WO2007128169A1 - SOUCHE DE FAIBLE VIRULENCE DU VACCIN RECOMBINANT LaSota DE LA MALADIE DE NEWCASTLE EXPRIMANT LA PROTÉINE HA DU VIRUS H5 DE LA GRIPPE AVIAIRE - Google Patents

SOUCHE DE FAIBLE VIRULENCE DU VACCIN RECOMBINANT LaSota DE LA MALADIE DE NEWCASTLE EXPRIMANT LA PROTÉINE HA DU VIRUS H5 DE LA GRIPPE AVIAIRE Download PDF

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WO2007128169A1
WO2007128169A1 PCT/CN2006/001626 CN2006001626W WO2007128169A1 WO 2007128169 A1 WO2007128169 A1 WO 2007128169A1 CN 2006001626 W CN2006001626 W CN 2006001626W WO 2007128169 A1 WO2007128169 A1 WO 2007128169A1
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newcastle disease
protein
vaccine strain
virus
avian influenza
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PCT/CN2006/001626
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French (fr)
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Zhigao Bu
Hualan Chen
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Zhigao Bu
Hualan Chen
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • 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
    • 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
    • C12N15/86—Viral vectors
    • 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
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18141—Use of virus, viral particle or viral elements as a vector
    • C12N2760/18143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to the field of recombinant viral vaccines, and more particularly to a recombinant Newcastle disease LaSota attenuated vaccine strain expressing a gene encoding a wild type or mutant avian influenza virus H5 subtype hemagglutinin (HA) protein, more specifically
  • the recombinant Newcastle disease LaSota attenuated vaccine strains were rL-QHwH5 and rL-QHmH5.
  • the invention also discloses a method for preparing the recombinant Newcastle disease LaSota attenuated vaccine strain and the application of the recombinant Newcastle disease LaSota attenuated vaccine strain in preparing a vaccine for preventing avian influenza. Background technique
  • Newcastle disease virus is a non-segmented single-stranded negative-strand RNA virus, which is an important member of the Paramyxoviridae family and a model virus.
  • Recombinant NDV has extraordinary advantages as a live virus vaccine vector: NDV attenuated vaccine including LaSota strain has been used for poultry epidemic prevention for a long time, and its safety and effectiveness have been fully proved; NDV inheritance is relatively stable, only one serotype, poison The possibility of recombination and virulence reintegration between plants is minimal; the replication process is completed in the cytoplasm, from RNA to RA, there is no possibility of DNA phase and cell genome integration; NDV attenuated vaccine can induce systemic humoral immunity, local The formation of mucosal immunity and cellular immunity forms a more comprehensive and accurate immune protection; it can be used in a variety of ways by drinking water, spraying, nasal drops, eye drops or injection; NDV has high titer of chicken embryo growth characteristics, Production costs are extremely low (1 ' 7 '
  • the reverse genetic operation of negative-strand RNA viruses is the process of making new viruses by manipulating viral genomic cDNA.
  • the basic process is: 1 assembling a complete viral genome (or recombinant genome) cDNA clone, 5, precisely at the end After being affixed to the T7 promoter, 3, the terminal is precisely fused to the self-cleaving nuclease sequence and the T7 transcription termination signal, constitutes a genomic cDNA transcription template; 2 is a genomic cDNA transcription template and a transcription-related functional structural protein necessary for initiating viral replication.
  • T7 polymerase such as nuclear protein (NP), phosphoprotein (P) and polymerase protein (L) expression plasmids (T7 promoter); 3 24-72 hours later harvest Culturing the supernatant, filtering and continuing the passage of sensitive cells or inoculation of the chicken embryoal allantoic cavity (rescue) virus.
  • NP nuclear protein
  • P phosphoprotein
  • L polymerase protein
  • T7 promoter 3 24-72 hours later harvest Culturing the supernatant, filtering and continuing the passage of sensitive cells or inoculation of the chicken embryoal allantoic cavity (rescue) virus.
  • the corresponding mutant or recombinant negative-strand RA virus (', 2 ' 3 ' 4 ') can be obtained by reverse genetic system (RGS system). 5 ' 6 ).
  • the NDV genome is 15186 nucleotides in length and, like other paramyxoviruses, includes nuclear protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), and lectin neuraminidase protein (HN). ), and a large polymerase protein (L) with six independent transcription coding units (Fig. 1A).
  • NP nuclear protein
  • P phosphoprotein
  • M matrix protein
  • F fusion protein
  • HN lectin neuraminidase protein
  • L large polymerase protein
  • Fig. 1A the IBDV VP2 protein was cloned between P and M to study the proper expression of foreign protein in NDV.
  • NDV has a minimal infectious unit of ribonucleoprotein complexes, and protein-free RNA itself is not infectious.
  • the genomic RNA of NDV forms a nuclear protein complex together with NP, P, L proteins, initiates the first round of transcription and translational synthesis of viral proteins, producing infectious progeny virus (7 ' 1Q) .
  • RGS system first reverse genetic system
  • Avian influenza is an important disease that jeopardizes the development of the world poultry industry. Highly pathogenic avian influenza can cause 100% of deaths in infected poultry and is classified as a Class A severe infectious disease by the OIE.
  • the H5 subtype has historically caused a highly pathogenic avian influenza outbreak. From the end of 2003 to the beginning of 2004, Asian countries such as South Korea, Japan, Vietnam, Thailand, Indonesia, Cambodia, Laos and mainland China have successively outbreaked H5 subtype highly pathogenic avian influenza, and existing avian influenza inactivated vaccines and live pox pox vaccines. It has the advantages of safe and immune protection, but it still has the disadvantages of high manufacturing cost and relatively inconvenient use. It is of great practical significance to develop a new generation of vaccines that are efficient, safe, low-cost and easy to use.
  • Avian Influenza is an avian infection and/or disease syndrome caused by Avian Influenza Virus (AIV).
  • AIV is taxonomically classified as: Viral (Vira)...-Orthomyxoviridae ( Orthomyxoviridae) - Influenza Virus A and B - Avian Influenza Virus.
  • the avian influenza virus belongs to the influenza A virus of the genus Influenza virus of the Orthomyxovirus family, and the genome consists of 8 single-stranded negative-strand RNA fragments.
  • the surface structural proteins hemagglutinin (HA) and neuraminidase (NA) are different in antigenicity and are classified into different subtypes.
  • Hemagglutinin is the main immunogenic protein of avian influenza virus, which induces the production of antibody-mediated specific humoral immune responses. Anti-HA antibodies can interfere with the binding of viruses to sialic acid receptors or viral envelopes. The process of fusion with the endocytic membrane thereby neutralizing the infection of the virus.
  • the virulence of AIV is closely related to the amino acid sequence of its surface structural protein HA cleavage site. The low virulence AIV HA cleavage site has only one basic amino acid arginine (R).
  • the HAO of the cleavage site containing a single basic amino acid arginine can be cleaved into active HA1 and HA2 to initiate the adsorption and replication cycle of the virus.
  • High pathogenicity H5 and H7 subtypes The AIV HA cleavage site contains a contiguous number of basic amino acid residues, RKKR, which can be recognized and cleaved by proteases widely present in various cells in the body, thus having a wide range of tissue tropism.
  • Hl, H2, H3 and H9 which are infecting humans
  • H5 and H7 subtypes AIV are potentially more harmful to humans because they may spread systemically once infected. And quickly died.
  • NDV B1 strain expressing H7 subtype HA gene is NDV and The survival protection of the H7 subtype highly pathogenic avian influenza lethal challenge is only 60% and 40%, respectively, and does not prevent the virus from replicating and discharging in the body.
  • the NDV genome inserts exogenous reporter genes or immunogenic genes at different sites, and maintains high genetic and expression stability through successive high passages of cells or chicken embryos.
  • the deficiency and defects of the live virus vector itself, and the cost of use, etc. it has not been widely applied in production. Summary of the invention
  • the present inventors further improved the immunogenicity of the avian influenza virus expressing antigen, and constructed a recombinant NDV live vector two attenuated vaccines rL-QHwH5 and rL-QHmH5 expressing the wild type and mutant avian influenza virus HA immunogen protein.
  • Animals are immunized by various methods such as nose drops, eye drops, intramuscular injection and even drinking water, spray inhalation, etc. to induce a protective immune response against avian influenza, and are used for the prevention of immunity against avian Newcastle disease and avian influenza.
  • a recombinant Newcastle disease LaSota attenuated vaccine strain which expresses a gene encoding a wild type or mutant avian influenza virus H5 subtype hemagglutinin (HA) protein.
  • the gene encoding the wild type HA protein has the nucleotide sequence set forth in SEQ ID No. 1.
  • the gene encoding the mutant HA protein has the nucleotide sequence shown in SEQ ID No. 2.
  • the Newcastle disease LaSota attenuated vaccine strain is AV1615.
  • the recombinant Newcastle disease LaSota attenuated vaccine strains are rL-QHwH5 and rL-QHmH5.
  • Still another object of the present invention is to provide a method for producing the above recombinant Newcastle disease LaSota attenuated vaccine strain, The method includes:
  • transcriptional helper plasmids comprising a cDNA sequence encoding the nuclear protein (NP) of the Newcastle disease LaSota attenuated vaccine strain, and a phosphoprotein (P) encoding the Newcastle disease LaSota attenuated vaccine strain a cDNA sequence, and a cDNA sequence encoding the large polymerase protein (L) of the Newcastle disease LaSota attenuated vaccine strain;
  • the gene of the HA protein was inserted into the artificially introduced Pmel site between the genomes P and M of the Newcastle disease LaSota attenuated vaccine strain.
  • the LaSota attenuated vaccine strain is AV1615.
  • the genomic cDNA sequence included in the transcription plasmid is located after the T7 promoter, and the genomic cDNA transcription is constituted before the sequence encoding the self-cleaving nuclease and the T7 transcription terminator. template.
