CN110559433B - Avian influenza virus-like particle vaccine, and preparation method and application thereof - Google Patents
Avian influenza virus-like particle vaccine, and preparation method and application thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5258—Virus-like particles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention relates to an avian influenza virus-like particle vaccine, which comprises an immune amount of H7 subtype avian influenza virus-like particle antigen and a pharmaceutically acceptable carrier, wherein the avian influenza virus-like particle antigen is assembled by H7 subtype avian influenza virus HA, NA antigen protein and bovine immunodeficiency virus Gag antigen protein. Compared with subunit vaccines, the avian influenza virus-like particle vaccine provided by the invention has greatly improved immune efficacy, and has better immune effect than inactivated vaccines with higher antigen content. Meanwhile, the compound can also protect against H9 subtype avian influenza virus.
Description
Technical Field
The invention relates to the field of biological pharmacy, in particular to a virus-like particle vaccine capable of protecting against avian influenza virus, and a preparation method and application thereof.
Background
Virus-like particles (VLPs) are hollow particles with a size of 15-400 nm, which are assembled from structural proteins of viruses. VLPs can be prepared by expressing one (or more) structural protein(s) of a virus efficiently in vitro, allowing them to self-assemble into hollow particles that are morphologically similar to native virus. The method mainly clones virus structural protein genes into expression vectors, and then transfers the vectors into prokaryotic or eukaryotic cells for expression.
Avian Influenza Virus (AIV) belongs to the family orthomyxoviridae, the genus Influenza Virus, influenza a Virus. Avian Influenza (AI) is an Avian infection and disease syndrome caused by this virus. The international veterinary Office (OIE) and the livestock and poultry epidemic prevention regulation of our country classify the disease as a type A virulent infectious disease. The disease is currently reported to occur in many countries and regions of the world, where Highly Pathogenic Avian Influenza (HPAI), particularly caused by strains of the H7 subtype, is more severe, characterized primarily by sudden morbidity and high mortality rates, often leading to total mortality in the feeding flock.
Up to now, vaccination with whole virus vaccines remains the most effective means for preventing and controlling the pandemic of avian influenza virus. However, because cross immune protection is hardly generated between different subtypes of the avian influenza virus, the current production of avian influenza vaccines cannot meet clinical needs, and the corresponding vaccines aiming at different subtype epidemic strains of the avian influenza virus have longer production period and higher production cost, which causes waste of manpower and material resources and may not achieve ideal prevention and control effects.
Although the avian influenza virus-like particle vaccine published in literature research in the prior art can generate better immune response, the avian influenza virus-like particle vaccine still cannot achieve the immune effect of the commercial whole virus vaccine, and the universal expression efficiency is lower, so that the avian influenza virus-like particle vaccine with good immune effect, safety and controllable cost is urgently needed.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides an avian influenza virus-like particle vaccine, wherein the avian influenza virus-like particle vaccine comprises an immune amount of H7 subtype avian influenza virus-like particle antigen and a pharmaceutically acceptable carrier, and the avian influenza virus-like particle antigen is assembled by H7 subtype avian influenza virus HA, NA antigen protein and bovine immunodeficiency virus Gag antigen protein.
The H7 subtype avian influenza virus-like particle vaccine HAs better immune effect than a commercial inactivated vaccine with the same HA antigen content; and not only provides complete protection for H7 subtype avian influenza, but also provides complete protection for H9 subtype avian influenza.
As an embodiment of the present invention, in the avian influenza virus-like particle vaccine according to the present invention, the H7 subtype avian influenza virus HA antigen protein is encoded by a sequence represented by SEQ ID No.1 or a degenerate sequence thereof, the H7 subtype avian influenza virus NA antigen protein is encoded by a sequence represented by SEQ ID No.2 or a degenerate sequence thereof, and the bovine immunodeficiency virus Gag antigen protein is encoded by a sequence represented by SEQ ID No.3 or a degenerate sequence thereof.
In a preferred embodiment of the present invention, in the avian influenza virus-like particle vaccine of the present invention, the H7 subtype avian influenza virus HA antigen protein is encoded by a sequence shown in SEQ ID No.1, the H7 subtype avian influenza virus NA antigen protein is encoded by a sequence shown in SEQ ID No.2, and the bovine immunodeficiency virus Gag antigen protein is encoded by a sequence shown in SEQ ID No. 3.
The avian influenza virus-like particle vaccine of the invention HAs antigens of surface antigen hemagglutinin HA, neuraminidase NA and self-assembly body of bovine immunodeficiency virus structural protein Gag of avian influenza virus. The vaccine can provide good immunogenicity, is greatly improved in immune efficacy compared with subunit vaccines with the same content, and has better immune efficacy compared with inactivated vaccines.
Meanwhile, the avian influenza virus-like particle vaccine has good persistence of the immune efficacy, can last 224 days after immunization, and can still realize complete protection on chickens.
In one embodiment of the invention, in the avian influenza virus-like particle vaccine of the invention, the content of the antigen of the H7 subtype avian influenza virus-like particle is HA titer more than or equal to 6log2.
The avian influenza virus-like particle antigen HAs good immunogenicity, when the content of the avian influenza virus-like particle antigen is 6log2 of HA titer, complete protection against avian influenza can be generated 14 days after immunization, and the immune effect of the inactivated vaccine is better than that of an inactivated vaccine with higher HA antigen titer.
The HA titer of the antigen content of the H7 subtype avian influenza virus-like particles in the H7 subtype avian influenza virus-like particle vaccine can be randomly selected from 6.0log2, 6.1log2, 6.2log2, 6.3log2, 6.4log2, 6.5log2, 6.6log2, 6.7log2, 6.8log2, 6.9log2, 6.0log2 7.0log2, 7.1log2, 7.2log2, 7.3log2, 7.4log2, 7.5log2, 7.6log2, 7.7log2, 7.8log2, 7.9log2, 8.0log2, 8.1log2, 8.2log2, 8.3log2, 8.4log2, 8.5log2, 8.6log2, 8.7log2, 8.8log2, 8.9log2, 9.0log2.
In a preferred embodiment of the present invention, in the avian influenza virus-like particle vaccine of the present invention, the content of the antigen of the H7 subtype avian influenza virus-like particle is HA titer 6log 2-9 log2.
In a more preferred embodiment of the present invention, in the avian influenza virus-like particle vaccine of the present invention, the content of the antigen of the H7 subtype avian influenza virus-like particle is 6log2 to 8log2 of the HA titer.
As an embodiment of the present invention, in the avian influenza virus-like particle vaccine of the present invention, the pharmaceutically acceptable carrier includes an adjuvant, and the adjuvant includes: (1) White 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, IMS 1314, muramyl dipeptide and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100; the concentration of the adjuvant ranges from 5% to 70% V/V, preferably from 30% to 70%, more preferably 66% V/V.
As an embodiment of the present invention, the pharmaceutically acceptable carrier includes an adjuvant, the adjuvant including: (1) White 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, IMS 1314, muramyl dipeptide and Gel adjuvant;
preferably, the saponin is Quil A, QS-21, GPI-0100;
preferably, the emulsion is an SPT emulsion, an MF59 emulsion, or an emulsion formed from an oil in combination with an emulsifier, which emulsion may be based on light liquid paraffin oil, isoprenoid oil resulting from the oligomerization of olefins (such as squalane or squalene oil, oils resulting from the oligomerization of olefins, 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 (particularly isostearate); the emulsifier is a nonionic surfactant (especially esters of polyoxyethylated fatty acids (e.g. oleic acid), sorbitan, mannide (e.g. anhydrous mannitol oleate), aliphatic diols, glycerol, polyglycerol, propylene glycol and oleic, isostearic, ricinoleic or hydroxystearic acid, which may be ethoxylated, ethers of fatty alcohols and polyhydric alcohols (e.g. oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers (especially
In particular L121));
preferably, the polymer of acrylic acid or methacrylic acid is a crosslinked polymer of acrylic acid or methacrylic acid, in particular a compound carbomer crosslinked with polyalkenyl ethers or polyalcohols of sugars, preferably carbopol 974P, 934P and 971P;
preferably, the copolymer of maleic anhydride and alkenyl derivative is a copolymer EMA of maleic anhydride and ethylene;
preferably, the adjuvant is a white oil adjuvant, which is used to prepare a water-in-oil emulsion;
the concentration of the adjuvant ranges from 5% to 70% V/V, preferably from 30% to 70%, more preferably 66% V/V.
