WO2019100686A1 - 区分免疫和感染动物h5亚型禽流感疫苗株及其制备方法和应用 - Google Patents

区分免疫和感染动物h5亚型禽流感疫苗株及其制备方法和应用 Download PDF

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WO2019100686A1
WO2019100686A1 PCT/CN2018/089519 CN2018089519W WO2019100686A1 WO 2019100686 A1 WO2019100686 A1 WO 2019100686A1 CN 2018089519 W CN2018089519 W CN 2018089519W WO 2019100686 A1 WO2019100686 A1 WO 2019100686A1
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sequence
homology
gene
influenza
virus
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宋家升
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宋家升
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Priority to ZA2020/02758A priority patent/ZA202002758B/en

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Definitions

  • the invention belongs to the technical field of genetic engineering vaccines, and relates to a method and an application for preparing a vaccine strain for differentiating immunity and infecting H5 subtype avian influenza.
  • the avian influenza virus belongs to the Orthomyxoviridae family and the influenza virus genus. Influenza viruses can be classified into A, B, and C types according to their antigenicity. Among them, influenza A hosts (including poultry, humans, pigs, etc.) have a strong pathogenicity. Influenza B only infects people and seals, and its virulence is relatively low. Influenza C virus is found only in humans and pigs.
  • the genomes of influenza A and B can be divided into PB2, PB1, PA, NP, HA, NA, M, NS has a total of 8 gene fragments. After the host is infected, it can be used for HA. NA, M1 and NP produce large amounts of antibodies.
  • Type A influenza is classified into a plurality of subtypes (HnNn) depending on the surface envelope protein hemagglutinin (HA) and neuraminidase NA of influenza A virions.
  • HnNn subtypes
  • HA hemagglutinin
  • NA neuraminidase NA of influenza A virions.
  • HA hemagglutinin
  • NA neuraminidase NA of influenza A virions.
  • there are 18 subtypes of HA and 11 subtypes of NA In theory, 198 (18x11) subtypes can be produced, but in reality there are only a few dominant subtypes in nature (such as H5N1, H7N9, H9N2, etc.).
  • influenza B virus is currently divided into only two groups, namely the Victoria group (named after B/Victoria/2/1987) and the Yamagata group (based on Named after B/Yamagata/16/1988). Almost all subtypes of influenza A in poultry have an important role in the storage and evolution of the virus.
  • Vaccine is currently one of the most effective ways to prevent and control avian influenza.
  • the replacement of the external gene (HA, NA) with the vaccine strain for the construction of the epidemic strain is safe, effective and inexpensive, and is most widely used in China, in the prevention of avian influenza vaccine.
  • Control plays an important role.
  • this whole-virus inactivated vaccine cannot serologically distinguish between immunized animals and infected animals, posing a huge obstacle to the monitoring and purification of avian influenza viruses.
  • the HA protein is responsible for binding to the receptor and assisting the virus to invade the cell; the NA protein disrupts the binding of the HA to the receptor and assists in the release of the virus from the cell.
  • the HA and NA genes are usually derived from the same strain of virus.
  • the introduction of heterologous NA may break the functional balance between HA-NAs, thereby reducing the ability of the virus to grow and replicate, and even lead to the inability of the recombinant virus to be rescued.
  • risks increase with the introduction of NA gene similarity (compared to homologous NA).
  • Substitutions between different subtypes of NA often affect biological properties such as replication and growth of rescued recombinant viruses.
  • Influenza A virus reassortants with surface glycoprotein genes of the avian Parent viruses effects of HA and NA gene combinations on virus aggregation. [J]. Archives of Virology, 1993, 133(3-4): 437-450. Since type B NA protein and type A are very different (similarity ⁇ 30%), the probability of success in introducing type B NA to obtain A/B type chimeric virus is small. Also rescued A/B Type NA chimeric viruses may have defects in growth performance and require continuous passage in vitro. Continuous passage introduces adaptive mutations, which cause the risk of antigenic drift, resulting in a large difference in the antigenicity of the prepared vaccine strains from the original wild-type strains.
  • H5 subtype whole virus inactivated vaccine has the advantages of immunization effect and low price, it is impossible to serologically distinguish between immunized animals and infected animals (DIVA), which seriously affects the monitoring of virus epidemics, hinders H5 subtype avian influenza is thoroughly purified in the farm.
  • DIVA immunized animals and infected animals
  • the high pathogenic H5 virus poses a continuing threat to public health safety because it cannot completely eliminate the presence of highly pathogenic avian influenza in farms. Therefore, it is currently required to prepare a novel avian influenza vaccine strain of H5 subtype capable of distinguishing between immunity and infection.
  • the present application has developed a method for preparing a novel avian influenza vaccine strain of H5 which is different from immunity and infection by using the NA gene of influenza B as a marker.
  • Vaccine strains prepared using the chimeric NA gene do not require passage in vitro, thereby avoiding the risk of antigenic variation caused by serial passage.
  • the present invention makes the rescued vaccine strain safer than the conventional vaccine strain by partial deletion of the NS1 protein and weak modification of the HA. Therefore, the present invention provides a method for preparing a H5 avian influenza vaccine which is safe and effective, has low production cost and can distinguish between immunized and infected animals by serological methods, and has important application value and outstanding public health safety significance.
  • Another object of the present invention is to provide a method for preparing an H5 subtype avian influenza vaccine strain which distinguishes between immunity and infection.
  • the tagged gene sequence comprises a DNA sequence encoding the amino acid sequence of the extracellular region of the influenza B virus NA gene, or contains at least 90% homology, or at least 92% homology, or at least 95 with the DNA sequence. % homology, or a sequence of at least 98% homology;
  • the tag gene sequence is a DNA sequence encoding a influenza B virus NA protein, or is encoded to have at least 90% homology, or at least 92% homology, or at least 95% homology to the NA protein amino acid sequence. a DNA sequence of, or at least 98% homologous amino acid sequence;
  • the tag gene sequence is the DNA sequence of the influenza B virus NA gene, or is at least 90% homologous, or at least 92% homologous, or at least 95% homologous, or at least homologous to the DNA sequence. 98% homologous sequence.
  • the H5 subtype avian influenza vaccine strain further comprises an H5 subtype HA gene or a mutant H5 subtype HA gene; the mutant H5 subtype HA gene is capable of mutating the amino acid sequence RERRRKRGLF in the wild type HA protein. For RETRGLF.
