CN114573708B - Avian bacillus paragallinarum HA fusion protein and trimer thereof, vaccine composition prepared by using same, preparation method and application - Google Patents

Avian bacillus paragallinarum HA fusion protein and trimer thereof, vaccine composition prepared by using same, preparation method and application Download PDF

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CN114573708B
CN114573708B CN202011380504.2A CN202011380504A CN114573708B CN 114573708 B CN114573708 B CN 114573708B CN 202011380504 A CN202011380504 A CN 202011380504A CN 114573708 B CN114573708 B CN 114573708B
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CN114573708A (en
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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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Abstract

The invention relates to a avian secondary bacillus HA fusion protein which consists of avian secondary bacillus HA protein fragments and a trimerization promoting structure which are sequentially arranged from N end to C end; the avian infectious microbe HA protein fragment is an A-type, B-type or C-type avian infectious microbe HA protein fragment, the A-type avian infectious microbe HA protein fragment is shown as SEQ ID No.1, the B-type avian infectious microbe HA protein fragment is shown as SEQ ID No.2, and the C-type avian infectious microbe HA protein fragment is shown as SEQ ID No. 3. The trimer formed after the avian bacterium paragallinarum HA fusion protein is trimerized HAs good immunogenicity, and the prepared vaccine can not only completely protect chickens with low age, but also completely protect chickens with low age, thereby solving the early immunization problem of meat chickens in the prior art.

Description

Avian bacillus paragallinarum HA fusion protein and trimer thereof, vaccine composition prepared by using same, preparation method and application
Technical Field
The invention relates to a paragallinarum fusion protein and a trimer thereof, a prepared vaccine composition, a preparation method and application thereof, and belongs to the field of medical preparations containing antigens or antibodies.
Background
Avian bacillus paragallinarum (Avibacterium paragallinarum, apg) is a pathogenic bacterium causing acute upper respiratory disease in chickens, and has three serotypes A, B, C. The disease was first found in Poland and the United states, and after the discovery, it was confirmed that it frequently occurred in other countries. In 1980, there have been reports of the disease in China, and at present, 3 serotypes are the main epidemic serotypes. The infected respiratory tract disease is commonly called chicken infectious rhinitis (Avian infectious coryza, IC), clinically takes facial edema, nasosinusitis and lacrimation as main characteristics, can cause the reduction of egg yield of laying hens, the delay of the growth and the open period of bred chickens, the reduction of the weight gain rate of broilers, and causes huge economic loss for poultry industry. Apg 3 serotypes do not have cross-protective power and there is also a large difference in protective power between different isolated strains of the same serotype.
Infectious rhinitis of chicken caused by avian bacterium paragallinarum is one of the important diseases which seriously endanger the development of poultry farming industry worldwide. Among the various control measures, vaccine immunization remains the most important measure. At present, whole-bacterium inactivated vaccines are commonly used in the poultry industry, and have high requirements on the number of bacteria per unit volume of fermentation culture and the risk of biosafety caused by incomplete inactivation.
Subunit vaccine is a new and feasible genetic engineering vaccine developed in recent years, and research on avian bacillus paragallinarum subunit vaccine is attempted to take HA protein as a research object, however, because the molecular weight is too large, high-efficiency expression is difficult to realize in vitro, the subunit vaccine is not developed into a product, and no avian bacillus paragallinarum subunit vaccine is marketed in the prior art. In addition, the disease is characterized by short incubation period and rapid transmission, and can reach the whole chicken flock in a short time, so that the 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 whole-bacterium antigen vaccine preparation process always cause side reactions such as appetite reduction, listlessness, tumor at the injection position and the like, and the whole-bacterium antigen vaccine cannot be applied to low-day-old broiler chicks, especially cannot be used for chickens below 21 days old, and the tumor at the injection position cannot be completely absorbed due to early slaughter of broiler chickens. How to solve the problem of early-stage avian bacterium paragallinarum immunization of broiler chicks is also a clinical urgent need to be solved.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a avian secondary bacillus fusion protein and a vaccine composition prepared from the same, and the vaccine composition can effectively prevent and/or treat avian secondary bacillus infection.
The invention provides a avian secondary bacillus HA fusion protein, which consists of avian secondary bacillus HA protein fragments and a trimerization promotion structure which are sequentially arranged from N end to C end; the avian secondary bacillus HA protein fragment is an A-type avian secondary bacillus HA protein fragment, a B-type avian secondary bacillus HA protein fragment or a C-type avian secondary bacillus HA protein fragment, wherein the A-type avian secondary bacillus HA protein fragment is shown as SEQ ID No.1, the B-type avian secondary bacillus HA protein fragment is shown as SEQ ID No.2, and the C-type avian secondary bacillus HA protein fragment is shown as SEQ ID No. 3.
The avian paragallinarum HA protein fragment used in the invention HAs good immunogenicity, and the vaccine prepared by the avian paragallinarum HA protein fragment HAs better immune effect than the vaccine prepared by the corresponding avian paragallinarum HA protein after immunization of chickens.
As one embodiment of the invention, the trimerization structure in the avian paragallinarum HA fusion protein is the C-terminal end of the avian paragallinarum HA protein, the C-terminal end of the T4 bacteriophage fibrin (T4 phage fibritin foldon trimerization), the yeast transcription activator GCN4 (Transcriptional activator GCN 4), or the CMP (CHICKEN CARTILAGE matrix protein); the C-terminal of the avian bacterium paragallinarum HA protein is shown as SEQ ID NO.4, the C-terminal of the T4 phage 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 avian infectious disease paratope HA fusion protein, a flexible peptide is further arranged between the avian infectious disease paratope HA protein fragment and the trimerization structure, and the flexible peptide is shown as SEQ ID NO.10 or SEQ ID NO. 11.
The avian paragallinarum fusion protein of the present invention may further comprise a flexible connecting peptide, wherein the length of the flexible connecting peptide is 4-10 amino acid sequences. The flexible linker peptide allows for a chimeric protein with better affinity, mainly because the flexible linker peptide increases the space between structural domains, eliminates or reduces the steric physical barrier between adjacent structural domains, increases the relative independence of the structural domains of the fusion protein, and also increases the structural stability and biological function of the functional domains of the fusion protein.
The invention also provides a avian secondary bacillus HA fusion protein trimer, wherein any one molecule of the avian secondary bacillus HA fusion protein trimer is formed by trimerizing three molecules of avian secondary bacillus HA fusion proteins.
The avian bacillus paragallinarum HA fusion protein trimer can provide complete protection after being prepared into a vaccine, and can provide complete protection for low-day-age chicks.
The invention also provides a vaccine composition, wherein the vaccine composition comprises an immune amount of the avibacterium paragallinarum HA fusion protein trimer and a pharmaceutically acceptable carrier.
The invention adopts the avian secondary bacillus HA protein fragment and the trimerization promotion structure to form the fusion protein for high-efficiency expression for the first time, forms the trimerization structure, prepares the vaccine composition, can prevent and/or treat infectious rhinitis epidemic situation of chickens, and can enable animal organisms to quickly generate antibodies after the vaccine composition containing the fusion protein is used for immunizing animals, thus having good prevention and control effects on independent or mixed infection of the currently popular avian secondary bacillus and good biological safety. Can provide complete protection and can provide complete protection for chicks of low age.
In one embodiment of the vaccine composition, the avian paragallinarum HA fusion protein trimer is an A-type avian paragallinarum HA fusion protein trimer and/or a B-type avian paragallinarum HA fusion protein trimer and/or a C-type avian paragallinarum HA fusion protein trimer.
As one embodiment of the invention, in the vaccine composition, the titer of the A-type avibacterium paragallinarum HA fusion protein trimer AGP is more than or equal to 1:8, the titer of the B-type avibacterium paragallinarum HA fusion protein trimer AGP is more than or equal to 1:16, and the titer of the C-type avibacterium paragallinarum HA fusion protein trimer AGP is more than or equal to 1:16.
As a preferred embodiment of the invention, in the vaccine composition of the invention, the titer of the A-type avibacterium paragallinarum HA fusion protein trimer AGP is 1:8-1:32, the titer of the B-type avibacterium paragallinarum HA fusion protein trimer AGP is 1:16-1:32, and the titer of the C-type avibacterium paragallinarum HA fusion protein trimer AGP is 1:16-1:32.
The A-type avibacterium paragallinarum HA fusion protein trimer AGP titer can be selected from 1:8, 1:16 and 1:32.
The B-type avibacterium paragallinarum HA fusion protein trimer AGP titer can be selected from 1:16 and 1:32.
