CN114573708A - Avibacterium paragallinarum HA fusion protein and tripolymer thereof, vaccine composition prepared from avibacterium paragallinarum HA fusion protein, preparation method and application of vaccine composition - Google Patents

Avibacterium paragallinarum HA fusion protein and tripolymer thereof, vaccine composition prepared from avibacterium paragallinarum HA fusion protein, preparation method and application of vaccine composition Download PDF

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CN114573708A
CN114573708A CN202011380504.2A CN202011380504A CN114573708A CN 114573708 A CN114573708 A CN 114573708A CN 202011380504 A CN202011380504 A CN 202011380504A CN 114573708 A CN114573708 A CN 114573708A
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avibacterium paragallinarum
type
fusion protein
antigen
protein
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田克恭
逄文强
金云云
张许科
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Pulaike Biological Engineering Co Ltd
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Pulaike Biological Engineering Co Ltd
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    • 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
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • 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
    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • 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

Abstract

The invention relates to a avibacterium paragallinarum HA fusion protein, which consists of avibacterium paragallinarum HA protein fragments and a trimer-promoting structure which are sequentially arranged from an N end to a C end; the avibacterium paragallinarum HA protein fragment is an A type, B type or C type avibacterium paragallinarum HA protein fragment, the A type avibacterium paragallinarum HA protein fragment is shown as SEQ ID No.1, the B type avibacterium paragallinarum HA protein fragment is shown as SEQ ID No.2, and the C type avibacterium paragallinarum HA protein fragment is shown as SEQ ID No. 3. The trimer formed after trimerization of the avibacterium paragallinarum HA fusion protein HAs good immunogenicity, and the vaccine prepared by the trimer can not only produce complete protection, but also completely protect chicks of low-day age, thereby solving the problem of early immunity of the broiler chicks in the prior art.

Description

Avibacterium paragallinarum HA fusion protein and trimer thereof, vaccine composition prepared from avibacterium paragallinarum HA fusion protein, preparation method and application of vaccine composition
Technical Field
The invention relates to avibacterium paragallinarum fusion protein and a trimer thereof, a prepared vaccine composition, a preparation method and application, belonging to the field of antigen or antibody-containing medical preparations.
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.
Infectious coryza of chicken caused by avibacterium paragallinarum is one of the important diseases which seriously jeopardize the development of poultry breeding industry in the world at present. Among the various preventive measures, vaccine immunization remains the most important measure. At present, the whole-bacterium inactivated vaccine is generally used in the poultry industry, and has higher requirements on the number of bacteria in unit volume of fermentation culture and the risk in the aspect of biological safety caused by incomplete inactivation.
The subunit vaccine is a novel genetic engineering vaccine which is feasible and developed in recent years, the research of the subunit vaccine of avibacterium paragallinarum tries to take HA protein as a research object, however, because the molecular weight is too large, the high-efficiency expression in vitro is difficult to realize, the subunit vaccine of avibacterium paragallinarum is not developed into a product, and the subunit vaccine of avibacterium paragallinarum is not on the market in the prior art. In addition, the disease has the characteristics of short incubation period, rapid spread and capability of spreading to whole chicken flocks in a short time, so that a subunit vaccine composition with good immune effect is urgently needed to be developed clinically, and the epidemic of the disease can be effectively prevented.
In the current industry, endotoxin and bacterial fragments existing in the preparation process of the whole-bacterium antigen vaccine cause side reactions such as appetite reduction, listlessness, injection part lumps and the like, and particularly, the whole-bacterium antigen vaccine can not be applied to chicks with age of less than 21 days, and in addition, the broiler is early in slaughter, the injection part lumps can not be completely absorbed, so that the whole-bacterium antigen vaccine can not be applied to the chicks with low age days. How to solve the problem that the immunization of the avibacterium paragallinarum in the early stage of the broiler chicken is also an urgent need to be solved in clinic.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides the avibacterium paragallinarum fusion protein and the vaccine composition prepared from the same, and the vaccine composition can effectively prevent and/or treat the infection of avibacterium paragallinarum.
The invention provides a avibacterium paragallinarum HA fusion protein, wherein the avibacterium paragallinarum HA fusion protein consists of avibacterium paragallinarum HA protein fragments and a trimer-promoting structure which are sequentially arranged from an N end to a C end; the avibacterium paragallinarum HA protein fragment is an A type avibacterium paragallinarum HA protein fragment, a B type avibacterium paragallinarum HA protein fragment or a C type avibacterium paragallinarum HA protein fragment, the A type avibacterium paragallinarum HA protein fragment is shown as SEQ ID No.1, the B type avibacterium paragallinarum HA protein fragment is shown as SEQ ID No.2, and the C type avibacterium paragallinarum HA protein fragment is shown as SEQ ID No. 3.
The avibacterium paragallinarum HA protein fragment used in the invention HAs good immunogenicity, and HAs better immune effect than vaccines prepared from the corresponding avibacterium paragallinarum HA protein after the vaccine is prepared to immunize chickens.
As an embodiment of the present invention, the trimerization promoting structure in the avibacterium paragallinarum HA fusion protein is C-terminal of avibacterium paragallinarum HA protein, C-terminal of T4 bacteriophage fibrin (T4 phase fibrin folding polymerization), yeast transcription activator GCN4(Transcriptional activator GCN4), or cmp (chicken tissue matrix protein); the C-terminal of the avibacterium paragallinarum HA protein is shown as SEQ ID NO.4, the C-terminal of the T4 bacteriophage fibrin is shown as SEQ ID NO.5, the yeast transcription activator GCN4 is shown as SEQ ID NO.6, and the CMP is shown as SEQ ID NO. 7.
As a preferred embodiment of the invention, in the avibacterium paragallinarum HA fusion protein, a flexible peptide segment is further arranged between the avibacterium paragallinarum HA protein fragment and the trimer-promoting structure, and the flexible peptide segment is shown as SEQ ID No.10 or SEQ ID No. 11.
The avibacterium paragallinarum fusion protein can also comprise flexible connecting peptide, and the length of the flexible connecting peptide is 4-10 amino acid sequences. The flexible linker peptide enables the chimeric protein to have better affinity, mainly because the flexible linker peptide increases the space between the structural domains, eliminates or reduces the physical barrier effect of the space between adjacent structural domains, increases the relative independence of each structural domain of the fusion protein, and also increases the structural stability and biological function of each functional domain of the fusion protein.
The invention also provides an avibacterium paragallinarum HA fusion protein trimer, wherein any molecule of the avibacterium paragallinarum HA fusion protein trimer is formed by trimerization of three molecules of the avibacterium paragallinarum HA fusion protein.
The avibacterium paragallinarum HA fusion protein trimer can provide complete protection after being prepared into a vaccine, and can provide complete protection for chicks of low-day-age.
The invention also provides a vaccine composition, wherein the vaccine composition comprises an immunizing amount of the avibacterium paragallinarum HA fusion protein trimer and a pharmaceutically acceptable carrier.
The invention adopts the avibacterium paragallinarum HA protein fragment and the trimer-promoting structure to form the fusion protein for high-efficiency expression for the first time, and then the fusion protein forms the trimer structure to prepare the vaccine composition, which can prevent and/or treat the infectious coryza epidemic situation of chicken, and the vaccine composition containing the fusion protein can enable an animal body to quickly generate antibodies after immunizing animals, HAs good prevention and control effects on the single or mixed infection of the currently popular avibacterium paragallinarum, and HAs good biological safety. Can provide complete protection and can provide complete protection for chicks of low day age.
As an embodiment of the present invention, in the vaccine composition of the present invention, the avibacterium paragallinarum HA fusion protein trimer is an avibacterium paragallinarum HA fusion protein trimer type a and/or an avibacterium paragallinarum HA fusion protein trimer type B and/or an avibacterium paragallinarum HA fusion protein trimer type C.
As an embodiment of the invention, in the vaccine composition, the AGP titer of the A type avibacterium paragallinarum HA fusion protein trimer is more than or equal to 1:8, the AGP titer of the B type avibacterium paragallinarum HA fusion protein trimer is more than or equal to 1:16, and the AGP titer of the C type avibacterium paragallinarum HA fusion protein trimer is more than or equal to 1: 16.
As a preferable embodiment of the invention, in the vaccine composition, the AGP titer of the A type avibacterium paragallinarum HA fusion protein trimer is 1: 8-1: 32, the AGP titer of the B type avibacterium paragallinarum HA fusion protein trimer is 1: 16-1: 32, and the AGP titer of the C type avibacterium paragallinarum HA fusion protein trimer is 1: 16-1: 32.
The AGP titer of the A type avibacterium paragallinarum HA fusion protein trimer can be selected from 1:8, 1:16 and 1: 32.
The AGP titer of the B type avibacterium paragallinarum HA fusion protein trimer can be selected from 1:16 and 1: 32.
The AGP titer of the C type avibacterium paragallinarum HA fusion protein trimer can be selected from 1:16 and 1: 32.