In one embodiment of the present invention, in the avian influenza virus-like particle vaccine of the present invention, the avian influenza virus-like particle vaccine further comprises an immunizing amount of H5 subtype avian influenza virus-like particle antigen, and the H5 subtype avian influenza virus-like particle antigen is assembled from H5 subtype avian influenza virus HA, NA antigen protein and bovine immunodeficiency virus Gag antigen protein.
When the H7 and H5 subtype bivalent vaccine is used 14 days after immunization, the H5 and H7 antigens in the bivalent vaccine can generate better immune effect than the inactivated vaccine with the antigen with higher HA content. Meanwhile, the bivalent vaccine can not only completely protect against H5 and H7 subtype avian influenza, but also completely protect against H9 subtype avian influenza; therefore, the compound can simultaneously protect against H5, H7 and H9 subtype avian influenza.
In a preferred embodiment of the present invention, in the avian influenza virus-like particle vaccine of the present invention, the H5 subtype avian influenza virus HA antigen protein is encoded by the sequence shown in SEQ ID No.4 or a degenerate sequence thereof, the H5 subtype avian influenza virus NA antigen protein is encoded by the sequence shown in SEQ ID No.5 or a degenerate sequence thereof, and the bovine immunodeficiency virus Gag antigen protein is encoded by the sequence shown in SEQ ID No.3 or a degenerate sequence thereof.
As a more preferred embodiment of the present invention, in the avian influenza virus-like particle vaccine of the present invention, the H5 subtype avian influenza virus HA antigen protein is encoded by a sequence shown in SEQ ID No.4, the H5 subtype avian influenza virus NA antigen protein is encoded by a sequence shown in SEQ ID No.5, and the bovine immunodeficiency virus Gag antigen protein is encoded by a sequence shown in SEQ ID No. 3.
As a preferable embodiment of the invention, in the avian influenza virus-like particle vaccine, the content of the H5 subtype avian influenza virus-like particle antigen is more than or equal to 6log2 of HA titer.
In the H7 and H5 subtype bivalent avian influenza virus-like particle vaccine, the HA titer of the antigen content of the H7 subtype avian influenza virus-like particle and the HA titer of the antigen content of the H5 subtype avian influenza virus-like particle can be randomly selected from 6.0log2, 6.1log2, 6.2log2, 6.3log2, 6.4log2, 6.5log2 6.6log2, 6.7log2, 6.8log2, 6.9log2, 7.0log2, 7.1log2, 7.2log2, 7.3log2, 7.4log2, 7.5log2, 7.6log2, 7.7log2, 7.8log2, 7.9log2, 8.0log2, 8.1log2, 8.2log2, 8.3log2, 8.4log2, 8.5log2, 8.6log2, 8.7log2, 8.8log2, 8.9log2, 9.0log2.
In a more preferred embodiment of the present invention, in the avian influenza virus-like particle vaccine of the present invention, the content of the antigen in the H5 subtype avian influenza virus-like particle is 6log2 to 9log2 of HA titer.
In a further preferred embodiment of the present invention, in the avian influenza virus-like particle vaccine of the present invention, the content of the antigen in the H5 subtype avian influenza virus-like particle is 6log2 to 8log2 of HA titer.
In one embodiment of the invention, in the H7, H5 subtype bivalent avian influenza virus-like particle vaccine of the present invention, the pharmaceutically acceptable carrier is an adjuvant; the adjuvant content is 5% to 70% V/V, preferably from 30% to 70%, more preferably 66% V/V.
In one embodiment of the present invention, the avian influenza virus-like particle vaccine further comprises a drug, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative; the immunostimulant includes alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 2 (IL 2).
To prepare such compositions, methods known in the art may be used.
The invention also provides a method for preparing the avian influenza virus-like particle vaccine, wherein the method comprises the following steps: cloning HA, NA antigen protein and Gag antigen protein genes of the H7 subtype avian influenza virus to the same vector, wherein the HA, NA and Gag antigen protein genes are respectively shown as sequences SEQ.ID No.1, SEQ.ID No.2 and SEQ.ID No. 3; transforming and recombining the vector obtained in the step (1) to obtain a recombinant baculovirus plasmid containing the HA, NA and Gag antigen protein genes; transfecting the recombinant baculovirus plasmid containing the HA, NA and Gag antigen protein genes obtained in the step (2) into an insect cell sf9, and expressing the HA, NA and Gag antigen proteins in series; separating avian influenza virus-like particle antigen which is released to the supernatant of an extracellular culture medium after the self-assembly in insect cells is finished and assembled by the HA, NA and Gag antigen proteins; and (5) adding an adjuvant into the avian influenza virus-like particle antigen obtained in the step (4), and uniformly mixing to obtain the avian influenza virus-like particle vaccine.
The invention utilizes an insect baculovirus expression system to produce the avian influenza virus-like particle antigen containing avian influenza HA, NA and bovine immunodeficiency virus Gag, and the HA, NA and Gag antigens can be self-assembled in insect cells and are released to the supernatant of an extracellular culture medium in the form of virus-like particle particles, so that the expression and assembly are efficient.
As a preferred embodiment of the present invention, in the method of the present invention, the vector in the step (1) is pFastBac I; the baculovirus plasmid in the step (2) is Bacmid.
The invention also relates to a method for preparing the avian influenza virus-like particle vaccine, wherein the method comprises the following steps: cloning the HA, NA antigen protein gene and the Gag antigen protein gene of the H7 subtype avian influenza virus to the same vector, wherein the HA, NA antigen protein gene and the Gag antigen protein gene of the H7 subtype avian influenza virus are respectively shown as sequences SEQ.ID No.1, SEQ.ID No.2 and SEQ.ID No.3, cloning the HA, NA antigen protein gene and the Gag antigen protein gene of the H5 subtype avian influenza virus to the same vector, and the HA, NA antigen protein gene and the Gag antigen protein gene of the H5 subtype avian influenza virus are respectively shown as sequences SEQ.ID No.4, SEQ.ID No.5 and SEQ.ID No. 3; transforming and recombining the vector obtained in the step (1) to obtain a recombinant baculovirus plasmid containing the HA, NA and Gag antigen protein genes of the H7 subtype avian influenza virus and a recombinant baculovirus plasmid containing the HA, NA and Gag antigen protein genes of the H5 subtype avian influenza virus; step (3) transfecting insect cells sf9 with the recombinant baculovirus plasmids containing the H7 subtype avian influenza virus HA, NA and Gag antigen protein genes and the recombinant baculovirus plasmids containing the H5 subtype avian influenza virus HA, NA and Gag antigen protein genes obtained in the step (2) respectively, and expressing the HA, NA and Gag antigen proteins in series; step (4) respectively separating avian influenza virus-like particle antigen assembled by the HA, NA and Gag antigen proteins of the H7 subtype avian influenza virus and avian influenza virus-like particle antigen assembled by the HA, NA and Gag antigen proteins of the H5 subtype avian influenza virus which are released into the supernatant of an extracellular culture medium after self-assembly in insect cells is completed; and (5) mixing the H7 subtype avian influenza virus-like particle antigen and the H5 subtype avian influenza virus-like particle antigen in proportion, adding an adjuvant, and uniformly mixing.
