CN110540579A - avibacterium paragallinarum antigen protein, vaccine composition containing avibacterium paragallinarum antigen, and preparation method and application thereof - Google Patents

avibacterium paragallinarum antigen protein, vaccine composition containing avibacterium paragallinarum antigen, and preparation method and application thereof Download PDF

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CN110540579A
CN110540579A CN201810534472.3A CN201810534472A CN110540579A CN 110540579 A CN110540579 A CN 110540579A CN 201810534472 A CN201810534472 A CN 201810534472A CN 110540579 A CN110540579 A CN 110540579A
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type
antigen
hmtp2102
avibacterium paragallinarum
region
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CN110540579B (en
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田克恭
金云云
孙进忠
张许科
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Luoyang Huizhong Biotech Co ltd
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Pulaike Biological Engineering Co Ltd
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    • 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
    • A61K39/02—Bacterial antigens
    • 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
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention relates to a fowl paragallibacterium antigen protein and a vaccine composition containing the fowl paragallibacterium antigen. The three antigen proteins in the vaccine composition have a synergistic effect, and can have a good immune effect at a low content. And can provide complete protection for different serotype strains from different regional sources, and has broad-spectrum immunogenicity. The vaccine composition containing the avibacterium paragallinarum antigen and the newcastle disease antigen has a treatment effect on chicken flocks infected with avibacterium paragallinarum, and can avoid the reduction of the yield of the chicken flocks.

Description

avibacterium paragallinarum antigen protein, vaccine composition containing avibacterium paragallinarum antigen, and preparation method and application thereof
Technical Field
The invention relates to an avibacterium paragallinarum antigen protein, a vaccine composition containing the avibacterium paragallinarum antigen, a preparation method and application, and belongs to the field of veterinary drugs.
Background
Avian paragallinarum (Avibacterium paragallinarum, Apg) is a pathogenic bacterium causing acute upper respiratory disease of chicken, and has A, B, C serotypes. The disease was first discovered in Poland and the United states, and after discovery, it was confirmed to occur frequently in other countries. Since 1980, China has been reported to suffer from the disease frequently, and at present, 3 serotypes are main epidemic serotypes. The infected respiratory disease is commonly called Infectious Coryza (IC), which is clinically mainly characterized by facial edema, nasosinusitis and lacrimation, can cause the reduction of the egg yield of laying hens, the obstruction of the growth of bred chickens, the delay of the laying period, the reduction of the weight gain rate of broilers and cause huge economic loss to the poultry industry. Apg 3 serotypes do not have cross-protection and are widely different in protection among different isolates of the same serotype. Most of vaccines for preventing the disease in the current market are A-type monovalent inactivated vaccines, A, C-type divalent inactivated vaccines and imported A, B, C-type trivalent inactivated vaccines, because of the prevalence of B-type strains, monovalent vaccines or divalent vaccines cannot generate a comprehensive protection effect, and the imported vaccines are not ideal for the protection effect of the disease due to the difference between the antigens of the currently prevalent strains and the vaccine-producing strains.
Because the disease is characterized by short latency, rapid spread and spread in a short time, the whole chicken flock can be reached, and therefore, the vaccine composition with the property of preventing and even treating is particularly necessary.
disclosure of Invention
In order to solve the defects of the prior art, the invention provides the avibacterium paragallinarum C-type HMTp2102 region antigen protein, and the avibacterium paragallinarum C-type HMTp2102 region antigen protein is coded by a sequence shown in SEQ ID NO. 3.
The avian paragallibacterium C-type HMTp2102 region antigen protein has good immunogenicity, and can generate synergistic effect on the avian paragallibacterium A-type HMTp2102 region antigen protein and the avian paragallibacterium B-type HMTp2102 region antigen protein.
The invention also relates to a construct, which comprises the avibacterium paragallinarum C-type HMTp2102 region antigen protein gene.
The invention also relates to a recombinant cell, wherein the recombinant cell is obtained by transforming a receptor cell with the construct; optionally, the recipient cell is selected from the group consisting of bacteria, yeast, animal cells, and plant cells.
The invention also relates to a chicken infectious rhinitis subunit vaccine composition, wherein the vaccine composition comprises the avibacterium paragallinarum immunoprotective antigen protein and a pharmaceutically acceptable carrier.
The invention also provides a vaccine composition containing the avibacterium paragallinarum antigen, wherein the vaccine composition contains an immunizing amount of avibacterium paragallinarum A type HMTp2102 region antigen protein, an immunizing amount of avibacterium paragallinarum B type HMTp2102 region antigen protein, an immunizing amount of avibacterium paragallinarum C type HMTp2102 region antigen protein and a pharmaceutically acceptable carrier.
The vaccine composition containing the avibacterium paragallinarum antigen can provide comprehensive protection for avibacterium paragallinarum infection and can completely protect each serotype of avibacterium paragallinarum.
As an embodiment of the invention, in the vaccine composition containing the avibacterium paragallinarum antigen, the antigenic protein of the avibacterium paragallinarum A-type HMTp2102 region is coded by a sequence shown in SEQ ID NO.1, the antigenic protein of the avibacterium paragallinarum B-type HMTp2102 region is coded by a sequence shown in SEQ ID NO.2, and the antigenic protein of the avibacterium paragallinarum C-type HMTp2102 region is coded by a sequence shown in SEQ ID NO. 3.
The vaccine composition containing the specific antigen has broad-spectrum immunogenicity, and can completely protect different serotype avibacterium paragallinarum from different regional sources.
As an embodiment of the invention, in the vaccine composition containing the avibacterium paragallinarum antigen, the content of the antigenic protein in the A-type HMTp2102 region of the avibacterium paragallinarum is more than or equal to 15 mu g/ml, the content of the antigenic protein in the B-type HMTp2102 region of the avibacterium paragallinarum is more than or equal to 25 mu g/ml, and the content of the antigenic protein in the C-type HMTp2102 region of the avibacterium paragallinarum is more than or equal to 15 mu g/ml.
As a preferred embodiment of the invention, in the vaccine composition containing the avibacterium paragallinarum antigen, the content of the antigenic protein in the HMTp2102 area of the avibacterium paragallinarum type A is 15 mu g/ml-100 mu g/ml, the content of the antigenic protein in the HMTp2102 area of the avibacterium paragallinarum type B is 25 mu g/ml-100 mu g/ml, and the content of the antigenic protein in the HMTp2102 area of the avibacterium paragallinarum type C is 15 mu g/ml-100 mu g/ml.
The avian infectious coryza subunit vaccine composition provided by the invention is characterized in that the content of the antigen protein in the A-type HMTp2102 area of the avibacterium paragallinarum can be arbitrarily selected from 15 mu g/ml, 20 mu g/ml, 25 mu g/ml, 30 mu g/ml, 35 mu g/ml, 40 mu g/ml, 45 mu g/ml, 50 mu g/ml, 55 mu g/ml, 60 mu g/ml, 65 mu g/ml, 70 mu g/ml, 75 mu g/ml, 80 mu g/ml, 85 mu g/ml, 90 mu g/ml, 95 mu g/ml and 100 mu g/ml.
The avian infectious coryza subunit vaccine composition provided by the invention is characterized in that the content of the antigen protein in the B-type HMTp2102 area of the avibacterium paragallinarum can be arbitrarily selected from 25 mu g/ml, 30 mu g/ml, 35 mu g/ml, 40 mu g/ml, 45 mu g/ml, 50 mu g/ml, 55 mu g/ml, 60 mu g/ml, 65 mu g/ml, 70 mu g/ml, 75 mu g/ml, 80 mu g/ml, 85 mu g/ml, 90 mu g/ml, 95 mu g/ml and 100 mu g/ml.
The avian infectious coryza subunit vaccine composition of the invention, wherein the content of the antigen protein in the C-type HMTp2102 area of the avibacterium paragallinarum can be arbitrarily selected from 15 mu g/ml, 20 mu g/ml, 25 mu g/ml, 30 mu g/ml, 35 mu g/ml, 40 mu g/ml, 45 mu g/ml, 50 mu g/ml, 55 mu g/ml, 60 mu g/ml, 65 mu g/ml, 70 mu g/ml, 75 mu g/ml, 80 mu g/ml, 85 mu g/ml, 90 mu g/ml, 95 mu g/ml and 100 mu g/ml.
The antigen protein in the vaccine composition has good immunogenicity, and can stimulate good immune response when the antigen content is low, so that the vaccine composition only can completely protect chickens.
As a more preferred embodiment of the invention, in the vaccine composition containing the avibacterium paragallinarum antigen, the content of the antigenic protein in the HMTp2102 area of the avibacterium paragallinarum type A is 25 to 100 mu g/ml, the content of the antigenic protein in the HMTp2102 area of the avibacterium paragallinarum type B is 50 to 100 mu g/ml, and the content of the antigenic protein in the HMTp2102 area of the avibacterium paragallinarum type C is 25 to 100 mu g/ml.