  • the self-shearing nuclease is a hepatitis D virus ribozyme (Rib).
  • the cDNA sequence encoding the nuclear protein (NP) of the Newcastle disease LaSota attenuated vaccine strain in the transcriptional helper plasmid, and the phosphoric acid encoding the Newcastle disease LaSota attenuated vaccine strain are included.
  • the cDNA sequence of the protein (P) and the cDNA sequence of the large polymerase protein (L) encoding the Newcastle disease LaSota attenuated vaccine strain are located after the T7 promoter.
  • the transcription plasmid is pBRN-FL-QHwH5 or pBR-FL-QHmH5, and the transcriptional helper plasmid is plasmid pBSNP, pBSP and pBSLc.
  • the host cell is BHK-21.
  • the present invention also provides the use of the above-mentioned recombinant Newcastle disease LaSota attenuated vaccine strain (especially rL-QHwH5 and rL-QHmH5) for preparing a vaccine for preventing avian influenza.
  • NP genomic protein
  • P phosphoprotein
  • L large polymerase protein
  • NDV has potential as a vaccine live vector, laying a foundation for the development of a new avian influenza vaccine.
  • the above recombinant NDV can be used not only as a bivalent attenuated vaccine against the H5 subtype highly pathogenic avian influenza and Newcastle disease and avian influenza, but also as a second inactivated vaccine strain, and does not interfere with the current widespread Applied avian influenza epidemiological serological surveillance.
  • HA as an RNA viral envelope protein disease, may be involved in the surface of the viral envelope of recombinant NDV, and may play a role in cell invasion and cell invasion.
  • the present invention manually deletes four consecutive amino acids (-RKKR-) at the cleavage site by PCR, and mutates another amino acid to form a mutant low pathogenic form of the H5 subtype HA gene (QHmH5 gene). , SEQ ID No. 2), for construction of a recombinant Newcastle disease LaSota bivalent vaccine strain expressing the H5 subtype avian influenza virus HA antigen.
  • FIG. 1 Subgenome overlapping cDNA fragments generated from high fidelity RT-PCR assembled with full-length NDV cDNA.
  • the cDNA fragments were ligated at a consensus restriction site and assembled in the transcription plasmid pBR322, and the RBZ and T7 terminator sequences were pre-cloned between the Eco?/ and ra// sites in the transcription plasmid pBR322 (see instructions). .
  • (A) shows the first and last nucleotides of the entire full-length genome of the parental NDV.
  • B A cDNA clone of NDV containing the wild type or mutant HA gene is displayed at the top, and the horizontal line below the genetic map shows the position of a single cDNA.
  • FIG. 3A and D Recombinant Newcastle disease virus rL-QHwH5 and rL-QHmH5 expression Immunofluorescence analysis of H5 subtype HA antigen.
  • FIG. 3B and E rL-QHwH5 infected BHK-21 cells with MOI of 1
  • FIG. 3C and F NDV
  • LaSota parent strain AV1615 BHK-21 cells were infected with MOI of 1 and infected BHK cells were fixed in methanol 20 hours after infection, respectively, with chicken anti-H5 subtype avian influenza virus high serum ( Figures 3A, B and C) and chicken anti-Newcastle disease virus.
  • High-free serum (Fig. 3D, E and F) was indirect immunofluorescence detection of primary antibody, FITC-conjugated rabbit anti-chicken IgG as secondary antibody, and cells were observed under Leica DMIRES2 fluorescence microscope. The results showed that both wild-type and mutant H5 subtype HA antigens were correctly expressed in the recombinant Newcastle disease LaSota attenuated vaccine strain. - Figure 4. Comparison of kinetics of chicken embryo growth in recombinant Newcastle disease virus live vector vaccine.
  • FIG. 1 Recombinant Newcastle disease virus rL-QHwH5 and rL-QHmH5 immunization SPF chicks H5 avian influenza virus specific HI antibody response.
  • H5 subtype avian influenza Newcastle disease virus live vector bivalent vaccine (rL-QHwH5 and rL-QHmH5) immunization 1 week old SPF chicks induced H5 subtype avian influenza specific HI antibody immune response.
  • the vaccine was immunized with 7-day-old chicks at a dose of 100 ⁇ each.
  • FIG. 6 Recombinant Newcastle disease virus rL-QHwH5 and rL-QHmH5 immunization SPF chick Newcastle disease virus specific HI antibody response.
  • H5 subtype avian influenza Newcastle disease virus live vector bivalent vaccine (rL-QHwH5 and rL-QHmH5) immunized 1 week ⁇ SPF chicks induced Newcastle disease specific HI antibody immune response.
  • the recombinant Newcastle disease virus was immunized with 7-day-old chicks by intranasal and eye-dropping methods, each of which was ⁇ volume. After 19 days (26 days of age), sputum serum was collected for NDV and ⁇ 5 subtype AIV specific HI antibody detection.
  • Figure 8 DNA sequence of the pBTRT plasmid.
  • the first italic part T7 promoter; underlined part: ribozyme sequence; second underlined italic part: T7 terminator.
  • FIG. 1 DNA sequence of the P BRN-FL-QHwH5 plasmid. Underlined italic bold portion: QHwH5 gene sequence (SEQ ID No. 1).
  • Figure 13 AV1615-based gene sequence, in which 122 to 1591 bp is the coding sequence of the gene NP, The 1887 to 3074 bp is the coding sequence of the gene P, and the 8381 to 14995 bp is the coding sequence of the gene L.
  • Figure 14 Sequence of plasmid pBSNP, the underlined italic portion is the coding sequence for the NP gene.
  • Example 1 Recombinant Newcastle disease expressing wild-type or mutant avian influenza virus H5 subtype hemagglutinin (HA) protein
  • BHK-21 cells milk hamster kidney cell ATCC CCL-10
  • medium is DMEM containing 10% fetal bovine serum (Hyclone) and lg/ml G418 (Dulbecco's modified Eagle's medium); NDV Lasota vaccine strain AV1615 (purchased From China Veterinary Microbial Culture Collection Management Center (CVCC).
  • CVCC Veterinary Microbial Culture Collection Management Center
  • H5 subtype highly pathogenic avian influenza virus HPAIV
  • H5N1 H5 subtype highly pathogenic avian influenza virus
  • H5N1 H5 subtype highly pathogenic avian influenza virus
  • H5N1 H5 subtype highly pathogenic avian influenza virus
  • H5N1 H5 subtype highly pathogenic avian influenza virus
  • H5N1 H5 subtype highly pathogenic avian influenza virus
  • H5N1 H5 subtype highly pathogenic avian influenza virus
  • H5N1 H5N1
  • H5N1 H5 subtype avian influenza virus strain QH/05
  • GD/96 reverse-genetic operation rescued wild-type Newcastle disease virus LaSota
  • the vaccine rLaSota was purchased from the Harbin Veterinary Research Institute. Construction of transcription vectors
  • the genomic RNA transcription vector pBTRT was constructed with the low-copy cloning vector pBR322 (Invitrogen) and inserted into the T7 promoter (T7 promotor), the hepatitis D virus ribozyme (Rib) and the T7 transcription termination signal (T7 terminal ) at the EcoRIAra/I site.
  • T7 promotor T7 promoter
  • Rib hepatitis D virus ribozyme
  • T7 terminal T7 transcription termination signal
  • the full-length cDNA clone of the corresponding genome must be constructed first as the genomic negative-strand RNA.
  • the transcription template was used to construct ten cDNA clones covering the entire genome. Using the cleavage site of the overlapping portions of each fragment, a complete cDNA clone of 15186 nt was obtained by ligation of the low-copy plasmid transcription vector plasmid pBTRT.
  • the 6178th base of the F protein coding region in the genomic cDNA was synonymously changed from T to C by the PCR genome and used as a molecular marker for rescue of the virus.
  • Genomic RNA was extracted by conventional method (animal virology, second edition); the whole gene component was RT-PCR amplified by 10 fragments (F1-F10) with overlapping ends.
  • the cDNA fragment was cloned into the pBluescript (Clontech) Smal site and confirmed by sequence analysis to be identical to the viral genomic RNA sequence; the sequence assay results were registered in GenBank under accession number AY845400.
  • the methylation of the genomic cDNA of Lasota vaccine strain was selected at 6172 bp; the ⁇ site, the sequence was TCTAGATCA, which was mutated to TCTAGACCA by PCR, so that it was no longer methylated.
  • the enzyme is recognized and thus recognized by the restriction enzymes «1; the restriction enzyme cleavage sites present in overlapping portions of adjacent fragments are ligated into assembled NDV genomic cDNAs (Fig. 1A), and H5 subtypes are respectively Wild-type and mutant HA genes of influenza virus QHwH5 and QHmH5 genes (QHwH5: The avian influenza virus genome was proposed by Trizol (Invitrogen). After reverse transcription, the gene was amplified by PCR. The following primers were added to the system.
  • IB containing the wild type and mutant HA genes QHwH5 and QHmH5 of H5 subtype avian influenza virus were constructed on the vector pBTRT;
  • ORP open reading frame
  • cDNAs of (NP), phosphoprotein (P) and large polymerase protein (L) genes were cloned next to the Sma I site downstream of the pBluescript ll SK (+/-) plasmid T7 promoter, respectively.
  • the transcriptional helper plasmid pBSNP is constructed.
  • BHK-21 cells were first co-transfected with pBRN-FL-QHwH5 and pBRN-FL-QHmH5 and a helper plasmid expressing NDV NP, P, L proteins, respectively.
  • the fusion protein F0 of NDV must be lysed into F1 and F2 to be infectious.
  • BHK-21 cells can not secrete the trypsin required for cleavage of F0 protein, so the medium should be replaced with serum-free culture at this time.