The invention utilizes the insect baculovirus expression system to produce the avian influenza virus-like particles, and has the advantages of high yield, low production cost, good immunogenicity, no biological safety risk and the like.
As an embodiment of the present invention, in the preparation method of the present invention, the vector in the step (1) is pFastBac I; the baculovirus plasmid in the step (2) is Bacmid.
The invention also provides application of the avian influenza virus-like particle vaccine in preparation of a medicament for preventing and/or treating diseases caused by avian influenza virus.
As an embodiment of the present invention, in the use of the present invention, the avian influenza virus is an H7 subtype, an H5 subtype and/or an H9 subtype avian influenza virus.
The administration object for preparing the medicament for preventing and/or treating the avian influenza virus infection comprises chickens.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
"Virus-like particles (VLPs)" are particles assembled from one or more viral structural proteins and have similar external structure and antigenicity to viral particles, but do not contain viral genes.
The term "vaccine" as used herein refers to a pharmaceutical composition comprising an avian influenza virus-like particle antigen which induces, stimulates or enhances the immune response of a chicken against avian influenza alone.
The term "immunizing amount" is to be understood as an "immunologically effective amount", also known as an immunoprotective amount or an amount effective to produce an immune response, which is an amount of antigen effective to induce an immune response in a recipient sufficient to prevent or ameliorate the signs or symptoms of disease, including adverse health effects or complications thereof. The immune response may be sufficient for diagnostic purposes or other testing, or may be suitable for use in preventing signs or symptoms of disease, including adverse health consequences or complications thereof caused by infection by a pathogen. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with a wild-type strain, while the protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs such as mortality, reduction in morbidity, temperature values, overall physiological condition of the subject, and overall health and performance. The immune response may include, but is not limited to, induction of cellular and/or humoral immunity.
The term "pharmaceutically acceptable carrier" refers to all other ingredients in the vaccine composition of the present invention, except for the avian influenza virus antigen, which do not stimulate the body and do not hinder the biological activity and properties of the compound used, and is preferably an adjuvant.
The term "adjuvant" may include an alumina gel adjuvant; 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; a copolymer of maleic anhydride and an alkenyl (alkinyl) derivative. 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 isostearate. 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 practical application of adjuvants by Hunter et al (Ed. By DES Stewart-Tull, john Wiley and Sons, new York, 1995. For example, SPT emulsions described on page 147 and MF59 emulsions described on page 183 of Vaccine design, the Subunit and the 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 US patent 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 (aliphatic) radicals 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) and are particularly suitable. They are crosslinked with allyl sucrose or with allyl pentaerythritol. Among these, carbopols 974P, 934P, and 971P may be mentioned, with carbopol 971P being most preferably used. The term "copolymers of maleic anhydride and alkenyl derivatives" also envisages the copolymers EMA of maleic anhydride and ethylene (Monsanto), these polymers being dissolved in water to give an acidic solution, neutralized, preferably to physiological pH, in order to give an adjuvant solution, into which the immunogenic, immunogenic or vaccinal composition itself can be incorporated. The term "adjuvant" also includes, but is not limited to, the RIBI adjuvant system (Ribi Incorporation), block co-polymer (CytRx, atlanta GA), SAF-M (Chiron, emeryville CA), monophosphoryl lipid A (monophosphoryl lipid A), avridine lipoamine adjuvant, E.coli heat-labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, gel adjuvant, and the like. Preferably, the adjuvant comprises one or more of an alumina Gel adjuvant, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a water-in-oil-in-water emulsion, a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and an alkenyl (alkenyl) derivative, a RIBI adjuvant system, a Block co-polymer, SAF-M, monophosphoryl lipid a, an Avridine lipid-amine adjuvant, escherichia coli heat labile enterotoxin, cholera toxin, 1314, muramyl dipeptide, or Gel adjuvant.
The term "preventing and/or treating" when referring to an avian influenza virus infection refers to inhibiting replication of avian influenza virus, inhibiting transmission of avian influenza virus, or preventing colonization of avian influenza virus in its host, as well as alleviating the symptoms of an avian influenza virus infected disease or disorder. Treatment is considered to be therapeutically effective if the viral load is reduced, the condition is reduced and/or the food intake and/or growth is increased.
The present invention will be further described with reference to specific embodiments, and advantages and features of the present 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 chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group.
In order that the invention may be more readily understood, reference will now be made to the following examples. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
Example 1 expression of H7 subtype avian influenza HA protein
1. Construction of Donor plasmids
HA genes shown in a sequence table SEQ.ID NO.1 are synthesized by Jinzhi, and enzyme cutting sites of incision enzymes BamHI and HindIII are added at the upstream and the downstream of the genes respectively. The synthesized HA gene is cut by BamHI and HindIII enzyme and is connected with a pFastBacI vector cut by the same enzyme, a connection product is transformed into Escherichia coli DH5 alpha, and a correct plasmid is identified and named as pFastBac-HA.
2. Construction and identification of recombinant Bacmid
Adding 2 mu l of pFastBac-HA plasmid into DH10Bac competent cells, flicking and uniformly mixing, incubating for 30min on ice, thermally shocking for 45s at 42 ℃, incubating for 5min on ice, adding 400 mu l of SOC culture medium for 4h at 37 ℃, taking 100 mu l of bacterial liquid, coating the bacterial liquid on a plate containing IPTG/X-gal/kana/tetracycle/Qingda three-antibody, culturing for at least 48h at 37 ℃, and picking white single bacterial colony to 5ml of Kana/tetracycle/Qingda three-antibody liquid LB culture medium for shaking bacteria overnight when the blue-white bacterial colony is obvious. Taking 1 mul as a template for PCR identification of bacteria liquid the next day. The size of the PCR product is about 3900bp, and the recombinant Bacmid-HA is obtained by extracting the recombinant Bacmid by using a reagent in a small-extraction kit of the Tiangen plasmid.
3. Obtaining and passaging recombinant baculovirus:
the recombinant Bacmid-HA was transfected into insect cells sf9. Reference to
II Regent instructions for transfection, 72h after transfection, and harvesting cell supernatant labeled rBac-HA P1 after cytopathic effect.
Sf9 cells in logarithmic growth phase are arranged according to 0.9X 10 6 Inoculating the cell/dish with 10cm cell culture dish, and adding the recombinant baculovirus of the P1 generation into the cell culture dish paved with sf9 according to the volume ratio of 1And (4) continuously culturing at 27 ℃ in the dish until cytopathic effect is obvious for about 72 hours, harvesting the supernatant and marking as P2 generation recombinant baculovirus, wrapping the baculovirus with tinfoil paper, and keeping the baculovirus in a refrigerator at 4 ℃ in a dark place for later use. The steps are repeated, and the recombinant baculovirus of P3 and P4 generations is obtained by inoculating according to the proportion of 1.
4. Expression and characterization of proteins
The recombinant virus transferred to P4 is inoculated with Sf9 cells according to the volume ratio of 1 to 5-1. Cells were disrupted by resuspension and harvested by centrifugation. The HA content in the extracellular supernatant was determined to be 0 and the intracellular HA content was determined to be 12log2. The results of transmission electron microscopy showed that the protein (HA protein) fragments were aggregated and no virus-like particles were formed.
Example 2 expression of H7 subtype avian influenza Virus-like particles
1. Carrier engineering
NdeI, notI, salI and XbaI restriction enzyme sites are sequentially inserted into 4413-4414 positions of pFastBac I by using a commercial vector pFastBac I as a template and primers pFBmut-F and pFBmut-R, and the primers are shown in Table 1. Adding 1 mul of DpnI enzyme into the PCR product to digest away the template plasmid, taking 5 mul to transform DH5 alpha according to a conventional method, and naming the successfully transformed plasmid as pFastBac mut.
