CN113827715A - Vaccine composition for resisting H7 subtype and H5 subtype avian influenza viruses, and preparation method and application thereof - Google Patents

Vaccine composition for resisting H7 subtype and H5 subtype avian influenza viruses, and preparation method and application thereof Download PDF

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CN113827715A
CN113827715A CN202010582681.2A CN202010582681A CN113827715A CN 113827715 A CN113827715 A CN 113827715A CN 202010582681 A CN202010582681 A CN 202010582681A CN 113827715 A CN113827715 A CN 113827715A
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avian influenza
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田克恭
张盼涛
王同燕
张许科
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Pulaike Biological Engineering Co Ltd
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Abstract

The invention relates to a vaccine composition for resisting H7 subtype and H5 subtype avian influenza viruses, which comprises immunization amount of H7 subtype avian influenza virus-like particle antigen, immunization amount of H5 subtype avian influenza virus-like particle antigen and a pharmaceutically acceptable carrier, wherein the H7 subtype avian influenza virus-like particle antigen is assembled by H7 subtype avian influenza virus HA, NA and M1 antigen proteins, the H7 subtype avian influenza virus HA antigen protein is shown as Seq ID No.1, the H7 subtype avian influenza virus NA antigen protein is shown as Seq ID No.2, and the H7 subtype avian influenza virus M1 antigen protein is shown as Seq ID No. 3. The vaccine composition can protect the existing H7 subtype epidemic strains, has equivalent or better effect compared with the inactivated vaccine with the same antigen content, and generates synergistic interaction among the components of the vaccine composition, thereby further ensuring the immune effect.

Description

Vaccine composition for resisting H7 subtype and H5 subtype avian influenza viruses, and preparation method and application thereof
Technical Field
The invention relates to the field of biological pharmacy, in particular to a vaccine composition for resisting H7 subtype and H5 subtype 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 in vitro with high efficiency, allowing it to self-assemble into hollow particles that are morphologically similar to native viruses. 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, Influenza a Virus. Avian Influenza (AI) is a syndrome of infection and disease in birds caused by this virus. The international veterinary Office (OIE) and the national regulations on epidemic prevention of domestic animals and poultry classify the disease as class 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 subtype H5, subtype H7, is more severe, is characterized primarily by sudden morbidity and high mortality, often resulting in total mortality in the feeding flock.
Up to now, vaccination with whole virus vaccines remains the most effective means for preventing and controlling the avian influenza virus epidemics. However, because the variation speed of avian influenza virus is far greater than the research and development and preparation speed of corresponding vaccines of variant strains, and cross immune protection is hardly generated between different subtypes, the production of avian influenza vaccines needs to be updated constantly, and the production period of corresponding vaccines of influenza virus epidemic strains is longer, the production cost is higher, so that the waste of manpower and material resources is caused, and an ideal prevention and control effect can not be achieved. The avian influenza virus-like particle vaccine adopts a genetic engineering technology, has short research and development period, is particularly controllable in production cost of a eukaryotic expression system, a prokaryotic expression system and a yeast expression system, and can avoid the defects.
However, although the avian influenza virus-like particle vaccine published in the literature in the prior art can generate better immune response, the vaccine still cannot achieve the immune effect of the commercial whole virus vaccine, and the universal expression efficiency is lower, so that the preparation of the avian influenza virus-like particle vaccine with good immune effect, safety and controllable cost by screening an ideal avian influenza strain sequence is urgent, and the vaccine also meets the requirement of effective prevention and control of major animal diseases provided by the country and the healthy and sustainable development of the animal husbandry.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a vaccine composition for resisting H7 subtype and H5 subtype avian influenza viruses, wherein the vaccine composition comprises immune amount of H7 subtype avian influenza virus-like particle antigen, immune amount of H5 subtype avian influenza virus-like particle antigen and a pharmaceutically acceptable carrier, the H7 subtype avian influenza virus-like particle antigen is assembled by H7 subtype avian influenza virus HA, NA and M1 antigen proteins, the H7 subtype avian influenza virus HA antigen protein is shown as Seq ID No.1, the H7 subtype avian influenza virus NA antigen protein is shown as Seq ID No.2, and the H7 subtype avian influenza virus M1 antigen protein is shown as Seq ID No. 3.
The H7 subtype avian influenza virus-like particle antigen HAs good immunogenicity, can provide complete protection for H7 subtype avian influenza virus, and HAs the immune efficacy equivalent to or better than that of inactivated vaccine with the same HA antigen content.
As an embodiment of the present invention, in the vaccine composition of the present invention, the H5 subtype avian influenza virus-like particle antigen consists of an H5 subtype 2.3.4.4d branch virus-like particle antigen and an H5 subtype 2.3.2.1d branch virus-like particle antigen; the antigen of the H5 subtype 2.3.4.4d branched virus-like particle is assembled by HA, NA and bovine immunodeficiency virus Gag antigen protein, the HA antigen protein of the H5 subtype 2.3.4.4d branched avian influenza virus is shown as Seq ID No.4, the NA antigen protein of the H5 subtype 2.3.4.4d branched avian influenza virus is shown as Seq ID No.5, and the amino acid sequence of the bovine immunodeficiency virus Gag antigen protein is shown as Seq ID No. 8; and the antigen of the H5 subtype 2.3.2.1d branched virus-like particles is assembled by HA and NA of the antigen and bovine immunodeficiency virus Gag antigen protein, the HA antigen protein of the H5 subtype 2.3.2.1d branched virus is shown as Seq ID No.6, the NA antigen protein of the H5 subtype 2.3.2.1d branched virus is shown as Seq ID No.7, and the bovine immunodeficiency virus Gag antigen protein is shown as an amino acid sequence of Seq ID No. 8.
The H5 subtype avian influenza virus-like particle antigen HAs good immunogenicity, can provide complete protection for H5 subtype avian influenza virus, and HAs the immune efficacy equivalent to or better than that of inactivated vaccine with the same HA antigen content.
As one embodiment of the invention, in the vaccine composition of the invention, the content of the H7 subtype avian influenza virus-like particle antigen is HA titer more than or equal to 6log 2.
The H7 subtype avian influenza virus-like particle antigen HAs good immunogenicity, can generate complete protection when the content is HA titer 6log2, can generate complete protection against avian influenza 14 days after immunization, and HAs better immune effect than inactivated vaccine with higher HA antigen titer.
As a preferable embodiment, in the vaccine composition, the content of the H7 subtype avian influenza virus-like particle antigen is HA titer 6log 2-8 log 2.
The HA titer of the antigen content of the avian influenza virus-like particles of subtype H7 in the avian influenza virus-like particle vaccine of the present invention may be arbitrarily 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, 8.0log 2. In one embodiment of the invention, in the vaccine composition of the invention, the antigen content of the H7 subtype avian influenza virus-like particle is HA titer 6log 2-8 log2, the antigen content of the H5 subtype 2.3.4.4d branch virus-like particle is HA titer 6log 2-8 log2, and the antigen content of the H5 subtype 2.3.2.1d branch virus-like particle is HA titer 6log 2-8 log 2.
The H5 subtype avian influenza virus-like particle antigen HAs good immunogenicity, can generate complete protection when the content is HA titer 6log2, can generate complete protection against avian influenza 14 days after immunization, and HAs better immune effect than inactivated vaccine with higher HA antigen titer.
The HA titer of the antigen content of the H5 subtype 2.3.4.4d branched avian influenza virus-like particle in the avian influenza virus-like particle vaccine of the present invention may be arbitrarily 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, 8.0log 2.
The HA titer of the antigen content of the H5 subtype 2.3.2.1d branched virus-like particles in the avian influenza virus-like particle vaccine of the present invention may be arbitrarily 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, 8.0log 2.
The components of the vaccine composition generate synergistic interaction in the content range, so that the immune effect is further ensured.