The C-type avibacterium paragallinarum HA fusion protein trimer AGP titer 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 titer of the A-type avibacterium paragallinarum HA fusion protein trimer AGP is 1:8, the titer of the B-type avibacterium paragallinarum HA fusion protein trimer AGP is 1:16, and the titer of the C-type avibacterium paragallinarum HA fusion protein trimer AGP 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, aluminium gel adjuvant, saponin, alfulidine, DDA; (2) A water-in-oil emulsion, an oil-in-water emulsion, and a water-in-oil-in-water emulsion; or (3) a polymer of acrylic acid or methacrylic acid, a copolymer of 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, E.coli heat labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, ISA 206, gel adjuvant; the adjuvant content is 5% -70% V/V.
As 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, the adjuvant content in the vaccine composition of the present invention is 66% V/V.
The vaccine compositions of the present invention may further incorporate additional agents into the compositions of the present invention.
As an embodiment of the present invention, the pharmaceutically acceptable carrier includes a drug, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative; the immunostimulants include interferon-alpha, interferon-beta, interferon-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin 2 (IL 2).
Preferably, the immunostimulants include interferon-alpha, interferon-beta, interferon-gamma, 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, the vaccine composition of the present invention further comprises one or more of the group consisting of: newcastle disease virus antigen, avian influenza virus antigen, infectious bronchitis virus antigen, infectious bursal disease virus antigen, egg drop syndrome virus antigen, avian reovirus antigen, escherichia coli antigen, avian adenovirus antigen, mycoplasma synoviae, mycoplasma gallisepticum antigen, pasteurella multocida antigen, marek's virus antigen, avian encephalomyelitis virus antigen, infectious laryngotracheitis virus antigen.
The vaccine composition of the invention can contain various antigen components, and the avian paragallinarum HA fusion protein trimer of the invention can form a combined vaccine or a compound vaccine with various antigens, and can be immunized in the form of the combined vaccine or the compound vaccine so as to simplify the immunization procedure.
As a preferred embodiment of the present invention, the vaccine composition of the present invention further comprises one or more of the group consisting of: newcastle disease virus inactivated antigen, avian influenza virus inactivated antigen, avian infectious bronchitis virus inactivated antigen, avian infectious bursal disease virus subunit antigen, avian adenovirus inactivated antigen or subunit antigen, avian egg drop syndrome virus subunit antigen.
In a more preferred embodiment of 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 a Fiber-2 protein, and the avian egg drop syndrome virus subunit antigen is an avian egg drop syndrome virus tFiber protein fragment.
As a preferred embodiment of the vaccine composition, the content of the inactivated antigen of the newcastle disease virus is 10 8.0～109.0EID50/0.1 ml before inactivation, the content of the inactivated antigen of the avian influenza virus is 10 6.5～108.5EID50/0.1 ml before inactivation, the content of the inactivated antigen of the avian infectious bronchitis virus is 10 6.0～107.0EID50/0.1 ml before inactivation, the content of VP2 protein of the avian infectious bursal disease virus is AGP titer 1:16-1:128, the content of the protein of the avian adenovirus Penton is AGP titer 1:2-1:16, the content of the protein of the avian adenovirus Fiber-2 is AGP titer 1:2-1:16, and the content of the protein fragment of the avian egg drop syndrome virus tFiber is AGP titer 1:8-1:32.
As a more preferred embodiment of the vaccine composition, the content of the inactivated antigen of the newcastle disease virus is 10 8.0EID50/0.1 ml before inactivation, the content of the inactivated antigen of the avian influenza virus is 10 8.0EID50/0.1 ml before inactivation, the content of the inactivated antigen of the avian infectious bronchitis virus is 10 6.0EID50/0.1 ml before inactivation, the content of VP2 protein of the avian infectious bursal disease virus is AGP titer 1:16, the content of Penton protein of the avian adenovirus is AGP titer 1:4, the content of Fiber-2 protein of the avian adenovirus is AGP titer 1:4, and the content of tFiber protein fragments of the avian egg drop syndrome virus is AGP titer 1:8.
The invention also provides a method of preparing the vaccine composition, 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, wherein the expressed A-type avibacterium paragallinarum HA fusion protein, B-type avibacterium paragallinarum HA fusion protein and C-type avibacterium paragallinarum HA fusion protein are respectively trimerized to form an A-type avibacterium paragallinarum HA fusion protein trimer, a B-type avibacterium paragallinarum HA fusion protein trimer and a C-type avinarum HA fusion protein trimer; step (2), 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 of the step (1) by using a nonionic surfactant to remove endotoxin; and step (3) uniformly mixing the endotoxin-removed A-type avibacterium paragallinarum fusion protein trimer, B-type avibacterium paragallinarum HA fusion protein trimer, C-type avibacterium paragallinarum HA fusion protein trimer and an adjuvant to obtain the vaccine composition.
The method for preparing the vaccine composition uses the antigen with good immunogenicity, and simultaneously reduces endotoxin by using an endotoxin removal technology, so that early immunity of the meat chicks can be realized at early stage, and the problem to be solved clinically is solved.
The invention also provides application of the vaccine composition in preparing a medicament for preventing and/or treating avian bacillus paragallinarum infection.
In one embodiment of the present invention, the agent for preventing and/or treating a secondary avian bacterial infection is an agent for preventing and/or treating a secondary avian bacterial infection in a meat chicken of 21 days or less.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
Definition of the definition
The term "avibacterium paragallinarum" (Avibacterium paragallinarum, apg) is a pathogenic bacterium of infectious rhinitis in chickens, which is facultative anaerobic and can cause acute respiratory diseases, mainly manifested by the appearance of large amounts of serous or mucinous secretions, facial oedema and conjunctivitis in the nasal passages and sinuses. Can lead to the reduction of the laying rate and the hatching rate and the increase of the elimination rate, thereby causing serious economic loss. Avian paragallinarum can be divided into A, B, C serotypes by plate agglutination assay.
The term "avian secondary bacillus HA protein fragment" is a partial fragment of avian secondary bacillus hemagglutinin protein, which is an important component in avian secondary bacillus antigen and is also the basis of avian secondary bacillus typing.
The term "trimer of avian paragallinarum HA protein fragments" i.e. three identical avian paragallinarum HA protein fragments are polymerized into one protein fragment, called trimer, which is a low molecular weight polymer.
The term "avibacterium paragallinarum HA trimer fusion protein" consists of a avibacterium paragallinarum HA protein fragment and a trimerization promotion structure, wherein the trimerization promotion structure is the C-terminal of the avibacterium paragallinarum HA protein, the C-terminal of T4 bacteriophage fibrin, and yeast transcriptional activator GCN4 or CMP.
The term "C-terminal end of the avibacterium paragallinarum HA protein" is similar to the C-terminal membrane anchor region of the TAA (trimeric autotransporter Adhesin) family (membrane anchor domain), consisting of 229 amino acids.
The term "C-terminal of the T4 bacteriophage fibrin" is also known as the C-terminal trimerization sequence of the T4 bacteriophage fibrin (T4 phage fibritin foldon trimerization sequence), consisting of 27 amino acids, the domain consisting of 3 identical subunits, each subunit comprising 1 beta-hairpin structure.
The term "yeast transcriptional activator GCN4" is a protein that binds DNA via a leucine zipper (leucine zipper) structure.
The term "CMP" is chicken cartilage matrix protein (CHICKEN CARTILAGE matrix protein).
The AGP titer is also called as an agar diffusion test titer (Agar Gel Precipitation (test) titer), the agar diffusion test is a precipitation reaction, the antigen and the antibody can be freely diffused to the periphery in the agar gel containing the electrolyte, the antigen and the antibody are combined with each other, a precipitation line appears at the optimal ratio, and the highest dilution ratio of the precipitation line appears as the effective value, namely the calibrated antigen or antibody content.
"Vaccine composition" refers to a pharmaceutical composition containing avian paragallinarum immunogenicity that can induce, stimulate or enhance an immune response in chickens against avian paragallinarum alone. The vaccine composition includes an immunizing amount of an avian paragallinarum immunoprotection antigen protein.
The term "immunizing amount" should be understood to mean an "immunologically effective amount," also known as an immunoprotective amount, or an amount effective to produce an immune response, that is an amount of antigen effective to induce an immune response in a recipient sufficient to prevent or ameliorate a sign or symptom of the disease, including adverse health effects or complications thereof. The immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of disease, including adverse health consequences or complications thereof caused by infection by a pathogen. Humoral immunity or cell-mediated immunity, or both, may 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 a vaccine can be assessed by measuring, for example, clinical signs such as mortality, reduction in morbidity, temperature values, overall physiological condition and overall health and performance of the subject. 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 does not irritate the body of the subject vaccine compositions other than the antigen, which does not hinder the use of the biological activity and properties of the compound.