  • the trypsin (Sigma, Cat# T8802 ) ( ⁇ g / ml ) treated with TPCK (toluenesulfonylalanyl chloromethanone) was added and culture was continued for 2-3 days. The supernatant of the transfected cells was harvested and inoculated into 9- 1 1 day old SPF chicken embryo. After 4 days, the chicken embryo allantoic fluid was harvested, and the blood coagulation (HA) test results were positive. The HA value of different chicken embryos was between 28 and u; the NDV immune serum hemagglutination inhibition (HI) test also showed positive results.
  • TPCK toluenesulfonylalanyl chloromethanone
  • Viral positive allantoic fluid was harvested as the F1 generation of the rescued viruses rL-QHwH5 and rL-QHmH5. Further RT-PCR and sequence analysis showed that the 6178 site of the F1 generation rescued the viral genomic cDNA was C, but the T of the original LaSota parental strain was exactly the same as expected (Fig. 2). The results showed that the infective progeny viruses rL-QHwH5 and rL-QHmH5 were successfully rescued by NDV LaSota vaccine strain genomic cDNA clone by reverse genetic manipulation technique.
  • the experimental procedure is as follows: BHK-21 cells are seeded in a 35 mm six-well plate to grow 50-80% monolayer, and the transcription plasmid and helper plasmid pBRN-FL-QHwH5 or pBRN-FL-QHmH5, pBSNP, pBSP BHK-21 cells were co-transfected with pBSL at 5 g, 2.5 ⁇ ⁇ , 1.25 ⁇ ⁇ 1.25 ⁇ ⁇ , respectively, and the CaP0 4 transfection kit (Invitrogene) was used, and the operation was carried out according to the kit instructions.
  • the CaP0 4 transfection kit Invitrogene
  • the transfection mixture was discarded, cells were shocked with PBS containing 10% DMSO for 2.5 minutes, added to complete DMEM overnight, replaced with serum-free medium the next day, and added with TPCK (1 ⁇ ⁇ /ml) After continuing to incubate for 2-3 days, the culture supernatant was harvested, filtered through a 0.22 um pore filter, and inoculated with SP-1 embryoid sac cavity for 9-1 1 day; the SPF embryo after inoculation was further cultured, 3-5 days, and the chicken was taken.
  • Embryonic vesicle fluid 50 ⁇ 1 Newcastle disease virus hemagglutination (HA) and hemagglutination inhibition (HI) tests (Thayer SG, Nersessian BN, Rivetz B, Fletcher OJ. Comparison of serological tests for antibodies against Newcastle disease virus and infectious bronchitis virus using ImmunoComb solid -phase immunoassay, a commercial enzyme-linked immunosorbent assay, and the hemagglutination-inhibition assay. Avian Dis. 1987 Jul-Sep; 31(3): 459-63.
  • the HA and HI test results were positive for allantoic fluid, frozen at -70 °C, and the EID 50 and PFU virus content per ml (1Q) were titrated on 9-10 days of chicken embryo and chicken embryo fibroblasts according to conventional methods. Named rL-QHwH5 and rL-QHmH5, respectively.
  • Example 2 Recombinant NDV expression AVI HA protein indirect immunofluorescence assay (IFA) test
  • the NDV LaSota vaccine strain transiently infects mammalian cells cultured in vitro.
  • both of the allantoic sacs infected approximately 70-80% of monolayer BHK-21 cells with a MOI of 1 virus 3A and B
  • the NDV wild-type LaSota vaccine strain was used as a control (Fig. 3C)
  • early CPE cytopathic
  • NDV high-free SPF chicken positive serum was used as the detection antibody.
  • Indirect immunofluorescence staining showed that strong positive reactions were observed under fluorescence microscopy of the three virus-infected cells (Fig. 3A, B and C). More specifically, the experimental steps were as follows:
  • rL-QHwH5 and rL-QHmH5 infected cells After washing with PBST and fluorescence microscopy (Leica DMIRES2), rL-QHwH5 and rL-QHmH5 infected cells all showed strong positive reaction, while wild type Lasota infected cells were completely negative.
  • Example 3 Growth characteristics and pathogenic characteristics of rNDV in chicken embryos To determine the growth characteristics of chicken embryos of rL-QHwH5 and rL-QHmH5 rescued by reverse genetic manipulation and their effects on chicken embryos Pathogenicity, will be rescued virus chicken embryo expansion F1 generation according to lxlO 4 EID 5 () inoculated 9 to 10 days old SPF chicken embryo chorioallantoic cavity.
  • Newcastle disease virus LaSota vaccine strain (rLaSota, which was rescued by reverse genetic manipulation, using the transcription vector pBTRT and the transcriptional helper plasmids pBSNP, pBSP and pBSL, was rescued by the reverse genetic manipulation as described in the present invention.
  • Newcastle disease virus LaSota vaccine strain AV161 120 hours completely lethal SPF chicken embryos 24 hours after inoculation 48 hours, 72 hours and allantoic fluid was harvested 96 hours, per milliliter of allantoic fluid is EID 5Q respectively 10_ 8.5 , 1 0 -8'6, I 0 -IG'G and ⁇ 0 -9 ⁇ 4.
  • RL _Q HWH5 and R L-QHmH5 were dosed in the same dose route for 9 to 10 days old SPF chicken 'embryonic oocysts, and SPF chicken embryos were also not killed within 120 hours; rL-QHwH5 24 hours, 48 hours, 72 hours and 96 after inoculation h allantoic fluid was harvested, per milliliter of allantoic fluid EID 5Q respectively to 2 ⁇ 10-8, 2.8 ⁇ 10 6, 10 'and 10 9 8.5.
  • the allantoic fluid was harvested at 24 hours, 48 hours, 72 hours, and 96 hours after rL-QHmH5 inoculation, and the EID 5 o per milliliter of allantoic fluid was 10' 7 ⁇ 9 , 10- 8 - 5 , 10 - 92 , and 10 , respectively . — 8 6 .
  • the results in Figure 4 indicate that the chick embryo growth kinetics of the reverse genetic manipulation rescue virus rL-QHwH5 and rL-QHmH5 are similar to those of the wild strain NDV Lasota vaccine strain (rLaSota), and still maintain a high drop of the NDV LaSota vaccine parent strain in the chicken embryo. Degree of growth and low lethal biological characteristics.
  • rLaSota wild-type Lasota vaccine strain
  • IVPI intravenous pathogenicity index
  • MDT average embryonic lethal time
  • the chicken embryo amplifies the F1 generation of allantoic acid 2xl0 6 EID 5Q dose by eye drops.
  • 12 artificially immunized 12-day-old white henhen SPF chicks (provided by SPF Experimental Animal Center of Harbin Veterinary Research Institute), and another 8 non-immune control group; the immunized group and the non-immune control group were separately incubated in air negative pressure filtration.
  • the blood samples from the wing veins were routinely tested for specific hemagglutination inhibitory antibodies against Newcastle disease and H5 subtype avian influenza.
  • FIG. 5 shows a representative set of data for the recombinant antibody rL-QHwH5 and rL-QHmH5 viruses to induce a protective antibody immunoreactivity to H5 subtype AIV.
  • Figure 6 shows a representative set of data for the recombinant antibody rL-QHwH5 and rL-QHmH5 viruses to induce a protective antibody immunoreactivity to NDV.
  • the above data indicate that both the recombinant viruses rL-QHwH5 and rL-QHniH5 can simultaneously induce protective antibody immunoreactivity to NDV and AIV.
  • the vaccine is immunized with 7-day-old chicks by intranasal and eye-dropping, with a dose of 2 X 10 6 EID 5Q per dose of ⁇ ⁇ ; ** 21 days after immunization (28 days old), NDV virulent F48E9 strain 10 4 ELD 5Q dose was used for intramuscular injection and continued for 21 days.
  • the two H5 subtypes of avian influenza Newcastle disease virus live vector bivalent vaccine, rL-QHwH5 and rL-QHmH5 viruses were immunized against 1-week-old SPF chicks to assess their lethality against H5 subtype highly pathogenic avian influenza. Immune protection. The results are shown in Table 3.
  • H5 subtype avian influenza Newcastle disease virus live vector bivalent vaccine rL-QHwH5 and rL-QHmH5 immunization 1 week ⁇ SPF chicks immune protection against homologous and heterologous H5 subtype ⁇ pathogenic avian lethal challenge
  • the vaccine is immunized with 7-day-old chicks by intranasal and eye-dropping methods, with a dose of 2x10 6 EID 5Q per dose, a total volume of ⁇ ⁇ ;
  • Newcastle disease virus LaSota attenuated vaccine which has been widely used in China for many years and has been proven to have good immune effect as a parent strain.
  • the P gene and M gene in the genome were selected.
  • the non-coding region is a foreign gene insertion site, and a HA immunogen that expresses the wild type HA immunogen of H5 subtype highly pathogenic avian influenza virus and a mutant HA immunogen with artificial deletion cleavage site and multiple basic amino acids are constructed.
  • the recombinant NDV strains, rL-QHwH5 and rL-QHmH5, were used as bivalent attenuated vaccine candidates for the prevention of Newcastle disease and H5 subtype highly pathogenic avian influenza, and biosafety assessments were conducted. Studies have shown that the NDV genome inserts exogenous reporter genes or immunogenic genes at different sites, and the biological characteristics, low pathogenicity and genetic stability of the genome are maintained by successive passages of cells or chicken embryos.
  • the immunoassay results of recombinant viruses rL-QHwH5 and rL-QHmH5 showed that the rL-QHmH5 and rL-QHmH5 recombinant vaccines can form a 100% killing attack on H5 subtype highly pathogenic avian influenza and Newcastle disease virulent immunity.