As a more preferred embodiment of the invention, in the vaccine composition of the invention, the AGP titer of the A type avibacterium paragallinarum HA fusion protein trimer is 1:8, the AGP titer of the B type avibacterium paragallinarum HA fusion protein trimer is 1:16, and the AGP titer of the C type avibacterium paragallinarum HA fusion protein trimer is 1: 16.
As an embodiment of the present invention, in the vaccine composition of the present invention, the pharmaceutically acceptable carrier is an adjuvant selected from the group consisting of: (1) mineral oil, alumina gel adjuvant, saponin, alfudine, 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, IMS1314, muramyl dipeptide, ISA 206 and Gel adjuvant; the adjuvant content is 5% -70% V/V.
In a preferred embodiment of the present invention, the saponin in the vaccine composition of the present invention is Quil a, QS-21, GPI-0100; the adjuvant content is 30% to 70% V/V.
As a more preferred embodiment of the present invention, in the vaccine composition of the present invention, the adjuvant content is 66% V/V.
The vaccine composition of the present invention may further comprise other agents added to the composition of the present invention.
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).
Preferably, the immunostimulant comprises 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 of the present invention, 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, a chicken infectious laryngotracheitis virus antigen.
The vaccine composition of the invention can contain a plurality of antigen components, and the avibacterium paragallinarum HA fusion protein trimer of the invention can form a combined vaccine or a complex vaccine with a plurality of antigens, and immunizes in the form of the combined vaccine or the complex vaccine so as to simplify the immunization procedure.
As a preferred embodiment of the present invention, in the vaccine composition of the present invention, the vaccine composition further comprises one or more of the group consisting of: the antigen comprises a newcastle disease virus inactivated antigen, an avian influenza virus inactivated antigen, an avian infectious bronchitis virus inactivated antigen, an avian infectious bursal disease virus subunit antigen, an avian adenovirus inactivated antigen or subunit antigen and an avian egg drop syndrome virus subunit antigen.
As a more preferred embodiment of the present invention, in the vaccine composition of the present invention, the newcastle disease virus inactivated antigen is a whole virus inactivated antigen of N7a strain, the avian influenza virus inactivated antigen is a whole virus inactivated antigen of HF strain, the avian infectious bronchitis virus inactivated antigen is a whole virus inactivated antigen of M41 strain, the avian infectious bursal disease virus subunit antigen is an avian infectious bursal disease virus VP2 protein, the avian adenovirus inactivated antigen is a whole virus inactivated antigen of FAV-HN strain, the avian adenovirus subunit antigen is an avian adenovirus Penton protein or Fiber-2 protein, and the avian egg drop syndrome virus subunit antigen is an avian egg drop syndrome virus tFiber protein fragment.
As a preferable embodiment of the invention, in the vaccine composition of the invention, the content of the inactivated antigen of newcastle disease virus in chicken is 10 before inactivation8.0～109.0EID500.1ml, the content of the inactivated antigen of the avian influenza virus is 10 before inactivation6.5～108.5EID500.1ml, the content of the inactivated antigen of the avian infectious bronchitis virus is 10 before inactivation6.0～107.0EID500.1ml, the content of the chicken infectious bursal disease virus VP2 protein is AGP titer of 1: 16-1: 128, the content of the avian adenovirus Penton protein is AGP titer of 1: 2-1: 16, the content of the avian adenovirus Fiber-2 protein is AGP titer of 1: 2-1: 16, and the content of the avian egg drop syndrome virus tFiber protein fragment is AGP titer of 1: 8-1: 32.
AsIn a more preferred embodiment of the present invention, in the vaccine composition of the present invention, the content of the inactivated antigen of newcastle disease virus in chicken is 10 before inactivation8.0EID500.1ml, the content of the inactivated antigen of the avian influenza virus is 10 before inactivation8.0EID500.1ml, the content of the inactivated antigen of the avian infectious bronchitis virus is 10 before inactivation6.0EID500.1ml, the content of the chicken infectious bursal disease virus VP2 protein is AGP titer 1:16, the content of the avian adenovirus Penton protein is AGP titer 1:4, the content of the avian adenovirus Fiber-2 protein is AGP titer 1:4, and the content of the avian egg drop syndrome virus tFiber protein fragment is AGP titer 1: 8.
The invention also provides a method for preparing the vaccine composition, wherein the method comprises the following steps: respectively recombining and expressing the A-type avibacterium paragallinarum HA fusion protein, the B-type avibacterium paragallinarum HA fusion protein and the C-type avibacterium paragallinarum HA fusion protein by using an Ecoli expression system, and respectively trimerizing the expressed A-type avibacterium paragallinarum HA fusion protein, the B-type avibacterium paragallinarum HA fusion protein and the C-type avibacterium paragallinarum HA fusion protein to form an A-type avibacterium paragallinarum HA fusion protein trimer, a B-type avibacterium paragallinarum HA fusion protein trimer and a C-type avibacterium paragallinarum HA fusion protein trimer; respectively treating the A-type avibacterium paragallinarum HA fusion protein trimer, the B-type avibacterium paragallinarum HA fusion protein trimer and the C-type avibacterium paragallinarum HA fusion protein trimer in the step (1) by using a nonionic surfactant to remove endotoxin; and (3) uniformly mixing the endotoxin-removed A-type avibacterium paragallinarum fusion protein trimer, B-type avibacterium paragallinarum HA fusion protein trimer and C-type avibacterium paragallinarum HA fusion protein trimer with an adjuvant to obtain the vaccine composition.
According to the method for preparing the vaccine composition, the antigen with good immunogenicity is used, and the endotoxin is reduced by using an endotoxin removal technology, so that early immunity of the broiler chicken can be realized at an early stage, and the problem which needs to be solved clinically is solved.
The invention also provides application of the vaccine composition in preparation of a medicament for preventing and/or treating avian paragallinarum infection.
In one embodiment of the present invention, the agent for preventing and/or treating an avian paragallinarum infection is an agent for preventing and/or treating an avian paragallinarum infection in a broiler chick aged less than 21 days.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
Definition of
The term "avian paragallinarum" (Avibacterium paragallinarum, Apg) is the causative bacterium of infectious coryza in chickens, which is facultative anaerobic and can cause acute respiratory diseases, mainly manifested by the presence of large amounts of serous or mucoid secretions in the nasal passages and sinuses, facial edema and conjunctivitis. Can lead to the reduction of laying rate and hatching rate and the increase of elimination rate, thereby causing serious economic loss. Avibacterium paragallinarum can be classified into A, B, C three serotypes by a plate agglutination test.
The term "avibacterium paragallinarum HA protein fragment" is a partial fragment of avibacterium paragallinarum hemagglutinin protein, and the hemagglutinin protein is an important component in avibacterium paragallinarum antigen and is also the basis of avibacterium paragallinarum typing.
The term "trimer of an HA protein fragment of avibacterium paragallinarum", i.e. three identical HA protein fragments of avibacterium paragallinarum are polymerized into one protein fragment, which is called trimer, and is a low molecular weight polymer.
The term "avibacterium paragallinarum HA trimer fusion protein" is composed of avibacterium paragallinarum HA protein fragments and a trimer-promoting structure, wherein the trimer-promoting structure is C-terminal of avibacterium paragallinarum HA protein, C-terminal of T4 bacteriophage fibrin, yeast transcription activator GCN4 or CMP.
The term "C-terminal of the HA protein of avibacterium paragallinarum" is similar to the C-terminal membrane anchoring region (membrane anchor domain) of the taa (tertiary autotransporter adhesin) family and consists of 229 amino acids.
The term "C-terminal of the fibrin of T4 phage", also known as the C-terminal trimerization sequence of fibrin of T4 phage (T4 phase fibrosis fusion sequence), consists of 27 amino acids, and this domain is composed of 3 identical subunits, each of which contains 1 β -hairpin structure.
The term "yeast transcriptional activator GCN 4" is a protein that binds DNA via a leucine zipper (Leucine zipper) structure.
The term "CMP" refers to chicken cartilage matrix protein (chicken cartilage matrix protein).
The AGP titer is also called "Agar Gel Precipitation titer" (Agar diffusion test) titer), the Agar diffusion test is a Precipitation reaction, the antigen and antibody can be freely diffused to the periphery in the Agar Gel containing electrolyte, the antigen and antibody are combined with each other, a Precipitation line appears at the optimum proportion, and the highest dilution proportion of the Precipitation line is the efficacy value, namely the calibrated antigen or antibody content.
"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, inducing 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 group may itself 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, IMS1314, 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 Block co-polymer, SAF-M, a monophosphoryl lipid A, Avridine lipid-amine adjuvant, escherichia coli heat labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide or Gel adjuvant.
The term "mineral oil" is also named as paraffin oil, white oil and white oil, and mineral oil is a wider adjuvant used in inactivated vaccines, and has the functions of delaying the retention time of immunogen in the body, enabling the immunogen to be continuously and slowly released, and enhancing the phagocytosis and bactericidal capacity of macrophages.