  • influenza B virus comprises a medieval group and a Yamagata group of influenza B virus.
  • influenza B virus specifically includes, but is not limited to, strain B/Massachusets/2/2012, B/Brisbane/60/2008, B/Yamagata/16/1988, B/Malaysia/2506/04.
  • the tag gene sequence further comprises a packaging signal sequence at both ends, wherein the 5' end packaging signal sequence comprises a non-coding sequence, an intracellular sequence, and a transmembrane sequence.
  • the intracellular region sequence encodes 5 to 7 amino acids, and the amino acid sequence is located intracellularly.
  • transmembrane region sequence encodes 24 to 32 amino acids, and the amino acid sequence is located in the transmembrane region.
  • the tag gene sequence further comprises a packaging signal sequence at both ends, wherein the 3' end packaging signal sequence is SEQ ID NO: 4, or a sequence having at least 80% homology, or at least 85% homology, or at least 90% homology, or at least 95% homology to SEQ ID NO:4.
  • a method for preparing an H5 subtype avian influenza vaccine strain for differentiating and infecting influenza A virus comprises the steps of: tagging a gene sequence with an HA gene of a H5 subtype avian influenza virus or a mutant H5 subtype HA gene; The reverse genetic operating system was rescued to obtain a recombinant vaccine strain, that is, an H5 subtype avian influenza vaccine strain that differentiates between immunity and infection with influenza A virus;
  • the mutated H5 subtype HA gene is capable of mutating the amino acid sequence RERRRKRGLF in the wild type HA protein to RETRGLF;
  • the tagged gene sequence contains a DNA sequence encoding the amino acid sequence of the extracellular region of the influenza B virus NA protein, or contains at least 90% homology, or at least 92% homology, or at least the amino acid sequence encoding the extracellular region. a DNA sequence of 95% homology, or at least 98% homologous amino acid sequence;
  • the tag gene sequence is a DNA sequence encoding a influenza B virus NA protein, or is encoded to have at least 90% homology, or at least 92% homology, or at least 95% homology to the NA protein amino acid sequence. a DNA sequence of, or at least 98% homologous amino acid sequence;
  • the tag gene sequence further comprises a packaging signal sequence at both ends.
  • the 5'-end packaging signal sequence of the tag gene sequence is SEQ ID NO: 3, or is SEQ ID NO: 3 sequences having at least 80% homology, or at least 85% homology, or at least 90% homology, or at least 95% homology.
  • the 3'-end packaging signal sequence of the tag gene sequence is SEQ ID NO: 4, or is SEQ ID NO:4 A sequence having at least 80% homology, or at least 85% homology, or at least 90% homology, or at least 95% homology.
  • the reverse genetic operating system is rescued by using six PR8 internal genes, namely wild type NS or mutant ⁇ NS gene and PB2, PB1, PA, NP, M; wherein ⁇ NS is a mutant NS gene , ⁇ NS nucleotide sequence such as SEQ ID NO: 5 is shown.
  • H5 subtype avian influenza vaccine strain that is immune to and infected with influenza A virus is named H5 subtype avian influenza vaccine candidate strain Re-MuH5-DIVA- ⁇ NS, which has been deposited in the China Center for Type Culture Collection under the accession number CCTCC. NO: V201741.
  • Applicants deposited the vaccine strain Re-MuH5-DIVA- ⁇ NS of the present invention in the China Center for Type Culture Collection.
  • the depository address was Wuhan University, Wuhan, China, and the deposit center received the vaccine strain provided by the applicant on October 19, 2017.
  • the deposit number given to the culture by the deposit center is CCTCC NO: V201741, the proposed classification is named H5 subtype avian influenza vaccine candidate strain Re-MuH5-DIVA- ⁇ NS, and the vaccine strain identified as being deposited on October 28, 2017 is alive.
  • This application uses the NA gene of influenza B virus as a label to develop a method for preparing a novel avian influenza vaccine of H5 that distinguishes between immunity and infection.
  • the present invention successfully constructs an H5 subtype avian influenza vaccine strain which distinguishes immunized animals from infected animals, in which the NA gene and the HA gene have good compatibility and exhibit good replication and growth.
  • Such biological characteristics do not require transgenic adaptation in vitro, avoiding the shortcomings of antigenic variation caused by passage adaptation. Even after three generations of transmission, the characteristics of low virulence and high titer growth of chicken embryos were maintained.
  • the invention has important application value and outstanding public health safety significance.
  • H5 subtype avian influenza not only brings huge economic losses to the livestock industry, but also seriously threatens the safety of public health, although the conventional H5 subtype avian influenza whole virus inactivated vaccine is effective, but in serology It is impossible to distinguish between antibodies produced by immunity and infection, which poses a huge obstacle to the monitoring and purification of avian influenza.
  • the invention uses the NA of influenza B as a label, and successfully constructs a vaccine strain for differentiating immunity and infecting H5 subtype avian influenza, and has important significance and application value for the prevention and purification of H5 subtype avian influenza.
  • Figure 1 is a schematic view showing the structure of a synthetic A/B chimeric NA gene
  • Figure 2 is a schematic diagram showing the pFLu vector map and the cloning of influenza virus gene fragments
  • Figure 3 is a measure of the reactivity of serum against anti-Re-MuH5-DIVA- ⁇ NS (A/B chimeric NA) with influenza A by immunofluorescence.
  • Example 1 Method for preparing avian influenza vaccine strain Re-MuH5-DIVA- ⁇ NS virus
  • the pFlu vector is a bidirectional transcription vector that synthesizes viral RNA in the human polI promoter prior to transcription of the entire viral RNA and in the CMV promoter to synthesize viral proteins (Hoffmann) Et al., PNAS, USA 97, 6108-6113, 2000).
  • a synthetic wild-type H5 gene (A/Duck/Hubei/49/2005), which mutates the highly conserved sequence of the highly pathogenic wild-type HA amino acid sequence (RERRRKRGLF) into a low-pathogenic amino acid sequence by site-directed mutagenesis (RETRGLF), the corresponding low pathogenic MuH5HA gene sequence was obtained.
  • the modified MuH5HA gene was cloned into pFlu by BsmBI site.
  • the vector was obtained as a recombinant plasmid pFlu-MuH5HA, and the construction principle is shown in Fig. 2.