As a further preferred embodiment of the present invention, in the vaccine composition containing the avibacterium paragallinarum antigen, the content of the antigenic protein in the HMTp2102 region of avibacterium paragallinarum type A is 15 μ g/ml, the content of the antigenic protein in the HMTp2102 region of avibacterium paragallinarum type B is 25 μ g/ml, and the content of the antigenic protein in the HMTp2102 region of avibacterium paragallinarum type C is 15 μ g/ml.
When the content of each antigen component in the vaccine composition containing the avibacterium paragallinarum antigen is selected, the antigen components generate synergistic action, and the immune protection effect is equivalent to that of a monovalent vaccine containing the same antigen component with higher dose.
as a further preferred embodiment of the present invention, in the vaccine composition containing the avibacterium paragallinarum antigen, the content of the antigenic protein in the HMTp2102 region of avibacterium paragallinarum type A is 25 μ g/ml, the content of the antigenic protein in the HMTp2102 region of avibacterium paragallinarum type B is 50 μ g/ml, and the content of the antigenic protein in the HMTp2102 region of avibacterium paragallinarum type C is 25 μ g/ml.
When the content of each antigen component in the vaccine composition containing the avibacterium paragallinarum antigen is selected, the vaccine composition can completely protect the avibacterium paragallinarum of different serotypes from different regional sources.
As an embodiment of the present invention, in the vaccine composition containing an avibacterium paragallinarum antigen of the present invention, the pharmaceutically acceptable carrier includes an adjuvant, and the adjuvant includes: (1) alumino-gel adjuvant, saponin, avridine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of 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 dipeptide and Gel adjuvant;
Preferably, the saponin is Quil A, QS-21, GPI-0100;
Preferably, the emulsion is an SPT emulsion, an MF59 emulsion, or an emulsion formed from an oil in combination with an emulsifier, the emulsion may be based on light liquid paraffin oil, isoprenoid oil resulting from the oligomerization of olefins (such as squalane or squalene oil, oil resulting from the oligomerization of olefins, in particular isobutene or decene), linear alkyl-containing esters of acids or alcohols (more particularly vegetable oil, ethyl oleate, propylene glycol di- (caprylate/caprate), glycerol tri- (caprylate/caprate) or propylene glycol dioleate), esters of branched fatty acids or alcohols (in particular isostearate); the emulsifier is a nonionic surfactant (especially esters of polyoxyethylated fatty acids (e.g. oleic acid), sorbitan, mannide (such as anhydrous mannitol oleate), aliphatic diols, glycerol, polyglycerol, propylene glycol and oleic, isostearic, ricinoleic or hydroxystearic acid, which may be ethoxylated, ethers of fatty alcohols and polyhydric alcohols (e.g. oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers (especially L121));
Preferably, the polymer of acrylic acid or methacrylic acid is a crosslinked polymer of acrylic acid or methacrylic acid, in particular a compound carbomer crosslinked with polyalkenyl ethers or polyalcohols of sugars, preferably carbopol 974P, 934P and 971P;
Preferably, the copolymer of maleic anhydride and alkenyl derivative is a copolymer EMA of maleic anhydride and ethylene;
Preferably, the adjuvant is a white 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.
as one embodiment of the present invention, the pharmaceutically acceptable carrier includes drugs, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives; the immunostimulant includes alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 2(IL 2).
To prepare such compositions, methods well known in the art may be used.
As an embodiment of the present invention, in the vaccine composition containing an avibacterium paragallinarum antigen of the present invention, the vaccine composition further includes one or more of the following antigens: a chicken newcastle disease virus antigen, an avian influenza virus antigen, an infectious bronchitis virus antigen, a chicken infectious bursal disease virus antigen, an egg drop syndrome virus antigen, an avian reovirus antigen, an escherichia coli antigen, an avian adenovirus antigen, a mycoplasma synoviae, a mycoplasma gallisepticum antigen, a pasteurella multocida antigen, a marek's virus antigen, an avian encephalomyelitis virus antigen, or a chicken infectious laryngotracheitis virus antigen.
The vaccine composition of the present invention may contain various antigen components, and may be used in combination vaccine or composite vaccine for immunization to simplify the immunization process.
The vaccine composition of the present invention may further comprise other pathogens to be used in combination to prepare a composite vaccine against various diseases including avian paragallinarum infection. The term "combination vaccine" refers to vaccines prepared from bacterial and viral antigens. For example, the avibacterium paragallinarum subunit antigen of the present invention may be combined with a newcastle disease virus antigen. In one embodiment of the present invention, in the vaccine composition of the present invention, the newcastle disease virus antigen is a gene type vii inactivated whole virus antigen, subunit antigen or synthetic peptide antigen.
As an embodiment of the present invention, in the vaccine composition of the present invention, the newcastle disease virus antigen is a gene type vii newcastle disease virus N7a inactivated whole virus antigen, and the newcastle disease virus (gene type vii) N7a strain has a preservation number of CCTCC NO: v201545.
newcastle Disease Virus (gene vii) strain N7a (Newcastle Disease Virus (genotype vii), strain N7a) deposited in the chinese type culture collection with a deposition number of CCTCCNO: v201545, preservation date of 2015, 10 months and 19 days, and preservation address of wuhan, wuhan university, china, disclosed in chinese patent application CN 107281479A.
As a more preferred embodiment of the present invention, in the vaccine composition containing an avibacterium paragallinarum antigen of the present invention, the newcastle disease virus antigen is an inactivated whole virus antigen of newcastle disease virus (gene vii) N7a strain, and the newcastle disease virus (gene vii) N7a strain has a preservation number of CCTCC NO: v201545; the content of the inactivated whole virus antigen of the Newcastle disease virus N7a strain is 108.0EID50/0.1 ml-109.0 EID50/0.1ml before inactivation.
As a more preferable embodiment of the invention, in the vaccine composition containing the avibacterium paragallinarum antigen, the content of the antigenic protein in the A-type HMTp2102 region of the avibacterium paragallinarum is 25 mug/ml, the content of the antigenic protein in the B-type HMTp2102 region of the avibacterium paragallinarum is 50 mug/ml, the content of the antigenic protein in the C-type HMTp2102 region of the avibacterium paragallinarum is 25 mug/ml, and the content of the inactivated whole virus antigen of the N7a strain of the Newcastle disease virus is 108.0EID50/0.1ml before inactivation.
In the combined vaccine composition of the present invention, the pharmaceutically acceptable carrier may include an adjuvant, and the adjuvant may have the same selection range as the monovalent vaccine composition of the present invention.
Preferably, the adjuvant is a white oil adjuvant, and the dosage of the adjuvant is 66% by volume.
The vaccine composition containing the avibacterium paragallinarum antigen and the newcastle disease virus N7a inactivated whole virus antigen can completely protect the avibacterium paragallinarum and the newcastle disease virus and only has a treatment effect on infected chickens.
The invention also provides a method for preparing the vaccine composition containing the avibacterium paragallinarum antigen, wherein the method comprises the following steps: respectively cloning and recombining an antigen protein gene of the A-type HMTp2102 region of the avibacterium paragallinarum shown in SEQ ID NO.1, an antigen protein gene of the B-type HMTp2102 region of the avibacterium paragallinarum shown in SEQ ID NO.2 and an antigen protein gene of the C-type HMTp2102 region of the avibacterium paragallinarum shown in SEQ ID NO.3 to obtain a recombined antigen protein expression vector of the A-type HMTp2102 region of the avibacterium paragallinarum, a recombined antigen protein expression vector of the B-type HMTp2102 region of the avibacterium paragallinarum and a recombined antigen protein expression vector of the C-type HMTp2102 region of the avibacterium paragallinarum; step (2) respectively expressing the recombinant expression vector of the A-type HMTp2102 region antigen protein of the avibacterium paragallinarum, the recombinant expression vector of the B-type HMTp2102 region antigen protein of the avibacterium paragallinarum and the recombinant expression vector of the C-type HMTp2102 region antigen protein of the avibacterium paragallinarum in the step (1) to obtain the expressed recombinant A-type HMTp2102 region antigen protein of the avibacterium paragallinarum, the recombinant B-type HMTp2102 region antigen protein of the avibacterium paragallinarum and the recombinant C-type HMTp2102 region antigen protein of the avibacterium paragallinarum; and step (3) mixing the recombinant avibacterium paragallinarum A type HMTp2102 region antigen protein, the recombinant avibacterium paragallinarum B type HMTp2102 region antigen protein and the recombinant avibacterium paragallinarum C type HMTp2102 region antigen protein expressed in the step (2) according to the content ratio; an adjuvant is added.