TABLE 1 vector modified primer Table
2. Construction of three expression cassette Donor plasmids
HA gene shown in a sequence table SEQ ID NO.1, NA gene shown in SEQ ID NO.2 and Gag gene shown in SEQ ID NO.3 are synthesized by Jinzhi, and endonuclease BamHI and HindIII enzyme cutting sites are respectively added at the upstream and downstream of the genes. The synthesized HA gene, NA gene and Gag gene are respectively cut by BamHI and HindIII enzyme and connected with pFastBac mut cut by the same enzyme, the connection product is converted into DH5 alpha, and the correct plasmids are identified and named as pFastBac mut-HA, pFastBac mut-NA and pFastBac mut-Gag.
The method comprises the steps of taking pFastBac mut-HA and pFastBac mut-NA as templates, amplifying HA and NA expression cassettes by using primers PH-F (NdeI) + PA-R (NotI) and PH-F (SalI) + PA-R (XbaI), inserting the HA expression cassette into a pFastBac mut-Gag plasmid through NdeI + NotI, and naming the plasmid as pFastBac mut-Gag-HA; the NA expression cassette was inserted into pFastBac mut-Gag-HA by SalI + XbaI and the plasmid was named pFastBac mut-Gag-HA-NA.
3. Construction and identification of recombinant Bacmid
Adding 2 mu l of pFastBac mut-Gag-HA-NA plasmid into DH10Bac competent cells, flicking and uniformly mixing, incubating on ice for 30min, thermally shocking at 42 ℃ for 45s, incubating on ice for 5min, adding 400 mu l of SOC culture medium at 37 ℃ for 200rpm for 4h, taking 100 mu l of bacterial liquid, coating the bacterial liquid on a plate containing IPTG/X-gal/kana/tetracyclic/Qingda three-antibody, culturing at 37 ℃ for at least 48h, and picking white single bacterial colony to 5ml of Kana/tetracyclic/Qingda three-antibody liquid LB culture medium for shaking bacteria overnight when cyanobacteria colony is obvious. Taking 1 microliter as a template for carrying out PCR identification on bacteria liquid on the next day. The size of the PCR product is about 8300bp, and the reagent in the Tiangen plasmid miniprep kit is used for extracting the recombinant Bacmid, which is named Bacmid-Gag.
4. Obtaining and passaging recombinant baculovirus:
the recombinant Bacmid-Gag was transfected into insect cells sf9. Reference to
II Regent instructions for transfection, 72h after transfection, and harvesting cell supernatant labeled rBac-Gag P1 after cytopathic effect.
Sf9 cells in logarithmic growth phase are arranged according to 0.9X 10 6 And (3) inoculating the cell/dish with 10cm of cell culture dish, after the cells are completely attached to the wall, adding the P1 generation recombinant baculovirus into the sf9 paved cell culture dish according to the volume ratio of 1. The steps are repeated, and the P3 and P4 generations of recombinant baculoviruses are obtained by inoculation according to the proportion of 1.
5. Expression and characterization of proteins
The recombinant virus transferred to P4 is inoculated with Sf9 cells according to the volume ratio of 1 to 5-1. Cells were disrupted by resuspension and harvested by centrifugation. The HA content in the extracellular supernatant was determined to be 12log2, and the HA content in the cells was determined to be 2log2. The observation of a transmission electron microscope shows that the protein harvested from the extracellular supernatant presents a virus-like particle shape, is basically uniform in size and presents a hollow particle state; while the protein fragments harvested intracellularly (HA + NA + Gag protein) aggregate and no virus-like particles are formed.
Example 3 preparation of subunit vaccine and Virus-like particle vaccine for H7 subtype avian influenza
The HA protein obtained from the intracellular harvest in example 1, the HA, NA and Gag protein obtained from the intracellular harvest in example 2 and the virus-like particles obtained from the extracellular supernatant in example 2 with different contents are respectively added into a white oil adjuvant to prepare various vaccine compositions, and the specific mixture ratio is shown in Table 2.
TABLE 2H7 subtype avian influenza vaccine composition ratio
| Components | Vaccine 1 | Vaccine 2 | Vaccine 3 | Vaccine 4 |
| Example 1 protein (HA content) | 8log2 | - | - | - |
| Example 2 protein (HA content) | - | 8log2 | - | - |
| Example 2VLPs (HA content) | - | - | 6log2 | 8log2 |
| White oil adjuvant (V/V%) | 66％ | 66％ | 66％ | 66％ |
Example 4 immunogenicity testing of H7 subtype avian influenza vaccines
60 SPF chickens of 21 days old are divided into 6 groups of 10, the 1 st to 4 th groups are respectively injected subcutaneously into the neck to immunize the vaccines 1 to 4 prepared in the example 3, the 5 th group is injected subcutaneously with commercial inactivated vaccine (H5 (Re-8), H7 (H7-Re 1) bivalent inactivated vaccine, H7HA content 8log 2), the immunizing dose is 0.3ml, and the 6 th group is injected subcutaneously with 0.3ml physiological saline as a blank control. All test chickens were kept separately, blood was collected 14 days and 21 days after immunization, serum was separated, and HI antibody titer was determined. The results of the different HI antibody tests after immunization are shown in table 3.
TABLE 3 results of immunogenicity tests on H7 subtype avian influenza vaccines
* Remarking: the H7 (H7-Re 1) standard antigen of Kazakhstan research is used
The results show that the 14-day HI antibody titers in both the group 1 and group 2 immunization groups were below 6.0log2, and were not effective in providing immune protection against H7 subtype infection; the average value of the HI antibody titer of the 1 st group and the 2 nd group of immune groups at 21 days reaches more than 6.0log2, and immune protection against H7 subtype infection can be effectively provided; the 14-day HI antibody titer average values of the 3 rd group immunization group and the 4 th group immunization group are both higher than 6.0log2, so that the immune protection against H7 subtype infection can be effectively provided, and the synchronous immune effect of the H7 subtype infection is higher than that of the 5 th group commercial vaccine immunization group; the average value of 14-day HI antibody titers of the 5-group commercial vaccine immunization group reaches more than 6.0log2, so that immune protection against H7 subtype infection can be effectively provided; in group 3, under the condition of HA dose immunization lower than that of inactivated vaccine, the average value of HI antibody titer in 14 days still can exceed the same-phase immunization effect of commercial vaccine immunization groups; when the group 4 immunization group and the commercial vaccine group are immunized at the same dose, the 4 th group immune response is generated more quickly, and the average value of HI antibodies of the commercial vaccine for 21 days can be reached at 14 days. It was shown that the subunit vaccine prepared in example 3 of the present invention (vaccine 1) and the subunit vaccine containing a different antigenic component (vaccine 2) did not provide the desired immunogenic efficacy at 14 days; the virus-like particle vaccine composition provided by the invention can provide ideal immune efficacy in 14 days, and has better immune effect and quicker immune response compared with the commercial whole virus inactivated vaccine.