The term "adjuvant" is a non-specific immunopotentiator that, when injected or pre-injected with an antigen, enhances the body's immune response to the antigen or alters the type of immune response.
Adjuvants may include aluminum gel adjuvants; saponins (saponines) such as Quil A、QS-21(Cambridge Biotech Incorporation,Cambridge MA)、GPI-0100(Galenica Pharmaceuticals Incorporation,Birmingham AL); water-in-oil emulsions; an oil-in-water emulsion; a water-in-oil-in-water emulsion; polymers of acrylic acid or methacrylic acid; a compound selected from copolymers of maleic anhydride and alkenyl (alk) derivatives. The term "emulsion" may be based in particular on light liquid paraffin oil (European Pharmacopea types); isoprenoid oils (isoprenoid oil) resulting from olefin oligomerization, 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, esters of mannitol (mannide) (such as anhydrous mannitol oleate), esters of aliphatic diols (glycerol), esters of polyglycerol (polyglycitol), esters of propylene glycol and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which are optionally ethoxylated, and also polyoxypropylene-polyoxyethylene block copolymers, in particular of Pluronic products, in particular of L121. See also "The theory and practical application of adjuvants"(Ed.by DES Stewart-Tull,John Wiley and Sons,New York,1995:51-94) written by Hunter et al and "Vaccine" written by Todd et al (1997, 15:564-570). For example, SPT emulsions described on page 147 and MF59 emulsions described on page 183 of the SPT emulsions described on page VACCINE DESIGN, the Subunit and adiuvant approach (Plenum Press, 1995) written by Powell M and Newman M may be used. The term "polymer of acrylic acid or methacrylic acid" is preferably a crosslinked acrylic acid or methacrylic acid polymer, in particular with polyalkenyl ethers or polyols of sugars (sugam), known as carbomers (trade name Carbopol) (Phameuropa, 1996,8 (2)). Those skilled in the art are also referred to U.S. patent No. 2909462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably no more than 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are replaced by unsaturated aliphatic groups (ALIPHATIC RADICAL) 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 GROUP). The unsaturated group may itself contain other substituents, such as methyl. These products are sold in the name carbopol, (BF Goodrich, ohio, USA) which is particularly suitable. They are crosslinked with allyl sucrose or with allyl pentaerythritol (allyl pentaerythritol). Among these, carbopol 974P, 934P, and 971P may be mentioned, and carbopol 971P is most preferably used. The term "copolymers of maleic anhydride and alkenyl derivatives" also contemplates copolymers of maleic anhydride and ethylene EMA (Monsanto) which dissolve in water to give an acidic solution, which is neutralized, preferably to physiological pH, in order to give an adjuvant solution into which the immunogenic, immunogenic or vaccine composition itself can be incorporated. The term "adjuvant" also includes, but is not limited to, RIBI adjuvant system (Ribi Incorporation), block co-polymer (CytRx, atlanta GA), SAF-M (Chiron, EMERYVILLE CA), monophosphoryl lipid A (monophosphoryl lipid A), avridine lipid-amine adjuvants, E.coli heat labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, gel adjuvants, and the like. Preferably, the adjuvant comprises one or more of an aluminum 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 (alkinyl) derivative, a RIBI adjuvant system, a Block co-polymer, SAF-M, a monophosphoryl lipid A, avridine lipid-amine adjuvant, an E.coli thermolabile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, or Gel adjuvant.
The term "mineral oil" is also named paraffin oil, white oil and white oil, and the mineral oil is widely used as an adjuvant in inactivated vaccines, and has the effects of delaying the residence time of immunogen in a body, enabling the immunogen to be released continuously and slowly, and enhancing the phagocytosis and sterilization capability of macrophages.
The term "composite vaccine" refers to vaccines prepared from bacterial and viral antigens. For example, the avian subunit antigen of the present invention may be combined with an E.coli antigen.
The term "combination vaccine" refers to vaccines made of viruses and viral antigens. For example, the avian paragallinarum subunit antigen of the present invention may be combined with newcastle disease virus antigen.
The term "endotoxin" is a component of the cell wall of gram-negative bacteria called lipopolysaccharide. Lipopolysaccharide is toxic to the host and is released when bacteria die and dissolve or when bacterial cells are destroyed by artificial means, and is therefore called endotoxin.
The term "preventing" as used herein refers to all actions of inhibiting infectious rhinitis or delaying onset of a disease in a chicken by administering a vaccine composition according to the present invention. The term "treatment" refers to all actions that lead to a alleviation or improvement of symptoms caused by avian infection in chickens by administration of the vaccine composition according to the present invention.
The advantages and features of the present invention will become more apparent from the following description of the embodiments. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
The sample treatment solution used in the examples of the present invention was PBS buffer (pH 7.4,0.02 mol/L), and the formulation of 1L volume PBS buffer was exemplified as follows: na 2HPO4·12H2O 5.8g、NaH2PO4·2H2 O0.59 g, but is not limited to this formulation; unless otherwise indicated, all were diluted with PBS buffer.
The chemical reagents used in the invention are all analytically pure and purchased from the national drug group. The experimental methods provided by the invention are conventional methods unless specified; the biological material, unless otherwise specified, is commercially available.
EXAMPLE 1 construction of BL21 (DE 3) -A-GCN4-tHA expression engineering Strain
1.1 Extraction of avian A-type DNA from avian bacteria
According to the specification of the virus DNA extraction kit, a strain HN3 of avian bacterium paragallinarum A (Avibacterium paragallinarum Serotype A STRAIN HN) which is preserved in China Center for Type Culture Collection (CCTCC) for 20 days in 01 month of 2015, the preservation number is CCTCC NO: M2015151, the preservation address is the pure culture of Chinese university of Wuhan and Wuhan, disclosed in Chinese patent application CN 106267176A) is selected, a plurality of colonies are picked up in 100 mu L of sterile double distilled water, boiled for 10 minutes, ice-bath for 10 minutes and centrifugation is carried out for 1 minute in 12000r/min, and the supernatant is taken as a template for subsequent experiments.
1.2A-tHA Gene amplification of protein fragments
Oligonucleotide primers were synthesized based on the sequences of the conserved regions at the 5 'and 3' ends of tHA protein genes 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 sequenced by Invitrogen company, and the amino acid sequence of the A-tHA protein fragment is shown as 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 named pET28a-GCN4. And (3) carrying out electrophoresis on a avian bacterium paragallinarum A-type tHA protein PCR product, purifying by using a DNA gel recovery kit, carrying out double digestion on the obtained DNA fragment by using BamHI and XhoI, connecting with pET28a-GCN4 plasmid subjected to the same double digestion treatment, transforming the connection product into escherichia coli DH5 alpha, and screening positive clones. The plasmid extraction kit is used for extracting the transformant plasmid and carrying out double enzyme digestion identification, the plasmid with correct enzyme digestion identification is subjected to sequencing analysis, and the recombinant plasmid with correct verification is named pET28a-A-GCN4-tHA.
Construction of 1.4BL21 (DE 3) -A-GCN4-tHA expression engineering Strain
The recombinant plasmid pET28a-A-GCN4-tHA with correct sequence is transformed into the competence of escherichia coli BL21 (DE 3) to construct the expression engineering bacterium BL21 (DE 3) -A-GCN4-tHA. Single colonies were picked from LB plates (containing 50. Mu.g/mL kanamycin), plasmids were extracted after expansion culture in LB liquid medium, double restriction enzyme identification was performed using BamHI and XhoI restriction enzymes, and sequencing analysis was performed on plasmids with correct restriction enzyme identification. The strain identified correctly by enzyme digestion and sequencing is stored at-70 ℃ for standby.
