WO2020093674A1 - 一种重组h7n9亚型禽流感病毒株、灭活标记疫苗及其制备方法 - Google Patents

一种重组h7n9亚型禽流感病毒株、灭活标记疫苗及其制备方法 Download PDF

Info

Publication number
WO2020093674A1
WO2020093674A1 PCT/CN2019/086620 CN2019086620W WO2020093674A1 WO 2020093674 A1 WO2020093674 A1 WO 2020093674A1 CN 2019086620 W CN2019086620 W CN 2019086620W WO 2020093674 A1 WO2020093674 A1 WO 2020093674A1
Authority
WO
WIPO (PCT)
Prior art keywords
avian influenza
virus
influenza virus
subtype avian
recombinant
Prior art date
Application number
PCT/CN2019/086620
Other languages
English (en)
French (fr)
Inventor
彭大新
陈素娟
孙志豪
秦涛
王秋霞
刘秀梵
Original Assignee
扬州大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 扬州大学 filed Critical 扬州大学
Priority to US16/960,222 priority Critical patent/US11376319B2/en
Publication of WO2020093674A1 publication Critical patent/WO2020093674A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39516—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum from serum, plasma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16161—Methods of inactivation or attenuation
    • C12N2760/16162—Methods of inactivation or attenuation by genetic engineering