  • Fully immunoprotective, the ability to induce protective antibodies is comparable to existing inactivated vaccines, and has an advantage in inducing important mucosal and cellular immunity; maintaining a safe, effective, high titer of the parental LaSota vaccine strain on newborn chicks
  • the advantages of chicken embryo growth characteristics, ease of use, etc.; environmental and social benefits are significant, compared with the traditional avian influenza vaccine, the same dose of vaccine production is only one percent of the chicken embryo, the product volume is only one thousandth, and the production
  • the use of mineral oil is not required, and the effect of traditional oil emulsion inactivated vaccine injection on immunization against commercial chickens is completely avoided.
  • Newcastle disease virus as a live virus vector has great advantages in constructing a double-valent vaccine against avian influenza and Newcastle disease.
  • Avian influenza and Newcastle disease have been classified as Class A severe infectious diseases by the OIE. They are an important disease that jeopardizes the development of the world poultry industry. Avian influenza also has extremely important public health significance.
  • the attenuated vaccine used in Newcastle disease prevention in China is at least 10 billion feathers per year.
  • the application of NDV attenuated vaccine, especially LaSota vaccine strain is an indispensable immunization program for all newborn chicks in China.
  • Newcastle disease virus (NDV) is a non-segmented single-stranded RA virus with clear genomic structure and functional background. Only one serotype is genetically stable.
  • the foreign gene inserted in recombinant NDV is degenerated in cells or chicken embryos. Stable expression can still be maintained after sub-passage, and is very suitable as an expression or vaccine vector.
  • the safety and efficacy of the NDV attenuated LaSota vaccine strain has been fully proved; the live vaccination vaccine can induce the formation of systemic humoral immunity, local mucosal immunity and cellular immunity, and form a more comprehensive and accurate immune protection; , spray, nose, eye or injection in a variety of ways to the seedlings, the use of extremely convenient; NDV has a high titer of chicken embryo growth characteristics, production costs are extremely low.
  • the application of the rL-QHmH5 recombinant vaccine will make the H5 subtype highly pathogenic avian influenza vaccine control almost no additional cost of manufacturing and use.
  • the country can save at least tens of millions of dollars of epidemic prevention funds and a large number of society every year. Labor costs, and reduce the adverse stress response of immunized subjects.
  • Available data indicate that the vaccine is linked to the existing new city Compared with the avian influenza vaccine, the disease has great social, economic and environmental benefits, and the domestic and international markets have broad prospects.
  • Newcastle disease virus as a vaccine carrier is an internationally advanced new technology. If the progress is further accelerated, the H5 subtype avian influenza Newcastle disease virus live vector vaccine will be expected to become the first live carrier negative carrier RNA virus vaccine in the world.

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Description

表达禽流感病毒 H5亚型 HA蛋白的重组新城疫 LaSota弱毒疫苗株
技术领域
本发明涉及重组病毒疫苗领域， 更具体地， 本发明涉及一种表达编码野生型或突 变型禽流感病毒 H5亚型血凝素 （HA) 蛋白的基因的重组新城疫 LaSota弱毒疫苗株， 更具体地， 重组新城疫 LaSota弱毒疫苗株是 rL-QHwH5和 rL-QHmH5。本发明还公开 了制备所述重组新城疫 LaSota弱毒疫苗株的方法和该重组新城疫 LaSota弱毒疫苗株 在制备预防禽流感的疫苗中的应用。 背景技术
新城疫病毒（Newcastle disease virus, DV) 为不分节段单股负链 RNA病毒， 作 为副粘病毒科的重要成员和模型病毒， 得到深入研究。 重组 NDV 作为活病毒疫苗载 体具有非凡的优点：包括 LaSota株在内的 NDV弱毒疫苗长期以来一直用于家禽防疫， 其安全有效性已被充分证明； NDV遗传相对稳定， 仅有一个血清型， 毒株间发生重组 及毒力返强可能性极小； 复制过程在细胞浆内完成， 从 RNA到 R A， 不存在 DNA阶 段及细胞基因组整合的可能； NDV弱毒疫苗可同时诱导全身性体液免疫、局部粘膜免 疫及细胞免疫的形成， 形成更加全面、 确实的免疫保护； 可通过饮水、 喷雾、 滴鼻、 点眼或注射多种方式给苗， 使用极为方便； NDV具有高滴度的鸡胚生长特性， 生产成 本极为低廉 (1'7'8)。 NDV为高度传染性和高度致死性的家禽疫病原， 我国每年用于新城 疫防制的弱毒疫苗至少在百亿羽份以上。 NDV作为活病毒疫苗载体应用的经济意义十 分巨大。
负链 RNA病毒的反向遗传操作 （Reverse genetic) 是通过操作病毒基因组 cDNA 制造新病毒的过程，其基本过程是：①组装完整的病毒基因组（或重组型基因组） cDNA 克隆， 5，末端精确地缀于 T7启动子后， 3，末端精确缀于自我剪切的核酸酶序列和 T7 转录终止信号之前， 构成基因组 cDNA转录模板； ②以基因组 cDNA转录模板与启动 病毒复制必须的转录相关功能结构蛋白如核蛋白 （NP)、 磷酸蛋白 （P)和聚合酶蛋白 (L)的表达质粒（T7启动子）一起，共转染整合表达 T7聚合酶的病毒复制许可细胞； ③ 24-72 小时后收获培养上清， 过滤后继续敏感细胞传代或接种鸡胚尿囊腔救获 (rescue) 病毒。 对基因组 cDNA 进行突变、 缺失或外源基因插入修饰后， 通过反向 遗传操作系统 （reverse genetic system, RGS 系统） 可获得相应的突变或重组的负链 R A病毒 ('，2'3'4'5'6)。