As an embodiment of the present invention, in the vaccine composition of the present invention, the pharmaceutically acceptable carrier is an adjuvant selected from the group consisting of: (1) mineral oil, alumina gel adjuvant, saponin, 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, Montanide ISA 206, and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100; preferably, the adjuvant is a mineral oil adjuvant, which is used to prepare a water-in-oil emulsion;
the adjuvant content is 5% -70% V/V, preferably from 30% -70% V/V, more preferably 66% V/V.
As one embodiment of the present invention, the pharmaceutically acceptable carrier includes drugs, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives; the immunostimulant includes alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 2(IL 2).
To prepare such compositions, methods well known in the art may be used.
The invention also relates to a method for preparing the vaccine composition, wherein the method comprises the following steps: cloning HA, NA and M1 antigen protein genes of the H7 subtype avian influenza virus to the same vector, and cloning HA, NA antigen protein genes and bovine immunodeficiency virus Gag antigen protein genes of the H5 subtype avian influenza virus to another same vector; transforming and recombining the vector obtained in the step (1) to obtain a recombinant baculovirus plasmid containing the H7 subtype avian influenza virus HA, NA and M1 antigen protein genes, and transforming and recombining the vector obtained in the step (1) to obtain a recombinant baculovirus plasmid containing the H5 subtype avian influenza virus HA, NA antigen protein genes and bovine immunodeficiency virus Gag antigen protein genes; step (3) transfecting insect cells sf9 with the recombinant baculovirus plasmids containing the HA, NA and M1 antigen protein genes of the H7 subtype avian influenza virus obtained in the step (2), expressing the HA, NA and M1 antigen proteins of the H7 subtype avian influenza virus in series, transfecting insect cells sf9 with the recombinant baculovirus plasmids containing the HA, NA antigen protein genes and bovine immunodeficiency virus Gag antigen protein genes of the H5 subtype avian influenza virus obtained in the step (2), and expressing the HA, NA antigen protein and bovine immunodeficiency virus Gag antigen protein of the H5 subtype avian influenza virus in series; and (4) separating the H7 subtype avian influenza virus-like particle antigen assembled by the HA, NA and M1 antigen proteins of the H7 subtype avian influenza virus released into the supernatant of the extracellular culture medium after the self-assembly in the insect cell is completed, and separating the H5 subtype avian influenza virus-like particle antigen assembled by the HA, NA antigen proteins and bovine immunodeficiency virus Gag antigen proteins of the H5 subtype avian influenza virus released into the supernatant of the extracellular culture medium after the self-assembly in the insect cell is completed; adding an adjuvant, and uniformly mixing to obtain the vaccine composition.
As an embodiment of the present invention, in the method for preparing the vaccine composition according to the present invention, the vectors in the step (1) are both pFastBac I; the baculovirus plasmid in the step (2) is Bacmid.
The invention also relates to application of the vaccine composition in preparing a medicament for preventing and/or treating diseases caused by the 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 and/or an H5 subtype avian influenza virus.
The administration object for preparing the medicine for preventing and/or treating the avian influenza virus infection comprises chicken.
The antigen of the avian influenza virus-like particle vaccine is an self-assembly body of surface antigen hemagglutinin HA, neuraminidase NA and matrix protein M1 of H7 subtype avian influenza virus or the self-assembly body of surface antigen hemagglutinin HA, neuraminidase NA and bovine immunodeficiency virus Gag of H5 subtype avian influenza virus. The invention utilizes an insect baculovirus expression system to produce the avian influenza virus-like particle antigen, has the advantages of high yield, low production cost, good immunogenicity, no biological safety risk and the like, can provide protective activity for H7 subtype avian influenza, and can also provide protective activity for H5 subtype 2.3.4.4d branch avian influenza and H5 subtype 2.3.2.1d branch avian influenza.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
Definition of
"Antigen" refers to a substance that induces an immune response in the body, i.e., a substance that is specifically recognized and bound by an Antigen receptor (TCR/BCR) on the surface of T/B lymphocytes, activates T/B cells, proliferates and differentiates the T/B cells, produces an immune response product (sensitized lymphocytes or antibodies), and specifically binds to the corresponding product in vitro or in vivo.
"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", "vaccine composition" 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.
The term "immunizing amount" shall be understood as an "immunologically effective amount," also referred to as an immunoprotective amount or an amount effective to produce an immune response, of antigen effective to induce an immune response in a recipient, sufficient to prevent or ameliorate the signs or symptoms of disease, including adverse health effects or complications thereof. The immune response may be sufficient for diagnostic purposes or other testing, or may be suitable for use in preventing signs or symptoms of disease, including adverse health consequences or complications thereof caused by infection by a pathogen. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with a wild-type strain, while the protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs such as mortality, reduction in morbidity, temperature values, overall physiological condition of the subject, and overall health and performance. The immune response may include, but is not limited to, induction of cellular and/or humoral immunity.
The term "pharmaceutically acceptable carrier" refers to all other ingredients in the vaccine composition of the present invention, except for the avian influenza virus antigen, that do not stimulate the body and do not hinder the biological activity and properties of the compound used, and preferably are adjuvants. 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; maleic anhydride and alkenyl (alkenyl) derivatives. The term "emulsion" may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils (isoprenoid oils) resulting from the oligomerization of olefins, such as squalane (squalane) or squalene oil (squalene oil), in particular isobutene or decene; linear alkyl-containing esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di- (caprylate/caprate), glycerol tri- (caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearic acid esters. The oil is used in combination with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (such as, for example, anhydrous mannitol oleate), of aliphatic diols (glycols), of polyglycerols, of propylene glycol and of oleic acid, of isostearic acid, of ricinoleic acid or of hydroxystearic acid, which are optionally ethoxylated, and also polyoxypropylene-polyoxyethylene block copolymers, in particular the Pluronic products, in particular L121. See The description of The same and The reactive application of adjuvants by Hunter et al (Ed. by DES Stewart-Tull, John Wiley and Sons, New York,1995:51-94) and The description of Vaccine by Todd et al (1997,15: 564-570). For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of Vaccine design, the Subunit and adivant propaach (Plenum Press,1995) written by Powell M and Newman M can be used. The term "polymer of acrylic or methacrylic acid" is preferably a crosslinked polymer of acrylic or methacrylic acid, in particular a polyalkenyl ether or polyalcohol crosslinked with a sugar (sugar), these compounds being known under the name Carbomer (Carbopol, trade name Carbopol) (Phameuropa,1996,8 (2)). Those skilled in the art can also see US2909462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are substituted by unsaturated aliphatic hydrocarbon groups (aliphatic radial) having at least 2 carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups (ethylenically unsaturated groups). The unsaturated groups may themselves contain other substituents, such as methyl. These products are sold under the name carbopol, (BF Goodrich, Ohio, USA) are particularly suitable. They are crosslinked with allyl sucrose or with allyl pentaerythritol. Among these, mention may be made of carbopols 974P, 934P and 971P, the most preferred being the use of carbopol 971P. The term "copolymers of maleic anhydride and alkenyl derivative" also contemplates the maleic anhydride and ethylene copolymers ema (monsanto), which are dissolved in water to give an acidic solution, neutralized, preferably to physiological pH, in order to give an adjuvant solution into which the immunogenic, immunogenic or vaccinal composition itself can be incorporated. The term "adjuvant" also includes, but is not limited to, the RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A (monophosphoryl lipid A), Avridine lipoamine adjuvant, E.coli heat labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant, and the like. Preferably, the adjuvant comprises one or more of mineral oil, an alumina Gel adjuvant, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a water-in-oil-in-water emulsion, a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and an alkenyl (alkenyl) derivative, a RIBI adjuvant system, a Block co-polymer, SAF-M, a monophosphoryl lipid A, Avridine lipid-amine adjuvant, escherichia coli heat labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, or Gel adjuvant.