Example 5H7 subtype avian influenza vaccine Cross-protection assay
60 SPF chickens of 21 days old are divided into 6 groups, each group comprises 10 SPF chickens, the 7 th group to the 10 th group are respectively injected subcutaneously through the neck to immunize vaccines 1 to 4 prepared in the embodiment 3, the 11 th group is injected subcutaneously with commercial inactivated vaccine (H5 (Re-8), H7 (H7-Re 1) bivalent inactivated vaccine, the H7HA content is 8log 2), the immunizing dose is 0.3ml, and the 12 th group is injected subcutaneously with 0.3ml physiological saline to be used as blank control. All test chickens were kept separately and challenged with H9 subtype avian influenza SZ strain virus solution by intravenous injection 21 days after immunization, each 0.2ml (containing 10) 7.0 EID 50 ). Collecting cloaca swab 5 days after toxin attack, treating,inoculating 5 SPF (specific pathogen free) chick embryos of 10-11 days in allantoic cavity, incubating and observing for 5 days, determining the agglutination value of chick embryo fluid erythrocytes in both dead embryos and live embryos, and judging that the virus is separated positively if only 1 chick embryo fluid agglutination value in the 5 chick embryos inoculated by each swab sample is not less than 1. For samples negative to virus isolation, the judgment should be made after blind transmission once. The immune group should be negative to isolate at least 9 chicken viruses; the control group should isolate at least 4 chicken viruses as positive. The results are shown in Table 4.
TABLE 4H7 subtype avian influenza vaccine Cross-protection test results
The results show that vaccine 1, vaccine 2 and the commercial vaccine immunization group can not resist the attack of the strong virus of the H9 subtype avian influenza 21 days after immunization; the vaccine 3 and vaccine 4 immunization groups can resist the attack of the strong virus of the H9 subtype avian influenza 21 days after immunization. This indicates that the avian influenza subunit vaccine prepared in example 3 of the present invention could not provide protection against heterotypic H9 subtype avian influenza, whereas the avian influenza virus-like particle vaccine provided by the present invention could provide protection against heterotypic H9 subtype avian influenza infection.
Example 6 duration of immunization test with H7 subtype avian influenza Virus-like particle vaccine
30 SPF chickens of 21 days old were divided into 3 groups of 10, and the vaccine 3 prepared in example 3 was injected subcutaneously into the neck of group 13, commercial inactivated vaccine (bivalent inactivated vaccine H5 (Re-8) and H7 (H7-Re 1) with 8log2 of H7HA content) was injected subcutaneously into group 14, and the immunization dose was 0.3ml, and 0.3ml of physiological saline was injected subcutaneously into group 15 as a blank control. All test chickens were kept separately, blood was collected 21 days, 56 days, 84 days, 112 days, 140 days, 168 days, 196 days, and 224 days after immunization, serum was separated, and HI antibody titer was measured. The results of the different HI antibody tests after immunization are shown in table 5.
TABLE 5 duration of immunization test results for subtype H7 avian influenza virus-like particle vaccine
| Group of | 21 days | For 56 days | 84 days | For 112 days | 140 days | 168 days | 196 days | 224 days |
| 13 | 10.2 | 10.2 | 9.3 | 9.0 | 8.7 | 8.3 | 7.6 | 7.3 |
| 14 | 9.1 | 8.6 | 7.3 | 7.1 | 6.8 | 6.5 | 5.5 | 5.1 |
| 15 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
* Remarking: the H7 (H7-Re 1) standard antigen was used
The results show that the duration of immunization in the vaccine 3 immunization group is longer than that in the commercial vaccine immunization group, and the average HI antibody titer at 32 weeks (224 days) is still higher than 6.0log2, so that complete protection can be achieved. The high-efficiency expression virus-like particle vaccine provided by the invention is shown to have good immunogenicity, and maintain long-term immunity.
Example 7 expression of subtype H5 Virus-like particles
The HA gene shown in a sequence table SEQ ID NO.4, the NA gene shown in SEQ ID NO.5 and the Gag gene shown in SEQ ID NO.3 are synthesized by Jinzhi, the method of the embodiment 2 is referred to carry out high-efficiency expression of the H5 subtype avian influenza virus-like particles, and the HA content in the extracellular supernatant is determined to be 12log2, and the HA content in the cells is determined to be 2log2. The results of observation by a transmission electron microscope show that the protein harvested from the extracellular supernatant presents a virus-like particle shape, is uniform in size and presents a hollow particle state; and the protein fragments harvested in the cells are aggregated, and no virus-like particles are formed.
Example 8 preparation of H7, H5 subtype bivalent avian influenza Virus-like particle vaccine
The virus-like particles harvested from the extracellular supernatant of example 2 and the virus-like particles harvested from the extracellular supernatant of example 7 were added to white oil adjuvant to prepare vaccine compositions, the specific ratios are shown in table 6.
TABLE 6 proportioning of H7, H5 subtype bivalent avian influenza virus-like particle vaccine compositions
| Components | Vaccine 5 | Vaccine 6 |
| Example 2VLPs (HA content) | 6log2 | 8log2 |
| Example 7VLPs (HA content) | 6log2 | 8log2 |
| White oil adjuvant (V/V%) | 66％ | 66％ |
Example 9 immunogenicity test of H7, H5 subtype bivalent avian influenza Virus-like particle vaccine
Partial immunogenicity test for H7 subtype avian influenza
40 SPF chickens of 21 days old were divided into 4 groups of 10, 16 th and 17 th groups, and were injected subcutaneously through the neck to immunize vaccine 5 and vaccine 6 prepared in example 8, respectively, and 18 th group was injected subcutaneously with commercial inactivated vaccine (H5 (Re-8), H7 (H7-Re 1) bivalent inactivated vaccine, H7HA content 8log 2), the immunization dose was 0.3ml, and 19 th group was injected subcutaneously with 0.3ml physiological saline as a blank control. All test chickens were kept separately, blood was collected 14 days and 21 days after immunization, serum was separated, and HI antibody titer was determined. The results of the different HI antibody tests after immunization are shown in table 7.
TABLE 7 partial immunogenicity test results for H7, H5 subtype bivalent avian influenza vaccine H7 subtype
* Remarking: the H7 (H7-Re 1) standard antigen of Kazakhstan research is used
The results show that the 14-day HI antibody titer average values of the 16 th immunization group and the 17 th immunization group are higher than 6.0log2, so that the immune protection against H7 subtype infection can be effectively provided, and the synchronous immune effect of the commercial vaccine immunization group of the 18 th group is exceeded; the average value of 14-day HI antibody titer of the 18 th group of commercial vaccine immunization groups reaches more than 6.0log2, so that immune protection against H7 subtype infection can be effectively provided; under the condition of lower dose immunization compared with inactivated vaccine, the average value of the HI antibody titer in 14 days still can exceed the average value of HI antibodies in a commercial vaccine immunization group; when the group 17 immunization group and the commercial vaccine group are immunized at the same dose, the group 17 immune response is generated more quickly, and the immune effect of the commercial vaccine can be achieved within 21 days within 14 days. The bivalent avian influenza virus-like particle vaccine composition provided by the invention has a better immune effect and a quicker immune response compared with a commercial whole virus inactivated vaccine.
Partial immunogenicity test for H5 subtype avian influenza
40 SPF chickens of 21 days old were divided into 4 groups of 10 chickens, the 20 th group and the 21 th group were injected subcutaneously through the neck to immunize vaccine 5 and vaccine 6 prepared in example 8, the 22 th group was injected subcutaneously with commercial inactivated vaccine (H5 (Re-8), H7 (H7-Re 1) bivalent inactivated vaccine, H7HA content 8log 2), the immunizing dose was 0.3ml, and the 23 th group was injected subcutaneously with 0.3ml physiological saline as a blank control. All test chickens were kept separately, blood was collected 14 days and 21 days after immunization, serum was separated, and HI antibody titer was determined. The results of the different HI antibody tests after immunization are shown in table 8.