NDV基因组全长 15186核苷酸， 与其它副粘病毒一样， 包括核蛋白 （NP) ， 磷 蛋白 （P) ， 基质蛋白 （M) ， 融合蛋白 （F) ， 凝集素神经氨酸酶蛋白 （HN) ， 和大 聚合酶蛋白 （L) 六个独立转录编码单元 （图 1A) 。 本研究将 IBDV VP2蛋白克隆到 P和 M之间， 来研究外源蛋白在 NDV中的表达适当位置。 NDV和其它负链 RNA病 毒一样， 其最小感染单位是核糖核蛋白复合物， 无蛋白包裹的 RNA本身并无感染性。 NDV的基因组 RNA通过与 NP、 P、 L蛋白一起组成核蛋白复合体， 启动 的首轮 转录及病毒蛋白的翻译合成、 产生感染性子代病毒 (7'1Q)。 根据这一原理， 1999 年欧洲 学者率先建立了第一个高致病性 NDV的反向遗传操作系统 （reverse genetic system, RGS系统）（2)。 目前在欧、 美至少有 4个实验室利用 NDV的 RGS技术在基础与应用 研究方面开展激烈的竞争性研究。
禽流感是危害世界养禽业发展的重要疾病，高致病力禽流感可导致感染禽群 100% 的死亡， 被国际兽疫局列为 A类烈性传染病。 H5亚型历史上引起高致病力禽流感暴 发。 2003年末至 2004年初， 韩国、 日本、 越南、 泰国、 印尼、 柬埔寨、 老挝以及中 国大陆等亚洲国家相继暴发 H5 亚型高致病力禽流感， 现有禽流感灭活疫苗和禽痘活 载体疫苗具安全、 免疫保护效果好的优点， 但仍存在着制造成本高、 使用相对不便的 不足。 研制高效安全、 成本低廉、 使用方便的新一代疫苗具有重要的现实意义。
禽流感 （Avian Influenza, AI) 是由禽流感病毒 （Avian Influenza Virus, AIV) 引 起的禽类感染和 /或疾病综合征， AIV在分类学上属于： 病毒界 （Vira)…-正粘病毒科 (Orthomyxoviridae) ——流感病毒属 （ Influenza Virus A and B ) ——禽流感病毒 (Avian Influenza Virus )。禽流感病毒属于正粘病毒科流感病毒属的 A型流感病毒，基因组由 8 个单股负链 RNA片段组成。 其表面结构蛋白血凝素（HA)和神经氨酸酶 （NA)抗原 性不同， 被划分为不同亚型。 血凝素 （HA)是禽流感病毒主要的免疫原蛋白， 它可诱 导机体产生抗体介导的特异性体液免疫应答， 抗 HA的抗体可以通过干扰病毒与唾液 酸受体的结合或者病毒囊膜与内吞体膜的融合过程从而中和病毒的感染。 AIV的致病 力与其表面结构蛋白 HA裂解位点的氨基酸序列密切相关。 低致病力 AIV的 HA裂解 位点只有一个碱性氨基酸精氨酸 （R)， 这一结构决定了这些病毒感染动物后只能在呼 吸系统内繁殖，因为只有呼吸道上皮细胞内含有一种精氨酸特异的类似胰酶的蛋白酶， 可将裂解位点含单一碱性氨基酸精氨酸的 HAO裂解为有活性的 HA1和 HA2, 启动病 毒的吸附和复制周期。高致病力 H5和 H7亚型 AIV HA裂解位点含有连续的多个碱性 氨基酸残基一 RKKR—， 可被体内多种细胞内广泛存在的蛋白酶识别和裂解， 因此具 有广泛的组织嗜性，一旦感染便会造成全身性扩散并导致迅速死亡。与可感染人的 Hl、 H2、 H3及 H9等亚型流感病毒相比， 高致病性的 H5和 H7亚型 AIV对人类的潜在危 害更为巨大， 因为一旦感染人后即可能全身性扩散和迅速致死。
2001-2002年， Palese. P.等相继构建表达 HI亚型流感病毒 HA免疫原基因的重组 NDV B1株和表达 H7亚型流感病毒 HA免疫原基因的重组 NDVB1株， 免疫试验表明 这两种 NDV活载体疫苗可分别在小鼠和禽类诱导保护性免疫反应。但是由于 B1本身 高度致弱， 在免疫接种鸡体内的复制能力较差， 因而诱导免疫鸡形成有效免疫保护的 能力也相对较弱， 试验表明， 表达 H7亚型 HA基因的 NDV B1株对 NDV及 H7亚型 高致病力禽流感致死性攻击的存活保护分别仅为 60%和 40%，且不能阻止病毒在体内 的复制和排放。 研究表明， NDV基因组在不同位点插入外源报告基因或免疫原基因， 经细胞或鸡胚连续高代次传代仍保持高度的遗传和表达稳定性。但由于上述表达系统、 活病毒载体本身的不足与缺陷以及使用成本等原因，均未实现在生产实际的广泛应用。 发明内容
针对上述研究背景， 本发明人为进一步提高禽流感病毒表达抗原的免疫原性， 构 建表达野生型和突变型禽流感病毒 HA免疫原蛋白的重组 NDV活载体二联弱毒疫苗 rL-QHwH5和 rL-QHmH5， 通过滴鼻、 点眼、 肌肉注射甚至饮水、 喷雾吸入等多种途 径免疫动物以诱导对禽流感的保护免疫反应， 用于禽类新城疫与禽流感的预防免疫。
因此， 本发明的一个目的是提供一种表达编码野生型或突变型禽流感病毒 H5亚 型血凝素 （HA) 蛋白的基因的重组新城疫 LaSota弱毒疫苗株。
在一个实施方案中， 所述编码野生型 HA蛋白的基因具有 SEQ ID No. 1所示的核 苷酸序列。
在另一个实施方案中， 所述编码突变型 HA蛋白的基因具有 SEQ ID No. 2所示的 核苷酸序列。
优选所述新城疫 LaSota弱毒疫苗株是 AV1615。
更优选所述重组新城疫 LaSota弱毒疫苗株是 rL-QHwH5和 rL-QHmH5。
本发明还有一个目的是提供一种生产上述重组新城疫 LaSota弱毒疫苗株的方法， 该方法包括：
( 1 ) 构建转录质粒， 该转录质粒包括其中插入编码野生型或突变型禽流感病毒 H5亚型 HA蛋白的基因（SEQ ID No. 1或 SEQ ID No. 2 )的所述新城疫 LaSota弱毒疫 苗株的基因组 cDNA序列；
(2 ) 构建一个或多个转录辅助质粒， 该辅助质粒包括编码所述新城疫 LaSota弱 毒疫苗株的核蛋白 （NP ) 的 cDNA序列、 编码所述新城疫 LaSota弱毒疫苗株的磷酸 蛋白 （P ) 的 cDNA序列、 和编码所述新城疫 LaSota弱毒疫苗株的大聚合酶蛋白 （L ) 的 cDNA序列；
( 3 )将所述转录质粒和转录辅助质粒共转染所述病毒复制许可的宿主细胞，培养 转染后的宿主细胞；
(4) 收获上清液， 过滤后继续敏感细胞传代或接种鸡胚尿囊腔救获重组病毒株。 在上述生产方法的一个实施方案中， 将编码野生型或突变型禽流感病毒 H5亚型
HA蛋白的基因插入到新城疫 LaSota弱毒疫苗株的基因组 P， M之间人工引入的 Pmel 位点。 优选所述 LaSota弱毒疫苗株是 AV1615。
在上述生产方法的另一个实施方案中， 包括在所述转录质粒中的基因组 cDNA序 列位于 T7启动子之后， 而在编码自我剪切的核酸酶的序列和 T7转录终止子之前， 构 成基因组 cDNA转录模板。 优选所述自我剪切的核酸酶是丁肝病毒核酶 （Rib)。
在上述生产方法的另一个实施方案中， 包括在所述转录辅助质粒中的编码所述新 城疫 LaSota弱毒疫苗株的核蛋白 （NP ) 的 cDNA序列、 编码所述新城疫 LaSota弱毒 疫苗株的磷酸蛋白 （P ) 的 cDNA序列、 和编码所述新城疫 LaSota弱毒疫苗株的大聚 合酶蛋白 （L ) 的 cDNA 序列都位于 T7 启动子之后。 优选所述转录质粒是 pBRN-FL-QHwH5或 pBR -FL-QHmH5， 所述转录辅助质粒是质粒 pBSNP, pBSP禾口 pBSLc 在一个优选实施方案中， 所述宿主细胞是 BHK-21。
本发明还提供了上述重组新城疫 LaSota 弱毒疫苗株 （特别是 rL-QHwH5 和 rL-QHmH5 ) 在制备预防禽流感的疫苗中的应用。
本发明通过 RT-PCR扩增了 NDV疫苗株 LaSota 10个 cDNA片段，利用片段之间 互相重叠的部分进行拼接， 装配成全长 cDNA克隆。 序列测定结果已经登录 GenBank, 登录号为 AY845400。 接着分别将禽流感病毒 （Avian influenza virus ) H5 亚型， A/Bar-headed goose/Qinghai/3/2005 (H5N1) (2005年青海湖死亡斑头雁分离 H5亚型禽 流感病毒株， 购自中国哈尔滨兽医研究所。 参考文献， Li Y， Chen H, 等， Journal of Virology, 2006, in press; 野生型 HA基因， 已登记于流感病毒序列数据库： http: II www.flu.lanl.gov. ISDN138009) 分离株野生型 （QHwH5， 保留 HA蛋白酶裂解位点， SEQ ID No. 1 )和突变型（QHmH5，蛋白酶裂解位点缺失 4个碱性氨基酸， SEQ ID No. 2 ) HA基因分别重组到 NDV疫苗株 LaSota的 P和 M之间。 将其与核蛋白 （NP) 、 磷蛋白 （P ) 和大聚合酶蛋白 （L) 辅助质粒共转染表达 T7聚合酶的痘病毒感染的细 胞内， 从而合成反基因组 RNA。 此 R A在 NP、 P和 L蛋白的作用下， 进行转录和复 制。 将转染上清接种 SPF胚， 得到来自 cDNA的具有感染性的病毒。 通过碱基突变， 产生了带有遗传标签的 Lasota的派生株 rL-QHwH5和 rL-QHmH5， 救获的病毒在鸡胚 上增殖特征与野毒相近，血凝价高达 212， 以上结果显示，两种类型的 HA均得到表达， 再一次证实 NDV 具有作为疫苗活载体的潜力， 为研制新型禽流感疫苗奠定基础。 以 上重组 NDV不仅可以作为预防 H5亚型高致病力禽流感和新城疫和禽流感的二联弱毒 疫苗双价弱毒疫苗， 也可作为二联灭活疫苗种毒株， 并且完全不干扰目前普遍应用的 常规禽流感流行病学血清学监测。