In one embodiment of the present invention, in the method for preparing the vaccine composition containing the avibacterium paragallinarum antigen according to the present invention, the recombinant avibacterium paragallinarum a-type HMTp2102 region antigen protein, the recombinant avibacterium paragallinarum B-type HMTp2102 region antigen protein, and the recombinant avibacterium paragallinarum C-type HMTp2102 region antigen protein expressed in the step (2) are active proteins.
The HMTp2102 region antigen protein active protein can be prepared by recombination expression by using a conventional method, and the prepared recombinant protein has biological or physiological activity and often has a three-level or four-level spatial structure. Conventional recombinant expression methods such as secretory expression of a prokaryotic expression system, expression of soluble protein by a eukaryotic expression system and expression of soluble protein by a yeast expression system. Artificially synthesized proteins having biological activity may also be used; or expressing the active protein formed by denaturation and renaturation of the formed inclusion body.
the invention also provides application of the vaccine composition containing the avibacterium paragallinarum antigen in preparing a medicament for preventing and treating diseases caused by avibacterium paragallinarum infection.
A non-exhaustive list of diseases associated with avibacterium paragallinarum to which the present invention may be applied includes, for example, facial edema, sinusitis, lacrimation, decreased egg production, hindered growth of the growing chicken, delayed laying hen laying period, decreased broiler weight gain, and the like.
Detailed Description
"vaccine composition" refers to a pharmaceutical composition comprising the immunogenicity of avibacterium paragallinarum that induces, stimulates or enhances the immune response of a chicken against avibacterium paragallinarum. The vaccine composition includes an immunizing amount of an immunoprotective antigenic protein of avibacterium paragallinarum.
The term "immunizing amount" shall be understood as an "immunologically effective amount," also referred to as an immunoprotective amount or an amount effective to produce an immune response, of antigen effective to induce an immune response in a recipient, sufficient to prevent or ameliorate the signs or symptoms of disease, including adverse health effects or complications thereof. The immune response may be sufficient for diagnostic purposes or other testing, or may be suitable for use in preventing signs or symptoms of disease, including adverse health consequences or complications thereof caused by infection by a pathogen. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with a wild-type strain, while the protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs such as mortality, reduction in morbidity, temperature values, overall physiological condition of the subject, and overall health and performance. The immune response may include, but is not limited to, induction of cellular and/or humoral immunity.
The term "pharmaceutically acceptable carrier" refers to a carrier or diluent, preferably an adjuvant, that is a component other than the antigen in the vaccine composition of the present invention that does not stimulate the body and does not hinder the biological activity and properties of the compound used.
The term "adjuvant" is a non-specific immunopotentiator that, when injected with or pre-injected into a body with an antigen, enhances the body's immune response to the antigen or alters the type of immune response.
Adjuvants may include aluminous adjuvants; saponins (saponin), such as Quil A, QS-21(Cambridge Biotech Incorporation, Cambridge MA), GPI-0100(Galenica Pharmaceuticals Incorporation, Birmingham AL); a water-in-oil emulsion; an oil-in-water emulsion; a water-in-oil-in-water emulsion; polymers of acrylic acid or methacrylic acid; maleic anhydride and alkenyl (alkenyl) derivatives. The term "emulsion" may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils (isoprenoid oils) resulting from the oligomerization of olefins, such as squalane (squalane) or squalene oil (squalene oil), in particular isobutene or decene; linear alkyl-containing esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di- (caprylate/caprate), glycerol tri- (caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearic acid esters. The oil is used in combination with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (such as, for example, anhydrous mannitol oleate), of aliphatic diols (glycols), of polyglycerols, of propylene glycol and of oleic acid, of isostearic acid, of ricinoleic acid or of hydroxystearic acid, which are optionally ethoxylated, and also polyoxypropylene-polyoxyethylene block copolymers, in particular the Pluronic products, in particular L121. See The description of The same and The reactive application of adjuvants by Hunter et al (Ed. by DES Stewart-Tull, John Wiley and Sons, New York,1995:51-94) and The description of Vaccine by Todd et al (1997,15: 564-570). For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of Vaccine design, the Subunit and adivant propaach (Plenum Press,1995) written by Powell M and Newman M can be used. The term "polymer of acrylic or methacrylic acid" is preferably a crosslinked polymer of acrylic or methacrylic acid, in particular a polyalkenyl ether or polyalcohol crosslinked with a sugar (sugar), these compounds being known under the name Carbomer (Carbopol, trade name Carbopol) (Phameuropa,1996,8 (2)). Those skilled in the art can also see US2909462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are substituted by unsaturated aliphatic hydrocarbon groups (aliphatic radial) having at least 2 carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups (ethylenically unsaturated groups). The unsaturated groups may themselves contain other substituents, such as methyl. These products are sold under the name carbopol, (BF Goodrich, Ohio, USA) are particularly suitable. They are crosslinked with allyl sucrose or with allyl pentaerythritol. Among these, mention may be made of carbopols 974P, 934P and 971P, the most preferred being the use of carbopol 971P. The term "copolymers of maleic anhydride and alkenyl derivative" also contemplates the maleic anhydride and ethylene copolymers ema (monsanto), which are dissolved in water to give an acidic solution, neutralized, preferably to physiological pH, in order to give an adjuvant solution into which the immunogenic, immunogenic or vaccinal composition itself can be incorporated. The term "adjuvant" also includes, but is not limited to, the RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A (monophosphoryl lipid A), Avridine lipoamine adjuvant, E.coli heat labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant, and the like. Preferably, the adjuvant comprises one or more of an alumina Gel adjuvant, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a water-in-oil-in-water emulsion, a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and an alkenyl (alkenyl) derivative, a RIBI adjuvant system, a Blockco-polymer, SAF-M, a monophosphoryl lipid A, Avridine lipid-amine adjuvant, escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide or Gel adjuvant.
The term "combination vaccine" as used herein is intended to mean a vaccine prepared from a viral mixture of a recombinant virus of the invention and at least one different virus.
The term "composite vaccine" as used herein refers to a vaccine prepared from viruses and bacteria. For example, the recombinant viruses of the present invention may be mixed or combined with newcastle disease virus, avian adenovirus virus and/or escherichia coli, mycoplasma synoviae.
The term "active protein" as used herein refers to a protein having a spatial structure, biological or physiological activity, and may be a recombinant protein, such as a soluble protein secreted by a prokaryotic expression system, a soluble protein expressed by a eukaryotic expression system, or a soluble protein expressed by a yeast expression system. Or artificially directly synthesizing protein with biological activity; or expressing the active protein formed by denaturation and renaturation of the formed inclusion body.
The term "prevention" as used in the present invention refers to all actions of suppressing infectious rhinitis or delaying the onset of disease in chickens by administering the vaccine composition according to the present invention. The term "treatment" refers to all actions leading to a reduction or improvement of the symptoms caused by an avibacterium paragallinarum infection by administration of the vaccine composition according to the invention.
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The sample treatment solution used in the examples of the present invention was PBS buffer (pH7.4, 0.02mol/L), and the formulation of 1L volume of PBS buffer was exemplified by: na2HPO4 & 12H2O 5.8.8 g, NaH2PO4 & 2H2O 0.59.59 g, but not limited to this formulation; unless otherwise specified, the samples were diluted with PBS buffer.
The chemical reagents used in the invention are all analytically pure and purchased from the national pharmaceutical group.
The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
example 1 construction of Gene recombinant plasmid in region 2 of avian Paragallinarum HMTp2102
1.1 primer design
According to Apg A type, B type and C type HMTp2102 area 2 gene sequences, 1 pair of universal primers are designed by using Primer5.0 software, BamH I enzyme cutting sites and Hind III enzyme cutting sites and protective bases are respectively introduced into the 5' ends of upstream and downstream primers and are used for amplifying the full-length gene sequences of the HMTp2102 area 2, the sequences of the primers are shown in a table 1, wherein the drawn line part is the introduced enzyme cutting sites, and the primers are synthesized by Jinzhi company.
TABLE 1 primer sequences
1.2 extraction of avian Paragallinarum genomic DNA
Apg A type HN3 strain, B type HN5 strain and C type SD3 strain are respectively streaked and inoculated on a TSA plate, the culture is carried out for 24 to 36 hours in a 5 percent CO2 incubator at 37 ℃, 3 to 5 typical colonies are selected and inoculated in a test tube containing 5ml of chicken soup culture medium, the shake culture is carried out for 10 to 12 hours at 37 ℃ and 180rpm, and the obtained bacterial liquid is respectively extracted from genome DNA of Apg different serotype strains by a kit.