  • the synthetic A/B chimeric NA gene shown in Figure 1 which contains the amino acid sequence encoding the extracellular region of the influenza B virus NA (SEQ ID NO: 1) DNA sequence (SEQ ID NO: 2) as a tag gene sequence, SEQ ID
  • the sequence containing the extracellular domain of B-type NA shown by NO: 2 is derived from B/Massachusets/2/2012 in the Yamagata group of influenza B virus (Ping J et al, PNAS, 2016, 113(51): E8296-E8305), the tagged gene sequence also contains a packaging signal sequence at both ends, wherein the 5'-end packaging signal sequence (SEQ ID) NO: 3) includes a non-coding sequence, an intracellular region sequence, and a transmembrane region sequence, and the 3'-end packaging signal sequence is SEQ ID NO: 4. Inserting chimeric NA into pFlu via BsmBI site The vector obtained the recombinant plasmid pFlu-PR8-BNA.
  • the virus containing the mutant gene ⁇ NS loses its function of antagonizing interferon, and therefore can only grow and multiply in cells in which interferon-deficient cells or interferon systems are underdeveloped, and thus has good safety.
  • Recombinant vaccine strain Re-MuH5-DIVA- ⁇ NS was rescued using the classic "6+2" influenza reverse genetics operating system.
  • PR8 internal genes pFlu-PR8-PB2, pFlu-PR8-PB1, pFlu-PR8-PA, pFlu-PR8-NP, pFlu-PR8-M, pFlu-PR8- ⁇ NS and two external genes, pFlu-MuH5HA, and 0.5 ⁇ g of pFlu-PR8-BNA were co-transfected into 293T cells (Lipofectamine). 3000).
  • the rescued Re-MuH5-DIVA- ⁇ NS strain became low pathogenic or non-virulent and could only grow and reproduce on cell lines lacking the interferon system or low-age chicken embryos with impaired interferon system. Security.
  • After the harvested virus-containing allantoic fluid is inactivated with formalin, it is further prepared into an inactivated oil seedling.
  • Applicants deposited the vaccine strain Re-MuH5-DIVA- ⁇ NS of the present invention in the China Center for Type Culture Collection.
  • the depository address was Wuhan University, Wuhan, China, and the deposit center received the vaccine strain provided by the applicant on October 19, 2017.
  • the deposit number given to the culture by the deposit center is CCTCC NO: V201741, the proposed classification is named H5 subtype avian influenza vaccine candidate strain Re-MuH5-DIVA- ⁇ NS, and the vaccine strain identified as being deposited on October 28, 2017 is alive.
  • the preparation method in the second embodiment is the same as that in the first embodiment except that in constructing the artificial A/B chimeric NA gene shown in Fig. 1, the DNA sequence encoding the amino acid sequence of the extracellular region protein of influenza B virus NA and its implementation The same applies to Example 1 except for the difference in Example 1.
  • the DNA sequence encoding the extracellular region protein amino acid sequence (SEQ ID NO: 6) in the influenza B virus NA is as SEQ.
  • ID NO: 7 as the tag gene sequence, the sequence shown in SEQ ID NO: 7 is derived from B/Brisbane/60/2008 (Ping J) in the medieval influenza virus group B. Et al, PNAS, 2016, 113(51): E8296-E8305).
  • the cells were serially passaged on 8-day-old chicken embryos at 0.2 ml/piece, and the inoculated chicken embryos were cultured in a 37 ° C incubator for 48 hours.
  • Chicken embryo allantoic fluid (F0 generation) was collected and its blood coagulation titer was measured.
  • the F0 generation virus was diluted and inoculated with 10 SPF chicken embryos. After 48 hours of culture, the harvested virus was defined as F1 generation. In the same way, the F1 generation virus was successively passaged to the F3 generation.
  • Example 1 having the Massachusetts/2/2012 (Yamagata group) NA gene
  • the Re-MuH5-DIVA- ⁇ NS vaccine strain has a titer of 7 log2 HA on chicken embryos
  • Example 2 with the NA gene of B/Brisbane/60/2008
  • the titer of Re-MuH5-DIVA- ⁇ NS vaccine strain on chicken embryo can reach 5.5log2 HA potency.
  • the F0 and F3 viruses were amplified by RT-PCR, and the chimeric NA gene was stably sequenced to progeny virus.
  • the homology of the NA full-length nucleotide sequence from the different populations of influenza B virus representative strains B/Brisbane/60/2008 (Victoria group) and Massachusetts/2/2012 (Yamagata group) was 94.9%, amino acid sequence The homology was 94.9%; the homology of the DNA sequences encoding the extracellular region of the NA protein was 95.1%, and the amino acid sequence homology of the extracellular region of the NA protein was 94.6%. Since influenza B is only divided into Victoria and Yamagata populations, the present invention demonstrates that representative strains of NA from both populations (Example 1 and Example 2) are compatible with HA of H5, indicating B.
  • the NA gene of the influenza virus can be used to prepare an H5 subtype avian influenza vaccine strain that is immune to and infected with influenza A virus.
  • the inactivated oil seedling is a milky white water-in-oil emulsion with low viscosity, uniform particle size and good stability, and is suitable for injection.
  • the NheI site was cloned into the pCAGGS eukaryotic expression plasmid and designated as pCAGGS-N1, pCAGGS-N2, pCAGGS-N6, pCAGGS-N9. Put 1 ⁇ g Each pCAGGS-N1, pCAGGS-N2, pCAGGS-N6, pCAGGS-N9
  • the plasmid was transfected into 293T cells previously plated on 24-well cell culture plates. At 30 h after transfection, the reactivity of the following 7 groups of chicken serum and N1, N2, N6, N9 was detected by immunofluorescence.
  • Anti-MuH5-DIVA- ⁇ NS chicken serum chicken serum immunizing only the Re-MuH5-DIVA- ⁇ NS inactivated vaccine of the present invention
  • Anti-H5N1 standard H5N1 standard serum, purchased from Harbin Veterinary Research Institute.
  • Anti-H5+H7 serum clinical serum of immunized H5N1 Re-8 strain + H7N9 Re-1 strain whole virus inactivated vaccine.
  • Anti-N2 chicken serum weekly age SPF chicken immunization (intramuscular injection) 100 ⁇ g pCAGGS-N2, whole blood was collected 4 weeks after immunization, and serum was prepared.
  • Anti-N6 chicken serum weekly age SPF chicken immunization (intramuscular injection) 100 ⁇ g pCAGGS-N6, whole blood was collected 4 weeks after immunization, and serum was prepared.