HA蛋白裂解位点连续多个碱性氨基酸是决定 H5 亚型高致病力禽流感必须的分 子基础。 HA作为 RNA病毒囊膜蛋白病表达后有可能嵌合到重组 NDV的病毒囊膜表 面， 有可能发挥其细胞膜受体结合与融合等细胞侵入相关功能的。 通过人工突变删除 HA裂解位点连续多个碱性氨基酸，使其转变为低致病力禽流感病毒 HA蛋白的基因形 式， 将完全避免潜在的生物安全隐患。 为此， 本发明通过 PCR方法， 人工删除了裂解 位点连续 4个碱性氨基酸 (-RKKR-), 并突变了另外一个氨基酸， 形成突变的低致病力 形式 H5亚型 HA基因 (QHmH5基因， SEQ ID No. 2), 用于构建表达 H5亚型禽流感病 毒 HA抗原的重组新城疫 LaSota双价疫苗株。 附图说明
图 1. 从高保真 RT-PCR产生的亚基因组重叠 cDNA片段装配全长 NDV cDNA。 将 cDNA片段在共有的限制位点连接， 并且在转录质粒 pBR322中装配， 在转录质粒 pBR322中将 RBZ和 T7终止子序列预先克隆在 Eco ?/和 ra//位点之间（详见说明书）。
(A) 显示亲代 NDV的整个全长基因组的第一个和最后一个核苷酸。 （B ) 在顶部显 示含有野生型或突变型 HA基因的 NDV的 cDNA克隆， 在遗传图谱之下的水平线显 示单个 cDNA的位置。
图 2. 通过 RT-PCR产生引入修饰酶位点的核苷酸变化， 并通过使用 PRISM试剂 盒 (Perkin-Elmer)和 Applied Biosystems ABB 10自动测序仪测序。加框的是通过 PCR诱 变在 pBRNl-10中引入的一个核苷酸替代 (由 A突变为 G)。
图 3. 重组新城疫病毒 rL-QHwH5和 rL-QHmH5表达 H5亚型 HA抗原的免疫荧 光分析。 （图 3A和 D) rL-QHwH5以 MOI为 1感染 BHK-21细胞， （图 3B和 E)和 rL-QHmH5以 MOI为 1感染 BHK-21细胞， （图 3C和 F) NDV LaSota亲本株 AV1615 对照以 MOI为 1感染 BHK-21细胞， 感染后 20小时将感染的 BHK细胞甲醇固定， 分 别以鸡抗 H5亚型禽流感病毒高免血清 (图 3A、 B和 C)和鸡抗新城疫病毒高免血清 (图 3D、 E和 F)为一抗、 FITC-偶联的兔抗-鸡 IgG为二抗进行间接免疫荧光检测， Leica DMIRES2荧光显微镜下观察细胞。 结果显示野生型和突变型 H5亚型 HA抗原均可在 重组新城疫 LaSota弱毒疫苗病毒株获得正确表达。 - 图 4. 重组新城疫病毒活载体疫苗鸡胚生长动力测定比较。
图 5. 重组新城疫病毒 rL-QHwH5及 rL-QHmH5免疫 SPF雏鸡 H5禽流感毒特异 HI抗体反应。 H5 亚型禽流感新城疫病毒活载体双价疫苗 (rL-QHwH5及 rL-QHmH5) 免疫 1周龄 SPF雏鸡诱导 H5亚型禽流感特异 HI抗体免疫反应。疫苗经滴鼻加点眼途 径免疫 7日龄雏鸡， 每羽共 100 μΐ体积。
图 6. 重组新城疫病毒 rL-QHwH5及 rL-QHmH5免疫 SPF雏鸡新城疫病毒特异 HI 抗体反应。 H5亚型禽流感新城疫病毒活载体双价疫苗 (rL-QHwH5及 rL-QHmH5)免疫 1周齢 SPF雏鸡诱导新城疫特异 HI抗体免疫反应。重组新城疫病毒经滴鼻加点眼途径 免疫 7日龄雏鸡， 每羽共 ΙΟΟμΙ体积。 免疫后 19天 (26日龄） 釆集血清进行 NDV和 Η5亚型 AIV特异 HI抗体检测。
图 7. pBTRT的质粒图谱。
图 8. pBTRT质粒的 DNA序列。 第一个斜体部分： T7启动子； 带下划线部分： 核酶序列； 第二个带下划线的斜体部分： T7终止子。
图 9. pBRN-FL-QHwH5的质粒图谱。
图 10. PBRN-FL-QHwH5质粒的 DNA序列。 带下划线斜体粗体部分： QHwH5基 因序列 （SEQ ID No. 1 )。
图 l l. pBRN-FL-QHmH5的质粒图谱。
图 12. pBRN-FL-QHmH5质粒的 DNA序列。 带下划线斜体粗体部分： QHmH5基 因序列 （SEQ ID No. 2)。
图 13. AV1615基.因组 cDNA序列，其中第 122至 1591 bp为基因 NP的编码序列， 第 1887至 3074 bp为基因 P的编码序列， 第 8381至 14995 bp为基因 L的编码序列。 图 14. 质粒 pBSNP的序列， 带下划线的斜体部分是 NP基因的编码序列。
图 15. 质粒 pBSP的序列， 带下划线的斜体部分是 P基因的编码序列。
图 16. 质粒 pBSL的序列，， 带下划线的斜体部分是 L基因的编码序列。 具体实施方式
下文将参考实施例详细描述本发明， 所述实施例仅是意图举例说明本发明， 而不 是意图限制本发明的范围。 本发明的范围由后附的权利要求具体限定。 实施例 1 表达野生型或突变型禽流感病毒 H5亚型血凝素 （HA) 蛋白的重组新城疫
LaSota弱毒疫苗株的构建 细胞、 病毒及试验材料
BHK-21 细胞 （乳仓鼠肾细胞 ATCC CCL-10 ) , 培养基为含 10 %胎牛血清 (Hyclone )及 l g/ml G418的 DMEM(Dulbecco's 改良的 Eagle's 培养基）； NDV Lasota 疫苗株 AV1615 (购自中国兽医微生物菌种保藏管理中心 （CVCC) )。 接种 9-10 日龄 SPF鸡胚尿囊腔扩增后 -70°C冻存备用； 鸡抗 NDV高免性血清由本研究室制备 （Chu, H.P., G Snell, D. J. Alexander, 禾卩 G. C. Schild. 1982. Avian Pathol 11 :227-234)； SPF鸡胚 及 SPF鸡雏均由哈尔滨兽医研究所 SPF实验动物中心提供。 H5亚型高致病力禽流感 病毒 （HPAIV ) A/Bar-headed goose/Qinghai/3/2005 (H5N1) (2005年青海湖死亡斑头雁 分离 H5亚型禽流感病毒株 QH/05 ,购自中国哈尔滨兽医研究所)及其 SPF鸡高免血清、 H5亚型 HPAIV分离株 A/Goose/Guangdong/l/1996/H5Nl (GD/96)、 反向遗传操作救获 的野生型新城疫病毒 LaSota疫苗 （rLaSota) 分别购自哈尔滨兽医研究所。 转录载体的构建
基因组 RNA转录载体 pBTRT以低拷贝克隆载体 pBR322 (Invitrogen) 为骨架并 在 EcoRIAra/I位点插入 T7启动子 （T7 promotor)、 丁肝病毒核酶 （Rib ) 和 T7转录终 止信号（T7 terminal ) , 由本实验室自行构建。 克隆在 T7启动子和核酶之间的 DNA片 段可以在 T7 RNA聚合酶的作用下得到转录， 并且由于 Rib的自身催化功能， 可以保 证转录产物的 3'末端与克隆的 DNA片断精确一致。 插入野生型和突变型 HA基因的重组 NDV LaSota株基因组全长 cDNA的构建 为建立 NDV新城疫 Lasota疫苗株的反向遗传操作系统， 必须首先构建相应基因 组的全长 cDNA克隆， 作为基因组负链 RNA转录模板， 为此构建了覆盖整个基因组 的十个 cDNA克隆片段， 利用各个片断间重叠部分的酶切位点， 在低拷贝质粒转录载 体质粒 pBTRT连接组装获得了 15186 nt的完整 cDNA克隆， 序列测定结果已经登录 GenBank,登录号为 AY845400,并将 H5亚型禽流感病毒的野生型和突变型 HA基因， QHwH5 (图 10中带下划线斜体部分， 序列表 SEQ ID No. 1 ) 和 QHmH5基因 （基因 全长 DNA序列见图 12中带下划线斜体部分， 序列表 SEQ ID No. 2 ) ， 克隆到 P， M 之间。 在全长 cDNA片段 5'末端前缀 T7 RNA聚合酶启动子， 在 cDNA片断后连有具 有自我催化功能的肝炎 δ核酶 （GenBank X04451 ) 和 T7转录终止信号。 构建完成的 质粒分别命名为 pBRN-FL-QHwH5和 pBRN-FL-QHmH5的质粒图谱及其 DNA全序列 分别见图 9、 10和图 1 1、 12 ) 。 为避免 _¾α Ι位点的甲基化， 通过 PCR基因组将基因 组 cDNA中 F蛋白编码区第 6178位碱基由 T同义突变为 C， 并作为拯救病毒的分子 标记。 与其他研究者一样， 我们同时在 T7聚合酶启动子于基因组 cDNA的 5'末端引 入两个多余的 G， 这可能有助于副粘病毒反向遗传操作的病毒拯救。 具体如.下：
NDV Lasota疫苗株病毒鸡胚接种尿囊液经常规方法（动物病毒学， 第二版）提取 基因组 RNA;整个基因组分为末端部分重叠的 10个片段（F1-F10 )进行 RT-PCR扩增， cDNA片段克隆至 pBluescript ( Clontech) Smal位点并经序列分析确证与病毒基因组 RNA序列完全一致；序列测定结果已经登录 GenBank，登录号为 AY845400。为引入特 异的分子遗传标签， 选择 Lasota疫苗株基因组 cDNA 6172 bp处存在一甲基化的;》αΙ 位点， 序列为 TCTAGATCA, 利用 PCR手段将其突变为 TCTAGACCA, 使其不再受 甲基化酶识别， 因而能够被限制性内切酶》«1所识别； 利用相邻片段重叠部分存在的 限制酶切位点连接成组装完整的 NDV基因组 cDNA (图 1A)， 并分别将 H5亚型禽流 感病毒的野生型和突变型 HA 基因 QHwH5 和 QHmH5 基因 （QHwH5 : 用 Trizol ( Invitrogen)提出禽流感病毒基因组， 反转录后， 通过 PCR扩增该基因。 体系中加入 如 下 引 物 。 上 游 引 物 5 ，