EXAMPLE 2 investigation of protein expression Properties of BL21 (DE 3) -A-GCN4-tHA expression engineering Strain
2.1 Expression of the GCN4-tHA protein
The identified correct expression engineering bacteria were streaked onto LB plates (containing 50. Mu.g/mL kanamycin) and incubated at 37℃for 14 hours. Single colonies were picked, inoculated into 3mL of LB liquid medium (containing 50. Mu.g/mL kanamycin), cultured for 14 hours at 37℃on a shaker (220 r/min), transferred into a 250mL triangular flask containing 100mL of LB liquid medium (containing 50. Mu.g/mL kanamycin) according to 1 mL/flask, cultured for 4 hours on a shaker (37 ℃,220 r/min), cooled to 28℃and added with isopropyl thioβ -D-galactoside (IPTG) at a final concentration of 0.2mmol/L, and cultured for 5 hours. Centrifuging the culture at 10000r/min at 4deg.C for 5min, collecting thallus, and re-suspending in 10mL sterile physiological saline, and performing ultrasonic crushing in ice-water bath for 35 min under the following conditions: the ultrasonic power is 400 watts after 5 seconds of operation and 15 seconds of intermittent operation. Centrifuging the crushed solution at 4 ℃ and 10000r/min for 10 minutes, and taking out supernatant for subsequent detection.
2.2 Potency determination of expressed proteins
Taking the supernatant of the thallus lysate in the 2.1 item, and carrying out AGP titer determination by using A-type avibacterium paragallinarum positive serum as an antibody according to a conventional method. AGP potency of the A-GCN4-tHA protein was 1:256, endotoxin content was 0.56X10: 10 5 EU/mL.
2.3 Removal of endotoxin
To a 1.5mL centrifuge tube, 0.5mL of the solution to be treated and 1% (v/v) final concentration of Triton X-114 (Triton X-114) (5. Mu.L) were added, and vortexed. Immediately after the sample was placed on ice for 5 minutes and vortex-cooled, the centrifuge tube was put into a 37 ℃ water bath for 5 minutes, so that a new two-phase was generated. The sample was then centrifuged at 37℃for 60s. After centrifugation, the target protein will remain in the upper layer, while the detergent containing endotoxin will remain in the bottom of the centrifuge tube in the form of oil droplets. The entire operation was cycled 3 times. The AGP titer of the A-GCN4-tHA protein was determined to be 1:256 and the endotoxin content was determined to be 0.01X10: 10 5 EU/mL.
2.4 Identification of expressed proteins
SDS-PAGE and Western Blot results show that A-GCN4-tHA has a molecular weight of about 60kDa, while non-reduced SDS-PAGE and Western Blot results show that protein bands with a molecular weight of about 180kDa form a trimeric structure, and can specifically react with rabbit anti-Apg A positive serum.
EXAMPLE 3 construction of BL21 (DE 3) -A-tHA expression engineering Strain and A-tHA protein expression
And (3) carrying out electrophoresis on a avian bacterium paragallinarum A-type tHA protein PCR product, purifying by using a DNA gel recovery kit, carrying out double digestion on the obtained DNA fragment by using BamHI and XhoI, connecting with pET28a plasmid subjected to the same double digestion treatment, transforming the connection product into escherichia coli DH5 alpha competence, and screening positive clones. The plasmid extraction kit is used for extracting the transformant plasmid and carrying out double enzyme digestion identification, the plasmid with correct enzyme digestion identification is subjected to sequencing analysis, and the recombinant plasmid with correct verification is named pET28a-A-tHA. The expression engineering strain BL21 (DE 3) -A-tHA was constructed by the method of example 1, and the correct strain was identified by digestion and sequencing and stored at-70℃for further use.
Referring to example 2, tHA proteins were expressed, the supernatant of the cell lysate was used to conduct AGP titer measurement by a conventional method using A-type avibacterium paragallinarum positive serum as an antibody. AGP potency of A-tHA protein was 1:256, endotoxin content was 0.58X10 5 EU/mL.
Endotoxin was removed by the method of example 2, and the AGP titer of the A-tHA protein was determined to be 1:256, and the endotoxin content was 0.01X10: 10 5 EU/mL.
EXAMPLE 4 preparation of avian Bacillus paragallinarum subunit A vaccine
The A-GCN4-tHA fusion protein prepared in example 2 and the A-tHA fusion protein prepared in example 3 were added to mineral oil adjuvants slowly, the motor was started at the same time, the stirring was continued for 5min at 17500r/min, and 1% thiomersal solution was added to a final concentration of 0.01% before the stirring was stopped. The specific proportions are shown in Table 1.
TABLE 1 avian bacillus paragallinarum A subunit vaccine formulation
Component (A) Vaccine 1 Vaccine 2 Vaccine 3
A-GCN4-tHA protein (AGP potency) 1:8 1:32 -
A-tHA protein (AGP potency) - - 1:32
Mineral oil adjuvant (V/V%) 66％ 66％ 66％
EXAMPLE 5 comparison of efficacy of avian A subunit vaccine against avian Paramygdalin
40 SPF chickens of 8 weeks of age were randomly divided into 4 groups, 10 per group, and the avian paragallinarum type A subunit vaccine prepared in example 4 was immunized. The 1 st to 3 rd groups are immunized with vaccine 1 to 3 respectively, and the 4 th group is not immunized as a virus-counteracting control. Each immune group was injected with 0.5 mL/dose of vaccine, the challenge control group was injected with 0.5 mL/dose of PBS, and each immune group together with the challenge control group was injected with 0.2mL (the viable count was about 2.0X10 4 CFU) of strain A HN 3. After continuous observation for 7 days, the onset of any symptom of facial swelling or nasal discharge is judged as the onset of the disease, and the toxicity attack protection result is shown in table 2.
TABLE 2 results of efficacy comparisons of avian Bacillus paragallinarum A subunit vaccine
The results show that vaccine 3 cannot provide effective immune protection against avian bacillus paragallinarum type a, and vaccine 1 and vaccine 2 can provide complete immune protection; all the attack control groups are ill. The result shows that the avian paragallinarum A type tHA protein still cannot provide effective immune protection when the antigen content is AGP titer 1:32, and the avian paragallinarum A type GCN4-tHA fusion protein provided by the invention can provide complete immune protection when the antigen content is AGP titer 1:8.
EXAMPLE 6 preparation of avian B subunit vaccine
6.1 Extraction of avian B-type DNA from avian bacteria
According to the specification of the virus DNA extraction kit, a strain HN5 of avian B-type chicken (avian B-type chicken strain HN5 (Avibacterium paragallinarum Serotype B Strain HN) which is preserved in China Center for Type Culture Collection (CCTCC) on the 22 th month of 2017, the preservation number is CCTCC NO: M2017056, the preservation address is pure culture of Chinese university of Wuhan and Wuhan, disclosed in Chinese patent application CN 108624522A) is selected, a plurality of colonies are picked up in 100 mu L of sterile double distilled water, boiled for 10 minutes, 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 Gene amplification of protein fragments
Oligonucleotide primers were synthesized based on the conserved region sequences of the 5 'and 3' ends of tHA protein genes, the upstream primer tHA-F was shown as SEQ ID NO.8, and the downstream primer tHA-R was shown as SEQ ID NO.9, and PCR was performed.
The PCR product was sequenced by Invitrogen company, and the amino acid sequence of the B-tHA protein fragment was shown as SEQ ID NO. 2.
6.3 Expression of the B-GCN4-tHA protein
BL21 (DE 3) -B-GCN4-tHA expression engineering strain was constructed by the method of example 1, B-GCN4-tHA protein was expressed by the method of example 2, and after endotoxin removal, the AGP titer of B-GCN4-tHA protein was determined to be 1:256 and the endotoxin content was determined to be 0.01X10 5 EU/mL.
Identification of the 4B-GCN4-tHA protein
SDS-PAGE and Western Blot results show that B-GCN4-tHA has a molecular weight of about 60kDa, while non-reduced SDS-PAGE and Western Blot results show that a protein band with a molecular weight of about 180kDa appears, indicating that B-GCN4-tHA forms a trimeric structure and can specifically react with rabbit anti-Apg B positive serum.
Construction of 6.5BL21 (DE 3) -B-tHA expression engineering strain and B-tHA protein expression
The B-type tHA protein PCR product of the avian bacillus paragallinarum is used for electrophoresis and then purified by using a DNA gel recovery kit to obtain a DNA fragment, an expression engineering strain BL21 (DE 3) -B-tHA is constructed by the method of the reference example 3, the B-tHA protein is expressed by the method of the reference example 2, after endotoxin is removed, the AGP titer of the B-tHA protein is determined to be 1:256, and the endotoxin content is 0.01X10 5 EU/mL.
6.6 Preparation of avian B subunit vaccine
The prepared B-GCN4-tHA fusion protein and B-tHA fusion protein are respectively and slowly added into mineral oil adjuvants, meanwhile, a motor is started, stirring is carried out for 5min at 17500r/min, and 1% merthiolate solution is added before stirring is stopped, so that the final concentration is 0.01%. The specific proportions are shown in Table 3.