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 an escherichia coli antigen.
The term "combination vaccine" refers to vaccines prepared from viruses and viral antigens. For example, the avibacterium paragallinarum subunit antigen of the present invention may be combined with a newcastle disease virus antigen.
The term "endotoxin" is a component of the cell wall of gram-negative bacteria, called lipopolysaccharide. Lipopolysaccharides are toxic to the host and are released when bacteria die and lyse or artificially destroy bacterial cells, and are called endotoxins.
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: na (Na)2HPO4·12H2O 5.8g、NaH2PO4·2H2O0.59 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 BL21(DE3) -A-GCN4-tHA expression engineering strains
1.1 extraction of A-type DNA of Acinetobacter paragallinarum
According to the instruction of a virus DNA extraction kit, a pure culture of an avian paragallinarum A type Strain HN3 Strain (avian paragallinarum A type HN3 Strain (Avibacterium paragallinarum Serotype A Strain HN3), which has been preserved in a China typical culture preservation center (CCTCC) at 2015 01-20 days, the preservation number is CCTCC NO: M2015051, the preservation address is China Wuhan university and is disclosed in China patent application CN106267176A), a plurality of colonies are picked into 100 mu L sterile double distilled water, boiled for 10 minutes, subjected to ice bath for 10 minutes, centrifuged for 1 minute at 12000r/min, and the supernatant is taken as a template for subsequent experiments.
1.2A-tHA protein fragment Gene amplification
Oligonucleotide primers were synthesized based on the conserved region sequences at the 5 'and 3' ends of the tHA protein gene, and PCR was performed. The upstream primer tHA-F is shown as SEQ ID NO.8, and the downstream primer tHA-R is shown as SEQ ID NO. 9.
The PCR product was sent to Invitrogen corporation for sequencing, and the amino acid sequence of the A-tHA protein fragment is shown in SEQ ID NO. 1.
1.3 construction of A-tHA fusion protein expression plasmid
The yeast transcription activator GCN4 gene shown in SEQ ID NO.6 is recombined into a pET28a vector and is named as pET28a-GCN 4. And (2) carrying out electrophoresis on a PCR product of the avibacterium paragallinarum type A tHA protein, purifying by using a DNA gel recovery kit, carrying out double enzyme digestion on the obtained DNA fragment by using BamHI and XhoI, connecting with pET28a-GCN4 plasmid subjected to the same double enzyme digestion treatment, transforming escherichia coli DH5 alpha competence by using a connecting product, and screening positive clones. And (3) extracting a transformant plasmid by using a plasmid extraction kit, carrying out double enzyme digestion identification, carrying out sequencing analysis on the plasmid with correct enzyme digestion identification, and naming the recombinant plasmid with correct verification as pET28a-A-GCN 4-tHA.
1.4BL21(DE3) -A-GCN4-tHA construction of expression engineering strain
The recombinant plasmid pET28a-A-GCN4-tHA with correct sequencing is transformed into escherichia coli BL21(DE3) competence to construct expression engineering bacteria BL21(DE3) -A-GCN 4-tHA. Single colonies were picked from LB plates (containing 50. mu.g/mL kanamycin), plasmids were extracted after extensive culture in LB liquid medium, double restriction enzyme identification was performed with BamHI and XhoI restriction enzymes, and the plasmids with correct restriction enzyme identification were subjected to sequencing analysis. The strains with correct enzyme cutting and sequencing identification are stored at-70 ℃ for later use.
Example 2BL21(DE3) -A-GCN4-tHA expression engineering strain protein expression characteristic study
2.1 expression of A-GCN4-tHA protein
Correctly identified engineered bacteria were streaked onto LB plates (containing 50. mu.g/mL kanamycin) and cultured at 37 ℃ for 14 hours. A single colony was picked, inoculated into 3mL of LB liquid medium (containing 50. mu.g/mL kanamycin), cultured at 37 ℃ for 14 hours with a shaker (220r/min), transferred to a 250mL Erlenmeyer flask containing 100mL of LB liquid medium (containing 50. mu.g/mL kanamycin) with a shaker (37 ℃, 220r/min) for 4 hours, cooled to 28 ℃, added with isopropylthio β -D-galactoside (IPTG) at a final concentration of 0.2mmol/L, and cultured for another 5 hours. Centrifuging the culture at 4 ℃ and 10000r/min for 5 minutes, collecting thalli, suspending the thalli in 10mL sterile physiological saline, and carrying out ultrasonic crushing in an ice water bath for 35 minutes under the crushing conditions: the operation is carried out for 5 seconds, the pause is 15 seconds, and the ultrasonic power is 400 watts. The crushed liquid is centrifuged at 10000r/min for 10 minutes at 4 ℃, and then the supernatant is taken out for subsequent detection.
2.2 measurement of titer of expressed protein
Taking the supernatant of the thallus lysate in item 2.1, and using A type avibacterium paragallinarum positive serum as an antibody to carry out AGP titer determination by a conventional method. AGP titer of A-GCN4-tHA protein is 1:256, endotoxin content is 0.56 × 105EU/mL。
2.3 removal of endotoxin
A1.5 mL centrifuge tube was charged with 0.5mL of the solution to be treated and Triton X-114 (5. mu.L) at a final concentration of 1% (v/v) and vortexed. The sample was placed on ice for 5 minutes and after vortexing the cooled sample, the centrifuge tube was immediately placed in a 37 ℃ water bath for 5 minutes to allow a new two phase to develop. The sample was then centrifuged for 60s at 37 ℃. After centrifugation, the target protein will remain in the upper layer, and the detergent containing endotoxin will remain in the bottom of the centrifuge tube in the form of oil droplets. The whole operation cycle was 3 times. The AGP titer of the A-GCN4-tHA protein is 1:256, and the endotoxin content is 0.01 × 105EU/mL。
2.4 identification of expressed proteins
SDS-PAGE and Western Blot result shows that A-GCN4-tHA has molecular weight of about 60KDa, and non-reduction SDS-PAGE and Western Blot result shows that a protein band with molecular weight of about 180KDa appears, which indicates that A-GCN4-tHA forms a trimer structure and can perform specific reaction with rabbit anti-Apg A type positive serum.
Example 3 construction of BL21(DE3) -A-tHA expression engineering strains and A-tHA protein expression
And (2) carrying out electrophoresis on a PCR product of the avibacterium paragallinarum type A tHA protein, purifying by using a DNA gel recovery kit, carrying out double enzyme digestion on the obtained DNA fragment by using BamHI and XhoI, connecting with pET28a plasmid subjected to the same double enzyme digestion treatment, transforming escherichia coli DH5 alpha competence by using a connecting product, and screening positive clone. And (3) extracting a transformant plasmid by using a plasmid extraction kit, carrying out double enzyme digestion identification, carrying out sequencing analysis on the plasmid with correct enzyme digestion identification, and naming the recombinant plasmid with correct verification as pET28 a-A-tHA. An expression engineering strain BL21(DE3) -A-tHA was constructed according to the method of example 1, and the correct strain was identified by digestion and sequencing and stored at-70 ℃ for further use.
tHA protein was expressed by the method of example 2, and AGP titer was determined by a conventional method using a positive serum of avian paragallibacterium type A as an antibody. The A-tHA protein has AGP titer of 1:256 and endotoxin content of 0.58 × 105EU/mL。
By removing endotoxin according to the method of example 2, the AGP titer of A-tHA protein was determined to be 1:256, inThe toxin content is 0.01 × 105EU/mL。
Example 4 preparation of avian paragallibacterium type a subunit vaccine
The A-GCN4-tHA fusion protein prepared in example 2 and the A-tHA protein prepared in example 3 were each slowly added to mineral oil adjuvant while the motor was turned on, stirred at 17500r/min for 5min, and 1% thimerosal solution was added to the solution until the stirring was stopped, to give a final concentration of 0.01%. The specific ratio is shown in table 1.
TABLE 1 avian paragallibacterium A-type subunit vaccine ratios
Components Vaccine 1 Vaccine 2 Vaccine 3
A-GCN4-tHA protein (AGP titer) 1:8 1:32 -
A-tHA protein (AGP titer) - - 1:32
Mineral oil adjuvant (V/V%) 66％ 66％ 66％
Example 5 comparison of efficacy of avian Paragallinarum type A subunit vaccines
40 SPF chickens 8 weeks old were randomly divided into 4 groups, 10 chickens/group, and the avian paragallinarum type A subunit vaccine prepared in example 4 was immunized. The 1 st to 3 rd groups of immune vaccines are 1 to 3 respectively, and the 4 th group of immune vaccines are used as challenge control. Each immune group is injected with 0.5 mL/vaccine, the challenge control group is injected with 0.5 mL/PBS, 28 days after immunization, each immune group and the challenge control group are injected with 0.2mL of A-type HN3 strain bacterial liquid (the number of viable bacteria contained is about 2.0 multiplied by 10)4CFU). 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 2.
