙基-β-D-硫代吡喃半乳糖苷(IPTG)，使其终浓度为1.0mM，28℃振荡培养24小时。
培养结束后，收集菌体，并用PBS(氯化钠8g，氯化钾0.2g，磷酸氢二钠1.44g，磷酸二氢钾0.24g，调节pH为7.4,定容至1L)重悬菌体，超声破碎，离心取上清。收集Fiber-2蛋白液。
参照实施例3方法进行内毒素清除，经测定，内毒素含量下降为0.008×10 5EU/ml。
实施例15禽减蛋综合征病毒联合疫苗的制备
将实施例3纯化后的禽减蛋综合征病毒tFiber蛋白片段抗原分别和实施例10制备的鸡新城疫抗原、实施例11制备的禽流感抗原、实施例12制备的鸡传染性支气管炎抗原、实施例13制备的传染性法氏囊抗原、实施例14制备的禽腺病毒抗原按比例混合，加入到矿物油佐剂中，同时开动电机，17500r/min搅拌5min，在终止搅拌前加入1％硫柳汞溶液，使其终浓度为0.01％。具体配比见表11、12、13、14。
表11禽减蛋综合征病毒二联疫苗配比
组分 疫苗5 疫苗6 疫苗7 疫苗8 疫苗9
tFiber蛋白片段(AGP效价) 1：8 1：16 1：32 1：8 1：16
N7a株抗原(EID 50/0.1ml) 10 8.0 — — — —
HF株抗原(EID 50/0.1ml) — 10 8.0 — — —
M41株抗原(EID 50/0.1ml) — — 10 6.0 — —
VP2蛋白(AGP效价) — — — 1：16 —
Fiber-2蛋白(AGP效价) — — — — 1：4
矿物油佐剂(V/V％) 66％ 66％ 66％ 66％ 66％
表12禽减蛋综合征病毒三联疫苗配比
组分 疫苗10 疫苗11 疫苗12 疫苗13
tFiber蛋白片段(AGP效价) 1：32 1：8 1：16 1：32
N7a株抗原(EID 50/0.1ml) 10 8.0 10 8.0 10 8.0 10 8.0
HF株抗原(EID 50/0.1ml) 10 8.0 — — —
M41株抗原(EID 50/0.1ml) — 10 6.0 — —
VP2蛋白(AGP效价) — — 1：16 —
Fiber-2蛋白(AGP效价) — — — 1：4
矿物油佐剂(V/V％) 66％ 66％ 66％ 66％
表13禽减蛋综合征病毒四联疫苗配比
Figure PCTCN2020101961-appb-000006
表14禽减蛋综合征病毒五联疫苗配比
组分 疫苗19 疫苗20
tFiber蛋白片段(AGP效价) 1：16 1：16
N7a株抗原(EID 50/0.1ml) 10 8.0 10 8.0
HF株抗原(EID 50/0.1ml) 10 8.0 10 8.0
M41株抗原(EID 50/0.1ml) 10 6.0 10 6.0
VP2蛋白(AGP效价) 1：16 —
Fiber-2蛋白(AGP效价) — 1：4
矿物油佐剂(V/V％) 66％ 66％
实施例16禽减蛋综合征病毒联合疫苗免疫原性试验
16.1禽减蛋综合征病毒部分免疫原性试验
取21日龄的SPF鸡170只，分成17组，每组10只，第16组～第31组分别经颈部皮下注射免疫实施例15制备的疫苗5～疫苗20，0.5ml/只；第32组皮下注射0.5ml生理盐水，作为空白对照。所有试验鸡均隔离饲养，免疫后21日，每只鸡分别采血，分离血清，测定血清中禽减蛋综合征HI抗体效价。结果见表15。
表15禽减蛋综合征病毒联合疫苗禽减蛋综合征病毒部分免疫原性试验结果
Figure PCTCN2020101961-appb-000007
结果显示，疫苗5～疫苗20免疫组在免疫后21天均产生了较高的HI抗体效价，可有效保护鸡群产蛋综合征的发生。表明本发明提供的 禽减蛋综合征病毒tFiber蛋白片段，作为抗原制备的油乳剂联苗可对鸡群提供完全保护。
16.2新城疫病毒部分免疫原性试验