"Gene recombination": refers to the recombination of genes that control different traits. Modern genetic engineering techniques carry out genetic recombination, also called recombinant DNA, in vitro by artificial design, with the aim of transferring a genetic gene in one individual cell to another individual cell DNA molecule of a different character, causing genetic variation. After the target gene from the donor is transferred into the recipient bacterium, the expression of the gene product can be carried out, thereby obtaining a product which is difficult to obtain by a common method.
"transformation" refers to the acquisition of a new genetic phenotype in a cell or a cultured recipient cell by the automated acquisition or artificial supply of exogenous DNA.
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 invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
Example 1 expression of subtype H7 avian influenza Virus-like particles
1. Carrier engineering
A commercial vector pFastBac I is taken as a template, and restriction sites of XhoI, SphI, NheI, MluI, EcoRI and XbaI are inserted at 4413-4414 position in sequence. 1 mu l of Dpn I enzyme is added into the PCR product to digest away the template plasmid, 5 mu l of the PCR product is taken to transform DH5 alpha according to a conventional method, and the successfully transformed plasmid is named as pFastBac mut.
2. Construction of three expression cassette Donor plasmids
An HA gene shown in a sequence table Seq ID No.1, an NA gene shown in a sequence table Seq ID No.2 and an M1 gene shown in a sequence table Seq ID No.3 are synthesized by Suzhou Jinzhi Biotechnology Limited, 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, NA gene and M1 gene are cut by BamHI and HindIII enzyme and are connected with pFastBac mut cut by the same enzyme, the connection product is transformed into DH5 alpha, and the correct plasmids are identified and named as pFastBac mut-HA, pFastBac mut-NA and pFastBac mut-M1.
Amplifying an NA expression cassette by taking pFastBac mut-NA as a template, carrying out double enzyme digestion by SphI and MluI, and then connecting the amplified NA expression cassette into a pFB-M1 plasmid to obtain a pFastBac mut-M1-NA plasmid; and then taking pFastBac mut-HA as a template, carrying out double enzyme digestion on the amplified HA expression cassette by XhoI and SphI, and connecting the amplified HA expression cassette into pFastBac mut-M1-NA plasmid to obtain donor plasmid expressed by three genes of pFastBac mut-M1-HA-NA in series.
3. Construction and identification of recombinant Bacmid
Adding 2 mu l of pFastBac mut-M1-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 ℃ and 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 the blue-white bacterial colony is obvious. Taking 1 mul as a template for PCR identification of bacteria liquid the next day. And (3) identifying the PCR product correctly, extracting the recombinant bacmid by using a reagent in the small Tiangen plasmid extraction kit, and naming the recombinant bacmid-MNH as the rBacmid-MNH.
4. Acquisition and passage of recombinant baculovirus
Recombinant bacmid-MNH was transfected into insect cells sf 9. Reference to
Figure BDA0002552973370000101
II Regent instructions for transfection, 72h after transfection, harvest cell supernatant marker rBac-MNH P1 after cytopathic.
Sf9 cells in logarithmic growth phase were grown according to 2.5X 106After cells are completely attached to the wall, adding the P1 generation recombinant baculovirus into a cell culture bottle paved with sf9 according to the volume ratio of 1:20-1:40, continuously culturing at 27 ℃, harvesting the supernatant and marking as the P2 generation recombinant baculovirus when the cytopathic effect is obvious about 72 hours, wrapping the recombinant baculovirus with tin foil paper, and keeping the recombinant baculovirus in a refrigerator at 4 ℃ in a dark place for later use. Inoculating the P2 generation recombinant baculovirus into suspended Sf-9 cells, inoculating according to the ratio of 1: 500-1: 2000, harvesting the P3 generation virus when cytopathic effect is obvious, and repeating the steps to obtain the P4 generation recombinant baculovirus.
5. Expression and characterization of proteins
Inoculating Sf9 cells to the recombinant virus transferred to P4 according to the MOI of 0.1-1, inoculating for about 72-96h to obtain cells, and centrifuging to obtain a supernatant. The HA content of the extracellular supernatant was determined to be 12log 2. The results of transmission electron microscope observation show that the harvested protein presents a virus-like particle shape, is basically uniform in size and is in a hollow particle state.
Example 2 preparation of subtype H7 avian influenza Virus-like particle vaccine
The virus-like particles harvested in example 1 were added to mineral oil adjuvant to prepare each vaccine composition, the specific ratios are shown in table 1.
TABLE 1 proportioning of subtype H7 avian influenza virus-like particle vaccine composition
Components Vaccine 1 Vaccine 2
rBac-MNH (HA content) 6log2 8log2
Adjuvant (V/V%) 66％ 66％
Example 3 stability test of subtype H7 avian influenza Virus-like particle vaccine
The vaccine 1 and the vaccine 2 prepared in the example 2 are respectively stored at the temperature of 2-8 ℃ for 12 months, and the vaccine 1 and the vaccine 2 after being stored for 12 months are respectively named as vaccine 1-1 and vaccine 2-1. The observation of an electron microscope shows that after 12 months of storage, the vaccine 1-1 and the vaccine 2-1 are still stable.
60 SPF chickens of 21 days old are divided into 6 groups, each group comprises 10 SPF chickens, the 1 st group and the 2 nd group are respectively injected with vaccine 1 and vaccine 2 prepared in immunization example 2 through neck subcutaneous injection, the 3 rd group and the 4 th group are respectively injected with vaccine 1-1 and vaccine 2-1 after immune preservation for 12 months through neck subcutaneous injection, the 5 th group is injected with commercial inactivated vaccine (H5(Re-11+ Re-12) and H7(H7-Re2) through neck subcutaneous injection, the content of H7HA is 8log2), the immune dose is 0.3ml, the 6 th group is injected with 0.3ml of physiological saline through subcutaneous injection to serve as 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 2.
TABLE 2H7 subtype avian influenza Virus-like particle vaccine stability test results
Figure BDA0002552973370000111
Figure BDA0002552973370000121
Remarks are as follows: the standard antigen of H7(H7-Re2) was used
The results show that the 14-day HI antibody titer mean values of the 1 st group, the 2 nd group, the 3 rd group, the 4 th group and the 5 th group of immunization groups are all higher than 6log2, namely, the immune protection against the H7 subtype infection can be effectively provided; the average value of the HI antibody titer of the immunization groups of the 3 rd and the 4 th day is equivalent to the average value of the HI antibody titer of the immunization groups of the 1 st and the 2 nd day 14; the mean value of HI antibody titers at day 14 in the 5 th immunization group was lower than the mean value of HI antibody titers at day 14 in the 1 st, 2 nd, 3 rd and 4 th immunization groups. The H7 subtype avian influenza virus-like particle vaccine prepared in the embodiment 2 of the invention has good stability, is stable after being stored for 12 months at the temperature of 2-8 ℃, and has no influence on HI antibody titer; compared with the commercial whole virus inactivated vaccine, the virus-like particle vaccine composition provided by the invention has better immune effect and quicker immune response.
Example 4 expression of subtype H5 avian influenza Virus-like particles
4.1 expression of H5 subtype 2.3.4.4d branched Virus-like particles
An HA gene shown in sequence table Seq ID No.4, an NA gene shown in sequence table Seq ID No.5, and a Gag gene shown in sequence table Seq ID No.8 were synthesized by Kingzhi Biotech, Suzhou, and a recombinant baculovirus named rBac-GNH-1 was constructed by the method of example 1. Inoculating Sf9 cells to the recombinant virus transferred to P4 according to the MOI of 0.1-1, inoculating for about 72-96h to obtain cells, and centrifuging to obtain a supernatant. The HA content of the extracellular supernatant was determined to be 12log 2. The results of transmission electron microscope observation show that the harvested protein presents a virus-like particle shape, is basically uniform in size and is in a hollow particle state.