TABLE 2 comparison of the efficacy of the A-type subunit vaccine of avibacterium paragallinarum
Figure BDA0002808342750000151
The results show that vaccine 3 can not provide effective immune protection against avibacterium paragallinarum type a, and vaccine 1 and vaccine 2 can provide complete immune protection; all disease occurred in the control group. The result shows that the A-type tHA protein of the avibacterium paragallinarum still can not provide effective immune protection when the antigen content is 1:32 of AGP titer, and the A-type GCN4-tHA fusion protein of the avibacterium paragallinarum can provide complete immune protection when the antigen content is 1:8 of AGP titer.
Example 6 preparation of a B-type subunit vaccine of Acinetobacter paragallinarum
6.1 extraction of B-type DNA of Acinetobacter paragallinarum
According to the specification of a virus DNA extraction kit, a B-type avian paragallinarum Strain HN5 Strain (an Avibacterium paragallinarum Serotype B Strain HN5) is taken, a pure culture which is preserved in a China typical culture preservation center (CCTCC) at 22.02.2017 and has the preservation number of CCTCC NO: M2017056 and the preservation address of Wuhan university in Wuhan, Wuhan university and disclosed in China patent application CN108624522A is selected, a plurality of colonies are selected in 100 mu L sterile double distilled water, boiled for 10 minutes, subjected to ice bath for 10 minutes, centrifuged for 1 minute at 12000r/min, and the supernatant is taken as a template for subsequent experiments.
6.2B-tHA protein fragment Gene amplification
Oligonucleotide primers were synthesized based on the conserved region sequences at the 5 'and 3' ends of tHA protein gene, with the upstream primer tHA-F shown as SEQ ID NO.8 and the downstream primer tHA-R shown as SEQ ID NO.9, for PCR.
The PCR product was sent to Invitrogen corporation for sequencing, and the amino acid sequence of the B-tHA protein fragment is shown in SEQ ID NO. 2.
6.3 expression of B-GCN4-tHA protein
BL21(DE3) -B-GCN4-tHA expression engineering strain is constructed by the method of reference example 1, B-GCN4-tHA protein is expressed by reference example 2, after endotoxin is removed, the AGP titer of the B-GCN4-tHA protein is 1:256, and the endotoxin content is 0.01 multiplied by 105EU/mL。
6.4 identification of B-GCN4-tHA protein
SDS-PAGE and Western Blot result shows that B-GCN4-tHA has molecular weight of about 60KDa, but non-reduction SDS-PAGE and Western Blot result shows that a protein band with molecular weight of about 180KDa appears, which indicates that B-GCN4-tHA forms a trimer structure and can perform specific reaction with rabbit anti-Apg B-type positive serum.
6.5BL21(DE3) -B-tHA construction of expression engineering strain and B-tHA protein expression
PCR products of B-type tHA protein of avibacterium paragallinarum are electrophoresed and purified by using a DNA gel recovery kit to obtain DNA fragments, BL21(DE3) -B-tHA expression engineering strains are constructed by referring to the method in example 3, B-tHA protein is expressed by referring to example 2, after endotoxin is removed, the AGP titer of the B-tHA protein is 1:256, and the endotoxin content is 0.01 multiplied by 105EU/mL。
6.6 preparation of B-type subunit vaccine of avibacterium paragallinarum
The prepared B-GCN4-tHA fusion protein and B-tHA protein are respectively and slowly added into a mineral oil adjuvant, a motor is started simultaneously, stirring is carried out at 17500r/min for 5min, and a 1% thimerosal solution is added before the stirring is stopped, so that the final concentration is 0.01%. The specific ratio is shown in Table 3.
TABLE 3 avian paragallibacterium B type subunit vaccine ratios
Components Vaccine 4 Vaccine 5 Vaccine 6
B-GCN4-tHA protein (AGP titer) 1:16 1:32 -
B-tHA protein (AGP titer) - - 1:32
Mineral oil adjuvant (V/V%) 66％ 66％ 66％
Example 7 comparison of efficacy of B-type subunit vaccine against avian pulleyi paragallinarum
40 SPF chickens 8 weeks old were randomly divided into 4 groups, 10 chickens/group, and immunized with the avian paragallinarum type B subunit vaccine prepared in example 6. The 5 th to 7 th groups of immune vaccines are 4 to 6 respectively, and the 8 th group of immune vaccines are used as challenge control. Each immune group is injected with 0.5mL of vaccine, each challenge control group is injected with 0.5mL of PBS, 28 days after immunization, each immune group and the challenge control group are injected with B-type HN5 in the infraorbital sinusBacterial strain liquid 0.2mL (containing about 1.0X 10 viable count)4CFU). After continuous observation for 7 days, the disease is judged to be the onset of any symptom of face swelling or rhinorrhea, and the toxic counteracting protection results are shown in table 4.
TABLE 4 comparison of the efficacy of B-type subunit vaccine of avibacterium paragallinarum
Figure BDA0002808342750000171
The results show that vaccine 6 can not provide complete immune protection against avibacterium paragallinarum type B, and vaccines 4 and 5 can provide complete immune protection; all disease occurred in the control group. The result shows that the avibacterium paragallinarum B type tHA protein still can not provide complete immune protection when the antigen content is AGP titer is 1:32, and the avibacterium paragallinarum B type GCN4-tHA fusion protein provided by the invention can provide complete immune protection when the antigen content is AGP titer is 1: 16.
EXAMPLE 8 preparation of Acinetobacter paragallinarum type C subunit vaccine
8.1 extraction of C-type DNA of Acinetobacter paragallinarum
According to the instruction of a virus DNA extraction kit, a pure culture of an Avibacterium paragallinarum C-type Strain HN5 Strain (Avibacterium paragallinarum Serotype C Strain SD3) which is preserved in China typical culture collection center (CCTCC) 20 months and 2015, the preservation number is CCTCC NO: M2015052, the preservation address is Wuhan, Wuhan university, China published in China patent application CN106267176A) is taken, a plurality of colonies are picked up in 100 mu L sterile double distilled water, boiled for 10 minutes, subjected to ice bath for 10 minutes, centrifuged for 1 minute at 12000r/min, and the supernatant is taken as a template for subsequent experiments.
8.2C-tHA protein fragment Gene amplification
Oligonucleotide primers were synthesized based on the conserved region sequences at the 5 'and 3' ends of tHA protein gene, with the upstream primer tHA-F shown as SEQ ID NO.8 and the downstream primer tHA-R shown as SEQ ID NO.9, for PCR.
The PCR product was sent to Invitrogen corporation for sequencing, and the amino acid sequence of the C-tHA protein fragment is shown in SEQ ID NO. 3.
8.3 expression of C-GCN4-tHA protein
BL21(DE3) -C-GCN4-tHA expression engineering strain is constructed by the method of reference example 1, C-GCN4-tHA protein is expressed by reference example 2, after endotoxin is removed, the AGP titer of the C-GCN4-tHA protein is 1:256, and the endotoxin content is 0.01 multiplied by 105EU/mL。
8.4 identification of C-GCN4-tHA protein
SDS-PAGE and Western Blot result shows that C-GCN4-tHA has molecular weight of about 60KDa, and non-reduction SDS-PAGE and Western Blot result shows that a protein band with molecular weight of about 180KDa appears, which indicates that C-GCN4-tHA forms a trimer structure and can perform specific reaction with rabbit anti-Apg C type positive serum.
8.5BL21(DE3) -C-tHA construction of expression engineering strain and C-tHA protein expression
PCR products of the avian paragallibacterium C-type tHA protein are electrophoresed and purified by using a DNA gel recovery kit to obtain DNA fragments, BL21(DE3) -C-tHA expression engineering strains are constructed by the method in reference example 3, C-tHA protein is expressed by reference example 2, after endotoxin is removed, the AGP titer of the C-tHA protein is 1:256, and the endotoxin content is 0.01 multiplied by 105EU/mL。
8.6 preparation of avian Paragallinarum C type subunit vaccine
The prepared C-GCN4-tHA fusion protein and C-tHA protein are respectively and slowly added into a mineral oil adjuvant, a motor is started simultaneously, stirring is carried out at 17500r/min for 5min, and a 1% thimerosal solution is added before the stirring is stopped, so that the final concentration is 0.01%. The specific ratio is shown in Table 5.
TABLE 5 avian Paragallibacterium C subunit vaccine ratios
Components Vaccine 7 Vaccine 8 Vaccine 9
C-GCN4-tHA protein (AGP titer) 1:16 1:32 -
C-tHA protein (AGP titer) - - 1:32
Mineral oil adjuvant (V/V%) 66％ 66％ 66％
Example 9 comparison of efficacy of avian Paragallinarum type C subunit vaccines
40 SPF chickens 8 weeks old were randomly divided into 4 groups, 10 chickens/group, and the avian paragallibacterium type C subunit vaccine prepared in example 8 was immunized. The 9 th to 11 th groups are immune vaccines 7 to 8 respectively, and the 12 th group is immune and serves as a challenge control. Each immune group is injected with 0.5mL of vaccine, the challenge control group is injected with 0.5mL of PBS, 28 days after immunization, each immune group and the challenge control group are injected with 0.2mL of C-type SD3 bacterial liquid (the number of viable bacteria is about 1.0 multiplied by 10)4CFU). 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 6.