1.3 PCR amplification of the Gene in the 2 region of HMTp2102
The PCR reaction system is as follows: 1.0ul of each upstream and downstream primer, 25ul of 2 × TransStart FastPfu PCR Supermix, 21ul of sterile double distilled water and 2ul of template; the PCR reaction program is: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 20s, renaturation at 55 ℃ for 20s, extension at 72 ℃ for 2min, 30 cycles, and final extension at 72 ℃ for 10 min. The amplification products were detected electrophoretically using a 1% gel.
As a result: the sizes of target genes amplified by the Apg A and B, C type strain DNA templates are respectively 1635bp, 1611bp and 1620bp theoretically, the sizes of the amplified target genes are consistent with the theory, the amplified A, B, C type target gene fragments are respectively sent to Invitrogen company for sequencing, codon optimization is carried out according to the sequencing result, and the optimized gene sequences of the 2 region of the A, B, C type HMTp2102 region are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 of the sequence table.
1.4 construction of recombinant plasmid of avibacterium paragallinarum
The optimized A, B, C type HMTp2102 region 2 gene is sent to Suzhou Jinzhi biotechnology limited for complete sequence synthesis and is connected to pET-32a vector, and the connection system is as follows: 20 ul of target gene, 0.5 ul of pET-32a vector, 2.0 ul of ligase buffer, 1.0ul of T4DNA ligase and 8.5 ul of sterilized double distilled water. The ligation reaction conditions were 16 ℃ for 30 min.
1.5 identification of recombinant plasmid of avibacterium paragallinarum
Taking out E.coli DH5 alpha competent cells from a refrigerator at the temperature of-80 ℃, slowly unfreezing the E.coli DH5 alpha competent cells on ice, adding 10 mu l of the ligation product after the E.coli DH5 alpha competent cells are completely thawed, uniformly mixing, and carrying out ice bath for 30 min; heating in a 42 deg.C water bath for 45s, ice-cooling for 1min, adding 890 μ l LB liquid medium, shake-culturing at 37 deg.C for 60min, inoculating the culture to LB agar plate containing 100 μ g/ml ampicillin, and culturing at 37 deg.C overnight. Typical colonies were picked, inoculated into LB liquid medium containing 100. mu.g/ml ampicillin, and cultured at 200rpm for 10 hours. Boiling 1ml of bacterial liquid for 10min, centrifuging at 12000rpm for 5min, taking the supernatant as a template, and performing PCR identification, wherein the PCR procedure is the same as that in example 1.3; and simultaneously extracting plasmids, carrying out double enzyme digestion identification by using BamH I/Hind III, detecting the enzyme digestion product by 1% gel electrophoresis, and recovering a target gene fragment. The plasmid with positive PCR and enzyme cutting identification is sent to Jinzhi corporation for sequencing. The recombinant plasmids with correct sequences identified by sequencing are respectively named as PET-A, PET-B, PET-C.
Example 2 construction of recombinant expression strains BL21-A, BL21-B and BL21-C
2.1 transformation of E.coli BL-21 competent cells by recombinant plasmids and expression of recombinant proteins
e.coli BL-21 competent cells were transformed with recombinant plasmids whose sequences were identified by sequencing in example 1 to obtain recombinant expression strains BL21-A, BL21-B, BL21-C, respectively, and E.coli BL-21 competent cells were transformed with empty plasmid pET-32a in the same manner, spread on LB solid medium containing ampicillin, cultured at 37 ℃ for 16 hours, picked single colonies that grew well, inoculated on 10ml LB liquid medium containing ampicillin, and cultured at 37 ℃ for 2 hours with shaking at 200 rpm. And when the OD600 value of the bacterial liquid reaches 0.6-0.8, respectively taking 1ml of uninduced expression bacterial liquid and empty plasmid bacterial liquid as samples before induction, adding IPTG (isopropyl thiogalactoside) with the final concentration of 1.0mmol/L into the residual bacterial liquid for induction expression for 3-4 h, and respectively taking 1ml of induced expression bacterial liquid and empty plasmid bacterial liquid as samples after induction.
2.2 SDS-PAGE detection of recombinant proteins
Centrifuging 1ml of the sample taken in example 2.1 at 12000rpm for 1min, discarding the supernatant, adding 0.5ml of PBS with the pH value of 7.4 into the precipitate for resuspension, carrying out ultrasonic lysis at 2-8 ℃ in an ice bath, centrifuging at 12000rpm for 30min, taking out the whole supernatant, resuspending the precipitate with 0.5ml of PBS with the pH value of 7.4, respectively adding 2 xSDS Loading Buffer with the same volume into part of the supernatant and the precipitate, uniformly mixing, boiling in boiling water for 5-10 min, centrifuging, and taking 10ul of the supernatant for SDS-PAGE.
As a result: the induced BL21-A, BL21-B, BL21-C recombinant expression bacteria all have bands near 60KDa, the sizes of the bands are basically consistent with the sizes of the three expected recombinant proteins, expression products are soluble expressions in the cracked supernatant, and the non-induced recombinant expression bacteria and the induced empty plasmid bacteria do not have target bands.
2.3 Western Blot detection of recombinant proteins
(1) Performing SDS-PAGE on the induced expression product; (2) transferring to a PVDF membrane by using a protein membrane transfer instrument; (3) after transfer printing is finished, washing the PVDF membrane by PBST, then putting the PVDF membrane into ponceau dyeing solution for dyeing for 5-10 min, and after a protein band appears, washing the PVDF membrane for a plurality of times by PBST until the ponceau dyeing solution is washed away; (4) sealing the PVDF membrane by PBST sealing liquid containing 5% skim milk at room temperature for about 2h in a shaking way; (5) after washing with PBST, rabbit anti-Apg A-type HN3 strain, B-type HN5 strain or C-type SD3 strain polyclonal antiserum was added and reacted at room temperature for 2 hours, PBST was washed 4 times, HRP-labeled goat anti-rabbit IgG-HRP diluted at 1:2000 was added and reacted at 37 ℃ for 2 hours, followed by color development.
As a result: the p-A, p-B, p-C protein expressed by the recombinant BL21-A, BL21-B, BL21-C can respectively and specifically react with rabbit anti-Apg A-type, B-type and C-type positive serum.
2.4 purification of the recombinant protein p-A, p-B, p-C
Respectively inducing and expressing 500mL of BL21-A, BL21-B, BL21-C recombinant expression bacteria by the method in example 2.1, centrifuging and collecting thalli, suspending in 50mL of thalli lysate, acting at room temperature for 30min, carrying out ice bath ultrasonic lysis at 2-8 ℃, centrifuging at 12000rpm for 30min, and collecting supernatant containing soluble protein. Purifying the recombinant protein from the supernatant by His affinity chromatography, filling the purified recombinant protein into a dialysis bag, dialyzing the purified recombinant protein by PBS with the pH value of 7.4 at the temperature of 2-8 ℃ under the stirring condition, collecting the dialyzed recombinant protein, and performing SDS-PAGE detection and protein concentration determination on the recombinant protein, wherein the concentrations of the p-A, p-B, p-C protein after analysis and purification are 1.5mg/mL, 1.0mg/mL and 1.0mg/mL respectively.
EXAMPLE 3 preparation of infectious coryza vaccine for chickens
3.1 preparation of recombinant Chicken infectious rhinitis subunit vaccine
Combining the p-A, p-B, p-C recombinant proteins according to a certain proportion, adding tween-80 to prepare a water phase, mixing and emulsifying the water phase and imported white oil according to the volume ratio of 1:2 to prepare the chicken infectious rhinitis subunit vaccines containing three recombinant protein components with different concentrations, wherein the concentration of the three recombinant protein components in each vaccine is shown in table 2.
TABLE 2 different vaccine protein content
Example 4 Chicken infectious rhinitis vaccine test
4.1 safety inspection
90 SPF chickens of 8 weeks old are divided into 9 groups and 10 groups, and the chicken infectious rhinitis vaccine prepared in the example 3 is immunized. Each group was administered with 1.0ml of vaccine intramuscularly in the legs for 14 consecutive days. The results show that no adverse reaction is seen in 14 days of observation of the SPF chickens immunized by the vaccines, the mental state is good, the food intake and the water drinking are normal, and no death situation exists. Partial vaccine residues can be seen in the vaccine injection part by the autopsy, but adverse reactions such as induration, abscess, ulceration and the like are not seen. The chicken infectious rhinitis subunit vaccine is proved to have better safety to SPF chickens.