4.2 expression of H5 subtype 2.3.2.1d branched Virus-like particles
An HA gene shown in sequence table Seq ID No.6, an NA gene shown in sequence table Seq ID No.7, and a Gag gene shown in sequence table Seq ID No.8 were synthesized by Kinzhi Biotech, Suzhou, and a recombinant baculovirus named rBac-GNH-2 was constructed by the method of example 1. Inoculating Sf9 cells to the recombinant virus transferred to P4 according to the MOI of 0.1-1, inoculating for about 72-96h to obtain cells, and centrifuging to obtain a supernatant. The HA content of the extracellular supernatant was determined to be 12log 2. The results of transmission electron microscope observation show that the harvested protein presents a virus-like particle shape, is basically uniform in size and is in a hollow particle state.
Example 5 preparation of H7, H5 subtype bivalent avian influenza Virus-like particle vaccine
The virus-like particles harvested in example 1 and example 4 were added to mineral oil adjuvant to prepare vaccine compositions, and the specific ratios are shown in table 3.
TABLE 3 proportioning of the H7, H5 subtype bivalent avian influenza virus-like particle vaccine composition
Figure BDA0002552973370000131
Example 6 immunogenicity test of bivalent avian influenza Virus-like particle vaccine subtype H7, H5
80 SPF chickens of 21 days old are divided into 8 groups, each group comprises 10 SPF chickens, the 7 th group to the 12 th group are respectively injected with vaccines 3 to 8 prepared in immunization example 5 through neck subcutaneous injection, the 13 th group is injected with commercial inactivated vaccine (H5(Re-11+ Re-12) and H7(H7-Re2) trivalent inactivated vaccine subcutaneously, the H7HA content is 8log2), the immunization dose is 0.3ml, and the 14 th group is injected with 0.3ml of physiological saline subcutaneously to be used as 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 detection after immunization are shown in tables 4, 5 and 6.
TABLE 4 immunogenicity test results 1 for H7, H5 subtype bivalent avian influenza virus-like particle vaccine
Figure BDA0002552973370000141
Remarks are as follows: the standard antigen of H7(H7-Re2) was used
The results show that the 14-day HI antibody titer mean values of the 11 th group and the 12 th group of immunization groups are both higher than 6log2, so that the vaccine can effectively provide immune protection against H7 subtype infection, and the synchronous immune effect is better than that of the 13 th group of commercial vaccine immunization groups; the average value of the HI antibody titer of the group 13 commercial vaccine immunization group at 14 days is more than 6log2, thus effectively providing immune protection against H7 subtype infection; under the condition of HA dose immunization lower than that of inactivated vaccine in the 11 th group, the average value of HI antibody titer in 14 days can still exceed the synchronous immunization effect of the commercial vaccine immunization group in the 7 th group; when the group 12 immunization group and the group 13 commercial vaccine group are immunized at the same dose, the group 12 immune response is generated more quickly, and the average value of HI antibodies of the group 13 commercial vaccine on 21 days can be reached on 14 days. The multicomponent virus-like particle vaccines (vaccine 7 and vaccine 8) prepared in the example 5 of the invention can provide ideal immune efficacy; compared with the commercial whole virus inactivated vaccine, the virus-like particle vaccine composition provided by the invention has better immune effect and quicker immune response.
TABLE 5 immunogenicity test results 2 for H7, H5 subtype bivalent avian influenza virus-like particle vaccine
Figure BDA0002552973370000151
Remarks are as follows: the standard antigen of H5(H5-Re-11) was used
The results show that the 14-day HI antibody titer mean values of 7 th, 8 th, 11 th and 12 th immunization groups are all higher than 6log2, so that the vaccine can effectively provide immune protection against H5 subtype 2.3.4.4d branched infection, and the immune protection exceeds the synchronous immune effect of 13 th commercial vaccine immunization group; the average value of the HI antibody titer of the group 13 commercial vaccine immunization group at 14 days is more than 6log2, thus effectively providing immune protection against H5 subtype 2.3.4.4d branched infection; under the immunization of the groups 7 and 11 with HA dose lower than that of inactivated vaccine, the average value of HI antibody titer at 14 days can still exceed the synchronous immunization effect of the group 13 commercial vaccine immunization group; when the 8 th group, the 12 th group and the 13 th group of commercial vaccines are immunized at the same dose, the 8 th group and the 12 th group have faster immune response, and the average value of HI antibodies of the 13 th group of commercial vaccines at 21 days can be reached at 14 days; under the same HA dose immunization, the HI antibody titer mean value of the multi-component immunization groups of 11 and 12 is higher than that of the single-component immunization groups of 7 and 8. It is shown that the virus-like particle vaccines (vaccine 3 and vaccine 4) prepared in example 5 of the present invention, which are single-component vaccines, or multi-component vaccines (vaccine 7 and vaccine 8), provide ideal immune efficacy; and synergistic effect is generated among the components in the multi-component virus-like particle vaccines (vaccine 7 and vaccine 8), the immunization effect of the H5 subtype 2.3.4.4d branched virus-like particle antigen is better than that of the single-component virus-like particle vaccines (vaccine 3 and vaccine 4), because the other components generate synergistic effect on the H5 subtype 2.3.4.4d branched virus-like particle antigen. Compared with the commercial whole virus inactivated vaccine, the virus-like particle vaccine composition provided by the invention has better immune effect and quicker immune response; the multicomponent virus-like particle vaccines (vaccine 7 and vaccine 8) provided by the invention have better immune effect.
TABLE 6 immunogenicity test results of H7, H5 subtype bivalent avian influenza virus-like particle vaccine 3
Figure BDA0002552973370000161
Figure BDA0002552973370000171
Remarks are as follows: the standard antigen of H5(H5-Re-12) was used
The results show that the 14-day HI antibody titer mean values of the 9 th, 10 th, 11 th and 12 th immunization groups are all higher than 6log2, so that the vaccine can effectively provide immune protection against H5 subtype 2.3.2.1d branched infection, and the immune protection exceeds the synchronous immune effect of the 13 th commercial vaccine immunization group; the average value of the HI antibody titer of the group 13 commercial vaccine immunization group at 14 days is more than 6log2, thus effectively providing immune protection against H5 subtype 2.3.2.1d branched infection; under the immunization of the groups 9 and 11 with HA dose lower than that of inactivated vaccine, the average value of HI antibody titer at 14 days can still exceed the synchronous immunization effect of the group 13 commercial vaccine immunization group; when the 10 th group, the 12 th group and the 13 th group of commercial vaccines are immunized at the same dose, the 10 th group and the 12 th group have faster immune response, and the average value of HI antibodies of the 13 th group of commercial vaccines at 21 days can be reached at 14 days; under the same HA dose immunization, the HI antibody titer mean value of the multi-component immunization groups of 11 and 12 is higher than that of the single-component immunization groups of 9 and 10. It is shown that both the single-component virus-like particle vaccine (vaccine 5, vaccine 6) and the multi-component virus-like particle vaccine (vaccine 7, vaccine 8) prepared in example 2 of the present invention can provide ideal immune efficacy, and a synergistic effect is generated between the components in the multi-component virus-like particle vaccine (vaccine 7, vaccine 8), so that the immune effect of the H5 subtype 2.3.2.1d branched virus-like particle antigen is better than that of the single-component virus-like particle vaccine (vaccine 5, vaccine 6), because the other components generate a synergistic effect on the immune effect of the H5 subtype 2.3.2.1d branched virus-like particle antigen. Compared with the commercial whole virus inactivated vaccine, the virus-like particle vaccine composition provided by the invention has better immune effect and quicker immune response; the multicomponent virus-like particle vaccines (vaccine 7 and vaccine 8) provided by the invention have better immune effect.