取21日龄的SPF鸡130只，分成13组，每组10只，第33组～第44组分别经颈部皮下注射免疫实施例15制备的疫苗5、疫苗10～疫苗20，20μl/只；第45组皮下注射20μl生理盐水，作为攻毒对照。所有试验鸡均隔离饲养，免疫后21日，将第33组～第44组免疫鸡，连同第45组攻毒对照鸡，采血并分离血清。检测新城疫病毒HI抗体，同时用新城疫强毒HN1101株病毒液通过肌肉注射攻击，观察14日，记录发病、死亡及保护数。结果见表16。
表16禽减蛋综合征病毒联合疫苗新城疫病毒部分免疫原性试验结果
Figure PCTCN2020101961-appb-000008
注：HI抗体测定为免疫鸡抗体的几何平均数。
结果显示，疫苗5、疫苗10～疫苗20免疫组在免疫后21天均能产生较高的新城疫抗体，而且免疫组和对照相比，可以完全保护强毒的攻击。表明以本发明提供的N7a株新城疫病毒液，作为抗原制备的油乳剂联苗可对鸡群提供完全保护。
16.3禽流感部分免疫原性试验
取21日龄的SPF鸡80只，分成8组，每组10只，第46组～第52组分别经颈部皮下注射免疫实施例15制备的疫苗6、疫苗10、疫苗14、疫苗17～疫苗20，0.3ml/只；第53组皮下注射0.3ml生理盐水，作为攻毒对照。所有试验鸡均隔离饲养，免疫后21日，将第46组～第52组免疫鸡，连同第53组对照鸡，采血并分离血清。检测H9亚型禽流感HI抗体效价，同时用HF株病毒液静脉注射攻击，每只0.2ml(含10 7.0EID 50)。于攻毒后5日，采集泄殖腔拭子，经处理后，尿囊腔接种10～11日龄SPF鸡胚5个，孵育观察5日，无论死胚、活胚均应测定鸡胚液红细胞凝集价，每个拭子样品接种的5个鸡胚中只要有1个鸡胚液的凝集价不低于1:16(微量法)，即可判为病毒分离阳性。对病毒分离阴性的样品，应盲传一次后再进行判定。免疫组应至少9只鸡病毒分离为阴性；对照组应至少4只鸡病毒分离为阳性。结果见表17。
表17禽减蛋综合征病毒联合疫苗禽流感部分免疫原性试验结果
Figure PCTCN2020101961-appb-000009
注：HI抗体测定为免疫鸡抗体的几何平均数。
结果显示，疫苗6、疫苗10、疫苗14、疫苗17～疫苗20在免疫后21天均能产生较高的禽流感抗体，而且免疫组和对照相比，可以完全保护强毒的攻击。表明本发明提供的H9亚型禽流感病毒液，作为抗原制备的油乳剂联苗可对鸡群提供完全保护。
16.4鸡传染性支气管炎部分免疫原性试验
取21日龄的SPF鸡80只，分成8组，每组10只，第54组～第60组各点眼、滴鼻接种鸡传染性支气管炎活疫苗(H120株)1羽份(0.05ml)。接种后21日，连同第61组对照鸡，采血并分离血清。同时，第54组～第60组各组经颈部皮下注射免疫实施例15制备的疫苗7、疫苗11、疫苗14、疫苗15、疫苗16、疫苗19、疫苗20，0.3ml/只。接种后28日，连同第61组攻毒对照鸡分别采血并分离血清；第54～第60组免疫鸡在活苗首免后21日、灭活苗免后28日两次采集的血清(第61组攻毒对照鸡同样时间采集血清)测HI抗体效价。免疫组二免血清HI抗体效价几何平均值不低于首免血清HI抗体效价几何平均值的4倍，未免疫对照组血清HI抗体效价的几何平均值不高于1:8(微量法)。同时用鸡传染性支气管炎M41强毒每羽滴鼻攻毒10 3.0EID 50，作攻毒实验。结果见表18。
表18禽减蛋综合征病毒联合疫苗及传染支气管炎部分免疫原性试验结果
Figure PCTCN2020101961-appb-000010
Figure PCTCN2020101961-appb-000011
结果显示，疫苗7、疫苗11、疫苗14、疫苗15、疫苗16、疫苗19、疫苗20二免血清HI抗体效价几何平均值均不低于首免血清HI抗体效价几何平均值的4倍，攻毒后全部免疫鸡的气管内未分离出病毒，可以完全保护强毒的攻击。表明本发明提供的鸡传染性支气管炎病毒液，作为抗原制备的油乳剂联苗可对鸡群提供完全保护。