TABLE 8 partial immunogenicity test results for H7, H5 subtype bivalent avian influenza vaccine H5 subtype
* Remarking: the H5 (Re-8) standard antigen was developed using Kazakhstan research
The results show that the 14-day HI antibody titer average values of the 20 th group immunization group and the 21 st group immunization group are higher than 6.0log2, so that the immune protection against H5 subtype infection can be effectively provided, and the synchronous immune effect of the group of commercial vaccine immunization groups is higher than that of the 22 th group immunization group; the 14-day HI antibody titer average value of the 22-group commercial vaccine immunization group reaches more than 6.0log2, so that immune protection against H5 subtype infection can be effectively provided; the average value of the HI antibody titer of the group 20 at 14 days can still exceed the average value of the HI antibody titer of the commercial vaccine immunization group under the condition of lower dose immunization compared with the inactivated vaccine; when the group 21 immunization group and the commercial vaccine group are immunized at the same dose, the group 21 immune response is generated more quickly, and the immune effect of the commercial vaccine can be achieved within 21 days within 14 days. The bivalent avian influenza virus-like particle vaccine composition provided by the invention has better immune effect and quicker immune response compared with the commercialized whole virus inactivated vaccine.
Example 10H7, H5 subtype bivalent avian influenza Virus-like particle vaccine Cross-protection assay
30 SPF chickens of 21 days old were divided into 3 groups of 10, 24 groups were immunized by cervical subcutaneous injection with vaccine 5 prepared in EXAMPLE 8, 25 groups were subcutaneously injected with commercial inactivated vaccine (H5 (Re-8), H7 (H7-Re 1) bivalent inactivated vaccine, both H7 and H5HA contents of 8log 2), the immunization dose was 0.3ml, and 26 groups were subcutaneously injected with 0.3ml physiological saline as a blank control. All test chickens were kept separately and challenged with H9 subtype avian influenza SZ strain virus solution by intravenous injection 21 days after immunization, each 0.2ml (containing 10) 7.0 EID 50 ). Collecting cloaca swabs 5 days after the virus attack, inoculating 5 SPF (specific pathogen free) chick embryos of 10-11 days after the treatment of the cloaca swabs, incubating and observing for 5 days, wherein the agglutination value of the erythrocyte of the chick embryo liquid is determined whether the dead embryo and the live embryo exist, and the virus can be judged to be positive if the agglutination value of 1 chick embryo liquid in the 5 chick embryos inoculated by each swab sample is not less than 1. For samples negative to virus isolation, the judgment should be made after blind transmission once. The immune group should be isolated from at least 9 chicken virusesIs negative; the control group should isolate at least 4 chicken viruses as positive. The results are shown in Table 9.
TABLE 9 results of cross-protection test for H7, H5 subtype bivalent avian influenza virus-like particle vaccine
The results show that the commercial vaccine immunization group can not resist the attack of the strong virus of the H9 subtype avian influenza 21 days after immunization; vaccine 5 immunization group was resistant to challenge by virulent H9 subtype avian influenza virus 21 days after immunization. It is shown that the H7 and H5 subtype bivalent avian influenza virus-like particle vaccine prepared in the embodiment 8 of the invention can provide protection against heterotypic H9 subtype avian influenza infection.
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> Luoyang Huzhong Biotechnology Co., ltd
<120> avian influenza virus-like particle vaccine, and preparation method and application thereof
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 1695
<212> DNA
<213> subtype H7 Avian Influenza Virus (Avian Influenza Virus, H7 subtype)
<400> 1
atgaacaccc agatcctcgt gttcgccctc atcgctatca tccccaccaa cgccgacaag 60
atctgtttag gccaccacgc tgtcagcaac ggtaccaagg tgaacactct gaccgagcgt 120
ggcgtggaag tcgtgaacgc taccgagacc gtcgagcgca ctaacacccc ccgtatctgc 180
tccaaaggca aacgcaccgt ggatttaggt cagtgcggtt tattaggtac catcaccggt 240
ccccctcagt gcgaccaatt tttagagttc tccgccgatt taatcatcga gcgccgcgaa 300
ggcagcgacg tgtgctaccc cggtaagttc gtgaacgagg aagccctccg tcagatttta 360
cgtgagagcg gcggtatcga caaggagccc atgggtttca cctacaacgg catccgcacc 420
aacggtgtga ccagcgcttg tcgtcgttcc ggtagcagct tttacgccga gatgaagtgg 480
ctcctctcca acaccgacaa tgctgccttc ccccagatga ccaagtccta caagaacact 540
cgtgagtccc ccgctatcat cgtgtggggc atccaccatt ccgtgtccac cgctgaacag 600
accaaactct acggctccgg taacaagctg gtcaccgtcg gcagctccaa ctaccagcag 660
tcctttgtcc cctcccccgg tgctcgtccc caagttaatg gtcaaagcgg tcgcatcgac 720
ttccactggc tgattttaaa ccccaacgac accgtgacct tctcctttaa cggcgctctg 780
attgcccccg atcgcgcttc ctttctgcgt ggcaagagca tgggcatcca gagcggtgtg 840
caagttgacg ctaactgtga aggtgattgc taccactccg gcggtactat catttccaat 900
ttacctttcc agaatattga cagccgcgct gtcggcaagt gtcctcgtta cgtgaagcaa 960
cgctctttat tattagccac tggtatgaag aacgtccccg aagtccctaa gggtaagcgt 1020
accgctcgcg gtctgttcgg cgctatcgct ggcttcatcg aaaacggttg ggagggttta 1080
atcgatggtt ggtacggctt tcgccaccag aacgcccaag gtgagggtac cgccgctgac 1140
tacaagtcca cccagtccgc catcgaccag attaccggca agctcaatcg tctgatcgcc 1200
aagaccaacc aacagtttaa gctgattgac aacgagttca acgaggtgga gaagcagatc 1260
ggcaacgtga ttaactggac ccgcgactcc atcaccgaag tgtggtccta taacgccgaa 1320
ctgctcgtgg ccatggagaa ccagcacact atcgatttag ctgacagcga gatggacaag 1380
ctgtacgagc gcgtcaagcg ccaactgcgt gagaacgctg aagaagacgg cactggctgt 1440
ttcgagattt tccacaaatg cgacgacgac tgcatggcta gcatccgcaa caacacctac 1500
gaccatcgca agtaccgcga agaggctatg cagaaccgta tccagatcga ccccgttaag 1560
ctgagctccg gctacaaaga cgtcatcctc tggttcagct tcggcgcttc ttgtttcatt 1620
ctgctcgcca tcgtcatggg tctggtgttc atttgtgtca agaacggtaa catgcgttgt 1680
actatctgca tctaa 1695
<210> 2
<211> 1398
<212> DNA
<213> subtype H7 Avian Influenza Virus (Avian Influenza Virus, H7 subtype)
<400> 2
atgaacccta accagaagat tttatgcact agcgccaccg ctatcaccat cggcgctatc 60
accgtgctga tcggtatcgc taatttaggt ttaaacatcg gtttacatct caaaagcggt 120
tgcaactgca gccgtagcca gcccgagact accaacacca gccagactat catcaacaac 180