TABLE 3 avian bacillus paragallinarum B subunit vaccine formulation
Component (A) Vaccine 4 Vaccine 5 Vaccine 6
B-GCN4-tHA protein (AGP potency) 1:16 1:32 -
B-tHA protein (AGP potency) - - 1:32
Mineral oil adjuvant (V/V%) 66％ 66％ 66％
Example 7 comparison of efficacy of avian B subunit vaccine against avian bacillus paragallinarum
40 SPF chickens of 8 weeks of age were randomly divided into 4 groups, 10 per group, and the avian B-subunit vaccine of paragallinarum prepared in example 6 was immunized. The 5 th to 7 th groups are immunized with vaccines 4 to 6 respectively, and the 8 th group is not immunized as a virus-counteracting control. Each immune group was injected with 0.5 mL/dose of vaccine, the challenge control group was injected with 0.5 mL/dose of PBS, and each immune group together with the challenge control group was injected with 0.2mL (the viable count was about 1.0X10 4 CFU) of strain B HN 5. After continuous observation for 7 days, the onset of any symptom of facial swelling or nasal discharge is judged as the onset of the disease, and the toxicity attack protection result is shown in table 4.
TABLE 4 comparison of efficacy of avian B subunit vaccine against avian bacillus paragallinarum
The results show that vaccine 6 can not provide complete immune protection against avian bacillus paragallinarum type B, and vaccine 4 and vaccine 5 can provide complete immune protection; all the attack control groups are ill. The result shows that the avian secondary bacillus B-type tHA protein still cannot provide complete immune protection when the antigen content is AGP titer 1:32, and the avian secondary bacillus B-type GCN4-tHA fusion protein provided by the invention can provide complete immune protection when the antigen content is AGP titer 1:16.
EXAMPLE 8 preparation of avian Bacillus paragallinarum C subunit vaccine
8.1 Extraction of avian Bacillus paragallinarum C-type DNA
According to the specification of the virus DNA extraction kit, a strain HN5 of avian bacillus paragallinarum type C (strain SD3 of avian bacillus paragallinarum (Avibacterium paragallinarum Serotype C STRAIN SD) which is preserved in China Center for Type Culture Collection (CCTCC) for 20 days in 01 month of 2015, the preservation number is CCTCC NO: M2015052, the preservation address is pure culture of university of Wuhan and Wuhan in China, disclosed in China patent application CN 106267176A) is selected, a plurality of colonies are picked up in 100 mu L of sterile double distilled water, boiled for 10 minutes, ice-bath for 10 minutes, centrifugation is carried out for 1 minute in 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 of the 5 'and 3' ends of tHA protein genes, the upstream primer tHA-F was shown as SEQ ID NO.8, and the downstream primer tHA-R was shown as SEQ ID NO.9, and PCR was performed.
The PCR product was sequenced by Invitrogen company, and the amino acid sequence of the C-tHA protein fragment is shown as SEQ ID NO. 3.
8.3 Expression of the C-GCN4-tHA protein
BL21 (DE 3) -C-GCN4-tHA expression engineering strain was constructed by the method of example 1, C-GCN4-tHA protein was expressed by the method of example 2, and after endotoxin removal, the AGP titer of C-GCN4-tHA protein was determined to be 1:256 and the endotoxin content was determined to be 0.01X10 5 EU/mL.
Identification of the 8.4C-GCN4-tHA protein
SDS-PAGE and Western Blot results show that C-GCN4-tHA has a molecular weight of about 60kDa, while non-reduced SDS-PAGE and Western Blot results show that a protein band with a molecular weight of about 180kDa appears, indicating that C-GCN4-tHA forms a trimeric structure and can react specifically with rabbit anti-Apg C positive serum.
Construction of 8.5BL21 (DE 3) -C-tHA expression engineering strain and C-tHA protein expression
The avian bacillus paragallinarum C tHA protein PCR product is used for electrophoresis, then a DNA gel recovery kit is used for purification to obtain a DNA fragment, BL21 (DE 3) -C-tHA expression engineering strain is constructed according to the method of example 3, C-tHA protein is expressed according to the method of example 2, after endotoxin is removed, the AGP titer of the C-tHA protein is determined to be 1:256, and the endotoxin content is 0.01X10 5 EU/mL.
8.6 Preparation of avian bacillus C subunit vaccine
The prepared C-GCN4-tHA fusion protein and the prepared C-tHA fusion protein are respectively and slowly added into mineral oil adjuvants, meanwhile, a motor is started, stirring is carried out for 5min at 17500r/min, and 1% merthiolate solution is added before stirring is stopped, so that the final concentration is 0.01%. The specific proportions are shown in Table 5.
TABLE 5 avian bacillus paragallinarum C subunit vaccine formulation
Component (A) Vaccine 7 Vaccine 8 Vaccine 9
C-GCN4-tHA protein (AGP potency) 1:16 1:32 -
C-tHA protein (AGP potency) - - 1:32
Mineral oil adjuvant (V/V%) 66％ 66％ 66％
Example 9 comparison of efficacy of avian paragallinarum subunit C vaccine
40 SPF chickens of 8 weeks of age were randomly divided into 4 groups, 10 per group, and the avian bacterium paragallinarum type C subunit vaccine prepared in example 8 was immunized. The 9 th to 11 th groups are immunized with 7 to 8 th groups of vaccines respectively, and the 12 th group is not immunized as a virus-counteracting control. Each immune group was injected with 0.5 mL/dose of vaccine, the challenge control group was injected with 0.5 mL/dose of PBS, and each immune group together with the challenge control group was injected with 0.2mL of C-type SD3 strain bacterial liquid (containing about 1.0×10 4 CFU) in the infraorbital sinus 28 days after immunization. After continuous observation for 7 days, the onset of any symptom of facial swelling or nasal discharge is judged as the onset of the disease, and the toxicity attack protection result is shown in Table 6.
TABLE 6 comparison of efficacy of avian bacillus paragallinarum C subunit vaccine
The results show that vaccine 9 cannot provide effective immune protection against avian bacillus paragallinarum type C, and vaccine 7 and vaccine 8 can provide complete immune protection; all the attack control groups are ill. The result shows that the avian secondary bacillus C-type tHA protein still cannot provide effective immune protection when the antigen content is AGP titer 1:32, and the avian secondary bacillus C-type GCN4-tHA fusion protein provided by the invention can provide complete immune protection when the antigen content is AGP titer 1:16.
EXAMPLE 10 preparation of avian subunit vaccine against avian Paramygdalin
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 into a mineral oil adjuvant, and simultaneously, a motor was started, the mixture was stirred for 5min at 17500r/min, and a 1% merthiolate solution was added to a final concentration of 0.01% before the stirring was stopped. The specific proportions are shown in Table 7.
TABLE 7 avian bacillus subunit vaccine formulation
Component (A) Vaccine 10 Vaccine 11
A-GCN4-tHA protein (AGP potency) 1:8 1:32
B-GCN4-tHA protein (AGP potency) 1:16 1:32
C-GCN4-tHA protein (AGP potency) 1:16 1:32
Mineral oil adjuvant (V/V%) 66％ 66％
Example 11 comparison of efficacy of avian subunit vaccine against SPF chickens with imported vaccine
The vaccine 10 prepared in immunization example 10 of group 13, group 17 and group 21, the vaccine 11 prepared in immunization example 10 of group 14, group 18 and group 22, and the trivalent inactivated vaccine (containing A type W strain with the content of more than or equal to 6X 10 8 CFU/mL before inactivation, B type Spross strain with the content of more than or equal to 6X 10 8 CFU/mL before inactivation, C type Modesto strain with the content of more than or equal to 6X 10 8 CFU/mL before inactivation) for the infectious rhinitis of the 8-week-old SPF chickens and the 16 th, 20 th and 24 th non-immunized as the challenge control are randomly divided into 12 groups, 10 groups, 13 groups, 17 groups and 21 groups. Each immunization group is injected with 0.5 mL/dose of vaccine, the challenge control group is injected with 0.5 mL/dose of PBS, and 40 chickens in 13, 14, 15 and 16 groups are injected with 0.2mL (the viable count is about 2.0X10 4 CFU) of HN3 strain bacteria liquid in the infraorbital sinus 28 days after immunization; group 17, group 18, group 19 and group 20 40 chickens were injected with 0.2mL of strain B HN5 (containing about 1.0 x 10 4 CFU of viable bacteria); group 21, group 22, group 23 and group 24 40 chickens were injected with 0.2mL of strain C SD3 strain solution (containing about 1.0X10 4 CFU viable count) in the infraorbital sinus. The results of the toxicity attack protection are shown in Table 8 after continuous observation for 7 days.