TABLE 6 comparison of the efficacy of the type C subunit vaccine of avibacterium paragallinarum
Figure BDA0002808342750000191
The results show that vaccine 9 can not provide effective immune protection against avibacterium paragallinarum type C, and vaccine 7 and vaccine 8 can provide complete immune protection; all disease occurred in the control group. The result shows that the avibacterium paragallinarum C-type tHA protein still cannot provide effective immune protection when the antigen content is AGP titer is 1:32, and the avibacterium paragallinarum C-type GCN4-tHA fusion protein provided by the invention can provide complete immune protection when the antigen content is AGP titer is 1: 16.
Example 10 preparation of avibacterium paragallinarum subunit vaccine
The A-GCN4-tHA fusion protein prepared in example 2, the B-GCN4-tHA fusion protein prepared in example 6 and the C-GCN4-tHA fusion protein prepared in example 8 were slowly added to a mineral oil adjuvant, while the electric motor was turned on, the mixture was stirred at 17500r/min for 5min, and a 1% thimerosal solution was added to the mixture before the stirring was stopped, so that the final concentration was 0.01%. The specific ratio is shown in Table 7.
TABLE 7 avian paragallibacterium subunit vaccine ratios
Components Vaccine 10 Vaccine 11
A-GCN4-tHA protein (AGP titer) 1:8 1:32
B-GCN4-tHA protein (AGP titer) 1:16 1:32
C-GCN4-tHA protein (AGP titer) 1:16 1:32
Mineral oil adjuvant (V/V%) 66％ 66％
Example 11 comparison of the efficacy of the avian pulleyi paragallinarum subunit vaccine and the imported vaccine against SPF chickens
Randomly dividing 120 SPF chickens of 8 weeks old into 12 groups, 10 groups, 13 groups, 17 groups and 21 groups to immunize the vaccine 10 prepared in the example 10, 14 groups, 18 groups and 22 groups to immunize the vaccine 11 prepared in the example 10, and 15 groups, 19 groups and 23 groups to immunize imported chicken infectious rhinitis trivalent inactivated vaccines (respectively containing A type W strains, the content of which is more than or equal to 6 multiplied by 10 before inactivation)8CFU/mL; contains B type Spross strain with the content of more than or equal to 6 multiplied by 10 before inactivation8CFU/mL; contains C type Modesto strain with content of not less than 6 × 10 before inactivation8CFU/mL), 16 th, 20 th and 24 th groups were not immunized as challenge controls. Each immunization group was injected with 0.5mL of vaccine, the challenge control group was injected with 0.5mL of PBS, 28 days after immunization, 40 chickens in groups 13, 14, 15 and 16, and the infraorbital sinus was injected with 0.2mL of the bacterial solution of HN3 strain A (containing about 2.0X 10 viable cell count)4CFU); 40 chickens in groups 17, 18, 19 and 20 were injected into the infraorbital sinus with 0.2mL of bacterial suspension of HN5 strain B (containing about 1.0X 10 viable cell count)4CFU); 40 chickens in 21 st, 22 nd, 23 nd and 24 th groups were injected with 0.2mL of bacterial suspension of type C SD3 strain (viable count about 1.0X 10)4CFU). The results of the challenge protection are shown in Table 8 after 7 days of continuous observation.
TABLE 8 comparison of the efficacy of the avian paragallibacterium subunit vaccine and the imported vaccine against SPF chickens
Figure BDA0002808342750000201
Figure BDA0002808342750000211
The results show that compared with the immunization group of the vaccine 10, the vaccine 11 and the imported vaccine and the non-immune challenge control group, the protection rate of the immunization group and the challenge control group is obviously different, the protection effect of the immunization group of the vaccine 10 and the vaccine 11 is superior to that of the imported vaccine, and further, the subunit vaccine has good immune protection effect.
Example 12 efficacy testing of avibacterium paragallinarum subunit vaccine on small day old SPF chickens
60 SPF chickens of 3 weeks old were randomly divided into 6 groups, 10 groups, 25 th group, 27 th group and 29 th group to immunize the vaccine 10 prepared in example 10, and 26 th group, 28 th group and 30 th group to be used as challenge control. Each immune group is injected with 0.5mL of vaccine, the control group is injected with 0.5mL of PBS, 28 days after immunization, 20 chickens in 25 th and 26 th groups are injected with 0.2mL of A-type HN3 strain liquid (the number of viable bacteria is about 1000CFU) in the infraorbital sinus; 20 chickens in 27 th group and 28 th group are injected with 0.2mL of bacterial liquid of type B HN5 strain (containing about 500CFU of viable count) in the infraorbital sinus; in groups 29 and 30, 20 chickens were injected into the infraorbital sinus with 0.2mL of bacterial suspension of type C SD3 strain (containing about 1000CFU viable count). The results of the challenge protection are shown in Table 9 after 7 days of continuous observation.
TABLE 9 results of efficacy test of avibacterium paragallinarum subunit vaccine on small day-old SPF chickens
Figure BDA0002808342750000212
The result shows that compared with the non-immune control group, the immune group and the control group of the vaccine 10 have obvious protection rate difference, and further shows that the subunit vaccine has good immune protection effect.
The avian bacterium paragallinarum subunit vaccine can completely protect 21-day-old chickens, so that the problem that the broiler chickens below 21 days old cannot be subjected to immune protection in actual production is solved, and the food safety of the broiler chickens is ensured.
Example 13 preparation of Newcastle disease antigen
Newcastle Disease Virus (Gene VII) strain N7a (Newcastle Disease Virus (genotype VII), strain N7a) (deposited in the China center for type culture Collection,the preservation number is CCTCC NO: v201545, preservation date of 2015, 10 months and 19 days, preservation address of Wuhan, Wuhan university, China, published in Chinese patent application CN107281479A, sterilized normal saline for proper dilution (10)-4Or 10-5) Inoculating 0.1mL of susceptible chick embryos of 10-11 days old, and after inoculation, placing at 37 ℃ for continuous incubation. Selecting dead and alive chick embryos 48-120 hours after inoculation, harvesting allantoic fluid, and measuring the virus content to be 108.0EID500.1 mL. Adding a formaldehyde solution (v/v) with the final concentration of 0.1%, inactivating at 37 ℃, stirring once every 4-6 h, and inactivating for 16h for later use after complete inactivation.
Example 14 preparation of avian influenza antigen
Taking H9 Subtype Avian Influenza Virus HF Strain (Avian Influenza Virus (H9 Subtype) HF Strain (Avian Influenza Virus (Subtype H9), Strain HF), with the preservation number of CCTCC NO: V201941, the preservation date of 2019, 06, 19 days and the preservation address of Wuhan university of Wuhan, China) Virus seed, diluting to 10 with sterile physiological saline-3(0.1 mL of virus liquid is added into 0.9mL of sterile physiological saline, shaken and mixed uniformly and then diluted for 2 times in sequence), 10-day-old susceptible chick embryos (self-hatching with SPF hatching eggs purchased from Beijing Meiliyangtong laboratory animal technology Co., Ltd.) are inoculated into the allantoic cavity, and each embryo is 0.1mL (containing 10 SPF hatching eggs)5EID50). After inoculation, the pinholes are sealed, and incubation is continued at 36-37 ℃ without turning eggs. And taking out the mixture for 96 hours, standing the mixture in an upward air chamber, and cooling the mixture for 12 to 24 hours at the temperature of between 2 and 8 ℃. And (5) harvesting the embryo liquid of the cooled chick embryo. The virus content was determined to be 108.5EID500.1 mL. Adding a formaldehyde solution (v/v) with the final concentration of 0.1%, inactivating at 37 ℃, stirring once every 4-6 h during inactivation for 24h, and completely inactivating for later use.
Example 15 preparation of infectious bronchitis antigen
The avian infectious bronchitis virus M41 strain (purchased from China institute of veterinary drugs) was taken and diluted with sterilized normal saline (10)-2Or 10-3) Inoculating 0.1mL of susceptible chick embryos of 10-11 days old, and continuously incubating at 36-37 ℃ after inoculation. Selecting dead and alive chick embryos 24-48 hours after inoculation, harvesting allantoic fluid, and determining virus contentIs 106.0EID500.1 mL. Adding a formaldehyde solution (v/v) with the final concentration of 0.1%, placing at 37 ℃ for inactivation, stirring once every 4-6 h, and inactivating for 16h for later use after complete inactivation.