4.2 efficacy test
180 SPF chickens (8-week-old) were randomly divided into 18 groups and 10 chickens/group, and the infectious coryza vaccine prepared in example 3 was immunized. The 1 st to 6 th groups of immune vaccines are respectively 1 to 6, 7 th to 9 th groups of immune vaccines 7, 10 th to 12 th groups of immune vaccines 8, 13 th to 15 th groups of immune vaccines 9, and 16 th to 18 th groups of immune vaccines are used as controls. Each immunization group is injected with 0.5ml of vaccine, the control group is injected with 0.5ml of PBSl, 28 days after immunization, the 1 st, 2 nd, 7 th, 10 th, 13 th and 16 th groups are injected with 0.2ml of A-type HN3 strain liquid (the number of live bacteria is about 2.0 multiplied by 104 CFU); group 3, 4, 8, 11, 14, 17, 0.2ml of bacterial suspension of HN5 strain B (containing about 1.0 × 104CFU viable count) was injected into the infraorbital sinus; in groups 5, 6, 9, 12, 15 and 18, 0.2ml of bacterial suspension of type C SD3 strain (containing about 1.0 × 104CFU viable count) was injected into the infraorbital sinus. After continuously observing for 7 days, the disease is judged to occur when any symptom of face swelling or rhinorrhea appears, and the toxic attack protection result is shown in table 3. The results show that vaccine 1 can not provide effective immune protection against avian paragallinarum type a, and vaccine 2, vaccine 7, vaccine 8 and vaccine 9 can provide complete immune protection; aiming at the avibacterium paragallinarum type B, the vaccine 3 can not provide effective immune protection, and the vaccine 4, the vaccine 7, the vaccine 8 and the vaccine 9 can provide complete immune protection; aiming at the avibacterium paragallinarum type C, the vaccine 5 can not provide effective immune protection, and the vaccine 6, the vaccine 7, the vaccine 8 and the vaccine 9 can provide complete immune protection; all the control groups with all offensive toxin were attacked. The avian paragallinarum A-type recombinant protein provided by the invention is proved to have good immunogenicity, and 100% immune protection can be generated on homologous strains when the content of the recombinant protein of a monovalent vaccine is more than or equal to 25 mug/ml or the content of the recombinant protein of a trivalent vaccine is more than or equal to 15 mug/ml; the B-type recombinant protein of the avibacterium paragallinarum has good immunogenicity, and 100 percent of immune protection can be generated on homologous strains when the content of the recombinant protein of a monovalent vaccine is more than or equal to 50 mu g/ml or the content of the recombinant protein of a trivalent vaccine is more than or equal to 25 mu g/ml; the C-type recombinant protein of the avibacterium paragallinarum has good immunogenicity, and 100 percent of immune protection can be generated to homologous strains when the content of the recombinant protein of a monovalent vaccine is more than or equal to 25 mu g/ml or the content of the recombinant protein of a trivalent vaccine is more than or equal to 15 mu g/ml. Further indicates that the trivalent vaccine provided by the invention has better immune effect.
Compared with the corresponding monovalent vaccines, the trivalent vaccine provided by the invention has the same immune effect at lower content, and the three subunit antigen proteins in the trivalent vaccine generate a synergistic interaction effect, so that the immune effect of each subunit antigen protein is improved.
TABLE 3 immunoprotection results of chicken infectious rhinitis vaccines
example 5 Cross-protection assay for infectious coryza vaccines against heterologous strains of chicken
The vaccine 8 prepared in example 3 was immunized by randomly dividing 240 SPF chickens 8 weeks old into 24 groups, 10 groups/group. The 19 th, 21 st, 23 rd, 25 th, 27 th, 29 th, 31 th, 33 th, 35 th, 37 th, 39 th, 41 th groups were immunization groups, and the 20 th, 22 th, 24 th, 26 th, 28 th, 30 th, 32 th, 34 th, 36 th, 38 th, 40 th, 42 th groups were immunization groups. 0.5 ml/vaccine is injected into each immunization group, 0.5 ml/PBS is injected into the control group, 28 days after immunization, 0.2ml of A-type Hpg-8 bacterial liquid of an early-stage isolate A is injected into 19 th and 20 th groups of chickens, and the infraorbital sinus is injected into the early-stage isolate (the number of live bacteria is about 2.0 multiplied by 106 CFU); in 21 st and 22 nd groups of 20 chickens, 0.2ml of A-type HB2 strain liquid newly separated from Hebei province of China (the number of viable bacteria is about 2.5 multiplied by 105CFU) is injected into infraorbital sinus; in 23 rd and 24 th groups of 20 chickens, 0.2ml of A-type JS6 strain liquid (with viable count of about 2.5 × 105CFU) newly separated from Jiangsu province in China is injected into suborbital sinus; in groups 25 and 26, 20 chickens were injected into the infraorbital sinus, and 0.2ml of a type-A GD11 strain (containing about 2.5X 105CFU of viable bacteria) newly isolated from Guangdong province in China was injected into the infraorbital sinus; in groups 27 and 28, 20 chickens were injected into the infraorbital sinus of 0.2ml of bacterial liquid of the B-type BJ4 strain newly isolated from Beijing City of China (the viable count is about 5.0X 105 CFU); in groups 29 and 30, 20 chickens were injected into the infraorbital sinus, and 0.2ml of bacterial liquid (containing about 2.5X 105CFU of viable bacteria) of the type B JX16 strain newly isolated from Jiangxi province in China was injected into the infraorbital sinus; in groups 31 and 32, 20 chickens were injected into the infraorbital sinus, and 0.2ml of bacterial suspension (containing about 2.2X 105CFU of viable bacteria) of type B SD4 strain newly isolated from Shandong province in China was injected into the infraorbital sinus; injecting 0.2ml of B type ZJ3 strain liquid newly separated from Zhejiang province of China into the infraorbital sinus of 20 chickens in 33 th and 34 th groups (the number of live bacteria is about 2.5 multiplied by 105 CFU); in 35 th and 36 th groups of 20 chickens, 0.2ml of bacterial liquid of the C-type Hpg-668 strain of the early isolate is injected into the infraorbital sinus (the number of the live bacteria is about 2.0 multiplied by 106 CFU); in groups 37 and 38, 20 chickens were injected into the infraorbital sinus, and 0.2ml of bacterial liquid (containing about 2.0X 105CFU of viable bacteria) of strain C AH1 newly isolated from Anhui province of China was injected into the infraorbital sinus; in groups 39 and 40, 20 chickens were injected into the infraorbital sinus, and 0.2ml of bacterial suspension (containing about 2.0 × 105CFU of viable count) of C-type HN1 strain newly isolated from Henan province, China was injected into the infraorbital sinus; in groups 41 and 42, 20 chickens were injected into the infraorbital sinus, and 0.2ml of the bacterial suspension of the newly isolated strain C-GD 12 strain (containing about 2.0X 105CFU viable cell count) isolated from GD province of China was injected into the infraorbital sinus. After continuously observing for 7 days, the disease is judged to occur when any symptom of face swelling or rhinorrhea appears, and the toxic attack protection result is shown in table 4. The result shows that the chicken infectious rhinitis subunit vaccine can generate better immune protection effect for the virus attack of heterologous strains.
TABLE 4 Cross-protection results of chicken infectious rhinitis subunit vaccine against heterologous strains
Example 6 comparison of the efficacy of the Chicken infectious rhinitis subunit vaccine and the imported vaccine against SPF chickens
90 SPF chickens of 8 weeks old are randomly divided into 9 groups, 10 groups, 43 groups, 46 groups and 49 groups to immunize the vaccine 8 prepared in the example 3, and 44 groups, 47 groups and 50 groups to immunize imported chicken infectious rhinitis trivalent inactivated vaccine 10 (containing type A W strains, the content of which is more than or equal to 6 x 108CFU/ml before inactivation, containing type B Spross strains, the content of which is more than or equal to 6 x 108CFU/ml before inactivation, and containing type C modelsto strains, the content of which is more than or equal to 6 x 108CFU/ml before inactivation), and the 45 th group, 48 groups and 51 groups are used as challenge control groups. On 28 days after immunization, 30 chickens in groups 43, 44 and 45 were injected into the infraorbital sinus with 0.2ml of the A-type HN3 strain (containing about 2.0X 104CFU viable cell count); 30 chickens in groups 46, 47 and 48, 0.2ml of bacterial liquid of HN5 strain B (with the viable count of about 1.0 × 104CFU) is injected into the infraorbital sinus; 30 chickens in 49, 50 and 51 groups were injected with 0.2ml of bacterial suspension of type C SD3 strain (containing about 1.0X 104CFU viable count) into the infraorbital sinus. The results of the challenge protection are shown in Table 5 after 7 days of continuous observation. The results show that compared with an attacking contrast group, the immune group of the vaccine 8 and the imported vaccine 10 have very obvious difference in protection rate, the immune group of the vaccine 8 has better protection effect than the imported vaccine, and the subunit vaccine of the invention has better immune protection effect.