tactacaacg agaccaatat cactaacatc caaatggagg aacgtaccag ccgcaacttc 240
aacaatttaa ctaagggtct gtgtaccatc aactcttggc acatctacgg caaggacaat 300
gccgtgcgca tcggtgaatc cagcgacgtg ctggtgactc gcgagcccta cgtgtcttgt 360
gaccccgatg agtgccgttt ctacgctctc tcccaaggta ctaccatccg cggcaaacac 420
tccaacggca ccatccacga ccgtagccag taccgcgctt taatcagctg gcctctgtcc 480
tcccccccta ccgtctacaa cagccgcgtg gagtgcatcg gttggtccag cacttcttgt 540
cacgacggta agtcccgcat gtccatctgc atcagcggtc ccaataacaa cgctagcgct 600
gtcatctggt acaaccgccg ccccgtcgcc gaaatcaaca cttgggcccg taatatcctc 660
cgcacccaag aatccgagtg tgtgtgccat aacggcgtgt gccccgttgt cttcaccgac 720
ggccccgcta ctggtcccgc tgacacccgc atctattact tcaaggaggg caagatttta 780
aagtgggaga gcctcaccgg taccgctaag cacatcgagg agtgctcttg ttacggtaag 840
cgtactggca ttacttgtac ttgtcgcgac aactggcaag gtagcaaccg tcccgttatc 900
cagatcgacc ccgtcgccat gactcacacc agccagtaca tctgctcccc cgttctgacc 960
gatagccccc gccctaacga ccctaacatc ggcaagtgca acgaccccta ccccggcaat 1020
aacaacaatg gcgtcaaggg tttcagctat ttagatggtg acaatacttg gctcggtcgt 1080
accatctcca ccgcttcccg ttccggctac gagatgctca aggtgcccaa cgctctgacc 1140
gacgaccgtt ccaagcctat ccaaggtcaa accattgtcc tcaacgccga ctggtccggc 1200
tactccggca gcttcatgga ctactgggct gagggcgact gctaccgcgc ttgcttctac 1260
gtggagctga tccgcggcaa gcccaaggag gacaaggtgt ggtggacttc caactccatc 1320
gtgtccatgt gctccagcac cgagttcctc ggccagtgga actggcccga tggcgccaaa 1380
attgagtact ttttataa 1398
<210> 3
<211> 1431
<212> DNA
<213> Bovine immunodeficiency virus (Bovine immunodeficiency virus)
<400> 3
atgaagagac gtgagctcga aaaaaagctg cgtaaggtgc gtgtcaccct ccaacaagat 60
aagtactaca ccatcggcaa cctgcaatgg gccatccgta tgatcaacct catgggcatc 120
aaatgcgtgt gcgatgagga atgctctgct gctgaggtgg ccctgatcat cacccagttc 180
tctgctctgg acctggagga cagccccatc aagggtaagg aagaagtcgc tattaagaac 240
accctcaagg tcttttggtc tctgctcgct ggttacaagc ccgagagcac cgaaaccgcc 300
ctcggttact gggaggcctt cacttacaga gagcgtgagg cccgtaccga gaaggaaggc 360
gagatcaaga gcatctaccc cagcctgact cagaacaccc agaataagaa gcaaacctct 420
aaccagacca acacccagag cctgcccgct atcactaccc aggacggtac cccccgtttc 480
gatcctgacc tcatgaagca gctcaagatc tggagcgatg ccaccgagcg taacggcatc 540
gacctccacg ctgtcaacat cctgggcgtc atcactgcta acctggtgca ggaggaaatc 600
aagctgctgc tcaacagcac ccccaagtgg cgtctcgacg tccagctgat cgaaagcaag 660
gtccgtgaga aagagaacgc ccatcgtacc tggaagcaac accaccccga agcccccaag 720
accgatgaaa tcatcggtaa gggcctgagc agcgccgaac aggctactct gatctctgtg 780
gagtgcagag agacctttcg tcagtgggtc ctccaagctg ccatggaagt cgctcaagcc 840
aaacacgcta cccctggtcc catcaacatc caccagggcc ccaaagagcc ctacaccgac 900
tttatcaaca gactggtcgc cgccctggaa ggtatggctg ctcccgaaac caccaaggag 960
tacctgctgc agcacctctc tatcgaccac gccaacgaag actgccagag catcctccgt 1020
cccctgggtc ctaacacccc tatggagaaa aaactggagg cctgccgtgt ggtcggctct 1080
cagaagagca agatgcagtt cctggtcgcc gccatgaagg agatgggtat ccagagccct 1140
atccctgctg tcctccctca tactcccgag gcttatgcca gccagactag cggccctgag 1200
gacggtagaa gatgctacgg ctgcggtaag accggccatc tgaagaaaaa ctgcaagcag 1260
cagaagtgct accattgcgg caagcctggt caccaggcca gaaactgtcg tagcaaaaac 1320
ggcaagtgct ctagcgcccc ctacggccaa cgtagccagc ctcagaacaa cttccaccag 1380
tctaacatgt ctagcgtcac cccttctgcc cctcctctca tcctcgatta a 1431
<210> 4
<211> 1704
<212> DNA
<213> subtype H5 Avian Influenza Virus (Avian Influenza Virus, H5 subtype)
<400> 4
atggaaaaga ttgtcctgct gctcgctgtg gtcagcctcg tgaagagcga ccagatctgc 60
atcggttacc acgctaacaa ttctaccgag caggtggaca ccatcatgga gaaaaacgtg 120
actgtgaccc acgcccagga cattctcgaa aagacccaca acggcagact ctgtgacctc 180
aacggtgtca agcccctcat cctgaaggac tgttctgtcg ccggctggct gctgggcaac 240
cctatgtgcg acgaattcat tcgtgtgccc gagtggagct acattgtcga gagagccaac 300
ccctctaacg acctctgtta ccccggcaac ctgaacgact acgaggaact gaagcacctg 360
ctgagcagaa ttaaccactt cgagaagact ctcatcattc ccaaaagcag ctggcccgat 420
catgacacct ctctgggtgt cagcgccgct tgcccctacc aaggcatgcc cagcttcttc 480
agaaacgtcg tctggctgat caagaagaac gacacctacc ccaccatcaa gatgagctac 540
aataacacca accgtgagga cctgctgatt ctgtggggca tccatcacag caacaacgct 600
gccgagcaga ctaacctcta caagaacccc accacctacg tgtctgtcgg taccagcacc 660
ctgaaccagc gtctggtccc caagattgcc actcgttctc aggtcaacgg tcagcgtggc 720
agaatggatt tcttctggac catcctgaag cccaacgatg ccatccattt cgagagcaac 780
ggtaacttca tcgctcccga gtacgcctac aagatcgtca agaagggcga cagcaccatc 840
atgaagagcg aaatggagta tggccactgc aacaccaaat gccagacccc tattggcgcc 900
attaacagca gcatgccctt ccacaacatc caccccctga ccatcggcga atgtcccaag 960
tacgtcaagt ctaacaagct cgtgctggcc accggtctcc gtaacaaccc cctccgtgaa 1020
agaagacgta agagaggtct gttcggtgct atcgccggct tcatcgaggg tggctggcag 1080
ggcatggtcg atggttggta cggttaccac catagcaacg aacagggtag cggctacgcc 1140
gctgacaagg agtctactca gaaggctatc gacggcgtca ccaacaaggt gaactctatc 1200
atcgacaaga tgaacactca attcgaggcc gtgggccgtg agttcaataa cctggaaaga 1260
agaattgaaa atctgaacaa gaagatggaa gacggcttcc tggacgtctg gacctacaac 1320
gccgaactgc tcgtcctgat ggaaaacgag agaaccctcg acttccacga cagcaatgtg 1380
aagaacctgt acgacaaagt cagactccaa ctgcgtgaca acgccaagga gctgggcaac 1440
ggctgctttg agttctacca caaatgcgac aacgaatgca tggaaagcgt ccgtaacggt 1500
acctacgact accctcaata cagcgaggaa gcccgtctca aacgtgaaga gatcagcggc 1560
gtgaaactcg agagcatcgg cacttaccag attctgtcta tctatagcac cgtcgctagc 1620
agcctggctc tggctatcat tgtcgctggc ctctctctct ggatgtgcag caacggcagc 1680
ctgcagtgca gaatttgcat ctaa 1704
<210> 5
<211> 1350
<212> DNA
<213> subtype H5 Avian Influenza Virus (Avian Influenza Virus, H5 subtype)
<400> 5
atgaacccta acaagaagat cgtcaccatc ggctctatct gcatcgtgat cggcatcatc 60
tctctgatgc tccagatcgg caacatcatc agcatttgga tctctcacag catccagacc 120
gtcaaccagc accagaccga gcccatccgt aacaccaatt ttctgaccga gaacgccgtg 180
gcttctgtga ctctggctgg caacagctct ctgtgcccca ttagaggctg ggccgtgcat 240
agcaaggaca atagcatccg tatcggcagc aagggtgacg tcttcgtcat ccgtgagccc 300
tttatcagct gctctcacct ggagtgccgt accttcttcc tcacccaggg tgccctgctg 360
aacgacaagc acagcaacgg taccgtgaag gaccgttctc ctcaccgtac tctgatgtct 420
tgccccattg gcgaggcccc tagcccctac aactctcgtt tcgagagcgt cgcctggagc 480
gcttctgctt gtcacgacgg caccagctgg ctgatcatcg gtatcagcgg tcccgacaac 540
ggtgccgtgg ctgtcctcaa gtacaacggc atcatcaccg acaccatcaa gtcttggcgt 600
aacaacatcc tgagaactca ggagagcgag tgcgcctgcg tcaacggcag ctgcttcact 660
gtgatgactg acggcccctc taacggccag gccagctaca agatcttcaa gatcgaaaag 720
ggtaaggtcg tcaagtctgt cgagctcaac gctcccaact accactacga agagtgctct 780
tgctaccccg atgctggcga aatcatctgc gtctgccgtg acaactggca cggctctaac 840