Definitions

  • the invention relates to the technical field of animal vaccines, in particular to a recombinant H7N9 subtype avian influenza virus strain, a labeled vaccine and a preparation method thereof.
  • the H7N9 subtype avian influenza virus is an emerging zoonotic pathogen that has caused 5 waves of epidemics in humans since it was first reported in China in 2013.
  • the early H7N9 subtype avian influenza virus is of low pathogenicity to poultry, with no obvious symptoms after infection, and human infections are related to exposure to infected poultry or contaminated live poultry trading markets.
  • the H7N9 subtype avian influenza virus had become a highly pathogenic virus due to the insertion of 4 amino acids at the HA cleavage site so that it has continuous basic amino acids, and it has a high lethality rate for chickens.
  • H7 subtype avian influenza virus inactivated vaccine In order to control the spread of H7N9 subtype avian influenza virus in poultry and reduce the risk of human infection caused by contact with infected poultry and polluting the environment, China has approved the use of H7 subtype recombinant avian influenza virus inactivated vaccine, and has achieved good results Prevention and control effect.
  • H7 subtype recombinant avian influenza virus vaccine After immunization with an inactivated recombinant avian influenza virus vaccine, it is not possible to distinguish between vaccine immunization and naturally infected animals, and it is impossible to confirm the infected chickens by serological methods, so as to take measures to fight the infected chickens. Therefore, there is an urgent need to develop a H7 subtype avian influenza to distinguish between natural infection and vaccine-immunized animal (DIVA) vaccine to meet the technical needs of H7 subtype avian influenza purification and extermination.
  • DIVA vaccine-immunized animal
  • the object of the present invention is to provide a recombinant H7N9 subtype avian influenza virus strain, a labeled vaccine and a preparation method thereof.
  • the labeled vaccine can not only accurately distinguish between vaccine immunization and naturally infected animals, but also Used for effective prevention and control and purification of H7N9 subtype avian influenza.
  • the invention provides a recombinant H7N9 subtype avian influenza virus strain, taking the H7N9 subtype avian influenza virus JD / 17 virus strain as the parental virus strain, and replacing the HA of the JD / 17 virus strain with the amino acid sequence of the H3 subtype Amino acid sequence in protein;
  • amino acid sequence of the H3 subtype is shown in SEQ ID No. 1 in the sequence table;
  • the amino acid sequence of the HA protein in the JD / 17 virus strain is shown in SEQ ID No. 2 in the sequence table;
  • the deposit number of the H7N9 subtype avian influenza virus JD / 17 strain is CCTCC No. V201862.
  • the invention provides a method for preparing the recombinant H7N9 subtype avian influenza virus strain, which includes the following steps:
  • the HA-1 gene fragment was amplified with the primer pair KS-H7-1 and JDH7H3-1-R, and the HA- was amplified with the primer pair JDH7H3-2-F and KS-H7-2 2 gene fragments;
  • the nucleotide sequence of the KS-H7-1 is shown in SEQ ID No. 3 in the sequence table;
  • JDH7H3-1-R The nucleotide sequence of JDH7H3-1-R is shown in SEQ ID No. 4 in the sequence table;
  • the nucleotide sequence of JDH7H3-2-F is shown in SEQ ID No. 5 in the sequence table;
  • KS-H7-2 The nucleotide sequence of KS-H7-2 is shown in SEQ ID No. 6 in the sequence table;
  • step (3) Using the HA-1 gene fragment obtained in step (2) and the HA-2 gene fragment as templates to perform overlapping PCR amplification to obtain sequence-replaced HA gene fragments;
  • the amplification procedures of the HA-1 gene fragment and the HA-2 gene fragment are independently:
  • the amplification systems of the HA-1 gene fragment and the HA-2 gene fragment are independently: 2.5 ⁇ L 10 ⁇ PCR buffer, 0.5 ⁇ L 10 mM dNTP, 0.5 ⁇ L 25 mM upstream primer, 0.5 ⁇ L 25 mM downstream primer, 0.5 ⁇ L high-fidelity Enzyme, 2 ⁇ L DNA template and 18.5 ⁇ L ultrapure water.
  • the overlapping PCR amplification procedure is:
  • the preparation method of the recombinant H7N9 subtype avian influenza virus labeled vaccine provided by the present invention includes the following steps:
  • the recombinant H7N9 subtype avian influenza virus strain or the recombinant H7N9 subtype avian influenza virus strain prepared by the preparation method is inoculated into SPF chicken embryos and incubated to obtain viral allantoic fluid;
  • the virus allantoic fluid and formaldehyde solution are mixed, and the resulting mixed fluid is shaken and inactivated for 24 hours under the condition of 4 ° C to obtain an inactivated viral allantoic fluid;
  • the inactivated virus allantoic fluid is sequentially mixed with Tween 80 and white oil to obtain an inactivated virus allantoic fluid mixture;
  • the volume concentration of the formaldehyde solution is 4%.
  • the volume ratio of viral allantoic fluid to formaldehyde solution is 43: 7.
  • the volume ratio of the inactivated virus allantoic fluid, Tween 80 and white oil is 24: 1: 75.
  • the invention provides a recombinant H7N9 subtype avian influenza virus labeled vaccine prepared by the preparation method.
  • the invention provides a recombinant H7N9 subtype avian influenza virus strain, taking the H7N9 subtype avian influenza virus JD / 17 virus strain as the parental virus strain, and replacing the HA of the JD / 17 virus strain with the amino acid sequence of the H3 subtype
  • the amino acid sequence in the protein; the amino acid sequence of the H3 subtype is shown in SEQ ID No. 1 in the sequence table; the amino acid sequence in the HA protein in the JD / 17 virus strain is shown in SEQ ID No. 2 in the sequence table; the H7N9
  • the deposit number of subtype avian influenza virus JD / 17 strain is CCTCC No. V201862.
  • the present invention transforms the HA protein, one of the major surface glycoproteins of the avian influenza virus, and replaces the specific surface antigen epitope of the HA protein with the corresponding sequence of the H3 subtype to achieve successful virus rescue.
  • the invention recombinant H7N9 avian influenza virus strains provided by measuring the HA titer, EID 50, TCID 50, the results show rescue of virus strains maintain parental strain similar biological properties, it has a high HA titer monovalent and EID 50, and high antibody levels after immunization inactivated emulsified chickens produced distinguished antibody and the antibody can be produced by the avian influenza virus subtype H7N9 infected chickens, as a candidate for a marker vaccine strain.
  • the recombinant H7N9 subtype avian influenza virus strain provided by the present invention is selected to be transformed in HA2 protein. Since the HA2 protein itself is relatively conservative and is not prone to chimeric recombination, the epitope in the recombined HA protein is not prone to mutation. To ensure that the new vaccine prepared subsequently maintains the vaccine effect for a long time.
  • the invention provides a recombinant H7N9 subtype avian influenza virus labeled vaccine prepared by the preparation method, which is obtained by inactivation on the basis of the recombinant H7N9 subtype avian influenza virus strain described in the above scheme, and the challenge protection test shows that: immunity After the high pathogenic and low pathogenic H7N9 subtype strains were challenged, the virus was not detected in the laryngeal or cloaca cotton swabs on days 1, 3, 5 and 7 and the protection rate was 100%.
  • the maternal strain (JD / 17 virus strain) only detected the detoxification of highly pathogenic or low pathogenic strains on days 1 and 3, with a protection rate of 90%; this indicates that
  • the inactivated marker vaccine provided by the present invention has an immune protection effect not lower than that of the vaccine prepared by the parent strain, and has a very good protection rate no matter for the highly pathogenic H7N9 subtype AIV or the low pathogenic H7N9 subtype AIV.
  • the H7N9 avian influenza virus strain (Orthomyxoviridae Alphainfluenza virus), the H7N9 avian influenza virus strain is deposited at the Chinese Type Culture Collection, and the deposit time is October 23, 2018.
  • the address is 299 Bayi Road, Wuchang District, Wuhan City, Hubei province, Wuhan University, China.
  • the unit is abbreviated as CCTCC, the biological deposit number is CCTCC No. V201862, and the strain number is JD / 17.
  • FIG. 1 is an experimental procedure of a labeled vaccine provided by the present invention
  • Figure 2 is a schematic diagram of spotting information of a protein chip
  • Figure 3 is a bar graph of the response of various HA subtype avian influenza sera to H7-12 peptide
  • Figure 4 is a graph showing the specific results of indirect immunofluorescence test to verify the chicken serum immunized with peptide-conjugate
  • Figure 5 is a sequence replacement scheme for vaccine strain construction
  • Figure 6 is a histogram of the response of vaccine candidate and maternal strain sera to H7-12.
  • the invention provides a recombinant H7N9 subtype avian influenza virus strain, taking the H7N9 subtype avian influenza virus JD / 17 virus strain as the parental virus strain, and replacing the HA of the JD / 17 virus strain with the amino acid sequence of the H3 subtype Amino acid sequence in protein;
  • amino acid sequence of the H3 subtype is shown in SEQ ID No. 1 in the sequence table;
  • the amino acid sequence of the HA protein in the JD / 17 virus strain is shown in SEQ ID No. 2 in the sequence table;
  • the deposit number of the H7N9 subtype avian influenza virus JD / 17 strain is CCTCC No. V201862.
  • the construction idea of the recombinant H7N9 subtype avian influenza virus strain is as follows: the HA2 specific epitope of the JD / 17 virus strain is identified by the polypeptide chip method, and the HA2 antigen table is deleted or modified by reverse genetic technology In particular, the HA2 specific epitope is replaced by the corresponding sequence of any subtype of influenza virus with low homology except H7 subtype, due to the use of other low homology epitopes other than H3 subtype The amino acid sequence of the subtype influenza virus cannot successfully achieve virus rescue, so the homologous amino acid sequence of the H3 subtype influenza virus was selected to replace the HA2 specific epitope of the JD / 17 virus strain, thereby developing the H7N9 subtype avian influenza DIVA vaccine and Its matching detection technology can effectively distinguish vaccine immunization and naturally infected animals through serological detection methods, and is used for the prevention, control and purification of H7N9 subtype avian influenza.
  • the method for identifying the HA2-specific epitope of the JD / 17 virus strain preferably includes the following steps:
  • SEQ ID No. 2 is the HA2-specific epitope of JD / 17 virus strain.
  • the types of the avian influenza viruses of different subtypes are not particularly limited, and it is sufficient to use the avian influenza virus subtypes common in the art.
  • the method for preparing the serum antibodies of the different subtypes of avian influenza virus is not particularly limited, and it is sufficient to use the preparation scheme of serum antibodies well known in the art.
  • the invention provides a method for preparing the recombinant H7N9 subtype avian influenza virus strain, which includes the following steps:
  • the HA-1 gene fragment was amplified with the primer pair KS-H7-1 and JDH7H3-1-R, and the HA- was amplified with the primer pair JDH7H3-2-F and KS-H7-2 2 gene fragments;
  • the nucleotide sequence of the KS-H7-1 is shown in SEQ ID No. 3 in the sequence table;
  • JDH7H3-1-R The nucleotide sequence of JDH7H3-1-R is shown in SEQ ID No. 4 in the sequence table;
  • the nucleotide sequence of JDH7H3-2-F is shown in SEQ ID No. 5 in the sequence table;
  • KS-H7-2 The nucleotide sequence of KS-H7-2 is shown in SEQ ID No. 6 in the sequence table;
  • step (3) Using the HA-1 gene fragment obtained in step (2) and the HA-2 gene fragment as templates to perform overlapping PCR amplification to obtain sequence-replaced HA gene fragments;
  • the invention extracts the total RNA of the H7N9 subtype avian influenza virus JD / 17 virus strain, reverse transcribes it to obtain cDNA.
  • the method for extracting the total RNA and the method for reverse transcription are not particularly limited, and the method of the kit well known in the art may be used.
  • the present invention uses the cDNA as a template to amplify the HA-1 gene fragment with the primer pair KS-H7-1 and JDH7H3-1-R, and the primer pair to expand JDH7H3-2-F and KS-H7-2 Increase the HA-2 gene fragment;
  • the nucleotide sequence of the KS-H7-1 is shown in SEQ ID No. 3 in the sequence table;
  • JDH7H3-1-R The nucleotide sequence of JDH7H3-1-R is shown in SEQ ID No. 4 in the sequence table;
  • the nucleotide sequence of JDH7H3-2-F is shown in SEQ ID No. 5 in the sequence table;
  • the nucleotide sequence of the KS-H7-2 is shown in SEQ ID No. 6 in the sequence table.
  • the amplification procedures of the HA-1 gene fragment and the HA-2 gene fragment are preferably independently:
  • the amplification systems of the HA-1 gene fragment and the HA-2 gene fragment are independently: 2.5 ⁇ L 10 ⁇ PCR buffer, 0.5 ⁇ L 10 mM dNTP, 0.5 ⁇ L 25 mM upstream primer, 0.5 ⁇ L 25 mM downstream primer, 0.5 ⁇ L high-fidelity Enzyme, 2 ⁇ L DNA template and 18.5 ⁇ L ultrapure water.
  • the quality test is identified by agarose gel electrophoresis. After the band size is correct, the gel is cut and recovered with a gel recovery kit (see the instructions for details). The concentration and purity are measured. The test results OD 260 / OD 280 are 1.8 to 2.0 Qualified for subsequent experiments.
  • the present invention uses the HA-1 gene fragment and the HA-2 gene fragment obtained in step (2) as templates to perform overlapping PCR amplification to obtain a sequence-replaced HA gene Fragment.
  • the primers used in the overlapping PCR amplification are KS-H7-1 and KS-H7-2.
  • the overlapping PCR amplification procedure is preferably: pre-denaturation at 94 ° C for 5 min; denaturation at 94 ° C for 30 s, annealing at 54 ° C for 40 s, extension at 72 ° C for 1 min and 40 s, 35 cycles; extension at 72 ° C for 10 min.
  • the overlapping PCR amplification system is preferably 25 ⁇ L, except that the DNA template is 4 ⁇ L (the upper and lower genes are 2 ⁇ L each), and ultrapure water is reduced to 16.5 ⁇ L, and the other is the same as in the above PCR amplification system.
  • the present invention transfects the sequence-replaced HA plasmid with the other 7 gene expression plasmids of the JD / 17 virus strain to rescue the recombinant H7N9 subtype avian influenza virus strain.
  • the sequence-replaced HA gene fragment is connected to the Blunt3 vector to obtain the intermediate transition plasmid.
  • it is digested with BsmBI enzyme, and the resulting digested target product is cloned into pHW2000 vector and extracted Plasmids; Transfect the extracted plasmids with the expression plasmids constructed with the other 7 genes of the JD / 17 virus strain pHW2000 vector.
  • the other 7 gene types include PB2, PB1, PA, NP, NA, M and NS.
  • the method of constructing the recombinant plasmid with the extracted plasmid and the pHW2000 vector of the other 7 genes of the JD / 17 virus strain can be found in the following documents: Lu Jianhong, Long Jinxue, Shao Weixing, etc. Using reverse genetic manipulation technology to produce weak Recombinant influenza virus of subtype H5 [J] .Journal of Microbiology, 2005,45: 53-57.