AGTGCTTCTT 3，， 下游弓 |物 5， GTTTAAACTTAAA TGCAAATTCTGC ATTGT3，。 QHmH5 ： 上 游 引 物 5 ， GTTTAAACC 下游弓 I物 GTTTAAACTTAAATGCAAATTCTGCATTGT5 , ) 克隆到 P， M之间人工弓 | 入 的 Pmei 位 点 ， 并在前缀基 因 终止和基 因起始序列 （ GE/GS ) ( TTAAGAAAAAA/T/ACGGGTAGAA ) , 并克隆在转录载体 pBTRT上， 分别构建成 含有 H5亚型禽流感病毒的野生型和突变型 HA基因 QHwH5和 QHmH5基因的病毒基 因组转录质粒 pBR -FL-QHwH5和 pBRN-FL-QHmH5 (图 IB ) ; 表达核蛋白 （NP)、 磷酸蛋白 （P ) 及大聚合酶蛋白 （L ) 基因的开放阅读框架 （ORP ) cDNA分别紧接着 克隆在 pBluescript ll SK ( +/— ) 质粒 T7启动子下游 Sma I位点， 分别构成转录辅助 质粒 pBSNP。 从重组全长 cDNA克隆救获感染性 NDV (病毒拯救）
为了从克隆的 cDNA 中拯救感染性 NDV,首先分别以 pBRN-FL-QHwH5 和 pBRN-FL-QHmH5及表达 NDV NP、 P、 L蛋白的辅助质粒共转染 BHK-21细胞。 NDV 的融合蛋白 F0必须裂解成 F1和 F2才具有感染性， 对于 Lasota弱毒株而言， BHK-21 细胞不能分泌裂解 F0蛋白所需的胰酶蛋白酶，因此培养基此时应换成无血清培养基并 加入 TPCK (甲苯磺酰苯丙氨酰氯甲酮）处理的胰酶（Sigma, Cat# T8802 ) ( ^g/ml )， 继续培养 2-3天， 收获转染细胞上清接种于 9-1 1 日龄的 SPF鸡胚。 4天后收获鸡胚尿 囊液， 血凝 （HA) 试验结果阳性， 不同鸡胚的 HA价介于 28— u ; NDV免疫血清血凝 抑制（HI)试验分析同样呈现阳性结果。收获病毒阳性尿囊液作为救获病毒 rL-QHwH5 和 rL-QHmH5的 F1代。 进一步的 RT-PCR及序列分析结果显示， F1代救获病毒基因 组 cDNA的 6178位点碱基为 C，而非原 LaSota亲本株的 T，和预期完全相符（图 2 )。 结果表明， 通过反遗传操作技术， 利用 NDV LaSota疫苗株基因组 cDNA克隆成功地 救获具有感染性的子代病毒 rL-QHwH5和 rL-QHmH5。 更具体地， 实验步骤如下： BHK-21细胞接种于 35mm六孔板内生长达 50-80 %单层时，将转录质粒及辅助质 粒 pBRN-FL-QHwH5或 pBRN-FL-QHmH5、 pBSNP、 pBSP和 pBSL分别以 5 g、 2.5μδ、 1.25μ§ 1.25μβ, 共转染 BHK-21细胞， 釆用 CaP04转染试剂盒（Invitrogene ) ， 操作按 试剂盒说明书进行。 转染后 8-12小时， 弃去转染混合物， 用含 10 %DMSO的 PBS液 休克细胞 2.5 分钟， 加入完全 DMEM过夜孵育， 第二天换成无血清培养基， 并加入 TPCK ( 1 μ§ /ml ) 继续孵育 2-3天后， 收获培养物上清， 0.22um孔径滤器过滤后接种 9-1 1天的 SPF胚尿囊腔； 接种后的 SPF胚继续培养， 3-5天， 取鸡胚尿囊液 50μ1进行 按常规进行新城疫病毒的血凝（HA)和血凝抑制（HI)试验（Thayer SG, Nersessian BN, Rivetz B, Fletcher OJ. Comparison of serological tests for antibodies against Newcastle disease virus and infectious bronchitis virus using ImmunoComb solid-phase immunoassay, a commercial enzyme-linked immunosorbent assay, and the hemagglutination-inhibition assay. Avian Dis. 1987 Jul-Sep; 31(3): 459-63. ) 。 收获 HA及 HI试验结果阳性尿囊液， -70°C冻存， 并按常规方法分别于 9-10 日齢鸡胚及鸡胚成纤维细胞滴定每毫升 EID50 及 PFU病毒含量 (1Q)。 分别命名为 rL-QHwH5和 rL-QHmH5。 实施例 2 重组 NDV表达 AVI HA蛋白间接免疫荧光试验 （IFA) 试验
NDV LaSota疫苗株能一过性感染体外培养的哺乳动物细胞。 为证明 rL-QHwH5 和 rL-QHmH5病毒在 BHK-21细胞内的复制及病毒抗原表达，二者尿囊毒以 MOI为 1 的病毒量分别感染约 70-80 %的单层 BHK-21细胞 （图 3A和 B ) ， 同时以 NDV野生 型 LaSota疫苗株感染细胞为对照（图 3C ) ， 感染后 20小时细胞出现早期 CPE (细胞 病变） 现象， 立即以 NDV高免 SPF鸡阳性血清为检测抗体进行间接免疫荧光染色， 结果三种病毒感染细胞荧光显微镜下观察到强阳性反应 (图 3A、 B和 C)更具体地， 实 验步骤如下：
分别以鸡胚接种传代二代次尿囊病毒液 rL-QHwH5、 rL-QHmH5和野生型 LaSota 疫苗株 AV1615 (图 3D、 E和 F ) DMEM适当倍数稀释， 按 ΜΟΙ=5、 50μ1体积感染生 长于 24孔板的 BHK-21， 37°C，孵育 lh后用 DMEM洗涤三遍， 然后加入完全 DMEM 继续培养， 24h后用 95 %乙醇固定细胞 5min， PBST (含有 0.05 %吐温 20的磷酸盐缓 冲液） 洗细胞后用 SPF鸡血清进行封闭 1小时后， 以鸡抗 H5亚型高致病力禽流感病 毒高免 SPF鸡阳性血清为一抗， 作用 30分钟后 PBST洗涤后， 加入 1 : 160稀释荧光素 (FITC )标记的兔抗鸡 IgG二抗（Sigma)，作用 30min， PBST洗涤后荧光显微镜（Leica DMIRES2 )观察， rL-QHwH5和 rL-QHmH5感染细胞全部出现强阳性反应， 而野生型 Lasota感染细胞则完全阴性。
结果表明， 重组新城疫病毒 rL-QHwH5和 rL-QHmH5成功表达了 H5亚型禽流感 病毒 HA抗原蛋白。 实施例 3 rNDV在鸡胚的生长特性及致病特性 为确定反向遗传操作救获 rL-QHwH5和 rL-QHmH5的鸡胚生长特性及其对鸡胚的 致病性， 将救获病毒鸡胚扩增 F1代按 lxlO4 EID5()接种 9〜10日龄 SPF鸡胚尿囊腔。 结果反向遗传操作救获的野生型新城疫病毒 LaSota疫苗株（rLaSota, 即使用转录载体 pBTRT和转录辅助质粒 pBSNP, pBSP和 pBSL,通过如本发明所述的反向遗传操作救 获的野生型新城疫病毒 LaSota疫苗株 AV161 ) 120小时内完全不致死 SPF鸡胚， 接种 后 24小时、 48小时、 72小时及 96小时收获尿囊液，每毫升尿囊液 EID5Q则分别为 10_8·5、 10-8'6、 I0-IG'G和 ι0-9·4。 RL_QHWH5和 RL-QHmH5相同剂量途径接种 9〜10日龄 SPF鸡' 胚尿囊腔， 120小时内同样不致死 SPF鸡胚； rL-QHwH5接种后 24小时、 48小时、 72 小时及 96小时收获尿囊液， 每毫升尿囊液 EID5Q则分别为 10—8·2、 10·8·6、 10'9 和 10— 8·5。 rL-QHmH5接种后 24小时、 48小时、 72小时及 96小时收获尿囊液， 而每毫升尿囊液 EID5o则分别为 10'7·9、 10-8-5, 10— 92和 10—8 6。 图 4的结果表明反向遗传操作救获病毒 rL-QHwH5及 rL-QHmH5的鸡胚生长动力学与野生株 NDV Lasota疫苗株 （rLaSota) 类似， 仍然保持 NDV LaSota疫苗亲本株在鸡胚的高滴度生长及低致死的生物学特性。
接下来将重组病毒 rL-QHwH5 及 rL-QHmH5 与反向遗传操作救获病毒野生型 Lasota疫苗株 （rLaSota), 即 rLaSota进行致病性比较分析。 具体按国际动物卫生组织 (O.I.E.) 推荐标准进行脑内致病指数 （ICPI)、 静脉内致病指数 （IVPI) 及鸡胚平均 致死时间 （MDT) 测定。 用于新生刍鸡的 LaSota活毒疫苗株 ICPI应在 0.4左右或低 于 0.4。 结果， rL-QHmH5病毒不仅保持 NDV Lasota疫苗株 AV1615的低致病性， 而 且被更加致弱。 上述结果表明了该重组 NDV保持了亲本 LaSota疫苗株在 SPF鸡胚的 高滴度生长特性及低致病性。 表 1.重组新城疫病毒致病性分析
Figure imgf000012_0001
** 按 O.I.E推荐标准进行。
常规 RT - PCR及序列分析。 实施例 4诱导保护性抗体的免疫效果
为测定反向遗传操作救获病毒 rL-QHwH5和 rL-QHmH5对 SEF鸡雏的免疫原性， 以 rL-QHwH5为例， 鸡胚扩增 F1代尿囊毒 2xl06 EID5Q剂量经滴鼻加点眼途径人工免 疫 12羽七日龄白色来亨 SPF鸡雏 （哈尔滨兽医研究所 SPF实验动物中心提供） ， 另 设非免疫组对照组 8羽； 免疫组和非免疫对照组分别饲养于空气负压过滤隔离器中。 3 周以后翅静脉采血分离血清按常规检测新.城疫和 H5亚型禽流感的特异血凝抑制抗体。
实验结果： rL-QHwH5和 rL-QHmH5尿囊病毒液 F1代分别以 2xl06 EID50剂量经 滴鼻加点眼途径人工免疫七日龄白色来亨 SPF鸡雏， 免疫后观察 3周， 期间免疫组所 有雏鸡无任何异常， 饲料消耗及生长发育与非免疫对照组无明显差异； 结果， 两种重 组病毒弱毒一次免疫雏鸡后 3周， 即可诱导高水平的 NDV和 H5亚型 AIV特异 HI抗 体水平反应， 并且持续时间长。 结果表明， 重组具有良好的免疫原性， 并且保留低致 病性 LaSota疫苗株良好的安全性。 图 5显示重组病毒 rL-QHwH5和 rL-QHmH5病毒 诱导对 H5亚型 AIV 的保护性抗体免疫反应的一组代表性数据。 图 6显示重组病毒 rL-QHwH5和 rL-QHmH5病毒诱导对 NDV的保护性抗体免疫反应的一组代表性数据。 上述数据表明重组病毒 rL-QHwH5和 rL-QHniH5病毒都能够同时诱导对 NDV和 AIV 的保护性抗体免疫反应。