EXAMPLE 16 preparation of avian adenovirus antigen
16.1 preparation of Fiber-2cDNA
FADV virus DNA is extracted from infected Fowl adenovirus FAV-HN strain (fowladenovirus, FAV-HN strain (strain FAV-HN) with the preservation number of CCTCC NO: V201609, the preservation unit is China center for type culture Collection, the preservation address is Wuhan university in Wuhan, China, the preservation time is 2016, 2 and 29 days, and the DNA is disclosed in Chinese patent application CN107523556A) according to the operation of a virus RNA extraction kit. Oligonucleotide primers are synthesized according to the conserved region sequences at the 5 'and 3' ends of the Fiber-2 protein gene, an upstream primer Fiber-2-F is shown as SEQ ID NO.12, a downstream primer Fiber-2-R is shown as SEQ ID NO.13, PCR amplification is carried out, and the PCR product is recovered by an agarose gel recovery kit and stored at-20 ℃.
16.2 expression vector construction
The optimized Fiber-2 protein gene was sent to Sovizhou Jinzhi Biotechnology Ltd for complete sequence synthesis and separately ligated to pET28a plasmid. The connected plasmid is transformed into Escherichia coli BL21(DE3), a single clone is selected to be cultured in an LB liquid culture medium (containing 50 mu g/mL kanamycin) overnight, plasmid is extracted, sequencing analysis is carried out, and a positive clone is the BL21(DE3) -FADV-Fiber-2 expression strain.
16.3 preparation of Fiber-2 protein
BL21(DE3) -FADV-Fiber-2 expression strain was inoculated into LB liquid medium (containing 50. mu.g/mL kanamycin) at an inoculum size of 1% (V/V), and cultured with shaking at 37 ℃. When the OD600 value reaches 0.4-0.6, the mixture is placed at 28 ℃ for 30 minutes. Isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0mmol/L, and shaking culture was carried out at 28 ℃ for 24 hours. After the culture, the cells were collected, resuspended in PBS, sonicated, and centrifuged to obtain the supernatant. Collecting the Fiber-2 protein solution. Endotoxin removal was performed according to the method of example 2, and it was determined that the endotoxin content was reduced to 0.008X 105EU/mL。
Example 17 preparation of a Complex vaccine against avian bacterium paragallinarum
The avibacterium paragallinarum a-GCN4-tHA fusion protein antigen prepared in example 2, the avibacterium paragallinarum B-GCN4-tHA fusion protein antigen prepared in example 6, the avibacterium paragallinarum C-GCN4-tHA fusion protein antigen prepared in example 8, the newcastle disease antigen prepared in example 13, the avian influenza antigen prepared in example 14, the infectious bronchitis antigen prepared in example 15, and the avian adenovirus antigen prepared in example 16 were mixed in proportion, added to a mineral oil adjuvant, while a motor was started, stirred at 17500r/min for 5min, and a 1% thimerosal solution was added before the stirring was terminated to make the final concentration 0.01%. The concrete proportions are shown in tables 10, 11, 12 and 13.
TABLE 10 avian paragallibacterium composite vaccine ratio 1
Components Vaccine 12 Vaccine 13 Vaccine 14 Vaccine 15
A-GCN4-tHA protein (AGP titer) 1:8 1:8 1:8 1:8
B-GCN4-tHA protein (AGP titer) 1:16 1:16 1:16 1:16
C-GCN4-tHA protein (AGP titer) 1:16 1:16 1:16 1:16
N7a strain antigen (EID50/0.1mL) 108.0 — — —
HF strain antigen (EID50/0.1mL) — 108.0 — —
M41 strain antigen (EID50/0.1mL) — — 106.0 —
Fiber-2 protein (AGP potency) — — — 1:4
Mineral oil adjuvant (V/V%) 66％ 66％ 66％ 66％
TABLE 11 avian paragallibacterium composite vaccine ratio 2
Components Vaccine 16 Vaccine 17 Vaccine 18
A-GCN4-tHA protein (AGP titer) 1:8 1:8 1:8
B-GCN4-tHA protein (AGP titer) 1:16 1:16 1:16
C-GCN4-tHA protein (AGP titer) 1:16 1:16 1:16
N7a strain antigen (EID50/0.1mL) 108.0 108.0 108.0
HF strain antigen (EID50/0.1mL) 108.0 — —
M41 strain antigen (EID50/0.1mL) — 106.0 —
Fiber-2 protein (AGP potency) — — 1:4
Mineral oil adjuvant (V/V%) 66％ 66％ 66％
TABLE 12 compounding ratio of avibacterium paragallinarum compound vaccine 3
Components Vaccine 19 Vaccine 20
A-GCN4-tHA protein (AGP titer) 1:8 1:8
B-GCN4-tHA protein (AGP titer) 1:16 1:16
C-GCN4-tHA protein (AGP titer) 1:16 1:16
N7a strain antigen (EID50/0.1mL) 108.0 108.0
HF strain antigen (EID50/0.1mL) 108.0 108.0
M41 strain antigen (EID50/0.1mL) 106.0 —
Fiber-2 protein (AGP potency) — 1:4
Mineral oil adjuvant (V/V%) 66％ 66％
TABLE 13 avian paragallibacterium composite vaccine ratio 4
Figure BDA0002808342750000251
Figure BDA0002808342750000261
Example 18 Acinetobacter paragallinarum composite vaccine immunogenicity assay
18.1 partial immunogenicity assay for avibacterium paragallinarum
3 weeks old SPF chickens 330 were randomly divided into 33 groups, 10 groups, 31 groups to 33 groups of vaccine 12 prepared in immune example 17, 34 groups to 36 groups of vaccine 13 prepared in immune example 17, 37 groups to 39 groups of vaccine 14 prepared in immune example 17, 40 groups to 42 groups of vaccine 15 prepared in immune example 17, 43 groups to 45 groups of vaccine 16 prepared in immune example 17, 46 groups to 48 groups of vaccine 17 prepared in immune example 17, 49 groups to 51 groups of vaccine 18 prepared in immune example 17, 52 groups to 54 groups of vaccine 19 prepared in immune example 17, 55 groups to 57 groups of vaccine 20 prepared in immune example 17, 58 groups to 60 groups of vaccine 21 prepared in immune example 17, and 61 groups to 63 groups of non-immune as controls. Each immunization group is injected with 0.5mL of vaccine, the control group is injected with 0.5mL of PBS, and 28 days after immunization, 110 chickens in 31 st, 34 th, 37 th, 40 th, 43 th, 46 th, 49 th, 52 th, 55 th, 58 th and 61 st groups are injected with 0.2mL of A-type HN3 strain liquid (the number of viable bacteria is about 1000CFU) in the infraorbital sinus; 110 chickens in 32 th, 35 th, 38 th, 41 th, 44 th, 47 th, 50 th, 53 th, 56 th, 59 th and 62 th groups were injected into the infraorbital sinus with 0.2mL of HN5 strain B (containing about 500CFU viable count); 110 chickens in 33 rd, 36 th, 39 th, 42 th, 45 th, 48 th, 51 th, 54 th, 57 th, 60 th and 63 th groups were injected into the infraorbital sinus with 0.2mL of SD3 strain C (containing about 1000CFU viable count). The results of the toxin-counteracting protection are shown in tables 14, 15 and 16 after 7 days of continuous observation.
TABLE 14 results of partial immunogenicity of A-type avian Paragallinarum vaccine complex
Figure BDA0002808342750000271
TABLE 15 partial immunogenicity test results for B-type avian paragallibacterium composite vaccine
Figure BDA0002808342750000272
TABLE 16 results of partial immunogenicity of avian paragallibacterium C vaccine
Figure BDA0002808342750000281
The results show that the vaccine 12-21 immunization groups can provide immune protection against A type, B type and C type of avibacterium paragallinarum 28 days after immunization, and infectious rhinitis caused by A type, B type and C type of avibacterium paragallinarum of chicken groups can be completely protected. The avian paragallibacterium fusion protein provided by the invention is shown to be used as an antigen to prepare the avian paragallibacterium composite vaccine which can provide complete protection for chicken flocks.
18.2 partial immunogenicity assay for Newcastle disease Virus
80 SPF chickens of 3 weeks old are taken and divided into 8 groups, each group comprises 10 SPF chickens, and the 64 th group to the 70 th group are respectively injected with vaccine 12, vaccine 16 to vaccine 21 and 20 mu L/SPF chicken prepared in the immunization example 17 by neck subcutaneous injection; group 71 was injected subcutaneously with 20 μ L of saline as challenge control. All test chickens were kept separately, and 21 days after immunization, the 64 th to 70 th groups of immunized chickens, together with the 71 th group of challenge control chickens, were bled and serum was separated. Detecting the HI antibody of the Newcastle disease virus, meanwhile, attacking by intramuscular injection with the HN1101 strain virus liquid of the Newcastle disease virulent virus, observing for 14 days, and recording the morbidity, mortality and protection number. The results are shown in Table 17.