TABLE 8 results of efficacy comparison of avian subunit vaccine against imported vaccine against SPF chickens
The results show that the vaccine 10, the vaccine 11 and the imported vaccine immune group have obvious protective rate difference from the immune group and the virus attack control group compared with the non-immune virus attack control group, and the protective effect of the vaccine 10 and the vaccine 11 immune group is better than that of the imported vaccine, so that the subunit vaccine has good immune protective effect.
EXAMPLE 12 efficacy test of avian subunit vaccine against Small day-old SPF chickens
SPF chickens 60 of 3 weeks of age were randomly divided into 6 groups, 10 groups/group, 25 groups, 27 groups, 29 groups, immunization of the vaccine 10 prepared in example 10, and 26 groups, 28 groups, 30 groups were not immunized as a challenge control. Each immunized group was injected with 0.5 mL/dose of vaccine, the control group was injected with 0.5 mL/dose of PBS, and the 25 th and 26 th groups of 20 chickens were injected with 0.2mL (containing about 1000CFU of viable bacteria) of HN3 strain solution in the infraorbital sinus 28 days after immunization; group 27 and group 28 20 chickens, infraorbital sinus injected with 0.2mL of strain B HN5 (viable count approximately 500 CFU); group 29 and group 30 20 chickens were injected with 0.2mL (containing about 1000CFU viable bacteria) of strain C SD 3. The results of the toxicity attack protection are shown in Table 9 after continuous observation for 7 days.
TABLE 9 results of efficacy test of avian subunit vaccine against Small day-old SPF chickens
The result shows that the immune group of the vaccine 10 has obvious difference in protection rate from the control group compared with the non-immune control group, and further shows that the subunit vaccine has good immune protection effect.
The avian bacillus paragallinarum subunit vaccine can provide complete protection for 21-day-old chickens, so that the problem that chickens below 21-day-old chickens cannot be immune-protected in actual production is solved, and the food safety of broilers is ensured.
EXAMPLE 13 preparation of newcastle disease antigen
The N7a strain (NEWCASTLE DISEASE Virus (genotype VII), strain N7 a) of newcastle disease Virus (gene VII type) is preserved in China center for type culture collection (preservation number is CCTCC NO: V20145, preservation date is 2015, 10 month and 19 days, preservation address is university of Wuhan and Wuhan in China, and disclosed in China patent application CN 107281479A), 10-11 days old susceptible chick embryos are inoculated with 0.1mL of sterilized normal saline by proper dilution (10 -4 or 10 -5), and each embryo is continuously incubated at 37 ℃. Selecting dead and alive chick embryos 48-120 hours after inoculation, harvesting allantoic fluid, and determining the virus content to be 10 8.0EID50/0.1 mL. Adding formaldehyde solution (v/v) with the final concentration of 0.1%, inactivating at 37 ℃ while stirring for 16h every 4-6 h, and completely inactivating for later use.
EXAMPLE 14 preparation of avian influenza antigen
The H9 Subtype avian influenza virus HF Strain (avian influenza virus (H9 Subtype) HF Strain (Avian Influenza Virus (Subtype H9), stress HF) with the preservation number of CCTCC NO: V201941 and the preservation date of 2019, 06 month and 19 days, the preservation address of Wuhan university, wuhan) is diluted to 10 -3 by sterile normal saline (0.1 mL of virus liquid is added into 0.9mL of sterile normal saline, and diluted for 2 times in sequence after shaking and mixing uniformly) is adopted, and 10-day-old susceptible chick embryos (which are automatically hatched by SPF eggs purchased from Beijing Mei Weitong laboratory animal technologies Co., ltd.) are inoculated through allantoic cavities, wherein each embryo is 0.1mL (containing 10 5EID50). Sealing the pinhole after inoculation, and placing at 36-37 ℃ for continuous incubation without turning over the eggs. Taking out until 96 hours, standing the air chamber upwards, and cooling for 12-24 hours at the temperature of 2-8 ℃. And (5) harvesting embryo liquid from the cooled chick embryo. The virus content was determined to be 10 8.5EID50/0.1 mL. Adding formaldehyde solution (v/v) with the final concentration of 0.1%, inactivating at 37 ℃ for 24 hours after stirring every 4-6 hours.
EXAMPLE 15 preparation of antigen for infectious bronchitis in chickens
Taking a chicken infectious bronchitis virus M41 strain (purchased from Chinese veterinary drug monitoring institute), properly diluting (10 -2 or 10 -3) with sterile physiological saline, inoculating susceptible chicken embryos of 10-11 days old, 0.1mL each embryo, and continuously incubating at 36-37 ℃. The chick embryo which dies and survives 24 to 48 hours after inoculation is selected, allantoic fluid is harvested, and the virus content is measured to be 10 6.0EID50/0.1 mL. Adding formaldehyde solution (v/v) with the final concentration of 0.1%, placing at 37 ℃ for inactivation, stirring for 16h every 4-6 h during the inactivation, and completely inactivating for later use.
EXAMPLE 16 preparation of avian adenovirus antigen
16.1Fiber-2cDNA preparation
FADV viral DNA was extracted from an infected avian adenovirus FAV-HN strain (avian adenovirus, FAV-HN strain (Fowl aviadenovirus, STRAIN FAV-HN), accession No. CCTCC NO: V201609, accession No. China center for type culture Collection, accession No. university of Wuhan and Wuhan, china, accession No. 2016, 2 months and 29 days, disclosed in China patent application CN 107523556A) according to the viral RNA extraction kit procedure. Oligonucleotide primers are synthesized according to the sequences of conserved regions 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 oligonucleotide primers are recovered by using an agarose gel recovery kit and stored at the temperature of minus 20 ℃.
16.2 Expression vector construction
The optimized Fiber-2 protein gene was subjected to full sequence synthesis by Jin Weizhi Biotechnology Inc. of Suzhou and ligated into pET28a plasmid, respectively. E.coli BL21 (DE 3) is transformed by the plasmid after connection, single clone is selected and cultured in LB liquid medium (containing 50 mug/mL kanamycin) overnight, the plasmid is extracted and sequenced, positive clone is BL21 (DE 3) -FADV-Fiber-2 expression strain.
16.3 Preparation of Fiber-2 protein
BL21 (DE 3) -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 cultured with shaking at 28℃for 24 hours. After the culture, the cells were collected, resuspended in PBS, sonicated, and centrifuged to obtain the supernatant. Collecting Fiber-2 protein solution. Endotoxin removal was performed as described in example 2, and the endotoxin content was found to be reduced to 0.008X 10 5 EU/mL.
EXAMPLE 17 preparation of avibacterium paragallinarum composite vaccine
The avian secondary bacillus A-GCN4-tHA fusion protein antigen prepared in example 2, the avian secondary bacillus B-GCN4-tHA fusion protein antigen prepared in example 6, the avian secondary bacillus 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 avian infectious bronchitis antigen prepared in example 15 and the avian adenovirus antigen prepared in example 16 are mixed in proportion, added into a mineral oil adjuvant, simultaneously, a motor is started, the stirring is carried out for 5min at 17500r/min, and 1% merthiolate solution is added before the stirring is stopped, so that the final concentration is 0.01%. The specific proportions are shown in tables 10, 11, 12 and 13.