  • Anti-N9 chicken serum weekly age SPF chicken immunization (intramuscular injection) 100 ⁇ g pCAGGS-N9, whole blood was collected 4 weeks after immunization, and serum was prepared.
  • the immunofluorescence method is as follows:
  • the primary antibody was diluted with PBS containing 1% BSA (anti-Re-MuH5-DIVA- ⁇ NS, anti-H5N1 standard, anti-H5+H7, 100-fold; anti-N1/N2/N6/N9, 20-fold), and Add 0.5ml to each well Incubate for 1 hour at 37 ° C in a wet box and then wash three times with PBS.
  • BSA anti-Re-MuH5-DIVA- ⁇ NS, anti-H5N1 standard, anti-H5+H7, 100-fold; anti-N1/N2/N6/N9, 20-fold
  • Chicken secondary antibody (Alexa Fluor 594 ⁇ anti-chicken IgY) contains 1% BSA was diluted 200-fold in PBS, 0.5 ml was added to each well, incubated for 0.5 hour at room temperature, and then washed three times with PBS.
  • Example 6 Method for preparing H5 subtype avian influenza vaccine strain Re-MuH5-DIVA- ⁇ NS which is immune to and infected with influenza A virus
  • the preparation method in the present Example 6 is the same as that in Example 1, except that in constructing the artificial A/B chimeric NA gene shown in Fig. 1, the NA sequence of the influenza B virus used is a DNA sequence encoding the NA whole protein sequence.
  • the other is the same as in Example 1, wherein the DNA sequence of the NA is derived from the NA whole gene sequence of B/Massachusets/2/2012 in the Yamagata group of the influenza B virus (Ping). J et al, PNAS, 2016, 113(51): E8296-E8305).

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Abstract

提供了一种区分免疫和感染动物H5亚型禽流感疫苗株及其制备方法和应用。所述疫苗株用B型流感的NA蛋白作为标签，对H5亚型禽流感的防控和净化具有应用价值和公共卫生学意义。

Description

区分免疫和感染动物H5亚型禽流感疫苗株及其制备方法和应用
技术领域
本发明属于基因工程疫苗技术领域，涉及一种制备区分免疫和感染H5亚型禽流感疫苗株的方法及应用。
背景技术
禽流感病毒属于正黏病毒科，流感病毒属。流感病毒根据抗原性不同可分为A，B，C型，其中A型流感宿主广泛（包括禽类，人，猪等）致病力较强危害巨大。 B型流感仅感染人和海豹，致病力相对较低。C型流感病毒，仅在人与猪中发现。A型和B型流感的基因组可以分为PB2, PB1, PA, NP, HA, NA, M，NS 共8个基因片段。宿主被感染后，可以对HA， NA，M1和NP产生大量抗体。其中HA可以直接诱导主要中和抗体。以往的研究发现A型和B型病毒诱导的针对HA，NA，M1和NP的四种主要抗体在血清学上无交叉反应性。根据A型流感病毒粒子的表面囊膜蛋白血凝素（HA）和神经氨酸酶NA不同，将A型流感分为众多亚型（HnNn）。其中HA有18种亚型，NA有11种亚型，理论上可以产生198（18x11）种亚型，但是实际上自然界只存在少数优势亚型（如H5N1，H7N9， H9N2等）。不同亚型HA 蛋白间序列同源性在 40%-80%之间 (Air G M. Proceedings of the National Academy of Sciences of the United States of America, 1981, 78(12):7639. Nobusawa E, et al. Virology, 1991, 182(2):475-485)。B 型流感没有亚型之分，各个毒株基因相似性较高。根据HA的抗原性和基因型不同，B型流感病毒目前仅分为两个群，分别为Victoria群（依据B/Victoria/2/1987命名）和Yamagata群（依据 B/Yamagata/16/1988命名)。禽类中几乎存在所有A型流感的各种亚型，在病毒的储存和进化中伴有重要作用。禽流感全球肆虐给养禽业带来巨大经济损失，随着禽流感病毒对人类的逐渐适应，禽流感感染人的事例也逐渐增多。相比季节性型人流感，禽流感感染人具有发病严重，死亡率高的特征，严重威胁公共卫生安全。在众多禽流感亚型中， H5危害及其严重，不仅造成巨大经济损失而且可以直接感染人。高致病性H5亚型禽流感可以在数天内导致家禽100%死亡，而且可以直接感染人。人感染后症状严重，死亡率高。禽流感在养殖场的彻底净化，是杜绝禽流感对食品安全和公共卫生风险的最有效方式。