TABLE 17 partial immunogenicity test results for the complex vaccine of avian paragallibacterium newcastle disease virus
Figure BDA0002808342750000291
Note: HI antibodies were determined as geometric means of immune chicken antibodies.
The results show that the vaccine 12 and 16-21 immune groups can generate higher newcastle disease antibodies 21 days after immunization, and compared with a control, the immune groups can completely protect virulent attacks. The N7a strain Newcastle disease virus liquid provided by the invention is used as an antigen to prepare the avibacterium paragallinarum compound vaccine which can provide complete protection for chicken flocks.
18.3 partial immunogenicity assay for avian influenza
Taking 60 SPF chickens of 3 weeks old, dividing the SPF chickens into 6 groups, each group comprises 10 SPF chickens, and 72 th to 76 th groups respectively carry out neck subcutaneous injection immunization on the vaccine 13, the vaccine 16 and the vaccine 19-21 prepared in the embodiment 17, wherein each group comprises 0.3 mL; group 77 was injected subcutaneously with 0.3mL of saline as a challenge control. All test chickens were kept separately, and 21 days after immunization, the 72 th to 76 th immunized chickens were collected with the 77 th group control chicken, and blood was taken and serum was separated. Detecting the HI antibody titer of H9 subtype avian influenza virus, and simultaneously using HF strain virus solution for intravenous injection to challenge, wherein each 0.2mL (containing 10 percent of the virus solution)7.0EID50). Collecting cloacal swabs 5 days after virus challenge, inoculating 5 SPF (specific pathogen free) chick embryos of 10-11 days after treatment in an allantoic cavity, incubating and observing for 5 days, determining the agglutination value of the erythrocyte of the chick embryo fluid whether the dead embryo and the live embryo are dead embryos, and judging that the virus is separated positively if the agglutination value of 1 chick embryo fluid in the 5 chick embryos inoculated in each swab sample is not less than 1:16 (a micro method). For samples negative to virus isolation, the judgment should be made after blind transmission once. The immune group should be negative for at least 9 chicken viruses isolated; the control group should isolate at least 4 chicken viruses as positive. The results are shown in Table 18.
TABLE 18 partial immunogenicity test results for avian influenza virus of avian paragallibacterium composite vaccine
Figure BDA0002808342750000301
Note: HI antibodies were determined as geometric means of antibodies in immunized chickens.
The results show that the vaccine 13, the vaccine 16 and the vaccines 19 to 21 can generate higher avian influenza antibodies 21 days after immunization, and compared with a control group, the immune group can completely protect against virulent attacks. The H9 subtype avian influenza virus liquid provided by the invention is shown to be used as an antigen for preparing the avibacterium paragallinarum compound vaccine which can provide complete protection for chicken flocks.
18.4 partial immunogenicity test for infectious bronchitis in chickens
50 SPF chickens of 3 weeks old are taken and divided into 5 groups, each group comprises 10 SPF chickens, and the live vaccine (H120 strain) for infectious bronchitis is inoculated to the 78 th group to the 81 th group by eye dropping and nose dropping to obtain 1 feather (0.05 mL). On day 21 post inoculation, along with group 82 control chickens, blood was collected and serum was isolated. Meanwhile, the vaccine 14, the vaccine 17, the vaccine 19 and the vaccine 21 prepared in the immunization example 17 were injected subcutaneously into the neck of each of the 78 th to 81 th groups, and the volume of each of the groups was 0.3 mL. Collecting blood and separating serum of 82 th group of challenge control chicken 28 days after inoculation; serum collected from the 78 th to 81 th group of immunized chickens 21 days after the live vaccine first immunization and twice 28 days after the live vaccine immunization (serum collected from the 82 th group of challenge control chickens at the same time) is used for measuring the HI antibody titer. The geometric mean value of the antibody titer of the hyperimmune serum HI in the immune group is not less than 4 times of the geometric mean value of the antibody titer of the hyperimmune serum HI, and the geometric mean value of the antibody titer of the serum HI in the nonimmune control group is not more than 1:8 (micro-dose method). Simultaneously, the infectious bronchitis M41 with strong toxicity and each feather is used for treating the toxin by dripping the nose to 103.0EID50 for toxicity test. The results are shown in Table 19.
TABLE 19 partial immunogenicity test results for avian paragallibacterium composite vaccine avian infectious bronchitis
Figure BDA0002808342750000311
The results show that the geometric mean value of the antibody titer of the secondary immune serum HI of the vaccine 14, the vaccine 17, the vaccine 19 and the vaccine 21 is not lower than 4 times of the geometric mean value of the antibody titer of the primary immune serum HI, and the virus is not separated from the trachea of all immunized chickens after challenge, so that the strong-virus challenge can be completely protected. The avian paragallibacterium compound vaccine prepared by the avian infectious bronchitis virus liquid serving as the antigen can completely protect chickens.
18.5 avian adenovirus partial immunogenicity assay
Taking 50 SPF chickens of 3 weeks old, dividing the SPF chickens into 5 groups, wherein each group comprises 10 SPF chickens, and the 83 th group to the 86 th group are respectively injected with 0.3 mL/SPF chicken by neck subcutaneous injection for immunizing the vaccine 15, the vaccine 18, the vaccine 20 and the vaccine 21 prepared in the embodiment 17; group 87 was injected subcutaneously with 0.3mL of saline as a challenge control. All test chickens were kept separately, challenged with FAV-HN strain virus solution by intramuscular injection 21 days after immunization, observed for 14 days, and recorded for morbidity, mortality, and protection. The results are shown in Table 20.
TABLE 20 partial immunogenicity test results for avian adenovirus of avian paragallibacterium composite vaccine
Figure BDA0002808342750000312
The results show that the 87 th group of control group is completely killed, while the 83 th to 86 th groups of immune group have good immune protection effect on the immunized chickens, and the immune effect is good. The avian adenovirus antigen provided by the invention is shown to be used as a paragallibacterium avium compound vaccine prepared by the antigen and can provide complete protection for chicken flocks.
Proved by verification, the avibacterium paragallinarum compound vaccine provided by the invention can effectively resist the invasion of relevant pathogens, shows good immunogenicity, and can effectively control the prevalence of the avibacterium paragallinarum relevant diseases in China.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Puleco bioengineering GmbH
<120> avibacterium paragallinarum HA fusion protein and trimer thereof, vaccine composition prepared from same, preparation method and application
<130> 20NAP0197C
<141> 2020-11-30
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<213> A type Avibacterium paragallinarum (Avibacterium paragallinarum A subtype)
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Ile His Asp Ala Ile Asn Asn Val Leu Thr Lys Gly Ile Tyr Leu Lys
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Ala Asp Gln Asn Asp Pro Thr Gly Asn Gln Gly Gln Lys Val Glu Leu
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Gly Asn Ala Ile Thr Leu Ser Ala Thr Asn Gln Trp Ala Asn Asn Gly
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Val Asn Tyr Lys Thr Asn Asn Leu Thr Thr Tyr Asn Ser Gln Asn Gly
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Thr Ile Leu Phe Gly Met Arg Glu Asp Pro Ser Val Lys Gln Ile Thr
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Ala Gly Thr Tyr Asn Thr Thr Gly Asp Ala Asn Asn Lys Asn Gln Leu
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Asn Asn Thr Leu Gln Gln Thr Thr Leu Glu Ala Thr Gly Ile Thr Ser
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Ser Val Gly Ser Thr Asn Tyr Ala Gly Phe Ser Leu Gly Ala Asp Ser
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Val Thr Phe Ser Lys Gly Gly Ala Gly Thr Val Lys Leu Ser Gly Val
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Ser Asp Ala Thr Ala Asp Thr Asp Ala Ala Thr Leu Lys Gln Val Lys
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Asp Thr Ile Thr Glu Gly Asp Ala Thr Asp Asp Lys Lys Leu Thr Thr
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<213> type B avian bacterium paragallinarum (Avibacterium paragallinarum B subtype)
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Gly Asn Ala Ala Thr Asp Gly Ile Lys Val Ser Gly Val Lys Ala Gly
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Ser Ile Val Ile Asn Asn Gly Gly Ile His Ala Gly Asn Lys Val Ile
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180 185 190
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<213> Yeast (yeast)
<400> 6
Arg Met Lys Gln Leu Glu Asp Lys Val Glu Glu Leu Leu Ser Lys Asn
1 5 10 15
Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val Gly Glu
20 25 30
Arg
<210> 7
<211> 44
<212> PRT
<213> chicken (fowls)
<400> 7
Ile Glu Glu Asp Pro Cys Glu Cys Lys Ser Ile Val Lys Phe Gln Thr
1 5 10 15
Lys Val Glu Glu Leu Ile Asn Thr Leu Gln Gln Lys Leu Glu Ala Val
20 25 30
Ala Lys Arg Ile Glu Ala Leu Glu Asn Lys Ile Ile
35 40
<210> 8
<211> 30
<212> DNA
<213> Artificial sequence (synthetic sequence)
<400> 8
cgcggatccg atggcacaat tacatttaca 30
<210> 9
<211> 39
<212> DNA
<213> Artificial sequence (synthetic sequence)
<400> 9
ccgctcgagt taaccttgag tgctagatgc tgtaggtgc 39
<210> 10
<211> 4
<212> DNA
<213> Artificial sequence (synthetic sequence)
<400> 10
gssg 4
<210> 11
<211> 4
<212> DNA
<213> Artificial sequence (synthetic sequence)
<400> 11
gggs 4
<210> 12
<211> 17
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 12
ctccgggccc ctaaaag 17
<210> 13
<211> 15
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 13
cgggacggag gccgc 15

Claims (10)

1. An avibacterium paragallinarum HA fusion protein, wherein the avibacterium paragallinarum HA fusion protein consists of avibacterium paragallinarum HA protein fragments and a trimer-promoting structure which are sequentially arranged from an N end to a C end; the avibacterium paragallinarum HA protein fragment is an A type avibacterium paragallinarum HA protein fragment, a B type avibacterium paragallinarum HA protein fragment or a C type avibacterium paragallinarum HA protein fragment, the A type avibacterium paragallinarum HA protein fragment is shown as SEQ ID No.1, the B type avibacterium paragallinarum HA protein fragment is shown as SEQ ID No.2, and the C type avibacterium paragallinarum HA protein fragment is shown as SEQ ID No. 3.