  • the virus rescue method includes the following steps: one day before transfection, 293T and MDCK cells are mixed in equal amounts, and then plated into a 6-well cell culture plate (about 6 ⁇ 10 5 cells / well). Transfection was performed when the cell coverage reached 80%.
  • the transfection steps refer to the instructions of Polyjet Transfection Reagent.
  • Each transfection system contains 8 fragments of transcription / expression plasmid (300ng / plasmid, HA fragment is HA after sequence replacement, and the rest are 7 fragments of JD / 17) .
  • 48-72h after transfection, freezing and thawing were repeated 3 times, the transfection supernatant was collected, and inoculated with 10-day-old SPF chicken embryo, 0.3mL / embryo.
  • Inoculated chicken embryos were tested for potency by hemagglutination test (HA) to verify whether the virus was successfully rescued.
  • Positive chicken embryo allantoic fluid was used to extract the total RNA of the virus, and 8 fragments were amplified by PCR for sequencing. If the sequence was correct, the virus allantoic fluid was stored in the refrigerator at -70 °C for later use.
  • the H7-12 peptide microarray chip was used to detect the immune serum of recombinant H7N9 subtype avian influenza virus strain and the original virus strain.
  • the results showed that the immune serum of the original virus strain JD / 17 had a higher level of H7-12 peptide.
  • the positive response (6.39 ⁇ 0.13), and the immune sera of the recombinant H7N9 subtype avian influenza virus strains were all negative responses (0.44 ⁇ 0.14). This indicates that the vaccine candidate has lost the epitope H7-12, and the DIVA strategy was successful.
  • the HA titer, EID 50 and TCID 50 of the recombinant H7N9 subtype avian influenza virus strains were determined, and the results showed that the biological characteristics of the rescued virus strains were similar to the parental strains.
  • the preparation method of the recombinant H7N9 subtype avian influenza virus labeled vaccine provided by the present invention includes the following steps:
  • the recombinant H7N9 subtype avian influenza virus strain or the recombinant H7N9 subtype avian influenza virus strain prepared by the preparation method is inoculated into SPF chicken embryos and incubated to obtain viral allantoic fluid;
  • the virus allantoic fluid and formaldehyde solution are mixed, and the resulting mixed solution is shaken and inactivated for 24 hours under the condition of 4 ° C to obtain an inactivated viral allantoic fluid;
  • the inactivated virus allantoic fluid is sequentially mixed with Tween 80 and white oil to obtain an inactivated virus allantoic fluid mixture;
  • the volume concentration of the formaldehyde solution is preferably 4%.
  • the volume ratio of the viral allantoic fluid to the formaldehyde solution is preferably 43: 7.
  • the volume ratio of the inactivated virus allantoic fluid, Tween 80 and white oil is preferably 24: 1: 75.
  • the invention provides a recombinant H7N9 subtype avian influenza virus labeled vaccine prepared by the preparation method.
  • the challenge protection experiment showed that the immune protection effect of the prepared inactivated labeled vaccine is not lower than that of the vaccine prepared by the parent strain, and whether it is directed against the highly pathogenic H7N9 subtype AIV or the low pathogenic H7N9 subtype AIV A good protection rate does not change its biological characteristics after replacement.
  • HI hemagglutination inhibition
  • the hanging line indicates that the serum and virus have fully reacted, indicating positive.
  • the dilution of the serum from the reading to the complete hanging line is the hemagglutination inhibition value of the serum against the virus, that is, the HI titer, and the titer of 4 or more is regarded as effective, and the virus is identified according to the HI titer.
  • the positive control of virus in hole 11 is not hanging, and the negative control of hole 12 in PBS is hanging.
  • Viral allantoic fluid was serially diluted 10 times with four antibodies (penicillin, streptomycin, kanamycin, and gentamicin) PBS, and 6 dilutions (10 -5 -10 -10 ) were inoculated for 10 days.
  • SPF chicken embryos were inoculated with 5 embryos per dilution, 0.2 mL / embryo. Seeded at 35 °C chick embryo culture, embryo as once every 12h, 72h until, finally EID 50 is calculated according to Reed-Muench Method.
  • Viral allantoic fluid and diluted 1:50 formaldehyde aqueous solution were mixed in the ratio of 43: 7, evenly placed at 4 ° C on a shaker, and shaken and inactivated for 24h.
  • H7N9 subtype avian influenza strain A / Chicken / Huadong / JD / 17 was isolated from a chicken farm in 2017 H7N9) (JD / 17), see the supplementary material for the gene sequence of the 8 fragments.
  • Biological characteristics measurement results showed (Table 1), which has a high strain HA titer and EID 50, and after inactivating emulsion produced high antibody levels in vaccinated flocks, it is suitable as a vaccine candidate.
  • RNA of JD / 17 allantoic fluid was extracted by the Trizol method, and then 8 gene fragments of the virus were amplified by reverse transcription PCR (RT-PCR) respectively.
  • RT-PCR reverse transcription PCR
  • PCR amplification program pre-denaturation at 94 °C for 5min; denaturation at 94 °C for 30s, annealing at 54 °C for 40s, extension at 72 °C for 1min and 30s, 35 cycles; extension at 72 °C for 10min
  • the PCR products were electrophoresed with 1% agarose, and after the electrophoresis was completed, the target bands were recovered according to the instructions of the DNA Gel Extraction Kit. Determine the DNA concentration of the recovered PCR product with a spectrophotometer. When the concentration reaches ⁇ 50ng / ⁇ L, which meets the sequencing requirements, send the PCR recovered product and primers to the company for two-way sequencing; if the recovered PCR product concentration is low, the recovered product It is connected to T3EasyVector and transformed into DH5 ⁇ E. coli competent cells. For the specific method, please refer to the instruction of T3EasyVector.
  • H1A Label virus Subtype Hemagglutination titer (HAlog2) H1 A / Duck / EasternChina / 103/03 H1N1 7 H3 A / Duck / EasternChina / 852/03 H3N2 6 H4 A / Duck / EasternChina / 160/02 H4N6 5 H5-1 A / Mallard / Huadong / S / 2005 H5N1 7 H5-2 A / Chicken / Huadong / 1111/16 H5N6 7 H5-3 A / Chicken / Huadong / ZJ0104 / 16 H5N2 6 H6 A / Duck / EasternChina / 58/03 H6N2 7 H9-1 A / Chicken / Shanghai / F / 98 H9N2 9 H9-2 A / Chicken / Fujian / SN / 14 H9N2 6 H10 A / Chicken / Huadong / RD5 / 13 H10N9 6 H11 A / Duck /
  • the modified silica gel mold (iPDMS) was purchased from SJ Biomaterisls, and the peptide was synthesized by GL Biochem (Shanghai, China). According to the deduced amino acid sequence, the HA2 protein of H7N9 subtype avian influenza virus JD / 17 strain was synthesized into overlapping polypeptides (10 amino acids overlapped between each adjacent 2 peptides), and spotted on the modified silica gel mold for total synthesis 13 peptides (Table 4) (where the positive quality control point is goat anti-chicken IgY; the negative control point is the spotting buffer) were prepared for the peptide chip, and the microarray spotting information is shown in Table 5 and Figure 2.
  • H7-2 SEQ ID No. 32 ENGWEGLIDGWYGFRHQNAQ H7-3 SEQ ID No. 33 WYGFRHQNAQGEGTAADYKS H7-4 SEQ ID No. 34 GEGTAADYKSTQSAIDQITG H7-6 SEQ ID No. 35 KLNRLIAKTNQQFELIDNEF H7-7 SEQ ID No. 36 QQFELIDNEFNEVEKQIGNV H7-8 SEQ ID No. 37 NEVEKQIGNVINWTRDSITE H7-9 SEQ ID No. 38 INWTRDSITEVWSYNAELLV H7-10 SEQ ID No. 39 VWSYNAELLVAMENQHTIDL H7-12 SEQ No.
  • the serum antibodies of different subtypes of avian influenza virus prepared in Experiment 2 were combined with the peptide chip to obtain an epitope that could only bind to antibodies of the H7N9 subtype of avian influenza virus.
  • the specific steps are as follows:
  • serum samples were diluted 1: 100 in serum dilution buffer, and 200 ⁇ L was added to each microarray, and incubated on a shaker for 2 h (150 r / min, 4 ° C.).
  • Figure 3 shows the response of various HA subtype avian influenza sera to H7-12 peptide.
  • the results showed that only the H7 subtype avian influenza serum antibody had a specific positive response to the H7-12 peptide (SNR> 2), while the other HA subtype serum antibodies had a negative response to the H7-12 peptide (SNR ⁇ 2) ( Figure 3) (P ⁇ 0.01), indicating that the H7-12 peptide is an epitope that can only specifically bind to the H7N9 subtype avian influenza serum antibody; and each HA subtype serum antibody pair except the H7-12 peptide The peptides showed varying degrees of response (Table 6).
  • the screened H7-12 peptide was coupled with BSA to obtain H7-12-BSA conjugate.
  • the conjugate was used to prepare immune antigen and immunized SPF chicken to prepare multiple antiserum.
  • the immunization procedure is as follows:
  • CEF cells are cultured in a 96-well plate. After the cells grow to 80%, the medium is discarded and washed 3 times with PBS.
  • Dilute chicken serum 1 1000, add it to a 96-well plate, 200 ⁇ L / well, and incubate at 37 °C for 1.5h. And add H7 monoclonal antibody and negative serum as positive control and negative control, respectively.
  • Results The results showed that the chicken serum prepared by the peptide-conjugate showed specific fluorescence only for H7 subtype avian influenza, and no specific fluorescence for other subtype virus samples, indicating that the polypeptide epitope was H7 subtype Avian influenza virus specific antigen epitope, the results are shown in Figure 4.
  • the HA2-12 peptide in its HA was replaced, and the amino acids in the H7-12 peptide region of the JD / 17 strain (ADSEMDKLYERVKRQLRENA) were used in Overlap-PCR technology (primers are shown in the table 7) Replaced with the H3 subtype sequence (ADSEMNKLFEKTKKQLRENA), as shown in FIG. 5.
  • PCR was performed to amplify the target fragment.
  • Configure 25 ⁇ L system 2.5 ⁇ L 10 ⁇ PCR buffer, 0.5 ⁇ L dNTP (10mM), 0.5 ⁇ L 25mM upstream primer, 0.5 ⁇ L 25mM downstream primer, 0.5 ⁇ L high-fidelity enzyme, 2 ⁇ L DNA template and 18.5 ⁇ L ultrapure water.
  • the PCR amplification procedure is as follows: 94 ° C pre-denaturation for 5 min; 94 ° C denaturation for 30 s, 54 ° C annealing for 40 s, 72 ° C extension for 1 min and 30 s, 35 cycles; 72 ° C extension for 10 min.
  • PCR products HA-1 and HA-2 were identified by agarose gel electrophoresis. After the bands were correct, the gel was cut and recovered with a gel recovery kit (see the instructions for details), and the concentration and purity were measured (OD 260 / OD 280 was 1.8 ⁇ 2.0), then use the recovered product of the upper and lower sections of the HA gene as a template for Overlap-PCR, the system is still 25 ⁇ L, except for the DNA template is 4 ⁇ L (upper and lower section gene 2 ⁇ L), ultrapure water is reduced to 16.5 ⁇ L, other and above In the PCR system, the amplification program at 72 ° C is extended to 1 min and 40 s, and the other is the same as the above PCR amplification program.
  • the sequence-replaced HA gene fragment was connected to the Blunt3 vector to obtain the intermediate transition plasmid.
  • the enzyme was digested with BsmBI enzyme.
  • the obtained target product was cloned into the pHW2000 vector and the plasmid was extracted.
  • the plasmid was transfected with the expression plasmid constructed by pHW2000 vector of 7 other genes of JD / 17 virus strain.
  • the other 7 gene types include PB2, PB1, PA, NP, NA, M and NS.
  • Virus rescue method One day before transfection, 293T and MDCK cells were mixed in equal amounts and plated in a 6-well cell culture plate (approximately 6 ⁇ 10 5 cells / well). Transfection was performed when the cell coverage area reached 80%. The transfection steps refer to the instructions of Polyjet transfection reagent. Each transfection system contains 8 fragments of transcription / expression plasmid (300ng / plasmid, HA fragment is the HA gene fragment after sequence replacement, and the rest are 7 of JD / 17 Fragment). From 48 to 72h after transfection, freeze-thaw was repeated three times, and the transfection supernatant was collected and inoculated with 10-day-old SPF chicken embryos at 0.3mL / embryo.
  • Inoculated chicken embryos were tested for potency by hemagglutination test (HA) to verify whether the virus was successfully rescued.
  • Positive chicken embryo allantoic fluid was used to extract the total RNA of the virus, and 8 fragments were amplified by PCR for sequencing. If the sequence was correct, the virus allantoic fluid was stored in the refrigerator at -70 °C for later use.
  • CEF cells were plated into 96-well cell culture plates the day before the inoculation. After the cells formed a monolayer, the culture supernatant was aspirated and washed 3 times with sterile PBS, then the virus allantoic fluid was serially diluted 10 times to the cells. On the surface, the dilution of inoculation is 10 -4 ⁇ 10 -9 . Each dilution is inoculated with 4 wells, 0.1 mL / well. The infected cells were continuously cultured at 37 ° C and 5% CO 2. After 72h, the number of positive infected wells was counted by hemagglutination test, and the TCID 50 was calculated according to the Reed-Muench method.
  • the HA titer, EID 50 and TCID 50 of cHA H7 / H3 were measured. The results showed that the rescued vaccine candidate cHA H7 / H3 had biological characteristics similar to the maternal strain, and its biological characteristics were not changed after replacement (Table 8).
  • the SPF chicken was immunized with the obtained recombinant virus to prepare serum, and the wild-type strain JD / 17 was used as a control at the same time.
  • Viral allantoic fluid and formaldehyde solution with a volume concentration of 4% were mixed at a ratio of 43: 7, evenly placed on a shaker at 4 ° C, and shaken and inactivated for 24 hours.
  • 21-day-old SPF chickens were randomly divided into 7 groups, 10 birds / group, including 4 immunized groups, 2 challenge control groups and 1 healthy control group.
  • the challenge control group of low pathogenic strains started detoxification on day 1 and reached a peak on days 3 and 5; high pathogenicity
  • the strain control group also started detoxification on the first day, and all died on the third day.
  • the cHAH7 / H3 immunization group had no virus detected in the laryngeal or cloaca cotton swabs on days 1, 3, 5 and 7 after challenge with the low pathogenic and high pathogenic H7N9 subtype strains. It was 100%; and the maternal immunization group strains only detected the detoxification of highly pathogenic or low pathogenic AIV on Day 1 and Day 3, respectively.
  • the inactivated vaccine prepared by the candidate vaccine strain cHAH7 / H3 has an immune protection effect not lower than that of the vaccine prepared by the parental strain, and it has very good effects on the strong H7N9 subtype AIV or the weak H7N9 subtype AIV Protection rate.
  • Hemagglutination inhibition test was performed on naturally infected chicken serum to determine its HI titer.
  • the peptide chip prepared with H7-12 peptide was used to detect naturally infected chicken serum and calculate the SNR value.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • Pulmonology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种重组H7N9亚型禽流感病毒株，以保藏编号为CCTCC No.V201862的H7N9亚型禽流感病毒JD/17病毒株为母本病毒株，用H3亚型的氨基酸序列替换所述JD/17病毒株的HA蛋白中氨基酸序列。通过测定HA效价、EID 50、TCID 50，结果显示拯救的病毒株保持了母本病毒株相似的生物学特性，具有较高的HA效价和EID 50。还提供了利用该病毒株经灭活乳化后制备的标记疫苗及其制备方法。该疫苗免疫鸡群产生的抗体水平较高，且该抗体能与鸡自然感染H7N9亚型禽流感病毒后产生的抗体相区别。