Table 10 avian bacillus composite vaccine ratio 1
Component (A) Vaccine 12 Vaccine 13 Vaccine 14 Vaccine 15
A-GCN4-tHA protein (AGP potency) 1:8 1:8 1:8 1:8
B-GCN4-tHA protein (AGP potency) 1:16 1:16 1:16 1:16
C-GCN4-tHA protein (AGP potency) 1:16 1:16 1:16 1:16
N7a strain antigen (EID 50/0.1 mL) 108.0 — — —
HF strain antigen (EID 50/0.1 mL) — 108.0 — —
M41 Strain antigen (EID 50/0.1 mL) — — 106.0 —
Fiber-2 protein (AGP potency) — — — 1:4
Mineral oil adjuvant (V/V%) 66％ 66％ 66％ 66％
Table 11 avian bacillus paragallinarum composite vaccine ratio 2
Component (A) Vaccine 16 Vaccine 17 Vaccine 18
A-GCN4-tHA protein (AGP potency) 1:8 1:8 1:8
B-GCN4-tHA protein (AGP potency) 1:16 1:16 1:16
C-GCN4-tHA protein (AGP potency) 1:16 1:16 1:16
N7a strain antigen (EID 50/0.1 mL) 108.0 108.0 108.0
HF strain antigen (EID 50/0.1 mL) 108.0 — —
M41 Strain antigen (EID 50/0.1 mL) — 106.0 —
Fiber-2 protein (AGP potency) — — 1:4
Mineral oil adjuvant (V/V%) 66％ 66％ 66％
Table 12 avian bacillus pair composite vaccine mixture ratio 3
Component (A) Vaccine 19 Vaccine 20
A-GCN4-tHA protein (AGP potency) 1:8 1:8
B-GCN4-tHA protein (AGP potency) 1:16 1:16
C-GCN4-tHA protein (AGP potency) 1:16 1:16
N7a strain antigen (EID 50/0.1 mL) 108.0 108.0
HF strain antigen (EID 50/0.1 mL) 108.0 108.0
M41 Strain antigen (EID 50/0.1 mL) 106.0 —
Fiber-2 protein (AGP potency) — 1:4
Mineral oil adjuvant (V/V%) 66％ 66％
Table 13 avian bacillus paragallinarum composite vaccine mixture ratio 4
EXAMPLE 18 avian bacterial composite vaccine immunogenicity test
18.1 Avian bacillus paragallinarum partial immunogenicity test
The SPF chicken 330 of 3 weeks of age was randomly divided into 33 groups, 10 groups, vaccine 12 prepared by immunization example 17 of 31 st to 33 th groups, vaccine 13 prepared by immunization example 17 of 34 th to 36 th groups, vaccine 14 prepared by immunization example 17 of 37 th to 39 th groups, vaccine 15 prepared by immunization example 17 of 40 th to 42 th groups, vaccine 16 prepared by immunization example 17 of 43 th to 45 th groups, vaccine 17 prepared by immunization example 17 of 46 th to 48 th groups, vaccine 18 prepared by immunization example 17 of 49 th to 51 th groups, vaccine 19 prepared by immunization example 17 of 52 th to 54 th groups, vaccine 20 prepared by immunization example 17 of 55 th to 57 th groups, vaccine 21 prepared by immunization example 17 of 58 th to 60 th groups, and no immunization of 61 th to 63 th groups as controls. Each immunization group was injected with 0.5 mL/dose of vaccine, the control group was injected with 0.5 mL/dose of PBS, and the total number of bacteria contained in the strain A type HN3 was 0.2mL (the number of viable bacteria was about 1000 CFU) in the group 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 and 61 groups of chickens, and the infraorbital sinus was injected with the strain A type HN3 at 28 days after immunization; group 32, group 35, group 38, group 41, group 44, group 47, group 50, group 53, group 56, group 59, group 62, 110 chickens, 0.2mL of strain B HN5 (containing about 500 CFU) was injected into the infraorbital sinus; group 33, group 36, group 39, group 42, group 45, group 48, group 51, group 54, group 57, group 60, group 63, 110 chickens were injected with 0.2mL (the viable count is about 1000 CFU) of the C-type SD3 strain solution. The results of the toxicity attack protection are shown in tables 14, 15 and 16 after continuous observation for 7 days.
TABLE 14 results of partial immunogenicity test of avian paragallinarum composite vaccine avian paragallinarum A type
TABLE 15 results of partial immunogenicity test of avian secondary bacillus compound vaccine against avian secondary bacillus B
TABLE 16 results of test for C-type partial immunogenicity of avian secondary bacteria composite vaccine
The results show that 28 days after the immunization groups of the vaccine 12-21 are immunized again, the immunity protection against the avian infectious coryza A type, B type and C type of the avian infectious coryza of the chicken flock can be completely protected. The paragallinarum bacillus fusion protein provided by the invention is shown to be used as an antigen to prepare the paragallinarum bacillus composite vaccine, so that complete protection can be provided for chicken flocks.
18.2 New castle disease Virus partial immunogenicity assay
80 SPF chickens of 3 weeks of age are divided into 8 groups of 10 chickens, and the 64 th to 70 th groups are respectively injected into the neck to immunize the vaccine 12, the vaccine 16 to the vaccine 21 and 20 mu L/chicken prepared in the example 17; group 71 was subcutaneously injected with 20 μl of physiological saline as an challenge control. All test chickens were kept separate, and on 21 days after immunization, the 64 th to 70 th immunized chickens, along with the 71 st challenge control chickens, were collected and serum was isolated. Detecting the HI antibody of the newcastle disease virus, simultaneously using the strong HN1101 strain virus liquid of the newcastle disease virus to attack through intramuscular injection, observing for 14 days, and recording the number of morbidity, mortality and protection. The results are shown in Table 17.
Table 17 results of immunogenicity test of Newcastle disease virus portion of avian bacillus composite vaccine
Note that: HI antibodies were determined as the geometric mean of immunized chicken antibodies.
The results show that the vaccine 12, the vaccine 16-vaccine 21 immune groups can generate higher newcastle disease antibodies 21 days after immunization, and compared with the control, the immune groups can completely protect against virulent attack. The N7a strain newcastle disease virus liquid provided by the invention is used as an antigen to prepare the parachicken bacillus composite vaccine which can provide complete protection for chicken flocks.
18.3 Avian influenza partial immunogenicity assay
Taking 60 SPF chickens of 3 weeks of age, dividing the SPF chickens into 6 groups of 10 chickens, and respectively carrying out neck subcutaneous injection on vaccine 13, vaccine 16, vaccine 19-vaccine 21 prepared in immunization example 17 and 0.3 mL/chicken 72-76; group 77 was subcutaneously injected with 0.3mL of saline as an challenge control. All test chickens were kept separate, and on day 21 post immunization, the 72 th to 76 th immunized chickens, along with the 77 th control chickens, were collected and serum was isolated. H9 subtype avian influenza HI antibody titers were measured while challenged with HF strain virus solution by intravenous injection, each at 0.2mL (containing 10 7.0EID50). Collecting cloacal swabs 5 days after virus attack, inoculating 5 SPF chick embryos of 10-11 days old into allantoic cavities after treatment, incubating and observing for 5 days, and determining the erythrocyte agglutination value of chick embryo liquid no matter dead embryo or living embryo, wherein the agglutination value of 1 chick embryo liquid in 5 chick embryos inoculated by each swab sample is not lower than 1:16 (micro method), and judging that the virus separation is positive. Samples negative for virus isolation should be determined after blind transmission. The immune group should be negative for at least 9 chicken viruses to isolate; the control group should be positive for at least 4 chicken viruses. The results are shown in Table 18.
Table 18 results of partial immunogenicity test of avian influenza Virus of avian Paramygdalin composite vaccine
Note that: HI antibodies were determined as the geometric mean of immunized chicken antibodies.
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, the immune group can completely protect against virulent attack. The H9 subtype avian influenza virus liquid provided by the invention is shown to be used as an antigen to prepare the parachicken avian bacillus composite vaccine which can provide complete protection for chicken flocks.
18.4 Avian infectious bronchitis partial immunogenicity test
50 SPF chickens of 3 weeks old are divided into 5 groups of 10 chickens, and each eye of 78 th to 81 th groups is inoculated with 1 feather fraction (0.05 mL) of live vaccine (H120 strain) of infectious bronchitis through nose drops. 21 days after inoculation, along with group 82 control chickens, blood was collected and serum was isolated. Meanwhile, each of the 78 th to 81 th groups was immunized by subcutaneous injection into the neck of each of the vaccine 14, vaccine 17, vaccine 19, vaccine 21 prepared in example 17, 0.3 mL/dose. 28 days after inoculation, blood is taken and serum is separated respectively along with the 82 nd group of virus-fighting control chickens; the serum collected twice on 21 days after the first immunization of the live vaccine and 28 days after the immunization of the inactivated vaccine (the serum collected by the 82 nd virus-attack control chicken at the same time) of the 78 th to 81 th groups of immunized chickens is used for measuring HI antibody titer. The geometric mean of the immune group di-immune serum HI antibody titers is not lower than 4 times that of the primary immune serum HI antibody titers, and the geometric mean of the non-immune control group serum HI antibody titers is not higher than 1:8 (micro method). Meanwhile, the experiment of the virulent infectious bronchitis M41 virus 10 3.0 EID50 per feather drop nose is used for the virus attack. The results are shown in Table 19.