cgtccttgga tcagcttcaa ccagaacctg gagtaccaaa tcggctacat ctgcagcggc 900
gtgttcggtg acaaccctcg tcccaacgac ggtactggtt cttgcggtcc cgtcagctct 960
aacggtgcct atggcgtgaa gggtttcagc ttcaaatacg gcaacggtgt gtggatcggc 1020
cgtaccaaga gcacccatag cagaagcggc ttcgagatga tctgggaccc caatggttgg 1080
accggcaccg actctgagtt cagcatgaag caggacatcg tggccattac cgattggagc 1140
ggttacagcg gcagcttcgt ccaacaccct gagctcaccg gtctcgactg tatccgtccc 1200
tgcttctggg tggagctgat ccgtggtcgt cccaaggagt ctaccatctg gacctctggc 1260
agcagcatca gcttctgcgg tgtcaatagc gacactgtgt cttggagctg gcccgacggt 1320
gccgaactcc ctttcaccat cgacaaataa 1350
Claims (17)
1. The avian influenza virus-like particle vaccine comprises an immunizing amount of H7 subtype avian influenza virus-like particle antigen and a pharmaceutically acceptable carrier, wherein the avian influenza virus-like particle antigen is assembled by H7 subtype avian influenza virus HA, NA antigen protein and bovine immunodeficiency virus Gag antigen protein;
the H7 subtype avian influenza virus HA antigen protein is coded by a sequence shown by SEQ ID NO.1, the H7 subtype avian influenza virus NA antigen protein is coded by a sequence shown by SEQ ID NO.2, and the bovine immunodeficiency virus Gag antigen protein is coded by a sequence shown by SEQ ID NO. 3.
2. The avian influenza virus-like particle vaccine of claim 1, wherein the antigen content of the H7 subtype avian influenza virus-like particle HAs an HA titer of more than or equal to 6log2.
3. The avian influenza virus-like particle vaccine according to claim 2, wherein the content of the H7 subtype avian influenza virus-like particle antigen is HA titer 6log2 to 9log2.
4. The avian influenza virus-like particle vaccine according to claim 3, wherein the content of the H7 subtype avian influenza virus-like particle antigen is HA titer 6log2 to 8log2.
5. The avian influenza virus-like particle vaccine of claim 1 wherein said pharmaceutically acceptable carrier comprises an adjuvant comprising one or more of white oil, alumina Gel adjuvant, saponin, avridine, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, block co-polymer, SAF-M, monophosphoryl lipid a, avrie lipid-amine adjuvant, escherichia coli heat labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, gel adjuvant, said adjuvant being in a concentration ranging from 5% to 70 din V/V.
6. The avian influenza virus-like particle vaccine according to claim 5, wherein the saponin is Quil A, QS-21 or GPI-0100.
7. The avian influenza virus-like particle vaccine of claim 5, wherein the concentration of said adjuvant ranges from 30% to 70%.
8. The avian influenza virus-like particle vaccine of claim 7, wherein the concentration range of said adjuvant is 66% V/V.
9. The avian influenza virus-like particle vaccine of claim 1, further comprising an immunizing amount of H5 subtype avian influenza virus-like particle antigen, wherein the H5 subtype avian influenza virus-like particle antigen is assembled from H5 subtype avian influenza virus HA, NA antigen protein and bovine immunodeficiency virus Gag antigen protein.
10. The avian influenza virus-like particle vaccine as claimed in claim 9, wherein the H5 subtype avian influenza virus HA antigen protein is encoded by a sequence shown in SEQ ID No.4, the H5 subtype avian influenza virus NA antigen protein is encoded by a sequence shown in SEQ ID No.5, and the bovine immunodeficiency virus Gag antigen protein is encoded by a sequence shown in SEQ ID No. 3.
11. The avian influenza virus-like particle vaccine according to claim 9, wherein the content of the H5 subtype avian influenza virus-like particle antigen is HA titer no less than 6log2.
12. The avian influenza virus-like particle vaccine of claim 11, wherein the content of the H5 subtype avian influenza virus-like particle antigen is HA titer 6log2 to 9log2.
13. The avian influenza virus-like particle vaccine according to claim 12, wherein the content of the H5 subtype avian influenza virus-like particle antigen is HA titer 6log2 to 8log2.
14. A method of making the avian influenza virus-like particle vaccine of claim 1, wherein the method comprises:
cloning HA, NA antigen protein genes and Gag antigen protein genes of the H7 subtype avian influenza virus to the same vector, wherein the HA, NA and Gag antigen protein genes are respectively shown as sequences SEQ.ID No.1, SEQ.ID No.2 and SEQ.ID No. 3;
transforming and recombining the vector obtained in the step (1) to obtain a recombinant baculovirus plasmid containing the HA, NA and Gag antigen protein genes;
transfecting the recombinant baculovirus plasmid containing the HA, NA and Gag antigen protein genes obtained in the step (2) into an insect cell sf9, and expressing the HA, NA and Gag antigen proteins in series;
separating avian influenza virus-like particle antigen which is released to the supernatant of an extracellular culture medium after the self-assembly in insect cells is finished and assembled by the HA, NA and Gag antigen proteins; and
and (5) adding an adjuvant into the avian influenza virus-like particle antigen obtained in the step (4), and uniformly mixing to obtain the avian influenza virus-like particle vaccine.
15. The method according to claim 14, wherein the vector in the step (1) is pFastBac I; the baculovirus plasmid in the step (2) is Bacmid.
16. The use of the avian influenza virus-like particle vaccine according to claim 1 in the preparation of a medicament for the prevention of a disease caused by avian influenza virus.
17. The use according to claim 16, wherein the avian influenza virus is an H7 subtype avian influenza virus.
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| CN113827713A (en) * | 2020-06-23 | 2021-12-24 | 普莱柯生物工程股份有限公司 | Vaccine composition for resisting H7 subtype avian influenza virus, and preparation method and application thereof |
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| CN104721817A (en) * | 2013-12-19 | 2015-06-24 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and application thereof |
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| CN104721817A (en) * | 2013-12-19 | 2015-06-24 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Preparation of Quadri-Subtype Influenza Virus-Like Particles Using Bovine Immunodeficiency Virus Gag Protein;Irina Tretyakova等;《Virology》;20160131;第487卷;第1页摘要，第2-9页材料和方法，表1 * |
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