目前疫苗是防控禽流感最有效的方法之一。以鸡胚高度适应株PR8内部基因为背景，外部基因（HA，NA）替换为针对流行株的构建的疫苗株具有安全，有效和廉价等优点，在中国使用最为广泛，在禽流感疫苗的防控中起到重要作用。但是这种全病毒灭活疫苗不能在血清学上区分免疫动物和感染动物，给禽流感病毒的监测和净化带来巨大障碍。在病毒的生命循环中，HA蛋白负责与受体结合，协助病毒入侵细胞；NA蛋白是破坏HA与受体的结合，协助病毒从细胞释放。因此保持HA-NA之间的功能平衡（HA-NA的匹配性），直接影响流感病毒的复制能力，生长特性（Mitnaul L J, Matrosovich M N, Castrucci M R, et al. Balanced Hemagglutinin and Neuraminidase Activities Are Critical for Efficient Replication of Influenza A Virus[J]. Journal of Virology, 2000, 74(13):6015-20.）。因此选择匹配度较好的HA和NA是研制优异疫苗株的关键之一（Murakami S, et al. Growth Determinants for H5N1 Influenza Vaccine Seed Viruses in MDCK Cells[J]. Journal of Virology, 2008, 82(21):10502.）。为了保证疫苗株HA和NA有较好的匹配度，通常情况下HA和NA基因都来源于同一株病毒。引入异源的NA可能打破HA-NA之间的功能性平衡，从而降低病毒的生长和复制能力，甚至导致重组病毒无法救获。一般而言，这样的风险会随着引入NA基因相似性（与同源NA相比）降低而不断增加。不同亚型的NA之间的替换，往往会影响获救重组病毒的复制和生长等生物学特性。这也是自然界里为什么只存在少数优势亚型组合（如常见H9N2，H5N1， H7N9等）而不是HA-NA的随机组合（如罕见H9N1，H5N9等）（Wagner R et al，Functional balance between haemagglutinin and neuraminidase in influenza virus infections[J]. Reviews in Medical Virology, 2002, 12(3):159）。Rudneva et al 用N1基因和不同亚型HA组合，发现获救的H3，H4，H10和H13的重组病毒在鸡胚上的生长特性比其野生型的病毒更差（Rudneva I A et al. Influenza A virus reassortants with surface glycoprotein genes of the avian parent viruses: effects of HA and NA gene combinations on virus aggregation. [J]. Archives of Virology, 1993, 133(3-4):437-450. 由于B型NA蛋白和A型差异很大（相似性<30%），引入B型NA获得A/B型嵌合病毒的成功几率很小。此外获救的A/B 型NA嵌合病毒可能存在生长性能的缺陷，需要在体外连续传代适应。而连续传代会引入适应性的突变，引起抗原漂移的风险，从而导致制备的疫苗株的抗原性与原有的野生流行株有较大差异。
现有的H5亚型全病毒灭活疫苗虽然具有免疫效果确实，价格低廉等的优点，但是无法在血清学上区分免疫动物和感染动物（DIVA）严重影响了对病毒流行性的监测，阻碍对H5亚型禽流感在养殖场的彻底净化。由于无法彻底杜绝高致病性禽流感病在养殖场的存在，使得高致病性H5病毒具有持续威胁公共卫生安全的风险。因此，制备出能够区分免疫和感染的H5亚型新型禽流感疫苗株为目前所需。
发明内容
为了解决上述存在的问题，本申请用B型流感的NA基因作为标记，研制出一种制备区分免疫和感染的H5新型禽流感疫苗株的方法。使用嵌合型NA基因制备的疫苗株不需要在体外传代适应，从而避免连续传代引起抗原变异的风险。此外，本发明通过对NS1蛋白的部分缺失和HA的致弱修饰使得获救的疫苗株的安全性明显优于普通的疫苗株。因此本发明提供一种制备安全有效，生产成本低而且可以通过血清学方法区分免疫和感染动物的H5禽流感疫苗的方法，具有重要的应用价值和突出的公共卫生安全意义。
本发明的目的在于提供一种区分免疫和感染的H5亚型禽流感疫苗株及其应用。
本发明的另一目的在于提供一种制备区分免疫和感染的H5亚型禽流感疫苗株方法。
本发明所采取的技术方案是：
标签基因序列在制备区分免疫和感染A型流感病毒的H5亚型禽流感疫苗株中的应用，所述标签基因序列含有编码B型流感病毒NA蛋白胞外区氨基酸序列的DNA序列，或者含有编码与该胞外区氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列；
或者，所述标签基因序列含有B型流感病毒NA基因中编码胞外区氨基酸序列的DNA序列，或者含有与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列；
或者，所述标签基因序列为编码B型流感病毒NA蛋白的DNA序列，或者为编码与该NA蛋白氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列；
或者，所述标签基因序列为B型流感病毒NA基因的DNA序列，或者为与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列。
进一步的，所述H5亚型禽流感疫苗株中还含有H5亚型HA基因或突变的H5亚型HA基因；所述突变的H5亚型HA基因能够将野生型HA蛋白中的氨基酸序列RERRRKRGLF突变为RETRGLF。
进一步的，所述B型流感病毒包括Victoria群和Yamagata群的B型流感病毒。
进一步的，所述B型流感病毒具体包括但不限于病毒株B/Massachusetts/2/2012，B/Brisbane/60/2008，B/Yamagata/16/1988，B/Malaysia/2506/04。
进一步的，所述标签基因序列两端还含有包装信号序列，所述包装信号为H1亚型NA的包装信号，或者为与H1亚型NA的包装信号具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的包装信号序列。
进一步的，所述标签基因序列两端还含有包装信号序列，其中5′端包装信号序列包括非编区序列、胞内区序列、跨膜区序列。
进一步的，所述胞内区序列编码5～7个氨基酸，氨基酸序列位于胞内。
进一步的，所述跨膜区序列编码24～32个氨基酸，氨基酸序列位于跨膜区。
进一步的，所述标签基因序列的5′端包装信号序列为SEQ ID NO:3，或者为与SEQ ID NO:3具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
进一步的，所述标签基因序列两端还含有包装信号序列，其中3′端包装信号序列为SEQ ID NO:4，或者为与SEQ ID NO:4具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
一种制备区分免疫和感染A型流感病毒的H5亚型禽流感疫苗株的方法，包括以下步骤：将标签基因序列与H5亚型禽流感病毒的HA基因或突变的H5亚型HA基因；通过反向遗传操作系统获救得到重组疫苗株，即区分免疫和感染A型流感病毒的H5亚型禽流感疫苗株；
所述突变的H5亚型HA基因能够将野生型HA蛋白中的氨基酸序列RERRRKRGLF突变为RETRGLF；
所述标签基因序列含有编码B型流感病毒NA蛋白胞外区氨基酸序列的DNA序列，或者含有编码与该胞外区氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列；