TABLE 5 comparison of the potency of chicken infectious rhinitis subunit vaccine and imported vaccine against SPF chickens
Example 7 preparation of antigen of Newcastle disease Gene type VII N7a Strain
The NDV N7a strain was diluted 10,000 times with sterile physiological saline, and 20 SPF embryos of 10 days old were inoculated, 0.1ml of each embryo was inoculated, and incubation was continued at 37 ℃. And (3) discarding the dead embryos within 24 hours after inoculation, immediately placing the dead embryos for 24-120 hours at 4 ℃, collecting the mixed sample for 120 hours, and determining that HA and EID50 for preparing the vaccine poison are 9.6 and 109.5EID50/0.1ml respectively. Introducing the newcastle disease virus liquid with the determined titer into an inactivation tank, metering and adding 10% formaldehyde solution, starting a stirrer to stir so as to fully mix the solution, wherein the final concentration of formaldehyde is 0.1%, and inactivating the solution for 16 hours at 37 ℃.
EXAMPLE 8 preparation of Chicken infectious rhinitis, Newcastle disease Gene type VII bivalent inactivated vaccine
Preparing vaccine 11 from the avian paragallibacterium p-A, p-B, p-C recombinant protein prepared in examples 2 and 7 and the antigen of the VII-type N7a strain of the Newcastle disease gene, preparing four antigen solutions into mixed antigen solutions according to the final antigen content of combined vaccine, mixing the antigen solutions with imported white oil according to the volume ratio of 1:2, stirring at 800rpm for 40min, and adding 1% thimerosal solution before stopping stirring to ensure that the final concentration of the thimerosal solution is not more than 0.01%. The specific formulation of vaccine 11 is shown in table 6.
TABLE 6 chicken infectious rhinitis and Newcastle disease gene VII combined inactivated vaccine components and contents
composition (I) Vaccine 11
p-A(μg/ml) 25
p-B(μg/ml) 50
p-C(μg/ml) 25
50n7a strain EID50(EID50/0.1ml) 108.0
White oil adjuvant (V/V) 66％
Example 9 immune Effect test of Chicken infectious rhinitis and Newcastle disease Gene type VII bivalent inactivated vaccine
1. Newcastle disease component
20 SPF chickens of 30 days old are selected and divided into two groups, 10 chickens per group. Group 52 was immunized with 20. mu.l/vaccine prepared in example 8, and group 53 was used as a control group. On day 21 after immunization, each chicken was bled, serum was separated, and HI antibody titer was determined. The immunisation group and the control were challenged intramuscularly with the dose of newcastle disease virus HN1101 strain 105EID 50/mouse, observed for 14 days, and the number of morbidity, mortality and protection rate were recorded, and the results are shown in table 7. Therefore, the geometric mean values of the antibody titers of the immunized chicken HI are all larger than or equal to 4log2, and the geometric mean values of the antibody titers of the control chicken HI are all smaller than or equal to 2log2, so that the effect detection standard is met.
TABLE 7 partial potency test results of the chicken infectious rhinitis and Newcastle disease gene type VII bivalent inactivated vaccine Newcastle disease
Note: HI antibodies were determined as geometric means of the immunized chicken antibodies, indicated as X ± SD, with X representing the mean and SD representing the standard deviation.
The results show that vaccine 11 group produced higher antibodies 21 days after immunization, and completely protected against the challenge of newcastle disease virulent virus compared to the control.
The vaccine 11 is proved to be capable of protecting the lethal dose of the Newcastle disease virulent virus attack, and the complete protection of the chicken flock can be provided by the content of the vaccine not less than 108.0EID50/0.1 ml.
2. Infectious coryza parts of chicken
Selecting 60 SPF chickens of 56 days old, randomly dividing into 6 groups, 10 groups, 54 th to 56 groups to immunize the vaccine 11 prepared in the embodiment 8, and 57 th to 59 th groups to be control groups. Each immunization group was injected with 0.5ml of vaccine, 28 days after immunization, the 54 th and 57 th groups were injected with 0.2ml of culture diluent (about 2 ten thousand live bacteria) of HN3 chicken soup for 12 hours; injecting HN5 chicken soup into infraorbital sinus of 55 th group and 58 th group for 12 hours, and diluting the culture with 0.2ml (about 2 ten thousand live bacteria); SD3 chicken soup was injected into the infraorbital sinus of 56 th and 59 th groups for 12 hours, and 0.2ml of culture diluent (about 2 ten thousand live bacteria) was added. After 7 days of observation, the toxic substance counteracting protection results are shown in Table 8. The control group had all the disease (swollen face or running nose) and the immunized group had all the protection.
TABLE 8 partial potency test results of chicken infectious rhinitis and newcastle disease gene type VII bivalent inactivated vaccine for chicken infectious rhinitis
the results prove that the chicken infectious rhinitis and Newcastle disease gene VII combined inactivated vaccine meets the product requirements, has a good immune protection effect, and can effectively prevent outbreak of two epidemic diseases.
Example 10 test of the use of the type VII bivalent inactivated vaccine for infectious coryza and Newcastle disease genes in laying hens
1000 laying hens are selected from a chicken farm with infectious coryza of chickens, the clinical manifestations of the chickens in the chicken farm are obviously delayed in laying period and accompanied with symptoms of face swelling, rhinorrhea, conjunctivitis and the like of partial chickens, although the symptoms are treated by antibiotics urgently, the symptoms are not obviously improved, the infected strains in the chicken farm are drug-resistant strains, then pathogenic bacteria of the diseased chickens are separated, pathogenic bacteria of paragallinaceous fowl bacilli can be separated, the pathogenic bacteria are randomly divided into two groups, 500 chickens are selected, the group A is a vaccine inoculation group, and the group B is a blank control group. Group A on the day of test initiation (day 0), vaccine 11 prepared in example 8 was administered at a dose of 0.5 ml/tube. Group B was not vaccinated as a blank control. Laying rates were recorded and the trial was terminated at week 12 after immunization. In the early stages of the experiment, the two groups had comparable laying rates and no difference. Two groups of chickens had already seen a significant difference in egg production from week 2 onwards until the end of the trial. Compared with the blank control group, the laying rate of the test chicken in the group A is gradually recovered after the immunization, and the laying rate is recovered to a normal level around 8 weeks. The egg production statistics are shown in table 9.
TABLE 9 egg laying rate statistics for chickens
Number of weeks tested Group A (average) Group B (mean value) Difference value P value
1 64.9％ 64.4％ 0.5％ 0.34ns
2 69.0％ 62.1％ 6.9％ ＜0.01
3 72.1％ 60.1％ 12.0％ ＜0.01
4 75.2％ 59.1％ 16.1％ ＜0.01
5 78.8％ 60.7％ 18.1％ ＜0.01
6 82.7％ 60.1％ 22.6％ ＜0.01
7 86.8％ 61.5％ 25.3％ ＜0.01
8 89.2％ 65.2％ 24.0％ ＜0.01
9 88.5％ 64.0％ 24.5％ ＜0.01
10 90.1％ 60.7％ 29.4％ ＜0.01
11 91.0％ 60.5％ 30.5％ ＜0.01
12 91.0％ 61.8％ 29.2％ ＜0.01
Denotes the P value of the comparison t test among groups, ns denotes that the difference is not significant, and P is less than or equal to 0.01 denotes that the difference is extremely significant.
The field experiments prove that the chicken infectious rhinitis and Newcastle disease gene VII bigeminy inactivated vaccine has a good immune protection effect on chicken flocks infected with the chicken infectious rhinitis, and the chicken flocks are infected with the chicken infectious rhinitis, so that the vaccine composition has a treatment effect on the yield reduction of mixed infection of the chicken infectious rhinitis, can shorten the course of disease and reduce loss.