16.5法氏囊部分免疫原性试验
取21日龄的SPF鸡60只，分成6组，每组10只，第62组～第66组分别经颈部皮下注射免疫实施例15制备的疫苗8、疫苗12、疫苗15、疫苗17、疫苗19，0.3ml/只；第67组皮下注射0.3ml生理盐水，作为攻毒对照。所有试验鸡均隔离饲养，免疫后21日，第62组～第67组，每只点眼途径接种100倍稀释的鸡传染性法氏囊病强毒BC6-85((CVCC AV7株)购于中国兽医药品监察所)株病毒液0.1ml(实含毒量≥100个BID)。攻毒后，每天观察鸡只的临床表现，记录发病和死亡鸡数，至72～96小时，扑杀存活鸡，逐只解剖，观察法氏囊等病变。免疫鸡应至少8只正常，不出现法氏囊病变；对照鸡应至少4只鸡发病，出现明显的法氏囊病变(如胸肌或腿肌条状出血、法氏囊肿大或萎缩、发黄、内有胶冻样分泌物等一种以上病变)。结果见表19。
表19禽减蛋综合征病毒联合疫苗法氏囊部分免疫原性试验结果
Figure PCTCN2020101961-appb-000012
结果显示，疫苗8、疫苗12、疫苗15、疫苗17、疫苗19在免疫后21天，可以完全保护鸡传染性法氏囊病强毒的攻击。
16.6禽腺病毒部分免疫原性试验
取21日龄的SPF鸡60只，分成6组，每组10只，第68组～第72组分别经颈部皮下注射免疫实施例15制备的疫苗9、疫苗13、疫苗16、疫苗18、疫苗20，0.3ml/只；第73组皮下注射0.3ml生理盐水，作为攻毒对照。所有试验鸡均隔离饲养，免疫后21日，用FAV-HN株病毒液通过肌肉注射攻击，观察14日，记录发病、死亡及保护数。结果见表20。
表20禽减蛋综合征病毒联合疫苗腺病毒部分免疫原性试验结果
Figure PCTCN2020101961-appb-000013
结果显示，第73组对照组全部发病死亡，而第68组～第72组免疫组对免疫鸡均产生了较好的免疫保护效果，免疫效果良好。表明本发明提供的禽腺病毒抗原，作为抗原制备的油乳剂联苗可对鸡群提供完全保护。
证明了本发明提供的禽减蛋综合征病毒联合疫苗能够抵抗相关病原的侵袭，显示出良好的免疫原性，可有效控制我国禽减蛋综合征病毒相关疾病的流行。由于禽减蛋综合征病毒tFiber蛋白片段的亚单位抗原成功研制，以及大肠杆菌表达亚单位蛋白后，内毒素的清除技术的应用使得针对禽类主要疫病的多联苗的研制成为现实；制备的禽减蛋综合征病毒tFiber蛋白片段的亚单位抗原、和鸡新城疫抗原、和禽流感抗原、和鸡传染性支气管炎抗原、和传染性法氏囊亚单位抗原五联苗，以及禽减蛋综合征病毒tFiber蛋白片段的亚单位抗原、和鸡新城疫抗原、和禽流感抗原、和鸡传染性支气管炎抗原、和禽腺病毒亚单位抗原五联苗实 现了首次同时免疫五种抗原，省略了繁复的免疫程序。
以上所述仅是本发明的优选实施例而已，并非对本发明做任何形式上的限制，虽然本发明已以优选实施例揭露如上，然而并非用以限定本发明，任何熟悉本专业的技术人员，在不脱离本发明技术方案的范围内，当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (14)

  1. 一种禽减蛋综合征病毒tFiber蛋白片段，其中，所述禽减蛋综合征病毒tFiber蛋白片段的基因如SEQ ID NO.2或其简并序列所示。
  2. 一种疫苗组合物，其中，所述疫苗组合物包括免疫量的权利要求1所述的禽减蛋综合征病毒tFiber蛋白片段或免疫量的重组有权利要求1所述的禽减蛋综合征病毒tFiber蛋白片段的基因的活载体以及药学上可接受的载体。
  3. 根据权利要求2所述的疫苗组合物，其中，所述禽减蛋综合征病毒tFiber蛋白片段含量为AGP效价≥1:8。