Description

一种重组H7N9亚型禽流感病毒株、灭活标记疫苗及其制备方法
本申请要求于2018年11月05日提交中国专利局、申请号为201811308245.5、发明名称为“一种重组H7N9亚型禽流感病毒株、灭活标记疫苗及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及动物疫苗技术领域，尤其涉及一种重组H7N9亚型禽流感病毒株、标记疫苗及其制备方法。
背景技术
H7N9亚型禽流感病毒是一种新出现的人兽共患病病原，自从2013年首次在我国报道以来，在人类已引起5波流行。早期的H7N9亚型禽流感病毒对家禽为低致病性的，家禽感染后无明显症状，人类感染与接触感染家禽或污染的活禽交易市场有关。至2017年初，H7N9亚型禽流感病毒由于在HA裂解位点插入4个氨基酸使其具有连续的碱性氨基酸而变为高致病性的病毒，对鸡有很高的致死率。
为了控制H7N9亚型禽流感病毒在家禽中的传播，降低人因接触感染家禽及污染环境而导致感染的风险，我国已批准使用H7亚型重组禽流感病毒灭活疫苗，并取得了较好的防控效果。但是重组禽流感病毒灭活疫苗免疫后不能区分疫苗免疫和自然感染动物，无法通过血清学方法确认感染鸡群，从而对感染鸡群采取扑杀措施。因此，迫切需要研制出H7亚型禽流感区分自然感染和疫苗免疫动物(DIVA)疫苗，满足H7亚型禽流感净化扑灭的技术需求。
发明内容
为了克服现有技术的上述缺点，本发明的目的在于提供一种重组H7N9亚型禽流感病毒株、标记疫苗及其制备方法，所述标记疫苗不仅能准确区分疫苗免疫和自然感染动物，而且能用于H7N9亚型禽流感的有效防控和净化。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种重组H7N9亚型禽流感病毒株，以H7N9亚型禽流感病毒JD/17病毒株为母本病毒株，用H3亚型的氨基酸序列替换所述JD/17病毒株的HA蛋白中氨基酸序列；
所述H3亚型的氨基酸序列见序列表中SEQ ID No.1；
所述JD/17病毒株中的HA蛋白中氨基酸序列见序列表中SEQ ID  No.2；
所述H7N9亚型禽流感病毒JD/17病毒株的保藏编号为CCTCC No.V201862。
本发明提供了所述的重组H7N9亚型禽流感病毒株的制备方法，包括以下步骤：
(1)提取H7N9亚型禽流感病毒JD/17病毒株的总RNA，反转录，得到cDNA；
(2)以所述cDNA为模板，用引物对KS-H7-1和JDH7H3-1-R扩增HA-1基因片段，用引物对JDH7H3-2-F和KS-H7-2扩增HA-2基因片段；
所述KS-H7-1的核苷酸序列如序列表中SEQ ID No.3所示；
所述JDH7H3-1-R的核苷酸序列如序列表中SEQ ID No.4所示；
所述JDH7H3-2-F的核苷酸序列如序列表中SEQ ID No.5所示；
所述KS-H7-2的核苷酸序列如序列表中SEQ ID No.6所示；
(3)以步骤(2)得到的HA-1基因片段和HA-2的基因片段为模板进行重叠PCR扩增，得到序列替换的HA基因片段；
(4)将所述步骤(3)中序列替换的HA基因片段与Blunt 3载体连接，得到的中间过渡质粒，经测序验证序列正确后，用BsmBI酶进行酶切，得到的酶切的目的产物克隆至pHW2000载体，提取质粒；将提取的质粒与JD/17病毒株的其他7个基因的pHW2000载体构建的表达质粒进行转染，拯救得到重组H7N9亚型禽流感病毒株。
优选的，所述HA-1基因片段和HA-2基因片段的扩增程序独立为：
94℃预变性5min；94℃变性30s，54℃退火40s，72℃延伸1min 30s，35个循环；72℃延伸10min。
优选的，所述HA-1基因片段和HA-2基因片段的扩增体系独立为：2.5μL 10×PCR缓冲液，0.5μL 10mM dNTP，0.5μL25mM上游引物，0.5μL25mM下游引物，0.5μL高保真酶，2μLDNA模板和18.5μL超纯水。
优选的，所述重叠PCR扩增的程序为：
94℃预变性5min；94℃变性30s，54℃退火40s，72℃延伸1min40s，35个循环；72℃延伸10min。
本发明提供的重组H7N9亚型禽流感病毒标记疫苗的制备方法，包括以下步骤：
A.将所述重组H7N9亚型禽流感病毒株或所述制备方法制备的重组H7N9亚型禽流感病毒株接种SPF鸡胚，孵育，得到病毒尿囊液；
B.将所述病毒尿囊液和甲醛溶液混合，得到的混合液转移4℃的条件 下振摇灭活24h，得到灭活病毒尿囊液；
C.当所述灭活病毒尿囊液的血凝效价>4log2时，将所述灭活病毒尿囊液依次与吐温80和白油混合，得到灭活病毒尿囊液混合物；
D.将所述灭活病毒尿囊液混合物乳化，得到重组H7N9亚型禽流感病毒标记疫苗。
优选的，所述甲醛溶液的体积浓度为4％。
优选的，所述病毒尿囊液和甲醛溶液的体积比为43:7。
优选的，所述灭活病毒尿囊液、吐温80和白油的体积比为24:1:75。
本发明提供了所述制备方法制备的重组H7N9亚型禽流感病毒标记疫苗。
本发明提供了一种重组H7N9亚型禽流感病毒株，以H7N9亚型禽流感病毒JD/17病毒株为母本病毒株，用H3亚型的氨基酸序列替换所述JD/17病毒株的HA蛋白中氨基酸序列；所述H3亚型的氨基酸序列见序列表中SEQ ID No.1；所述JD/17病毒株中的HA蛋白中氨基酸序列见序列表中SEQ ID No.2；所述H7N9亚型禽流感病毒JD/17病毒株的保藏编号为CCTCC No.V201862。本发明在全病毒的基础上，对禽流感病毒的主要表面糖蛋白之一的HA蛋白进行改造，将HA蛋白的特异性表面抗原表位替换为H3亚型相应序列，实现病毒成功拯救。同时本发明提供的重组H7N9亚型禽流感病毒株，通过测定HA效价、EID 50、TCID 50，结果显示拯救的病毒株保持了母本毒株相似的生物学特性，具有较高的HA效价和EID 50，且经灭活乳化后免疫鸡群产生的抗体水平较高，且该抗体能与H7N9亚型禽流感病毒感染鸡后产生的抗体相区别，适合作为标记疫苗的候选株。
同时，本发明提供的重组H7N9亚型禽流感病毒株选择在HA2蛋白进行改造，由于HA2蛋白本身比较保守，又不易发生嵌合性重组，重组后的HA蛋白中的抗原表位不易发生变异，保证后续制备的新疫苗长期稳定保持疫苗效果。
本发明提供了所述制备方法制备的重组H7N9亚型禽流感病毒标记疫苗，是在上述方案所述的重组H7N9亚型禽流感病毒株的基础上灭活得到，经攻毒保护试验表明：免疫组在高致病性和低致病性H7N9亚型毒株攻毒后，在第1、3、5和7天，均未在喉头或泄殖腔棉拭中检测出病毒，保护率为100％，而母本毒株(JD/17病毒株)也仅在第1天和第3天检测出了对高致病性毒株或低致病性毒株的排毒，保护率为90％；这表明本发明提供的灭活标记疫苗免疫保护效果不低于母本毒株制备的疫苗，且 无论针对高致病性H7N9亚型AIV还是低致病性H7N9亚型AIV均有非常好的保护率。
生物保藏信息
H7N9禽流感病毒株(Orthomyxoviridae Alphainfluenza virus)，H7N9禽流感病毒株保藏在中国典型培养物保藏中心，保藏时间为2018年10月23日。地址为湖北省武汉市武昌区八一路299号，中国武汉大学，单位简称，CCTCC，生物保藏编号为CCTCC No.V201862，菌株编号为JD/17。
附图说明
图1为本发明提供的标记疫苗的实验流程；
图2为蛋白芯片点样信息的示意图；
图3为各个HA亚型禽流感血清对H7-12肽的响应情况柱状图；
图4为间接免疫荧光试验验证多肽-偶联物免疫鸡血清的特异性结果图；
图5为疫苗株构建序列替换方案；
图6为疫苗候选株和母本毒株血清对H7-12的响应情况柱状图。
具体实施方式
本发明提供了一种重组H7N9亚型禽流感病毒株，以H7N9亚型禽流感病毒JD/17病毒株为母本病毒株，用H3亚型的氨基酸序列替换所述JD/17病毒株的HA蛋白中氨基酸序列；
所述H3亚型的氨基酸序列见序列表中SEQ ID No.1；
所述JD/17病毒株中的HA蛋白中氨基酸序列见序列表中SEQ ID No.2；
所述H7N9亚型禽流感病毒JD/17病毒株的保藏编号为CCTCC No.V201862。
在本发明中，所述重组H7N9亚型禽流感病毒株的构建思路如下：通过多肽芯片方法鉴定出JD/17病毒株的HA2特异性抗原表位，通过反向遗传技术缺失或修饰HA2抗原表位，具体是采用除H7亚型外与其同源性低的任何一种亚型流感病毒的相应序列替换HA2特异性抗原表位，由于采用除H3亚型之外的其他同源性低的各亚型流感病毒的氨基酸序列不能成功实现病毒拯救，故选择H3亚型流感病毒的同源氨基酸序列替换JD/17病毒株的HA2特异性抗原表位，从而研制出H7N9亚型禽流感DIVA疫苗及其配套的检测技术，通过血清学检测方法能有效地区分疫苗免疫和自然感染动物，用于H7N9亚型禽流感的防控和净化。
在本发明中，所述JD/17病毒株的HA2特异性抗原表位的鉴定方法，优选包括以下步骤：
①将JD/17病毒株进行全基因组序列测定，得到HA蛋白的编码基因；
②将所述HA蛋白的编码基因的核苷酸序列翻译为氨基酸序列，将所述氨基酸序列从5’端为起始位点，截取得到第一条含20个氨基酸的多肽，从5’端的第11位氨基酸为起始位点，截取得到第二条含20个氨基酸的多肽，如此重复合成多条多肽，使每相邻的2条多肽之间重叠10个氨基酸，直至得到第三条含20个氨基酸的多肽..........和第N条含20个氨基酸的多肽；
③将得到的N条多肽制备多肽微阵列芯片；
④将不同亚型禽流感病毒血清抗体在所述多肽微阵列芯片上微量点样，根据荧光结果筛选仅能与H7N9亚型禽流感病毒抗体结合的多肽，得到的多肽微阵列芯片上多肽序列(SEQ ID No.2)为JD/17病毒株的HA2特异性抗原表位。
在本发明中，所述不同亚型禽流感病毒的种类没有特殊限制，采用本领域常见的禽流感病毒亚型即可。所述不同亚型禽流感病毒血清抗体的制备方法没有特殊限制，采用本领域所熟知的血清抗体的制备方案即可。
本发明提供了所述的重组H7N9亚型禽流感病毒株的制备方法，包括以下步骤：
(1)提取H7N9亚型禽流感病毒JD/17病毒株的总RNA，反转录，得到cDNA；
(2)以所述cDNA为模板，用引物对KS-H7-1和JDH7H3-1-R扩增HA-1基因片段，用引物对JDH7H3-2-F和KS-H7-2扩增HA-2基因片段；
所述KS-H7-1的核苷酸序列如序列表中SEQ ID No.3所示；
所述JDH7H3-1-R的核苷酸序列如序列表中SEQ ID No.4所示；
所述JDH7H3-2-F的核苷酸序列如序列表中SEQ ID No.5所示；
所述KS-H7-2的核苷酸序列如序列表中SEQ ID No.6所示；
(3)以步骤(2)得到的HA-1基因片段和HA-2的基因片段为模板进行重叠PCR扩增，得到序列替换的HA基因片段；
(4)将所述步骤(3)中序列替换的HA基因片段与Blunt 3载体连接，得到的中间过渡质粒，经测序验证序列正确后，用BsmBI酶进行酶切，得到的酶切的目的产物克隆至pHW2000载体，提取质粒；将提取的质粒与JD/17病毒株的其他7个基因的pHW2000载体构建的表达质粒进行转染，拯救得到重组H7N9亚型禽流感病毒株。
本发明提取H7N9亚型禽流感病毒JD/17病毒株的总RNA，反转录，得到cDNA。
本发明对所述总RNA的提取方法和反转录的方法没有特殊限制，采用本领域所熟知的试剂盒的方法即可。
得到cDNA后，本发明以所述cDNA为模板，用引物对KS-H7-1和JDH7H3-1-R扩增HA-1基因片段，用引物对JDH7H3-2-F和KS-H7-2扩增HA-2基因片段；所述KS-H7-1的核苷酸序列如序列表中SEQ ID No.3所示；
所述JDH7H3-1-R的核苷酸序列如序列表中SEQ ID No.4所示；
所述JDH7H3-2-F的核苷酸序列如序列表中SEQ ID No.5所示；
所述KS-H7-2的核苷酸序列如序列表中SEQ ID No.6所示。
在本发明中，所述HA-1基因片段和HA-2基因片段的扩增程序独立优选为：
94℃预变性5min；94℃变性30s，54℃退火40s，72℃延伸1min 30s，35个循环；72℃延伸10min。
优选的，所述HA-1基因片段和HA-2基因片段的扩增体系独立为：2.5μL 10×PCR缓冲液，0.5μL 10mM dNTP，0.5μL25mM上游引物，0.5μL25mM下游引物，0.5μL高保真酶，2μLDNA模板和18.5μL超纯水。
得到HA-1基因片段和HA-2的基因片段后优选先进行质量检测。所述质量检测为经琼脂糖凝胶电泳鉴定，条带大小正确后，切胶并用胶回收试剂盒回收(步骤详见说明书)，测浓度和纯度，检测结果OD 260/OD 280为1.8～2.0合格，用于后续实验。
得到HA-1基因片段和HA-2的基因片段后，本发明以步骤(2)得到的HA-1基因片段和HA-2的基因片段为模板进行重叠PCR扩增，得到序列替换的HA基因片段。
在本发明中，所述重叠PCR扩增时所用的引物为KS-H7-1和KS-H7-2。
在本发明中，所述重叠PCR扩增的程序优选为：94℃预变性5min；94℃变性30s，54℃退火40s，72℃延伸1min40s，35个循环；72℃延伸10min。
在本发明中，所述重叠PCR扩增的体系优选为25μL，除了DNA模板为4μL(上下段基因各2μL)，超纯水减到16.5μL，其它和上述PCR扩增体系中一样。
得到序列替换的HA基因片段后，本发明将所述序列替换的HA质粒 与JD/17病毒株的其他7个基因表达质粒进行转染，拯救出重组H7N9亚型禽流感病毒株。