另外， 对于 rL-QHwH5和 rL-QHmH5病毒， 分别将它们与 NDV Lasota疫苗株 AV1615对新城疫强毒 F48E9 (购自 CVCC )致死性攻击的免疫保护进行比较。 结果在 表 2显示， 表明 rL-QHwH5和 rL-QHmH5病毒两者都与 AV1615具有对新城疫强毒致 死性攻击的相同的免疫保护作用。 表 2. H5亚型禽流感新城疫病毒活载体双价疫苗 rL-QHwH5和 rL-QHmH5免疫 1 周龄 SPF雏鸡对新城疫强毒致死性攻击的免疫保护
Figure imgf000013_0001
* 疫苗经滴鼻加点眼途径免疫 7日龄雏鸡， 每羽 2X106 EID5Q剂量共 ΙΟΟ μΙ体积; **免疫后 21天（28日龄）采用 NDV强毒 F48E9株 104 ELD5Q剂量经肌肉注射途 径攻击， 持续观察 21天。 最后， 分别将 rL-QHwH5和 rL-QHmH5病毒两种 H5亚型禽流感新城疫病毒活载 体双价疫苗对 1周龄 SPF雏鸡免疫， 评估其对 H5亚型高致病力禽流感致死性攻击的 免疫保护。 结果如表 3所示， 结果表明， rL-QHwH5和 rL-QHmH5病毒重组疫苗免疫 雏鸡对新城疫强毒株和 H5亚型高致病力禽流感病毒致死攻击形成 100%完全免疫保护 免疫保护， 不发病、 不死亡； H5亚型高致病力禽流感病毒攻击后完全阻止呼吸道、 消 化道病毒排放。
表 3. H5亚型禽流感新城疫病毒活载体双价疫苗 rL-QHwH5和 rL-QHmH5免疫 1周齢 SPF雏鸡对同源和异源 H5亚型咼致病力禽 感致死性攻击的免疫保护
Figure imgf000015_0001
*疫苗经滴鼻加点眼途径免疫 7日龄雏鸡， 每羽 2xl06 EID5Q剂量， 共 ΙΟΟ μΙ体积;
**免疫后 21 天 （ 28 日齢） 分别采用由哈尔滨兽医研究所提供的异源 Η5 亚型高致病力禽流感病 A/Goose/Guangdong/l/1996/H5Nl(GD/96)株和同源毒株 A/Bar-headed goose/Qinghai/3/2005/H5Nl(QH/05)经鼻腔感染途径攻击， 剂量均 100 LD50, 攻毒后持续观察 21天；
***攻毒后分别于第 3、 5、 7天采集喉头和泄殖腔拭子接种 9〜10日龄 SPF鸡胚尿囊腔进行常规病毒分离， D.P.I., 强毒感染后天数
本研究选择我国自行培育、 生产中广泛应用多年、 实践证明免疫效果良好的一株 新城疫病毒 LaSota弱毒疫苗作为亲本株， 通过负链 R A病毒的反基因操作技术， 选 择基因组内 P基因和 M基因间非编码区为外源基因插入位点， 构建了表达 H5亚型高 致病力禽流感病毒野生型的 HA免疫原基因及人工缺失裂解位点连续多个碱性氨基酸 的突变型 HA免疫原基因的重组 NDV株， rL-QHwH5和 rL-QHmH5,作为预防新城疫 和 H5亚型高致病力禽流感的双价弱毒疫苗候选株， 并进行了生物安全性评估。 研究 表明， NDV基因组在不同位点插入外源报告基因或免疫原基因， 经细胞或鸡胚连续高 代次传代仍保持生物学特性、 低致病性及基因组的遗传稳定性。
对重组病毒 rL-QHwH5和 rL-QHmH5的免疫试验结果表明， rL-QHmH5和 rL-QHmH5重组疫苗一次免疫即可对 H5亚型高致病力禽流感和新城疫强毒的致死攻 击形成 100%完全免疫保护， 诱导保护性抗体的能力与现有灭活疫苗相当， 而在诱导 具有重要意义的粘膜免疫和细胞免疫方面更具优势； 保持了亲本 LaSota疫苗株对新生 雏鸡安全有效、 高滴度鸡胚生长特性、 使用方便等优点； 环境社会效益显著， 与传统 禽流感疫苗相比， 同样剂量疫苗生产用鸡胚量仅为的百分之一， 产品体积仅为千分之 一， 并且生产中无需矿物油的使用， 完全避免了传统油乳剂灭活疫苗注射对免疫对商 品鸡体的影响。
新城疫病毒作为活病毒载体构建防治禽流感和新城疫双价疫苗具有巨大优越性。 禽流感和新城疫被国际兽疫局列为 A类烈性传染病， 是危害世界养禽业发展的重要疾 病， 禽流感同时具有极其重要公共卫生意义。 我国每年用于新城疫防制的弱毒疫苗至 少在百亿羽份以上， NDV弱毒疫苗特别是 LaSota疫苗株的应用在我国养禽业几乎是 所有新生雏鸡必不可少的免疫程序。 新城疫病毒 （NDV) 为不分节单股负链 R A病 毒， 基因组结构与功能背景清楚， 仅有一个血清型， 遗传相对稳定， 重组 NDV 中插 入的外源基因在细胞或鸡胚经髙代次传代后仍可保持稳定表达， 非常适合作为表达或 疫苗载体。 长期以来， NDV弱毒 LaSota疫苗株的安全有效性已被充分证明； 活毒疫 苗免疫可同时诱导全身性体液免疫、局部粘膜免疫及细胞免疫的形成， 形成更加全面、 确实的免疫保护； 可通过饮水、 喷雾、 滴鼻、 点眼或注射多种方式给苗， 使用极为方 便； NDV具有高滴度的鸡胚生长特性， 生产成本极为低廉。
rL-QHmH5重组疫苗的应用， 将使得 H5亚型高致病力禽流感的疫苗防制几乎不 再需要额外的制造和使用成本， 全国每年可至少节约数千万元以上的防疫经费和大量 社会劳动成本， 并减少免疫对象的不良应激反应。 已有的数据表明本疫苗与现有新城 疫和禽流感疫苗相比， 具有巨大社会、 经济和环境效益优势， 国、 内外市场前景广阔。 新城疫病毒作为疫苗载体是国际先进的新型技术， 如果进一步加快进度， H5亚型禽流 感新城疫病毒活载体疫苗将有望成为国际上第一个推向生产实际的负链 RNA病毒活 载体疫苗。 参考文献
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Claims

权 利 要 求
1. 一种表达编码野生型或突变型禽流感病毒 H5亚型血凝素（HA)蛋白的基因的 重组新城疫 LaSota弱毒疫苗株，其中所述编码野生型 HA蛋白的基因具有 SEQ ID No.
1所示的核苷酸序列， 并且所述编码突变型 HA蛋白的基因具有 SEQ ID No. 2所示的 核苷酸序列。
2. 根据权利要求 1的重组新城疫 LaSota弱毒疫苗株，其中所述用于表达 HA蛋白 的新城疫 LaSota弱毒疫苗株是 AV1615。
3. 根据权利要求 2 的重组新城疫 LaSota 弱毒疫苗株， 其为 rL-QHwH5 和
Figure imgf000019_0001
4. 根据权利要求 1-3中任何一项的重组新城疫 LaSota弱毒疫苗株在制备预防禽流 感的疫苗中的应用。
5.一种生产根据权利要求 1 的重组新城疫 LaSota弱毒疫苗株的方法， 该方法包 括：
( 1 ) 构建转录质粒， 该转录质粒包括其中插入编码野生型或突变型禽流感病毒 H5亚型 HA蛋白的基因（SEQ ID No. 1或 SEQ ID No. 2)的所述新城疫 LaSota弱毒疫 苗株的基因组 cDNA序列；
(2) 构建一个或多个转录辅助质粒， 该辅助质粒包括编码所述新城疫 LaSota弱 毒疫苗株的核蛋白 （NP) 的 cDNA序列、 编码所述新城疫 LaSota弱毒疫苗株的磷酸 蛋白 （P) 的 cDNA序列、 和编码所述新城疫 LaSota弱毒疫苗株的大聚合酶蛋白 （L) 的 cDNA序列；
(3 )将所述转录质粒和转录辅助质粒共转染所述病毒复制许可的宿主细胞，培养 转染后的宿主细胞；
(4) 收获上清液， 过滤后继续敏感细胞传代或接种鸡胚尿囊腔救获重组病毒株。
6. 根据权利要求 5 的方法， 其中所述转录质粒是 pBRN-FL-QHwH5 或 pBR -FL-QHmH5 c
7. 根据权利要求 5的方法，其中所述转录辅助质粒是质粒 pBSNP，pBSP和 pBSL。
8. 根据权利要求 5-7中任何一项的方法， 其中所述 LaSota弱毒疫苗株是 AV1615 (中国兽医微生物菌种保藏管理中心， cvcc)。
9. 根据权利要求 5-7中任何一项的方法， 其中所述宿主细胞是稳定表达 T7聚合 酶的细胞系， 如 BHK-21。
10. 根据权利要求 5 的方法制备的重组新城疫 LaSota 弱毒疫苗株 rL-QHwH5 和 rL-QHmH5o
PCT/CN2006/001626 2006-05-09 2006-07-10 SOUCHE DE FAIBLE VIRULENCE DU VACCIN RECOMBINANT LaSota DE LA MALADIE DE NEWCASTLE EXPRIMANT LA PROTÉINE HA DU VIRUS H5 DE LA GRIPPE AVIAIRE WO2007128169A1 (fr)

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CN112111503A (zh) * 2020-08-24 2020-12-22 河北省动物疫病预防控制中心 同时预防禽流感h5和h9亚型的腺病毒载体二价苗及其制备方法
CN113005099A (zh) * 2021-03-25 2021-06-22 江苏省农业科学院 一种W蛋白沉默的重组新城疫病毒rVII-NJ-Wko株及其制备方法和应用

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CN108060141B (zh) * 2017-12-14 2021-05-28 天津瑞普生物技术股份有限公司 Vp2基因和np基因重组腺病毒及其应用
CN110559434A (zh) * 2018-06-05 2019-12-13 普莱柯生物工程股份有限公司 一种禽流感病毒样颗粒疫苗、及其制备方法和应用
CN112111503A (zh) * 2020-08-24 2020-12-22 河北省动物疫病预防控制中心 同时预防禽流感h5和h9亚型的腺病毒载体二价苗及其制备方法
CN112111503B (zh) * 2020-08-24 2023-04-07 河北省动物疫病预防控制中心 同时预防禽流感h5和h9亚型的腺病毒载体二价苗及其制备方法
CN113005099A (zh) * 2021-03-25 2021-06-22 江苏省农业科学院 一种W蛋白沉默的重组新城疫病毒rVII-NJ-Wko株及其制备方法和应用

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