The virus-like particle vaccines provided by the invention have a good immune effect, and the multi-component virus-like particle vaccines generate a synergistic effect, so that the immune effect is better.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> Puleco bioengineering GmbH
<120> vaccine composition for resisting H7 subtype and H5 subtype avian influenza viruses, and preparation method and application thereof
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 560
<212> PRT
<213> subtype H7 Avian Influenza Virus (H7 subtype, Avian Influenza Virus)
<400> 1
Met Asn Thr Gln Ile Leu Val Phe Ala Leu Ile Ala Ile Ile Pro Thr
1 5 10 15
Asn Ala Asp Lys Ile Cys Leu Gly His His Ala Val Ser Asn Gly Thr
20 25 30
Lys Val Asn Thr Leu Thr Glu Lys Gly Val Glu Val Val Asn Ala Thr
35 40 45
Glu Thr Val Glu Arg Thr Asn Thr Pro Arg Ile Cys Ser Lys Gly Lys
50 55 60
Arg Thr Val Asp Leu Gly Gln Cys Gly Leu Leu Gly Thr Ile Thr Gly
65 70 75 80
Pro Pro Gln Cys Asp Gln Phe Leu Glu Phe Ser Ala Asp Leu Ile Ile
85 90 95
Glu Arg Arg Glu Gly Ser Asp Val Cys Tyr Pro Gly Lys Phe Val Asn
100 105 110
Glu Glu Ala Leu Arg Gln Ile Leu Arg Glu Ser Gly Gly Ile Asp Lys
115 120 125
Glu Pro Met Gly Phe Thr Tyr Asn Gly Ile Arg Thr Asn Gly Val Thr
130 135 140
Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp
145 150 155 160
Leu Leu Ser Asn Thr Asp Asn Ala Ala Phe Pro Gln Met Thr Lys Ser
165 170 175
Tyr Lys Asn Thr Arg Glu Ser Pro Ala Ile Val Val Trp Gly Ile His
180 185 190
His Ser Val Ser Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn
195 200 205
Lys Leu Val Thr Val Gly Ser Ser Asn Tyr Gln Gln Ser Phe Val Pro
210 215 220
Ser Pro Gly Ala Arg Pro Gln Val Asn Gly Gln Ser Gly Arg Ile Asp
225 230 235 240
Phe His Trp Leu Ile Leu Asn Pro Asn Asp Thr Val Thr Phe Ser Phe
245 250 255
Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Leu Arg Gly Lys
260 265 270
Ser Met Gly Ile Gln Ser Gly Val Gln Val Asp Ala Asn Cys Glu Gly
275 280 285
Asp Cys Tyr His Ser Gly Gly Thr Ile Ile Ser Asn Leu Pro Phe Gln
290 295 300
Asn Ile Asp Ser Arg Ala Val Gly Lys Cys Pro Arg Tyr Val Lys Gln
305 310 315 320
Arg Ser Leu Leu Leu Ala Thr Gly Met Lys Asn Val Pro Glu Val Pro
325 330 335
Lys Gly Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly
340 345 350
Trp Glu Gly Leu Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ala
355 360 365
Gln Gly Glu Gly Thr Ala Ala Asp Tyr Lys Ser Thr Gln Ser Ala Ile
370 375 380
Asp Gln Ile Thr Gly Lys Leu Asn Arg Leu Ile Ala Lys Thr Asn Gln
385 390 395 400
Gln Phe Lys Leu Ile Asp Asn Glu Phe Asn Glu Val Glu Lys Gln Ile
405 410 415
Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Ile Thr Glu Val Trp Ser
420 425 430
Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr Ile Asp
435 440 445
Leu Ala Asp Ser Glu Met Asp Lys Leu Tyr Glu Arg Val Lys Arg Gln
450 455 460
Leu Arg Glu Asn Ala Glu Glu Asp Gly Thr Gly Cys Phe Glu Ile Phe
465 470 475 480
His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile Arg Asn Asn Thr Tyr
485 490 495
Asp His Arg Lys Tyr Arg Glu Glu Ala Met Gln Asn Arg Ile Gln Ile
500 505 510
Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Val Ile Ser Phe Ala
515 520 525
Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp
530 535 540
Ala Cys Gln Cys Val Lys Asn Gly Asn Met Arg Cys Thr Ile Cys Ile
545 550 555 560
<210> 2
<211> 465
<212> PRT
<213> subtype H7 Avian Influenza Virus (H7 subtype, Avian Influenza Virus)
<400> 2
Met Asn Pro Asn Gln Lys Ile Leu Cys Thr Ser Ala Thr Ala Ile Thr
1 5 10 15
Ile Gly Ala Ile Thr Val Leu Ile Gly Ile Ala Asn Ile Gly Leu Asn
20 25 30
Ile Gly Leu His Leu Lys Ser Gly Cys Asn Cys Ser Arg Ser Gln Pro
35 40 45
Glu Thr Thr Asn Thr Ser Gln Thr Ile Ile Asn Asn Tyr Tyr Asn Glu
50 55 60
Thr Asn Ile Thr Asn Ile Gln Met Gly Glu Arg Thr Ser Arg Asn Phe
65 70 75 80
Asn Asn Leu Thr Lys Gly Leu Cys Thr Ile Asn Ser Trp His Ile Tyr
85 90 95
Gly Lys Asp Asn Ala Val Arg Ile Gly Glu Ser Ser Asp Val Leu Val
100 105 110
Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Glu Cys Lys Phe Tyr
115 120 125
Ala Leu Ser Gln Gly Thr Thr Ile Arg Gly Lys His Ser Asn Gly Thr
130 135 140
Ile His Asp Arg Ser Gln Tyr Arg Ala Leu Ile Ser Trp Pro Leu Ser
145 150 155 160
Ser Pro Pro Thr Val Tyr Asn Ser Arg Val Glu Cys Ile Gly Trp Ser
165 170 175
Ser Thr Ser Cys His Asp Gly Lys Ser Arg Met Ser Ile Cys Ile Ser
180 185 190
Gly Pro Asn Asn Asn Ala Ser Ala Val Ile Trp Tyr Asn Arg Arg Pro
195 200 205
Val Ala Glu Ile Asn Thr Trp Ala Arg Asn Ile Leu Arg Thr Gln Glu
210 215 220
Ser Glu Cys Val Cys His Asn Gly Val Cys Pro Val Val Phe Thr Asp
225 230 235 240
Gly Pro Ala Thr Gly Pro Ala Asp Thr Arg Ile Tyr Tyr Phe Lys Glu
245 250 255
Gly Lys Ile Leu Lys Trp Glu Ser Leu Thr Gly Thr Ala Lys His Ile
260 265 270
Glu Glu Cys Ser Cys Tyr Gly Lys Arg Thr Gly Ile Thr Cys Thr Cys
275 280 285
Arg Asp Asn Trp Gln Gly Ser Asn Arg Pro Val Ile Gln Ile Asp Pro
290 295 300
Val Ala Met Thr His Thr Ser Gln Tyr Ile Cys Ser Pro Val Leu Thr
305 310 315 320
Asp Ser Pro Arg Pro Asn Asp Pro Asn Ile Gly Lys Cys Asn Asp Pro
325 330 335
Tyr Pro Gly Asn Asn Asn Asn Gly Val Lys Gly Phe Ser Tyr Leu Asp
340 345 350
Gly Asp Asn Thr Trp Leu Gly Arg Thr Ile Ser Thr Ala Ser Arg Ser
355 360 365
Gly Tyr Glu Met Leu Lys Val Pro Asn Ala Leu Thr Asp Asp Arg Ser
370 375 380
Lys Pro Ile Gln Gly Gln Thr Ile Val Leu Asn Ala Asp Trp Ser Gly
385 390 395 400
Tyr Ser Gly Ser Phe Met Asp Tyr Trp Ala Glu Gly Asp Cys Tyr Arg
405 410 415
Ala Cys Phe Tyr Val Glu Leu Ile Arg Gly Lys Pro Lys Glu Asp Lys
420 425 430
Val Trp Trp Thr Ser Asn Ser Ile Val Ser Met Cys Ser Ser Thr Glu
435 440 445
Phe Leu Gly Gln Trp Asn Trp Pro Asp Gly Ala Lys Ile Glu Tyr Phe
450 455 460
Leu
465
<210> 3
<211> 252
<212> PRT
<213> subtype H7 Avian Influenza Virus (H7 subtype, Avian Influenza Virus)
<400> 3
Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro
1 5 10 15
Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe
20 25 30
Ala Gly Lys Asn Ala Asp Leu Glu Ala Leu Met Glu Trp Ile Lys Thr
35 40 45
Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe
50 55 60
Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val
65 70 75 80
Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala
85 90 95
Val Lys Leu Tyr Lys Lys Leu Lys Arg Glu Met Thr Phe His Gly Ala
100 105 110
Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met
115 120 125
Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Ala Glu Gly Ala Leu
130 135 140
Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ala Gln His Arg
145 150 155 160
Ser His Arg Gln Met Ala Thr Thr Thr Asn Pro Leu Ile Arg His Glu
165 170 175
Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met
180 185 190
Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln
195 200 205
Ala Arg Gln Met Val Gln Ala Met Arg Thr Val Gly Thr His Pro Asn
210 215 220
Ser Ser Thr Gly Leu Lys Asp Asp Leu Ile Glu Asn Leu Gln Ala Tyr
225 230 235 240
Gln Asn Arg Met Gly Val Gln Leu Gln Arg Phe Lys
245 250
<210> 4
<211> 563
<212> PRT
<213> subtype H5 Avian Influenza Virus (H5 subtype, Avian Influenza Virus)
<400> 4
Met Glu Lys Ile Val Leu Leu Leu Ser Val Val Ser Leu Val Lys Ser
1 5 10 15
Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val
20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45
Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asn Gly Val Lys
50 55 60
Pro Leu Ile Leu Lys Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn
65 70 75 80
Pro Met Cys Asp Glu Phe Ile Arg Val Pro Glu Trp Ser Tyr Ile Val
85 90 95
Glu Arg Ala Asn Pro Ser Asn Asp Leu Cys Tyr Pro Gly Asn Leu Asn
100 105 110
Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu
115 120 125
Lys Thr Gln Ile Ile Pro Lys Arg Ser Trp Ser Asn His Thr Ser Ser
130 135 140
Gly Val Ser Ala Ala Cys Pro Tyr Gln Gly Val Ala Ser Phe Phe Arg
145 150 155 160
Asn Val Val Trp Leu Thr Lys Lys Asn Asp Ala Tyr Pro Thr Ile Lys
165 170 175
Met Ser Tyr Asn Asn Thr Asn Lys Glu Asp Leu Leu Ile Leu Trp Gly
180 185 190
Ile His His Ser Asn Ser Ala Glu Glu Gln Thr Asn Leu Tyr Lys Asn
195 200 205
Pro Thr Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Gln Arg Leu
210 215 220
Val Pro Lys Ile Ala Thr Arg Ser Gln Val Asn Gly Gln Arg Gly Arg
225 230 235 240
Met Asp Phe Phe Trp Thr Ile Leu Arg Pro Asn Asp Ala Ile His Phe
245 250 255
Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile Ile
260 265 270
Lys Thr Gly Asp Ser Thr Ile Met Lys Ser Glu Ile Glu Tyr Gly Asn
275 280 285
Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser Met
290 295 300
Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr
305 310 315 320
Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro
325 330 335
Leu Arg Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu
340 345 350
Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser
355 360 365
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Arg Glu Ser Thr Gln Lys
370 375 380
Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met
385 390 395 400
Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Ser Leu Glu Arg
405 410 415
Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val
420 425 430
Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr
435 440 445
Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg
450 455 460
Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe Glu
465 470 475 480
Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly
485 490 495
Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys Arg Glu
500 505 510
Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln Ile Leu
515 520 525
Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Ala Ile Ile Val
530 535 540
Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg
545 550 555 560
Ile Cys Ile
<210> 5
<211> 459
<212> PRT
<213> subtype H5 Avian Influenza Virus (H5 subtype, Avian Influenza Virus)
<400> 5
Met Asn Pro Asn Gln Lys Ile Thr Cys Ile Ser Ala Thr Gly Val Thr
1 5 10 15
Leu Ser Val Val Ser Leu Leu Ile Gly Ile Ala Asn Leu Gly Leu Asn
20 25 30
Ile Gly Leu His Tyr Lys Val Ser Asp Ser Thr Thr Ile Asn Ile Pro
35 40 45
Lys Thr Asn Glu Thr Asn Pro Thr Thr Thr Asn Ile Thr Asn Ile Ile
50 55 60
Val Asn Lys Asn Glu Glu Lys Thr Phe Leu Lys Leu Thr Lys Pro Leu
65 70 75 80
Cys Glu Val Asn Ser Trp His Ile Leu Ser Lys Asp Asn Ala Ile Arg
85 90 95
Ile Gly Glu Asp Ala His Ile Leu Val Thr Arg Glu Pro Tyr Leu Ser
100 105 110
Cys Asp Pro Gln Gly Cys Arg Met Phe Ala Leu Ser Gln Gly Thr Thr
115 120 125
Leu Arg Gly Gln His Ala Asn Gly Thr Ile His Asp Arg Ser Pro Phe
130 135 140
Arg Ala Leu Ile Ser Trp Glu Met Gly Gln Ala Pro Ser Pro Tyr Asn
145 150 155 160
Thr Arg Val Glu Cys Ile Gly Trp Ser Ser Thr Ser Cys His Asp Gly
165 170 175
Ile Ser Arg Met Ser Ile Cys Ile Ser Gly Pro Asn Asn Asn Ala Ser
180 185 190
Ala Val Val Trp Tyr Arg Gly Arg Pro Val Thr Glu Ile Pro Ser Trp
195 200 205
Ala Gly Asn Ile Leu Arg Thr His Glu Ser Glu Cys Val Cys His Lys
210 215 220
Gly Ile Cys Pro Val Val Met Thr Asp Gly Pro Ala Asn Asn Lys Ala
225 230 235 240
Ala Thr Lys Ile Ile Tyr Phe Lys Glu Gly Met Ile Gln Lys Ala Glu
245 250 255
Glu Leu Gln Gly Asn Ala Gln His Ile Glu Glu Cys Ser Cys Tyr Gly
260 265 270
Ala Ala Arg Met Ile Lys Cys Val Cys Arg Asp Asn Trp Lys Gly Ala
275 280 285
Asn Arg Pro Val Ile Ile Ile Asp Pro Glu Met Met Thr His Thr Ser
290 295 300
Lys Tyr Leu Cys Ser Lys Ile Leu Thr Asp Thr Ser Arg Pro Asn Asp
305 310 315 320
Pro Thr Asn Gly Asn Cys Asp Ala Pro Ile Thr Gly Gly Ser Pro Asp
325 330 335
Pro Gly Val Lys Gly Phe Ala Phe Leu Asp Gly Glu Asn Ser Trp Leu
340 345 350
Gly Arg Thr Ile Ser Lys Asp Ser Arg Ser Gly Tyr Glu Met Leu Lys
355 360 365
Val Pro Asn Ala Glu Ile Asp Thr Gln Ser Gly Pro Ile Ser Tyr Gln
370 375 380
Leu Ile Val Asn Asn Gln Asn Trp Ser Gly Tyr Ser Gly Ala Phe Ile
385 390 395 400
Asp Tyr Trp Ala Asn Lys Glu Cys Phe Asn Pro Cys Phe Tyr Val Glu
405 410 415
Leu Ile Arg Gly Arg Pro Lys Glu Ser Gly Val Leu Trp Thr Ser Asn
420 425 430
Ser Met Val Ala Leu Cys Gly Ser Arg Glu Arg Leu Gly Ser Trp Ser
435 440 445
Trp His Asp Gly Ala Glu Ile Ile Tyr Phe Lys
450 455
<210> 6
<211> 564
<212> PRT
<213> subtype H5 Avian Influenza Virus (H5 subtype, Avian Influenza Virus)
<400> 6
Met Glu Lys Ile Val Leu Phe Phe Ala Thr Ile Ser Leu Val Lys Ser
1 5 10 15
Asp His Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val
20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Lys Asp Ile
35 40 45
Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asn Gly Val Lys
50 55 60
Pro Leu Ile Leu Lys Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn
65 70 75 80
Pro Leu Cys Asp Glu Phe Thr Asn Val Pro Glu Trp Ser Tyr Ile Val
85 90 95
Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110
Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu
115 120 125
Lys Ile Gln Ile Ile Pro Lys Asp Ser Trp Ser Asp His Glu Ala Ser
130 135 140
Leu Gly Val Ser Ala Ala Cys Ser Tyr Gln Gly Asn Ser Ser Phe Phe
145 150 155 160
Arg Asn Val Val Trp Leu Ile Lys Lys Asp Asn Ala Tyr Pro Thr Ile
165 170 175
Lys Lys Ser Tyr Asn Asn Thr Asn Arg Glu Asp Leu Leu Ile Leu Trp
180 185 190
Gly Ile His His Pro Asn Asp Glu Ala Glu Gln Thr Lys Leu Tyr Gln
195 200 205
Asn Pro Thr Thr Tyr Ile Ser Ile Gly Thr Ser Thr Leu Asn Gln Arg
210 215 220
Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Ile Asn Gly Gln Ser Gly
225 230 235 240
Arg Ile Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile His
245 250 255
Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile
260 265 270