在本发明中，序列替换的HA基因片段与Blunt 3载体连接，得到的中间过渡质粒，经测序验证序列正确后，用BsmBI酶进行酶切，得到的酶切的目的产物克隆至pHW2000载体，提取质粒；将提取的质粒与JD/17病毒株的其他7个基因的pHW2000载体构建的表达质粒进行转染。所述其他7个基因的种类包括PB2、PB1、PA、NP、NA、M和NS。将提取的质粒与JD/17病毒株的其他7个基因的pHW2000载体构建重组质粒的方法具体步骤参见如下文献：卢建红,龙进学,邵卫星,等.用反向遗传操作技术产生致弱的H5亚型重组流感病毒[J].微生物学报,2005,45:53-57.
在本发明中，所述病毒拯救的方法，包括以下步骤：转染前一天，将293T和MDCK细胞等量混合后铺入6孔细胞培养板(约6×10 5个细胞/孔)，待细胞覆盖面积达80％时进行转染。转染步骤参照Polyjet转染试剂说明书，每个转染体系中均包含8个片段的转录/表达质粒(300ng/质粒，HA片段为序列替换后的HA，其余为JD/17的7个片段)。转染后48-72h，反复冻融3次，收集转染上清，接种10日龄SPF鸡胚，0.3mL/胚。接种的鸡胚用血凝试验(HA)测定效价，验证病毒是否拯救成功。阳性鸡胚尿囊液提取病毒总RNA，PCR扩增8个片段进行测序，序列正确无误则将病毒尿囊液置于-70℃冰箱保存备用。
为了验证DIVA特性，采用H7-12多肽微阵列芯片检测重组H7N9亚型禽流感病毒株和原始病毒毒株免疫血清，结果发现原始病毒株JD/17的免疫血清对H7-12肽有较高的阳性响应(6.39±0.13)，而重组H7N9亚型禽流感病毒株的免疫血清则均为阴性响应(0.44±0.14)。这表明该疫苗候选株已失去了H7-12这个抗原表位，DIVA策略成功。
对重组H7N9亚型禽流感病毒株测定了HA效价、EID 50、TCID 50，结果显示拯救的病毒株的生物学特性与母本毒株相似。
本发明提供的重组H7N9亚型禽流感病毒标记疫苗的制备方法，包括以下步骤：
A.将所述重组H7N9亚型禽流感病毒株或所述制备方法制备的重组H7N9亚型禽流感病毒株接种SPF鸡胚，孵育，得到病毒尿囊液；
B.将所述病毒尿囊液和甲醛溶液混合，得到的混合液转移4℃的条件下振摇灭活24h，得到灭活病毒尿囊液；
C.当所述灭活病毒尿囊液的血凝效价>4log2时，将所述灭活病毒尿囊液依次与吐温80和白油混合，得到灭活病毒尿囊液混合物；
D.将所述灭活病毒尿囊液混合物乳化，得到重组H7N9亚型禽流感病毒标记疫苗。
在本发明中，所述甲醛溶液的体积浓度优选为4％。
在本发明中，所述病毒尿囊液和甲醛溶液的体积比优选为43:7。
在本发明中，所述灭活病毒尿囊液、吐温80和白油的体积比优选为24:1:75。
本发明提供了所述制备方法制备的重组H7N9亚型禽流感病毒标记疫苗。
经攻毒保护实验表明，制备的灭活标记疫苗免疫保护效果不低于母本毒株制备的疫苗，且无论针对高致病性H7N9亚型AIV还是低致病性H7N9亚型AIV均有非常好的保护率，经过替换后未改变其生物学特性。
下面结合实施例对本发明提供的一种重组H7N9亚型禽流感病毒株、标记疫苗及其制备方法进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
H7N9亚型禽流感病毒分离鉴定
1.1病毒分离
(1)在采集活禽交易市场的家禽棉拭样品时，用灭菌的棉拭子采集家禽的泄殖腔和喉头样品，泄殖腔棉拭应尽量带有粪便，而喉头棉拭应尽量带有粘液，然后将棉拭子折断存储于装有1mL运输液的2mL指形管中，采集的样品置冰盒中运输。
(2)病料组织样品处理：在超净台中将病料组织剪碎置于加有4mL四抗PBS 5mL的研磨管；用生物样品匀质器以6500rpm的匀质速度匀浆20s后暂停10s循环2次进行研磨；将研磨管放置于-70℃冰箱，10min后取出融化，如此反复冻融3次；将冻融后的病料以8000rpm离心10min，取上清分装于指形管备用。棉拭子样品挤压后弃去棉拭子，将盛有液体的指形管置于-70℃冰箱，10min后取出融化，如此反复冻融3次；将冻融后的样品以8000rpm离心10min，取上清分装于指形管备用。
(3)应用鸡胚尿囊腔接种法进行病毒的分离和传代培养，将上述得到的上清接种鸡胚后，每隔12h照胚一次，死亡鸡胚于4℃冰箱放置4h后收获其尿囊液。连续照胚5天后将所有未死亡鸡胚置于4℃冰箱放置4h至死亡，按如下方法测定HA效价，阳性样品无菌收获尿囊液-70℃保存备用，阴性样品无菌收集一定量尿囊液，用SPF鸡胚传代一次确定是否为血凝阳性。将尿囊液中的病毒定义为病毒株JD/17并进行生物保藏。
(4)血凝(HA)试验：
①在96孔血凝板中每孔加入25μL PBS。
②在96孔血凝板的第一列孔中加入25μL上述尿囊液，从左至右倍比稀释至第11孔，弃掉25μL。第12孔为阴性对照。
③每孔补加25μL PBS。
④在每孔中均加入25μL 1％红细胞，每孔中最后液体体积为75μL，轻轻震荡血凝板使孔内液体混匀，将血凝板放置室温(20℃)40min。
⑤放置规定时间后，将V形血凝板倾斜，使阴性对照孔红细胞挂线。然后观察其它实验孔，取红细胞完全不挂线的稀释度为该病毒的HA效价。
1.2病毒鉴定
用血凝抑制(HI)试验鉴定病毒亚型，具体方法如下：
(1)在做血凝抑制实验前先做血凝实验，确定当时待测病毒的HA效价。
(2)在96孔血凝板每孔中均加入25μL PBS。
(3)在96孔血凝板的第一列孔中分别加入25μL禽流感H1、H3、H4、H5、H6、H7、H9、H10、H11亚型病毒或新城疫病毒或减蛋综合征病毒标准阳性血清，从左至右倍比稀释至第10孔，弃掉25μL。第11孔为病毒阳性对照，第12孔为PBS阴性对照。
(4)在96孔血凝板的前11列孔中加入现配的4单位病毒，12列孔中补加25μL PBS，轻轻震荡混匀，将血凝板放置室温(25℃)40min，或4℃环境60min，使血清和抗原充分反应。
(5)之后每孔加入25μL 1％的鸡红细胞，震荡混匀放置在室温(25℃)。
(6)将血凝板倾斜，观察红细胞挂线情况。挂线说明血清和病毒充分反应，表示为阳性。读数到完全挂线的血清稀释度为该血清针对该病毒的血凝抑制价，即HI效价，效价为4以上记为有效，根据HI效价鉴定病毒。第11孔病毒阳性对照不挂线，第12孔PBS阴性对照挂线。
1.3鸡胚半数感染量(50％egg infectious dose，EID 50)测定
病毒尿囊液用四抗(青霉素、链霉素、卡那霉素及庆大霉素)PBS做连续10倍倍比稀释，取6个稀释度(10 -5～10 -10)接种10日龄SPF鸡胚，每个稀释度接种5个胚，0.2mL/胚。接种的鸡胚在35℃条件下培养，每12h照胚一次，直至72h，最后根据Reed-Muench方法计算EID 50。
1.4免疫血清制备和效价测定
(1)取JD/17病毒株以8000r/min离心10min后取上清测定病毒灭活前HA效价。
(2)病毒尿囊液与稀释好的1:50甲醛水溶液以43:7的比例混合均匀至4℃摇床放置，振摇灭活24h。
(3)取出灭活的病毒尿囊液，测定灭活后血凝效价(血凝效价>4log2时满足要求)。
(4)在灭活好的病毒尿囊液中以24:1比例加入吐温80，混匀后，以3:1比例将白油加入灭活病毒后乳化制备疫苗。
(5)用制成的疫苗颈部皮下注射3周龄SPF鸡，0.3mL/只，每组注射5只SPF鸡。
(6)免疫后，于14天和21天采集鸡血，分离血清，测定HI效价。
1.5分离和鉴定结果
通过病毒分离，HA和HI效价测定及病毒全基因组序列测定，从2017年的某鸡场中分离到了一株低致病性H7N9亚型禽流感毒株A/Chicken/Huadong/JD/17(H7N9)(JD/17)，8个片段的基因序列见补充材料。生物学特性测定结果发现(表1)，该毒株具有较高的HA效价和EID 50，且经灭活乳化后免疫鸡群产生的抗体水平较高，故适合作为疫苗候选株。
检测结果见表1。
表1 JD/17病毒株生物学特性测定
Figure PCTCN2019086620-appb-000001
实施例2
全基因序列测定
Trizol法提取JD/17病毒尿囊液总RNA，随后用反转录PCR(RT-PCR)分别扩增病毒的8个基因片段，扩增引物见表2。
表2 全基因组序列测定扩增引物
Figure PCTCN2019086620-appb-000002
Figure PCTCN2019086620-appb-000003
以上述8个基因片段的引物进行PCR扩增，配置25μL PCR体系:2.5μL 10×PCR缓冲液，0.5μLdNTP(10mM)，0.5μL25mM上游引物，0.5μL25mM下游引物，0.5μL高保真酶，2μLDNA模板和18.5μL超纯水。
PCR扩增程序：94℃预变性5min；94℃变性30s，54℃退火40s，72℃延伸1min 30s，35个循环；72℃延伸10min。
用1％浓度的琼脂糖对PCR产物进行电泳，电泳完毕后对目的条带进行回收，按DNA Gel ExtractionKit的说明书进行。对回收的PCR产物用分光光度计测定DNA浓度，当浓度达到≥50ng/μL符合测序要求时，将PCR回收产物连同引物送公司进行双向测序；若回收的PCR产物浓度较低，则将回收产物连接到T3EasyVector并转化至DH5α大肠杆菌感受态细胞，具体方法参见T3EasyVector说明书。在IPTG +、X-gal +和Amp +LB平板上挑取白色菌落，以常规方法小提质粒，质粒经EcoRI酶切鉴定 正确后，阳性质粒送公司测序，得到8个基因片段的核苷酸序列。
实施例3
不同HA亚型禽流感鸡免疫血清的制备
利用实验室现有的不同HA亚型的禽流感毒株，具体信息见表3。经灭活乳化后免疫3周龄SPF鸡(步骤同上)，制备不同HA亚型禽流感病毒的鸡免疫血清，用于H7亚型禽流感病毒的特异性抗原表位的筛选。
表3 制备免疫血清的不同HA亚型禽流感病毒信息
标号 病毒 亚型 血凝效价(HAlog2)
H1 A/Duck/Eastern China/103/03 H1N1 7
H3 A/Duck/Eastern China/852/03 H3N2 6
H4 A/Duck/Eastern China/160/02 H4N6 5
H5-1 A/Mallard/Huadong/S/2005 H5N1 7
H5-2 A/Chicken/Huadong/1111/16 H5N6 7
H5-3 A/Chicken/Huadong/ZJ0104/16 H5N2 6
H6 A/Duck/Eastern China/58/03 H6N2 7
H9-1 A/Chicken/Shanghai/F/98 H9N2 9
H9-2 A/Chicken/Fujian/SN/14 H9N2 6
H10 A/Chicken/Huadong/RD5/13 H10N9 6
H11 A/Duck/Eastern China/906/02 H11N2 7
H7-1 A/Chicken/Jiangsu/JT/13 H7N9 8
H7-2 A/Chicken/Jiangsu/JX05/14 H7N9 8
H7-3 A/Chicken/Jiangsu/W1-8/15 H7N9 7
H7-4 A/Chicken/Huadong/JD/17 H7N9 10
实施例4
多肽合成和多肽芯片制备
改性硅胶模(iPDMS)购自SJ Biomaterisls公司，多肽由GL Biochem(中国上海)公司合成。将H7N9亚型禽流感病毒JD/17株的HA2蛋白按照推导出的氨基酸序列合成重叠的多肽(每相邻2条肽之间重叠10个氨基酸)，点样在改性硅胶模上，总共合成了13条多肽(表4)(其中阳性质控点为羊抗鸡IgY；阴性质控点为点样缓冲液)，制备多肽芯片，微阵列点样信息见表5和图2。
表4 13条H7亚型HA2多肽及其序列
Peptides 序列编号 序列
H7-1 SEQ ID No.31 GLFGAIAGFIENGWEGLIDG
H7-2 SEQ ID No.32 ENGWEGLIDGWYGFRHQNAQ
H7-3 SEQ ID No.33 WYGFRHQNAQGEGTAADYKS
H7-4 SEQ ID No.34 GEGTAADYKSTQSAIDQITG
H7-6 SEQ ID No.35 KLNRLIAKTNQQFELIDNEF
H7-7 SEQ ID No.36 QQFELIDNEFNEVEKQIGNV
H7-8 SEQ ID No.37 NEVEKQIGNVINWTRDSITE
H7-9 SEQ ID No.38 INWTRDSITEVWSYNAELLV
H7-10 SEQ ID No.39 VWSYNAELLVAMENQHTIDL
H7-12 SEQ ID No.2 ADSEMDKLYERVKRQLRENA
H7-13 SEQ ID No.40 RVKRQLRENAEEDGTGCFEI
H7-14 SEQ ID No.41 EEDGTGCFEIFHKCDDDCMA
H7-15 SEQ ID No.42 FHKCDDDCMASIRNNTYDHR
表5 蛋白芯片点样信息
PC H7-1 H7-2 NC
H7-3 H7-4 H7-6 H7-7
H7-8 H7-9 H7-10 H7-12
H7-13 H7-14 H7-15 PC
实施例5
H7亚型禽流感病毒HA2蛋白特异性抗原表位的鉴定
利用实验2制备的不同亚型禽流感病毒血清抗体与多肽芯片进行结合筛选，获得仅能与H7N9亚型禽流感病毒抗体结合的抗原表位。具体操作步骤如下：
(1)首先将血清样品在血清稀释缓冲液中以1：100稀释，并且在每个微阵列中加入200μL，在振荡器上孵育2h(150r/min，4℃)。