或者，所述标签基因序列含有B型流感病毒NA基因中编码胞外区氨基酸序列的DNA序列，或者含有与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列；
或者，所述标签基因序列为编码B型流感病毒NA蛋白的DNA序列，或者为编码与该NA蛋白氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列；
或者，所述标签基因序列为B型流感病毒NA基因的DNA序列，或者为与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列。
进一步的，所述标签基因序列两端还含有包装信号序列。
进一步的，所述标签基因序列的5′端包装信号序列为SEQ ID NO:3，或者为与SEQ ID NO:3具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
进一步的，所述标签基因序列的3′端包装信号序列为SEQ ID NO:4，或者为与SEQ ID NO:4具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
进一步的，所述反向遗传操作系统获救过程中还用到6个PR8内部基因，即野生型的NS或突变的ΔNS基因与PB2、PB1、PA、NP、M；其中ΔNS为突变的NS基因，ΔNS的核苷酸序列如SEQ ID NO:5所示。
区分免疫和感染A型流感病毒的H5亚型禽流感疫苗株，其命名为H5亚型禽流感疫苗候选株Re-MuH5-DIVA-ΔNS，已保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:V201741。
上述所述的疫苗株在制备禽流感疫苗中的应用。
申请人将本发明疫苗株Re-MuH5-DIVA-ΔNS保藏于中国典型培养物保藏中心，保藏单位地址为中国武汉武汉大学，保藏中心于2017年10月19日收到申请人提供的疫苗株。保藏中心给予该培养物的保藏号为CCTCC NO:V201741，提议的分类命名为H5亚型禽流感疫苗候选株Re-MuH5-DIVA-ΔNS，已于2017年10月28日鉴定保藏的疫苗株是存活的。
本发明的有益效果是：
（1）本申请用B型流感病毒的NA基因作为标签，研制出一种制备区分免疫和感染的H5新型禽流感疫苗的方法。
（2）本发明成功构建了区分免疫动物和感染动物的H5亚型禽流感疫苗株，在该疫苗株中，NA基因和HA基因具用很好的兼容性，表现出很好的复制和生长等生物学特性，不需要在体外的传代适应，避免了传代适应引起的抗原变异的缺点。即使续传3代，仍然保持对鸡胚低致病力和高滴度生长的特性。本发明具有重要的应用价值和突出的公共卫生安全意义。
（3）高致病H5亚型禽流感不仅给畜牧业带来巨大经济损失，而且严重威胁公共卫生学的安全，虽然常规的H5亚型禽流感全病毒灭活疫苗效果确实，但在血清学上无法区分免疫和感染产生的抗体，给禽流感的监测和净化带来巨大障碍。本发明用B型流感的NA作为标签，成功构建区分免疫和感染H5亚型禽流感疫苗株，对H5亚型禽流感的防控和净化具有重要的意义和应用价值。
附图说明
图1为人工合成A/B嵌合型NA基因的结构示意图；
图2为 pFLu 载体图谱及流感病毒基因片段的克隆示意图；
图3为免疫荧光检测抗Re-MuH5-DIVA-ΔNS（A/B嵌合型NA）的血清与A型流感NA的反应性。
具体实施方式
下面结合具体实施例和附图，对发明进行详细说明，但本发明的实施方案不局限于此。未注明的常规实验方法请参考《分子克隆实验指南第三版》（萨姆布鲁克 主编， 科学出版社，2002）。
实施例1禽流感疫苗株Re-MuH5-DIVA-ΔNS病毒的制备方法
（1）构建低致病性HA突变基因
pFlu载体是一种双向转录载体，既可以在人polI启动子先转录完整病毒的RNA，又可以在CMV启动下转录病毒mRNA，从而合成病毒蛋白（Hoffmann et al., PNAS,USA 97, 6108-6113,2000）。
人工合成的野生型H5基因（A/Duck/Hubei/49/2005），通过定点突变技术将该高致病性的野生型HA氨基酸序列中高保守序列(RERRRKRGLF)突变为具有低致病性氨基酸序列（RETRGLF），获得相应的低致病性的MuH5HA基因序列。将修饰后的MuH5HA基因通过BsmBI位点克隆到pFlu 载体获得重组质粒pFlu-MuH5HA，构建原理图如图2所示。
（2）构建低致病性A/B嵌合型NA基因
构建图1所示人工合成A/B嵌合型NA基因，其中含有B型流感病毒NA中编码胞外区蛋白氨基酸序列（SEQ ID NO：1）的DNA序列（SEQ ID NO：2）作为标签基因序列，SEQ ID NO：2所示的含B型NA胞外区的序列来自B型流感病毒Yamagata群中的B/Massachusetts/2/2012（Ping J et al, PNAS, 2016, 113(51):E8296-E8305），标签基因序列两端还含有包装信号序列，其中5′端包装信号序列（SEQ ID NO:3）包括非编区序列、胞内区序列和跨膜区序列，3′端包装信号序列为SEQ ID NO:4。通过BsmBI位点将嵌合NA插入pFlu 载体获得重组质粒pFlu-PR8-BNA。
（3）Re-MuH5-DIVA-ΔNS疫苗株的获得
为了保证疫苗株的安全性，我们对病毒野生型NS1基因进行修改，修改后的突变基因ΔNS的核苷酸序列如SEQ ID NO:5所示，含突变基因ΔNS的病毒丧失拮抗干扰素的功能，因此只能在干扰素缺失的细胞或干扰素系统发育不全的鸡胚生长繁殖，因此具有良好的安全性。
采用经典“6+2”流感反向遗传操作系统拯救重组疫苗株Re-MuH5-DIVA-ΔNS。将6个PR8内部基因pFlu-PR8-PB2, pFlu-PR8-PB1, pFlu-PR8-PA, pFlu-PR8-NP, pFlu-PR8-M, pFlu-PR8-ΔNS和2个外部基因pFlu-MuH5HA，pFlu-PR8-BNA各0.5ug共转染到293T细胞（Lipofectamine 3000）。转然后24h更换含有终浓度为0.5ug/ml TPCK-Trypsin的培养液，并在转然后48h收集细胞上清，将细胞上清按照0.2ml/枚通过尿囊腔接种8日龄SPF鸡胚。接种后的鸡胚在37℃温箱内培养48h。收集鸡胚尿囊液（F0代），获得疫苗株Re-MuH5-DIVA-ΔNS，并测定其是否有血凝价。
获救的Re-MuH5-DIVA-ΔNS株变为低致病力或者无致病力，只能在干扰素系统缺失的细胞系或干扰素系统发育不全的低日龄鸡胚上生长繁殖，具有良好的安全性。在8日龄SPF鸡胚培养48小时，无需在鸡胚上连续传代适应，其HA效价可达到7 log2。由于Re-MuH5-DIVA-ΔNS株的NS1部分缺失，虽然在鸡胚上的生长滴度要低于非缺失的的病毒，但是其安全性比非缺失的病毒更好。将收获含病毒尿囊液用福尔马林灭活后，进一步制备成灭活的油苗。