2. The avibacterium paragallinarum HA fusion protein of claim 1, wherein the trimerization promoting structure is an avibacterium paragallinarum HA protein C-terminus, a T4 phage fibrin folding polymerization (T4 phase fibrin polymerization), a yeast Transcriptional activator GCN4(Transcriptional activator GCN4), or cmp (chicken cardiac tissue matrix protein); the C-terminal of the avibacterium paragallinarum HA protein is shown as SEQ ID NO.4, the C-terminal of the T4 bacteriophage fibrin is shown as SEQ ID NO.5, the yeast transcription activator GCN4 is shown as SEQ ID NO.6, and the CMP is shown as SEQ ID NO. 7;
preferably, a flexible peptide segment is also arranged between the avibacterium paragallinarum HA protein segment and the trimer-promoting structure, and the flexible peptide segment is shown as SEQ ID NO.10 or SEQ ID NO. 11.
3. An avibacterium paragallinarum HA fusion protein trimer, wherein any molecule of the avibacterium paragallinarum HA fusion protein trimer is formed by trimerization of three molecules of the avibacterium paragallinarum HA fusion protein according to any one of claims 1 to 2.
4. A vaccine composition, wherein the vaccine composition comprises an immunizing amount of avibacterium paragallinarum HA fusion protein trimer of claim 3, and a pharmaceutically acceptable carrier;
preferably, the avibacterium paragallinarum HA fusion protein trimer is an A-type avibacterium paragallinarum HA fusion protein trimer and/or a B-type avibacterium paragallinarum HA fusion protein trimer and/or a C-type avibacterium paragallinarum HA fusion protein trimer.
5. The vaccine composition of claim 4, wherein the AGP titer of the A type avibacterium paragallinarum HA fusion protein trimer is more than or equal to 1:8, the AGP titer of the B type avibacterium paragallinarum HA fusion protein trimer is more than or equal to 1:16, and the AGP titer of the C type avibacterium paragallinarum HA fusion protein trimer is more than or equal to 1: 16; preferably, the AGP titer of the A type avibacterium paragallinarum HA fusion protein trimer is 1: 8-1: 32, the AGP titer of the B type avibacterium paragallinarum HA fusion protein trimer is 1: 16-1: 32, and the AGP titer of the C type avibacterium paragallinarum HA fusion protein trimer is 1: 16-1: 32; more preferably, the AGP titer of the A type avibacterium paragallinarum HA fusion protein trimer is 1:8, the AGP titer of the B type avibacterium paragallinarum HA fusion protein trimer is 1:16, and the AGP titer of the C type avibacterium paragallinarum HA fusion protein trimer is 1: 16.
6. The vaccine composition according to claim 4, wherein the pharmaceutically acceptable carrier is an adjuvant selected from the group consisting of: (1) mineral oil, alumina gel adjuvant, saponin, alfvudine, 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, IMS1314, muramyl dipeptide, ISA 206 and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100;
the adjuvant content is 5% -70% V/V, and the adjuvant content is preferably 30% to 70% V/V, and more preferably 66% V/V.
7. The vaccine composition of claim 4, wherein the vaccine composition further comprises one or more of the group consisting of: 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, a chicken infectious laryngotracheitis virus antigen;
preferably, the vaccine composition further comprises one or more of the group consisting of: a chicken newcastle disease virus inactivated antigen, an avian influenza virus inactivated antigen, a chicken infectious bronchitis virus inactivated antigen, a chicken infectious bursal disease virus subunit antigen, an avian adenovirus inactivated antigen or subunit antigen, and an avian egg drop syndrome virus subunit antigen;
more preferably, the newcastle disease virus inactivated antigen is a whole virus inactivated antigen of an N7a strain, the avian influenza virus inactivated antigen is a whole virus inactivated antigen of an HF strain, the avian infectious bronchitis virus inactivated antigen is a whole virus inactivated antigen of an M41 strain, the avian infectious bursal disease virus subunit antigen is an avian infectious bursal disease virus VP2 protein, the avian adenovirus inactivated antigen is a whole virus inactivated antigen of an FAV-HN strain, the avian adenovirus subunit antigen is an avian adenovirus Penton protein or Fiber-2 protein, and the avian egg drop syndrome virus subunit antigen is an avian egg drop syndrome virus tFiber protein fragment.
8. The vaccine composition according to claim 7, wherein the content of the inactivated antigen of newcastle disease virus in chicken is 10 before inactivation8.0～109.0EID500.1ml, the content of the inactivated antigen of the avian influenza virus is 10 before inactivation6.5～108.5EID500.1ml, the content of the inactivated antigen of the avian infectious bronchitis virus is 10 before inactivation6.0～107.0EID500.1ml, the content of the chicken infectious bursal disease virus VP2 protein is AGP titer of 1: 16-1: 128, the content of the poultry adenovirus Penton protein is AGP titer of 1: 2-1: 16, and the poultry adenovirus FibThe content of er-2 protein is 1: 2-1: 16 of AGP titer, and the content of tFiber protein fragment of the avian egg-dropping syndrome virus is 1: 8-1: 32 of AGP titer;
preferably, the content of the inactivated antigen of the newcastle disease virus of the chicken is 10 before inactivation8.0EID500.1ml, the content of the inactivated antigen of the avian influenza virus is 10 before inactivation8.0EID500.1ml, the content of the inactivated antigen of the avian infectious bronchitis virus is 10 before inactivation6.0EID500.1ml, the content of the chicken infectious bursal disease virus VP2 protein is AGP titer 1:16, the content of the avian adenovirus Penton protein is AGP titer 1:4, the content of the avian adenovirus Fiber-2 protein is AGP titer 1:4, and the content of the avian egg drop syndrome virus tFiber protein fragment is AGP titer 1: 8.
9. A method of making the vaccine composition of claim 4, wherein the method comprises:
respectively recombining and expressing the A-type avibacterium paragallinarum HA fusion protein, the B-type avibacterium paragallinarum HA fusion protein and the C-type avibacterium paragallinarum HA fusion protein by using an Ecoli expression system, and respectively trimerizing the expressed A-type avibacterium paragallinarum HA fusion protein, the B-type avibacterium paragallinarum HA fusion protein and the C-type avibacterium paragallinarum HA fusion protein to form an A-type avibacterium paragallinarum HA fusion protein trimer, a B-type avibacterium paragallinarum HA fusion protein trimer and a C-type avibacterium paragallinarum HA fusion protein trimer;
respectively treating the A-type avibacterium paragallinarum HA fusion protein trimer, the B-type avibacterium paragallinarum HA fusion protein trimer and the C-type avibacterium paragallinarum HA fusion protein trimer in the step (1) by using a nonionic surfactant to remove endotoxin; and
and (3) uniformly mixing the endotoxin-removed A-type avibacterium paragallinarum fusion protein trimer, B-type avibacterium paragallinarum HA fusion protein trimer and C-type avibacterium paragallinarum HA fusion protein trimer with an adjuvant to obtain the vaccine composition.
10. Use of a vaccine composition according to any one of claims 4 to 8 in the manufacture of a medicament for the prevention and/or treatment of an avian paragallibacterium infection; preferably, the medicament for preventing and/or treating the avibacterium paragallinarum infection is a medicament for preventing and/or treating the avibacterium paragallinarum infection of broiler chicks with age below 21 days.
CN202011380504.2A 2020-11-30 2020-11-30 Avibacterium paragallinarum HA fusion protein and tripolymer thereof, vaccine composition prepared from avibacterium paragallinarum HA fusion protein, preparation method and application of vaccine composition Pending CN114573708A (en)

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