(2)然后用TBST(20mM Tris-HCl，pH 6.8，137mM NaCl，0.1％Tween 20)冲洗微阵列三次，加入200μL 1:25000稀释的辣根过氧化物酶(HRP)标记的羊抗鸡IgY孵育1h，然后进行与上述相同的洗涤步骤。
(3)将15μL化学发光底物加入到微阵列中，并使用LAS4000成像系统(GE，USA)通过CCD相机捕获化学发光信号，用于获取微阵列的每个点的信号。
(4)最后将信号保存为TIFF格式的图像，然后使用GenePix Pro 6.0软件处理每个肽点的化学发光强度和635nm波长的背景值。将化学发光强度转换为信噪比(SNR)。SNR＝(信号强度-背景强度)/背景强度， 并将SNR≥2判断为阳性响应。
图3为各个HA亚型禽流感血清对H7-12肽的响应情况。结果显示，仅有H7亚型禽流感血清抗体对H7-12肽有特异性的阳性响应(SNR>2)，而其他HA亚型血清抗体对H7-12肽均呈阴性响应(SNR<2)(图3)(P<0.01)，表明H7-12肽为仅能与H7N9亚型禽流感血清抗体特异性结合的抗原表位；而各个HA亚型血清抗体对除了H7-12肽之外的多肽呈现出不同程度的响应(表6)。
实施例6
H7-12肽免疫血清制备和表位的验证
1.H7-12肽免疫血清制备
将筛选到的H7-12肽与BSA进行偶联，得到H7-12-BSA偶联物，将该偶联物制备免疫抗原并免疫SPF鸡制备多抗血清。免疫程序如下：
(1)初次免疫，取50μg多肽-BSA偶联物(溶于250μLPBS)，加入等量的弗氏完全佐剂，进行反复乳化，直至水乳相不再分层。在鸡的颈部皮下进行多点注射免疫。
(2)初次免疫3周后进行再次免疫，取同样剂量的多肽-BSA偶联物，加入等量的弗氏不完全佐剂进行反复乳化，在鸡的颈部皮下进行多点注射免疫。
(3)3周后采集免疫鸡血清。
2.间接免疫荧光试验
(1)在96孔板培养CEF细胞，待细胞长至80％后，弃去培养基，用PBS洗3次。
(2)用无抗无血DMEM将各个HA亚型的AIV(H1，H3，H4，H5，H6，H7，H9，H10)进行稀释，将稀释好的病毒加入细胞孔中，每孔100μL。
(3)同时设未接毒的正常CEF细胞作为空白对照。
(4)培养12h后，弃去培养基，用PBST洗涤3遍，每次5min，再用预冷的甲醇4℃固定15min；用PBST洗涤3次，每次5min，在吸水纸上拍干。
(5)将鸡血清进行1:1000倍稀释，加入96孔板中，200μL/孔，37℃作用1.5h。并加入H7单抗和阴性血清，分别作为阳性对照和阴性对照。
(6)用PBST洗涤3次，每次5min，避光加入1:500稀释的羊抗鸡FITC-IgG,50μL/孔，作用1h。
(7)用PBST洗涤3次，每次5min，在荧光显微镜下观察，出现特 异性的亮绿色荧光孔为阳性，反之为阴性。
结果：结果显示，该多肽-偶联物制备的鸡血清仅对H7亚型的禽流感出现了特异性荧光，对其他亚型病毒样品均未出现特异性荧光，表明该多肽表位为H7亚型禽流感病毒特异性抗原表位，结果见图4。
实施例7
HA2蛋白抗原表位修饰的H7N9亚型重组禽流感病毒构建
抗原表位替换
以筛选出的疫苗候选株JD/17为骨架，对其HA中HA2-12肽进行替换，将JD/17毒株的H7-12肽区域的氨基酸(ADSEMDKLYERVKRQLRENA)利用Overlap-PCR技术(引物见表7)替换为H3亚型的序列(ADSEMNKLFEKTKKQLRENA)，如图5所示。
表7 Overlap PCR引物
Figure PCTCN2019086620-appb-000004
以JD/17的cDNA为模板，PCR进行扩增目的片段。配置25μL体系:2.5μL 10×PCR缓冲液，0.5μLdNTP(10mM)，0.5μL 25mM上游引物，0.5μL 25mM下游引物，0.5μL高保真酶，2μL DNA模板和18.5μL超纯水。
PCR扩增程序如下：94℃预变性5min；94℃变性30s，54℃退火40s，72℃延伸1min 30s，35个循环；72℃延伸10min。
PCR产物HA-1和HA-2通过琼脂糖凝胶电泳鉴定，条带正确后，切胶并用胶回收试剂盒回收(步骤详见说明书)，测浓度和纯度(OD 260/OD 280为1.8～2.0)，然后以HA基因上下两段的胶回收产物为模板，进行Overlap-PCR，体系仍为25μL，除了DNA模板为4μL(上下段基因2μL)，超纯水减至16.5μL，其它和上述PCR体系中一样，扩增程序72℃延伸改为1min40s，其它和上述PCR扩增程序一样。
序列替换的HA基因片段与Blunt 3载体连接，得到的中间过渡质粒， 经测序验证序列正确后，用BsmBI酶进行酶切，得到的酶切的目的产物克隆至pHW2000载体，提取质粒；将提取的质粒与JD/17病毒株的其他7个基因的pHW2000载体构建的表达质粒进行转染。所述其他7个基因的种类包括PB2、PB1、PA、NP、NA、M和NS。
病毒拯救方法：转染前一天，将293T和MDCK细胞等量混合后铺入6孔细胞培养板(约6×10 5个细胞/孔)，待细胞覆盖面积达80％时进行转染。转染步骤参照Polyjet转染试剂说明书，每个转染体系中均包含8个片段的转录/表达质粒(300ng/质粒，HA片段为序列替换后的HA基因片段，其余为JD/17的7个片段)。转染后48～72h，反复冻融3次，收集转染上清，接种10日龄SPF鸡胚，0.3mL/胚。接种的鸡胚用血凝试验(HA)测定效价，验证病毒是否拯救成功。阳性鸡胚尿囊液提取病毒总RNA，PCR扩增8个片段进行测序，序列正确无误则将病毒尿囊液置于-70℃冰箱保存备用。
实施例8
利用反向遗传技术拯救重组DIVA疫苗候选株
(1)先将293T和MDCK细胞置于35mm培养皿中长至70～80％丰度。
(2)按照Polyfect操作指南进行转染，6h后吸弃转染液，并向培养皿中添加细胞维持液(含1％胎牛血清、2μg/mLTPCK-胰酶的DMEM培养基)。
(3)于转染后48h取出转染培养皿冻融3次后吹匀，取悬液接种10日龄SPF鸡胚，0.3mL/胚。
(4)接种72h后收集胚液并通过血凝试验(HA)检测病毒效价，若有效价初步判定为拯救成功，命名为cHAH7/H3。
(5)对重组的疫苗候选株经鸡胚传5代后，对病毒基因组进行测序分析，以验证重组病毒的基因遗传稳定性。
(6)组织培养物半数感染量(50％tissue culture infectious dose，TCID 50)
接种前一天将CEF细胞铺至96孔细胞培养板中，待细胞形成单层，吸弃培养上清并用无菌PBS洗涤3次，随后将连续10倍倍比稀释的病毒尿囊液接种至细胞表面，接种的稀释度为10 -4～10 -9，每个稀释度接种4个孔，0.1mL/孔。感染后的细胞在37℃，5％CO 2条件下继续培养，72h后用血凝试验统计阳性感染孔数，根据Reed-Muench方法计算TCID 50。
实施例9
DIVA疫苗候选株cHA H7/H3的生物学特性测定
对cHA H7/H3测定了HA效价、EID 50、TCID 50，结果显示拯救的疫苗候选株cHA H7/H3的生物学特性与母本毒株相似，经过替换后未改变其生物学特性(表8)。
表8.疫苗候选株及其母本毒株的生物学特性测定
Figure PCTCN2019086620-appb-000005
实施例10
重组疫苗候选株DIVA特性的验证
1.试验设计
(1)用获得的重组病毒免疫SPF鸡制备血清，同时用野生型毒株JD/17作为对照，血清制备方法步骤同上。
(2)在一次免疫后的21天采集免疫鸡血清。
(3)用H7-12肽制备的多肽芯片对制备的血清进行测定，方法步骤同上。
2.试验结果
结果显示，野生型毒株JD/17的免疫血清对H7-12肽有较高的阳性响应(6.39±0.13)，而疫苗候选株的免疫血清则均为阴性响应(0.44±0.14)，见图6。这表明该疫苗候选株已失去了H7-12这个抗原表位，DIVA策略成功。
实施例11
灭活标记疫苗的制备
(1)取疫苗候选株cHA H7/H3以8000r/min离心10min后取上清测定病毒灭活前HA效价。
(2)病毒尿囊液和体积浓度4％的甲醛溶液按43:7的比例混合均匀至4℃摇床放置，振摇灭活24h。
(3)取出灭活的病毒尿囊液，测定灭活后血凝效价(血凝效价>4log2时满足要求)。
(4)在灭活好的病毒尿囊液中以24:1比例加入吐温80，混匀后，以3:1比例将白油加入灭活病毒后乳化制备得到疫苗。
实施例12
攻毒保护试验
1.试验设计和免疫保护测定
(1)将21日龄SPF鸡随机分为7组，10只/组，其中4个免疫组，2个攻毒对照组和1组健康对照组。
(2)将母本毒株JD/17和疫苗候选株cHAH7/H3按相同EID 50剂量进行乳化，方法步骤同上。
(3)经颈部皮下以0.3mL/只的剂量注射油乳剂灭活疫苗。
(4)于免疫后的14天和21天采集鸡血清，测定HI效价。
(5)在免疫后21天，将10 6EID 50的低致病性毒株JD/17(H7N9)和高致病性毒株XT/17(H7N9)病毒经滴鼻点眼途径对免疫组和攻毒对照组进行攻毒。
(6)攻毒后每天观察记录各组鸡的发病和死亡情况，连续观察14天，计算各组鸡的存活率。
(7)在攻毒后的第1、3、5和7天采集所有试验鸡的泄殖腔和喉头拭子。
(8)棉拭样品经处理后接种2枚10日龄SPF鸡胚，测定各组鸡的排毒情况。
2.试验结果
HI试验结果显示(表9)，一次免疫后21天，针对低致病性H7N9亚型毒株JD/17，其HI效价可达8.8±0.4～9.2±0.6；针对高致病性H7N9亚型毒株XT/17，其HI效价可达4.5±1.3～5.2±0.7。在免疫后21天后攻毒，在攻毒的第2天，高致病性毒株XT/17攻毒对照组开始出现精神沉郁现象，并出现部分死亡；在攻毒后的第3天全部死亡。而低致病性毒株攻毒对照组和4组免疫组在攻毒后均未出现发病现象，且精神状态良好。
检测各组试验鸡在攻毒后的排毒情况(表9)，低致病性毒株攻毒对照组在第1天开始排毒，在第3天和第5天达到高峰；高致病性毒株攻毒对照组也在第1天开始排毒，在第3天全部死亡。cHAH7/H3免疫组在低致病性和高致病性H7N9亚型毒株攻毒后，在第1、3、5和7天，均未在喉头或泄殖腔棉拭中检测出病毒，保护率为100％；而母本免疫组毒株也仅在第1天和第3天分别检测出了对高致病性或低致病性AIV的排毒。
表9 免疫攻毒后各组鸡的排毒和存活情况
Figure PCTCN2019086620-appb-000006
Figure PCTCN2019086620-appb-000007
a针对毒本毒株的HI；b针对XT/17毒株的HI效价；C无存活。
以上结果表明，该疫苗候选株cHAH7/H3制备的灭活疫苗免疫保护效果不低于母本毒株制备的疫苗，且无论对强毒H7N9亚型AIV还是弱毒H7N9亚型AIV均有非常好的保护率。
实施例13
多肽芯片对自然感染鸡血清的检测
1.试验设计
(1)准备5只3周龄的SPF鸡，并做好脚标，同时设置3只SPF鸡作为对照组。
(2)测定野生型毒本毒株JD/17的EID 50，并用10 6EID 50的剂量以滴鼻点眼的方式对5只SPF鸡进行攻毒。
(3)在攻毒后的第3天、5天、7天、14天、21天和28天分别采集这5只SPF鸡和对照组的血清，置于-20℃保存备用。
(4)对自然感染的鸡血清进行血凝抑制试验(HI)，测定其HI效价。
(5)用H7-12肽制备的多肽芯片对自然感染的鸡血清进行检测，并计算SNR值。
2.试验结果
HI试验结果显示，在感染病毒后第7天，成功检测出HI效价(4.6±0.5)；多肽芯片结果显示，在感染病毒的第3天，测定的SNR值就表明已经出现阳性响应(SNR＝2.33±0.15)，随着感染时间增加，SNR值越来越大，具体结果见表10。
表10.HI试验和多肽芯片检测不同感染时间段的SPF鸡血清
Figure PCTCN2019086620-appb-000008
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Figure PCTCN2019086620-appb-000009
Figure PCTCN2019086620-appb-000010
Figure PCTCN2019086620-appb-000011
Figure PCTCN2019086620-appb-000012
Figure PCTCN2019086620-appb-000013
Figure PCTCN2019086620-appb-000014
Figure PCTCN2019086620-appb-000015
Figure PCTCN2019086620-appb-000016
Figure PCTCN2019086620-appb-000017
Figure PCTCN2019086620-appb-000018
Figure PCTCN2019086620-appb-000019
Figure PCTCN2019086620-appb-000020
Figure PCTCN2019086620-appb-000021
Figure PCTCN2019086620-appb-000022
Figure PCTCN2019086620-appb-000023
Figure PCTCN2019086620-appb-000024
Figure PCTCN2019086620-appb-000025
Figure PCTCN2019086620-appb-000026
Figure PCTCN2019086620-appb-000027