Table 19 results of the partial immunogenicity test of avian infectious bronchitis in chicken with the avian bacillus complex vaccine
The results show that the geometric average value of the titer of the di-immune serum HI antibodies of the vaccine 14, the vaccine 17, the vaccine 19 and the vaccine 21 is not lower than 4 times of the geometric average value of the titer of the di-immune serum HI antibodies, and viruses are not separated from the air pipes of all immunized chickens after the challenge, so that the challenge of strong viruses can be completely protected. The chicken infectious bronchitis virus liquid provided by the invention is used as an antigen to prepare the secondary chicken avian bacillus composite vaccine, so that complete protection can be provided for chicken flocks.
18.5 Avian adenovirus partial immunogenicity test
50 SPF chickens of 3 weeks of age are divided into 5 groups of 10 chickens, and the 83 th to 86 th groups are respectively subjected to neck subcutaneous injection to obtain vaccine 15, vaccine 18, vaccine 20, vaccine 21 and 0.3 mL/chicken which are prepared in immunization example 17; group 87 was subcutaneously injected with 0.3mL of saline as an 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 the number of morbidity, mortality and protection was recorded. The results are shown in Table 20.
Table 20 results of avian adenovirus partial immunogenicity test of avian Paramygdalin composite vaccine
The results show that the 87 th control group is all ill and dead, and the 83 th to 86 th immune groups have better immune protection effect on immunized chickens and good immune effect. The avian adenovirus antigen provided by the invention is used as the parachicken avian bacillus composite vaccine prepared by the antigen, so that complete protection can be provided for chicken flocks.
Proved by the invention, the composite vaccine of the paragallinarum can effectively resist the invasion of related pathogens, shows good immunogenicity, and can effectively control the epidemic of related diseases of the paragallinarum in China.
The present invention is not limited to the above-mentioned embodiments, but is capable of modification and variation in all embodiments without departing from the spirit and scope of the present invention.
Sequence listing
<110> Protect bioengineering Co., ltd
<120> Avian bacterium paragallinarum HA fusion protein and trimer thereof, vaccine composition prepared therefrom, preparation method and application thereof
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<141> 2020-11-30
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<211> 15
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 13
cgggacggag gccgc 15

Claims (17)

1. The vaccine composition is composed of fusion proteins consisting of A-type avibacterium paragallinarum HA protein fragments and yeast transcription activating factors GCN4, B-type avibacterium paragallinarum HA protein fragments and GCN4, and C-type avibacterium paragallinarum HA protein fragments and GCN4, wherein the A-type avibacterium paragallinarum HA protein fragments are shown as SEQ ID No. 1, the B-type avibacterium paragallinarum HA protein fragments are shown as SEQ ID No. 2, the C-type avinarum HA protein fragments are shown as SEQ ID No. 3, and the yeast transcription activating factors GCN4 are shown as SEQ ID No. 6.
2. The vaccine composition of claim 1, wherein the vaccine composition comprises a pharmaceutically acceptable carrier.
3. The vaccine composition of claim 1, wherein the AGP titer of the fusion protein consisting of the a-type avibacterium paragallinarum HA protein fragment and the yeast transcriptional activator GCN4 is not less than 1:8, the AGP titer of the fusion protein consisting of the B-type avibacterium paragallinarum HA protein fragment and the GCN4 is not less than 1:16, and the AGP titer of the fusion protein consisting of the C-type avibacterium paragallinarum HA protein fragment and the GCN4 is not less than 1:16.
4. The vaccine composition of claim 1, wherein the AGP titer of the fusion protein consisting of the a-type avibacterium paragallinarum HA protein fragment and the yeast transcriptional activator GCN4 is 1:8-1:32, the AGP titer of the fusion protein consisting of the B-type avibacterium paragallinarum HA protein fragment and the GCN4 is 1:16-1:32, and the AGP titer of the fusion protein consisting of the C-type avibacterium paragallinarum HA protein fragment and the GCN4 is 1:16-1:32.
5. The vaccine composition of claim 1, wherein the AGP titer of the fusion protein consisting of the a-type avibacterium paragallinarum HA protein fragment and the yeast transcriptional activator GCN4 is 1:8, the AGP titer of the fusion protein consisting of the B-type avibacterium paragallinarum HA protein fragment and the GCN4 is 1:16, and the AGP titer of the fusion protein consisting of the C-type avibacterium paragallinarum HA protein fragment and the GCN4 is 1:16.
6. The vaccine composition of claim 2, wherein the pharmaceutically acceptable carrier is an adjuvant selected from the group consisting of: (1) Mineral oil, aluminium gel adjuvant, saponin, alfulidine, DDA; (2) A water-in-oil emulsion, an oil-in-water emulsion, and a water-in-oil-in-water emulsion; or (3) a polymer of acrylic acid or methacrylic acid, a copolymer of 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, E.coli heat labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, ISA 206, gel adjuvant.
7. The vaccine composition of claim 6, wherein the adjuvant content is 5% -70% V/V.
8. The vaccine composition of claim 6, wherein the adjuvant content is 30% to 70% V/V.
9. The vaccine composition of claim 6, wherein the adjuvant content is 66% V/V.
10. The vaccine composition of claim 1, wherein the vaccine composition further comprises one or more of the group consisting of: newcastle disease virus antigen, avian influenza virus antigen, infectious bronchitis virus antigen, infectious bursal disease virus antigen, egg drop syndrome virus antigen, avian reovirus antigen, escherichia coli antigen, avian adenovirus antigen, mycoplasma synoviae, mycoplasma gallisepticum antigen, pasteurella multocida antigen, marek's virus antigen, avian encephalomyelitis virus antigen, infectious laryngotracheitis virus antigen.
11. The vaccine composition of claim 1, wherein the vaccine composition further comprises one or more of the group consisting of: newcastle disease virus inactivated antigen, avian influenza virus inactivated antigen, avian infectious bronchitis virus inactivated antigen, avian infectious bursal disease virus subunit antigen, avian adenovirus inactivated antigen or subunit antigen, avian egg drop syndrome virus subunit antigen.
12. The vaccine composition of claim 11, wherein the newcastle disease virus-inactivated antigen is a whole virus-inactivated antigen of strain N7a, the avian influenza virus-inactivated antigen is a whole virus-inactivated antigen of strain HF, the avian infectious bronchitis virus-inactivated antigen is a whole virus-inactivated antigen of strain M41, 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 strain FAV-HN, the avian adenovirus subunit antigen is an avian adenovirus Penton protein or a Fiber-2 protein, and the avian egg drop syndrome virus subunit antigen is an avian egg drop syndrome virus tFiber protein fragment.
13. The vaccine composition of claim 10, wherein the chicken newcastle disease virus inactivated antigen content is 10 8.0~109.0EID50/0.1 ml before inactivation, the avian influenza virus inactivated antigen content is 10 6.5~108.5 EID50/0.1 ml before inactivation, the avian infectious bronchitis virus inactivated antigen content is 10 6.0~107.0 EID50/0.1 ml before inactivation, the avian infectious bursal disease virus VP2 protein content is AGP titer 1:16-1:128, the avian adenovirus Penton protein content is AGP titer 1:2-1:16, the avian adenovirus Fiber-2 protein content is AGP titer 1:2-1:16, and the avian egg drop syndrome virus tFiber protein fragment content is AGP titer 1:8-1:32.
14. The vaccine composition of claim 10, wherein the chicken newcastle disease virus inactivated antigen content is 10 8.0 EID50/0.1 ml before inactivation, the avian influenza virus inactivated antigen content is 10 8.0 EID50/0.1 ml before inactivation, the avian infectious bronchitis virus inactivated antigen content is 10 6.0 EID50/0.1 ml before inactivation, the avian infectious bursal disease virus VP2 protein content is AGP titer 1:16, the avian adenovirus Penton protein content is AGP titer 1:4, the avian adenovirus Fiber-2 protein content is AGP titer 1:4, and the avian egg drop syndrome virus tFiber protein fragment content is AGP titer 1:8.
15. A method of preparing the vaccine composition of claim 1, 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, wherein the expressed A-type avibacterium paragallinarum HA fusion protein, B-type avibacterium paragallinarum HA fusion protein and C-type avibacterium paragallinarum HA fusion protein are respectively trimerized to form an A-type avibacterium paragallinarum HA fusion protein trimer, a B-type avibacterium paragallinarum HA fusion protein trimer and a C-type avinarum HA fusion protein trimer;
Step (2), 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 of 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.
16. Use of a vaccine composition according to any one of claims 1 to 14 in the manufacture of a medicament for the prevention and/or treatment of avian bacterial infection in chickens.
17. The use according to claim 16, wherein the medicament for preventing and/or treating a secondary avian bacterial infection is a medicament for preventing and/or treating a secondary avian bacterial infection of a meat chicken of 21 days old or less.
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