Val Lys Lys Gly Asp Ser Thr Ile Met Arg Ser Glu Val Glu Tyr Gly
275 280 285
Asn Cys Asn Thr Arg Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser
290 295 300
Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys
305 310 315 320
Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335
Pro Gln Arg Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile
340 345 350
Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His
355 360 365
Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln
370 375 380
Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys
385 390 395 400
Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu
405 410 415
Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp
420 425 430
Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg
435 440 445
Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val
450 455 460
Arg Leu Gln Leu Lys Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe
465 470 475 480
Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met Glu Ser Val Lys Asn
485 490 495
Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys Arg
500 505 510
Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Ile Tyr Gln Ile
515 520 525
Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Ala Ile Met
530 535 540
Met Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gln Cys
545 550 555 560
Arg Ile Cys Ile
<210> 7
<211> 449
<212> PRT
<213> subtype H5 Avian Influenza Virus (H5 subtype, Avian Influenza Virus)
<400> 7
Met Asn Pro Asn Gln Lys Ile Val Thr Ile Gly Ser Ile Cys Met Val
1 5 10 15
Ile Gly Ile Ile Ser Leu Met Leu Gln Ile Gly Asn Ile Ile Ser Ile
20 25 30
Trp Val Ser His Ser Ile Gln Thr Gly Asn Gln His Gln Thr Glu Pro
35 40 45
Ile Arg Asn Thr Asn Phe Leu Thr Glu Asn Thr Val Ala Ser Val Thr
50 55 60
Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Lys Gly Trp Ala Val His
65 70 75 80
Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val
85 90 95
Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe
100 105 110
Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr
115 120 125
Val Lys Asp Arg Ser Pro His Arg Thr Leu Met Ser Cys Pro Ile Gly
130 135 140
Glu Ala Pro Ser Pro Tyr Asn Ser Lys Phe Glu Ser Val Ala Trp Ser
145 150 155 160
Ala Ser Ala Cys His Asp Gly Thr Ser Trp Leu Val Ile Gly Ile Ser
165 170 175
Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile
180 185 190
Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln Glu
195 200 205
Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp
210 215 220
Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Phe Lys Ile Glu Lys
225 230 235 240
Gly Lys Val Val Lys Ser Val Glu Leu Asn Ala Pro Asn Tyr His Tyr
245 250 255
Glu Glu Cys Ser Cys Tyr Pro Glu Ala Gly Glu Val Met Cys Val Cys
260 265 270
Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gln
275 280 285
Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp
290 295 300
Asn Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Met Ser Ser
305 310 315 320
Asn Gly Ala Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly
325 330 335
Val Trp Ile Gly Arg Thr Lys Ser Thr His Ser Arg Ser Gly Phe Glu
340 345 350
Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp Ser Glu Phe Ser
355 360 365
Met Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser Gly Tyr Ser Gly
370 375 380
Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp Cys Ile Arg Pro
385 390 395 400
Cys Phe Trp Val Glu Leu Ile Arg Gly Gln Pro Lys Glu Ser Thr Ile
405 410 415
Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr
420 425 430
Val Ser Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp
435 440 445
Lys
<210> 8
<211> 476
<212> PRT
<213> Bovine immunodeficiency virus (Bovine immunodeficiency virus)
<400> 8
Met Lys Arg Arg Glu Leu Glu Lys Lys Leu Arg Lys Val Arg Val Thr
1 5 10 15
Leu Gln Gln Asp Lys Tyr Tyr Thr Ile Gly Asn Leu Gln Trp Ala Ile
20 25 30
Arg Met Ile Asn Leu Met Gly Ile Lys Cys Val Cys Asp Glu Glu Cys
35 40 45
Ser Ala Ala Glu Val Ala Leu Ile Ile Thr Gln Phe Ser Ala Leu Asp
50 55 60
Leu Glu Asp Ser Pro Ile Lys Gly Lys Glu Glu Val Ala Ile Lys Asn
65 70 75 80
Thr Leu Lys Val Phe Trp Ser Leu Leu Ala Gly Tyr Lys Pro Glu Ser
85 90 95
Thr Glu Thr Ala Leu Gly Tyr Trp Glu Ala Phe Thr Tyr Arg Glu Arg
100 105 110
Glu Ala Arg Thr Glu Lys Glu Gly Glu Ile Lys Ser Ile Tyr Pro Ser
115 120 125
Leu Thr Gln Asn Thr Gln Asn Lys Lys Gln Thr Ser Asn Gln Thr Asn
130 135 140
Thr Gln Ser Leu Pro Ala Ile Thr Thr Gln Asp Gly Thr Pro Arg Phe
145 150 155 160
Asp Pro Asp Leu Met Lys Gln Leu Lys Ile Trp Ser Asp Ala Thr Glu
165 170 175
Arg Asn Gly Ile Asp Leu His Ala Val Asn Ile Leu Gly Val Ile Thr
180 185 190
Ala Asn Leu Val Gln Glu Glu Ile Lys Leu Leu Leu Asn Ser Thr Pro
195 200 205
Lys Trp Arg Leu Asp Val Gln Leu Ile Glu Ser Lys Val Arg Glu Lys
210 215 220
Glu Asn Ala His Arg Thr Trp Lys Gln His His Pro Glu Ala Pro Lys
225 230 235 240
Thr Asp Glu Ile Ile Gly Lys Gly Leu Ser Ser Ala Glu Gln Ala Thr
245 250 255
Leu Ile Ser Val Glu Cys Arg Glu Thr Phe Arg Gln Trp Val Leu Gln
260 265 270
Ala Ala Met Glu Val Ala Gln Ala Lys His Ala Thr Pro Gly Pro Ile
275 280 285
Asn Ile His Gln Gly Pro Lys Glu Pro Tyr Thr Asp Phe Ile Asn Arg
290 295 300
Leu Val Ala Ala Leu Glu Gly Met Ala Ala Pro Glu Thr Thr Lys Glu
305 310 315 320
Tyr Leu Leu Gln His Leu Ser Ile Asp His Ala Asn Glu Asp Cys Gln
325 330 335
Ser Ile Leu Arg Pro Leu Gly Pro Asn Thr Pro Met Glu Lys Lys Leu
340 345 350
Glu Ala Cys Arg Val Val Gly Ser Gln Lys Ser Lys Met Gln Phe Leu
355 360 365
Val Ala Ala Met Lys Glu Met Gly Ile Gln Ser Pro Ile Pro Ala Val
370 375 380
Leu Pro His Thr Pro Glu Ala Tyr Ala Ser Gln Thr Ser Gly Pro Glu
385 390 395 400
Asp Gly Arg Arg Cys Tyr Gly Cys Gly Lys Thr Gly His Leu Lys Lys
405 410 415
Asn Cys Lys Gln Gln Lys Cys Tyr His Cys Gly Lys Pro Gly His Gln
420 425 430
Ala Arg Asn Cys Arg Ser Lys Asn Gly Lys Cys Ser Ser Ala Pro Tyr
435 440 445
Gly Gln Arg Ser Gln Pro Gln Asn Asn Phe His Gln Ser Asn Met Ser
450 455 460
Ser Val Thr Pro Ser Ala Pro Pro Leu Ile Leu Asp
465 470 475

Claims (8)

1. A vaccine composition for resisting H7 subtype and H5 subtype avian influenza viruses comprises an immunizing amount of H7 subtype avian influenza virus-like particle antigen, an immunizing amount of H5 subtype avian influenza virus-like particle antigen and a pharmaceutically acceptable carrier, wherein the H7 subtype avian influenza virus-like particle antigen is assembled by H7 subtype avian influenza virus HA, NA and M1 antigen proteins, the H7 subtype avian influenza virus HA antigen protein is shown as Seq ID No.1, the H7 subtype avian influenza virus NA antigen protein is shown as Seq ID No.2, and the H7 subtype avian influenza virus M1 antigen protein is shown as Seq ID No. 3.