申请人将本发明疫苗株Re-MuH5-DIVA-ΔNS保藏于中国典型培养物保藏中心，保藏单位地址为中国武汉武汉大学，保藏中心于2017年10月19日收到申请人提供的疫苗株。保藏中心给予该培养物的保藏号为CCTCC NO:V201741，提议的分类命名为H5亚型禽流感疫苗候选株Re-MuH5-DIVA-ΔNS，已于2017年10月28日鉴定保藏的疫苗株是存活的。
实施例2禽流感疫苗株Re-MuH5-DIVA-ΔNS病毒的制备方法
本实施例2中的制备方法同实施例1，除了在构建图1所示人工合成A/B嵌合型NA基因时，B型流感病毒NA中编码胞外区蛋白氨基酸序列的DNA序列与实施例1中的不同外，其他均同实施例1。
在本实施例中，B型流感病毒NA中编码胞外区蛋白氨基酸序列（SEQ ID NO：6）的DNA序列如SEQ ID NO：7所示，作为标签基因序列，SEQ ID NO：7所示的序列来自B型流感病毒Victoria群中的B/Brisbane/60/2008（Ping J et al, PNAS, 2016, 113(51):E8296-E8305）。
下面对本发明制备的Re-MuH5-DIVA-ΔNS疫苗株作进一步的效果检测。
方法：分别将实施例1和2获得的Re-MuH5-DIVA-ΔNS疫苗株、对照组1的PR8-ΔNS（NS缺陷的PR8病毒）、对照组2的PR8-WT（PR8野生型病毒）分别按0.2ml/枚接种在8日龄鸡胚上连续传代，接种后的鸡胚在37℃温箱内培养48h。收集鸡胚尿囊液（F0代），测定其血凝效价。将F0代病毒稀释后接种10枚SPF鸡胚，培养48h后收获病毒定义为F1代。用同样的方法，将F1代病毒连续传代致F3代。
结果：检测结果如表1所示。为了证明不同分支的B型NA基因是否能与H5亚型的HA（H5-BNA）良好匹配，我们选取了不同群的代表株B/Brisbane/60/2008（Victoria群）和Massachusetts/2/2012（Yamagata群）的NA基因进行研究，结果发现来自B型不同分支NA的基因（Victoria和Yamagata群）与H5均表现出良好的匹配性。其中，拥有Massachusetts/2/2012（Yamagata群）NA基因的实施例1 Re-MuH5-DIVA-ΔNS疫苗株在鸡胚上滴度为7 log2 HA 效价；拥有B/Brisbane/60/2008的NA基因的实施例2 Re-MuH5-DIVA-ΔNS疫苗株在鸡胚上滴度可达到5.5log2 HA 效价。取F0和F3病毒通过RT-PCR扩增嵌合NA基因，经过测序证明嵌合NA基因可以稳定遗传给子代病毒。
从表1中还可看出，虽然含突变型ΔNS的疫苗株比野生型的生长滴度低2 log2～3 log2，但是含突变型ΔNS的疫苗株的安全性更好。
Figure PCT180082GZ-appb-I000002
来自不同群的B型流感病毒代表株B/Brisbane/60/2008（Victoria群）和Massachusetts/2/2012（Yamagata群）二者NA全基因核苷酸序列的同源性为94.9%，氨基酸序列同源性为94.9%；二者编码NA蛋白胞外区的DNA序列的同源性为95.1%，NA蛋白胞外区的氨基酸序列的同源性为94.6%。由于B型流感只分为Victoria和Yamagata群，本发明证明了来自这两个群的NA的代表株（实施例1和实施例2）均能和H5的HA有较好的兼容性，说明B型流感病毒的NA基因均可用于制备区分免疫和感染A型流感病毒的H5亚型禽流感疫苗株。
实施例3 Re-MuH5-DIVA-ΔNS灭活疫苗的制备
收取上述实施例制备的Re-MuH5-DIVA-ΔNS疫苗株F0、F1、F2或F3代尿囊液50ml，用终浓度0.25%的福尔马林溶液37℃灭活24h。将灭活后的尿囊液加入2%的Tween-80,待充分溶解后与含有3%Span 80的白油乳化，乳化比例为1：3，剪切乳化速度12000rpm，3min。经剂型检验、粒度检验、粘度检验、稳定性检验，确定灭活油苗为乳白色的油包水型乳剂，粘度低，颗粒大小均匀，稳定性好，适宜注射。
实施例4 Re-MuH5-DIVA-ΔNS灭活疫苗免疫动物的效果检测
方法：将10只3周龄SPF鸡免疫本发明制备的Re-MuH5-DIVA-ΔNS疫苗，0.3ml/只，颈部皮下注射，免疫后21天采血，分离血清，测定HI抗体。
结果：实验表明Re-MuH5-DIVA-ΔNS刺激机体产生高水平的HI抗体，其HI效价在第3周的平均效价（log2）为9.5±0.85。HA和HI试验参考GBT 18936-2003（高致病性禽流感诊断技术）。
实施例5 血清学实验
将现有A型流感的N1，N2，N6，和N9基因通过KpnI 和NheI位点克隆到pCAGGS真核表达质粒，命名为pCAGGS-N1, pCAGGS-N2, pCAGGS-N6, pCAGGS-N9。把1μg 各个pCAGGS-N1, pCAGGS-N2, pCAGGS-N6, pCAGGS-N9 质粒转染到预先在铺24孔细胞培养板上293T细胞。转染后30h，用免疫荧光方法检测以下7组鸡血清和N1，N2，N6，N9的反应性。
7组鸡血清的情况如下：
抗Re-MuH5-DIVA-ΔNS鸡血清：只免疫本发明Re-MuH5-DIVA-ΔNS灭活疫苗的鸡血清；
抗H5N1标准:H5N1标准血清，购于哈尔滨兽医研究所。
抗H5+H7血清：免疫H5N1 Re-8株+H7N9 Re-1株全病毒灭活疫苗的临床血清。
抗N1鸡血清：周龄SPF鸡分别免疫（肌肉注射）100μg pCAGGS-N1，免疫4周后采集全血，制备血清。
抗N2鸡血清：周龄SPF鸡分别免疫（肌肉注射）100μg pCAGGS-N2，免疫4周后采集全血，制备血清。
抗N6鸡血清：周龄SPF鸡分别免疫（肌肉注射）100μg pCAGGS-N6，免疫4周后采集全血，制备血清。
抗N9鸡血清：周龄SPF鸡分别免疫（肌肉注射）100μg pCAGGS-N9，免疫4周后采集全血，制备血清。
免疫荧光方法如下：
1）在每细胞加入0.5ml 的4%多聚甲醛固定20分钟，然后用PBS洗三遍。
2）0.2%Triton X 100通透10分钟，然后用PBS洗三遍。
3）5% BSA 封闭 1小时，然后用PBS洗三遍。
4）一抗用含有1%BSA的PBS稀释相应倍数（抗Re-MuH5-DIVA-ΔNS，抗H5N1标准，抗H5+H7，100倍；抗N1/N2/N6/N9，20倍），并在每孔加入0.5ml ，37℃ 湿盒内孵育1 小时，然后用PBS洗三遍。
5）鸡二抗（Alexa Fluor 594 驴抗鸡IgY）用含有1% BSA的PBS稀释200倍，加入0.5 ml 于每孔，室温孵育0.5小时，然后用PBS洗三遍。
6）用荧光显微镜观察。
结果：在293T 细胞分别表达流感N1, N2, N6和N9神经氨酸酶，用免疫荧光的方法，检测Re-MuH5-DIVA-ΔNS免疫后3周的血清是否与N1、N2、N6和N9反应，结果发现抗Re-MuH5-DIVA-ΔNS的血清不与N1,N2, N6和N9蛋白发生交叉反应（如表2和图3所示），现有全A型病毒疫苗（H5N1 Re-8株+H7N9 Re-1株）免疫的临床血清和抗H5N1标准血清能与N1蛋白发生强烈反应。此实验证实免疫Re-MuH5-DIVA-ΔNS疫苗不仅可以诱导高水平HI抗体而且可以区分免疫和感染动物，而且克服了现有H5亚型全病毒疫苗不能区分免疫和感染动物的缺点。