Claims (10)

  1. 一种重组H7N9亚型禽流感病毒株，其特征在于，以H7N9亚型禽流感病毒JD/17病毒株为母本病毒株，用H3亚型的氨基酸序列替换所述JD/17病毒株的HA蛋白中氨基酸序列；
    所述H3亚型的氨基酸序列见序列表中SEQ ID No.1；
    所述JD/17病毒株中的HA蛋白中氨基酸序列见序列表中SEQ ID No.2；
    所述H7N9亚型禽流感病毒JD/17病毒株的保藏编号为CCTCC No.V201862。
  2. 权利要求1所述的重组H7N9亚型禽流感病毒株的制备方法，其特征在于，包括以下步骤：
    (1)提取H7N9亚型禽流感病毒JD/17病毒株的总RNA，反转录，得到cDNA；
    (2)以所述cDNA为模板，用引物对KS-H7-1和JDH7H3-1-R扩增HA-1基因片段，用引物对JDH7H3-2-F和KS-H7-2扩增HA-2基因片段；
    所述KS-H7-1的核苷酸序列如序列表中SEQ ID No.3所示；
    所述JDH7H3-1-R的核苷酸序列如序列表中SEQ ID No.4所示；
    所述JDH7H3-2-F的核苷酸序列如序列表中SEQ ID No.5所示；
    所述KS-H7-2的核苷酸序列如序列表中SEQ ID No.6所示；
    (3)以步骤(2)得到的HA-1基因片段和HA-2的基因片段为模板进行重叠PCR扩增，得到序列替换的HA基因片段；
    (4)将所述步骤(3)中序列替换的HA基因片段与Blunt 3载体连接，得到的中间过渡质粒，经测序验证序列正确后，用BsmBI进行酶切，得到的酶切的目的产物克隆至pHW2000载体，提取质粒；将提取的质粒与JD/17病毒株的其他7个基因的pHW2000载体构建的表达质粒进行转染，拯救得到重组H7N9亚型禽流感病毒株。
  3. 根据权利要求2所述的制备方法，其特征在于，所述HA-1基因片段和HA-2基因片段的扩增程序独立为：
    94℃预变性5min；94℃变性30s，54℃退火40s，72℃延伸1min 30 s，35个循环；72℃延伸10min。
  4. 根据权利要求2或3所述的制备方法，其特征在于，所述HA-1基因片段和HA-2基因片段的扩增体系独立为：2.5μL 10×PCR缓冲液，0.5μL 10mM dNTP，0.5μL 25mM上游引物，0.5μL 25mM下游引物，0.5μL高保真酶，2μL DNA模板和18.5μL超纯水。
  5. 根据权利要求2所述的制备方法，其特征在于，所述重叠PCR扩增的程序为：
    94℃预变性5min；94℃变性30s，54℃退火40s，72℃延伸1min40s，35个循环；72℃延伸10min。
  6. 一种重组H7N9亚型禽流感病毒标记疫苗的制备方法，其特征在于，包括以下步骤：
    A.将权利要求1所述重组H7N9亚型禽流感病毒株或权利要求2～5任意一项所述制备方法制备的重组H7N9亚型禽流感病毒株接种SPF鸡胚，孵育，得到病毒尿囊液；
    B.将所述病毒尿囊液和甲醛溶液混合，得到的混合液转移4℃的条件下振摇灭活24h，得到灭活病毒尿囊液；
    C.当所述灭活病毒尿囊液的血凝效价>4log2时，将所述灭活病毒尿囊液依次与吐温80和白油混合，得到灭活病毒尿囊液混合物；
    D.将所述灭活病毒尿囊液混合物乳化，得到重组H7N9亚型禽流感病毒标记疫苗。
  7. 根据权利要求6所述的制备方法，其特征在于，所述甲醛溶液的体积浓度为4％。
  8. 根据权利要求6所述的制备方法，其特征在于，所述病毒尿囊液和甲醛溶液的体积比为43:7。
  9. 根据权利要求6所述的制备方法，其特征在于，所述灭活病毒尿囊液、吐温80和白油的体积比为24:1:75。
  10. 权利要求6～9任意一项所述制备方法制备的重组H7N9亚型禽流感病毒标记疫苗。
PCT/CN2019/086620 2018-11-05 2019-05-13 一种重组h7n9亚型禽流感病毒株、灭活标记疫苗及其制备方法 WO2020093674A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/960,222 US11376319B2 (en) 2018-11-05 2019-05-13 Recombinant H7N9 subtype avian influenza virus, inactivated marked vaccine and preparation method thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811308245.5A CN109402070B (zh) 2018-11-05 2018-11-05 一种重组h7n9亚型禽流感病毒株、灭活标记疫苗及其制备方法
CN201811308245.5 2018-11-05