  4. 根据权利要求2所述的疫苗组合物，其中，禽减蛋综合征病毒tFiber蛋白片段含量为AGP效价1:8～1:32。
  5. 根据权利要求2所述的疫苗组合物，其中，所述药学上可接受的载体为佐剂，所述佐剂包括：(1)矿物油、铝胶佐剂、皂苷、阿夫立定、DDA；(2)油包水乳剂、水包油乳剂、水包油包水乳剂；或(3)丙烯酸或甲基丙烯酸的聚合物、顺丁烯二酸酐和链烯基衍生物的共聚物；以及RIBI佐剂系统、Block co-polymer、SAF-M、单磷酰脂质A、Avridine脂质-胺佐剂、大肠杆菌不耐热肠毒素、霍乱毒素、IMS 1314、胞壁酰二肽、Montanide ISA 206、Gel佐剂中的一种或几种；
    所述佐剂含量为5％-70％V/V。
  6. 根据权利要求5所述的疫苗组合物，其中，皂苷为Quil A、QS-21、GPI-0100；所述佐剂含量为30％到70％。
  7. 根据权利要求5所述的疫苗组合物，其中，所述佐剂含量为66％V/V。
  8. 根据权利要求2所述的疫苗组合物，其中，所述活载体为重组减毒沙门氏菌、重组新城疫病毒、重组痘病毒。
  9. 根据权利要求2所述的疫苗组合物，其中，所述疫苗组合物进一步包含由以下抗原组成的组的一种或多种：鸡新城疫病毒抗原、禽流感病毒抗原、鸡传染性支气管炎病毒抗原、鸡传染性法氏囊病病毒抗原、禽腺病毒抗原、禽呼肠孤病毒抗原、大肠杆菌抗原、副鸡禽杆菌抗原、 滑液囊支原体抗原、鸡毒支原体抗原、多杀性巴氏杆菌抗原、马立克氏病毒抗原、禽脑脊髓炎病毒抗原、鸡传染性喉气管炎病毒抗原。
  10. 根据权利要求9所述的疫苗组合物，其中，所述疫苗组合物还包括由以下抗原组成的组中的一种或多种：鸡新城疫病毒灭活抗原、禽流感病毒灭活抗原、鸡传染性支气管炎病毒灭活抗原、鸡传染性法氏囊病病毒亚单位抗原、禽腺病毒灭活抗原或亚单位抗原。
  11. 根据权利要求10所述的疫苗组合物，其中，所述鸡新城疫病毒灭活抗原为N7a株灭活抗原、所述禽流感病毒灭活抗原为HF株灭活抗原、所述鸡传染性支气管炎病毒灭活抗原为M41株灭活抗原、所述鸡传染性法氏囊病病毒亚单位抗原为鸡传染性法氏囊病病毒VP2蛋白、所述禽腺病毒灭活抗原为FAV-HN株灭活抗原、所述禽腺病毒亚单位抗原为禽腺病毒Penton蛋白或Fiber-2蛋白。
  12. 根据权利要求11所述的疫苗组合物，其中，所述禽减蛋综合征病毒tFiber蛋白片段含量为AGP效价1:8～1:32，所述鸡新城疫病毒灭活抗原含量为灭活前10 8.0～10 9.0EID 50/0.1ml，所述禽流感病毒灭活抗原含量为灭活前10 6.5～10 8.5EID 50/0.1ml，所述鸡传染性支气管炎病毒灭活抗原含量为灭活前10 6.0～10 7.0EID 50/0.1ml，所述鸡传染性法氏囊病病毒VP2蛋白含量为AGP效价1:16～1:128，所述禽腺病毒Penton蛋白含量为AGP效价1:2～1:16，所述禽腺病毒Fiber-2蛋白含量为AGP效价1:2～1:16。
  13. 根据权利要求11所述的疫苗组合物，其中，所述禽减蛋综合征病毒tFiber蛋白片段含量为AGP效价1:8～1:32，所述鸡新城疫病毒灭活抗原含量为灭活前10 8.0EID 50/0.1ml，所述禽流感病毒灭活抗原含量为灭活前10 8.0EID 50/0.1ml，所述鸡传染性支气管炎病毒灭活抗原含量为灭活前10 6.0EID 50/0.1ml，所述鸡传染性法氏囊病病毒VP2蛋白含量为AGP效价1:16，所述禽腺病毒Penton蛋白含量为AGP效价1:4，所述禽腺病毒Fiber-2蛋白含量为AGP效价1:4。
  14. 根据权利要求2～13任一项所述的疫苗组合物在制备预防和/或治疗禽减蛋综合征的药物中的应用。
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