Figure PCT180082GZ-appb-I000003
实施例6 区分免疫和感染A型流感病毒的H5亚型禽流感疫苗株Re-MuH5-DIVA-ΔNS的制备方法
本实施例6中的制备方法同实施例1，除了在构建图1所示人工合成A/B嵌合型NA基因时，所用的B型流感病毒NA序列为编码NA全蛋白序列的DNA序列之外，其他均同实施例1，其中，该NA的DNA序列来自B型流感病毒Yamagata群中的B/Massachusetts/2/2012的NA全基因序列（Ping J et al, PNAS, 2016, 113(51):E8296-E8305）。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (17)

  1. 标签基因序列在制备区分免疫和感染A型流感病毒的H5亚型禽流感疫苗株中的应用，所述标签基因序列含有编码B型流感病毒NA蛋白胞外区氨基酸序列的DNA序列，或者含有编码与该胞外区氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列；
    或者，所述标签基因序列含有B型流感病毒NA基因中编码胞外区氨基酸序列的DNA序列，或者含有与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列；
    或者，所述标签基因序列为编码B型流感病毒NA蛋白的DNA序列，或者为编码与该NA蛋白氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列；
    或者，所述标签基因序列为B型流感病毒NA基因的DNA序列，或者为与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列。
  2. 根据权利要求1所述的应用，其特征在于，所述H5亚型禽流感疫苗株中还含有H5亚型HA基因或突变的H5亚型HA基因；所述突变的H5亚型HA基因能够将野生型HA蛋白中的氨基酸序列RERRRKRGLF突变为RETRGLF。
  3. 根据权利要求1任一所述的应用，其特征在于，所述B型流感病毒包括Victoria群和Yamagata群的B型流感病毒。
  4. 根据权利要求3所述的应用，其特征在于，所述B型流感病毒具体包括但不限于病毒株B/Massachusetts/2/2012，B/Brisbane/60/2008，B/Yamagata/16/1988，B/Malaysia/2506/04。
  5. 根据权利要求1所述的应用，其特征在于，所述标签基因序列两端还含有包装信号序列，所述包装信号为H1亚型NA的包装信号，或者为与H1亚型NA的包装信号具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的包装信号序列。
  6. 根据权利要求1所述的应用，其特征在于，所述标签基因序列两端还含有包装信号序列，其中5′端包装信号序列包括非编区序列、胞内区序列、跨膜区序列。
  7. 根据权利要求6所述的应用，其特征在于，胞内区序列编码5～7个氨基酸，氨基酸序列位于胞内。
  8. 根据权利要求6所述的应用，其特征在于，跨膜区序列编码24～32个氨基酸，氨基酸序列位于跨膜区。
  9. 根据权利要求6所述的应用，其特征在于，所述标签基因序列的5′端包装信号序列为SEQ ID NO:3，或者为与SEQ ID NO:3具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
  10. 根据权利要求1所述的应用，其特征在于，所述标签基因序列两端还含有包装信号序列，其中3′端包装信号序列为SEQ ID NO:4，或者为与SEQ ID NO:4具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
  11. 一种制备区分免疫和感染A型流感病毒的H5亚型禽流感疫苗株的方法，其特征在于，包括以下步骤：将标签基因序列与H5亚型禽流感病毒的HA基因或突变的H5亚型HA基因；通过反向遗传操作系统获救得到重组疫苗株，即区分免疫和感染A型流感病毒的H5亚型禽流感疫苗株；
    所述突变的H5亚型HA基因能够将野生型HA蛋白中的氨基酸序列RERRRKRGLF突变为RETRGLF；
    所述标签基因序列含有编码B型流感病毒NA蛋白胞外区氨基酸序列的DNA序列，或者含有编码与该胞外区氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列；
    或者，所述标签基因序列含有B型流感病毒NA基因中编码胞外区氨基酸序列的DNA序列，或者含有与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列；
    或者，所述标签基因序列为编码B型流感病毒NA蛋白的DNA序列，或者为编码与该NA蛋白氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列；
    或者，所述标签基因序列为B型流感病毒NA基因的DNA序列，或者为与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列。
  12. 根据权利要求11所述的方法，其特征在于，所述标签基因序列两端还含有包装信号序列。
  13. 根据权利要求12所述的方法，其特征在于，所述标签基因序列的5′端包装信号序列为SEQ ID NO:3，或者为与SEQ ID NO:3具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
  14. 根据权利要求12所述的方法，其特征在于，所述标签基因序列的3′端包装信号序列为SEQ ID NO:4，或者为与SEQ ID NO:4具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
  15. 根据权利要求11所述的方法，其特征在于，所述反向遗传操作系统获救过程中还用到6个PR8内部基因，即野生型的NS或突变的ΔNS基因与PB2、PB1、PA、NP、M；其中ΔNS为突变的NS基因，ΔNS的核苷酸序列如SEQ ID NO:5所示。
  16. 区分免疫和感染A型流感病毒的H5亚型禽流感疫苗株，其命名为H5亚型禽流感疫苗候选株Re-MuH5-DIVA-ΔNS，已保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:V201741。
  17. 权利要求16所述的疫苗株在制备禽流感疫苗中的应用。
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