SEQUENCE LISTING
<110> Puleco bioengineering GmbH
<120> an avibacterium paragallinarum antigen protein, vaccine composition containing avibacterium paragallinarum antigen, and preparation method thereof
Method and application
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1635
<212> DNA
<213> avian paragallinarum A type HN3 strain (Avibacterium paragallinarum A serotype, strain HN3)
<400> 1
gatggcacca ttacctttac caacattggc ggcaccggtc aagctaccat ccacgatgcg 60
attaacaacg ttctgactaa aggtatctac ctgaaagcgg atcagaacga tccaaccggt 120
aaccaaggtc agaaagtgga actgggtaac gcaatcaccc tgtctgcaac caaccaatgg 180
gcgaacaacg gcgtaaacta taaaaccaac aacctgacca cttataactc tcaaaacggc 240
accattctgt ttggtatgcg tgaagatcca tctgtaaaac aaattaccgc gggtacctat 300
aacaccaccg gtgatgcgaa caacaaaaac caactgaaca acaccctgca acaaaccacc 360
ctggaagcaa ctggtatcac ctctagcgta ggttctacta actacgcggg cttcagcctg 420
ggggcagaca gcgttacctt ctctaaaggt ggtgctggca ccgtgaaact gtctggcgta 480
agcgatgcca ccgccgacac cgacgctgcc actctgaaac aagtgaaaga ataccgcacc 540
accctggtgg gtgataacga catcaccgca gcagatcgtt ctggcggcac cagcaacggc 600
attacctaca acctgagcct gaacaaaggt accgtttctg caaccgaaga aaaagtggtg 660
tctggtaaaa ctgtttatga agccattcgt aacgccatca ccggcaacat cttcaccatt 720
ggcctggacg ataccaccct gaacaaaatc aacaacccgg cggatcaaga tctgtctaac 780
ctgtctgaat ctggcaaaaa cgccattacc ggcctggtgg atgtggtgaa aaaaaccaac 840
tctccgatca ccgttgagcc gtctaccgat agcaacaaga aaaaaacctt cactgtaggc 900
gtggatttca ccgataccat taccgaaggt gacgcaaccg atgataaaaa actgaccact 960
tctaaatccg ttgaaagcta tgttaccaac aaactggcga acttctctac cgatattctg 1020
ctgtctgatg gtcgttctgg taacgcaacc accgcaaacg atggtgtggg taaacgtcgt 1080
ctgtctgatg gctttaccat caaatctgaa aactttaccc tgggttctaa acaatataac 1140
ggctctgata gcctgggtgt aatgtatgac gatcaaaacg gtgtttttaa actgagcctg 1200
aacatgaccg cactgaccac ttctctggct aacactttcg cgaagctgga tgcctctaac 1260
ctgactgatg atagcaacaa agagaaatgg cgtactgcgc tgaacgtgta ttctaaaacc 1320
gaagtagatg cagaaattca aaaatccaag gtaaccctga ccccagattc gggcctgatc 1380
ttcgcaacca aacaagctgg ttctggtaac aacgcaggta ttgatgctgg taacaagaaa 1440
atttctaacg ttgccgatgg tgatatttct ccaacctctg gtgatgtagt gaccggtcgt 1500
cagctgtacg ccctgatgca gaaaggtatt cgcgtgtatg gtgatgaagt ttctccaacc 1560
aagactcaaa ccaccgcacc gaccaacgca aacccaactg cgaccaccgc accgaccgca 1620
tctagcactc aaggt 1635
<210> 2
<211> 1620
<212> DNA
<213> Acidobacterium paragallinarum type B HN5 strain (Avibacterium paragallinarum B serotype, strain HN5)
<400> 2
gatggcacca ttacctttac caacattggc ggcaccggtc aagataccat ccacgatgcg 60
attaacaacg ttctgaccaa actgatctct ctgtctgcaa ccgaagaaga agtggtgtcg 120
ggtgagccgg tgtacgaacc actgaaaggt gcaaaaccaa ccgtttctgc agaagccaac 180
aaagacatta ctggcctggt ggatgtggtg aaaaaagcaa actctccgat caccgttgag 240
ccgtctaccg ataacaacaa gaaaaaaacc ttcactgttg gcctgatgaa agacattgaa 300
ggtgtaaaca gcattacctt tgataagtct ggtcaagatc caaaccaagt taccggccgt 360
atgagctctg cgggtctgac cttcaaaaaa ggcgacacca ccaacggttc taccaccact 420
tttgcagaag atggcctgac cattgatagc accaccaact ctgctcaaac caacctggtg 480
aaagtatctc gtgatggctt ctctgtgaaa aacggcagcg atgaaagcaa actggccccg 540
accaaactgt ctatcggtgc ggaaaacgca gaacacgttg aagtaactaa atctggcatc 600
gccctgaaag cggataacac ctccgataaa tctcgcatca ccctggccca agatgcgatt 660
actctggcgg gtaacgcaac cggtaccgcg attaaactga ctggtgttgc agatggcaac 720
attaccgcaa actctaaaga tgcggtaaac ggtggtcagc tgcgtaccct gctgggtgtt 780
gatagcggtg ctaaaattgg cggtactgag aaaaccacca tctctgaagc catttctgat 840
gtgaagcaag ctctgaccga tgcgaccctg gtatataaag cggacaacaa aaacggtaaa 900
accgttaaac tgactgacgg tctgaacttt actagcacca ccaacattgg cgcctctgta 960
gaagattctg gtgtggtgaa attcaccctg aaagatcgtc tgaccggcct gaaaactatc 1020
gtaactgagt ctctgaacgc ttctcaaaac attattgctg gcggcaccgt aaccgtgggc 1080
ggcgagaccg agggcattgt gctgaccaaa tctggctctg gtaacgaccg cactctgtct 1140
ctgtctggtg caggcaacgc agcaaccgat ggcattaaag tttctggcgt gaaagcaggt 1200
accgcagaca ccgatgcggt gaacaaaggt cagctggata aactgtttaa agcgatcaac 1260
gacgcactgg gcaccaccga tctggcggta accaaaaacc caaaccaaac ctctatcttt 1320
aacccgatca acggcaccgc tccaaccacc tttaaagacg cggtggataa actgaccacc 1380
gctgtgaaca ccggttgggg ttctaaggta ggtattctgg caaccggtat tgatggtatt 1440
gatgctggta acaagaaaat ttctaacgtt gccgatggtg atatttctcc aacctctggt 1500
gatgtagtga ccggtcgtca gctgtacgcc ctgatgcaga aaggtattcg cgtgtatggt 1560
gatgaagttt ctccaaccaa gactcaaacc accgcaccga ccgcatctag cactcaaggt 1620
<210> 3
<211> 1620
<212> DNA
<213> Acidobacterium paragallinarum type C SD3 strain (Avibacterium paragallinarum C serotype, strain SD3)
<400> 3
gatggcacca ttacctttac caacattggc ggcaccggtc aagataccat ccacgatgcg 60
attaacaacg ttctgaccaa actgatctct ctgtctgcaa ccgaagaaga agtggtgtct 120
ggtgaagctg tttatgatgc actgaaaggt gcaaaaccaa ccgtttctgc agaagccaac 180
aaaggcatta ctggcctggt ggatgtggtg aaaaaagcaa actctccgat caccgttgag 240
ccgtctaccg ataacaacaa gaaaaaaacc ttcactgttg gcctgatgaa agacattgaa 300
ggtgtaaaca gcattacctt tgataagtct ggtcaagatc tgaaccaagt taccggccgt 360
atgagctctg cgggtctgac cttcaaaaaa ggcgacacca ccaacggttc taccaccact 420
tttgcagaag atggcctgac cattgatagc accaccaact ctgctcaaac caacctggtg 480
aaagtatctc gtgatggctt ctctgtgaaa aacggcagcg atgaaagcaa actggcctct 540
accaaactgt ctatcggtgc ggaaaacgca gaacacgttg aagtaactaa atctggcatc 600
gccctgaaag cggataacac ctccgataaa tctagcatca ccctggccca agatgcgatt 660
actctggcgg gtaacgcaac cggtaccgcg attaaactga ctggtgttgc agatggcaac 720
attaccgtaa actctaaaga tgcggtaaac ggtggtcagc tgcgtaccct gctgggtgtt 780
gatagcggtg ctaaaattgg cggtactgag aaaaccacca tctctgaagc catttctgat 840
gtgaagcaag ctctgaccga tgcgaccctg gcatataaag cggacaacaa aaacggtaaa 900
accgttaaac tgactgacgg tctgaacttt actagcacca ccaacattga tgcttctgtg 960
gaagataacg gtgtggtgaa attcaccctg aaagataaac tgaccggcct gaaaactatc 1020
gcaactgaat ctctgaacgc ttctcaaaac atcatcgctg gcggtaccgt aaccgtgggc 1080
ggcgagaccg agggcattgt gctgaccaaa tctggctctg gtaacgaccg cactctgtct 1140
ctgtctggtg caggcaacgc agcaaccgat ggcattaaag tttctggcgt gaaagcaggt 1200
accgcagaca ccgatgcggt gaacaaaggt cagctggata aactgtttaa agcgatcaac 1260
gacgcactgg gcaccaccga tctggcggta accaaaaacc caaaccaaac ctctatcttt 1320
aacccgatca acggcaccgc tccaaccacc tttaaagacg cggtggataa actgaccacc 1380
gctgtgaaca ccggttgggg ttctaaggta ggtattctgg caaccggtat tgatggtatt 1440
gatgctggta acaagaaaat ttctaacgtt gccgatggtg atatttctcc aacctctggt 1500
gatgtagtga ccggtcgtca gctgtacgcc ctgatgcaga aaggtattcg cgtgtatggt 1560
gataaagttt ctccaaccaa gactcaaacc accgcaccga ccgcatctag cactcaaggt 1620

Claims (11)

1. An antigenic protein of the C-type HMTp2102 region of the avibacterium paragallinarum, wherein the antigenic protein of the C-type HMTp2102 region of the avibacterium paragallinarum is coded by a sequence shown in SEQ ID NO. 3.