Publications (1)

Publication Number Publication Date
WO2020093674A1 true WO2020093674A1 (zh) 2020-05-14

Family

ID=65471378

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/086620 WO2020093674A1 (zh) 2018-11-05 2019-05-13 一种重组h7n9亚型禽流感病毒株、灭活标记疫苗及其制备方法

Country Status (3)

Country Link
US (1) US11376319B2 (zh)
CN (1) CN109402070B (zh)
WO (1) WO2020093674A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113913394A (zh) * 2021-10-19 2022-01-11 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 人工重组的h5n6流感病毒及其制备方法和应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109402070B (zh) * 2018-11-05 2020-07-31 扬州大学 一种重组h7n9亚型禽流感病毒株、灭活标记疫苗及其制备方法
CN111154728A (zh) * 2020-01-13 2020-05-15 扬州大学 用于h7n9亚型禽流感病毒ha多肽竞争抑制elisa抗体检测的单克隆抗体及方法
CN113913396B (zh) * 2021-10-19 2022-06-07 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 人工重组的h7n9流感病毒及其制备方法和应用
CN114836390B (zh) * 2022-06-13 2023-05-02 华南农业大学 一株h9n2亚型禽流感病毒mdck细胞冷适应减毒活疫苗株及其应用
CN116063465B (zh) * 2022-08-09 2023-08-04 华南农业大学 针对h7亚型禽流感病毒的纳米抗体m111及其应用
CN117912568B (zh) * 2024-01-05 2024-08-06 中山大学 H9n2亚型禽流感毒株抗原性快速鉴别方法及装置

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107142280A (zh) * 2017-06-29 2017-09-08 扬州大学 一种表达h9亚型禽流感病毒ha基因的重组火鸡疱疹病毒株
CN107449912A (zh) * 2017-08-29 2017-12-08 扬州大学 抗h7n9亚型禽流感病毒单克隆抗体抗原表位及其筛选方法和应用
CN107765001A (zh) * 2017-10-24 2018-03-06 中国科学院苏州纳米技术与纳米仿生研究所 禽流感病毒感染或禽流感疫苗免疫分型试剂盒
CN109402070A (zh) * 2018-11-05 2019-03-01 扬州大学 一种重组h7n9亚型禽流感病毒株、灭活标记疫苗及其制备方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100571775C (zh) * 2006-11-09 2009-12-23 中国农业科学院哈尔滨兽医研究所 禽流感病毒标记疫苗及其制备方法和应用
US20180303928A1 (en) * 2017-03-07 2018-10-25 Sandra Newbury H7n2 influenza a virus
CN108018300B (zh) * 2017-11-21 2020-08-28 浙江迪福润丝生物科技有限公司 区分免疫和感染动物h7亚型禽流感疫苗株及其制备方法和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107142280A (zh) * 2017-06-29 2017-09-08 扬州大学 一种表达h9亚型禽流感病毒ha基因的重组火鸡疱疹病毒株
CN107449912A (zh) * 2017-08-29 2017-12-08 扬州大学 抗h7n9亚型禽流感病毒单克隆抗体抗原表位及其筛选方法和应用
CN107765001A (zh) * 2017-10-24 2018-03-06 中国科学院苏州纳米技术与纳米仿生研究所 禽流感病毒感染或禽流感疫苗免疫分型试剂盒
CN109402070A (zh) * 2018-11-05 2019-03-01 扬州大学 一种重组h7n9亚型禽流感病毒株、灭活标记疫苗及其制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SCHMEISSER F ET AL: "Antibodies to Antigenic Site A of Influenza H7 Hemaggluti- nin Provide Protection against H7N9 Challenge", PLOS ONE, vol. 10, no. 1, e0117108, 28 January 2015 (2015-01-28), pages 1 - 11, XP055706216, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0117108 *
WANG, Y ET AL: "A recombinant H7N9 influenza vaccine with the H7 hemagglutinin transmembrane domain replaced by the H3 domain induces increased cross-rea- ctive antibodies and improved interclade protection in mice", ANTIVIRAL RESEARCH, vol. 143, 31 July 2017 (2017-07-31), pages 97 - 105, XP085032218, ISSN: 0166-3542, DOI: 10.1016/j.antiviral.2017.03.029 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113913394A (zh) * 2021-10-19 2022-01-11 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 人工重组的h5n6流感病毒及其制备方法和应用
CN113913394B (zh) * 2021-10-19 2022-06-21 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 人工重组的h5n6流感病毒及其制备方法和应用

Also Published As

Publication number Publication date
US11376319B2 (en) 2022-07-05
CN109402070B (zh) 2020-07-31
US20210244810A1 (en) 2021-08-12
CN109402070A (zh) 2019-03-01

Similar Documents

Publication Publication Date Title
WO2020093674A1 (zh) 一种重组h7n9亚型禽流感病毒株、灭活标记疫苗及其制备方法
Kwon et al. Isolation and characterization of avian metapneumovirus from chickens in Korea
WO2019047608A1 (zh) 一种鸭坦布苏病毒e蛋白截短蛋白及应用
Lee et al. Immunoprophylactic effect of chicken egg yolk antibody (IgY) against a recombinant S1 domain of the porcine epidemic diarrhea virus spike protein in piglets
KR102336158B1 (ko) 돼지유행성설사병바이러스 및 돼지로타바이러스에 대한 백신 조성물
Gao et al. Serotype, antigenicity, and pathogenicity of a naturally recombinant TW I genotype infectious bronchitis coronavirus in China
JP2017212976A (ja) Ｉｂ−ｑｘ様株に由来する伝染性気管支炎ワクチン
CN111849923A (zh) 分泌抗犬瘟热病毒h蛋白单克隆抗体的杂交瘤细胞2d12株
CN110423269A (zh) 一种串联优势表位的重组猪圆环病毒2型Cap蛋白及其应用
WO2021098521A1 (zh) 一种快速制备流行性传染性支气管炎疫苗的方法
CN110872578A (zh) 一株变异株传染性法氏囊病毒、亚单位疫苗及其制备方法和应用
JP4953256B2 (ja) 鳥類免疫系によって認識されるｈｎ蛋白質のエピトープ及び前記エピトープ突然変異を含むニューカッスル病ウイルス
CN109289047A (zh) 一种通用型h5亚型禽流感亚单位疫苗及其制备方法
CN113817687B (zh) 一种杂交瘤细胞株、a型流感病毒核蛋白单克隆抗体及其应用
Jiang et al. Recombinant infectious bronchitis coronavirus H120 with the spike protein S1 gene of the nephropathogenic IBYZ strain remains attenuated but induces protective immunity
Xia et al. Evolution of prevalent H9N2 subtype of avian influenza virus during 2019 to 2022 for the development of a control strategy in China
CN104830811B (zh) H9n2亚型禽流感病毒ns1基因缺失减毒活疫苗候选株及其构建方法和应用
CN108348596A (zh) 用牛免疫缺陷病毒Gag蛋白的重组病毒样颗粒
CN108300702B (zh) 一株鸡源h9n2禽流感病毒冷适应株筛选方法及其应用
CN108640981A (zh) H7n9亚型流感病毒重组蛋白及病毒疫苗
CN102373184A (zh) 禽流感和传染性支气管炎混合病毒样颗粒、制备方法和应用
CN111647610B (zh) 一种互换ha和ns1缺失基因包装信号的h9n2亚型禽流感病毒及其构建方法和应用
KR102239927B1 (ko) 불활화된 소 로타바이러스를 함유하는 백신 조성물
CN102373181B (zh) 猪流感病毒和猪蓝耳病病毒混合病毒样颗粒、制备方法和应用
CN102370976B (zh) 猪流感病毒和猪口蹄疫病毒的混合病毒样颗粒、制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19882646

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19882646

Country of ref document: EP

Kind code of ref document: A1