2. The vaccine composition of claim 1, wherein the subtype H5 avian influenza virus-like particle antigen consists of a subtype H5 2.3.4.4d branched virus-like particle antigen and a subtype H5 2.3.2.1d branched virus-like particle antigen;
the antigen of the H5 subtype 2.3.4.4d branched virus-like particle is assembled by HA, NA and bovine immunodeficiency virus Gag antigen protein, the HA antigen protein of the H5 subtype 2.3.4.4d branched avian influenza virus is shown as Seq ID No.4, the NA antigen protein of the H5 subtype 2.3.4.4d branched avian influenza virus is shown as Seq ID No.5, and the amino acid sequence of the bovine immunodeficiency virus Gag antigen protein is shown as Seq ID No. 8; and
the antigen of the H5 subtype 2.3.2.1d branched virus-like particle is assembled by HA and NA of the antigen and bovine immunodeficiency virus Gag antigen protein, the HA antigen protein of the H5 subtype 2.3.2.1d branched virus is shown as Seq ID No.6, the NA antigen protein of the H5 subtype 2.3.2.1d branched virus is shown as Seq ID No.7, and the bovine immunodeficiency virus Gag antigen protein is shown as an amino acid sequence of Seq ID No. 8.
3. The vaccine composition according to claim 1, wherein the antigen content of the avian influenza virus-like particles of subtype H7 is HA titer is more than or equal to 6log 2; preferably, the antigen content of the H7 subtype avian influenza virus-like particle is HA titer 6log 2-8 log 2.
4. The vaccine composition according to claim 2, wherein the antigen content of the H7 subtype avian influenza virus-like particles is HA titer 6log 2-8 log2, the antigen content of the H5 subtype 2.3.4.4d branch virus-like particles is HA titer 6log 2-8 log2, and the antigen content of the H5 subtype 2.3.2.1d branch virus-like particles is HA titer 6log 2-8 log 2.
5. The vaccine composition of claim 1, wherein the pharmaceutically acceptable carrier is an adjuvant selected from the group consisting of: (1) mineral oil, alumina gel adjuvant, saponin, alfvudine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100; preferably, the adjuvant is a mineral oil adjuvant, which is used to prepare a water-in-oil emulsion;
the adjuvant content is 5% -70% V/V, preferably from 30% -70% V/V, more preferably 66% V/V.
6. A method of making the vaccine composition of claim 1, wherein the method comprises:
cloning HA, NA and M1 antigen protein genes of the H7 subtype avian influenza virus to the same vector, and cloning HA, NA antigen protein genes and bovine immunodeficiency virus Gag antigen protein genes of the H5 subtype avian influenza virus to another same vector;
transforming and recombining the vector obtained in the step (1) to obtain a recombinant baculovirus plasmid containing the H7 subtype avian influenza virus HA, NA and M1 antigen protein genes, and transforming and recombining the vector obtained in the step (1) to obtain a recombinant baculovirus plasmid containing the H5 subtype avian influenza virus HA, NA antigen protein genes and bovine immunodeficiency virus Gag antigen protein genes;
step (3) transfecting insect cells sf9 with the recombinant baculovirus plasmids containing the HA, NA and M1 antigen protein genes of the H7 subtype avian influenza virus obtained in the step (2), expressing the HA, NA and M1 antigen proteins of the H7 subtype avian influenza virus in series, transfecting insect cells sf9 with the recombinant baculovirus plasmids containing the HA, NA antigen protein genes and bovine immunodeficiency virus Gag antigen protein genes of the H5 subtype avian influenza virus obtained in the step (2), and expressing the HA, NA antigen protein and bovine immunodeficiency virus Gag antigen protein of the H5 subtype avian influenza virus in series; and
separating the H7 subtype avian influenza virus-like particle antigen assembled by the HA, NA and M1 antigen proteins of the H7 subtype avian influenza virus released into the supernatant of an extracellular culture medium after the self-assembly in the insect cell is completed, and separating the H5 subtype avian influenza virus-like particle antigen assembled by the HA, NA antigen proteins and bovine immunodeficiency virus Gag antigen proteins of the H5 subtype avian influenza virus released into the supernatant of the extracellular culture medium after the self-assembly in the insect cell is completed; adding an adjuvant, and uniformly mixing to obtain the vaccine composition.
7. The method according to claim 6, wherein the vectors in step (1) are each pFastBac I; the baculovirus plasmid in the step (2) is Bacmid.
8. Use of the vaccine composition according to claims 1-5 in the preparation of a medicament for preventing and/or treating diseases caused by H7 subtype and/or H5 subtype avian influenza virus.
CN202010582681.2A 2020-06-23 2020-06-23 Vaccine composition for resisting H7 subtype and H5 subtype avian influenza viruses, and preparation method and application thereof Pending CN113827715A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101678096A (en) * 2007-04-20 2010-03-24 葛兰素史密丝克莱恩生物有限公司 oil-in-water emulsion influenza vaccine
US20100074915A1 (en) * 2006-07-27 2010-03-25 Ligocyte Pharmaceuticals, Inc. Chimeric influenza virus-like particles
CN110559433A (en) * 2018-06-05 2019-12-13 洛阳惠中生物技术有限公司 Avian influenza virus-like particle vaccine, and preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100074915A1 (en) * 2006-07-27 2010-03-25 Ligocyte Pharmaceuticals, Inc. Chimeric influenza virus-like particles
CN101678096A (en) * 2007-04-20 2010-03-24 葛兰素史密丝克莱恩生物有限公司 oil-in-water emulsion influenza vaccine
CN110559433A (en) * 2018-06-05 2019-12-13 洛阳惠中生物技术有限公司 Avian influenza virus-like particle vaccine, and preparation method and application thereof

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