2. A vaccine composition containing an avian paragallinarum antigen, wherein the vaccine composition contains an immunizing amount of avian paragallinarum A type HMTp2102 region antigen protein, an immunizing amount of avian paragallinarum B type HMTp2102 region antigen protein, an immunizing amount of avian paragallinarum C type HMTp2102 region antigen protein and a pharmaceutically acceptable carrier.
3. the vaccine composition containing the avibacterium paragallinarum antigen as claimed in claim 2, wherein the antigenic protein of the avibacterium paragallinarum type a HMTp2102 region is encoded by a sequence shown in SEQ ID No.1, the antigenic protein of the avibacterium paragallinarum type B HMTp2102 region is encoded by a sequence shown in SEQ ID No.2, and the antigenic protein of the avibacterium paragallinarum type C HMTp2102 region is encoded by a sequence shown in SEQ ID No. 3.
4. The vaccine composition containing the avibacterium paragallinarum antigen as claimed in claim 2, wherein the content of the antigenic protein in the A-type HMTp2102 region of avibacterium paragallinarum is more than or equal to 15 μ g/ml, the content of the antigenic protein in the B-type HMTp2102 region of avibacterium paragallinarum is more than or equal to 25 μ g/ml, and the content of the antigenic protein in the C-type HMTp2102 region of avibacterium paragallinarum is more than or equal to 15 μ g/ml; preferably, the content of the antigen protein in the A-type HMTp2102 area of the avibacterium paragallinarum is 15 to 100 mu g/ml, the content of the antigen protein in the B-type HMTp2102 area of the avibacterium paragallinarum is 25 to 100 mu g/ml, and the content of the antigen protein in the C-type HMTp2102 area of the avibacterium paragallinarum is 15 to 100 mu g/ml; more preferably, the content of the antigen protein in the A-type HMTp2102 area of the avibacterium paragallinarum is 25 to 100 mu g/ml, the content of the antigen protein in the B-type HMTp2102 area of the avibacterium paragallinarum is 50 to 100 mu g/ml, and the content of the antigen protein in the C-type HMTp2102 area of the avibacterium paragallinarum is 25 to 100 mu g/ml; further preferably, the content of the antigen protein in the A-type HMTp2102 area of the avibacterium paragallinarum is 15 microgram/ml, the content of the antigen protein in the B-type HMTp2102 area of the avibacterium paragallinarum is 25 microgram/ml, and the content of the antigen protein in the C-type HMTp2102 area of the avibacterium paragallinarum is 15 microgram/ml; further preferably, the content of the antigen protein in the A-type HMTp2102 area of the avibacterium paragallinarum is 25 mu g/ml, the content of the antigen protein in the B-type HMTp2102 area of the avibacterium paragallinarum is 50 mu g/ml, and the content of the antigen protein in the C-type HMTp2102 area of the avibacterium paragallinarum is 25 mu g/ml.
5. The avibacterium paragallinarum antigen-containing vaccine composition according to claim 2, wherein the pharmaceutically acceptable carrier comprises an adjuvant comprising: (1) alumino-gel adjuvant, saponin, avridine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of 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 dipeptide and Gel adjuvant;
Preferably, the saponin is Quil A, QS-21, GPI-0100;
Preferably, the emulsion is an SPT emulsion, an MF59 emulsion, or an emulsion formed from an oil in combination with an emulsifier, the emulsion may be based on light liquid paraffin oil, isoprenoid oil resulting from the oligomerization of olefins (such as squalane or squalene oil, oil resulting from the oligomerization of olefins, in particular isobutene or decene), linear alkyl-containing esters of acids or alcohols (more particularly vegetable oil, ethyl oleate, propylene glycol di- (caprylate/caprate), glycerol tri- (caprylate/caprate) or propylene glycol dioleate), esters of branched fatty acids or alcohols (in particular isostearate); the emulsifier is a nonionic surfactant (especially esters of polyoxyethylated fatty acids (e.g. oleic acid), sorbitan, mannide (such as anhydrous mannitol oleate), aliphatic diols, glycerol, polyglycerol, propylene glycol and oleic, isostearic, ricinoleic or hydroxystearic acid, which may be ethoxylated, ethers of fatty alcohols and polyhydric alcohols (e.g. oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers (especially L121));
Preferably, the polymer of acrylic acid or methacrylic acid is a crosslinked polymer of acrylic acid or methacrylic acid, in particular a compound carbomer crosslinked with polyalkenyl ethers or polyalcohols of sugars, preferably carbopol 974P, 934P and 971P;
Preferably, the copolymer of maleic anhydride and alkenyl derivative is a copolymer EMA of maleic anhydride and ethylene;
Preferably, the adjuvant is a white 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.
6. The avibacterium paragallinarum antigen-containing vaccine composition according to claim 2, wherein the vaccine composition further comprises one or more of the following antigens: a chicken newcastle disease virus antigen, an avian influenza virus antigen, an infectious bronchitis virus antigen, a chicken infectious bursal disease virus antigen, an egg drop syndrome virus antigen, an avian reovirus antigen, an escherichia coli antigen, an avian adenovirus antigen, a mycoplasma synoviae, a mycoplasma gallisepticum antigen, a pasteurella multocida antigen, a marek's virus antigen, an avian encephalomyelitis virus antigen, or a chicken infectious laryngotracheitis virus antigen.
7. The vaccine composition containing the avibacterium paragallinarum antigen as claimed in claim 6, wherein the newcastle disease virus antigen is an inactivated whole virus antigen of newcastle disease virus (gene type VII) N7a strain, and the newcastle disease virus (gene type VII) N7a strain has a preservation number of CCTCC NO: v201545; the content of the inactivated whole virus antigen of the Newcastle disease virus N7a strain is 108.0EID50/0.1 ml-109.0 EID50/0.1ml before inactivation.
8. The vaccine composition containing the avibacterium paragallinarum antigen as claimed in claim 7, wherein the content of the antigenic protein in the A-type HMTp2102 region of avibacterium paragallinarum is 25 μ g/ml, the content of the antigenic protein in the B-type HMTp2102 region of avibacterium paragallinarum is 50 μ g/ml, the content of the antigenic protein in the C-type HMTp2102 region of avibacterium paragallinarum is 25 μ g/ml, and the content of the inactivated whole virus antigen of the N7a strain of Newcastle disease virus is 108.0EID50/0.1ml before inactivation.
9. A method of preparing the avibacterium paragallinarum antigen-containing vaccine composition of claim 2, wherein the method comprises:
Respectively cloning and recombining an antigen protein gene of the A-type HMTp2102 region of the avibacterium paragallinarum shown in SEQ ID NO.1, an antigen protein gene of the B-type HMTp2102 region of the avibacterium paragallinarum shown in SEQ ID NO.2 and an antigen protein gene of the C-type HMTp2102 region of the avibacterium paragallinarum shown in SEQ ID NO.3 to obtain a recombined antigen protein expression vector of the A-type HMTp2102 region of the avibacterium paragallinarum, a recombined antigen protein expression vector of the B-type HMTp2102 region of the avibacterium paragallinarum and a recombined antigen protein expression vector of the C-type HMTp2102 region of the avibacterium paragallinarum;
Step (2) respectively expressing the recombinant expression vector of the A-type HMTp2102 region antigen protein of the avibacterium paragallinarum, the recombinant expression vector of the B-type HMTp2102 region antigen protein of the avibacterium paragallinarum and the recombinant expression vector of the C-type HMTp2102 region antigen protein of the avibacterium paragallinarum in the step (1) to obtain the expressed recombinant A-type HMTp2102 region antigen protein of the avibacterium paragallinarum, the recombinant B-type HMTp2102 region antigen protein of the avibacterium paragallinarum and the recombinant C-type HMTp2102 region antigen protein of the avibacterium paragallinarum; and
Mixing the recombinant avian paragallinarum A-type HMTp2102 region antigen protein, the recombinant avian paragallinarum B-type HMTp2102 region antigen protein and the recombinant avian paragallinarum C-type HMTp2102 region antigen protein expressed in the step (2) according to the content ratio; an adjuvant is added.
10. The method according to claim 9, wherein the expressed recombinant avibacterium paragallinarum A-type HMTp2102 region antigenic protein, recombinant avibacterium paragallinarum B-type HMTp2102 region antigenic protein and recombinant avibacterium paragallinarum C-type HMTp2102 region antigenic protein of the step (2) are active proteins.
11. Use of the vaccine composition containing an avibacterium paragallinarum antigen as claimed in any one of claims 2 to 8 for the preparation of a medicament for the prevention and treatment of diseases caused by avibacterium paragallinarum infection.
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